Here we show that transplantation of autologous human hematopoietic fetal liver CD34+ cells into NOD/SCID mice previously implanted with human fetal thymic and liver tissues results in long-term, systemic human T-cell homeostasis. In addition, these mice show systemic repopulation with human B cells, monocytes and macrophages, and dendritic cells (DCs). T cells in these mice generate human major histocompatibility complex class I- and class II-restricted adaptive immune responses to Epstein-Barr virus (EBV) infection and are activated by human DCs to mount a potent T-cell immune response to superantigens. Administration of the superantigen toxic shock syndrome toxin 1 (TSST-1) results in the specific systemic expansion of human Vbeta2+ T cells, release of human proinflammatory cytokines and localized, specific activation and maturation of human CD11c+ dendritic cells. This represents the first demonstration of long-term systemic human T-cell reconstitution in vivo allowing for the manifestation of the differential response by human DCs to TSST-1.

Primary cultures of rat spermatogenic cells that did not bind to collagen matrices were able to colonize and form mature spermatozoa when transferred to testes of recipient males. Up to 73% of the progeny from matings with recipient males were derived from the transferred spermatogenic cells. Subsequently, two populations of germ cells were obtained by selection on laminin matrices. Both populations expressed the spermatogenic cell marker, DAZL, but not the somatic cell marker, vimentin. The cells that bound to laminin represented ≈5% of the total population and were greatly enriched in ability to colonize a recipient testis, suggesting an enrichment in germ-line stem cells. The colonization potential was maintained for at least 7 days in culture. These cells were subsequently transduced with a lentiviral enhanced GFP reporter vector and then transferred to WT recipient males. After mating, 26 of 44 pups were derived from the cultured donor germ cells, and 13 pups carried the lentiviral transgene. Based on Southern analysis, the transgene was integrated at a different genetic locus in each animal and was transmitted to ≈50% of pups in the F 2 generation. Thus, by using these procedures, ≈30% of pups in the F 1 generation inherited and stably transmitted a lentiviral transgene that integrated at various genomic sites.

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APD334 was discovered as part of our internal effort to identify potent, centrally available, functional antagonists of the S1P 1 receptor for use as next generation therapeutics for treating multiple sclerosis (MS) and other autoimmune diseases. APD334 is a potent functional antagonist of S1P 1 and has a favorable PK/PD profile, producing robust lymphocyte lowering at relatively low plasma concentrations in several preclinical species. This new agent was efficacious in a mouse experimental autoimmune encephalomyelitis (EAE) model of MS and a rat collagen induced arthritis (CIA) model and was found to have appreciable central exposure.

The discovery of in vitro culture systems yielding mouse 3 and human [4][5][6] DCs accelerated studies on their biology and led us to understand their complexity. Indeed, DCs have different functions at different stages of maturation. While immature (nonactivated) DCs that capture self-antigens (eg, apoptotic cells) induce tolerance, 7,8 mature antigen-loaded DCs induce antigen-specific immunity. Furthermore, several DC subsets exist that have different functions. 1 In humans, 2 major DC pathways have been described. A myeloid pathway includes Langerhans cells (LCs), found in stratified epithelia such as skin, and interstitial DCs (intDCs), found in all other tissues. While both subsets produce interleukin-12 (IL-12), only intDCs make IL-10 and induce B-cell proliferation and differentiation. 9 The other pathway includes the plasmacytoid DCs (pDCs), 10 which secrete large amounts of type I interferons within a few hours following viral encounter. 11,12 Depending on the maturation signal that they receive, pDCs modulate T-cell differentiation into either interferon-␥ (IFN-␥) or IL-4-producing CD4 T cells 13 or into regulatory CD8 T cells. 14 Thus, distinct human DC subsets differentially control lymphocytes in vitro.The in vitro generation of human DCs prompted their use in immunotherapy, particularly for the treatment of cancer. Early trials in humans have shown the safety of tumor-associated antigens (TAA)-loaded DCs as well as some clinical and immune responses (reviewed in Banchereau et al 15 ). We also are beginning to unravel the role of DCs in pathogenesis of human diseases, for example, thymic stroma lymphopoietin (TSLP)-triggered mDCs may play a key role in the initiation of allergic inflammation, 16 while IFN-␣-triggered mDCs appear as a major factor driving the autoimmune reaction in systemic lupus erythematosus. 17 Thus, DCs can be considered both as vectors and targets for immunomodulation.Understanding the specific functions of DC subsets and their interplay in vivo will be critical to understand the launching and modulation of immune responses. Hence, the need for preclinical models of the human immune system. Indeed, conclusions from studies in mice cannot be directly extrapolated to humans because of biologic differences as exemplified by the CD1 antigen presentation system that is different between species. 18 SCID mice reconstituted with human cells represent interesting candidates for human disease models [19][20][21] For personal use only. on May 12, 2018. by guest www.bloodjournal.org From Many of the difficulties encountered in these models could be due to insufficient reconstitution of human DCs, a parameter that has not been extensively studied. Given the key role that DCs play in T-cell homeostasis, 36 DC reconstitution might facilitate human T-lymphocyte reconstitution. Indeed, recent studies with ex vivogenerated monocyte-derived DCs suggest their capacity to support CD4 T-cell differentiation as well as humoral responses in vivo. 37,38 However, these adoptive transfer models do not p...

The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptormediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine 2B (5-HT 2B ) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Ga qmediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signalregulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT 2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptordissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT 2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.

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