bluuebunny/crossref_metadata_2025 huggingface dataset:
Total cell content of CR3 (CD11b/CD18) and LFA-1 (CD11a/CD18) in neonatal neutrophils: relationship to gestational age
Authors: McEvoy LT , Zakem-Cloud H , Tosi MF
Published: October 2019

Neonatal neutrophils (PMN) exhibit a well-documented defect in chemotaxis that is associated with several abnormalities of PMN structure and function, including deficient surface expression of CR3 (CD11b/CD18), a critical adhesion molecule, on chemoattractant- activated PMN. We recently documented that deficient surface expression of CR3 on stimulated neonatal PMN is due principally to a deficiency in total cell content of CR3. In the current studies, we tested the hypothesis that total cell CR3 content of PMN is even more profoundly deficient in premature infants and that PMN CR3 content is directly related to gestational age. A sandwich enzyme-linked immunosorbent assay for CR3 showed that PMN lysates from term neonates ( > or = 37 weeks gestation) contain about 60% of adult PMN levels of CR3, whereas PMN from premature infants (range of 27 to 36 weeks gestation) contained a mean of about 30%, ranging from 10% to 48% (P < .001 for term [n = 6] v premature [n = 11] by unpaired t-test). When the relationship between total cell CR3 and gestational age (n = 15) was analyzed, the correlation coefficient was .94 by linear regression, and the Spearman rank correlation was significant with P < .001. PMN content of LFA-1 (CD11a/CD18) was similarly measured for 14 neonates. Term neonates were equivalent to adults in LFA-1 content of their PMN (99.4% +/- 3.2% of paired adult values, n = 6), whereas prematures (28 to 36 weeks gestation) were deficient, overall (69.1% +/- 10.4%, n = 8, P = .035). Below 35 weeks gestation, LFA-1 values ranged from 26% to 65% of paired adult control values, but no infant of > or = 35 weeks gestation had PMN LFA-1 content that was less than 85% of its adult control. We concluded that CR3 total cell content is more profoundly deficient in premature than in term neonates, that at birth there is a direct relationship between PMN CR3 content and gestational age, and that LFA-1 is deficient only in prematures less than 35 weeks of gestational age. Below 30 weeks gestation, CR3 content of PMN approached that seen in genetic deficiency of the CD18 family of leukocyte integrins, or type 1 leukocyte adhesion deficiency, underscoring the severity of this host impairment in very early preterm neonates.
Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation
Authors: Baklouti F , Huang SC , Tang TK , Delaunay J , Marchesi VT , Benz EJ Jr
Published: October 2019

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5′ end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD “spectrin/actin binding domain” (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5′ end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.
Catalysis of soluble hemoglobin oxidation by free iron on sickle red cell membranes
Authors: Shalev O , Hebbel RP
Published: October 2019

Abnormal deposition of hemichrome on the inner aspect of the sickle red cell membrane promotes premature cell demise. The steps proximate to hemichrome formation in these cells are poorly understood. To test the hypothesis that the pathologic deposits of free ferric iron located on the inner aspect of sickle cell membranes would be redox active and promote oxidation of soluble oxyhemoglobin, we incubated native versus iron-stripped sickle or normal ghost membranes with oxyhemoglobin S. We found that sickle membranes exerted an exaggerated effect on methemoglobin formation in solution, an effect completely accounted for by their abnormal content of free iron. This ability of sickle membranes to promote hemoglobin oxidation was not diminished by catalase or by presence of a high-affinity, iron-inactivating chelator that is unable to remove membrane iron. Examination of those membranes likewise revealed that their free iron content promoted deposition of additional heme-protein. These results establish that the potential redox couple formed by membrane-associated ferric iron and cytoplasmic oxyhemoglobin is promotive of hemoglobin oxidation and deposition of hemichrome on the membrane. This predicts that removal of pathologic membrane iron might help prevent the detrimental formation of methemoglobin and hemichrome in vivo, insofar as this is accelerated by transition metal.
Quantitation of mafosfamide-resistant pre-colony-forming units in allogeneic bone marrow transplantation: relationship with rate of engraftment and evidence for long-lasting reduction in stem cell numbers
Authors: Kirkland MA , Spencer A , Davidson RJ , McDonald C , Goldman JM
Published: October 2019

Current assays of human committed-stem cells are of limited value in predicting the rate of engraftment or in assessing the integrity of the stem cell pool after allogeneic bone marrow (BM) transplantation (BMT). We have used a limiting dilution assay of mafosfamide-resistant progenitors (pre-colony-forming units [CFU]), which are ancestral to committed progenitors such as CFU-granulocyte-macrophage (GM) to analyze the kinetics of myeloid engraftment after BMT and to assess the size of the stem cell pool at intervals up to 66 months thereafter. In 24 patients transplanted for chronic myeloid leukemia in chronic phase (eight with matched unrelated donors and 16 with sibling donors), the rate of neutrophil engraftment correlated strongly with the number of pre-CFU transfused per kilogram recipient body weight (r = .7, P < .005) but not with CFU-GM per kilogram or nucleated cells per kilogram. In 25 patients studied 6 to 66 months after allogeneic BMT, the mean number of pre-CFU in the marrow was 3.1/10(5) mononuclear cells (MNC) (median, 3.47; range, 0.4 to 23.3), compared with 24.7/10(5) MNC (median, 27.3; range, 4.2 to 180) in 25 normal subjects. CFU-GM were also reduced in these patients, but with considerable overlap into the normal range (mean +/- SD: 54 +/- 45.6 per 10(5) MNC; normal, 129 +/- 61.6). Low pre-CFU but not low CFU-GM levels were associated with reduced peripheral blood white blood cell counts in post-BMT patients. Pre-CFU and CFU-GM levels were not related to the interval posttransplant and remained low for up to 66 months. We conclude that the pre-CFU assay measures a population of stem/progenitor cells that are important in the kinetics of engraftment after allogeneic BMT. Our data suggest that pre-CFU levels may remain low for some years after BMT in humans.
In vivo inhibition of cytokine responsiveness and graft-versus-host disease mortality by rapamycin leads to a clinical-pathological syndrome discrete from that observed with cyclosporin A
Authors: Blazar BR , Taylor PA , Panoskaltsis-Mortari A , Sehgal S , Vallera DA
Published: October 2019

Rapamycin (RAPA) has been shown to be a highly effective means of reducing the lethality of graft-versus-host disease (GVHD) in B10.BR recipients of allogeneic C57BL/6 donor cells. RAPA-treated mice had no clinical (e.g., weight loss, diarrhea, lethargy) or histologic evidence of classical acute or chronic GVHD but did develop a clinical- pathological syndrome consisting of ulcerative dermatitis, bile duct proliferation, and a nondestructive peribronchiolar pulmonary infiltration. Because RAPA was found to interfere with the deletion of self-reactive T cells, we wondered whether the RAPA-induced syndrome was related to failed negative selection or altered alloreactivity. We now show that the RAPA-induced syndrome is due to effects on mature, donor-derived alloreactive T cells. By titering the number of T cells infused we were able to vary the syndrome incidence. In contrast to the syndrome seen after cyclosporin A (CsA) administration, the RAPA syndrome did not require an intact thymus and the disease could not be adoptively transferred. The addition of CsA (which blocks T-cell cytokine production) to RAPA (which blocks T-cell cytokine response) prevented the generation of this syndrome, suggesting that the tissue manifestations seen in RAPA only treated recipients were caused by cytokine production and release. RAPA also caused this alloimmune syndrome in recipients of minor histocompatibility antigen disparate donor cells, showing that the RAPA effects were not restricted to a single donor-recipient strain combination or to instances in which the donor and recipient were fully major histocompatibility complex disparate. We conclude that RAPA is a highly effective means of preventing murine acute GVHD, and that when combined with CsA, warrants consideration for human investigations.
Hematopoietic growth factors as adjuncts to the treatment of acute myeloid leukemia
Authors: Schiffer CA
Published: October 2019

A number of randomized trials have recently been completed evaluating the effect of hematopoietic growth factors (granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor) as adjuncts to the treatment of patients with acute myeloid leukemia. Most studies used the growth factors to decrease the duration of neutropenia with the hope of reducing infectious morbidity and mortality. The results of these trials are generally quite consistent. Virtually all trials showed a modest reduction in the duration of severe neutropenia with a variable effect on the incidence of severe infections, antibiotic usage, and the duration of hospitalization. There was no consistent benefit in terms of improvements in complete response rate, complete response duration, or overall survival. However, it is important that there does not appear to be an increase in the incidence of drug-resistant leukemia in trials in which the growth factor was begun after completion of the chemotherapy. Other trials administered growth factors either before or simultaneous with the chemotherapy in an attempt to enhance chemosensitivity and decrease drug resistance. None of these trials, whether conducted as part of initial induction therapy or in relapse, showed improvements in response rate or survival. Lastly, some anecdotal reports have suggested that occasional patients who receive growth factors as the only therapy for overt leukemia can achieve remission, possibly through a differentiating effect of the growth factor. However, there are very few such reports, and growth factor use in this situation is potentially dangerous and should be performed only in the context of a clinical trial. In summary, there appears to be no role at this time for priming of leukemia cells by growth factors to enhance the effect of chemotherapy, and more in vitro studies should be performed before further clinical trials of this approach. It is clear that growth factors administered after induction and possibly consolidation chemotherapy can shorten the duration of neutropenia, without a significant effect on treatment outcome. It is as yet unclear whether the use of growth factors in this fashion is cost effective.
A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis
Authors: Poort SR , Rosendaal FR , Reitsma PH , Bertina RM
Published: October 2019

We have examined the prothrombin gene as a candidate gene for venous thrombosis in selected patients with a documented familial history of venous thrombophilia. All the exons and the 5′- and 3′-UT region of the prothrombin gene were analyzed by polymerase chain reaction and direct sequencing in 28 probands. Except for known polymorphic sites, no deviations were found in the coding regions and the 5′-UT region. Only one nucleotide change (a G to A transition) at position 20210 was identified in the sequence of the 3′-UT region. Eighteen percent of the patients had the 20210 AG genotype, as compared with 1% of a group of healthy controls (100 subjects). In a population-based case-control study, the 20210 A allele was identified as a common allele (allele frequency, 1.2%; 95% confidence interval, 0.5% to 1.8%), which increased the risk of venous thrombosis almost threefold odds ratio, 2.8; 95% confidence interval, 1.4 to 5.6. The risk of thrombosis increased for all ages and both sexes. An association was found between the presence of the 20210 A allele and elevated prothrombin levels. Most individuals (87%) with the 20210 A allele are in the highest quartile of plasma prothrombin levels (> 1.15 U/mL). Elevated prothrombin itself also was found to be a risk factor for venous thrombosis.
Granulocytic and monocytic differentiation of CD34hi cells is associated with distinct changes in the expression of the PU.1- regulated molecules, CD64 and macrophage colony-stimulating factor receptor
Authors: Olweus J , Thompson PA , Lund-Johansen F
Published: October 2019

The present study investigated the possibility that macrophage colony- stimulating factor (M-CSF) responsiveness of hematopoietic progenitor cells is regulated at the level of receptor expression and that M-CSF receptor (M-CSFR) may be used as an early marker of monocyte lineage commitment. Immunofluorescence measurements with an anti-M-CSFR antibody showed that 44% +/- 5% of CD34hi cells expressed the receptor. The M-CSFR was present on progenitor cells that were positive for the granulo-monocytic marker CD64, but not on primitive, erythroid, or lymphoid progenitors. The CD34hiCD64+ population could be divided into subsets of M-CSFRhi and M-CSFRlo cells. In addition, a subset of CD34hiCD64-M-CSFRhi cells was found. CD34+ cells that were positive for M-CSFR, CD64, or both gave rise exclusively to granulo-monocytic cells, and 65% of the granulomonocytic colony-forming cells in the CD34+ population were recovered from these cells. Approximately 70% of the colony-forming cells (CFCs) derived from CD34hiM-CSFRhi cells were macrophage colony-forming units (CFU-M), whereas 91% of the CFCs in the CD34hiCD64+M-CSFRlo population were granulocyte colony-forming units (CFU-G). The M-CSFRhi cells with the highest frequency of colony- forming and bipotent cells and largest average colony size were found in the CD64- subset, indicating that M-CSFR appears earlier than CD64 during monocyte development. After 60 hours in culture, a subset of the CD34hiM-CSFRhi cells had downmodulated M-CSFR (29% to 38%). This population gave rise almost exclusively to granulocytes, whereas the cells that remained M-CSFRhi gave rise exclusively to monocytes. In all experiments, the M-CSFRhi population responded to M-CSF, whereas minimal responses were observed among M-CSFRlo cells. These results suggest that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M- CSFR and show that M-CSFR is a useful marker to discriminate between monocytic and granulocytic progenitor cells.
A dinucleotide deletion in exon 4 of the PlA2 allelic form of glycoprotein IIIa: implications for the correlation of serologic versus genotypic analysis of human platelet alloantigens
Authors: Skogen B , Wang R , McFarland JG , Newman PJ
Published: October 2019

Platelets from a patient with a suspected case of posttransfusion purpura were subjected to alloantigen phenotyping and found to express the PlA1, but not the PlA2, allelic form of human platelet membrane glycoprotein (GP) IIIa on the platelet surface. However, genotyping showed unambiguously that the patient carried the genes for both of these GPIIa alleles. Based on these results, we postulated that the PlA2 allele was silent, ie, that this patient was a carrier for Glanzmann thrombasthenia (GT). Quantitative analysis of GPIIb-IIIa surface expression showed only 20,000 GPIIb-IIIa receptors/platelet, approximately half of the value obtained with control platelets. Southern blot analysis showed no large deletions or insertions within the GPIIIa gene, and amplification of all 14 exons encoding GPIIIa resulted in the production of normal sized polymerase chain reaction (PCR) products in all cases. DNA-sequence analysis showed an AG dinucleotide deletion affecting codons 210 and 211 within exon 4 of the GPIIIa gene, leading to a change in reading frame and the creation of a stop codon 38 nucleotides down-stream. The predicted truncated protein consists of only the first 223 of the normal 762 amino acids, thus accounting for the failure to express the PlA2 allele on the platelet surface. While encountered only rarely, carriers of either GT or Bernard Soulier syndrome that are at the same time heterozygous for human platelet alloantigenic epitopes found on GPIb, GPIIb, or GPIIIa have the possibility to give discrepant results when comparing genotypic versus phenotypic analysis. In such situations, the combination of serologic and DNA-based evaluation contributes complementary and beneficial diagnostic information than either one alone are able to provide.
Interleukin-10 is a growth factor for human myeloma cells by induction of an oncostatin M autocrine loop
Authors: Gu ZJ , Costes V , Lu ZY , Zhang XG , Pitard V , Moreau JF , Bataille R , Wijdenes J , Rossi JF , Klein B
Published: October 2019

We have a previously reported that interleukin-10 (IL-10) is a potent but IL-6-unrelated growth factor for freshly explanted myeloma cells (Lu et al, Blood 85:2521, 1995). We have also shown that exogenous IL-10 supported the growth of XG-1 and XG-2 human myeloma cell lines (HMCL) through an IL-6-independent mechanism. (Lu et al, Blood 85:2521, 1995). Because the IL-10 receptor does not involve the gp 130 IL-6 transducer, we have attempted to elucidate the mechanisms of IL-10 action on myeloma cells. Our results indicate that the myeloma cell growth factor activity of IL-10 was abrogated by an antibody to the gp 130 IL-6 transducer, indicating that it was mediated through one of the gp 130-activating cytokines. We found that myeloma cells from XG-1 and XG-2 HMCL and from 5 of 6 patients' tumoral samples produced oncostatin M (OM) constitutively but failed to produce IL-6, IL-11 and leukemia- inhibitory factor (LIF). The autocrine OM was inactive in the absence of IL-10 due to lack of a functional OM receptor on myeloma cells. IL- 10, by inducing the receptor for LIF (LIFR), produced a functional autocrine OM loop in XG-1 and XG-2 cells and in primary myeloma cells from 2 patients. We also found that some myeloma cell lines (XG-4, XG- 6, and XG-7) an fresh myeloma cells from 3 of 6 patients produced an autocrine IL-10 and that these cells constitutively expressed LIFR. One HMCL (XG-7) produced IL-10, OM, and IL-6 an expressed LIFR. The XG-7 cells used OM and IL-6 as autocrine growth factors. We have previously shown that IL-10 could induce IL-11 receptor in myeloma cells and confer on them sensitivity to IL-11 (Lu et al, FEBS Lett 377:515, 1995). Taken together, these results show that IL-10 is a key cytokine for inducing the expression of LIFR and IL-11R and possibly another uncharacterized OM coreceptor on myeloma cells and that OM and IL-10 might be produced by myeloma cells. They also emphasize that all myeloma cell growth factors reported to data involve an activation of the gp130 IL-6 transducer.
Hepatocyte growth factor and its receptor c-met in multiple myeloma
Authors: Borset M , Hjorth-Hansen H , Seidel C , Sundan A , Waage A
Published: October 2019

We have examined whether the hepatocyte growth factor (HGF)/c-met receptor-ligand pair is expressed in freshly isolated and highly purified myeloma cells and whether HGF can be found in the sera of myeloma patients. Myeloma cells were purified with an immunomagnetic method using the syndecan 1-specific antibody B-B4. HGF and c-met mRNA in these cells were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). HGF and c-met proteins were detected by enzyme- linked immunosorbent assay (ELISA) and Western blot, respectively. Serum from 13 myeloma patients was obtained at diagnosis and the levels of HGF were determined by ELISA. HGF and c-met mRNA were expressed in all examined samples (n = 7). HGF was detected in the supernatants of 17 of 20 primary cultures of myeloma cells, whereas bone marrow mononuclear cells from normal controls did not produce detectable amounts of HGF (n = 3). The mean HGF level in serum of myeloma patients at diagnosis was more than fourfold higher than the mean level in normal controls. Possible implications of HGF/c-met expression for the pathophysiology of multiple myeloma are discussed.
Mortality hazard functions as related to neutropenia at different times after marrow transplantation
Authors: Offner F , Schoch G , Fisher LD , Torok-Storb B , Martin PJ
Published: October 2019

We characterized the relationship between severe neutropenia and risk of death in 2,276 patients after marrow transplantation to define objective and clinically relevant criteria that could be used to judge the timing and potential value of interventions designed to improve survival in patients with delayed initial engraftment. Proportional hazard models were used to estimate the relative risk of death before day 100 among patients alive on any given day with an absolute neutrophil count (ANC) less than 100/microL compared with those alive on the same day with an ANC > or = 100/microL. Between day 10 and 14, the risk ratio remained close to 1.0, indicating that the risk of death before day 100 for patients with an ANC less than 100/microL was similar to that for patients with an ANC > or = 100/microL. Between day 15, when 38% of patients had an ANC less than 100/microL, and day 26, when 3.8% of patients had an ANC less than 100/microL, the risk ratio showed an overall upward trend, indicating that patients with an ANC less than 100/microL had a higher risk of death before day 100 than those with an ANC > or = 100/microL. Thereafter, the risk ratio fluctuated between 2.01 and 5.78, indicating consistently higher risks of mortality in patients with severe neutropenia. However, allogeneic and autologous transplant recipients each had distinctive risk ratio patterns. These results could be helpful in deciding the appropriate timing for treatment given to improve graft function after marrow transplantation.
Multiple signaling pathways induced by granulocyte colony-stimulating factor involving activation of JAKs, STAT5, and/or STAT3 are required for regulation of three distinct classes of immediate early genes
Authors: Tian SS , Tapley P , Sincich C , Stein RB , Rosen J , Lamb P
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of proliferation and differentiation of neutrophilic granulocyte precursor cells. G-CSF activates multiple signaling molecules, including the JAK1 and JAK2 kinases and the STAT transcription factors. To investigate G-CSF signaling events regulated by the JAK-STAT pathway, we have generated UT7-epo cells stably expressing either wild-type (wt) G-CSF receptor or a series of C-terminal deletion mutants. Gel mobility shift and immunoprecipitation/Western analysis showed that STAT5 is rapidly activated by G-CSF in cells expressing the wt G-CSF receptor, in addition to the previously reported STAT3 and STAT1. Mutants lacking any tyrosine residues in the cytoplasmic domain maintain their ability to activate STAT5 and STAT1 but cannot activate STAT3, implying that STAT5 and STAT1 activation does not require receptor tyrosine phosphorylation. We also observed significant changes in the ratio of STAT1:STAT3:STAT5 activated by various G-CSF receptor C-terminal deletion mutants. These mutant receptors were further used to investigate the role of JAKs and STATs in G-CSF-mediated responses in these cells. We found that JAK activation correlates with G-CSF-induced cell proliferation, whereas STAT activation is not required. We have also identified three classes of G-CSF immediate early genes, whose activation correlates with the activation of distinct JAK-STAT pathways. Our data show that, whereas c-fos is regulated through a pathway independent of STAT activation, oncostatin M, IRF-1, and egr-1 are regulated by an STAT5-dependent pathway and fibrinogen is regulated by an STAT3-dependent pathway. In conclusion, our results suggest that G-CSF regulates its complex biologic activities by selectively activating distinct early response genes through different JAK-STAT signaling molecules.
Retroviral transduction of human progenitor cells: use of granulocyte colony-stimulating factor plus stem cell factor to mobilize progenitor cells in vivo and stimulation by Flt3/Flk-2 ligand in vitro
Authors: Elwood NJ , Zogos H , Willson T , Begley CG
Published: October 2019

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony- stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.
Brain endothelium lack one of two pathways of P-selectin-mediated neutrophil adhesion
Authors: Barkalow FJ , Goodman MJ , Gerritsen ME , Mayadas TN
Published: October 2019

P-selectin, an endothelial leukocyte adhesion receptor, is rapidly translocated to the cell surface upon release from storage granules called Weibel-Palade bodies and is also transcriptionally upregulated upon cytokine stimulation of endothelial cells (ECs). These two pathways of surface expression are coincident with the rapid and cytokine-inducible pathway of neutrophil adhesion to ECs. Constitutive P-selectin expression is largely absent in cultured murine brain microvascular EC (BMEC) monolayers, but interleukin-1beta and tumor necrosis factor-alpha stimulation for 4 hours leads to dramatic P-selectin upregulation. The functional relevance of differential P-selectin expression in these cells was examined by studying BMECs derived from wild-type mice and P-selectin-deficient mice. We show that P-selectin deficiency does not affect Weibel-Palade body formation or their release in response to short-acting agonists. However, in the absence of P-selectin, the brain endothelium is unable to support neutrophil adhesion after stimulation with these agonists, which may contribute to the immune privilege status of the brain. We show that P- selectin does play a major role in supporting neutrophil adhesion in the cytokine-induced pathway in BMECs in the context of other cytokine- inducible endothelial-leukocyte adhesion receptors, E-selectin, ICAM-1, and VCAM-1.
Characterization of peripheral blood stem cells in mice
Authors: Yamamoto Y , Yasumizu R , Amou Y , Watanabe N , Nishio N , Toki J , Fukuhara S , Ikehara S
Published: October 2019

Peripheral blood stem cells (PBSCs) were mobilized in mice by treatment with cytosine-arabinoside on day 0, followed by the administration by injection of granulocyte colony-stimulating factor for 4 days. There were remarkable increases in the numbers of cells with lineage-negative (Lin-) c-kit+ markers, cells with colony-forming unit-cell (CFU-C) and colony-forming unit-spleen (CFU-S) activities, and cells with marrow- repopulating ability (MRA) in the extramedullary sites (the spleen, peripheral blood, and liver) on day 5, whereas the number of these immature hematopoietic cells decreased in the bone marrow (BM) on day 5. This finding suggests the mobilization of immature hematopoietic cells from the BM to the extramedullary sites. Three-color flow cytometric analyses showed that CD4 antigen was not expressed on the Lin-Sca-1+ cells in the mobilized PB cells (PBCs), although CD4lo cells were found in those of normal BM cells. Lin-c-kit+ cells in the mobilized PBCs contained more cells with immature phenotypes (Sca-1+, Thy1.2lo, CD71-, and Rh123dull) than in normal BMCs, indicating an alteration of the hierarchical composition of the Lin-c-kit+ cells. The Lin-c-kit+Sca-1+ cells in the mobilized PBCs had similar CFU-C and CFU- S activities to those in normal BMCs. Electron microscopic studies of these cells in the mobilized PBCs showed that only 10% to 20% of these cells had a thin rim of cytoplasm with poorly developed organelles. Allogeneic transplantation [B6 --> C3H] of PBSCs showed long-term reconstituting activity across the major histocompatibility complex barrier 24 weeks after transplantation, although longer observation is necessary.
Granulocyte-macrophage colony-stimulating factor receptor: stage- specific expression and function on late B cells
Authors: Till KJ , Burthem J , Lopez A , Cawley JC
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors (GMR) are expressed on myeloid cells throughout their maturational sequence. During myelopoiesis, GM-CSF induces the proliferation of precursors and has multiple effects on more mature cells; such effects include induction of maturation and priming for subsequent stimulation. GMR is expressed on a range of other cell types including acute leukemic blasts of myeloid and lymphoid lineage, but has been little studied on more mature lymphoid cells. Using sensitive triple-layer immunophenotypic techniques, we show here that both the alpha and beta c chains of the GMR are expressed on hairy cells (HCs) and myelomatous plasma cells (PCs), but not on chronic lymphocytic leukemia (CLL) or prolymphocytic leukemia (PLL) lymphocytes. The receptor was demonstrable on normal PCs in tonsil, but not on either activated or resting tonsillar B cells or on circulating normal B lymphocytes. The expression of the receptor is therefore stage specific, rather than a feature of activation. Perhaps, surprisingly, in view of its effects on myeloid cells, GM-CSF did not stimulate the proliferation or differentiation of HCs and did not protect them from apoptosis. However, the cytokine had a profound effect on the interaction of the HC with its environment. Thus, the cytokine caused a major cytoskeletal reorganization resulting in the inhibition of motility and loss of adhesion to cellular and matrix ligands. These studies indicate the importance of GM-CSF outside myelopoiesis and demonstrate a previously unrecognized stage specific role for the cytokine in B-cell biology. Taken together with our previous report that M-CSF enhances B-cell motility, the present findings indicate that myeloid growth factors act in concert to facilitate the controlled migration of certain B cells into and within tissues.
Moderation of hemophilia A phenotype by the factor V R506Q mutation
Authors: Nichols WC , Amano K , Cacheris PM , Figueiredo MS , Michaelides K , Schwaab R , Hoyer L , Kaufman RJ , Ginsburg D
Published: October 2019

Although many examples of unrelated hemophilia A patients carrying identical point mutations in the factor VIII (FVIII) gene have been reported, the clinical phenotype is not always the same among patients sharing the same molecular defect. Possible explanations for this discrepancy include undetected additional mutations in the FVIII gene or coinheritance of mutations at other genetic loci that modulate FVIII function. We report molecular genetic analysis of potential modifying genes in two sets of unrelated patients carrying common FVIII missense mutations but exhibiting different levels of clinical severity. Both mutations (FVIII R1689C and R2209Q) are associated with severe hemophilia A in some patients and mild/moderate disease in others. The common von Willebrand disease type 2N mutation (R91Q) was excluded as a modifying factor in these groups of patients. However, analysis of the recently described factor V (FV) R506Q mutation (leading to activated protein C resistance) identified a correlation of inheritance of this defect with reduced hemophilia A severity. Two moderately affected hemophilia A patients, each with either of two FVIII gene mutations, were heterozygous for FV R506Q, whereas two severely affected patients and two moderately affected patients were homozygous normal at the FV locus. Our results suggest that coinheritance of the FV R506Q mutation may be an important determinant of clinical phenotype in hemophilia A and that modification of the protein C pathway may offer a new strategy for the treatment of FVIII deficiency.
The proto-oncogene c-myc blocks myeloid differentiation independently of its target gene ornithine decarboxylase
Authors: Selvakumaran M , Liebermann D , Hoffman B
Published: October 2019

Ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine biosynthesis, has been shown to be required for entry into and progression through the cell cycle and to be a transcriptional target of the proto-oncogene, c-myc. We show that ODC transcripts and enzyme activity are down-regulated following induction of myeloid differentiation, using M1 myeloblastic leukemic cells and normal cells from bone marrow (BM), and fail to be suppressed when c-myc expression is deregulated. In M1mycer cells, when endogenous c-myc expression has been suppressed following stimulation by interleukin-6 (IL-60), treatment with estrogen and cycloheximide results in induction of ODC transcripts. These data demonstrate that ODC is a c-myc target gene in M1 cells. It was of interest to determine whether deregulated ODC expression would alter the myeloid differentiation program. To answer this question, M1-ODC cell lines constitutively expressing ODC were established. These cells can undergo terminal differentiation and growth arrest following IL-6 stimulation, exactly like parental M1 cells, demonstrating that deregulated ODC expression is not sufficient to block myeloid differentiation. Another question to be answered was whether ODC expression is necessary for the c-myc-mediated block in differentiation. The use of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC enzyme activity, indicates that ODC is not necessary for the c-myc-mediated differentiation block.
Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities
Authors: Uchida N , Combs J , Chen S , Zanjani E , Hoffman R , Tsukamoto A
Published: October 2019

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy- 1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.
Defective lymphocyte glycosidases in the macrophage activation cascade of juvenile osteopetrosis
Authors: Yamamoto N , Naraparaju VR , Orchard PJ
Published: October 2019

Generation of macrophage-activating factor requires a precursor protein, Gc protein (serum vitamin D3-binding protein), as well as participation of beta-galactosidase of inflammation-primed B lymphocytes and sialidase of T lymphocytes. The treatment of human peripheral blood mononuclear cells with an inflammatory lysophospholipid induced beta-galactosidase and sialidase activity of lymphocytes, leading to the generation of macrophage-activating factor and activation of monocytes/macrophages. However, lysophospholipid treatment of peripheral blood mononuclear cells from three infantile patients with osteopetrosis resulted in no significant activation of monocytes/macrophages. The lysophospholipid-inducible beta- galactosidase activity of B lymphocytes as well as that of the sialidase of T lymphocytes was found to be defective in these patients.
Evidence for a graft-versus-tumor effect in a patient treated with marrow ablative chemotherapy and allogeneic bone marrow transplantation for breast cancer
Authors: Eibl B , Schwaighofer H , Nachbaur D , Marth C , Gachter A , Knapp R , Bock G , Gassner C , Schiller L , Petersen F , Niederwieser D
Published: October 2019

Graft-versus-leukemia (GvL) has been shown to be an important immune- mediated antitumor effect in hematologic malignancies. It is still unknown whether such an immunemediated antitumor effect has clinical implications in patients with solid tumors. A 32-year-old woman with inflammatory breast cancer received a bone marrow transplant (BMT) from her HLA-identical sibling. During graft-versus-host disease (GvHD) cytotoxic T lymphocytes were grown and tested in a chromium-release assay against B and T lymphocytes of the patient and donor and against a panel of breast cancer cell lines. Resolution of liver metastases was observed simultaneously with clinical GvHD in the first weeks after transplant. In addition, minor histocompatibility antigen (MiHA)- specific and major histocompatibility complex (MHC) class I antigen- restricted cytotoxic T lymphocytes recognizing breast carcinoma target cells were isolated from the blood of the patient. Pretreatment of such target cells with tumor necrosis factor (TNF)-alpha but not with interferon (IFN)-alpha or IFN-gamma increased susceptibility of these cells to lysis by cytotoxic T lymphocytes. Clinical course and in vitro results suggest that a graft-versus-tumor (GvT) effect might exist after allogeneic BMT for breast cancer. However, clinical experience on a larger scale would be required to determine the clinical efficacy of GvT effects in patients with solid tumors.
Activation of the CPP32 protease in apoptosis induced by 1-beta-D- arabinofuranosylcytosine and other DNA-damaging agents
Authors: Datta R , Banach D , Kojima H , Talanian RV , Alnemri ES , Wong WW , Kufe DW
Published: October 2019

The response of human myeloid leukemia cells to treatment with 1-beta- arabinofuranosylcytosine (ara-C) includes the induction of apoptosis. Ara-C induced apoptosis is associated with proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and protein kinase C (PKC) delta. However, the signals involved in this response are unknown. The present studies show that ara-C treatment of U-937 cells is associated with induction of a protease activity that cleaves the tetrapeptides Ac-DEVD- pNA and Ac-DMOD-pNA found at the cleavage sites of PARP and PKC delta, respectively. The ara-C-induced protease activity was sensitive to overexpression of the anti-apoptotic protein Bcl-xL and the baculovirus protein p35. By contrast, overexpression of the cowpox virus protein CrmA blocked apoptosis induced by engagement of the Fas receptor but not that induced by ara-C. CrmA overexpression also had no detectable effect on ara-C-induced cleavage of PKC delta. The results further show that ara-C induces activation of the CPP32 protease by a CrmA- insensitive and p35-sensitive mechanism. Similar results were obtained with cisplatinum, etoposide, and camptothecin. These findings indicate that ara-C and other DNA-damaging agents activate a CrmA-insensitive apoptotic pathway involving CPP32 and that these signals differ from those associated with apoptosis induced by the Fas receptor.
The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis
Authors: Maeno M , Mead PE , Kelley C , Xu RH , Kung HF , Suzuki A , Ueno N , Zon LI
Published: October 2019

Vertebrate embryonic blood formation is regulated by factors that participate in dorsal-ventral patterning and mesoderm induction. The GATA-binding transcription factors are required for normal hematopoiesis and are expressed during gastrulation when ventral mesoderm (VM) is induced to form blood. Based on the recent demonstration that bone morphogenetic protein (BMP-4) is a potent ventralizing factor and inducer of hematopoietic tissue, we hypothesized that GATA-2 could be induced or activated by BMP-4. Here we demonstrate that BMP-4 can stimulate GATA-2 expression, and that expression of a dominant negative BMP-4 receptor can suppress GATA-2 induction by BMP-4 in ventral mesoderm. Over-expression of GATA-2 in ventral mesoderm leads to increased globin production and forced expression of GATA-2 in primitive ectoderm adjacent to ventral mesoderm also stimulates globin expression. Our results suggest that BMP-4 and GATA-2 can function in two adjacent germ layers, mesoderm and ectoderm, to participate in blood cell formation during embryogenesis.
Regulation of thrombomodulin expression by all-trans retinoic acid and tumor necrosis factor-alpha: differential responses in keratinocytes and endothelial cells
Authors: Raife TJ , Demetroulis EM , Lentz SR
Published: October 2019

Thrombomodulin is a cell-surface anticoagulant glycoprotein expressed by vascular endothelial cells and epidermal keratinocytes. Thrombomodulin expression in endothelial cells is regulated by retinoic acid and tumor necrosis factor-alpha (TNF), agents that also modulate epidermal differentiation. We examined thrombomodulin function and regulation of thrombomodulin expression by all-trans retinoic acid (ATRA) and TNF in human keratinocytes and endothelial cells. Untreated keratinocytes and endothelial cells expressed thrombomodulin of comparable activity and apparent thrombin affinity. Incubation of keratinocytes with 10 mumol/L ATRA for 24 hours increased thrombomodulin activity 5.4 +/- 0.9-fold (mean +/- SE), with equivalent increases observed in thrombomodulin protein (5.5 +/- 2.1-fold) and mRNA (4.2 +/- 1.2-fold). Incubation of keratinocytes with 1.0 nmol/L TNF markedly increased expression of keratinocyte transglutaminase, but had no effect on thrombomodulin activity, protein, or mRNA. In endothelial cells, ATRA produced a small increase in thrombomodulin activity (1.9 +/- 0.1-fold), and incubation with TNF for 24 hours decreased thrombomodulin activity 83% +/- 7%. The activity profile of keratinocyte thrombomodulin exhibited a distinct maximum near 1.0 mmol/L Ca2+. These results demonstrate that keratinocyte thrombomodulin is regulated by retinoids and Ca2+, but not by TNF, and that regulation of thrombomodulin expression differs in keratinocytes and endothelial cells.