In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fully or minimally myeloablated setting

Many nonmalignant hematologic disorders could potentially be treated by genetic correction of as few as 5-10% of target lineage cells. However, immune system clearance of cells expressing gene products perceived as foreign could be limiting. There is evidence that tolerance to foreign proteins can result when myeloablative conditioning is used, but this limits the overall applicability of such techniques. Therefore, we sought to evaluate the engraftment of hematopoietic stem cells carrying a foreign transgene after low-dose irradiation by comparing in vivo survival of murine long-term repopulating cells (LTRC) transduced with either a retroviral vector expressing the bacterial neomycin phosphotransferase gene (neo) or a vector containing neo gene sequences but modified to prevent protein expression (nonexpression). First, marrow cells from congenic donors were transduced with either vector and transplanted into recipients treated with standard dose irradiation of 800 rads. High-level engraftment and gene marking resulted, without differences in the marking levels or pattern of persistence of the cells between cells transduced with either vector. Low-dose irradiation at 100 rads was tested using higher cell doses. Marking levels as high as 10% overall were obtained, again with no differences between mice receiving cells transduced with the neo versus the nonexpression vectors. To investigate a potentially more immunogenic protein, marrow cells were transduced with a vector containing the green fluorescent protein (GFP) gene, and their persistence was studied in recipient mice receiving 100 rads. Stable GFP expression in 5-10% of circulating cells was observed long term. We conclude that even with very low dose conditioning, engraftment by genetically modified LTRC cells at clinically significant levels can be achieved without evidence for clearance of cells known to be expressing immunogenic proteins.     

Effect of bombesin on extragastric gastrin in man.

The effect of a protein test meal and a bombesin infusion on extragastric gastrin levels was studied in patients with truncal vagotomy, antrectomy, and gastroduodenostomy or gastrojejunostomy and in patients with total gastrectomy. In patients with vagotomy, antrectomy, and gastroduodenostomy and in patients with total gastrectomy the gastrin levels were raised by 33% and 35%, respectively, from basal after test meal, while during BBS infusion gastrin values decreased by 25% and 30%, respectively, from basal. In patients with vagotomy, antrectomy, and gastrojejunostomy, test meal and BBS infusion did not significantly alter basal gastrin values. It is concluded that BBS does not stimulate extragastric gastrin.     

Evaluation of antifibrotic drugs in bleomycin-induced pulmonary fibrosis in hamsters

Endotracheal administration of bleomycin to hamsters causes severe pulmonary fibrosis. We have examined the utility of this response as a model for the screening of pulmonary antifibrotic agents. The time course of collagen synthesis after bleomycin administration was examined in neutral salt soluble and insoluble fractions by in vitro incubation of minced lung with [14C] proline. Collagen synthesis increased to approximately 250% above control in both neutral salt soluble and insoluble collagen fractions by day 6 after bleomycin. Noncollagenous protein synthesis was also increased but to a lesser amount. The early rise in collagen synthesis leads to accumulation of collagen that can be biochemically quantitated within 1 week. This time course is advantageous for short-term testing of antifibrotic agents. In the present study, beta-aminopropionitrile, D-penicillamine, and p-aminobenzoic acid were examined. All three agents were found to reduce significantly the accumulation of neutral salt insoluble collagen in bleomycin-treated animals.     

Multiple comparisons model-based clustering and ternary pattern tree numerical display of gene response to treatment: procedure and application to the preclinical evaluation of chemopreventive agents

Microarray technology has greatly aided the identification of genes that are expressed differentially. Statistical analysis of such data by multiple comparisons procedures has been slow to develop, in part, because methods to cluster the results of such comparisons in biologically meaningful ways have not been available. We isolated and analyzed, by Northern blot and GeneChip, replicate liver RNA samples (n = 4/group) from rats fed with control diet or diet containing one of three chemopreventive compounds, selected because their pharmacological activities, including RNA expression response, are relatively well understood. We report on a classification tree, based on the results of nonparametric multiple comparisons, which results in the bipolar hierarchical clustering of genes in relation to their response to treatment. In addition to identifying treatment-responsive genes, application of this procedure to our test study identified the known pharmacological relationships among the treatment groups without supervision. Also, small treatment-specific subsets of genes were identified that may be indicative of additional pharmacophores present in the test compounds.     

Interactive role of trauma cytokines and erythropoietin and their therapeutic potential for acute and chronic wounds

If controllable, stem cell activation following injury has the therapeutic potential for supporting regeneration in acute or chronic wounds. Human dermally-derived stem cells (FmSCs) were exposed to the cytokines interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) in the presence of erythropoietin (EPO). Cells were cultured under ischemic conditions and phenotypically characterized using flow cytometry. Topical EPO application was performed in three independent clinical wound healing attempts. The FmSCs expressed the receptor for EPO. EPO had a strong inhibitory effect on FmSC growth in the absence of IL-6 and TNF-α. With IL-6, the EPO effects were reversed to that of growth stimulation. TNF-α had the strongest stimulatory effect. In contrast, IL-1β had an inhibitory effect. Topically applied EPO considerably enhanced wound healing and improved wound conditions of acute and chronic wounds. Site specificity of stem cell activation is mediated by IL-6 and TNF-α. In trauma, EPO ceases its inhibitory role and reverts to a clinically relevant boosting function. EPO may be an important therapeutic tool for the topical treatment of acute and chronic wounds.     

Phosphorylation of cardiac native tropomyosin and troponin: inhibitory effect of actomyosin and possible presence of endogenous myofibrillar-located cyclic-AMP-dependent protein kinase

Canine cardiac native tropomyosin which inhibits Mg²⁺-ATPase and superprecipitation of desensitized actomyosin in the presence of EGTA contains closely associated protein kinase, as manifested by phosphorylation and stimulation by cyclic-AMP. Native tropomyosin functions as a substrate for the endogenous protein kinase. Native actomyosin and desensitized actomyosin showed no significant cyclic-AMP-dependent or independent phosphorylation either in the presence or absence of added protein kinase. Native tropomyosin was fractionated into components by using hydroxylapatite column chromatography. Of the three components, Peak II (troponin) bound Ca²⁺; sodium dodecyl sulfate (SDS) gel electrophoresis revealed four subunits; and Peak III (tropomyosin) was a homogeneous protein and did not bind Ca²⁺. Troponin (Peak II) added to tropomyosin (Peak III) in appropriate concentrations inhibited superprecipitation of desensitized actomyosin in the absence of Ca²⁺. Peak II (troponin) phosphorylation was catalyzed by bovine cardiac protein kinase. The phosphorylation was markedly stimulated by cyclic-AMP. Peak III (tropomyosin) was not phosphorylated by protein kinase in the presence or absence of cyclic-AMP.The addition of desensitized actomyosin completely inhibited the phosphorylation of native tropomyosin. The data suggest the possible involvement of cyclic-AMP and protein kinase in modulating cardiac contraction and relaxation through and action on troponin.