Regulatory T-cell development and function are impaired in mice lacking membrane expression of full length intercellular adhesion molecule-1.

To further investigate the contribution of intercellular adhesion molecule-1 (ICAM-1) to adaptive immune responses, we analysed T-cell development and function in mice lacking full-length ICAM-1 (ICAM-1(tm1Jcgr) ). Compared with wild-type (ICAM-1(WT) ) mice, ICAM-1(tm1Jcgr) mice have impaired thymocyte development. Proportions and numbers of double negative, double positive, mature CD4(+) and CD8(+) thymocytes, as well as of regulatory T (Treg) cells were also significantly decreased. In the periphery, ICAM-1(tm1Jcgr) mice had significantly decreased proportions and numbers of naive and activated/memory CD4(+) and CD8(+) T cells, as well as of Treg cells, in lymph nodes but not in the spleen. In vitro activation of CD4(+) and CD8(+) T cells from ICAM-1(tm1Jcgr) mice with anti-CD3 antibodies and antigen-presenting cells (APCs) resulted in a significantly weaker proliferation, whereas proliferation induced with anti-CD3 and anti-CD28 antibody-coated beads was normal. In vivo immunization of ICAM-1(tm1Jcgr) mice resulted in normal generation of specific effector and memory immune responses that protect against a viral challenge. However, contrary to ICAM-1(WT) mice, immunization-induced specific effectors could not eradicate immunogen-expressing tumours. Treg cells from ICAM-1(tm1Jcgr) mice have abnormal activation and proliferation induced by anti-CD3 antibody and APCs, and have markedly decreased suppressive activity in vitro. In contrast to ICAM-1(WT) mice, they were unable to control experimentally induced colitis in vivo. Hence, our results further highlight the pleiotropic role of ICAM-1 in T-cell-dependent immune responses, with a major role in Treg cell development and suppressive function.

Delivery of the bacterial nitroreductase gene into endothelial cells prolongs the survival of tumour-bearing mice by bystander mechanisms.

A current target of cancer gene therapy is tumour vasculature. We present a gene-directed enzyme prodrug therapy (GDEPT) approach to target tumours in vivo by modifying endothelial cells (ECs) with the Escherichia coli nitroreductase (ntr) gene. Firstly, we isolated two ntr-transfected clones of the human umbilical vein endothelial cell line (HUV-EC-C/ntr+) that showed a differential sensitivity in vitro to the prodrug, dinitroaziridinylbenzamide (CB1954), with respect to untransfected HUV-EC-C cells (HUV-EC-C/ntr-). Then, these cells were injected subcutaneously into nude mice, either in association with the murine melanoma cell line, B16-F10 ('co-injected' groups), or into tumour-bearing animals ('post-injected' groups). After intratumoural injection, we demonstrated, using PCR analysis, that human ECs resided in the site of the injection without spreading to other organs, such as the liver or lung. After the treatment of mice with CB1954, we observed a prolonged survival of animals carrying the HUV-EC-C/ntr+ clones with respect to control animals injected with HUV-EC-C/ntr- cells. Significant differences in tumour growth were also observed and, after immuno-histological analysis, tumours carrying HUV-EC-C/ntr+ clones showed large areas of tumour necrosis, probably due to tumour ischemia, as well as the presence of major histocompatibility complex class-II (MHC-II) positive cells. Collectively, our data indicate that targeting of the tumour vasculature by this GDEPT strategy may be an efficient approach for cancer treatment in vivo, depending on two possible bystander mechanisms based on tumour ischemia and immune cell activation.

Anti-angiogenic strategies for cancer therapy (Review).

Acquired drug resistance to chemotherapy is a major problem in the treatment of cancer. After primary surgical intervention, followed by chemotherapy treatments, the majority of patients develop disease recurrence. This is due to tumor cell heterogeneity and genetic instability. In contrast to tumor cells, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate. Furthermore, tumor angiogenesis plays an important role in tumor development, vascular invasion and hematogenous metastasis. Thus, anti-angiogenic therapy directed against tumor ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance. We review different therapeutic approaches directed against tumor angiogenesis, showing potent antitumor activity in vitro and in vivo. These strategies involve the inhibition of the expression of proangiogenic molecules, as well as the overexpression of anti-angiogenic molecules by either injection of recombinant proteins or transfer of genes encoding anti-angiogenic molecules. The gene therapy approach based on the gene-directed enzyme prodrug therapy (GDEPT) system, as well as the use of endothelial progenitor cells (EPCs) as angiogenesis-selective gene-targeting vectors, will be further discussed.

Microbiota and mucosal immunity in amphibians.

We know that animals live in a world dominated by bacteria. In the last 20 years, we have learned that microbes are essential regulators of mucosal immunity. Bacteria, archeas, and viruses influence different aspects of mucosal development and function. Yet, the literature mainly covers findings obtained in mammals. In this review, we focus on two major themes that emerge from the comparative analysis of mammals and amphibians. These themes concern: (i) the structure and functions of lymphoid organs and immune cells in amphibians, with a focus on the gut mucosal immune system; and (ii) the characteristics of the amphibian microbiota and its influence on mucosal immunity. Lastly, we propose to use Xenopus tadpoles as an alternative small-animal model to improve the fundamental knowledge on immunological functions of gut microbiota.

Aponecrotic, antiangiogenic and antiproliferative effects of a novel dextran derivative on breast cancer growth in vitro and in vivo.

1. Since the sodium phenylacetate (NaPa) was reported to enhance the inhibitory effect of carboxymethyl benzylamide dextran (CMDB) on the breast cancer growth, we performed the esterification of CMDB with NaPa to obtain a new drug carrying the characteristics of these two components. A new molecule, phenylacetate carboxymethyl benzylamide dextran, was named NaPaC. 2. We investigated in vitro and in vivo the effects of NaPaC on MCF-7ras cell growth as well as its apoptotic and antiangiogenic effects in comparison to NaPa and CMDB. In addition, we assessed in vitro the antiproliferative effects of these drugs on other breast cancer cells, including MDA-MB-231, MDA-MB-435 and MCF-7. 3. In vitro, NaPaC inhibited MCF-7ras cell proliferation by 40% at concentration lower than that of CMDB and NaPa (12 microM vs 73 microM and 10 mM). IC(50)s were 6 and 28 microM for NaPaC and CMDB, respectively. The similar results were obtained for three other breast cancer cell lines. NaPaC reduced the DNA replication and induced cell recruitment in G(0)/G(1) phase more efficiently than its components. Moreover, it induced a cell death at concentration 1000-fold lower than NaPa. 4. In vivo, CMDB (150 mg kg(-1)) and NaPa (40 mg kg(-1)) inhibited the MCF-7ras tumour growth by 37 and 57%, respectively, whereas NaPaC (15 mg kg(-1)) decreased tumour growth by 66% without toxicity. 5. NaPa or CMDB reduced the microvessel number in tumour by 50% after 7 weeks of treatment. NaPaC had the same effect after only 2 weeks. After 7 weeks, it generated a large necrosis area without detectable microvessels. In vitro, NaPaC inhibited human endothelial cell proliferation more efficiently than CMDB or NaPa. NaPaC interacts with vascular endothelial growth factor as observed by affinity electrophoresis. 6. NaPaC acts like NaPa and CMDB but in more potent manner than components used separately. Its antiproliferative, aponecrotic and anti-angiogenic actions make it a good candidate for a new anti-cancer drug.

Similar immune responses and antitumor effects of murine dendritic cells loaded with either exogenous or endogenous-synthesized protein.

Conceptually, dendritic cells (DCs) take up and process exogenous antigens that are presented on MHC (major histocompatibility complex) class II molecules to stimulate CD4+ T cells, and present endogenously-produced proteins to CD8+ T cells through an MHC class I-dependent pathway. In this study, we compared the antitumor effects generated in vivo after vaccinations with DCs either loaded with exogenous protein (DCs-exo) or presenting antigens derived from endogenous-synthesized protein (DCs-endo). We used the murine MC26SC31 colon carcinoma cell line expressing on the cell surface the human CD4 (hCD4) molecule as a model tumor-associated antigen (TAA). Bone marrow (BM)-derived DCs-endo were obtained from histocompatible transgenic mice constitutively expressing hCD4; BM-derived DCs-exo were obtained after in vitro loading DCs, from syngenic normal mice, with purified soluble hCD4 protein (shCD4). Altogether, our results show that intravenous (i.v.) administration of DCs-exo and DCs-endo can trigger similar cytotoxic and humoral protecting immune responses against a lethal tumor challenge. hCD4 antigen-specific T cell-mediated cytotoxic immune response was not accompanied by elevated INF-gamma serum levels. This suggests, in the case of DCs-exo, a possible in vivo CTL cross-priming triggering a CD8+ T cell-mediated immune response in the absence of de novo antigen synthesis within the DCs.

A new phenylacetate-bisphosphonate inhibits breast cancer cell growth by proapoptotic and antiangiogenic effects.

Sodium phenylacetate (NaPa) and some bisphosphonates demonstrated antiproliferative and proapoptotic properties against cancer. We have previously shown that NaPa inhibited cell proliferation of MCF7-ras tumor breast cells both in vitro and in vivo. On the other hand, bisphosphonate activities have only been demonstrated in vitro. Here we evaluated the antitumor effects of a new bisphosphonate, the phenylacetate-bisphosphonate (PaBp), on human breast cancer MCF7 and MCF7-ras cell lines, both in vitro and in vivo. To our knowledge, this is the first report indicating the use of a bisphosphonate derivative as a powerful cytostatic and cytotoxic agent, with proapoptotic and antiangiogenic properties on human breast cancer cells lines, with no animal toxicity.

Fetal pancreas transplants are dependent on prolactin for their development and prevent type 1 diabetes in syngeneic but not allogeneic mice.

Transplantation of adult pancreatic islets has been proposed to cure type 1 diabetes (T1D). However, it is rarely considered in the clinic because of its transient effect on disease, the paucity of donors, and the requirement for strong immunosuppressive treatment to prevent allogeneic graft rejection. Transplantation of fetal pancreases (FPs) may constitute an attractive alternative because of potential abundant donor sources, possible long-term effects due to the presence of stem cells maintaining tissue integrity, and their supposed low immunogenicity. In this work, we studied the capacity of early FPs from mouse embryos to develop into functional pancreatic islets producing insulin after transplantation in syngeneic and allogeneic recipients. We found that as few as two FPs were sufficient to control T1D in syngeneic mice. Surprisingly, their development into insulin-producing cells was significantly delayed in male compared with female recipients, which may be explained by lower levels of prolactin in males. Finally, allogeneic FPs were rapidly rejected, even in the context of minor histocompatibility disparities, with massive graft infiltration with T and myeloid cells. This work suggests that FP transplantation as a therapeutic option of T1D needs to be further assessed and would require immunosuppressive treatment.

Bystander cell killing spreading from endothelial to tumor cells in a three-dimensional multicellular nodule model after Escherichia coli nitroreductase gene delivery.

Tumor cells are elusive targets for standard anticancer chemotherapy due to their heterogeneity and genetic instability. On the other hand, proliferating host endothelial cells (ECs) are genetically stable and have a low mutational rate. Thus, antiangiogenic therapy directed against tumor's ECs should, in principle, improve the efficacy of antitumor therapy by inducing little or no drug resistance. Here we present a gene-directed enzyme prodrug therapy (GDEPT) strategy for targeting the tumor vasculature, using the Escherichia coli nitroreductase (ntr) gene delivery associated with the treatment with the prodrug CB1954. In a first time we demonstrated the ability of the ntr/CB1954 system to induce an apoptotic-mediated cell death on monolayer cultures of human umbilical vein ECs (HUV-EC-C). Then, when ntr-transfected HUV-EC-C cells (HUV-EC-C/ntr(+)) were associated in a three-dimensional (3-D) multicellular nodule model with untransfected B16-F10 murine melanoma cell line, we observed a CB1954-mediated bystander cell killing effect from endothelial to neighboring melanoma cells. To our knowledge, this is the first report indicating that GDEPT-based antiangiogenic targeting may be an effective approach for cancer treatment relied on the spreading of the bystander effect from endothelial to tumor cells.