Growth factors and stromal support generate very efficient retroviral transduction of peripheral blood CD34+ cells from Gaucher patients

We have achieved high-efficiency gene transfer into nonmobilized peripheral blood (PB) CD34+ cells from patients with Gaucher's disease using a clinically acceptable retroviral supernatant transduction protocol. In our studies, bone marrow (BM) and PB CD34+ cells were transduced using a high titer (10(8) particles/mL) retroviral supernatant once a day for 4 consecutive days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF), with or without an irradiated allogeneic BM stromal layer. The growth factors alone resulted in 29% +/- 10% gene transfer of PB CD34+ clonogenic cells in contrast with 71% +/- 17% gene transfer efficiency using stroma with the growth factors; a 2.5-fold increase. The increase in gene transfer efficiency was less prominent when BM CD34+ cells were used (40% +/- 16% without and 57% +/- 8% with stroma, a 1.5-fold increase). The overall transduction efficiency of both PB and BM CD34+ cells was lower when the cells were transduced over a stromal cell layer without added growth factors. The combination of IL-3, IL-6, and SCF with stroma transduced 75% of primitive long-term culture initiating cells (PB LTC- ICs) in comparison with 34% of LTC-ICs when IL-3, IL-6, and SCF were used without stromal support. Using this clinically acceptable supernatant/cytokines/stroma transduction protocol, correction of the glucocerebrosidase (GC) deficiency in the progeny cells of PBLTC-ICs from Gaucher's-disease patients has been accomplished. Efficient transduction of the PB CD34+ cells using this transduction protocol may allow repeated delivery of "GC-corrected" hematopoietic stem and progenitor cells to Gaucher's-disease patients.     

Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells

Patients with solid tumors are increasingly being treated by autologous bone marrow transplantation (BMT). Although response rates appear to be increased, disease recurrence is the commonest cause of treatment failure. Whether relapse is entirely due to residual disease in the patient or arises also from infiltrating malignant cells contained in the autologous marrow transplant has not been resolved. If the latter explanation is correct, then purging would be required as part of the transplantation procedure. We used retrovirally mediated transfer of the neomycin-resistance gene to mark BM harvested from eight patients with neuroblastoma in clinical remission. The marked marrow cells were subsequently reinfused as part of an autologous BMT. At relapse, we sought the marker gene in malignant cell populations. Three patients have relapsed, and in each the marker gene was detected by phenotypic and genetic analyses of resurgent malignant cells at medullary and extramedullary sites. Analysis of neuroblast DNA for discrete marker gene integration sites suggested that at least 200 malignant cells, each capable of tumor formation, were introduced with the autologous marrow transplant and contributed to relapse. Thus, autologous BMTs administered to patients with this solid tumor may contain a multiplicity of malignant cells that subsequently contribute to relapse. The marker-gene technique we describe should permit evaluation of the mechanisms of relapse and the efficacy of purging in patients receiving autologous marrow transplantation for other solid tumors that infiltrate the marrow.     

Chronic schistosomiasis during pregnancy epigenetically reprograms T‐cell differentiation in offspring of infected mothers

Schistosomiasis is a nontransplacental helminth infection. Chronic infection during pregnancy suppresses allergic airway responses in offspring. We addressed the question whether in utero exposure to chronic schistosome infection (Reg phase) in mice affects B‐cell and T‐cell development. Therefore, we focused our analyses on T‐cell differentiation capacity induced by epigenetic changes in promoter regions of signature cytokines in offspring. Here, we show that naïve T cells from offspring of schistosome infected female mice had a strong capacity to differentiate into T_H1 cells, whereas T_H2 differentiation was impaired. In accordance, reduced levels of histone acetylation of the IL‐4 promoter regions were observed in naïve T cells. To conclude, our mouse model revealed distinct epigenetic changes within the naïve T‐cell compartment affecting T_H2 and T_H1 cell differentiation in offspring of mothers with chronic helminth infection. These findings could eventually help understand how helminths alter T‐cell driven immune responses induced by allergens, bacterial or viral infections, as well as vaccines.     

Plasma P-selectin is increased in thrombotic consumptive platelet disorders

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P- selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P- selectin may be a useful new marker for thrombotic diseases.     

Three unrelated Rh D gene polymorphisms identified among blood donors with Rhesus CCee (r'r') phenotypes

Human red blood cells are traditionally typed as Rhesus (Rh)-positive or -negative depending on the presence or absence of the Rh D antigen. A recent report demonstrated that the Rh D gene is completely absent in Rh D-negative individuals. In this study, Rh D-negative blood donors with ccee (n = 25) and CCee (n = 3) phenotypes were examined for the presence of absence of the D gene. Polymerase chain reaction (PCR) probes that hybridize to the 5' and 3' regions of the Rh CcEe gene and the closely related D gene were used in a Southern analysis. The D gene was absent in all ccee phenotypes examined. The CCee phenotypes showed three Rh D polymorphisms: one donor lacked the D gene, one donor had a partial deletion on one D gene at the 3' region, and the remaining donor appeared to have one normal D gene within the intron/exon regions examined. We conclude that, while the D gene may be absent in the majority of Rh D-negative phenotypes, rarer polymorphisms also occur that prevent expression of the D antigen resulting in the Rh D-negative phenotype.     

B lymphoblastoid cell lines with normal and defective O-glycosylation established from an individual with blood group Tn

Individuals with the Tn blood group contain terminal serine/threonine- linked N-acetylgalactosamine residues in their blood cells. This is due to lack of UDP-D-galactose: D-N-acetyl galactosamine beta-D-galactosyl transferase from part of their red cells and probably from their leukocytes. We have established B lymphoblastoid cell lines from such an individual by in vitro infection of his lymphocytes with Epstein- Barr virus. The original line contained a mixture of cells reactive and nonreactive with Helix pomatia lectin (Hp). These cells were subcloned after staining with fluorescent Hp by a fluorescence-activated cell sorter (FACS) into homogeneous, phenotypically stable lines of Hp- positive (Hp+) and Hp-negative (Hp-) cells. The molecular differences between the membrane glycoproteins were characterized by carbohydrate- specific surface labeling techniques, Hp affinity chromatography, polyacrylamide slab gel electrophoresis and glycopeptide/oligosaccharide analysis. The major O-glycosidic membrane glycoprotein (GP105) was retained on Hp-Sepharose columns only from Hp+ cells, whereas the common leukocyte antigen (GP160-200) was partially retained on Hp columns from both lines. These proteins were isolated by immune precipitation with monoclonal antibodies and characterized. The results show that the GP105 glycoprotein from Hp+ cells contains terminal N-acetylgalactosamine residues but also more complex oligosaccharides. The common leukocyte antigen showed different electrophoretic mobilities in Hp+ and Hp- cells. UDP-galactose D-N- acetyl galactosamine beta-galactosyl transferase was almost absent in the Hp+ cells. These cell lines are useful for studies on the functional role and regulation of the biosynthesis of O-glycosidic carbohydrates.     

Myocardial infarct size quantification by MR imaging early after coronary artery occlusion in dogs

The feasibility of using magnetic resonance (MR) imaging to estimate myocardial infarct size was explored in an in vitro model using only the inherent differences in contrast between infarcted and noninfarcted myocardium. Eight dogs underwent coronary occlusion; their hearts were removed 6 hours later. Estimates of T2 for normal and infarcted myocardium were derived from MR images. Infarct size was quantified anatomically using triphenyltetrazolium-chloride (TTC) staining and compared with MR estimates. The T2 values derived from the images clearly discriminated between infarcted (126 +/- 22 msec) and normal myocardium (88 +/- 10 msec, P less than .05), providing images with good contrast between normal and infarcted myocardium. Comparable differences in T2 values were also noted from spectrometric determinations. Estimates of infarct size by MR imaging compared well with TTC estimates (r = 0.98) over a wide range of infarct sizes from 3% to 29% of the left ventricular mass. These results suggest the potential for in vivo quantification of infarct size based on the inherent contrast difference between infarcted and normal myocardium.     

Heterogeneity of glomerular size in normal donor kidneys: impact of race

Glomerular size has been the subject of many studies and, in a number of settings, has a direct association with the development of glomerular sclerosis. However, the normal distribution of glomerular size has not been thoroughly evaluated in the general population in the United States. To address this issue, we analyzed the baseline biopsy specimens of 103 human donor kidneys to determine the maximal planar area (MPA) of the glomerular tuft in a heterogeneous human population. The MPA of each glomerulus was determined by measurement of sections through the vascular pole and/or origin of the proximal tubule, and was determined on each section by two methods: point counting and computer planimetry. There was very high agreement between these two methods. Multivariate analysis was used to identify significant correlates with MPA. Overall, younger donors had smaller glomeruli (P < 0.0001). Black donors had a larger MPA (23.4+/-8.6 mm2 x 10(-3)) than white donors (17.9+/-6.7 mm2 x 10(-3); P < 0.001), independent of donor age. MPA was not significantly different between genders. This heterogeneity in glomerular size may confound clinical studies if not recognized and may help explain differences in glomerular structure and function in response to injurious processes.