Faculty Bibliography

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR

Pre-T cell antigen receptor (pre-TCR) signaling plays a crucial role in the development of immature T cells. Although certain aspects of proximal pre-TCR signaling have been studied, the intermediate signal transducers and the distal transcription modulators have been poorly characterized. We report here a correlation between pre-TCR signaling and a biphasic rise in the cytosolic Ca2+ concentration. In addition, we show that constitutive pre-TCR signaling is associated with an increased rate of Ca2+ influx through store-operated plasma membrane Ca2+ channels. We show also that the biphasic nature of the observed pre-TCR-induced rise in cytosolic Ca2+ differentially modulates the activities of the transcription factors NF-kappaB and NFAT in developing T cells

In this report, we examined whether nitric oxide (NO) is involved in early embryo death. We have chosen various experimentally defined embryotoxic stimuli in mice and determined their ability to induce NO production by 2-cell stage embryos. The embryotoxic factors used were interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), 5-Azacytidine (5-AzaC) and the murine embryotoxic antibody DF4. We showed that in all cases the embryotoxic stimuli induced NO production by early stage embryos that correlated with the induction of the inducible and/or endothelial isoforms of NO synthase. This study was also extended to the human system where sera from women who aborted were tested for their ability to act embryotoxically by inducing NO in early mouse embryos and mature murine placenta. The results obtained confirmed the embryotoxic character of NO found in these particular sera leading to the hypothesis that NO plays a potential role in early embryo death

The detection of corticotropin-releasing hormone (CRH) in the pregnant and non-pregnant uterus has driven research to determine the role of this 41 amino acid neuropeptide in the female reproductive system. As concentrations of CRH mRNA and its peptide product are greater in the implantation sites of the early pregnant uterus compared with the regions between implantation sites, CRH has been hypothesised to participate in blastocyst implantation. Using the mouse system as an experimental model, we studied the distribution of CRH in the uterus during the oestrus cycle and early gestational period, and now provide evidence for its involvement in embryo implantation using cell culture techniques. The percentage of CRH-positive uterine cells and the amount of CRH released during anoestrus, pro-oestrus and oestrus were determined by immunofluorescence and ELISA experiments respectively. The highest number of intracellularly CRH-positive cells was obtained during pro-oestrus, whereas the highest CRH concentration in uterine cell culture supernatants was detected during anoestrus. At early stages of gestation, CRH was detected in the endometrium on days 2, 3 and 4 of pregnancy and in the myometrium on days 3 and 4, whereas it was undetectable on day 5. The functional role of CRH during early gestation was evaluated by administering anti-CRH antibody to mice from day 3 to day 8 of pregnancy. This treatment resulted in implantation failure in 60% of the cases, in which implantation sites, although clearly present in the uterus, had failed to host an embryo. These results provide direct evidence about the involvement of CRH in murine embryo implantation and are in agreement with hypotheses postulated in humans

Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion

We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR

In this report, we examined the involvement of the cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 as well as nitric oxide (NO) in the lipopolysaccharide (LPS)-induced experimental abortion model in BALB/c mice. Although in vivo administration of LPS in pregnant mice showed a 72% decrease of serum IL-10, no significant difference in serum TNF-alpha, IFN-gamma, and IL-4 levels, compared to controls, could be detected. At the same time, a correlation of fetal abortion and maternal splenomegaly with an important increase of NO synthesis in the serum was obtained. Simultaneous administration of LPS and aminoguanidine (AG; an inhibitor to NO synthase) rescued the LPS-induced fetal abortion, reduced maternal spleen weight to physiological levels, and decreased serum NO concentration to control levels. In vitro experiments showed that LPS directly induced NO production in primary placental cells and the TPOPHO-1 trophoblast cell line by stimulating the inducible isoform of NO synthase, which ultimately could be blocked by the NO synthase inhibitors AG and L-NAME. The results indicate that LPS, despite its beneficial involvement in intracellular infections, participates in inflammatory/autoimmune damage during pregnancy, leading to embryotoxicity, which is closely linked to the NO pathway

The construction of various gene-deficient mice has facilitated the understanding of the role of various receptors and signaling pathways that control the generation of alphabeta lineage cells. A predominant role is occupied by the pre-TCR, which not only generates large numbers of alphabeta lineage cells but also controls TCRbeta allelic exclusion as well as commitment to the gammadelta lineage versus the alphabeta lineage

Intracellular signals emanating from cytokine and antigen receptors are integrated during the process of intrathymic development. Still, the relative contributions of cytokine receptor signaling to pre-T cell receptor (TCR) and TCR-mediated differentiation remain undefined. Interleukin (IL)-7 interactions with its cognate receptor complex (IL-7Ralpha coupled to the common cytokine receptor gamma chain, gammac) play a dominant role in early thymopoiesis. However, alpha/beta T cell development in IL-7-, IL-7Ralpha-, and gammac-deficient mice is only partially compromised, suggesting that additional pathways can rescue alpha/beta T lineage cells in these mice. We have investigated the potential interdependence of gammac- and pre-TCR-dependent pathways during intrathymic alpha/beta T cell differentiation. We demonstrate that gammac-dependent cytokines do not appear to be required for normal pre-TCR function, and that the rate-limiting step in alpha/beta T cell development in gammac- mice does not involve TCR-beta chain rearrangements, but rather results from poor maintenance of early thymocytes. Moreover, mice double mutant for both gammac and pre-Talpha show vastly reduced thymic cellularity and a complete arrest of thymocyte differentiation at the CD44(+)CD25(+) cell stage. These observations demonstrate that the pre-TCR provides the gammac-independent signal which allows alpha/beta T cell development in gammac- mice. Thus, a series of overlapping signals derived from cytokine and T cell receptors guide the process of alpha/beta thymocyte development