An integrated approach of genetic resistance to Salmonella carrier state in fowls: from genetics to genomics and modelling.

Increasing resistance to acute Salmonellosis (that is, contamination level shortly after infection) is not sufficient to reduce the risk for consumers to be contaminated by Salmonella. Indeed, animals may remain contaminated at a low level for weeks or months. Increased resistance to the Salmonella carrier state, i.e., animals' ability to clear bacteria, is needed; it involves measuring bacterial contamination several weeks after inoculation with a low dose. To study such resistance traits, three convergent approaches were used. A quantitative trait loci (QTL) study was performed, taking advantage of inbred lines differing in resistance. Several QTLs controlling resistance at a younger age were identified and are currently being confirmed in a new cross before finer mapping, using advanced intercross lines. These inbred lines are also presently being compared using functional genomics. In parallel, a selection experiment for increased or decreased resistance at a younger and a later age was undertaken. Besides providing genetic models differing in their levels of resistance, it underlined the importance of the choice of selection criterion, whether marker assisted or not. Indeed, genes controlling resistance are strongly dependant on age; selecting for resistance at a younger age might result in increased susceptibility at an older age. Finally, the results of this experiment were used in a model of the intra-flock propagation of Salmonella. It showed that introducing a proportion of resistant animals within a flock of susceptible hens could dramatically change the evolution of contamination. Moreover, it demonstrated the magnitude of synergy between selection and vaccination, which should enhance the interest of increased resistance. The results show that selection for increased resistance to the Salmonella carrier state may be efficient, providing that the appropriate criteria of selection are used.

A model of Salmonella infection within industrial house hens.

Salmonella is one of the major sources of toxi-infection in humans. Incidences of human salmonellosis have greatly increased over the past 20 years and this can largely be attributed to epidemics of Salmonella enteritidis phage type 4 within poultry. The main concern with this bacterium is the existence of silent carriers, i.e. animals harbouring S. enteritidis without expressing any visible symptoms. In this article, we formulate a model for S. enteritidis transmission in hen houses, considering both the hens and the environmental bacterium contamination. By considering the hen's individual development of the disease, we build a model for the production of eggs contaminated by S. enteritidis. The objectives are to analyse the dynamic of the disease, and to provide understanding of measures to avoid the endemicity of S. enteritidis in industrial hen houses.

Oncogenic effects of overexpression of the interleukin-3 receptor on hematopoietic cells.

To elucidate the relationship between malignant transformation and cytokine receptor expression, the interleukin-3 receptor (IL-3R) complex was examined in an IL-3-dependent parental line and cells transformed by cytokines and oncogenes. In IL-3-dependent cells grown in medium containing optimum amounts of IL-3, the IL-3R complex was detected at low levels indicating that the receptor was down-regulated in response to IL-3. However, upon depletion of IL-3, IL-3R levels increased documenting that its expression correlated inversely with the concentration of IL-3 provided. In contrast, more IL-3 receptors were observed constitutively in autocrine-transformed derivative lines, which secreted suboptimal amounts of IL-3. To examine the effects of activated oncogenes on IL-3R expression in autocrine-transformed cells, the cells were infected with retroviral vectors containing various oncogenes. Decreased levels of IL-3R expression were observed in the oncogene-infected cells. These studies imply that important regulatory cross-talk occurs between ligands and their cognate receptors in cytokine-dependent hematopoietic cells. Deregulation of this ligand-receptor interaction in the oncogene-infected cells may be a consequence of the cells using modified signal transduction pathways which bypass the IL-3:IL-3R interaction. To determine the effects of IL-3 receptor overexpression on the cytokine dependency of hematopoietic cells, IL-3R alpha and beta cDNAs were inserted into retroviral vectors. Overexpression of either the alpha or beta chains did not directly relieve factor dependency, however, constitutive expression of the IL-3R alpha allowed the cells to proliferate in suboptimal concentrations of IL-3. Moreover, factor-independent transformants were subsequently isolated from pools of cells infected with viruses containing either the alpha or beta receptor cDNAs at a frequency of approximately 1 in 10(3) to 10(4) cells whereas such cells were not recovered from control cells. Deregulation of IL-3 receptor chain gene expression may provide a proliferative advantage to hematopoietic cells growing under conditions in which the cytokine is limiting and allow the development of a leukemia.

Prostaglandin E2 promotes the induction of anergy during T helper cell recognition of myelin basic protein.

Remission of experimental autoimmune encephalomyelitis (EAE) in Lewis rats may involve mediators such as prostaglandins (PG) that are produced within demyelinating lesions and are known to potently inhibit T cell responses. In support, this study shows that PGE2 inhibited myelin basic protein (MBP)-specific responses of proliferation and IL-2 production by continuously propagated lines of T-helper cells. Simultaneous exposure to PGE2 and immunogenic MBP rendered T cells profoundly anergic. Even after several weeks of propagation in IL-2-containing medium, anergic T cells exhibited marked reductions in MBP-stimulated proliferation and IL-2 production responses when restimulated with optimal concentrations of MBP and irradiated splenocytes (SPL). PGE2 did not block other measures of MBP-dependent activation, including induction of postactivation refractoriness in IL-2 production pathways, activation-dependent decreases in MBP reactivity, and activation-dependent increases in PGE2 sensitivity. Proliferative responses by anergic T cells were reduced in magnitude but were not altered in their sensitivity to MBP. PGE2-mediated anergy was manifest as an intrinsic deficit rather than an acquired suppressive activity and was associated with reduced mitogenic responsiveness and a block in IL-2 production pathways. Anergic T cells were responsive to IL-2 and eventually regained full antigenic reactivity after extended propagation in IL-2-supplemented medium. In summary, a limited exposure to PG had long-lasting inhibitory effects on subsequent T cell responsiveness to the target autoantigen MBP. These findings support the hypothesis that PG may promote disease remission by inducing anergy in helper T cells.

Differential-effects of viral and cellular oncogenes on the growth factor-dependency of hematopoietic-cells.

The effects of different viral and cellular oncogenes on the cytokine-dependency of murine hematopoietic cell lines were compared. The myeloid FDC-P1 cell line was sensitive to abrogation of growth factor-dependency by the constitutive expression of viral oncogenes (v-abl, v-src, v-Ha-ras, and v-fms) and the activated cellular oncogene BCR-ABL and Delta Nraf. The Delta Nraf encoded serine-threonine kinase was approximately 100-fold less efficient in relieving the factor-dependency of FDC-P1 cells than the other oncogenes examined. The synthesis of autocrine cytokines was not detected in the factor-independent FDC-P1 lines, indicating that the oncogene-mediated transformation occurred by a non-autocrine mechanism. A low frequency of cells were isolated after infection with the chronic retrovirus, murine leukemia virus and approximately 40% of these clones synthesized the autocrine lymphokine, granulocyte-macrophage colony stimulating factor. In contrast, only the v-abl and BCR-ABL oncogenes relieved the cytokine-dependency of the lymphoid FL5.12 cell line. In all the transformed cell lines, the rate of glucose transport was elevated above the basal level seen in uninfected cells indicating that this pivotal growth-regulated protein was associated with malignant transformation. In summary, these cell lines varied with respect to abrogation of growth factor-dependency as the myeloid FDC-P1 line was sensitive to transformation by all oncogenes examined whereas only the abl-family members would relieve the cytokine-requirement of lymphoid FL5.12 cells.

Autocrine growth-factor secretion after transformation of human cytokine-dependent cells by viral and cellular oncogenes.

The effects of viral and cellular oncogenes on a human erythroleukemic cell line (TF-1) were investigated. The TF-1 cell line required granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth but this factor-dependency was abrogated by the constitutive expression of either viral (v-fms, v-Ha-ras and v-src) or cellular oncogenes (BCR-ABL and Delta N-raf). Furthermore the overexpression of the human insulin-like growth factor-1 (IGF-1) receptor could substitute the dependency on GM-CSF with a requirement for either IGF-1 or insulin as a proliferative signal. An autocrine cytokine, (GM-CSF) was found in the supernatant of cells transformed by Delta N-raf (and to a lesser extent in cells infected with other oncogenes. The level of GM-CSF secreted by the Delta N-raf transformants was sufficient to support the proliferation of the parental cell line. GM-CSF mRNA transcripts were detected in the Delta N-raf-infected but not in the parental cells. No structural alterations of the GMCSF locus were seen in these cells. Together these observations indicated that overexpression of a raf oncogene resulted in the expression of GM-CSF transcripts. The rates of glucose transport were elevated above basal levels by GMCSF and by oncogene expression indicating that this pivotal control point of metabolism correlated with mitogenesis and malignant transformation. These studies indicate the importance of raf in growth regulation as its deregulation can lead to autocrine synthesis of cytokines in certain hematopoietic cells. Furthermore these results suggest a synergy between oncogene and cytokine gene regulation leading to autocrine growth factor expression and tumor progression.

Differential-effects of tumor promoters and cytokines on protooncogene expression in a hematopoietic cytokine-dependent cell-line.

In order to investigate the mechanisms by which cytokines and tumor promoters stimulate cell growth, the expression of genes implicated in the regulation of cellular proliferation were examined in an interleukin-3 (IL-3) dependent hematopoietic cell line. Upon stimulation of factor-deprived cells with IL-3, mRNA transcripts encoding the immediate-early genes: c-myc, jun-B, krox-20, beta-actin, and the cytokine genes: IL-4 and IL-6 were detected within 1 h. In contrast mRNA transcripts encoding the delayed-early genes: ornithine decarboxylase, p53, the IL-2 receptor-alpha, IL-4 receptor, and the T cell receptor c-gamma chains were observed at highest levels later. The tumor promoter, phorbol 12-myristate 13-acetate also stimulated the expression of many immediate-early genes, however, c-myc and the delayed-early genes were only detected when IL-3 was present. We conclude that cytokines and tumor promoters have distinct effects on proto-oncogene expression in hematopoietic cells which may affect the ability of these agents to promote cellular growth versus differentiation.