Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.     

Expression of the interleukin-2 receptor gamma chain on cord blood mononuclear cells

Lymphocytes in umbilical cord blood and neonatal peripheral blood have been shown to have less ability in an immune reaction. In our present experimental approach to address this issue, we made use of the cord blood of full-term birth infants to investigate the expression of the interleukin- 2 receptor gamma (IL-2Rgamma) chain that is shared with receptors for IL-4, IL-7, IL-9, and IL-15 as well as IL-2. The gamma chain expression in cord blood lymphocytes was about one-third that in the lymphocytes of adults, whereas no significant difference between cord blood and adult monocytes was observed. A reduced expression of the gamma chain was observed in all of the CD4+ T cells, CD8+ T cells, gamma-delta T cells, B cells, CD16+ natural killer (NK) cells, and CD56(bright) NK cells of the cord blood lymphocytes. The reduced gamma chain expression reached two-thirds of that in adults after 3 days of culture in vitro and in infants 3 days after birth, thus implying that the increase in the gamma chain may significantly contribute to the prevention of neonatal infection.     

A neutralizing anti-TGF-beta1 antibody promotes proliferation of CD34+Thy-1+ peripheral blood progenitors and increases the number of transduced progenitors

To reduce the time of computation of a motor unit potential (MUP), the shape of intracellular action potential (IAP) and (or) MU anatomy are generally simplified. A method of MUP presentation is suggested. It provides accuracy of the MUPs calculated for any distance and size of rectangular electrodes together with considerably reduced computational load and time. No simplification of the IAP shape or location of muscle fibres of different diameters and lengths is required. The MUP generated by N temporally and spatially dispersed single fibre action potentials is considered to be the output signal of a linear time-shift invariant system for potential generation. The input signal is the first temporal derivative of the IAP. The common impulse response (CIR) is the sum of potentials produced at the electrode by N pairs of dipoles propagating from the motor end-plates to the ends of the corresponding fibres. The potentials of each dipole at the rectangular plate electrode are determined analytically. Thus, the MUP is calculated as a single convolution between the input signal and CIR for a rectangular electrode of any size.     

Expansion of megakaryocyte progenitors from human umbilical cord blood using a new two-step separation procedure

We examined interference competition for food in oystercatchers, Haematopus ostralegus L., feeding on cockles, Cerastoderma edule L., when kleptoparasitism was infrequent. These birds opened cockles by hammering a hole in the shell, searched for them by touch and experienced densities of feeding conspecifics that ranged from 0 to 2362.5 birds/ha. Handling times were not significantly correlated with competitor density, but the probability of successfully opening a cockle declined significantly as competitor density increased because birds were more likely to abandon cockles they had found. Birds were also significantly more likely to carry cockles away from where they were found prior to attempting to open them as competitor density increased. We used an optimal diet model to predict maximum energy intake rates achievable for birds feeding on a given prey population, and experiencing a range of competitor densities. Despite affecting foraging behaviour, the model showed that competitor density had a negligible impact on overall intake rates. Although kleptoparasitism was rare in our study population, only 1.5% (9/586) of cockles being lost to parasites, a recent model suggests that it was likely to be profitable, under the conditions experienced by our birds. We suggest that kleptoparasitism might be infrequent because birds could reduce its likelihood by adjusting their behaviour, with only a minimal cost in terms of a reduced intake rate. Behaviour-based models of interference competition, therefore, need to consider a range of potentially complex avoidance behaviours when attempting to describe the dynamics of this process. Copyright 1998 The Association for the Study of Animal Behaviour     

Effects of lipidic mediators on the growth of human myeloid and erythroid marrow progenitors

The influence of hyperthyroidism on the functional vascular responsiveness of isolated coronary and renal resistance vessels was investigated. Hyperthyroidism was established by feeding rats for 1 and 4 weeks with 5 mg/kg L-thyroxine (T4)-containing rat chow. Preparations of either coronary or renal resistance vessels were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for the effects of 5-hydroxytryptamine (5-HT), 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F2alpha (U46619) and isoproterenol in coronary vessels, and for those of methoxamine, U46619 and isoproterenol in renal vessel preparations. Our results indicate that hyperthyroidism does not induce major changes in the sensitivity of both coronary and renal resistance vessels towards 5-HT, U46619 and methoxamine. A clearly sensitizing influence of acute hyperthyroidism (1 week of T4 treatment) was found for isoproterenol-induced relaxant responses, whereas hyperthyroidism for 4 weeks did not influence the responses mediated by isoproterenol in coronary resistance arteries. Furthermore, the isoproterenol-induced relaxation in renal arteries was not influenced by the chronic hyperthyroid state of the animal. The present results indicate that in acute hyperthyroidism beta-adrenoceptor-mediated vasodilation is increased. However, in chronic hyperthyroidism changes in responsiveness to vasoconstrictor or vasodilator agents of coronary and renal resistance arteries appear not to play a major role. The influence of hyperthyroidism on the functional response of resistance arteries appears to be both tissue and time dependent.     

Ultrastructural morphology of human mast cell progenitors in sequential cocultures of cord blood cells and fibroblasts

The ontogeny of human mast cells (HMCs) is not known, but agranular precursors committed to HMC development are present in cord blood mononuclear cells. Individual cord bloods were cocultured with murine fibroblasts and prepared for light microscopy, immunofluorescent determination of tryptase and electron microscopy after sequential times in culture. Mast cells and their progenitors were identified and quantitated during their evolution from agranular precursors in human cord blood. Each of these methods revealed similar numbers of HMC progenitors. The earliest, visible granule contents were particulate; later, scrolls superimposed on particulate materials were noted. HMC progenitors did not contain crystal granules, but fully mature HMCs present in long-term cocultures did. The developmental sequence for the evolution of the HMC lineage in vitro, made available by this new culture system, is identical to that described for the development of HMCs in vivo in sequentially examined samples of human fetal tissues.     

Growth of macroscopic human megakaryocyte colonies from cord blood in culture with recombinant human thrombopoietin (c-mpl ligand) and the effects of gestational age on frequency of colonies

The purpose of this quasi-experimental study was to determine the effect of an experimental clinical teaching method on nursing students' knowledge and critical thinking skills during clinical rotations in psychiatric nursing. Ausubel's Assimilation Theory of Learning provided the theoretical framework for this research. The subjects (N = 83) were from the baccalaureate nursing graduating classes of 1992 and 1993 of a medium-sized state university in the mid-atlantic region of the United States. The experimental clinical teaching method used computer-assisted instruction (CAI) throughout and a collaboration model (student-faculty-service) during initial clinical experiences. Knowledge was measured by the score for the psychiatric nursing component of the Mosby Assess Test and two parts of the National League for Nursing's (NLN) Psychiatric Exam (theory and total scores). Critical thinking was measured by two parts of the NLN's Psychiatric Nursing Exam (the score for assess, analyze, and evaluate as one measure and planning and implementation for the other measure). The comparison group scored significantly higher than the control group on assessing, analyzing, and evaluating (t = 2.15; p < .03), as well as planning and implementation (t = 2.33; p < .02), measures for critical thinking skills. However, there was no significant difference between the study groups with respect to knowledge. The findings support the appropriateness of Ausubel's Assimilation Theory with this clinical teaching method. Discussion relevant to the application of the study findings to nursing education and future research is presented.     

Relative sedimentation of hematopoietic progenitors in human cord blood, peripheral blood, and bone marrow as determined by counterflow centrifugal elutriation

OBJECTIVE: Although there have been many studies of the outcome of anorexia nervosa, methodological weaknesses limit their interpretation. The authors used a case-control design to try to improve knowledge about the outcome of anorexia nervosa. METHOD: All new female patients referred to an eating disorders service between Jan. 1, 1981, and Dec. 31, 1984, who had probable or definite anorexia nervosa were eligible for inclusion. Of these women, 86.4% (N = 70) were located and agreed to participate. The comparison group (N = 98) was a random community sample. All subjects were interviewed with a structured diagnostic instrument. RESULTS: A minority of the patients (10%) continued to meet the criteria for anorexia nervosa a mean of 12 years after initial referral. Even among those who no longer met these criteria, relatively low body weight and cognitive features characteristic of anorexia nervosa (perfectionism and cognitive restraint) persisted. The rates of lifetime comorbid major depression, alcohol dependence, and a number of anxiety disorders were very high. CONCLUSIONS: In the managed care/brief treatment era, therapeutic approaches with an excessive focus on weight gain that neglect the detection and treatment of associated psychological features and comorbidity may be inappropriate. Anorexia nervosa is a serious psychiatric disorder with substantial morbidity.     

Purification and characterization of the human interleukin-18 receptor

Interleukin (IL)-18 was identified as a molecule that induces IFN-gamma production and enhances NK cell cytotoxicity. In this paper, we report upon the purification and characterization of human IL-18 receptor (hIL-18R). We selected the Hodgkin's disease cell line, L428, as the most strongly hIL-18R-expressing cell line based on the results of binding assays. This binding was inhibited by IL-18 but not by IL-1beta. The dissociation constant (Kd) of 125I-IL-18 binding to L428 cells was about 18.5 nM, with 18,000 binding sites/cell. After immunizing mice with L428 cells and cloning, a single monoclonal antibody (mAb) against hIL-18R was obtained (mAb 117-10C). Sequentially, hIL-18R was purified from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-extracted L428 cells by wheat germ lectin-Sepharose 4B chromatography and mAb 117-10C-Sepharose chromatography. The internal amino acid sequences of hIL-18R all matched those of human IL-1 receptor-related protein (IL-1Rrp), the ligand of which was unknown to date. When expressed in COS-1 cells, the cDNA of IL-1Rrp conferred IL-18 binding properties on the cells and the capacity for signal transduction. From these results, we conclude that a functional IL-18 receptor component is IL-1Rrp.     

T-cell receptor BJ gene segment expression in human umbilical cord blood CD4+ and CD8+ T-cell subsets

By employing RT-PCR-based technology, followed by Southern-blot analysis, patterns of relative TRC BJ gene segment usage in human CD4+ and CD8+ umbilical cord blood T cells (UCT) from ten children were determined in relation to seven recombined TCR BV gene (sub) families (BV 3, 5S1, 6S1-3, 8, 9, 12 and 18). Normal frequency of usage of individual BJ members was observed to be extremely nonrandom. BJ usage in association with each BV was ranked and mean ranking values were calculated for individual BJs. Moreover, BJ family usage and family ranges as well as individual BJ over-representations were determined. In all these aspects of BJ exon expression, CD4+ and CD8+ UCT displayed similar distribution patterns. Comparisons of BJ usage in UCT subpopulations and in the adult peripheral blood lymphocyte (PBL) counterparts were performed and many similarities were observed. However, discrepancies in two parameters were recorded; contrary to observations in PBL, individual BJ over-representations were virtually absent in UCT, and significantly less wide BJ family ranges were demonstrated in CD8+ UCT relative to CD8+ PBL T cells. These differences support the notion that UCT are in a less dynamic state than are PBL T cells. Hence, despite the fact that PBL T cells are subjected to continuous antigenic challenge, the striking resemblance of PBL and UCT with regard to the overall individual relative usage, ranking, mean ranking and family utilisation of BJ gene segments, irrespective of the choice of recombined BV exons, may suggest a relatively nondiscriminatory role for the BJ gene product in antigen recognition as compared to those encoded by the BV, (N) and BD gene segments.     

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 10(3)-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface IgM (sIgM) after 5 weeks of co-culture. CD34+CD19- cells also showed a similar development of CD19+ cells and CD19+sigM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19-CD13- CD33- cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19-CD13-CD33- progenitors require the cell-cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.     

Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood

Recombinant adeno-associated virus 2 (AAV) virions were constructed containing a gene for resistance to neomycin (neoR), under the control of either the herpesvirus thymidine kinase (TK) gene promoter (vTK-Neo), or the human parvovirus B19 p6 promoter (vB19-Neo), as well as those containing an upstream erythroid cell-specific enhancer (HS-2) from the locus control region of the human beta-globin gene cluster (vHS2-TK-Neo; vHS2-B19-Neo). These recombinant virions were used to infect either low density or highly enriched populations of CD34+ cells isolated from human umbilical cord blood. In clonogenic assays initiated with cells infected with the different recombinant AAV-Neo virions, equivalent high frequency transduction of the neoR gene into slow-cycling multipotential, erythroid, and granulocyte/macrophage (GM) progenitor cells, including those with high proliferative potential, was obtained without prestimulation with growth factors, indicating that these immature and mature hematopoietic progenitor cells were susceptible to infection by the recombinant AAV virions. Successful transduction did not require and was not enhanced by prestimulation of these cell populations with cytokines. The functional activity of the transduced neo gene was evident by the development of resistance to the drug G418, a neomycin analogue. Individual high and low proliferative colony-forming unit (CFU)-GM, burst-forming unit-erythroid, and CFU-granulocyte erythroid macrophage megakaryocyte colonies from mock-infected, or the recombinant virus-infected cultures were subjected to polymerase chain reaction analysis using a neo-specific synthetic oligonucleotide primer pair. A 276-bp DNA fragment that hybridized with a neo-specific DNA probe on Southern blots was only detected in those colonies cloned from the recombinant virus-infected cells, indicating stable integration of the transduced neo gene. These studies suggest that parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors for high efficiency gene transfer into slow or noncycling primitive hematopoietic progenitor cells, without the need for growth factor stimulation, which could potentially lead to differentiation of these cells before transplantation.     

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.     

The transactivation domain AF-2 but not the DNA-binding domain of the estrogen receptor is required to inhibit differentiation of avian erythroid progenitors

Insight into the origin of human rotaviruses carrying the AU-1 VP4 allele was gained by examining their genomic RNA constellation using RNA-RNA hybridization and by sequencing the VP8* portion (nucleotides 1-750) of their gene 4. AU-1 like viruses isolated in Israel from children attending outpatient clinics were classified into three sub-genogroups based on RNA-RNA hybridization analysis: Subgenogroup 1 consists of two strains (Ro-5829 and Ro-5960) which belong to the AU-1 genogroup, since all their 11 segments hybridized to AU-1 segments. Subgenogroup 2 consists of one reassortant virus (Ro-5193) of which seven RNA segments hybridized to AU-1 segments and the remaining four segments hybridized to NCDV (bovine rotavirus). Subgenogroup 3 consists of four reassortant viruses (Ro-6460, Ro-6584, Ro-6784 and Ro-7044) which had a common genome constellation: only four of their RNA segments hybridized to AU-1 and the other seven segments hybridized to NCDV segments. Sequence analysis of the VP8* gene also revealed a three level pattern of homology with the AU-1 prototype and the local AU-1 like strains which was consistent with the overall genomic (RNA-RNA) constellation: Subgenogroup 1 had 98-98.1% homology with the AU-1 prototype; Subgenogroup 2 had 96.8% homology with the AU-1 prototype and 95.6-96.7% homology with Subgenogroup 1; Subgenogroup 3 had 95.3-95.6% homology with the prototype AU-1 and 93.4-94.3% homology with Subgenogroup 1. Possible evolutionary pathways are discussed.     

Study on the collecting method of human umbilical cord blood

Anemia is responsible for an estimated 20% of maternal deaths in West Africa and contributes to still more deaths through obstetric hemorrhage. Anemia during pregnancy has been linked to iron and folate dietary deficiencies, the secondary effects of malaria and hookworm infestations, infections such as human immunodeficiency virus, and hemoglobinopathies. Parasitic infestations interfere with the normal increase (given a balanced diet) in iron absorption during pregnancy. An understanding of locally salient etiologic factors should form the basis of public health programs aimed at addressing anemia during pregnancy. There is a need for basic prevalence statistics, especially from West Africa's rural areas. Finally, reliable laboratory parameters that can be used in the assessment of iron and folate status and the degree of anemia attributable to malaria must be established. Although there is emerging evidence that serum transferrin receptor concentration is not affected by chronic disease or the physiological changes of pregnancy, further studies are needed to validate this measure.     

Effects of nonsteroidal anti-inflammatory drugs on interleukin-1 receptor antagonist production in cultured human peripheral blood mononuclear cells

The effects of nonsteroidal anti-inflammatory drugs (NSAIDs), mofezolac, indomethacin, sodium diclofenac, and zaltoprofen, on the production of interleukin-1 receptor antagonist (IL-1ra) were examined in cultured human peripheral blood mononuclear cells (PBMC). Among the NSAIDs tested, mofezolac and sodium diclofenac were found to stimulate the mRNA expression for IL-1ra without affecting the mRNA expression for IL-1 beta. These two drugs also stimulated the secretion of IL-1ra by PBMC in the absence of bacterial lipopolysaccharide (LPS), however, the stimulatory effect of sodium diclofenac diminished in the presence of LPS. Mofezolac suppressed the mRNA expression for IL-1 beta in PBMC stimulated with exogenous IL-1 beta, indicating the secreted IL-1ra in the presence of mofezolac to be biologically active. Since IL-1ra suppresses the function of IL-1, a pro-inflammatory cytokine, the stimulatory effect of such NSAIDs as mofezolac on IL-1ra production could also be one of the mechanisms involved in its anti-inflammatory and antinociceptive actions.     

Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver

Recombinant adeno-associated virus vectors (AAV) were prepared in high titer (10(12) to 10(13) particles/mL) for the expression of human factor IX after in vivo transduction of murine hepatocytes. Injection of AAV-CMV-F.IX (expression from the human cytomegalovirus IE enhancer/promoter) into the portal vein of adult mice resulted in no detectable human factor IX in plasma, but in mice injected intravenously as newborns with the same vector, expression was initially 55 to 110 ng/mL. The expression in the liver was mostly transient, and plasma levels decreased to undetectable levels within 5 weeks. However, long-term expression of human F.IX was detected by immunofluorescence staining in 0.25% of hepatocytes 8 to 10 months postinjection. The loss of expression was likely caused by suppression of the CMV promoter, because polymerase chain reaction data showed no substantial loss of vector DNA in mouse liver. A second vector in which F.IX expression was controlled by the human EF1alpha promoter was constructed and injected into the portal vein of adult C57BL/6 mice at a dose of 6.3 x 10(10) particles. This resulted in therapeutic plasma levels (200 to 320 ng/mL) for a period of at least 6 months, whereas no human F.IX was detected in plasma of mice injected with AAV-CMV-F.IX. Doses of AAV-EF1alpha-F. IX of 2.7 x 10(11) particles resulted in plasma levels of 700 to 3, 200 ng/mL. Liver-derived expression of human F.IX from the AAV-EF1alpha-F.IX vector was confirmed by immunofluorescence staining. We conclude that recombinant AAV can efficiently transduce hepatocytes and direct stable expression of an F.IX transgene in mouse liver, but sustained expression is critically dependent on the choice of promoter.     

The structure and organization of the human erythroid anion exchanger (AE1) gene

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans approximately 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3' noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.