bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of superoxide dismutase in leukocytes by paraquat: correlation with age and possible predictor of longevity
Authors: Niwa Y , Ishimoto K , Kanoh T
Published: October 2019

Reactive oxygen species (ROS) are thought to play a role in the aging process as well as in a number of human diseases states. Superoxide dismutase (SOD), an enzyme that scavenges the superoxide anion (O2-) is constitutively expressed in leukocytes and other tissues. When assayed in peripheral blood leukocytes (PBL), constitutive SOD activity shows little variation among individuals of different ages. We have found that significant induction of SOD activity occurs in PBL incubated in vitro with paraquat, an agent known to cause intracellular O2- production. This induction was found to be highly age dependent; lymphocytes from 36 healthy subjects aged 20 to 40 years showed an increase of 85% +/- 10%, versus an increase of only 8% +/- 1% for lymphocytes from 30 healthy subjects aged 65 to 79 years (P less than 10(-4)). Forty subjects, aged 67 to 73 years, who were healthy at the time of assay of leukocyte SOD induction were followed up 5 years later. Nineteen of these subjects had died; all 19 had shown SOD induction of less than 10% (range, 0% to 7%; mean, 2.4%). In contrast, of the 21 survivors (range, 2.5% to 50%; mean, 21%), 12 had shown SOD induction greater than 10%, and 7 had shown SOD induction greater than or equal to 35% (P less than 10(-3)). Thirteen of the 19 deaths were attributable to malignancy or cerebrocardiovascular disease. Preservation of leukocyte SOD inducibility appears to correlate with longevity in elderly individuals and may be of value in predicting resistance to malignancy or fetal cardiovascular events.
Induction of immune tolerance in patients with hemophilia A and inhibitors treated with porcine VIIIC by home therapy
Authors: Hay CR , Laurian Y , Verroust F , Preston FE , Kernoff PB
Published: October 2019

Home therapy with porcine factor VIIIC was safe and effective when administered to five hemophilic patients over periods of 8 1/2, 6, 4, 3 1/2, and 2 years. No significant transfusion reactions occurred. Before treatment with porcine factor VIIIC, all five had high-level, high- responding anti-human VIIIC inhibitors initially lacking anti-porcine factor VIIIC activity. Although specific anti-porcine VIIIC inhibitors arose in all patients, these were generally transient, and only one patient became refractory to treatment. We believe that porcine factor VIIIC is the treatment of choice in patients whose inhibitors do not cross-react. All five patients lost their original anti-human VIIIC inhibitors after starting treatment with porcine VIIIC, permitting the reintroduction of human VIIIC in three of them. There has been no recurrence of anti-human VIIIC inhibitor activity during 2 to 3 years of regular treatment with human VIIIC in these patients. This suggests that tolerance to human VIIIC has arisen as a result of treatment with porcine VIIIC. Porcine VIIIC may have a role in the desensitization of some factor VIIIC inhibitor patients.
Influence of recombinant hematopoietins and of fetal bovine serum on the globin synthetic pattern of human BFUe
Authors: Migliaccio AR , Migliaccio G , Brice M , Constantoulakis P , Stamatoyannopoulos G , Papayannopoulou T
Published: October 2019

We have studied the effects of recombinant hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-3 (IL-3) on the globin program of adult human erythroid progenitors (BFUe) stimulated to terminal differentiation by erythropoietin under fetal bovine serum (FBS)-supplemented or FBS- deprived culture conditions. Fetal globin production by BFUe-derived erythroblasts was assessed at the protein and mRNA level and its cellular distribution was evaluated by immunofluorescence. Although hemoglobinization and maturation of BFUe-derived erythroblasts was by and large comparable in FBS-replete versus FBS-deprived cultures, the latter had significantly less (up to 20-fold) gamma-globin and gamma- globin mRNA levels. Reduced gamma-globin in serum-deprived cultures was also reflected by a smaller proportion of erythroblasts with detectable gamma-globin by immunofluorescence. Erythroid bursts induced by either GM-CSF or IL-3 produced similar levels of gamma-globin both in FBS- supplemented and in FBS-deprived cultures. These results, obtained even in cultures of highly enriched BFUe, suggest that GM-CSF and IL-3, although they significantly increase the number and size of erythroid bursts, do not by themselves exert a direct influence on the level of fetal globin synthesis. By contrast, factor(s) present in FBS appear to exert a dominant influence on fetal globin synthesis in vitro. Although FBS-deprived conditions appear to largely abrogate the in vitro activation of fetal hemoglobin (Hb F) in normal samples, they do support increased Hb F production in samples from patients with hereditary persistence of fetal hemoglobin or from cord blood.
Thrombin induces c-fos expression in cultured human endothelial cells by a Ca2(+)-dependent mechanism
Authors: Lampugnani MG , Colotta F , Polentarutti N , Pedenovi M , Mantovani A , Dejana E
Published: October 2019

The proto-oncogene c-fos has been implicated in the modulation of various cell functions. We have found that thrombin, a pleiotropic activator of endothelial cells, induced c-fos mRNA in human umbilical vein endothelial cells (HEC). This effect was dose-related (0.05 to 1.0 U/mL) and transient (maximal after 1 hour and negligible within 4 hours). Since thrombin activates phosphoinositide (PI) turnover through a pertussis toxin (PT)-sensitive guanosine triphosphate-binding regulatory protein(s) (G-protein) with subsequent stimulation of protein kinase C (PKC) and Ca2+ movements, we investigated whether these intracellular pathways are also responsible for c-fos induction. PT inhibited thrombin's effect on c-fos expression, but had no effect on c-fos expression by phorbol myristate acetate (PMA). Down regulation of PKC by prolonged exposure to PMA had no effect on thrombin and ionomycin stimulation of c-fos, but inhibited PMA activation of this gene. Quenching of the Ca1(2+) increase in response to quin2 loading in the absence of external Ca2+ suppressed thrombin activity on c-fos transcription. Under the same conditions PMA activity was not inhibited or only partially inhibited. Interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) stimulation of c-fos mRNA level were not inhibited by quin2; on the contrary, ionomycin effect was blocked by this agent. These results indicate that thrombin-induced c-fos expression in HEC does not require a fully active PKC but is dependent on normal intracellular Ca2+ availability.
Distinct characteristics of lymphokine-activated killer (LAK) cells derived from patients with B-cell chronic lymphocytic leukemia (B-CLL). A factor in B-CLL serum promotes natural killer cell-like LAK cell growth
Authors: Santiago-Schwarz F , Panagiotopoulos C , Sawitsky A , Rai KR
Published: October 2019

We show that lymphokine-activated killer (LAK) cell precursors derived from patients with B-cell chronic lymphocytic leukemia (B-CLL) and cultured in the presence of recombinant interleukin-2 and normal human serum (NHS), develop into primarily NK cell-like (CD 57+) LAK cells, whereas identically prepared LAK cell precursors from normal subjects develop into mainly T cell-like (CD 3+, CD 8+) LAK cells. B-CLL LAK cells exhibited greater proliferative capacity than did normal LAK cells. When normal LAK cells were grown in B-CLL serum instead of NHS, their proliferation increased; NK cell levels also increased to those found in B-CLL LAK cells, suggesting that B-CLL serum contains a factor that promotes NK cell-like growth, LAK cells derived from normal or B- CLL patients demonstrated similar lytic activity toward K562 and Raji cells. Growth in B-CLL serum did not reduce their lytic potential. Thus, the altered phenotype and growth exhibited by B-CLL LAK cells and normal LAK cells grown in B-CLL serum does not lead to abnormalities in their cytolytic functions. We propose instead that the predominance of NK-like cells in B-CLL LAK cell populations and the presence of an NK cell-like growth factor in B-CLL serum reflect abnormalities related to NK cell-mediated B-cell regulation; ie, either inhibition of normal B- cell growth and/or growth stimulation of the leukemic clone in B-CLL.
Characterization of a factor-dependent acute leukemia cell line with translocation (3;3)(q21;q26)
Authors: Oval J , Jones OW , Montoya M , Taetle R
Published: October 2019

A strictly factor-dependent cell line (UCSD/AML1) was established from a patient with the syndrome of multilineage acute leukemia with high platelets. The patient's cells and the cell line karyotype were 45,XX,- 7,t(3;3)(q21;q26), typical of the syndrome of acute leukemia with high platelets. The cell line expresses CD34, CD7, TdT, and myeloid (CD13, CD14, CD33) and megakaryocyte/platelet (CD36, CD41, CD42b, CDw49b) antigens. In short-term culture, UCSD/AML1 cells proliferate in response to interleukin-3 (IL-3), IL-4, IL-6, macrophage colony- stimulating factor (M-CSF), and granulocyte-macrophage CSF (GM-CSF), but not IL-1, IL-2, IL-5, or G-CSF. In long-term culture, proliferation can be sustained by GM-CSF, IL-6, or M-CSF. When maintained in GM-CSF, a small percentage of cells form multinucleated megakaryocyte-like giant cells. Culture with GM-CSF combined with IL-6, but not with IL-6 alone, increased giant cell formation fourfold to sevenfold. IL-6 alone or in combination with GM-CSF increased expression of platelet-related antigens. In contrast, culture with phorbol ester induced formation of macrophage-like cells. UCSD/AML1 is the first human acute nonlymphocytic leukemia cell line established from a patient with an acute leukemia syndrome associated with a specific chromosome abnormality.
Production of laminin B2 chain protein and messenger RNA by a murine neutrophil precursor cell line, 32Dc13 [see comments]
Authors: Tweardy DJ , Sasaki M , Cardamone JJ Jr , McCoy JP Jr , Bonidie MJ , Signorella AP
Published: October 2019

Laminin is a heterotrimeric glycoprotein that plays a central role in promoting neutrophil chemotaxis, motility, and attachment to basement membrane. Rabbit peritoneal exudate neutrophils stain positively for laminin, which is presumed to be of exogenous origin and bound to laminin receptors on the cell surface. We examined 32Dc13 cells, a murine neutrophil precursor cell line, by immunoprecipitation. Northern blot analysis, flow cytometry, and electron microscopy for the endogenous production of laminin. Our results demonstrate that 32Dc13 cells endogenously produce a laminin B2 chain protein and messenger RNA (mRNA) without producing any detectable A or B1 chain protein or mRNA. The B2 chain protein was not secreted by the cells; rather it could be detected on the cell surface after treatment of cells with neuraminidase. These findings suggest the possibility of a novel role for the laminin B2 chain in myeloid development and function.
Reconstitution of antibody response after allogeneic bone marrow transplantation: effect of lymphocyte depletion by counterflow centrifugal elutriation on the expression of hemagglutinins
Authors: Bar BM , Santos GW , Donnenberg AD
Published: October 2019

The generation of ABO hemagglutinins was used as a model to assess the tempo of reconstitution of antibody responses in recipients of elutriated (CCE) and nonelutriated (nonCCE) HLA matched bone marrow allografts. The study included 29 CCE recipients (10 major, 6 minor, and 1 major/minor ABO-mismatched, and 12 ABO-matched) and 40 nonCCE recipients (14 major, 12 minor, 2 major/minor, and 12 matched). Plasma volume in the graft and in blood product transfusions was uncorrelated with changes in hemagglutinin titers and therefore was excluded as a significant source of antibody. Removal of graft lymphocytes by CCE did not result in prolongation of host-derived hemagglutinins in recipients of major ABO-mismatched grafts. However, CCE resulted in a complete abrogation of the adoptive transfer of donor-derived antibody as detected in recipients of minor ABO-mismatched grafts. Despite the absence of adoptively transferred donor immunity in recipients of CCE grafts, they had hemagglutinin levels comparable with those of recipients of nonCCE grafts by 6 months after transplantation. This demonstrates that recipients of elutriated marrow were competent to mount de novo responses at that time. The strong correlation between donor pretransplant hemagglutinin titer and recipient titer 1 year after bone marrow transplantation in recipients of nonCCE grafts suggests that even late after transplant, antibody remains the product of adoptively transferred memory cells in recipients of grafts containing large numbers of mature lymphocytes.
Trisomy 13: a new recurring chromosome abnormality in acute leukemia
Authors: Dohner H , Arthur DC , Ball ED , Sobol RE , Davey FR , Lawrence D , Gordon L , Patil SR , Surana RB , Testa JR
Published: October 2019

A new recurring chromosome abnormality was identified in 8 of 621 consecutive successfully karyotyped adults with de novo acute leukemia. These eight patients had trisomy 13 as the sole cytogenetic abnormality. On central morphologic review, five cases were classified as subtypes of acute myeloid leukemia, one as acute mixed lymphoid and myeloid leukemia, one as acute lymphoid leukemia, and one as acute undifferentiated leukemia. Blasts of all eight cases expressed one or more myeloid differentiation antigens. Three also expressed T-lineage- associated antigens; however, none of these had rearrangement of the T- cell receptor beta, gamma, or delta genes. Four of six cases tested were TdT positive. All eight patients with trisomy 13 were treated with intensive induction chemotherapy; only three entered a short-lived complete remission. Survival of patients with trisomy 13 ranged from 0.5 to 14.7 months, and was significantly shorter than that of the remaining patients (median 9.5 v 16.2 months, P = .007). We conclude that trisomy 13 is a rare, recurring clonal chromosome abnormality in acute leukemia associated with a poor prognosis. Malignant transformation of an immature hematopoietic precursor cell is suggested by the expression of antigens characteristic of both the myeloid and lymphoid lineage, the high incidence of TdT positivity, and the morphologic heterogeneity in these leukemias.
Abnormalities of the long arm of chromosome 6 in childhood acute lymphoblastic leukemia
Authors: Hayashi Y , Raimondi SC , Look AT , Behm FG , Kitchingman GR , Pui CH , Rivera GK , Williams DL
Published: October 2019

To determine the biologic significance of the structural rearrangements of the long arm of chromosome 6(6q) in acute lymphoblastic leukemia (ALL) at diagnosis, we studied 412 consecutive children whose leukemic cell chromosomes had been completely banded and identified 45 (11%) children with this abnormality. The 45 cases were divided into del(6q) only (n = 11), del(6q) and numerical abnormalities (n = 4), del(6q) and structural abnormalities (n = 23), and 6q translocations (n = 7). The breakpoints of del(6q) were subgrouped: del(6)(q15q21) in 11 cases, del(6) (q13q21) in six, del(6)(q21q23) in four, del(6)(q15) in four, del(6)(q15q23) in three, and other deletions in 10 cases. Notably, all these deletions encompassed the 6q21 band, suggesting that this might be the locus of a recessive tumor suppressor gene, the absence of which contributes to malignant transformation or proliferation. Among the seven children with 6q translocations, a previously unidentified nonrandom translocation, t(6;12)(q21;p13) was noted in two cases with an early pre-B immunophenotype. Clinical features and event-free survival were similar among children with or without 6q abnormalities. Overall, children with 6q abnormalities were less likely than those without the abnormality to have a pre-B immunophenotype (P = .03). T- cell immunophenotypes were equally represented in cases with or without 6q abnormalities. However, all four children with del(6q) and a 12p abnormality had early pre-B ALL and all three children with del(6q) and a 9p abnormality had a T-cell immunophenotype. The lack of specificity for a particular immunophenotype may imply that the gene or genes affected by 6q abnormalities are broadly active in the multistep process of lymphoid leukemogenesis. The relatively high frequency of microscopically visible del(6q) indicates the need for molecular studies to identify cases with submicroscopic deletions.
The role of cellular maturation in neutrophil heterogeneity
Authors: Krause PJ , Todd MB , Hancock WW , Pastuszak WT , Maderazo EG , Hild DH , Kosciol CM
Published: October 2019

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or “brightxyc PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or “dull” PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.
Human macrophage colony-stimulating factor induces macrophage colonies after L-phenylalanine methylester treatment of human marrow
Authors: Rosenfeld CS , Evans C , Shadduck RK
Published: October 2019

Macrophage-colony stimulating factor (M-CSF) has well-known effects on murine bone marrow, but its colony stimulating activity for human bone marrow is controversial. After treatment of human bone marrow with L- phenylalanine methylester (PME), macrophage-colonies (CFU-M) were induced by M-CSF in a dose-dependent fashion. The optimal concentration of recombinant human-macrophage colony stimulating factor (rhM-CSF) was 1,000 U/mL. Purified human urine M-CSF had colony stimulating activity similar to rhM-CSF. Further studies were performed to determine the factors responsible for the enhanced CFU-M formation from PME treated marrow. Compared with nylon wool and carbonyl iron monocyte depletion methods, PME eliminated significantly more monocytes and myeloid cells. This observation suggested that these cells may release hematopoietic inhibitory factors for CFU-M. Low concentrations (1%) but not normal (10%) concentrations of blood monocytes were inhibitory (mean inhibition, 48%) to CFU-M. High concentrations of monocytes (50%) augmented CFU-M colonies. HL-60 conditioned media was used to simulate secretory products of early myeloid cells. HL-60 conditioned media (1%) inhibited CFU-M formation but not granulocyte macrophage or granulocyte colonies. We conclude that M-CSF has colony stimulating activity for human marrow that can be recognized after removal of inhibitory cells by PME treatment.
Seven-day administration of recombinant human granulocyte colony- stimulating factor to newborn rats: modulation of neonatal neutrophilia, myelopoiesis, and group B Streptococcus sepsis
Authors: Cairo MS , Plunkett JM , Mauss D , Van de ven C
Published: October 2019

Single-pulse administration of rhG-colony-stimulating factor (CSF) to neonatal rats was previously demonstrated to induce peripheral neutrophilia and modulate bone marrow (BM) neutrophil storage and proliferative pools (NSP + NPP). In this study, we investigated the prolonged effects of 7 days of rhG-CSF therapy (5 micrograms/kg/per day). Sprague-Dawley newborn rats (less than or equal to 24 hours) were injected intraperitoneally (IP) (daily for 7 days) with rhG-CSF or phosphate-buffered saline/human serum albumin (PBS/HSA). RhG-CSF induced a significant early and late peripheral neutrophilia: 6,905 +/- 1,625 (day 1) and 9,223 +/- 515 microL (day 7) v 1,275 +/- 90/microL (P less than or equal to .0001). In addition, 7 days of rhG-CSF resulted in a significant increase in the BM NSP: 3,247 +/- 190/microL v 1,677 +/- 339/microL (P less than or equal to .001). There was, however, no depletion or significant change in the BM NPP. Seven days of rhG-CSF also induced a mild increase in BM CFU-GM colony formation (P less than or equal to .01). There was, however, no significant change in liver/spleen CFU-GM colonies or in the CFU-GM proliferative rate in either the BM or liver/spleen cultures. Finally, 7 days of prophylactic rhG-CSF therapy resulted in a synergistic response with antibiotic therapy and significantly modulated the mortality rate during experimental group B streptococcal sepsis (GBS) (100% v 50%) (GvsC) (P less than or equal to .001). Pulse rhG-CSF administered at 6 hours or 18 hours after GBS inoculation, however, failed to act synergistically with antibiotics to improve survival or prevent peripheral neutropenia. This study suggests that 7 days of prophylactic rhG-CSF therapy induces peripheral neutrophilia, myeloid maturation, increases neutrophil BM reserves and also may provide immunologic enhancement of neonatal host defense during experimental GBS in term neonatal rats.
Autoantibodies against CD4 cells are associated with CD4 helper defects in human immunodeficiency virus-infected patients
Authors: Weimer R , Daniel V , Zimmermann R , Schimpf K , Opelz G
Published: October 2019

To investigate whether autoantibodies against CD4-positive lymphocytes might induce helper dysfunction, autoantibody formation and T-cell function was examined simultaneously in 61 hemophilia patients. Twenty patients were human immunodeficiency virus (HIV)-negative, 26 HIV- positive stage CDC II or III, and 15 were HIV-positive stage CDC IV. T lymphocytes, CD4-positive, or CD8-positive T subsets were cocultured with B lymphocytes and pokeweed mitogen (PWM) for 6 days and Ig- secreting cells were assessed in a reverse hemolytic plaque assay. The presence of IgM, IgG, C3d, or gp120 on the surface of T cells or T subsets was analyzed by flow cytometry. Autoantibodies against CD4- positive T cells were not detected in controls or HIV-negative patients, but were common in HIV-positive patients (20 of 41 patients). In patients with autoantibodies we found an increased incidence of CD4 helper defects (P less than .0001 in CDC II or III patients; P less than .02 in CDC IV patients). 12 of 13 patients with IgM autoantibodies and 4 of 4 with IgG autoantibodies showed CD4 helper defects. Complement fixation had no relevance. Autoantibody formation against CD4 cells was not due to increased in vivo B-cell stimulation (spontaneous plaque formation: 611 +/- 204 PFC/10(6) B cells in autoantibody-negative patients v 650 +/- 202 PFC/10(6) B cells in autoantibody-positive patients; not significant). Thus, our results suggest that autoantibody formation is not caused by a general state of in vivo B-cell activation. Rather, the production of autoantibodies appears to coincide with defects in B-cell proliferation or differentiation, as shown by reduced mitogen-stimulated B-cell responses in CDC II and III patients (P less than .05). Autoantibodies against CD4 cells appear to be involved in the pathogenesis of CD4 helper defects of HIV-infected patients.
Allogeneic marrow transplantation in patients positive for hepatitis B surface antigen
Authors: Reed EC , Myerson D , Corey L , Meyers JD
Published: October 2019

Twenty patients who were positive for hepatitis B surface antigen (HBsAg) underwent allogeneic marrow transplant for malignancy or other underlying hematologic disease between 1975 and 1986. After transplant, one patient had serologic evidence of hepatitis B virus (HBV) reactivation whereas three patients had evidence of an immune response to HBV. Among four patients with serologic follow-up of 1 year or more, three remained positive for HBsAg and one became HBsAg negative. Six patients (30%) developed clinical evidence of venocclusive disease and seven patients (35%) developed acute graft-versus-host disease involving the liver, but the incidence of these complications was similar to that expected among patients who are not carriers of HBsAg. Three patients died with hepatorenal failure, but all three had venocclusive disease and the contribution of HBV infection to liver failure was unclear. Available liver specimens obtained at autopsy (six patients) or biopsy (two patients) all showed either HBsAg (one specimen) or hepatitis B core antigen (four specimens) or both (three specimens) by immunoperoxidase staining. Although HBV reactivation leading to hepatic failure has been reported among allogeneic marrow transplant recipients as well as other immunocompromised patients, we did not observe an increase in the incidence of severe liver disease after transplant among these 20 patients positive for HBsAg at the time of transplant, and do not consider positivity for HBsAg to be a contraindication to allogeneic marrow transplantation.
Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix
Authors: Chen CS , Hawiger J
Published: October 2019

Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to “cell adhesion site” of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400–411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). “Hybrid” peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys- Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His- His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.
Characterization and pharmacokinetic parameters of recombinant soluble interleukin-4 receptor
Authors: Jacobs CA , Lynch DH , Roux ER , Miller R , Davis B , Widmer MB , Wignall J , VandenBos T , Park LS , Beckmann MP
Published: October 2019

The interleukin-4 receptor (IL-4R) is expressed as a 140-Kd membrane glycoprotein that binds IL-4 with high affinity. Recently, cDNA clones for the murine IL-4R have been isolated. One clone encodes an integral membrane protein, while another encodes a protein in which translation is terminated before the transmembrane region, thus producing a soluble form of the IL-4R (sIL-4R). HeLa cell clones overexpressing sIL-4R were isolated using a novel filter-overlay and 125I-IL-4 ligand binding technique. Quantitative analysis demonstrated that the kinetics and affinity of IL-4 binding to the recombinant sIL-4R were similar to the native membrane-bound IL-4R. As low doses of sIL-4R specifically inhibited IL-4-induced proliferative responses in vitro, sIL-4R biodistribution and elimination parameters were evaluated to assess the pharmacokinetic potential of sIL-4R as a therapeutic agent. Pharmacokinetic studies demonstrated that radiolabeled sIL-4R had a distribution half-life of 9 minutes and an elimination half-life of 2.3 hours following intravenous (IV) administration. When administered by intraperitoneal or subcutaneous (SC) injection, the elimination half- lives were prolonged to 4.2 hours and 6.2 hours, respectively. Although the initial blood level of sIL-4R was reduced if administered by SC injection, the bioavailability was comparable with IV administration. The main sites of sIL-4R elimination were the liver and kidney.
Detection and characterization of human T-cell lymphotropic virus type I (HTLV-I) associated T-cell neoplasms in an HTLV-I nonendemic region by polymerase chain reaction
Authors: Chadburn A , Athan E , Wieczorek R , Knowles DM
Published: October 2019

Human T-cell lymphotropic virus type I (HTLV-I) associated adult T-cell leukemia/lymphoma (ATLL) occurs endemically in southwestern Japan, the Caribbean, and West Africa, but occurs sporadically in most of the rest of the world. However, because ATLL and non-HTLV-I associated T-cell neoplasms share overlapping clinicopathologic features, the prevalence of ATLL in nonendemic regions is unknown. In this study, 75 T-cell neoplasms randomly procured from the metropolitan New York City area were examined by polymerase chain reaction (PCR) for the presence of integrated HTLV-I proviral sequences. HTLV-I genomic sequences were detected by PCR in 6 of the 75 cases (8%); this result was confirmed by Southern blot hybridization. The clinicopathologic features of the HTLV- I positive and HTLV-I negative T-cell neoplasms were then compared. Although the clinicopathologic features of patients from these two groups overlapped, some findings were more commonly associated with HTLV-I positive neoplasms. Five of the six patients with HTLV-I positive neoplasms were from HTLV-I endemic areas, five were black, five were women, and five were less than 45 years of age, while the majority of the patients with HTLV-I negative T-cell malignancies were elderly white men. The incidence of hypercalcemia and lytic bone lesions was significantly more common among patients with HTLV-I positive T-cell neoplasms (P less than .001 and P = .004, respectively). The immunophenotypes of the HTLV-I positive and negative tumors were similar; however, all HTLV-I positive neoplasms were CD7 negative (P less than .001). In summary, our findings: (1) demonstrate the special clinicopathologic and immunophenotypic features of HTLV-I positive T-cell neoplasms, (2) suggest that most of the rare cases of HTLV-I-associated T-cell neoplasms occurring in HTLV-I nonendemic areas are actually endemic cases; and (3) that PCR is a sensitive, clinically useful technique for identifying HTLV-I associated T-cell neoplasms.
Acceleration of chronic myeloid leukemia correlates with calcitonin gene hypermethylation
Authors: Malinen T , Palotie A , Pakkala S , Peltonen L , Ruutu T , Jansson SE
Published: October 2019

Calcitonin gene methylation at CCGG sites were determined in 39 chronic myeloid leukemia patients by isoschizomeric restriction endonuclease analysis. A total of 27 patients were analyzed while still in the chronic phase: 20 patients had a normal gene, and seven had a hypermethylated gene. There were 12 patients initially studied in accelerated or blastic phases. All but one patient showed gene hypermethylation, suggesting a good correlation between gene methylation and disease stage. All five patients who, while still in the chronic phase, had a major 3.1-kb hypermethylated calcitonin gene fragment, accelerated within 2 to 27 months. In consecutively analyzed patients, the initially normal calcitonin gene changed to a hypermethylated state as the disease escalated. The hypermethylation predicted disease acceleration with a median lead time of 6 months before any morphologic or clinical signs of disease progression were seen. The disease progressed in 8 of 27 patients initially studied in the chronic phase: in only two patients this occurred without predictive methylation changes. The results suggest that the assessment of calcitonin gene methylation status may be a promising tool for monitoring chronic myeloid leukemia disease escalation.
Sequence variations in the 5' flanking and IVS-II regions of the G gamma- and A gamma-globin genes of beta S chromosomes with five different haplotypes
Authors: Lanclos KD , Oner C , Dimovski AJ , Gu YC , Huisman TH
Published: October 2019

We have amplified and sequenced the 5′ flanking and the second intervening sequence (IVS-II) regions of both the G gamma- and A gamma- globin genes of the beta S chromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5′ flanking regions (G gamma and A gamma) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and G gamma levels observed among SS patients. In vitro expression experiments involving the approximately 1.3-kb 5′ flanking regions of the G gamma- and A gamma-globin genes of the beta S chromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.
Multimerin: a series of large disulfide-linked multimeric proteins within platelets
Authors: Hayward CP , Warkentin TE , Horsewood P , Kelton JG
Published: October 2019

Platelets contain proteins with biochemical properties that are well adapted to promoting hemostasis. One important adhesive protein is von Willebrand factor (vWF), which is a very large protein comprised of a series of multimers, ranging from 860,000 to over 10 million daltons. In this report we describe a second platelet protein, p-155, which has a similar unique multimeric composition. Using agarose-acrylamide gel electrophoresis, platelet p-155 was shown to be composed of multimers ranging from less than 450 Kd to many million daltons. Based on this unique structure, we propose that the native molecule be designated as multimerin. Comparison with vWF showed that multimerin contained less of the very high molecular weight multimers. Differential reduction demonstrated that the smallest multimer is a trimer, composed of three 155-Kd subunits. Platelet releasate was demonstrated to contain mainly the smaller multimers, suggesting that the larger multimers bind to the platelet surface. Other studies indicate that multimerin and vWF are the two largest platelet proteins and the only two platelet proteins exhibiting a complex, disulfide-linked multimeric composition with variability in multimer size.
Characterization of the structure of the erythropoietin receptor by ligand blotting
Authors: Atkins HL , Broudy VC , Papayannopoulou T
Published: October 2019

Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd +/- 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding.
Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7
Authors: Gunji Y , Sudo T , Suda J , Yamaguchi Y , Nakauchi H , Nishikawa S , Yanai N , Obinata M , Yanagisawa M , Miura Y
Published: October 2019

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.
Characteristics of quinine- and quinidine-induced antibodies specific for platelet glycoproteins IIb and IIIa
Authors: Visentin GP , Newman PJ , Aster RH
Published: October 2019

Recent studies have shown that antibodies characteristic of quinine- and quinidine-induced thrombocytopenia sometimes recognize the platelet membrane glycoprotein (GP) complex IIb/IIIa in addition to their well known target, GPIb/IX. We have investigated the frequency with which drug-induced antibodies bind to GPIIb/IIIa and the nature of their target epitopes. In studies of sera from 13 patients sensitive to quinidine or quinine, we found that 10 contained IgG antibodies specific for both GPIb/IX and GPIIb/IIIa, two reacted with GPIb/IX alone, and one reacted with GPIIb/IIIa alone. In all cases, the presence of drug was required for binding of IgG to target GPs. By immunoabsorption, we found that each of five polyspecific sera contained at least two different antibodies, one reactive with GPb/IX and the other with GPIIb/IIIa. Further studies with eight drug- dependent antibodies (DDAb) specific for GPIIb/IIIa showed that three recognized the GPIIb/IIIa complex only, one recognized GPIIb alone, and three recognized GPIIIa alone. The eighth serum appeared to bind to both GPIIIa alone and to an epitope determined by the GPIIb/IIIa complex. The three antibodies specific for GPIIIa alone also reacted with GPIIIa deglycosylated with endo-H, and with the major (61 Kd) fragment obtained by chymotryptic digestion of GPIIIa but failed to react with reduced GPIIIa. These findings demonstrate that, in drug- induced, immunologic thrombocytopenia, the anti-platelet immune response is typically directed against epitopes on both GPIb/IX and GPIIb/IIIa. The three DDAb we studied that were specific for GPIIIa alone recognize epitopes resistant to chymotrypsin and endo-H treatment that are dependent on intrachain disulfide bonding.
The immunoregulatory effects of merocyanine 540 on in vitro human T- and B-lymphocyte functions
Authors: Lum LG , Yamagami M , Giddings BR , Joshi I , Schober SL , Sensenbrenner LL , Sieber F
Published: October 2019

Merocyanine 540 (MC 540) is a photoactive dye used to purge bone marrow of tumor cells in autologous bone marrow transplantation. The effects of MC 540 on the lymphoid components in the marrow are unknown. This study evaluates the treatment of lymphocytes by MC 540 (15 micrograms/mL) and light (70 W/m2) on: (1) phytohemagglutinin and Con A- induced proliferation; (2) allogeneic mixed lymphocyte cultures (MLC); (3) the regulation of Ig synthesis by T cells; and (4) the ability of B cells to produce polyclonal Igs as measured by an enzyme-linked immunosorbent assay-plaque assay. The results show that MC 540 and light treatment reduced Con A-stimulated T-cell proliferation greater than 50% after 30 minutes and greater than 80% after 60 minutes of MC 540-sensitized photoirradiation. Ninety minutes of MC 540 and light exposure (designated treatment) inhibited MLC greater than 90%. In polyclonal Ig synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited Epstein- Barr virus-stimulated Ig synthesis. These data show that T- and B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions.
Structure and expression of the cDNA encoding human neutrophil collagenase
Authors: Devarajan P , Mookhtiar K , Van Wart H , Berliner N
Published: October 2019

We have isolated and characterized a 2.4-kb cDNA clone encoding human neutrophil collagenase (HNC), a member of the family of matrix metalloproteinases restricted to secondary granules within neutrophils. Partial amino acid sequence was used to deduce oligonucleotide probes. These probes were used to screen a human granulocyte cDNA library derived from messenger RNA (mRNA) from a patient with chronic granulocytic leukemia. Cell-free translation of RNA produced from the cDNA produced a 52-Kd protein that was recognized by anti-HNC antibody. The cDNA clone was sequenced and shown to encode a 467-residue protein whose sequence matched those regions currently known for HNC. The enzyme exhibits 58% homology to human fibroblast collagenase and has the same domain structure. It consists of a 20-residue signal peptide, and an 80-residue propeptide that is lost on autolytic activation by cleavage of an M-L bond. Other regions identified include the autolytic degradation site, the “cysteine switch” residue that is involved in latency and activation, and a putative zinc binding sequence. HNC has six potential N-linked glycosylation sites. The cDNA hybridized to a 3.4-kb mRNA in RNA from a patient with chronic granulocytic leukemia, but not to RNA from uninduced HL60 cells or HL60 cells that had been induced to undergo granulocytic or monocytic maturation with dimethyl sulfoxide or 12-O-tetradecanoylphorbol 13-acetate, respectively. These results parallel those seen with lactoferrin and transcobalamin I, two other secondary granule proteins.
Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication
Authors: Navarro S , Debili N , Le Couedic JP , Klein B , Breton-Gorius J , Doly J , Vainchenker W
Published: October 2019

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.