Interferon gamma eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells.

In Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected wild-type mice, there was a large expansion of an activated (CD44(hi)) splenic CD4 T cell population followed by a rapid contraction of this population to normal numbers. Contraction of the activated CD4 T cell population in wild-type mice was associated with increased apoptosis of activated CD4 T cells. In BCG-infected interferon (IFN)-gamma knockout (KO) mice, the activated CD4 T cell population did not undergo apoptosis. These mice accumulated large numbers of CD4(+)CD44(hi) T cells that were responsive to mycobacterial antigens. Addition of IFN-gamma to cultured splenocytes from BCG-infected IFN-gamma KO mice induced apoptosis of activated CD4 T cells. IFN-gamma-mediated apoptosis was abolished by depleting adherent cells or Mac-1(+) spleen cells or by inhibiting nitric oxide synthase. Thus, IFN-gamma is essential to a regulatory mechanism that eliminates activated CD4 T cells and maintains CD4 T cell homeostasis during an immune response.

Failure to suppress the expansion of the activated CD4 T cell population in interferon gamma-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis.

Mice deficient in interferon (IFN)-gamma or IFN-gamma receptor develop progressive and fatal experimental autoimmune encephalomyelitis (EAE). We demonstrate that CD4 T cells lacking IFN-gamma production were required to passively transfer EAE, indicating that they were disease-mediating cells in IFN-gamma knockout (KO) mice. IFN-gamma KO mice accumulated 10-16-fold more activated CD4 T cells (CD4(+)CD44(hi)) than wild-type mice in the central nervous system during EAE. CD4(+)CD44(hi) T cells in the spleen and central nervous system of IFN-gamma KO mice during EAE showed markedly increased in vivo proliferation and significantly decreased ex vivo apoptosis compared with those of wild-type mice. IFN-gamma KO CD4(+)CD44(hi) T cells proliferated extensively to antigen restimulation in vitro and accumulated larger numbers of live CD4(+) CD44(hi) T cells. IFN-gamma completely suppressed proliferation and significantly induced apoptosis of CD4(+)CD44(hi) T cells responding to antigen and hence inhibited accumulation of live, activated CD4 T cells. We thus present novel in vivo and in vitro evidence that IFN-gamma may limit the extent of EAE by suppressing expansion of activated CD4 T cells.

A novel endothelial cell-based gene therapy platform for the in vivo delivery of apolipoprotein E.

A major focus in gene therapy has been the use of recombinant viruses to deliver genes in vivo. Although this approach shows much promise, there are many safety concerns associated with the use of viral materials in the treatment of human diseases. Our alternative cell-based gene therapy approach utilizes endothelial cells (Pro 175) isolated from the murine embryonic yolk sac. These endothelial cells were evaluated for their potential use in gene therapy as a gene delivery platform. As a test model, we used these cells to deliver apolipoprotein E (apoE) in the murine apoE knockout atherosclerosis model. The lack of apoE protein in these animals results in high levels of serum cholesterol and formation of severe aortic plaques and lesions at a young age. After transplantation of the apoE secreting Pro 175 endothelial cells into apoE-deficient mice, serum cholesterol levels were measured at 2 week intervals. During the 3 months after the initiation of these experiments, levels of cholesterol in the animals having received the apoE secreting endothelial cells were statistically lower compared with the levels of age-matched controls having received non-secreting endothelial cells. Concomitant with cholesterol reduction, atherosclerotic aortic plaques were noticeably reduced in the experimental apoE+ animals. These results highlight the potential of these unique endothelial cells as an efficient delivery platform for somatic gene therapy.

Tetracycline-regulatable expression vectors tightly regulate in vitro gene expression of secreted proteins.

The regulation of gene expression by the tetracycline system has attracted a high level of interest in the recent past. However, expression of secreted proteins has not been evaluated precisely. In this study, we constructed two versions of a one-plasmid system containing the elements necessary for the regulation of gene expression. The regulatable elements and the selectable marker (Neor) were set up in two different configurations, pTRIN31 and pTRIN76. With these two regulatable versions, the levels of protein expression after transfection into the NIH/3T3 cell line were measured by insertion of three different genes encoding the secreted proteins (hGH, ApoE3, hGM-CSF). The maximum levels of gene expression obtained with the pTRIN76-derived plasmids were 100ng/24h/106 cells for hGH, 427ng/24h/106 cells for ApoE3 and 108ng/24h/106 cells for hGM-CSF. For the pTRIN31-derived plasmids the maximum levels were 2.7ng/24h/106 cells for hGH and 47ng/24h/106 for ApoE3. Both plasmids give rise to an expression of the transfected gene that can be tightly regulated by three different molecules: tetracycline, minocycline and doxycycline. The levels of the secreted proteins are below the detectable level when the reporter genes are repressed. This repression is reversible within 48h after the regulator has been removed from the medium.

The expression of leptin and its receptors in pre-ovulatory human follicles.

The expression of leptin and its receptors was examined by reverse transcriptase-polymerase chain reaction and immunofluorescence in granulosa and cumulus cells of pre-ovulatory follicles and in meiotically mature oocytes obtained from women undergoing in-vitro fertilization. Leptin concentrations were measured in newly aspirated follicular fluids and in maternal serum before and after the administration of an ovulatory dose of human chorionic gonadotrophin. The findings demonstrate leptin expression at the mRNA and protein levels by granulosa and cumulus cells, and the presence of leptin in mature human oocytes. While an association between follicular leptin concentration and embryo development was not observed, a post-ovulatory increase in serum leptin concentration was associated with implantation potential. The results are discussed with respect to possible roles of leptin in early human development.