Search

Bovine herpesvirus-1 (BoHV-1) uses many mechanisms to elude the immune system; one of them is spreading intracellularly, even in the presence of specific antiviral antibodies. Cytotoxic T lymphocytes (CTLs) are necessary to eliminate the virus. The main preventive strategy is vaccination based on inactivated virus. These vaccines are poor inducers of cellular immune responses, and complicate serological diagnosis and determination of the real prevalence of infection. DNA vaccines are a good option because of the capacity of Differentiating Infected from Vaccinated Animals-(DIVA vaccine)-and may be the best way to induce cytotoxic responses. Although this type of vaccines leads to only weak "

Bovine leukemia virus (BLV) is a retrovirus that causes malignant B-cell lymphoma in up to ten-percent of infected cattle. To date, the mechanisms of BLV linked to malignant transformation remain elusive. Although BLV-encoded miRNAs have been associated with the regulation of different genes involved in oncogenic pathways, this association has not been evaluated in cattle naturally infected with BLV. The objective of this study was to determine the relative expression of BLV-encoded miRNA blv-miR-b4-3p, the host analogous miRNA bo-miR-29a and a couple of potential target mRNAs (HBP-1 and PXDN, with anti-tumorigenic function in B-cells), in cattle naturally infected with BLV compared to uninfected animals (control group). We observed that PXDN was significantly downregulated in BLV-infected cattle (P = 0.03). Considering the similar expression of endogenous bo-miR-29a in both animal groups, the downregulation of PXDN in BLV-naturally infected cattle could be linked to blv-miR-b4-3p expression in these animals. Knowing that PXDN is involved in anti-tumoral pathways in B-cells, the results presented here suggest that blv-miR-b4-3p might be involved in BLV tumorigenesis during natural infection with BLV in cattle.

Foot-and-Mouth Disease (FMD) is an acute viral disease that causes important economy losses. Vaccines with new low-cost adjuvants that stimulate protective immune responses are needed and can be assayed in a mouse model to predict their effectiveness in cattle. Immunostimulant Particle Adjuvant (ISPA), also known as cage-like particle adjuvant, consisting of lipid boxes of dipalmitoyl-phosphatidylcholine, cholesterol, sterylamine, alpha-tocopherol, and QuilA saponin, was shown to enhance protection of a recombinant vaccine against Trypanosoma cruzi in a mouse model. Thus, in the present work, we studied the effects on the magnitude and type of immunity elicited in mice and cattle in response to a vaccine based on inactivated FMD virus (iFMDV) formulated with ISPA. It was demonstrated that iFMDV–ISPA induced protection in mice against challenge and elicited a specific antibody response in sera, characterized by a balanced Th1/Th2 profile. In cattle, the antibody titers reached corresponded to an expected percentage of protection (EPP) higher than 80%. EPP calculates the probability that livestock would be protected against a 10,000 bovine infectious doses challenge after vaccination. Moreover, in comparison with the non-adjuvanted iFMDV vaccine, iFMDV–ISPA elicited an increased specific T-cell response against the virus, including higher interferon gamma (IFNγ)+/CD8+ lymphocyte production in cattle. In this work, we report for first time that an inactivated FMDV serotype A vaccine adjuvanted with ISPA is capable of inducing protection against challenge in a murine model and of improving the specific immune responses against the virus in cattle. Fil: Bidart, Juan Esteban. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Kornuta, Claudia Alejandra. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Gammella, Mariela Vanesa. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Gnazzo, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Puerto Iguazú; Argentina Fil: Soria, Ivana. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Langellotti, Cecilia Ana. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Mongini, Claudia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Galarza, Roxana. Instituto Nacional de Tecnologia Agropecuaria. Centro Regional Buenos Aires Sur. Estacion Experimental Agropecuaria Cuenca del Salado. Agencia de Extension Rural Chascomus.; Argentina Fil: Calvinho, Luis Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina Fil: Lupi, Giuliana Antonella. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; Argentina Fil: Quattrocchi, Valeria. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina Fil: Marcipar, Iván Sergio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Litoral; Argentina Fil: Zamorano, Patricia Ines. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentina

T-cell lymphomas include diverse malignancies. They are rare, some have low survival rates and they lack curative therapies. The aim of this work was to assess whether employing the TLR7 agonist imiquimod and the T-cell costimulatory molecule CD40 or the combination of both as adjuvants of a cell lysate vaccine could enhance the antitumor immune response using a murine T-cell lymphoma model. Immunization with LBC-lysate and imiquimod protected almost all vaccinated animals. A specific humoral and a Th1-type cellular immunity were induced in mice that rejected the lymphoma, characterized by an elevated number of CD4 + T-cells and secretion of IFN-γ, locally and systemically. In contrast, CD40 alone or in combination with imiquimod did not improve the protective response obtained with LBC-lysate and imiquimod. Systemic administration of imiquimod proved to have high potential to serve as a vaccine adjuvant for the treatment of T-cell lymphomas and was effective in this immunotherapy model. Fil: Di Sciullo, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Menay, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Cocozza, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Gravisaco, María José. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Mongini, Claudia. Instituto Nacional de Tecnología Agropecuaria; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina

Exosomes are 60–150 nm small extracellular vesicles (EVs) released by most cells. Tumor-cell-derived exosomes, used as a vaccine, elicit a specific cytotoxic response against tumor cells, usually with a greater immunogenicity than tumor-cell lysates. However, the number of exosomes isolated from culture cells is limited. In recent studies, it was observed that cells respond to different stressor stimuli such as cytotoxic drugs, hypoxia, acidosis, or radiation by increasing the release of EVs. In this study, using the murine LBC T-cell lymphoma, we found that cyclophosphamide significantly increased EVs yield. These EVs express exosome marker proteins such as TSG-101, CD9, CD81, and CD63. Furthermore, similar humoral and cellular immune responses were induced in vivo by EVs isolated from LBC-tumor cells whether they were grown under normal culture conditions (EVs C) or in the presence of cyclophosphamide (EVs CTX). Mice vaccinated either with EVs C or EVs CTX were similarly protected against an intraperitoneal challenge with LBC tumor cells. CD4+ and CD8+ IFN-γ secreting cells were induced in immunized mice and a specific cytotoxic cellular immune response was elicited in vitro. These results demonstrate that a Th1 response was induced by immunization with the EVs. Our findings suggest that treatment of tumor cells with cyclophosphamide is a useful method to enhance the secretion of EVs in sensitive cell lines without altering their antitumor properties and thus may be used to produce antigens for future design of cancer vaccines. Fil: Cocozza, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Menay, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Tsacalian, Rodrigo Farid. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Elisei, Analia. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit.; Argentina Fil: Sampedro, Pura Isabel. Universidad de Morón; Argentina Fil: Soria, Ivana. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit.; Argentina Fil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Gravisaco, María José. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Mongini, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit | Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas. Grupo Vinculado Incuinta Al Ivit.; Argentina. Universidad de Morón; Argentina

Extracellular vesicles (EVs), including endosome-derived nanovesicles (exosomes), are involved in cell–cell communication. Through transfer of their molecular contents, extracellular nanovesicles can alter the function of recipient cells. Due to these characteristics, EVs have shown potential as a new alternative for cancer immunotherapy. Tumor exosomes isolated from malignant ascites can activate dendritic cells, thereby priming the immune system to recognize and kill cancer cells. However, a suppressive role on tumor immune response has also been reported, suggesting that the neoplastic stage of carcinogenesis and the microenvironment where tumor cells grow may influence the amount of EVs released by the cell. This neoplastic stage and microenvironment may also impact EVs’ components such as proteins and miRNA, determining their biological behavior. Most T-cell lymphomas have an aggressive clinical course and poor prognosis. Consequently, complementary alternative therapies are needed to improve the survival rates achieved with conventional treatments. In this work, we have characterized EVs isolated from ascites of mice bearing a very aggressive murine T-cell lymphoma and have studied their immunogenic properties. Small EVs were isolated by differential centrifugation, ultrafiltration, and ultracentrifugation at 100,000 × g on a sucrose cushion. The EVs were defined as exosomes by their morphology and size analyzed by electron microscopy, their floating density on a sucrose gradient, as well as their expression of endosome marker proteins ALIX, TSG-101; the tetraspanins CD63, CD9, and CD81. In addition, they contain tumor antigens, the marker for malignancy CD24, the heat shock protein HSP-70, and an unusual surface expression of HSP-90 was demonstrated. The administration of EVs isolated from ascites (EVs A) into naïve-syngeneic mice induced both humoral and cellular immune responses that allowed the rejection of subsequent tumor challenges. However, the immunization had no effect on a non-related mammary adenocarcinoma, demonstrating that the immune response elicited was specific and also it induced immune memory. In vitro analysis demonstrated that T-cells from EVs A-immunized mice secrete IFN-γ in response to tumor stimulation. Furthermore, tumor-specific CD4+ and CD8+ IFN-γ secreting cells could be efficiently expanded from mice immunized with EVs A, showing that a T helper 1 response is involved in tumor rejection. Our findings confirm exosomes as promising defined acellular tumor antigens for the development of an antitumor vaccine. Inst. de Biotecnología Fil: Menay, Florencia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina, Aires; Argentina Fil: Herschlik, Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina, Aires; Argentina Fil: Cocozza, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina, Aires; Argentina Fil: Tsacalian, Rodrigo Farid. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina, Aires; Argentina Fil: Gravisaco, Marí­a José. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Di Sciullo, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina; Argentina Fil: Vendrell, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina, Aires; Argentina Fil: Waldner, Claudia Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina, Aires; Argentina Fil: Mongini, Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos. Facultad de Medicina; Argentina

Primary tumor excision is one of the most widely used therapies of cancer. However, the risk of metastases development still exists following tumor resection. The liver is a common site of metastatic disease for numerous cancers. Breast cancer is one of the most frequent sources of metastases to the liver. The aim of this work was to evaluate the efficacy of the orally administered Salmonella Typhi vaccine strain CVD 915 on the development of liver metastases in a mouse model of breast cancer. To this end, one group of BALB/c mice was orogastrically immunized with CVD 915, while another received PBS as a control. After 24 h, mice were injected with LM3 mammary adenocarcinoma cells into the spleen and subjected to splenectomy. This oral Salmonella-based vaccine produced an antitumor effect, leading to a decrease in the number and volume of liver metastases. Immunization with Salmonella induced an early cellular immune response in mice. This innate stimulation rendered a large production of IFN-γ by intrahepatic immune cells (IHIC) detected within 24 h. An antitumor adaptive immunity was found in the liver and celiac and portal lymph nodes (LDLN) 21 days after oral bacterial inoculation. The antitumor immune response inside the liver was associated with increased CD4(+) and dendritic cell populations as well as with an inflammatory infiltrate located around liver metastatic nodules. Enlarged levels of inflammatory cytokines (IFN-γ and TNF) were also detected in IHIC. Furthermore, a tumor-specific production of IFN-γ and TNF as well as tumor-specific IFN-γ-producing CD8 T cells (CD8(+)IFN-γ(+)) were found in the celiac and portal lymph nodes of Salmonella-treated mice. This study provides first evidence for the involvement of LDLN in the development of an efficient cellular immune response against hepatic tumors, which resulted in the elimination of liver metastases after oral Salmonella-based vaccination. Fil: Vendrell, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Mongini, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Gravisaco, Maria Jose Federica. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Canellada, Andrea Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentina Fil: Tesone, Agustina Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina

We investigated the use of a live, attenuated Salmonella enterica serovar Typhi vaccine strain as an antitumor immunotherapy. Mice bearing a subcutaneous tumor (LM3 mammary adenocarcinoma) were immunized on three occasions with S. Typhi strain CVD 915 by injection into the tumor, the peritumoral tissue and the draining lymph node areas; this procedure was termed Salmonella multiple treatment (Salmonella MT). Tumor-bearing mice subjected to the Salmonella MT exhibited reduced tumor growth, prolonged survival and reduced incidence of lung metastases, compared to untreated mice. We examined the mechanisms mediating this effect and found that Salmonella MT promoted an antitumor Th1-type response characterized by increased frequencies of IFN-γ-secreting CD4(+) T and CD8(+) T cells with reduction of regulatory T cells in tumor draining lymph nodes. The main cells infiltrating bacteria-treated tumors were activated neutrophils, which can exert an antitumor effect through the secretion of TNF-α. These results demonstrate for the first time the efficacy of an attenuated S. Typhi vaccine strain as a cancer immunotherapeutic agent. By potentiating the host antitumor immune response, this approach could be a powerful adjunct tool for cancer therapy.

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 (2AE5 · 10 8 CFU) were challenged i.p. with LBC cells (1AE0 · 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 IFNc, 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.

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.

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.

From the time when they were first described in the 1970s by the group of Johnstone and Stahl, exosomes are a target of constant research. Exosomes belong to the family of nanovesicles which are of great interest for their many functions and potential for diagnosis and therapy in multiples diseases. Exosomes originate from the intraluminal vesicles of late endosomal compartments named multivesicular bodies and the fusion of these late endosomes with the cell membrane result in the release of the vesicles into the extracellular compartment. Moreover, their generation can be induced by many factors including extracellular stimuli, such as microbial attack and other stress conditions. The primary role attributed to exosomes was the removal of unnecessary proteins from the cells. Now, several studies have demonstrated that exosomes are involved in cell–cell communication, even though their biological function is not completely clear. The participation of exosomes in cancer is the field of microvesicle research that has expanded more over the last years. Evidence proving that exosomes derived from tumor-pulsed dendritic cells, neoplastic cells, and malignant effusions are able to present antigens to T-cells, has led to numerous studies using them as cell-free cancer vaccines. Because exosomes derive from all cell types, they contain proteins, lipids, and micro RNA capable of regulating a variety of target genes. Much research is being conducted, which focuses on the employment of these vesicles as biomarkers in the diagnosis of cancer in addition to innovative biomarkers for diagnosis, prognosis, and management of cardiovascular diseases. Interesting findings indicating the role of exosomes in the pathogenesis of several diseases have encouraged researchers to consider their therapeutic potential not only in oncology but also in the treatment of autoimmune syndromes and neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, in addition to infectious diseases such as tuberculosis, diphtheria, and toxoplasmosis as well as infections caused by prions or viruses such as HIV. The aim of this review is to disclose the emerging roles of exosomes in normal and pathological conditions and to discuss their potential therapeutic applications.

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.

In this article I examine the concept of bio-info sensibility recently developed By Alessandro Sarti and Franco Berardi (Bifo) and frame it in the realm of Guattari’s analysis about concerning techno-mutative conditions and Simondon’s idea of technoaesthetics. These two concepts allowed to place idea of bio-info sensibility within the constitution of a morphogenetic field delimited by both microphysical and aesthetic conditions. I will subsequently depart by constructing the conditions for this morphogenetic field along lines of thought opened by Simondon, Deleuze, Deleuze and Guattari. The concept of asymmetry in its elaboration by Alberto Toscano serves as a juncture between the development of theoretical conditions of individuation and their pragmatics of realization. The elaboration of this point of view will subsequently allow me to move back to Simondon’s theory in terms of a concrete research for possible components leading towards the morphogenetic construction of the concept of bio-info sensibility.

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. Images Figure 5

In this study, we assessed the effectiveness of a live, attenuated Salmonella enterica serovar Typhi (S. Typhi) vaccine strain as a cancer immunotherapy in a mouse model of metastatic T-cell lymphoma. EL4 tumor-bearing C57BL/6J mice immunized with S. Typhi strain CVD 915, by injection into the tumor and the draining lymph node areas, displayed a significant decrease in tumor growth, a reduction in the mitotic index (MI) of tumors, a delayed development of palpable lymph node metastases and most importantly improved survival, compared to untreated mice. Besides, complete tumor regression was achieved in a small number of bacteria-treated mice. A successful therapeutic response associated with a significant reduction of tumor mass was evident as early as 5 days after treatment. The administration of Salmonella to tumor-bearing mice promoted early cellular infiltration (mainly neutrophils) within the tumor, and was accompanied by a decreased intratumoral interleukin 10 production as well as by leukocyte expansion in tumor draining lymph nodes. A tumor-specific memory immune response was induced in most of cured animals, as evidenced by the lack of tumor growth after a rechallenge with the same tumor. EL4 cells cultured with live Salmonella failed to proliferate and underwent apoptosis in a dose-dependent, timedependent, and contact-dependent manner. To our knowledge, these results demonstrate for the first time the efficacy of a S. Typhi vaccine strain as an oncolytic and immunotherapeutic agent against a highly malignant tumor and support the use of S. Typhi-based vaccine strains in cancer therapy. Fil: Vendrell, Alejandrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Gravisaco, María J.. Instituto Nacional de Tecnologia Agropecuaria. Centro Nacional de Inv. Agropecuarias; Argentina Fil: Goin, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Pasetti, Marcela F.. University of Maryland; Estados Unidos Fil: Herschllik, Leticia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: de Toro, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Rodriguez, Carla Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Comision Nacional de Energia Atomica; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Larotonda, Gerardo. Lavet Lab Argentina; Argentina Fil: Mongini, Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Waldner, Claudia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina

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.

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.

A live system to release heterologous antigens using an attenuated Salmonella strain was developed. We transformed Salmonella typhimurium LVR03 (S. LVR03) with a recombinant pTECH2 vector encoding 0, 1, 2, and 4 tandem copies of an imunogenic peptide of bovine herpes virus-1 (BoHV-1) glycoprotein D (gD). The system used yielded peptides fused to the non-toxic C fragment of the tetanus toxin (TetC), which has been shown to have adjuvant properties. Inoculation of BALB/c mice with the transformed Salmonella strains gave rise to a mild self-limited infection, with primary replication of bacteria occurring in Peyer's patches, even when the bacteria was administered intranasally. Humoral and cellular immune responses directed against the BoHV-1 antigens were evaluated after oral or intranasal administration of the recombinant bacteria. The results showed that the S. LVR03-dimer vaccine induced specific humoral (IgG in serum and IgG(1) and IgA in saliva), and cellular immune responses (lymphoproliferation and lymphokine secretion), against not only the selected peptide and whole gD, but also against BoHV-1, when administered intranasally. This is the first time Salmonella has been used as an expression vector to induce immunity against BoHV-1. This work demonstrates the feasibility of using this antigen-release system and encourages future experimentation with a bovine experimental model.

Two methods which employ whole cells are described and compared for the detection of human IgG and IgM anti-brucella antibodies. Dot ELISA and ELISA were shown to be suitable for a screening diagnosis of human brucellosis. Titres of antibodies obtained by dot ELISA showed 100% coincidence for IgG and 97% for IgM, compared with agglutination and complement fixation tests; when ELISA was used 11 % positive sera were not detected. The comparison of these two methods with the conventional serological test kit indicated that both dot ELISA and ELISA were sensitive, reproducible and specific for the quantification of IgG and IgM anti-brucella antibodies.

The equal importance of the qualitative and quantitative characteristics of antigen presentation as well as the set of costimulatory signals provided by antigen presenting cells to T-cells in determining the outcome of T-cell responses at the time of antigen recognition is now clear. Moreover, an important function in innate mechanisms has been recently attributed to costimulatory molecules demonstrating their relevant role in different stages of immune response. In this paper, we demonstrated the ability of CD40L (CD154) and CD80 costimulatory molecules expression in a T-cell lymphoma to induce both T-cell dependent and independent immune responses leading to an important anti-tumor effect. CD40 expression by LBC cells enhanced only T-cell dependent anti-tumor immune response resulting in tumor rejection. Furthermore, this work represents the first report to describe complete tumor rejection after co-inoculation of lymphoma cells transfected with CD40L and CD80 in either presence or absence of CD40 expressing lymphoma cells. In addition, this synergistic effect resulted in long lasting immunity to parental tumor cells. Co-inoculation of tumor cells each genetically modified to express a different costimulatory molecule circumvents the need to co-transfect genetically unstable tumor cells and represents an option for those weakly or non-immunogenic tumors where either treatment alone proved to be inefficient. This strategy represents a promising approach for inducing anti-tumor immunity and provides a new rational design of cancer therapies.

In transfection protocols, the expression levels of the transgene is important to define, still is difficult to obtain in certain cell lines such as those derived from T-lymphoma cells. In this study we evaluate transgene expression kinetics in the presence and absence of two well known transcription activators such as phorbol-12-myristate13-acetate (PMA) and Ionomicin (IO). Three murine T lymphoma cell lines (LBC, EL4 and BW5147) were transfected by electroporation using green fluorescent protein (GFP) as a reporter gene and analyzed by flow cytometry. Addition of PMA/IO resulted in a significant increase of the Mean Fluorescence Intensity but not in GFP-positive cell percentages, either in transient or stable transfected LBC and EL4 cells. Remarkable, BW5147 cells showed low GFP induction with a significant increment only in stable transfected cells. Our results demonstrated that CMV promoter activity can be enhanced in transfected lymphoma cells by PMA/IO suggesting that transgene expression levels can be optimized by means of the use of transcription activators.

We studied the role of IL-2, IL-15, IL-10, TNF and IL-2 receptor complexes (IL-2R) produced constitutively by a T-cell lymphoma line (LBC) on their own proliferation. The constitutive expression of surface alpha, beta and gamma chains IL-2R was detected in tumor cells by flow cytometry. Using reverse-transcription PCR, mRNA for IL-2, IL-15, IL-10 and TNF were found to be present in LBC. In addition, tumor cells were found to constitutively express intracellular IL-2, IL-15, IL-10 and TNF. Despite the production of these cytokines by tumor cells, specific neutralising antibodies did not inhibit LBC proliferation; surprisingly, anti-IL-15 increased LBC cell growth. We also demonstrated that recombinant IL-2 or IL-15 enhanced LBC cell proliferation. Our data suggest that endogenous IL-2 and IL-15 may trigger the proliferation of lymphoma LBC cells, and so their growth could be regulated, at least partly, by IL-2/IL-15/IL-2R system. In addition, IL-10 and TNF, immunosuppressor and pro-metastatic cytokines, respectively, may promote the in vivo growth of the tumor. The fact that leukaemia-lymphoma cells produce simultaneously both IL-2 and IL-15 should be taken into consideration in the design of immunotherapy protocols directed to IL-2R.

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.

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.

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. Images Figure 1

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 105–1.3 × 105 Mr serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsine 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.

We report the first characterization of a mouse T-lymphoma cell line that surprisingly expresses cytoplasmatic (cy) cyCD4. Phenotypically, LBC cells are CD5+, CD8+, CD16+, CD24+, CD25+, CD2−/dim, CD3−/dim, TCRβ−/dim, TCRγδ−, CD154−, CD40−, and CD45R−. Coexpress cyTCRβ, 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...

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.

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.