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Background The present study assesses the effect of the stem cell antigen1 positive (Sca-1 + ) cell-based human growth hormone (hGH) ex vivo gene transfer strategy on endosteal bone mass in the mouse. Methods Sublethally irradiated recipien tm ice were transplanted with Sca-1 + cells transduced with lentiviral vectors expressing hGH or βgalactosidase control genes. Bone parameters were assessed by microcomputed tomography and histomorphometry. Results This hGH strategy drastically increased hGH mRNA levels in bone marrow cells and serum insulin-like growth factor-I (IGF-I) (by nearly 50%, p < 0.002) in hGH recipient mice. Femoral trabecular bone volume of the hGH mice was significantly reduced by 35% (p < 0.002). The hGH mice also had decreased trabecular number (by 26%;p < 0.0001), increased trabecular separation (by 38%; p < 0.0002) and reduced trabecular connectivity density (by 64%; p < 0.001), as well as significantly more osteoclasts (2.5-fold; p < 0.05) and greater osteoclastic surface per bone surface (2.6-fold; p < 0.01). Conclusions Targeted expression of hGH in cells of marrow cavity through the Sca-1 + cell-based gene transfer strategy increased circulating IGF-I and decreased endosteal bone mass through an increase in resorption in recipient mice. These results indicate that high local levels of hGH or IGF-I in the bone marrow microenvironment enhanced resorption, which is consistent with previous findings in transgenic mice with targeted bone IGF-I expression showing that high local IGF-I expression increased bone remodeling, favoring a net bone loss. Thus, GH and/or IGF-I would not be an appropriate transgene for use in this Sca-1 + cell-based gene transfer strategy to promote endosteal bone formation. Published 2011 John Wiley & Sons, Ltd.

This study assessed whether a Sca-1+ cell-based ex vivo gene transfer strategy, which has been shown to promote robust endosteal bone formation with a modified fibroblast growth factor-2 (FGF2) gene, can be extended to use with bone morphogenetic protein (BMP)2/4 hybrid gene.Sublethally irradiated recipient mice were transplanted with lentiviral (LV)-BMP2/4-transduced Sca-1+ cells. Bone parameters were monitored by pQCT and microCT. Gene expression was assessed by the real-time reverse transcriptase-polymerase chain reaction.Recipient mice of LV-BMP2/4-transduced Sca-1+ cells yielded high engraftment and increased BMP4 mRNA levels in marrow cells; but exhibited only insignificant increases in serum and bone alkaline phosphatase activity compared to control mice. pQCT and microCT analyses of femurs showed that, with the exception of small changes in trabecular bone mineral density and cortical bone mineral content in LV-BMP2/4 mice, there were no differences in measured bone parameters between mice of the LV-BMP2/4 group and controls. The lack of large endosteal bone formation effects with the BMP4 strategy could not be attributed to ineffective engraftment or expansion of BMP4-expressing Sca-1+ cells, an inability of the transduced cells to secrete active BMP4 proteins, or to use of the LV-based vector.Sca-1+ cell-based BMP4 ex vivo strategy did not promote robust endosteal bone formation, raising the possibility of intrinsic differences between FGF2- and BMP4-based strategies in their ability to promote endosteal bone formation. It emphasizes the importance of choosing an appropriate bone growth factor gene for delivery by this Sca-1+ cell-based ex vivo systemic gene transfer strategy to promote bone formation.

Interferons (IFNs) play an important role in host antiviral responses, but viruses, including vaccinia viruses (VV), employ mechanisms to disrupt IFN activities, and these viral mechanisms are often associated with their virulence. Here, we explore an attenuation strategy with a vaccine strain of VV lacking a virus-encoded IFN-gamma receptor homolog (viroceptor).To facilitate the monitoring of virus properties, first we constructed a Lister vaccine strain derivative VV-RG expressing optical reporters. Further, we constructed a VV-RG derivative, VV-RG8, which lacks the IFN-gammaR viroceptor (B8R gene product). Replication, immunological and pathogenic properties of the constructed strains were compared.Viruses did not show significant differences in humoral and cellular immune responses of immune-competent mice. Replication of constructed viruses was efficient both in vitro and in vivo, but showed marked difference in kinetics of propagation. In cultured CV-1 epithelial cells, the VV-RG8 strain retained the propagation potential of the parental virus, while, in the C6 glial cells, significant delay was observed in the kinetics of the VV-RG8 replication cycle compared to VV-RG. The pathogenesis of the viruses was tested by survival assay and biodistribution in nude mice. High dose inoculation of nude mice with VV-RG8 caused less pronounced virus dissemination, improved weight gain, and increased survival rate, as compared with the VV-RG strain.The replication-competent virus VV-RG8 carrying a mutation at the B8R gene is less pathogenic for mice than the parental vaccine virus. We anticipate that step-wise inactivation of VV vaccine genes involved in evasion of host immune response may provide an alternative approach for generation of hyper-attenuated replication-competent vaccines.

The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT-PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS-118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age- and sex-matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5-fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up- or down-regulated by at least 1.5-fold after spaceflight (P < or = 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT-PCR were as follows: Rbm3 (up-regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down-regulated). QRT-PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla-4, IFN-alpha2a (up-regulated) and CD44 (down-regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space.

This study was undertaken to investigate the effects of a single infusion of radiolabelled murine monoclonal antibody (MAb) on peripheral blood leukocytes in cancer patients. Eleven patients with disseminated colon cancer, malignant melanoma, or lung adenocarcinoma were infused with 111In-labelled anti-ZCE 025, anti-p97 type 96.5c, or LA 20207 MAb, respectively. Blood samples were obtained before infusion, immediately after infusion (1 hr), and at 4 and 7 days postinfusion. Flow cytometry analysis of CD3+, CD4+, CD8+, CD16+, and CD19+ lymphocytes showed increasing CD4:CD8 ratios in seven patients after infusion. This phenomenon was not restricted to antibody subclass or to type of cancer. Two of the remaining patients exhibited a marked post-infusion increase in CD8+ cells. In all three patients with malignant melanoma, decreasing levels of CD16+ lymphocytes were noted after infusion and natural killer cell cytotoxicity showed fluctuations which paralleled the changes in the CD16+ subpopulation. Oxygen radical production by phagocytic cells was markedly affected in three subjects. These results suggest that a single infusion of radiolabelled murine MAb may alter the balance of critical lymphocyte subpopulations and modulate other leukocyte responses in cancer patients.

Peripheral blood samples from six cancer patients (five colon cancer, one lung cancer) and six healthy volunteers were tested in vitro for oxygen radical production by phagocytic cells and in assays of mitogen-induced lymphoblastogenesis at physiologic and pharmacologic concentrations of pyridoxine (PN, 1.8-96 nmol/ml) or pyridoxal (PL, 0.08-90 nmol/ml). Plasma levels of pyridoxal-5'-phosphate (PLP), 4-pyridoxic acid (4PA), pyridoxamine phosphate (PMP), and PL were also determined. Phagocytic cells from three patients showed significantly increased capacity for oxygen radical production when incubated in PL-, but not PN-supplemented media. Oxygen burst capacity of cells from healthy subjects was significantly enhanced by PN-, but not PL-enriched media. Lymphocyte responsiveness to phytohemagglutinin or pokeweed mitogen (PWM) stimulation showed a modest increase in cell activation in three patients as the concentration of PN was increased; with concanavalin A, two showed enhanced responsiveness. On the other hand, PL-supplementation resulted in greater cell proliferation only with PWM. The cancer patients had significantly lower plasma PLP, 4PA, and PMP levels when compared with the healthy volunteers. These data indicate that in the cancer patients and in a majority of the healthy volunteers, vitamin B-6 status was marginal or deficient and suggest that increasing PN or PL in vivo levels may augment functions related to cell-mediated immunity.