bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential regulation of human B-lymphocyte tumor necrosis factor- alpha (TNF-alpha) and lymphotoxin (TNF-beta) production by protein phosphatase 1 and 2A inhibitor
Authors: Xia HZ , Kannapell CC , Fu SM , Sung SS
Published: October 2019

Tumor necrosis factor (TNF) and lymphotoxin (LT; TNF-beta) are major cytokines produced by B lymphocytes. Stimulation by okadaic acid, a phosphatase 1 and 2A inhibitor, markedly increased TNF mRNA accumulation and cytokine production. On the other hand, the accumulation of LT mRNA was not affected by okadaic acid despite structural and functional similarities between TNF and LT. The increase in TNF mRNA accumulation was due to the stimulation of gene transcription and a marked stabilization of this mRNA. The binding activities of the transcription factors AP-1 and AP-2 and NF kappa B, which regulates TNF gene transcription, were also stimulated by okadaic acid. In addition, okadaic acid was shown to increase TNF production at the protein level. These results show the importance of protein phosphatases in the regulation of cytokine production in B cells, and further identifies differences in the regulation of TNF-alpha and LT production.
p53 and N-ras mutations in two new leukemia cell lines established from a patient with multilineage CD7-positive acute leukemia
Authors: Abo J , Inokuchi K , Dan K , Nomura T
Published: October 2019

Two new myeloid cell lines (K051 and K052) were established from a patient with multilineage CD7-positive acute leukemia. The K051 and K052 were established from the patient's bone marrow cells at diagnosis and at relapse, respectively. The K051 cell expressed myeloid- associated antigens (CD13 and CD33), a platelet-associated antigen (CD41), and an erythroid antigen (glycophorin A). The K052 cell expressed myeloid-associated antigens (CD13, CD14, and CD33), lymphoid markers (CD2, CD5, and CD7), and HLA-DR. Chromosome analysis of both cell lines showed a 17p- chromosome. Both cell lines were investigated for aberrations of the p53 gene and the N-ras gene. A p53 mutation detected in both cell lines consisted of a C-->T substitution in codon 248. An N-ras mutation detected only in the K052 cell consisted of a G-- >C substitution in codon 13. Expression of the multidrug resistance gene (MDR1) was also investigated by the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). MDR1-mRNA was more highly expressed by the K052 cell than the K051 cell, being equivalent to that in HEL cells. The functional MDR1-protein against vincristine was also observed, and its function was inhibited by verapamile and Cyclosporin A. The K052 cells were capable of phenotypic or morphologic differentiation after being incubated with granulocyte colony- stimulating factor, interleukin-2, phorbol 12-myristate 13-acetate, or 1,25-dihydroxy-vitamin D3. In contrast, the K051 cells responded phenotypically to retinoic acid. Thus, the K051 and K052 cell lines will be useful for investigating the cellular and molecular events in leukemogenesis and differentiation, and the mechanism of expression of the MDR1 gene.
Recombinant human interleukin-4 inhibits growth of some human lung tumor cell lines in vitro and in vivo
Authors: Topp MS , Koenigsmann M , Mire-Sluis A , Oberberg D , Eitelbach F , von Marschall Z , Notter M , Reufi B , Stein H , Thiel E
Published: October 2019

Cytokines play an important role in activating the immune system against malignant cells. One of these cytokines, interleukin-4 (IL-4) has entered clinical phase I trials because of its immunoregulatory potency. In the present study we report that recombinant human (rh) IL- 4 has major direct antiproliferative effects on one human lung cancer cell line (CCL 185) in vitro as measured by a human tumor cloning assay (HTCA), tritiated thymidine uptake, and counting cell numbers and marginal activity in a second cell line (HTB 56) in the HTCA. This activity could be abolished by neutralizing antibody against rhIL-4. The biological response of the tumor cells to the cytokine is correlated with expression of receptors for human IL-4 on both the mRNA level and the protein level. The responsive cell line, CCL 185, secretes IL-6 after being incubated with rhIL-4. On the other hand, neutralizing antibodies against IL-6 showed no influence on the growth modulatory efficacy of rhIL-4 in this cell line. Furthermore, CCL 185 does not show detectable production of IL-1, tumor necrosis factor alpha or interferon gamma after incubation with rhIL-4. Thus, the response to rhIL-4 is not mediated through autocrine production of these cytokines triggered by rhIL-4. In a next series of experiments some of the cell lines were xenotransplanted to BALB/c nu/nu mice. Subsequently, the mice were treated for 12 days with two doses of 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control in two of the non-small cell lung cancer cell lines being responsive in vitro (CCL 185, HTB 56). Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. In contrast, no tumor growth inhibition was found in the small cell lung cancer cell lines (HTB 119, HTB 120) being nonresponsive in vitro. We conclude that rhIL-4 has direct antiproliferative effects on the growth of some human non-small cell lung cancer cell lines in vitro and in vivo, which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment.
Stimulation of interleukin-6 and prostaglandin E2 secretion from peritoneal macrophages by polymers of albumin
Authors: Shacter E , Arzadon GK , Williams JA
Published: October 2019

Interleukin-6 (IL-6) is a multifunctional cytokine that is elevated in vivo during acute infection, chronic inflammation, and some hematopoietic malignancies. To understand how IL-6 becomes elevated in vivo, it is important to identify factors that can stimulate its secretion from effector cells. We found that commercial preparations of bovine serum albumin (BSA) stimulated murine macrophages to secrete high levels of IL-6. In fact, BSA was at least as potent as bacterial lipopolysaccharide (LPS) in stimulating IL-6 production. Stimulation was clearly visible at concentrations as low as 20 micrograms/mL and reached saturation at 0.5 to 1 mg/mL albumin, at which concentration 1.1 x 10(6) oil-elicited macrophages produced 6,000 +/- 700 B9 units of IL-6 in an overnight incubation. Prostaglandin E2 production was induced by the same concentrations of BSA. Both resident and oil- elicited peritoneal cells were responsive to the albumin. The stimulatory activity did not derive from contamination of the protein with Escherichia coli LPS; when compared directly with LPS, the response to BSA was more rapid, had a higher amplitude, and was not inhibitable by polymyxin B. In addition, macrophages isolated from C3H/HeJ mice, which have an inherited defect in their ability to respond to LPS, secreted IL-6 in response to BSA but not to LPS. The stimulatory activity was stable to heat, mild acid, and reduction/alkylation and copurified with albumin on Cibachron Blue agarose (Sigma, St Louis, MO) and anti-albumin immunoaffinity chromatography. Comparison of different sources and preparations of albumin showed differences in the levels of IL-6-inducing activity; three different lots of commercial fatty acid-free BSA and one lot of polymer-enhanced BSA stimulated IL-6 secretion by more than 100-fold over basal levels whereas other preparations showed more limited activity. A sample of BSA that was active in vitro caused a marked elevation of IL-6 when injected into BALB/c mice, thus demonstrating inflammatory activity in vivo. When the albumin preparations were fractionated by ion exchange and gel filtration chromatography and then analyzed by sodium dodecyl sulfate-gel electrophoresis and Western blot immunoassay, it was found that the IL-6-inducing activity resided in high molecular weight polymers of albumin. The ability of albumin polymers to stimulate IL-6 production represents a novel mechanism for modulation of this cytokine.
Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils
Authors: Georas SN , McIntyre BW , Ebisawa M , Bednarczyk JL , Sterbinsky SA , Schleimer RP , Bochner BS
Published: October 2019

The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.
The bcl-2 oncogene in Hodgkin's disease arising in the setting of follicular non-Hodgkin's lymphoma
Authors: LeBrun DP , Ngan BY , Weiss LM , Huie P , Warnke RA , Cleary ML
Published: October 2019

Expression of the bcl-2 proto-oncogene on chromosome 18 is deregulated by the 14; 18 chromosomal translocation, an abnormality that is consistently associated with follicular non-Hodgkin's lymphomas (NHL). Because bcl-2 is believed to function by prolonging cell survival rather than by increasing proliferation, the presence of t(14; 18) in Hodgkin's disease (HD) would have profound implications for the pathogenesis of this neoplasm. We evaluated 32 cases of HD for t(14; 18) by polymerase chain reaction (PCR). These results were correlated with expression of bcl-2 oncogenic protein by Hodgkin cells and with the presence of Epstein-Barr virus (EBV), as determined by immunohistochemistry or in situ hybridization. PCR provided evidence of t(14; 18) in only 2 HD cases (6%), both of which were associated with a prior history of follicular lymphoma, and both of which were among the 7 cases (22%) with strong bcl-2 expression in Hodgkin cells. In at least 1 of the cases, the translocation involved identical chromosomal breakpoints in both types of lymphoma. Furthermore, 7 additional cases of combined follicular NHL and HD showed strong bcl-2 staining in Hodgkin cells. Although EBV was detected in 6 of 30 cases, it was not associated with t(14; 18) and usually not with strong bcl-2 expression. These results suggest that a small proportion of HD cases might evolve from follicular NHL, possibly through molecular events superimposed on the t(14; 18). High-level bcl-2 expression in Hodgkin cells is a potentially useful but not definitive marker for these cases.
Frequent HLA class I and DP sequence mismatches in serologically (HLA- A, HLA-B, HLA-DR) and molecularly (HLA-DRB1, HLA-DQA1, HLA-DQB1) HLA- identical unrelated bone marrow transplant pairs [published erratum appears in Blood 1994 Jun 15;83(12):3834]
Authors: Santamaria P , Reinsmoen NL , Lindstrom AL , Boyce-Jacino MT , Barbosa JJ , Faras AJ , McGlave PB , Rich SS
Published: October 2019

The rates of graft-versus-host disease (GVHD) and rejection are significantly higher among recipients of unrelated donor marrow (BM) than in recipients of marrow from HLA-identical siblings, even when donors and recipients are mixed lymphocyte culture (MLC) compatible and serologically and Dw identical. It has been hypothesized that phenotypically silent HLA class I and DP sequence mismatches might be associated with these differences, but little is known about their incidence. We have sequenced the HLA-A, HLA-B, HLA-C, HLA-DPA1, and HLA- DPB1 genes expressed by 12 unrelated marrow transplant pairs, 11 of whom were molecularly matched at DRB, DQA1, and DQB1 loci. Nine of these pairs were also HLA-A and HLA-B matched by serology. Six of these nine “HLA-identical” pairs were HLA-A (2 of 6), HLA-B (1 of 6), and HLA- C (6 of 6) mismatched at the sequence level. The mismatched class I alleles of all these pairs had strikingly different sequence motifs in the six specificity pockets of their antigen recognition site, and in five pairs they also had sequence differences at positions implicated in T-cell receptor (TCR) binding. Two of the three pairs who were serologically mismatched for one HLA-A or HLA-B antigen were also sequence mismatched at HLA-C. Finally, 10 of 11 pairs tested expressed different DP sequences. These data indicate that HLA class I, especially HLA-C, and DP sequence mismatches are frequent among unrelated subjects defined as HLA identical by current typing methods. We speculate that these sequence differences may explain, at least in part, the higher incidence of acute GVHD and rejection in unrelated BM transplantation as opposed to transplantation between HLA-identical siblings. Because of their high frequency, the role of HLA-A, HLA-B, HLA-C, and HLA-DP mismatches in transplantation outcome is now amenable to direct study.
Human monocytes support factor X activation by factor VIIa, independent of tissue factor: implications for the therapeutic mechanism of high- dose factor VIIa in hemophilia [see comments]
Authors: Hoffman M , Monroe DM , Roberts HR
Published: October 2019

High doses of recombinant factor VIIa are useful in managing bleeding in hemophiliacs with inhibitors. Whether this therapeutic effect of factor VIIa is dependent on tissue factor (TF) is a matter of debate. We examined the ability of freshly isolated human monocytes (which lack TF) to support the activation of coagulation-factor X by factor VIIa. The rate of factor-X activation by factor VIIa was accelerated in the presence of monocytes compared with the rate of X activation in solution. This activation of factor X on monocytes was saturable with a K1/2 of about 400 to 600 pmol/L factor VIIa. The rate of activation was not inhibited by an excess of inhibitory anti-TF antibody or a Gla- containing fragment of prothrombin. In contrast to monocytes, an endothelial cell line did not support activation of factor X by factor VIIa. Our findings suggest that at least one cell type can accelerate activation for factor X by factor VIIa in the absence of TF. This activity requires higher concentrations of factor VIIa than does the TF mechanism. The concentrations of VIIa required are of a similar order of magnitude to those required for a therapeutic effect of VIIa in bleeding hemophiliacs with inhibitors.
Expression of platelet-derived growth factor and its receptors by two pre-B acute lymphocytic leukemia cell lines
Authors: Tsai LH , White L , Raines E , Ross R , Smith RG , Cushley W , Ozanne B
Published: October 2019

Platelet-derived growth factors (PDGF) are potent regulators of cell proliferation. The three isoforms of PDGF AA, AB, and BB are encoded by two genes: PDGF A and PDGF B. The v-sis oncogene is homologous to the PDGF-B gene. v-sis can transform cells that express the appropriate PDGF receptors. Two different types of receptors, PDGF-alpha and PDGF- beta, also encoded by two genes, have been identified. We show that two cell lines. SMS-SB and NALM-6, both derived from pre-B-cell acute lymphocytic leukemias, express the PDGF-A chain gene, and one of them, SMS-SB, releases PDGF-A chains into the media. The SMS-SB cells also express the PDGF-beta receptor, whereas NALM-6 cells express the PDGF- alpha receptor and bind PDGF. This extends the possible targets for PDGF to the B-cell lineage lymphocytes.
Transforming growth factor-beta regulates c-kit message stability and cell-surface protein expression in hematopoietic progenitors
Authors: Dubois CM , Ruscetti FW , Stankova J , Keller JR
Published: October 2019

The cell-surface receptor c-kit and its cognate ligand stem-cell factor (SCF) or steel factor (SLF) are important for the maintenance of hematopoiesis both in vitro and in vivo. Transforming growth factor- beta (TGF-beta) has been shown to be a potent inhibitor of SLF-mediated synergistic growth of murine Lin-Sca-1+ progenitor cells, as well as more committed progenitors. In the present study, we examined the regulation of c-kit mRNA and cell-surface expression by TGF-beta. Among the murine hematopoietic progenitor cells tested, the myeloid cell line FDC-P1 and the mast-cell line MC-6, as well as progenitor-enriched bone marrow cells, constitutively expressed functional cell-surface c-kit. Treatment of these progenitor cell lines and primary progenitor cells with TGF-beta resulted in downregulation of cell-surface c-kit expression. This effect was not a secondary event of cell-cycle status. TGF-beta inhibition was dose- and time-dependent, with 50% inhibition seen between 0.3 to 3 ng/mL TGF-beta and maximal inhibition at 30 ng/mL. Using the FDC-P1 cell line, we observed that the inhibition of cell-surface c-kit expression by TGF-beta is preceded by a marked reduction in c-kit mRNA levels starting 2 hours after TGF-beta treatment, and reaches a maximum by 6 hours. The inhibition in steady- state c-kit mRNA levels is explained, in part, by a decrease in the half-life of c-kit transcripts (2 to 4 hours for control cells v 0.5 to 1.5 hours for TGF-beta-treated cells). These findings suggest that TGF- beta regulates the responsiveness of murine hematopoietic progenitors to SLF through a decrease in c-kit message stability leading to decreased cell-surface expression.
Bifunctional effects of tumor necrosis factor alpha (TNF alpha) on the growth of mature and primitive human hematopoietic progenitor cells: involvement of p55 and p75 TNF receptors
Authors: Rusten LS , Jacobsen FW , Lesslauer W , Loetscher H , Smeland EB , Jacobsen SE
Published: October 2019

Tumor necrosis factor alpha (TNF alpha) has previously been reported to have both inhibitory and stimulatory effects on hematopoietic progenitor cells. Specifically, TNF alpha has been proposed to stimulate early hematopoiesis in humans. In the present study we show that TNF alpha, in a dose-dependent fashion, can potently inhibit the growth of primitive high proliferative potential colony-forming cells (HPP-CFCs) stimulated by multiple cytokine combinations. Using agonistic antibodies to the p55 and p75 TNF receptors or TNF alpha mutants specific for either of the two TNF receptors, we show that both receptors can mediate this inhibition. In contrast, the potent stimulation of interleukin-3 (IL-3) plus granulocyte-macrophage colony- stimulating factor (GM-CSF) induced HPP-CFC colony formation observed at low concentrations of TNF alpha (2 ng/mL) was only a p55-mediated event. Moreover, the stimulatory effects of TNF alpha on GM-CSF or IL-3- induced colony formation, as well as the inhibition of G-CSF-induced colony growth, were also exclusively signaled through the p55 TNF receptor. Taken together, our results suggest that the inhibitory effects of TNF alpha on primitive bone marrow progenitor cells are mediated through both p55 and p75 TNF receptors, whereas the p55 receptor exclusively mediates the bidirectional effects on more mature, single factor-responsive bone marrow progenitor cells as well as stimulation of IL-3 plus GM-CSF-induced HPP-CFC colony growth.
Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates
Authors: Cadroy Y , Hanson SR , Kelly AB , Marzec UM , Evatt BL , Kunicki TJ , Montgomery RR , Harker LA
Published: October 2019

The relative antithrombotic effectiveness of targeting glycoprotein (GP) Ib-dependent versus GPIIb-IIIa-dependent platelet interactions has been determined in baboons by measuring thrombus formation after infusing comparable antihemostatic doses of anti-von Willebrand factor (vWF) monoclonal antibody (MoAb) BB3-BD5, anti-GPIb MoAb AP1, and anti- GPIIb-IIIa MoAb LJ-CP8 under conditions of arterial and venous flow (shear rates of 750 to 1,000 seconds-1 and 100 seconds-1, respectively). Thrombus formation was quantified as 111In-platelet deposition and 125I-fibrin accumulation on segments of collagen-coated tubing interposed in chronic exteriorized arteriovenous (AV) shunts for 40 minutes. In vitro, anti-vWF MoAb BB3 BD5 (IgG) and anti-GPIb MoAb AP1 [IgG or F(ab)2 fragments] inhibited ristocetin-induced platelet aggregation (IC50 50 nmol/L and 1 mumol/L, respectively), but neither of these MoAbs blocked platelet aggregation induced by adenosine diphosphate (ADP) (P > .5). Conversely, anti-GPIIb-IIIa MoAb LJ-CP8 inhibited platelet aggregation induced by ADP (IC50 1 mumol/L, but failed to block ristocetin-induced platelet aggregation (P > .5). In vivo, the intravenous infusion of anti-vWF MoAb BB3 BD5 or anti-GPIIb- IIIa MoAb LJ-CP8 into baboons at doses that abolished corresponding agonist-induced aggregation ex vivo (bolus injections of 0.5 mg/kg and 10 mg/kg, respectively) prolonged template bleeding times from baseline values of 4.0 +/- 0.3 minutes to > 27 +/- 4 minutes, and to > 26 +/- 4 minutes, respectively (P 3 .001 in both cases), without affecting the peripheral platelet count (P > .5). However, injection of anti-GPIb MoAb AP1 [10 mg/kg as IgG or 1 mg/kg as F(ab)2 fragments] produced immediate irreversible thrombocytopenia (< 40,000 platelets/microL). Anti-GPIIb-IIIa MoAb LJ-CP8 abolished platelet deposition and fibrin accumulation on collagen segments under both arterial and venous flow conditions (P < .01 in all cases), whereas MoAb BB3 BD5 produced minimal inhibition of platelet deposition and no decrease in fibrin accumulation at arterial shear rates and undetectable antithrombotic outcomes at low shear. Thus, inhibiting GPIIb-IIIa-dependent platelet recruitment abrogates both thrombus formation and platelet hemostatic function at both venous and arterial shear rates. By contrast, interfering with GPIb-vWF-dependent platelet interactions abolishes platelet hemostatic function without producing corresponding antithrombotic effects.
Retinoic acid-resistant HL-60 cells exclusively contain mutant retinoic acid receptor-alpha
Authors: Li YP , Said F , Gallagher RE
Published: October 2019

Sequence analysis of the retinoic acid receptor-alpha (RAR alpha) gene from a subline of HL-60 cells (RA-res) stably resistant to all-trans retinoic acid (RA) disclosed a single-base change in codon number 411, the same C to T transition previously reported in an independently selected HL-60 RA resistant clone by Robertson et al (Blood 80:1885, 1992). This mutation eliminates a FokI restriction endonuclease site. Using primers framing this mutation in exon 9 of the RAR alpha gene, we showed that polymerase chain reaction products amplified from either mRNA or genomic DNA templates from the RA-res subline were completely resistant to FokI digestion whereas those from wild-type (wt) HL-60 cells could be digested to completion. The lack of a normal allele in the RA-res cells was confirmed by mixing experiments and hybridization analyses. Southern blot analysis of DNA from the RA-res and wt cells versus control placental DNA indicated that the RAR alpha gene is not haploid. The independent isolation of the same RAR alpha mutation in different laboratories suggests either that the mutation exits in a small subpopulation in the wt line or that this is a mutational “hot spot.” Furthermore, the results indicate that if a dominant negative mode of resistance is involved in the RA-res subline, this must involve interference with the function of heterologous receptor proteins such as the retinoid X receptors. The lack of any normal RAR alpha in this subline may facilitate studies of the mode of action of retinoids.
Macrophage colony-stimulating factor-induced bone marrow macrophages do not synthesize or release prostaglandin E2
Authors: Shibata Y , Bjorkman DR , Schmidt M , Oghiso Y , Volkman A
Published: October 2019

Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2–14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.
Phosphorylation of protein 4.1 in Plasmodium falciparum-infected human red blood cells
Authors: Chishti AH , Maalouf GJ , Marfatia S , Palek J , Wang W , Fisher D , Liu SC
Published: October 2019

The composition of the erythrocyte plasma membrane is extensively modified during the intracellular growth of the malaria parasite Plasmodium falciparum. It has been previously shown that an 80-kD phosphoprotein is associated with the plasma membrane of human red blood cells (RBCs) infected with trophozoite/schizont stage malaria parasites. However, the identity of this 80-kD phosphoprotein is controversial. One line of evidence suggests that this protein is a phosphorylated form of RBC protein 4.1 and that it forms a tight complex with the mature parasite-infected erythrocyte surface antigen. In contrast, evidence from another group indicates that the 80-kD protein is derived from the intracellular malaria parasite. To resolve whether the 80-kD protein is indeed RBC protein 4.1, we made use of RBCs obtained from a patient with homozygous 4.1(-) negative hereditary elliptocytosis. RBCs from this patient are completely devoid of protein 4.1. We report here that this lack of protein 4.1 is correlated with the absence of phosphorylation of the 80-kD protein in parasite- infected RBCs, a finding that provides conclusive evidence that the 80- kD phosphoprotein is indeed protein 4.1. In addition, we also identify and partially characterize a casein kinase that phosphorylates protein 4.1 in P falciparum-infected human RBCs. Based on these results, we suggest that the maturation of malaria parasites in human RBCs is accompanied by the phosphorylation of protein 4.1. This phosphorylation of RBC protein 4.1 may provide a mechanism by which the intracellular malaria parasite alters the mechanical properties of the host plasma membrane and modulates parasite growth and survival in vivo.
The -158 (C-->T) promoter mutation is responsible for the increased transcription of the 3' gamma gene in the Atlanta type of hereditary persistence of fetal hemoglobin
Authors: Efremov DG , Dimovski AJ , Huisman TH
Published: October 2019

The Atlanta type of hereditary persistence of fetal hemoglobin (HPFH) is characterized by a mild elevation of Hb F (2% to 5% in heterozygotes), almost exclusively of the G gamma type (more than 90%). Gene-mapping analysis has identified this condition as a -G gamma-G gamma- arrangement with the -158 (C-->T) substitution in the promoters of both G gamma genes. We have reevaluated this condition in members of two families. Sequence analysis identified only two changes in the 3′ gamma gene as compared with the A gamma gene from a chromosome with haplotype no. 3 (or Senegal), namely the -158 (C-->T) promoter mutation and the C-->G change in codon (CD) 136, which accounts for the -G gamma- G gamma- phenotype. The absence of any other nucleotide (nt) substitution provides genetic evidence that the -158 (C-->T) change is primarily responsible for the elevated Hb F levels associated with this condition. A quantitative reverse transcription/polymerase chain reaction (RT/PCR) procedure, presented in detail in this report, was developed to determine the effect of this mutation on the transcription of individual gamma genes in four individuals with the Atlanta type of HPFH. Both gamma-globin genes, ie, the (5′) G gamma and the (3′) G gamma-Atlanta genes of the Atlanta type of HPFH chromosome, expressed elevated amounts of transcripts, which were present in nearly equal amounts. This shows that the -158 (C-->T) mutation exerts its effect on the transcriptional rate of the gene with which it is associated.
Development of a retroviral construct containing a human mutated dihydrofolate reductase cDNA for hematopoietic stem cell transduction
Authors: Li MX , Banerjee D , Zhao SC , Schweitzer BI , Mineishi S , Gilboa E , Bertino JR
Published: October 2019

A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock- infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR-amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX-resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.
Identification of molecular defects in a subject with type I CD36 deficiency
Authors: Kashiwagi H , Tomiyama Y , Kosugi S , Shiraga M , Lipsky RH , Kanayama Y , Kurata Y , Matsuzawa Y
Published: October 2019

We performed a molecular analysis of a subject whose platelets and monocytes did not express any cell surface CD36 (designated as a type I CD36 deficiency). Amplification of the 5′ half of platelet and monocyte CD36cDNA (corresponding to nucleotide [nt] 191–1009 of the published CD36 cDNA sequence [Oquendo et al, Cell, 58:95, 1989]) showed that two different-sized CD36 cDNAs existed. One cDNA was of predicted normal size, whereas the other was about 150 bp smaller than that predicted for normal CD36 cDNA. Amplification of the 3′ region of CD36 cDNA (nt 962–1714) in this subject showed only normal-sized CD36 cDNA. Cloning and nt sequence analysis of the cDNAs showed that the smaller sized CD36 cDNA had 161-bp deletion (from nt 331 to 491), and a dinucleotide deletion starting at nt position 539. The same dinucleotide deletion was also detected in the normal sized CD36 cDNA. Both deletions caused a frameshift leading to the appearance of a translation stop codon. RNA blot analysis and quantitative assay using the reverse transcription- polymerase chain reaction (RT-PCR) showed that the CD36 transcripts in both platelets and monocytes were greatly reduced. Comparison of the determined cDNA sequences with the genomic DNA sequence for the human CD36 gene showed that the dinucleotide deletion was located in exon 5, and that the 161-bp deletion corresponded to a loss of exon 4. PCR- based analysis using genomic DNA showed that this subject was homozygous for the dinucleotide deletion in exon 5. Except for the dinucleotide deletion, we could not find any abnormalities around exon 3, 4, and 5 including the splice junctions. These results suggested that the deletions in CD36 mRNA were likely to be responsible for instability of the transcripts, and the dinucleotide deletion in exon 5 might affect the splicing of exon 4.
Hypoxic human umbilical vein endothelial cells induce activation of adherent polymorphonuclear leukocytes
Authors: Arnould T , Michiels C , Remacle J
Published: October 2019

Several pieces of evidence are reported for the accumulation of activated neutrophils in ischemic and reperfused tissues leading to the transformation of the ischemic tissue into an inflammatory territory and to an enhancement of tissue damages during reoxygenation. However, the molecular mechanisms responsible for these observations and the precise role played by endothelial cells in this process are still poorly understood. In this study, an in vitro model that mimics this situation was used to investigate the effects of hypoxia-incubated human umbilical vein endothelial cells (HUVEC) on polymorphonuclear leukocyte (PMN) functions. A strong PMN activation characterized by an increase in intracellular calcium concentration as well as by superoxide anion release and leukotriene B4 production was observed when these cells were coincubated with hypoxic HUVEC. On the other hand, conditioned medium from hypoxia-incubated HUVEC failed to activate PMN, as determined by the lack of PMN calcium concentration increase, the failure of superoxide anion production enhancement, as well as the absence of effects on the integrin CD18, CD11a, and CD11b expression. These results indicate that the presence of hypoxia- incubated HUVEC is necessary to obtain an activation of the PMN, probably via the adherence process. Once activated by coincubation with hypoxic HUVEC, PMN became cytotoxic, as evidenced by 51Cr released from prelabeled HUVEC. This cytotoxic effect of activated PMN for hypoxic endothelial cells could be prevented by a combination of superoxide dismutase and catalase (94% inhibition), whereas superoxide dismutase alone was inefficient. Antiprotease (alpha 2-macroglobulin) and a specific elastase inhibitor (MAAPV-CMK) were also inefficient. These results correlate very well with the fact that no increase in elastase release could be observed in supernatants from PMN coincubated with hypoxic HUVEC. Furthermore, when adherence process was blocked by oleic acid or by anti-ICAM-1 monoclonal antibodies, protection was, respectively, 90% and 72%. We thus evidenced that free radicals but not elastase released from activated PMN coincubated with hypoxic HUVEC are involved in HUVEC injury. We conclude from these results that PMN activation is initiated by PMN adherence to hypoxic HUVEC. These observations indicate that hypoxic HUVEC may be partly responsible for neutrophil activation observed in ischemic tissues, which is part of the amplification process of tissue damage.
Oxygen transport properties in malaria-infected rodents--a comparison between infected and noninfected erythrocytes
Authors: Schmidt W , Correa R , Boning D , Ehrich JH , Kruger C
Published: October 2019

This study was performed to investigate oxygen transport properties in whole blood (WB) of malaria-infected rats as well as in infected erythrocytes (IE) and noninfected erythrocytes (NIE) separated by density centrifugation. One week after inoculation with Plasmodium berghei, mean parasitemia was 26.5% and high correlations were found between parasitemia and hemoglobin concentration ([Hb]; r = -.902), mean cellular Hb concentration (MCHC; r = -.712), MetHb (r = .923), and base excess (r = -.922). Compared with control animals (C), the oxygen affinity was lower in WB under standard (pH 7.40) and simulated “in vivo” (pH 7.00) conditions (difference in P50, 5.7 and 5.1 mm Hg, respectively; 2P < .01, 2P < .05). In IE Hb and 2,3-biphosphoglycerate (2,3-BPG) concentrations were decreased (MCHC: IE 14.6 +/- 1.0, NIE 33.1 +/- 1.7 g/100 mL; [2,3-BPG]: IE 2.0 +/- 0.6, NIE 7.6 +/- 1.8 mmol/L), whereas [MetHb] and [ATP] were increased ([MetHb]: IE 19.0 +/- 3.7, NIE 0.7% +/- 0.8%; [ATP]: IE 33.5 +/- 2.4, NIE 6.2 +/- 1.0 mumol/g Hb). At pH 7.40, half-saturation oxygen tension (P50) was reduced in IE (29.6 +/- 2.6, NIE 39.2 +/- 5.4 mm Hg, 2P < .001), which correlates with lower [2,3-BPG], increased MetHb content, and higher intrinsic Hb- O2 affinity. However, at pH 7.00, the oxygen affinity was lower in IE when compared with NIE, which was most likely due to high [ATP] in IE. The resulting Bohr coefficients (BC) calculated for CO2 and lactic acid were extremely high in IE and low in NIE (at 50% O2-saturation BCCO2: IE -1.04 +/- 0.06, NIE -0.26 +/- 0.10, 2P < .001; BCLac: IE -0.82 +/- 0.16, NIE -0.47 +/- 0.07, 2P < .001), which was caused by different [2,3-BPG] and [ATP] as well as probably by structural changes of the Hb molecule. The O2 capacity was 14.1 mL per 100 mL erythrocytes in IE compared with 44.4 mL/100 mL in NIE. On the basis of the calculated arterio-venous O2 difference under “in vivo” conditions, the infected red blood cell fraction transports 30% of the O2 amount delivered to the tissues by the noninfected cells (IE 8.0, NIE 26.9 mL/100 mL red blood cells). We conclude that the O2 transport in malaria infected blood is not only affected by the degree of anemia but also by the percentage of infected erythrocytes.
Chromosomal assignment of genes involved in glycosylphosphatidylinositol anchor biosynthesis: implications for the pathogenesis of paroxysmal nocturnal hemoglobinuria
Authors: Ware RE , Howard TA , Kamitani T , Chang HM , Yeh ET , Seldin MF
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematologic disorder that affects both sexes equally. The biochemical defect in PNH resides in the incomplete enzymatic assembly of glycosylphosphatidylinositol (GPI) anchors used for surface protein attachment. In all PNH patients tested to date, the biosynthetic defect occurs at the addition of N-acetyl-glucosamine to the phosphatidylinositol molecule (class A defect). A human cDNA, Piga, that repairs cell lines with the class A GPI-anchor biosynthetic defect has been recently cloned. Mapping of Piga to the X chromosome suggests that a single acquired mutation within Piga could alter GPI-anchor synthesis and result in PNH. However, this finding does not explain why all PNH patients have the class A defect. In the current study, the chromosomal assignment of Piga, as well as of Pigf and Pigh, two additional genes involved in GPI-anchor biosynthesis, has been established using a mouse interspecific backcross mapping technique. In contrast to Piga, both human and mouse Pigf and Pigh genes map to autosomes. The location of Pigf and Pigh suggests that mutations on both alleles of these autosomal genes would be necessary to produce PNH. This helps to explain the predominant class A defect in PNH.
Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ hematopoietic stem cells
Authors: Uchida N , Aguila HL , Fleming WH , Jerabek L , Weissman IL
Published: October 2019

Hematopoietic stem cells (HSCs) are believed to play a critical role in the sustained repopulation of all blood cells after bone marrow transplantation (BMT). However, understanding the role of HSCs versus other hematopoietic cells in the quantitative reconstitution of various blood cell types has awaited methods to isolate HSCs. A candidate population of mouse HSCs, Thy-1.1lo Lin-Sca-1+ cells, was isolated several years ago and, recently, this population has been shown to be the only population of BM cells that contains HSCs in C57BL/Ka-Thy-1.1 mice. As few as 100 of these cells can radioprotect 95% to 100% of irradiated mice, resulting long-term multilineage reconstitution. In this study, we examined the reconstitution potential of irradiated mice transplanted with purified Thy-1.1lo Lin-Sca-1+ BM cells. Donor-derived peripheral blood (PB) white blood cells were detected as early as day 9 or 10 when 100 to 1,000 Thy-1.1lo Lin-Sca-1+ cells were used, with minor dose-dependent differences. The reappearance of platelets by day 14 and thereafter was also seen at all HSC doses (100 to 1,000 cells), with a slight dose-dependence. All studied HSC doses also allowed RBC levels to recover, although at the 100 cell dose a delay in hematocrit recovery was observed at day 14. When irradiated mice were transplanted with 500 Thy-1.1lo Lin-Sca-1+ cells compared with 1 x 10(6) BM cells (the equivalent amount of cells that contain 500 Thy-1.1lo Lin-Sca-1+ cells as well as progenitor and mature cells), very little difference in the kinetics of recovery of PB, white blood cells, platelets, and hematocrit was observed. Surprisingly, even when 200 Thy1.1lo Lin-Sca- 1+ cells were mixed with 4 x 10(5) Sca-1- BM cells in a competitive repopulation assay, most of the early (days 11 and 14) PB myeloid cells were derived from the HSC genotype, indicating the superiority of the Thy-1.1lo Lin-Sca-1+ cells over Sca-1- cells even in the early phases of myeloid reconstitution. Within the Thy-1.1lo Lin-Sca-1+ population, the Rhodamine 123 (Rh123)hi subset dominates in PB myeloid reconstitution at 10 to 14 days, only to be overtaken by the Rh123lo subset at 3 weeks and thereafter. These findings indicate that HSCs can account for the early phase of hematopoietic recovery, as well as sustained hematopoiesis, and raise questions about the role of non-HSC BM populations in the setting of BMT.
In vivo blockade of CD28/CTLA4: B7/BB1 interaction with CTLA4-Ig reduces lethal murine graft-versus-host disease across the major histocompatibility complex barrier in mice
Authors: Blazar BR , Taylor PA , Linsley PS , Vallera DA
Published: October 2019

We tested whether the in vivo infusion of recombinant, soluble CTLA4 fused with Ig heavy chains, as a surrogate ligand used to block CD28/CTLA4 T-cell costimulation, could prevent efficient T-cell activation and thereby reduce graft-versus-host disease (GVHD). Lethally irradiated B10.BR recipients of major histocompatibility complex disparate C57BL/6 donor grafts received intraperitoneal injections of human CTLA4-Ig (hCTLA4-Ig) or murine CTLA4-Ig (mCTLA4-Ig) in various doses and schedules beginning on day -1 or day 0 of bone marrow transplantation (BMT). In all five experiments, recipients of CTLA4-Ig had a significantly higher actuarial survival rate compared to mice injected with an irrelevant antibody control (L6) or saline alone. Survival rates in recipients of hL6 or PBS were 0% at 29 to 45 days post-BMT. In recipients of CTLA4-Ig, survival rates were as high as 63% mice surviving 3 months post-BMT. However, protection was somewhat variable and recipients of CTLA4-Ig were not GVHD-free by body weight, clinical appearance, and histopathologic examination. There were no significant differences in the survival rates in comparing injection dose, injection duration, or species of CTLA4-Ig (hCTLA4-Ig v mCTLA4- Ig). Splenic and peripheral blood flow cytometry studies of long-term hCTLA4-Ig-injected survivors showed a significant peripheral B-cell and CD4+ T-cell lymphopenia, consistent with GVHD. A kinetic study of splenic reconstitution was performed in mice that received hCTLA4-Ig and showed that mature splenic localized CD8+ T-cell repopulation was not significantly different in recipients of hCTLA4-Ig compared with hL6, despite the significant increase in actuarial survival rate in that experiment. These data suggest that the beneficial effect of hCTLA4-Ig on survival is not mediated by interfering with mature donor- derived T-cell repopulation post-BMT. Neither hCTLA4-Ig nor mCTLA4-Ig interfered with hematopoietic recovery post-BMT. We conclude that CTLA4- Ig (most likely in combination with other agents) may represent an important new modality for GVHD prevention.
Cytogenetic findings in peripheral T-cell lymphomas as a basis for distinguishing low-grade and high-grade lymphomas
Authors: Schlegelberger B , Himmler A , Godde E , Grote W , Feller AC , Lennert K
Published: October 2019

Cytogenetic studies on lymph node and skin biopsy specimens and peripheral blood in 104 patients with peripheral T-cell lymphomas (PTL) were compared with histopathologic diagnoses made according to the updated Kiel classification. Low-grade lymphomas presented normal metaphases more frequently than high-grade ones (P < .0001). This difference remained significant if cases with greater than 10% and greater than 50% normal metaphases in unstimulated cultures and in cultures stimulated by different mitogens were compared. On the other hand, high-grade lymphomas more often showed aberrant clones (P < .05), triploid to tetraploid clones (P < .0001), and complex clones with more than four chromosome changes (P < .01). Low-grade PTL showed consistent cytogenetic features. Clones with both inv(14)(q11q32.1) and trisomy 8q, mostly caused by i(8q)(q10), were found in all cases of T-cell chronic lymphocytic leukemia (T-CLL) and T-cell prolymphocytic leukemia (T-PLL). Trisomy 3 was observed only in angioimmunoblastic lymphadenopathy with dysproteinemia (AILD)-type PTL, T-zone lymphoma, and lymphoepithelioid lymphoma. Moreover, the proportion of normal metaphases in these PTL was higher than in the other low-grade PTL (P < .01). On the contrary, T-CLL, T-PLL, and cutaneous T-cell lymphomas (CTCL) showed complex clones (P < .0001), duplications in 6p (P <.01), deletions in 6q (P < .01), trisomy 8q (P < .00001), inv(14) (P < .00001), and monosomy 13 or changes of 13q14 (P < .001) more frequently than the other low-grade PTL. Trisomy 5 and + X predominated in AILD- type PTL. A cytogenetic feature characteristic of AILD-type PTL and CTCL was unrelated clones, which were found in 15% of AILD-type PTL and 17% of CTCL. The only chromosome aberration restricted to a certain high-grade PTL was t(2;5)(p23;q35) in large-cell anaplastic lymphoma. Deletions in 6q, total or partial trisomies of 7q, and monosomy 13 or changes of 13q14 turned out to be significantly more frequent in high- grade than in low-grade lymphomas (P < .01, P < .01, and P < .05, respectively). In summary, the cytogenetic findings in our series of 104 PTL enabled us to distinguish not only between low-grade and high- grade lymphomas but also between various entities of PTL. Thus, the cytogenetic findings paralleled the histopathologic diagnoses made according to the updated Kiel classification.