The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response. CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and delayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs. Upon activation, mature skin dendritic cells fail to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) gene family, has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes such as long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF gene. We then used hippocampal slices from these mice to investigate whether LTP was affected by this mutation. Homo-and heterozygous mutant mice showed significantly reduced LTP in the CAl region of the hippocampus. The magnitude of the potentiation, as well as the percentage of cases in which LTP could be induced successfully, was clearly affected. According to the criteria tested, important pharmacological, anatomical, and morphological parameters in the hippocampus of these animals appear to be normal. These results suggest that BDNF might have a functional role in the expression of LTP in the hippocampus.Neurotrophic factors, in particular the members of the nerve growth factor (NGF) gene family, have so far been considered predominantly with regard to their function in regulating survival and differentiation of specific neuronal populations during embryonic development and the maintenance of characteristic neuronal function in adulthood (1-3). There is, however, evidence that neurotrophins might also be involved in neuronal plasticity (4-10). Long-term potentiation (LTP) is the most widely used paradigm to study cellular and molecular events underlying neuronal plasticity (11). We therefore used this paradigm in slices of the hippocampus from mice with targeted deletion of the brain-derived neurotrophic factor (BDNF) gene to test whether BDNF has a role in this important phenomenon of synaptic plasticity. MATERIALS AND METHODSIn the gene-targeting construct, a 560-bp fragment from the BDNF protein-coding exon was replaced by the selection marker-a neomycin-resistance gene flanked by a glycerate kinase gene promoter and a polyadenylylation signal-thus deleting most of the mature BDNF coding sequence (Fig. 1). Embryonic stem cells (D3, 129Sv) containing the disrupted BDNF gene were injected into BALB/c mouse blastocysts for subsequent generation of chimeric mice. Chimeric males were crossed with NMRI females to produce heterozygotes. In keeping with previously published reports (12, 13), homozygous BDNF (-/-) mutant mice were retarded in growth and had reduced weight (down to only 25% of the wild type) from postnatal day 3 (P3) on. They displayed aberrant limb coordination and balance, showed a loss of neurons in the dorsal root ganglia, and usually died between 2 and 4 weeks after birth. Such abnormalities were never observed in heterozygous BDNF (+/ -) mice.Transverse hippocampal slices (400 ,um thick) were prepared and maintained by standard procedures (medium, 124 mM NaCl/3 mM KCl/1.25 mM...

We describe the phenotype of gene-targeted mice lacking the putative chemokine receptor BLR1. In normal mice, this receptor is expressed on mature B cells and a subpopulation of T helper cells. Blr1 mutant mice lack inguinal lymph nodes and possess no or only a few phenotypically abnormal Peyer's patches. The migration of lymphocytes into splenic follicles is severely impaired, resulting in morphologically altered primary lymphoid follicles. Furthermore, activated B cells fail to migrate from the T cell-rich zone into B cell follicles of the spleen, and despite high numbers of germinal center founder cells, no functional germinal centers develop in this organ. Our results identify the putative chemokine receptor BLR1 as the first G protein-coupled receptor involved in B cell migration and localization of these cells within specific anatomic compartments.

Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

In this study, we evaluated mitochondrial distribution and ATP content of individual bovine oocytes before and after in vitro maturation (IVM). Cumulus-oocyte complexes were classified according to morphological criteria: category 1, homogeneous oocyte cytoplasm, compact multilayered cumulus oophorus; category 2, cytoplasm with small inhomogeneous areas, more than five layers of compact cumulus; category 3, heterogeneous/vacuolated cytoplasm, three to five layers of cumulus including small areas of denuded zona pellucida; category 4, heterogeneous cytoplasm, completely or in great part denuded. In immature oocytes, staining with MitoTracker green revealed mitochondrial clumps in the periphery of the cytoplasm, with a strong homogenous signal in category 1 oocytes, a weaker staining in category 2 oocytes, allocation of mitochondria around vacuoles in category 3 oocytes, and poor staining of mitochondria in category 4 oocytes. After IVM, mitochondrial clumps were allocated more toward the center, became larger, and stained more intensive in category 1 and 2 oocytes. This was also true for category 3 oocytes; however, mitochondria maintained their perivacuolar distribution. No mitochondrial reorganization was seen for category 4 oocytes. Before IVM, the average ATP content of category 1 oocytes (1.8 pmol) tended to be higher than that of category 2 oocytes (1.6 pmol) and was significantly (P < 0.01) higher than in category 3 (1.4 pmol) and 4 oocytes (0.9 pmol). The IVM resulted in a significant (P < 0.01) increase in the average ATP content of all oocyte categories, with no difference between oocytes extruding versus nonextruding a polar body. After in vitro fertilization (IVF) and culture, significantly (P < 0.05) more category 1 and 2 than category 3 and 4 oocytes developed to the morula or blastocyst stage (determined 168 h after IVF). Total cell numbers of expanded blastocysts derived from category 1 and 2 oocytes were significantly (P < 0.05) higher than of those originating from category 3 and 4 oocytes. These data indicate that mitochondrial reorganization and ATP levels are different between morphologically good and poor oocytes and may be responsible for their different developmental capacity after IVF.