The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of alpha-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.

Nesfatin-1 is a novel satiety molecule in the hypothalamus and is also present in peripheral tissues. Here we sought to identify the active segment of nesfatin-1 and to determine the mechanisms of its action after peripheral administration in mice. Intraperitoneal injection of nesfatin-1 suppressed food intake in a dose-dependent manner. Nesfatin-1 has three distinct segments; we tested the effect of each segment on food intake. Injection of the midsegment decreased food intake under leptin-resistant conditions such as db/db mice and mice fed a high-fat diet. After injection of the midsegment, expression of c-Fos was significantly activated in the brainstem nucleus tractus solitarius (NTS) but not in the hypothalamic arcuate nucleus; the nicotinic cholinergic pathway to the NTS contributed to midsegment-induced anorexia. Midsegment injection significantly increased expression of proopiomelanocortin and cocaine- and amphetamine-regulated transcript genes in the NTS but not in the arcuate nucleus. Investigation of mutant midsegments demonstrated that a region with amino acid sequence similarity to the active site of agouti-related peptide was indispensable for anorexigenic induction. Our findings indicate that the midsegment of nesfatin-1 causes anorexia, possibly by activating POMC and CART neurons in the NTS via a leptin-independent mechanism after peripheral stimulation.

Mesenchymal stem cells (MSCs) are attractive cell sources in regenerative medicine. We examined the effects of topical MSCs implantation on an experimental model of inflammatory bowel disease. Putative MSCs, isolated from bone marrow aspirates of male rats by dish adherence and expanded in vitro, were characterized by flow cytometry, reverse transcriptionpolymerase chain reaction, enzyme-linked immunosorbent assay, and differentiation assays. Experimental colitis was induced by intraluminal instillation of 2,4,6-trinitrobonzene sulfonic acid (TNBS) in the colons of male rats. The putative MSCs and unselected fresh bone marrow cells were injected into the colonic submucosa surrounding the area exposed to TNBS. The healing process of the injury was examined macroscopically and immunohistologically. Multipotent MSCs positive for CD29 and CD90, and negative for CD31 and CD34, were implanted into colon tissue surrounding the lesion; a majority of the engrafted cells were positive for vimentin. The implantation significantly accelerated healing of the damaged mucosa compared with vehicle-injected controls. The MSCs expressed vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-␤1 in vitro and after the implantation. In conclusion, we found that MSCs were successfully topically implanted in the colon and that they were associated with accelerated healing of TNBS-induced colitis. The beneficial effects of the MSCs might be mediated, at least in part, by their ability to differentiate into colonic interstitial cells and by their ability to provide VEGF and TGF-␤1 to the injured area.

Xenotransplantation from pigs could provide a potential solution to the severe shortage of allogeneic donor organs. Because xenogeneic tissues are subject to vigorous immune rejection, tolerance induction is likely to be essential to the success of clinical xenotransplantation. Here we explore the possibility of inducing human T-cell tolerance to porcine xenografts through mixed chimerism. We previously showed that NOD/SCID-Tg mice expressing porcine cytokine transgenes permit the induction of durable porcine hematopoietic chimerism. In this study we achieved human T-cell development in these mice by engrafting human fetal thymus/liver tissues. In porcine hematopoietic chimeras, human thymus grafts were populated with porcine class II high cells in addition to human cells, and human T cells were tolerant of the porcine hematopoietic donor as measured by mixed lymphocyte reaction assay and skin grafting. This study proves the principle that porcine chimerism induces tolerance of xenoreactive human T cells. IntroductionThe severe shortage of allogeneic organ donors currently limits the number of transplantations performed. 1,2 This supply-demand disparity could be corrected by the ability to use organs from other species (xenografts). In view of the ethical issues and impracticalities associated with the use of nonhuman primates, interest has focused on other species, in particular the pig, as the most suitable organ donor species for humans. In addition to organ size and physiologic similarities to humans, the ability to rapidly breed and inbreed pigs makes them particularly amenable to genetic modifications that could improve their ability to function as organ donors to humans. [3][4][5][6][7] However, organ transplantations across discordant species barriers are subject to vigorous immunologic rejection. 1,8 One might, therefore, expect the amount of nonspecific immunosuppression that would be required to overcome xenograft rejection to be so great that recipients would succumb to infections. Thus, it would be highly desirable to eliminate the immune response to xenografts through the induction of tolerance.Mixed chimerism has been proven to be a powerful and reliable approach for tolerance induction across allogeneic and closely related xenogeneic barriers. 9-14 Our recent studies showed that this approach also induces tolerance in a pig-to-mouse, highly disparate xenogeneic combination. 15 However, the ability of mixed chimerism to induce human T-cell tolerance to porcine xenografts has yet to be explored because of the lack of a suitable model system. It has been unclear whether or not genetic incompatibilities in immune cytokines, 16,17 accessory molecules, [17][18][19] and other unknown molecules between the 2 species might impede the induction of human T-cell tolerance to porcine xenografts through mixed chimerism. To address these questions, we have developed a murine model that permits the induction of both sustained porcine hematopoietic chimerism and active human thymopoiesis. Our results demonstrate...