Effects of improved early-life conditions on health, welfare, and performance of pigs raised on a conventional farm.

Nowadays, most pigs are raised indoors, on intensive farms providing a poor environment. In these conditions, the risk of the occurrence of damaging behaviours is high, with dramatic consequences for animal health and welfare as well as economic losses for farmers. Early-life conditions may predispose individuals to develop damaging behaviours later in life. In contrast, reinforcing affiliative behaviours between piglets before weaning might help to prevent tail-biting episodes. In this field study, we aimed at improving early-life conditions of piglets on a commercial farm by completely suppressing painful procedures and staggering their exposure to weaning stress factors. The alternative early-life management strategy combined housing in free-farrowing pens with temporary crating of the sow, socialisation during the lactation period with whole-life maintenance of the hierarchical groups, and delayed transfer to the postweaning room after sow removal. Control conditions included birth in farrowing crates, tail docking, absence of socialisation during the lactation period, abrupt weaning with immediate transfer to the postweaning room and mixing with non-littermates. We evaluated the health, welfare, and performance of alternatively raised pigs (n = 80) as compared to controls (n = 75). Visits were made throughout the lifespan of individuals to evaluate their growth and health status. Body and tail lesions were scored as proxy measures of aggressiveness and impaired welfare. Blood and bristle samples were periodically collected to evaluate stress, inflammation and immune competence. While the whole-life performance of pigs was similar among groups, the alternative early-life conditions prevented the growth slowdown usually observed after weaning. In addition, alternatively raised pigs displayed more neutrophils, eosinophils and monocytes the day after weaning, as well as higher C-Reactive Protein levels. One week later, their monocytes displayed greater phagocytic capacity. Altogether, these data suggest an enhanced innate immune competence for alternatively raised pigs around weaning. Piglets reared under alternative conditions also exhibited fewer and less severe body lesions than standard pigs, one week after weaning. In contrast, they showed more tail lesions on days 36 and 66 associated with greater levels of acute phase proteins (C-Reactive Protein and haptoglobin). To conclude, alternative early-life management better prepared piglets for weaning. However, the whole-life maintenance of early-established social groups was not sufficient to prevent the occurrence of damaging behaviours in undocked pigs.

Fetal calf serum-primed dendritic cells induce a strong anti-fetal calf serum immune response and diabetes protection in the non-obese diabetic mouse.

In recent years, several investigators have shown that transfer of dendritic cells (DC) prevents diabetes development in non-obese diabetic (NOD) mice. Accumulating evidences showing that DC cultured in medium containing fetal calf serum (FCS) can induce a dominant unspecific immune response in tumor models after i.v. injection prompted us to investigate if the protecting effect of DC on diabetes development in NOD mice might be supported by the induction of an anti-FCS immune response in recipient mice. Five-week-old NOD mice were injected i.v. with FCS-cultured bone marrow-derived DC or PBS as control. Levels of anti-FCS and anti-bovine serum albumin (BSA) antibodies were measured in the serum of recipient mice. Anti-FCS cellular immune responses were also analysed after a single DC injection using in vitro proliferation of splenocytes either in RPMI supplemented with FCS, AIMV-BSA or RPMI containing autologous mouse serum or BSA as a read out. DC injection prevented diabetes development in NOD mice and high titers of anti-FCS and anti-BSA antibodies were detected in serum of all DC-injected mice. Besides, splenocytes isolated from DC-injected mice proliferated vigorously in the presence of bovine proteins in contrast to splenocytes isolated from control mice but removing bovine proteins abrogated the high level of proliferation of those splenocytes suggesting that lymphocytes have been primed against bovine proteins in vivo after DC injection. All together, our data show that DC transfer induced cellular and humoral anti-FCS immune responses in recipient NOD mice suggesting that the protective effect of DC relies on their unspecific immunostimulatory effects.

Culture medium modulates the behaviour of human dental pulp-derived cells: technical note.

In vitro approaches have extensively been developed to study reparative dentinogenesis. While dental pulp is a source of unidentified progenitors able to differentiate into odontoblast-like cells, we investigated the effect of two media; MEM (1.8 mM Ca and 1 mM Pi) and RPMI 1640 (0.8 mM Ca and 5 mM Pi) on the behaviour of human dental pulp cells. Our data indicate that MEM significantly increased cell proliferation and markedly enhanced the proportion of alpha-smooth muscle actin positive cells, which represent a putative source of progenitors able to give rise to odontoblast-like cells. In addition, MEM strongly stimulated alkaline phosphatase activity and was found to induce expression of transcripts encoding dentin sialophosphoprotein, an odontoblastic marker, without affecting that of parathyroid hormone/parathyroid hormone related protein-receptor and osteonectin. In conclusion, these observations demonstrate that not only proliferation but also differentiation into odontoblast-like cells was induced by rich calcium and poor phosphate medium (MEM) as compared to RPMI 1640. This study provides important data for the determination of the optimal culture conditions allowing odontoblast-like differentiation in human pulp cell culture.

Functional canine dendritic cells can be generated in vitro from peripheral blood mononuclear cells and contain a cytoplasmic ultrastructural marker.

For physiological and practical reasons the dog is a large animal model used increasingly to study the pathogenesis of human diseases and new therapeutic approaches, in particular for immune disorders. However, some immunological resources are lacking in this model, especially concerning dendritic cells. The aim of our study was to develop an efficient method to generate dendritic cells (DC) in vitro from dog peripheral blood mononuclear cells (PBMC) and to characterize their functional, structural and ultrastructural properties. PBMC were cultured in vitro with IL-4 and GM-CSF. After 1 week of culture, a great proportion of non-adherent cells displayed typical cytoplasmic processes, as evidenced both by optical and electron microscopy. Cytometric analysis revealed the presence of 41.7+/-24.6% CD14+ cells expressing both CD11c and MHC class II molecules. Allogeneic mixed lymphocyte reactions confirmed the ability of these cultures to stimulate the proliferation of allogeneic lymphocytes as already reported as a characteristic of DC in other species. In addition, we describe for the first time the presence in canine DC of cytoplasmic periodic microstructures (PMS) that could represent ultrastructural markers of canine DC. In conclusion, our study provides an easy method to generate DC from PBMC in sufficient numbers for immunological in vitro investigations in dogs, a pre-clinical model for many human diseases.

Antigen-presenting cells that phagocytose apoptotic tumor-derived cells are potent tumor vaccines.

We have reported recently that treatments combining injections of apoptotic bodies from tumor cells and interleukin 2 led to tumor regression and induced specific protection. In the present study, we show that tumor-bearing rats were cured with an 80% success rate by injection of antigen-presenting cells (APCs) that had phagocytosed apoptotic bodies derived from poorly immunogenic tumor cells, whereas phagocytic cells exposed to nonapoptotic tumor cell extracts were essentially without effect. In addition, curative vaccination using APCs that had phagocytosed apoptotic bodies generated a tumor-specific cytotoxic T-cell response and long-term protection from parental tumor challenge. Thus, systems using the processing and presentation of antigenic molecules by professional APCs after phagocytosis of apoptotic bodies appear to offer new possibilities for anticancer treatment.

Evidence for control of nitric oxide synthesis by intracellular transforming growth factor-beta1 in tumor cells. Implications for tumor development.

Transforming growth factor-beta1 (TGF-beta1) has been shown to down-regulate NO synthesis in a variety of normal cells. In the present study, we investigated the influence of TGF-beta1 upon NO production in tumor cells and its consequences for tumor development. During the growth of PROb colon carcinoma cells intraperitoneally injected in syngeneic BDIX rats, intratumoral concentration of TGF-beta1 increases while NO concentration stays very low. Tumor regression induced by intraperitoneal injections of a lipid A is associated with a decrease in TGF-beta1 and an increase in NO intratumoral concentration. In these tumors, PROb tumor cells are the NO- and TGF-beta1-secreting cells. Using PROb cells transfected with an expression vector coding for TGF-beta1 antisense mRNA, we demonstrate in vitro that there is an inverse correlation between the amount of TGF-beta1 secreted and the ability of PROb cells to secrete NO. As the same results were obtained in the presence of an anti-TGF-beta type II receptor neutralizing antibody, and as exogenous TGF-beta1 is without any effect on NO secretion by PROb cells, TGF-beta1 apparently down-regulates NO synthesis in PROb cells by an intracellular mechanism. These results suggest that endogenous TGF-beta1 constitutes a potential target in a search for new antitumoral agents.

Immunomodulatory effects of tumor-associated fibroblasts in colorectal-tumor development.

In order to elucidate the role of myofibroblasts in tumor development, we compared fibroblastic reactions and their implications in the immune response in progressive and regressive rat colorectal-tumor models. Immunohistochemical analyses revealed that T lymphocytes and monocytes/macrophages were found outside progressive tumors that were surrounded by a large sheath of myofibroblasts. In vitro experiments using fibroblast- vs. myofibroblast-containing collagen gels showed that the mechanical properties of these tumor-activated myofibroblasts prevent penetration of T lymphocytes and macrophages within tumor nodules. These results indicate that tumor-activated myofibroblasts may prevent physical contact between cancer-cells and immune cells, an essential phenomenon for effective destruction of cancer cells. Successful immunotherapy against cancer should therefore include complementary treatments against these tumor-associated fibroblasts.

Role of antigen-presenting cells in long-term antitumor response based on tumor-derived apoptotic body vaccination.

Cellular therapy prospects for cancer are based on the development of T cell response, resulting in efficient tumor rejection and long-term protection. We have previously shown that treatment combining injection of interleukin-2 and tumor-derived apoptotic bodies, but not tumor cell extracts, permits to reject parental tumor in 40% of rats. We observed the implication of antigen-presenting cells (APCs) and tumor-derived apoptotic bodies in the rejection of established peritoneal carcinomatosis. We demonstrated that apoptotic bodies could be efficiently phagocytosed by monocytes, triggering them to an APC phenotype. When using these phagocytosing APCs, derived from peritoneal or blood monocytes, the remission rate reached 80% of rats. However, due to the lack of specific markers of rat monocyte-derived cells, the precise role of APCs, dendritic cells and/or macrophages responsible for this therapeutic improvement remained to be clarified. In order to elucidate this question, we developed an in vivo preventive cellular therapy based on tumor-derived apoptotic bodies, where macrophages were either depleted or activated. We report here that in a preventive antitumoral apoptotic body vaccination that allows survival for 40% of treated rats, the antitumor response was characterized by a specific long-term memory (cured rats rejected a second parental tumor cell challenge). Depletion of resident macrophages with silica or clodronate liposomes appeared to promote apoptotic body vaccination efficiency, increasing the treatment to 66% of success. In this case, FACS analysis showed that peritoneal cells present are essentially immature APCs and freshly recruited NK cells. In contrast, the onset of peritoneal inflammation by thioglycollate, inducing massive recruitment and activation of macrophages, reduced the overall survival, whatever the treatment was. Also, even though the surviving rate was better in silica-treated rats than control, no long-term protection was elicited. Our data suggest that massive inflammation, recruiting numerous activated macrophages, could inhibit tumor antigen presentation by 'professional' APCs having phagocytosed apoptotic bodies, and defavor a specific antitumoral T cell response. Although effective responses were developed against parental tumor cells with silica/apoptotic body treatment, they seemed only partial, limited to primary cytotoxic efficiency. In conclusion, even if macrophages did not appear necessary for a primary response to tumor cells, these cells seemed to be implicated in the establishment of memory and long-term antitumor response.

Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells.

Targeted disruption of the single mutant K-ras allele in two human colorectal carcinoma cell lines (DLD-1 and HCT-116) leads to loss of tumorigenic competence in nude mice with retention of ability to grow indefinitely in monolayer culture. Because expression of the mutant K-ras oncogene in these cell lines is associated with marked up-regulation of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), we sought to determine whether this potent angiogenesis inducer plays a role in K-ras-dependent tumorigenic competence. Transfection of a VEGF121 antisense expression vector into DLD-1 and HCT-116 cells resulted in suppression of VEGF/VPF production by a factor of 3- to 4-fold. The VEGF/VPF-deficient sublines, unlike the parental population or vector controls, were profoundly suppressed in their ability to form tumors in nude mice for as long as 6 months after cell injection. In contrast, in vitro growth of these sublines was unaffected, thus demonstrating the critical importance of VEGF/VPF as an angiogenic factor for HCT-116 and DLD-1 cells. Transfection of a full-length VEGF121 cDNA into two nontumorigenic mutant K-ras knockout sublines resulted in a weak but detectable restoration of tumorigenic ability in vivo in a subset of the transfectants, with no consistent change in growth properties in vitro. The findings indicate that mutant ras-oncogene-dependent VEGF/VPF expression is necessary, but not sufficient, for progressive tumor growth in vivo and highlight the relative contribution of oncogenes, such as mutant K-ras, to the process of tumor angiogenesis.

[Induction of apoptosis, in vitro and in vivo, on colonic tumor cells of the rat after sodium butyrate treatment]. / Induction de l'apoptose, in vitro et in vivo, sur cellules tumorales coliques de rat après traitement par le butyrate de sodium.

Sodium butyrate (NaB) is known to induce the process of cell differentiation, particularly for epithelial colonic cells. We previously observed that treatment with NaB in association with interleukin 2 (IL2), cures 60% of peritoneal carcinomatosis induced by injection of DHDK12/TRb cells in syngenic rats [15]. In the present work, we evidenced in vitro metabolic alterations of the DHDK12/TRb cell line treated with NaB, followed by an apoptotic process. Flow cytometric analysis evidenced that the tumour cells were arrested in the G1 and G2 phases of the cell cycle for the adherent cells to the plastic. Biological analysis of cells and debris released in the culture medium were essentially apoptotic cells. Complementary, the NaB-induced apoptotic process was confirmed by the staining of the nucleus from releasing cells by Hoechst 33258 and the DNA fragmentation revealed by DNA electrophoresis. Mitochondrial activity and glucose consumption were significantly stimulated after NaB treatment, which reveal an alteration of the metabolic activity of the treated tumour cells. As a consequence, we measured a significant increase of the active TGF beta 1 production, a cytokine previously described to participate to the epithelial cell differentiation. These in vitro data were confirmed in vivo showing a significant expression of apoptotic tumour cells in NaB- or NaB/IL2-treated tumours. Thus, the present results in the rat peritoneal carcinomatosis treatment show that combination of apoptotic process induced by NaB with immunostimulation by IL2 has powerful therapeutic properties.

The role of fibroblasts in tumor behavior.

The prominent desmoplastic or stromal reaction seen in many invasive carcinomas suggests that stromal cells play a role in cancer pathogenesis. Investigations based on cell typing, using antibodies to cytoskeletal constituents, have revealed that most tumors contain various types of fibroblasts. Stromal cells with myofibroblastic differentiation features are the predominant cell type at the periphery of epithelial tumors. These tumor-activated fibroblasts play a major role in tumor development and spread, affecting the proliferation, differentiation, invasion or regression of cancer cells. This review considers the events inducing the different fibroblastic responses and the role of tumor-activated fibroblasts in both tumor development and anti-cancer treatments.

Optimisation of CCL64-based bioassay for TGF-beta.

Transforming growth factor beta (TGF-beta) is released by a variety of cells and known to be involved in many different processes including the immune response, wound healing and carcinogenesis. As most experimental investigations have been based on quantitative analysis of TGF-beta production using a bioassay, it seemed important to test the validity and limitations of this method. This paper analyses several parameters that may impair TGF-beta quantification by bioassay. Recommendations are made concerning the influence of technical parameters and the presence of other cytokines (EGF and bFGF) commonly released by cultured cells to which the Mv1Lu mink lung epithelial cell line (CCL64) is sensitive.

The role of transforming growth factor beta 1 in the fibroblastic reaction associated with rat colorectal tumor development.

Many tumors are surrounded by a highly fibrous stroma composed of fibroblasts and extracellular matrix. This desmoplastic response has been suggested to both inhibit and favor tumor progression. The present study deals with the effects of tumor cells on the fibroblastic reactions they cause and relates this to progression or regression of tumors. Two rat colon carcinoma cell lines, one which develops progressive tumors when injected s.c. in syngeneic animals (PROb cell line) and the other which develops regressive tumors in similar conditions (REGb cell line), were compared by the fibroblastic reaction which they cause. Comparative histological analysis of progressive and regressive tumors developed by the two cell lines showed a significant but opposite response of fibroblastic compartment. The progressive tumor nodules were observed to grow within a loose tissue, whereas the regressive tumor cells were surrounded by a fibrous capsule. Immunohistological labelings revealed the presence of alpha-smooth muscle actin-positive myofibroblasts during the tumor expansion, while these specific cells disappeared during the tumor regression. Immunostainings of transforming growth factor beta 1 showed an increasing staining of the progressive tumor cells during tumor development but a slight expression by tumor cells and stroma during the tumor regression. This growth factor was demonstrated to facilitate initial steps of the tumor progression by addition of active transforming growth factor beta 1 at the time of s.c. injection of PROb cells in syngeneic rat models. In vitro experimental analysis with the use of neutralizing antibody showed that active transforming growth factor beta produced by the progressive cells inhibited fibroblast proliferation and facilitated their differentiation into myofibroblasts. Since the number of myofibroblasts increased with time in progressive tumors, their presence may constitute a potential growth advantage for tumor growth. In contrast, our results indicated involvement of platelet-derived growth factor-like protein(s) in fibroblast proliferation under the control of regressive cells and the presence of an important sheath of alpha-smooth muscle actin-negative fibroblasts in regressive tumors may support a role for this growth factor in vivo. Thus, the ability of tumor cells to produce or induce the production of transforming growth factor beta or platelet-derived growth factor may give rise to a specific fibroblast reaction, which in turn may determine consequent tumor evolution.

[Role of TGF beta 1 in the stromal reaction to human colonic tumors]. / Implication du TGF beta 1 dans la réaction stromale aux tumeurs coliques humaines.

Two types of human fibroblasts have been isolated from a patient with a colon cancer with metastasis, one type was derived from a healthy part of the colon, and the other one isolated from a metastasized lymph node close to the intestine. These fibroblasts have been characterized for their expression of collagens type I, III and IV, vimentin, fibronectin, alpha-smooth muscle actin, laminin and desmin. The effects of conditioned media of human colon cancer cell lines, HT29, SW1116, LS180 and HCT8R, on the metabolism of these fibroblasts were tested. All the conditioned media stimulate both types of fibroblasts, as reflected by their incorporation of radiolabelled methionine and proline. Normal fibroblasts were highly sensitive to the conditioned media as compared to the activated fibroblasts. Additionally, the production of TGF beta 1 by the four colorectal cancer cell lines has been quantified, and significant qualitative (production of latent and/or active form) and quantitative differences were observed. The effects of the conditioned media of the four tumoral cell lines and exogenous TGF beta 1 on the proliferation of the two types of fibroblasts were compared. Our data indicated that the two types of fibroblasts respond differently to TGF beta 1 whereas they are both growth stimulated by the conditioned media, apart from the LS180 conditioned medium. We conclude that if TGF beta 1 acts in the fibroblastic reaction, additional factors are required.