Lentiviral Vector Delivery of Human Interleukin-7 (hIL-7) to Human Immune System (HIS) Mice Expands T Lymphocyte Populations

Genetically modified mice carrying engrafted human tissues provide useful models to study human cell biology in physiologically relevant contexts. However, there remain several obstacles limiting the compatibility of human cells within their mouse hosts. Among these is inadequate cross-reactvitiy between certain mouse cytokines and human cellular receptors, depriving the graft of important survival and growth signals. To circumvent this problem, we utilized a lentivirus-based delivery system to express physiologically relevant levels of human interleukin-7 (hIL-7) in Rag2-/-γc-/- mice following a single intravenous injection. hIL-7 promoted homeostatic proliferation of both adoptively transferred and endogenously generated T-cells in Rag2-/-γc-/- Human Immune System (HIS) mice. Interestingly, we found that hIL-7 increased T lymphocyte numbers in the spleens of HIV infected HIS mice without affecting viral load. Taken together, our study unveils a versatile approach to deliver human cytokines to HIS mice, to both improve engraftment and determine the impact of cytokines on human diseases

Endocrine disruptors and abnormalities of pubertal development

Onset and development of puberty is regulated by the neuroendocrine system. Population-based studies worldwide have observed secular trends towards earlier puberty development. These changes are apparently caused by environmental factors such as improved socio-economic status, improved health care and nutrition. However, they may also partly result from endocrine-disrupting chemicals in the environment. Epidemiological studies have investigated the relationship between pubertal development and exposure to endocrine-disrupting chemicals (polychlorinated biphenyls, polybrominated biphenyls, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, phthalate esters, furans and the pesticide endosulfan). Associations with both perinatal and postnatal exposure have been reported. Studies in experimental animals support some of these findings and point to differential endocrine regulatory mechanisms linked to pubertal development acting in the perinatal and the pre-pubertal period. Pubertal development is naturally associated with growth and body composition. There is increasing evidence for a link between prenatal development and pubertal onset. In girls born small for gestational age (SGA), pubertal onset and age at menarche often are advanced, especially if there has been an extensive catch-up growth during the first months of life. In utero growth retardation may have multiple causes including exposure to xenobiotic substances as was suggested for some endocrine-disrupting chemicals. An abnormal perinatal environment of children born SGA may alter the endocrine status and the sensitivity of the receptors for endocrine and metabolic signalling that may have effects on maturation of brain and gonads. However, the causal pathways and the molecular mechanisms that may link the pubertal growth pattern of children born SGA, pubertal development and endocrine-disrupting chemicals need further study

Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34+ CD133+ cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg−/− (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34+ CD133+ fraction of expanded cells and that CD34+ CD133+ cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.Singapore-MIT Alliance for Research and Technology ( Infectious Diseases research grant

A human in vitro model system for investigating genome-wide host responses to SARS coronavirus infection

10.1186/1471-2334-4-34BMC Infectious Diseases4-BIDM

Antibodies in HIV-1 Vaccine Development and Therapy

Despite 30 years of study, there is no HIV-1 vaccine and, until recently, there was little hope for a protective immunization. Renewed optimism in this area of research comes in part from the results of a recent vaccine trial and the use of single-cell antibody-cloning techniques that uncovered naturally arising, broad and potent HIV-1–neutralizing antibodies (bNAbs). These antibodies can protect against infection and suppress established HIV-1 infection in animal models. The finding that these antibodies develop in a fraction of infected individuals supports the idea that new approaches to vaccination might be developed by adapting the natural immune strategies or by structure-based immunogen design. Moreover, the success of passive immunotherapy in small-animal models suggests that bNAbs may become a valuable addition to the armamentarium of drugs that work against HIV-1

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein

The glycosylated membrane protein M of the severe acute respiratory syndrome associated coronavirus (SARS-CoV) is the main structural component of the virion and mediates assembly and budding of viral particles. The membrane topology of SARS-CoV M and the functional significance of its N-glycosylation are not completely understood as is its interaction with the surface glycoprotein S. Using biochemical and immunofluorescence analyses we found that M consists of a short glycosylated N-terminal ectodomain, three transmembrane segments and a long, immunogenic C-terminal endodomain. Although the N-glycosylation site of M seems to be highly conserved between group 1 and 3 coronaviruses, studies using a recombinant SARS-CoV expressing a glycosylation-deficient M revealed that N-glycosylation of M neither influence the shape of the virions nor their infectivity in cell culture. Further functional analysis of truncated M proteins showed that the N-terminal 134 amino acids comprising the three transmembrane domains are sufficient to mediate accumulation of M in the Golgi complex and to enforce recruitment of the viral spike protein S to the sites of virus assembly and budding in the ERGIC

Regulation of peripheral T cell activation by calreticulin

Regulated expression of positive and negative regulatory factors controls the extent and duration of T cell adaptive immune response preserving the organism's integrity. Calreticulin (CRT) is a major Ca2+ buffering chaperone in the lumen of the endoplasmic reticulum. Here we investigated the impact of CRT deficiency on T cell function in immunodeficient mice reconstituted with fetal liver crt−/− hemopoietic progenitors. These chimeric mice displayed severe immunopathological traits, which correlated with a lower threshold of T cell receptor (TCR) activation and exaggerated peripheral T cell response to antigen with enhanced secretion of inflammatory cytokines. In crt−/− T cells TCR stimulation induced pulsatile cytosolic elevations of Ca2+ concentration and protracted accumulation of nuclear factor of activated T cells in the nucleus as well as sustained activation of the mitogen-activated protein kinase pathways. These observations support the hypothesis that CRT-dependent shaping of Ca2+ signaling critically contributes to the modulation of the T cell adaptive immune response