Tbx2 mediates dorsal patterning and germ layer suppression through inhibition of BMP/GDF and Activin/Nodal signaling.

BACKGROUND: Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFß ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFß signaling network. RESULTS: Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression. CONCLUSION: Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA-binding activity is required for repression of TGFß signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling.

Repression of Inappropriate Gene Expression in the Vertebrate Embryonic Ectoderm.

During vertebrate embryogenesis, precise regulation of gene expression is crucial for proper cell fate determination. Much of what we know about vertebrate development has been gleaned from experiments performed on embryos of the amphibian Xenopus laevis; this review will focus primarily on studies of this model organism. An early critical step during vertebrate development is the formation of the three primary germ layers-ectoderm, mesoderm, and endoderm-which emerge during the process of gastrulation. While much attention has been focused on the induction of mesoderm and endoderm, it has become clear that differentiation of the ectoderm involves more than the simple absence of inductive cues; rather, it additionally requires the inhibition of mesendoderm-promoting genes. This review aims to summarize our current understanding of the various inhibitors of inappropriate gene expression in the presumptive ectoderm.

Heparanase prevents the development of type 1 diabetes in non-obese diabetic mice by regulating T-cell activation and cytokines production.

BACKGROUND: Heparanase is an endo-beta-D-glucuronidase that cleaves heparan sulfate saccharide chains. The enzyme promotes cell adhesion, migration and invasion, and was shown to play a significant role in cancer metastasis and angiogenesis. METHODS: The present study focuses on the involvement of heparanase in autoimmunity, applying the murine non-obese diabetic (NOD) model, a T-cell-dependent disease often used to investigate the pathophysiology of type 1 diabetes. RESULTS: It was found that intra-peritoneal administration of heparanase ameliorated the clinical signs of the disease. In vitro studies revealed that heparanase has an inhibitory effect on the activation of T-cells through modulation of their repertoire of cytokines indicated by a marked increase in the levels of IL-4 and IL-10, and a parallel decrease in IL-12, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). CONCLUSIONS: We suggest that heparanase induces a shift from a Th1- to Th2-phenotype, resulting in inhibition of diabetes in NOD mice and possibly other autoimmune disorders.

Cannabidiol arrests onset of autoimmune diabetes in NOD mice.

We have previously reported that cannabidiol (CBD) lowers the incidence of diabetes in young non-obese diabetes-prone (NOD) female mice. In the present study we show that administration of CBD to 11-14 week old female NOD mice, which are either in a latent diabetes stage or with initial symptoms of diabetes, ameliorates the manifestations of the disease. Diabetes was diagnosed in only 32% of the mice in the CBD-treated group, compared to 86% and 100% in the emulsifier-treated and untreated groups, respectively. In addition, the level of the proinflammatory cytokine IL-12 produced by splenocytes was significantly reduced, whereas the level of the anti-inflammatory IL-10 was significantly elevated following CBD-treatment. Histological examination of the pancreata of CBD-treated mice revealed more intact islets than in the controls. Our data strengthen our previous assumption that CBD, known to be safe in man, can possibly be used as a therapeutic agent for treatment of type 1 diabetes.

The oxidative status of blood cells in a murine model of graft-versus-host disease.

We studied the oxidative status of red and white blood cells during the development of graft vs host disease (GVHD) as well as the effects of treatment with antioxidants, both in vitro and in vivo. (BALB/c X C57BL/6) F1 mice were conditioned by total body radiation and, 1 day later, transplanted with semi-allogeneic C57BL/6 spleen cells. GVHD was followed by its clinical manifestations. Oxidative stress in red blood cells (RBC), neutrophils, and lymphocytes was assessed by measuring generation of reactive oxygen species and the content of reduced glutathione by flow cytometry after gating of the specific populations. Oxidative stress was noticed 3 weeks after transplantation. It was higher in mice receiving allogeneic spleen cells as compared with mice transplanted with syngeneic cells, suggesting that it was associated with GVHD. The results also demonstrated that treatment with the antioxidants N-acetylcysteine and a derivative of vitamin E (tocopherol succinate, propofol), both in vitro and in vivo, reduced the oxidative stress. The results indicate that various blood cells, including RBC, neutrophils, and lymphocytes, are under oxidative stress and that treatment with antioxidants reduced the stress and, thus, may be useful in ameliorating the severe consequences of GVHD.

N-acetylcysteine mildly inhibits the graft-vs.-leukemia effect but not the lymphokine activated cells (LAK) activity.

N-acetylcysteine (NAC) is a known antioxidant and induces modulation of glutathione cellular content effects. It has been suggested that in the context of stem cell transplantation (SCT), NAC can prevent and treat graft-vs.-host disease, veno-occlusive disease and idiopathic pneumonia syndrome. We investigated the possible effect of NAC on graft-vs.-leukemia effect (GVL) and lymphokine activated cells (LAK) activity in murine models. After 10 days of NAC treatment, the cytotoxic activity of the LAK cells did not significantly differ from LAK activity generated from spleen cells obtained from untreated controls. However, NAC mildly suppressed GVL (appearance of leukemia in 8/36 animals treated with NAC as compared to 0/20 in the SCT control group, p=0.023). In spite of this mild suppression of GVL, no negative effect on achievement of donor chimerism was seen. We conclude that NAC usage in SCT may be relatively safe with regard to the GVL effect, yet further clinical studies are warranted.

Induction of early post-transplant graft-versus-leukemia effects using intentionally mismatched donor lymphocytes and elimination of alloantigen-primed donor lymphocytes for prevention of graft-versus-host disease.

Graft-versus-leukemia (GVL) effects can be induced in tolerant mixed chimeras prepared with nonmyeloablative conditioning. GVL effects can be amplified by post-grafting donor lymphocyte infusion (DLI). Unfortunately, DLI is frequently associated with graft-versus-host disease (GVHD). We investigated the feasibility of induction of potent GVL effects by DLI using intentionally mismatched lymphocytes followed by elimination of alloreactive donor T cells by cyclophosphamide for prevention of lethal GVHD following induction of very short yet most potent GVL effects. Mice inoculated with B-cell leukemia (BCL1) and mismatched donor lymphocytes were treated 2 weeks later with low-dose or high-dose cyclophosphamide. All mice receiving cyclophosphamide 2 weeks after DLI survived GVHD, and no residual disease was detected by PCR; all control mice receiving DLI alone died of GVHD. Analysis of host (female) and donor (male) DNA showed that cyclophosphamide treatment eradicated most alloreactive donor cells, yet mixed chimerism was converted to full donor chimerism following transient self-limited GVHD. Our working hypothesis suggests that short-term yet effective and safe adoptive immunotherapy of leukemia may be accomplished early post-transplantation using alloreactive donor lymphocytes, with prevention of GVHD by elimination of GVL effector cells.

Immunotherapy of murine leukemia following non-myeloablative conditioning with naïve or G-CSF mobilized blood or bone marrow stem cells.

Allogeneic stem cell transplantation (SCT) is the treatment of choice for a large number of hematologic malignancies. Its major advantage over conventional chemotherapy lies in the graft-versus-leukemia (GVL) effects mediated by allo- or tumor-reactive donor lymphocytes given in the course of SCT or post transplantation as donor lymphocyte infusions (DLI). The benefits of cell-mediated immunotherapy over myeloablative radiochemotherapy have also made it possible to reduce the intensity of conditioning regimens. Mobilized peripheral blood has proved preferable to bone marrow (BM) as a source of stem cells for transplantation, since it provides a larger number of stem cells on the one hand and immunologically competent lymphocytes on the other. The use of granulocyte colony stimulating factor (G-CSF), which is necessary to mobilize and increase the number of stem cells, may down-regulate the GVL effect by suppression of donor effector T lymphocytes by inducing Th1-->Th2 cytokine switch. It has previously been shown that GVL effects may be amplified by both in vivo and in vitro activation of donor lymphocytes with human recombinant interleukin-2 (rIL-2). Our studies using a leukemic murine model prepared for transplantation with low intensity conditioning prior to infusion of G-CSF-mobilized peripheral blood stem cells (PBSC) have demonstrated that mobilization of blood cells with G-CSF and in vivo treatment with rIL-2 following low-intensity conditioning enhances the GVL effects and prolongs survival of recipients inoculated with BCL1. Activation of donor lymphocytes with rIL-2 may thus be useful for amplifying GVL effects following mobilization with G-CSF.

Cytokine production in Linomide-treated nod mice and the potential role of a Th (1)/Th(2) shift on autoimmune and anti-inflammatory processes.

Linomide prevents the development of autoimmune insulitis and insulin-deficient diabetes mellitus in female NOD mice. Linomide prevents development of autoimmune manifestations in other experimentally induced and spontaneous autoimmune diseases as well, but the mechanism of action is unknown. The present report summarizes our investigations on the effect of Linomide on different functional T cell subsets in NOD mice analyzed according to their cytokine profile. Supernatants from cultured splenocytes and peritoneal cells taken from Linomide-treated mice contained lower levels of TNFalpha, IL-1 beta, IFN gamma and IL-12 versus higher levels of IL-4, IL-6 and IL-10 in comparison with supernatants from cultures of untreated mice. Our results suggest that regulation of autoimmunity following oral Linomide administration in NOD mice induces a shift from Th(1) to Th(2) phenotype response, thereby preventing the development of diabetes by active cytokine-induced immunoregulation of T cell subsets, including downregulation of Th(1) and upregulation of Th(2).