Support of human cord blood progenitor cells on human stromal cell lines transformed by SV40 large T antigen under the influence of an inducible (metallothionein) promoter

We describe the development of a human bone marrow (BM) culture system which allows study of the interaction of stromal cell lines (SCL) and highly purified hematopoietic progenitor cells. Normal BM stromal cells were electroporated with a plasmid containing the simian virus 40 (SV40) large T antigen (SV40 T Ag) under the control of a synthetic metallothionein promoter (MT4); this construct is designated MT4 SV40 T Ag. SCL in which the rate of proliferation could be controlled by altering the zinc (Zn) concentration were characterized, demonstrating that the SCL were heterogeneous with respect to G-CSF and GM-CSF production. Suppression of SCL proliferation on removal of Zn made it possible to use these lines in coculture with purified CD34+ progenitor cells from umbilical cord blood. The ability to control proliferation of SCL has allowed us to maintain the survival and expansion of colony- forming cells in culture for up to 2 months. These lines have enabled us to test for stromal cell characteristics at a clonal level and provided us with a tool to analyze the events leading to lineage commitment and hematopoietic differentiation, as demonstrated by suppression of hematopoiesis by an antibody directed against the c-kit molecule.     

Differential responses of sex steroid target tissues of rats treated with 4-hydroxyandrostenedione

4-Hydroxyandrostene-3,17-dione (4-OHA) inhibits ovarian aromatase activity and causes regression of carcinogen-induced hormone-dependent mammary tumors in rats. Although estrogen levels were reduced, LH levels did not increase nor did uterine weight decline in 4-OHA-treated animals. These findings are in contrast to those in animals deprived of estrogen by ovariectomy. The possible direct action of 4-OHA on gonadotropin secretion and uterine growth was, therefore, investigated in ovariectomized rats not treated with the carcinogen. Treatment with 4-OHA for 2 weeks prevented regression of the uterus and the increase in gonadotropin secretion in ovariectomized rats in a dose-dependent manner. The effect on gonadotropin secretion of 4-OHA at 50 mg/kg.day was similar to that of dihydrotestosterone at 0.5 mg/kg.day and could be completely antagonized by administration of the antiandrogen flutamide. The stimulation of uterine growth by 4-OHA was also blocked by flutamide, but not by the antiestrogen enclomiphene. The trophic action of 4-OHA at 50 mg/kg.day was equivalent to that of 1.8 mg/kg.day dihydrotestosterone. Furthermore, treatment with 4-OHA caused a reduction in uterine estrogen receptor and progesterone receptor levels. The reduction in uterine estrogen and progesterone receptor levels was also counteracted by the concomitant injection of flutamide, but not by enclomiphene. The results suggest that in the rat 4-OHA has multiple actions on sex steroid target tissues in addition to inhibition of aromatase. The effects appear to be related to the androgenic rather than estrogenic activity of the compound. Inhibition of gonadotropins may help maintain reduced ovarian estrogen secretion and contribute to the antitumor activity of this compound.     

Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady- state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.     

Primary structure of human corticosteroid binding globulin, deduced from hepatic and pulmonary cDNAs, exhibits homology with serine protease inhibitors.

We have isolated and sequenced cDNAs for corticosteroid binding globulin (CBG) prepared from human liver and lung mRNAs. Our results indicate that CBG mRNA is relatively abundant in the liver but is also present in the lung, testis, and kidney. The liver CBG cDNA contains an open reading frame for a 405-amino acid (Mr 45,149) polypeptide. This includes a predominantly hydrophobic, leader sequence of 22 residues that precedes the known NH2-terminal sequence of human CBG. We, therefore, predict that the mature protein is composed of 383 amino acids and is a polypeptide of Mr 42,646. A second, in-frame, 72-base-pair cistron of unknown significance exists between the TAA termination codon for CBG and a possible polyadenylylation signal (AATAAA) located 16 nucleotides before the polyadenylylation site. The deduced amino acid sequence of mature CBG contains two cysteine residues and consensus sequences for the attachment of six possible N-linked oligosaccharide chains. The sequences of the human lung and liver CBG cDNAs differ by only one nucleotide within the proposed leader sequence, and we attribute this to a point mutation. No sequence homology was found between CBG and other steroid binding proteins, but there is a remarkable similarity between the amino acid sequences of CBG and of alpha 1-antitrypsin, and this extends to other members of the serpin (serine protease inhibitor) superfamily.     

Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.     

Respiratory muscle strength in congestive heart failure

In experimental animals, conditions which drastically decrease cardiac output may reduce the strength and endurance of respiratory muscles leading to hypercapnic respiratory failure. Because patients with chronic CHF have reduced cardiac output and vital capacity (FVC), we measured PImax and PEmax and maximal handgrip force in 16 patients with CHF and 18 AMNs. The patients with CHF had a mean left ventricular ejection fraction of 26 +/- 7 percent. Maximal respiratory pressures were significantly reduced; group mean values (+/- SD) for PImax at FRC were 41.4 +/- 5.6 cm H2O (CHF) and 102.1 +/- 27.4 cm H2O (AMN) (p less than 0.001), with PImax values in five patients with CHF as low as 20 to 30 cm H2O. In most patients, PEmax was comparably reduced. Handgrip force was less dramatically reduced, suggesting selective respiratory muscle weakness. Possible explanations include reduction in respiratory muscle blood flow or generalized muscular atrophy and weakness related to cardiac cachexia.     

Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that "niches" need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.     

Can a Minimal Intervention Reduce Secondhand Smoke Exposure Among Children with Asthma from Low Income Minority Families? Results of a Randomized Trial

We report on the results of a low-intensity behavioral intervention to reduce second hand smoke (SHS) exposure of children with asthma from low income minority households in Los Angeles, California. In this study, 242 child/adult dyads were randomized to a behavioral intervention (video, workbook, minimal counseling) or control condition (brochure). Main outcome measures included child’s urine cotinine and parental reports of child’s hours of SHS exposure and number of household cigarettes smoked. Implementation of household bans was also considered. No differences in outcomes were detected between intervention and control groups at follow-up. Limitations included high attrition and low rates of collection of objective measures (few children with urine cotinine samples). There continues to be a need for effective culturally and linguistically appropriate strategies that support reduction of household SHS exposure among children with asthma in low income, minority households.     

Combined fluorescent and electron microscopic imaging unveils the specific properties of two classes of meiotic crossovers

Crossovers (COs) shuffle genetic information and allow balanced segregation of homologous chromosomes during the first division of meiosis. In several organisms, mutants demonstrate that two molecularly distinct pathways produce COs. One pathway produces class I COs that exhibit interference (lowered probability of nearby COs), and the other pathway produces class II COs with little or no interference. However, the relative contributions, genomic distributions, and interactions of these two pathways are essentially unknown in nonmutant organisms because marker segregation only indicates that a CO has occurred, not its class type. Here, we combine the efficiency of light microscopy for revealing cellular functions using fluorescent probes with the high resolution of electron microscopy to localize and characterize COs in the same sample of meiotic pachytene chromosomes from wild-type tomato. To our knowledge, for the first time, every CO along each chromosome can be identified by class to unveil specific characteristics of each pathway. We find that class I and II COs have different recombination profiles along chromosomes. In particular, class II COs, which represent about 18% of all COs, exhibit no interference and are disproportionately represented in pericentric heterochromatin, a feature potentially exploitable in plant breeding. Finally, our results demonstrate that the two pathways are not independent because there is interference between class I and II COs.Eukaryotic sexual reproduction involves meiosis, a specialized cell division in which DNA duplication in a diploid cell is followed by two cell divisions to produce four haploid cells. The first division, Meiosis I, involves crossing over and chiasmata formation between each pair of homologous chromosomes, thereby ensuring separation of the homologs and formation of two haploid cells, each with one complete set of replicated chromosomes. The second division, Meiosis II, is a mitosis-like division in which the two sister chromatids separate to yield four haploid cells that directly or indirectly form gametes. Because these four products are genetically unique due to crossing over and independent segregation of homologous chromosomes during Meiosis I, meiosis plays an important role in creating genetic diversity in sexually reproducing organisms.Crossing over during meiosis is tightly controlled so each pair of homologs has at least one “obligate” crossover (CO) that ensures balanced reductional segregation, but the presence of a CO reduces the likelihood of another CO in its vicinity, a phenomenon referred to as CO interference (1, 2). Significant progress has been made recently in illuminating the molecular events of meiotic recombination and the control of crossing over (3–8). The initiating event of meiotic recombination in most organisms is formation of numerous DNA double-strand breaks (DSBs). Homolog-dependent repair of a DSB may follow any one of at least three pathways: (i) non-CO that may result in a short gene conversion; (ii) CO with interference (class I COs, produced by pathway P1); or (iii) CO without interference (class II COs, produced by pathway P2) (6, 7, 9). The interfering CO pathway involves the resolution of double Holliday junctions, which requires many proteins including the ZMM group (ZIP1-4, MSH4-5, MER3) and the MutL homolog 1 (MLH1)/MLH3 complex (6, 10). The noninterfering CO pathway depends primarily on the Mus81/Mms4 endonuclease complex in budding yeast (MUS81/EME1 complex in plants and animals) (5–7, 11–14).Meiotic COs occur in association with two cytological structures, synaptonemal complexes (SCs) that link each pair of homologous chromosomes throughout their lengths during pachytene and late recombination nodules (RNs) that are ellipsoidal structures on SCs (15). Every SC has at least one RN, each RN marks a CO site, and most RNs contain MLH1 protein (16–19). RNs are too small (50-100 nm) to be resolved using light microscopy (LM), but they can be readily visualized by transmission electron microscopy (EM), particularly in 2D spreads of SCs (18). Antibodies to MLH1 protein have been used as immunofluorescent probes to map class I COs on SCs (e.g., refs. 19 and 20). Pathway 2 (P2), which was revealed using mutants of the P1 pathway, produces class II COs, and these class II COs showed no interference in the marker intervals studied (21–23). The P1 pathway produces the majority of COs, and the P2 pathway accounts for ∼5–30% of COs (8, 11, 21). CO distributions have been effectively modeled by assuming that class II COs are independent from class I COs (24). However, class II COs have not been independently mapped on chromosomes (12), and little is known about the properties of each pathway or whether they interact in wild-type organisms.Here, we describe an advanced approach that uses SC spreads from wild-type tomato (Solanum lycopersicum, 2n = 2x = 24) to directly identify the pathway of origin for each CO in individual meiotic nuclei. For this, we superimposed the immunofluorescent LM image of an SC spread showing MLH1 foci (class I COs) onto an EM image of the same SC spread showing RN locations (all COs). RNs that coincide with MLH1 foci (MLH1-positive RNs) mark class I COs, and RNs that do not coincide with MLH1 foci (MLH1-negative RNs) are considered to mark class II COs. Because EM is time-consuming, this approach takes advantage of both the relative speed of LM and the high resolution of EM, allowing us to analyze RNs on 1882 tomato SCs.