bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alpha 2-macroglobulin-kallikrein complexes detect contact system activation in hereditary angioedema and human sepsis
Authors: Kaufman N , Page JD , Pixley RA , Schein R , Schmaier AH , Colman RW 
Published: October 2019

Abstract
Activation of the contact system has been documented in severe sepsis and hereditary angioedema, but a sensitive, specific, and quantitative assay for assessing the degree of involvement of this proteolytic enzyme cascade is not yet available. We have developed a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) for the alpha 2- macroglobulin-kallikrein (alpha 2M-Kal) complex using an F(ab')2 derivative of a monospecific polyclonal antibody against alpha 2 M as the capture antibody and a unique murine monoclonal antibody, 13G11, against the heavy chain of kallikrein as the detector antibody. The assay does not detect complexes in normal plasma but reacts with complexes generated by activating normal plasma with dextran sulfate at 4 degrees C in a range of 5 to 375 nmol/L. A close correlation of the ELISA with an amidolytic assay for alpha 2M-Kal was documented. Patients with sepsis syndrome but negative bacterial blood cultures did not show elevated plasma complexes, whereas a majority of those with positive blood cultures did show modest elevation and a single patient with septic shock showed a very high level of alpha 2M-Kal complex. Similarly, a patient with classic hereditary angioedema (HAE) showed increased concentration of complexes on three separate occasions during attacks but normal levels between attacks. Two other HAE patients did not show elevated levels at quiescent periods. The ELISA for alpha 2M- Kal appears to be sensitive, specific, and quantitative, and it can be used to reflect the degree of contact system activation in human sepsis and in HAE attacks.
'Role of bone marrow stromal cells in the growth of human multiple myeloma
Authors: Caligaris-Cappio F , Bergui L , Gregoretti MG , Gaidano G , Gaboli M , Schena M , Zallone AZ , Marchisio PC 
Published: October 2019

Abstract
We have verified the hypothesis that multiple myeloma (MM) may be disseminated by circulating clonogenic cells that selectively home to the bone marrow (BM) to receive the signal(s) leading to proliferation, terminal differentiation, and production of the osteoclast activating factors. Long-term cultures of stromal cells have been developed from the BM of nine patients with MM. These cells were mostly fibroblast- like elements, interspersed with a proportion of scattered macrophages and rare osteoclasts. BM stromal cells were CD54+, produced high levels of interleukin-6 (IL-6) and measurable amounts of IL-1 beta, and were used as feeder layers for autologous peripheral blood mononuclear cells (PBMC). After 3 weeks of cocultures, monoclonal B lymphocytes and plasma cells, derived from PBMC, developed and the number of osteoclasts significantly increased. Both populations grew tightly adherent to the stromal cell layer and their expansion was matched by a sharp increase of IL-6 and by the appearance of IL-3 in the culture supernatant. These data attribute to BM stromal cells a critical role in supporting the growth of B lymphocytes, plasma cells, and osteoclasts and the in vivo dissemination of MM.
Lipid hydroperoxides permit deformation-dependent leak of monovalent cation from erythrocytes
Authors: Sugihara T , Rawicz W , Evans EA , Hebbel RP 
Published: October 2019

Abstract
Subtle peroxidative perturbation of normal red blood cells (RBC) using t-butylhydroperoxide creates a leak pathway for monovalent cations that is reversibly activated by cell deformation. To determine what factor promotes expression of this unique membrane defect, we have dissected “peroxidation” into components that can be evaluated separately by comparing K leak from suitably modified RBC during elliptical deformation and parallel control incubation. Selective introduction of phospholipid hydroperoxides into normal RBC membranes successfully induces a deformation-dependent leak pathway having the same phenomenology as that previously documented for cells treated with t- butylhydroperoxide itself (fully recoverable; calcium-independent; inhibited at lower pH; K efflux balanced by Na influx). This leak pathway occurs in the absence of detectable secondary peroxidative change and appears to reflect a direct influence of lipid hydroperoxide. Using micropipette examination of vesicular bilayers reconstituted from RBC lipid extracts, we find that lipid from peroxidized RBC exhibits only a slight tendency to be less cohesive than normal lipid, apparently precluding isolated lipid properties as an explanation for altered permeability barrier function. However, addition of a hydrophobic membrane-spanning peptide to these same lipids significantly diminishes bilayer cohesion, an effect that is exacerbated further by the presence of peroxidized lipid. These observations suggest that lipid hydroperoxide is a necessary, but perhaps not sufficient, factor for induction of this unique leak pathway. Our results may be relevant to the abnormal cation homeostasis of sickle RBC in which deformation of an oxidatively perturbed membrane occurs during the sickling phenomenon.
Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement
Authors: Ezzell JL , Wilcox LA , Bernshaw NJ , Parker CJ 
Published: October 2019

Abstract
To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.
Posttransfusion purpura due to an alloantibody reactive with glycoprotein Ia/IIa (anti-HPA-5b)
Authors: Christie DJ , Pulkrabek S , Putnam JL , Slatkoff ML , Pischel KD 
Published: October 2019

Abstract
A 38-year-old woman (JT) was diagnosed with posttransfusion purpura and significant posthysterectomy vaginal bleeding 9 days after the transfusion of 2 U of packed red blood cells. Analysis of JT's serum by a monoclonal antibody-antigen capture enzyme-linked immunosorbent assay method showed the presence of anti-HPA-5b (anti-Bra) antibodies directed against an epitope on platelet glycoprotein (GP) la of the GPIa/IIa complex. The patient's serum immunoprecipitated two proteins from 125I-labeled HPA-5b positive platelets that migrated under both nonreducing and reducing conditions on sodium dodecyl sulfate polyacrylamide gels at molecular weights characteristic of GPIa (150 Kd and 165 Kd, respectively) and GPIIa (120 Kd and 145 Kd, respectively). These bands were not precipitated when 125I-labeled HPA-5b negative platelets were used. Platelet typings performed on JT and her three children showed that the patient was HPA-5b negative and one of her children was HPA-5b positive. Platelets obtained from one of the donors who provided blood for the inciting transfusion also typed as HPA-5b positive. These findings demonstrate that posttransfusion purpura may be induced by antibodies directed against an alloantigenic epitope, namely HPA-5b (Bra), located on GPIa/IIa. Moreover, clinically significant bleeding can be associated with antibody reactions directed against this GP complex.
Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events
Authors: Goldberg MA , Gaut CC , Bunn HF 
Published: October 2019

Abstract
The human hepatoma cell line, Hep3B, synthesizes large quantities of erythropoietin (Epo) mRNA and protein in a regulated manner in response to hypoxia and cobaltous chloride (CoCl2). To further understand the regulation of Epo gene expression, we studied the effects of hypoxia and CoCl2 on the rate of Epo gene transcription. While Northern blot analyses showed that steady-state Epo mRNA levels increase more than 50- fold in response to hypoxia or CoCl2, nuclear run-off experiments demonstrated only a 10-fold increase in Epo gene transcription in response to these stimuli. In the presence of either actinomycin D (Act D) or cycloheximide, the stability of biologically functional Epo mRNA was much greater than that observed in the absence of these agents and much greater than that which has been reported in vivo. These findings suggest that the stability of Epo mRNA is modulated by the transcription and translation of rapidly turning over gene product(s). Thus, Epo mRNA levels are determined by both the rate of gene transcription and posttranscriptional events. These experiments demonstrate a potential pitfall in estimating mRNA half-lives based on Act D chase experiments alone.
Fibrinogen is not synthesized by human megakaryocytes
Authors: Louache F , Debili N , Cramer E , Breton-Gorius J , Vainchenker W 
Published: October 2019

Abstract
The origin of platelet fibrinogen is controversial. It may arise from two sources: (a) exogenously by endocytosis of plasma fibrinogen, or (b) endogenously by synthesis. We explored the second possibility because we previously demonstrated that the first mechanism does occur. Fibrinogen synthesis by human megakaryocytes (MK) was investigated by in situ hybridization and the polymerase chain reaction (PCR) applied to mRNA. MK differentiating from marrow CFU-MK were cultured in suspension. In situ hybridization using the 35S alpha and beta fibrinogen chain anti-sense riboprobes was totally negative in MK in comparison with negative controls (lambda phage and alpha and beta fibrinogen chain sense riboprobes). In contrast, synthesis of fibrinogen was detected by this technique in a hepatoma cell line (HepG 2). Furthermore, mRNA for alpha and beta chains of fibrinogen was not detected by the PCR performed on mRNA from cultured MK enriched to 90% purity, by the immunomagnetic bead technique, even after Southern blotting of the amplified products. In addition, fibrinogen mRNA was undetected in marrow MK and in platelets by the same technique, whereas a specific megakaryocyte gene transcript (GPIb alpha) was easily detected. These observations demonstrate that the only mechanism responsible for the presence of fibrinogen in platelets is endocytosis of fibrinogen from plasma.
Synergistic inhibition by verapamil and quinine of P-glycoprotein- mediated multidrug resistance in a human myeloma cell line model
Authors: Lehnert M , Dalton WS , Roe D , Emerson S , Salmon SE 
Published: October 2019

Abstract
In an effort to develop a clinically useful approach to overcoming P- glycoprotein-mediated multidrug resistance (MDR1), we evaluated combined chemosensitization with verapamil and quinine in a multidrug- resistant (MDR) human myeloma cell line model. In clonogenic assay, verapamil was used at concentrations from 0.1 to 1.0 micrograms/mL, bracketing the plasma levels achieved by oral administration and high- dose intravenous (IV) infusion, respectively. The dose of quinine was held constant at 1.0 micrograms/mL, a plasma concentration readily achieved by oral administration. At each dose level of verapamil tested, the combination with quinine proved more effective than either drug individually in reversing resistance to doxorubicin and vinblastine and synergistic chemosensitizing interaction was observed. Verapamil at 0.1 microgram/mL combined with quinine was capable of restoring sensitivity to doxorubicin fully and reduced resistance to vinblastine as effectively as verapamil alone at 1.0 micrograms/mL. Furthermore, the combination of 1.0 mumol verapamil with 10 mumols quinine increased accumulation and retention of anthracycline in the resistant cells to a greater extent than did either drug individually (P less than .001) and inhibited drug efflux as effectively as verapamil alone at 10 mumols. Our findings suggest that combined chemosensitization with verapamil and quinine may prove useful for overcoming MDR1 in patients with drug-refractory B-cell neoplasms such as multiple myeloma or non-Hodgkin's lymphomas.
Anti-CD33 monoclonal antibody and etoposide/cytosine arabinoside combinations for the ex vivo purification of bone marrow in acute nonlymphocytic leukemia
Authors: Stiff PJ , Schulz WC , Bishop M , Marks L 
Published: October 2019

Abstract
Pharmacologic and immunologic methods of ex-vivo bone marrow (BM) purging for acute nonlymphocytic leukemia (ANLL) were combined to augment the effect of either method alone. Etoposide (VP16; 20 to 30 micrograms/mL) with or without cytosine arabinoside (Ara C; 10 mg/mL) was used in tandem with the anti-CD33 monoclonal antibody (MoAb), MY9, chosen because CD33 is found on the stem cell pool in the majority of patients with ANLL. The agents were tested singly or sequentially, with a 1-hour incubation of the drugs preceding complement-mediated lysis using MY9. VP16 combined with Ara C killed up to 3.9 +/- 0.3 and 5.11 +/- 0.4 logs of the human ANLL cell lines HL60 and K562 at drug concentrations that killed only 1.2 +/- 0.1 logs of normal committed granulocyte/macrophage stem cells (CFU-GM). Adding a single exposure of the MY9 and complement (C′) to the drug-treated cells, greater than 5.4 logs of HL60 were killed. Similar to other pharmacologic agents, no differential kill for clonagenic leukemic cells (colony-forming unit- leukemia; CFU-L) from patients with ANLL was seen for drug only treated blasts versus normal CFU-granulocyte-macrophage (CFU-GM), with less than 1 log CFU-L kill at drug concentrations that spared 1 log of CFU- GM. Similarly, only 1.1 +/- 0.3 logs of ANLL CFU-L were eliminated using MY9 and C′. However, with the sequential VP16/Ara C----MY9 + C′ treatment, synergy was demonstrated and 2.6 +/- 0.3 logs of CFU-L were eliminated. Because CD33 is also found on the normal CFU-GM pool, two- stage long-term BM cultures were performed to determine pluripotent stem cell elimination by the drug/MoAb purging combination. No difference of CFU-GM or BFU-E production at 4 to 6 weeks of culture for VP16/Ara C, MY9 + C′, or VP16/AraC----My9 + C′ treated cells was seen compared with untreated controls indicating sparing of early progenitor cells. Sequential ex vivo treatment of human ANLL CFU-L with VP16/Ara C followed by complement-mediated lysis using MY9 synergistically kills CFU-L while sparing early normal hematopoietic progenitor cells, and thus may be a more effective way to purge BM than either alone.
Typing of MNSs blood group specific sequences in the human genome and characterization of a restriction fragment tightly linked to S-s- alleles
Authors: Huang CH , Guizzo ML , McCreary J , Leigh EM , Blumenfeld OO 
Published: October 2019

Abstract
Human erythrocyte membrane alpha and delta glycophorins (glycophorins A and B) carry the antigens for the M,N,S,s blood group system. Synthetic oligonucleotides spanning coding regions for M,N,S, and s epitopes were used to examine DNAs from 50 individuals selected at random and from individuals known to exhibit S(-)s(-)U- or S(-)s(-)U+ blood group phenotypes. We showed that M,N,S,s, blood group-specific sequences occur as multicopies in the human genome and reside within the alpha and delta glycophorin genes and also within the third glycophorin gene (glycophorin E gene). DNA typing with M- and N-epitope-specific probes showed distinct patterns that allowed correlation of the genotypes with the blood group phenotypes. The correlation using S- and s-specific probes was less definite owing to cross-hybridization. An Mspl restriction fragment length polymorphism (RFLP) residing in the E gene was detected in the black population. This RFLP is also carried by all individuals tested who exhibit the S(-)s(-)U- and S(-)s(-)U+ blood groups phenotypes, thereby serving as a useful marker for the S-s- alleles. The site of cleavage resulting in this RFLP was localized to the second intron of the E gene, and cleavage could occur through differential methylation of its two alleles.
Neutrophil function and pyogenic infections in bone marrow transplant recipients
Authors: Zimmerli W , Zarth A , Gratwohl A , Speck B 
Published: October 2019

Abstract
In a consecutive entry trial, the incidence and time course of decreased neutrophil function was assessed in 20 patients treated with allogeneic bone marrow transplantation (BMT). The aim of the study was to assess the prognostic value of low neutrophil function for late pyogenic infections. Chemotaxis, superoxide production, and phagocytic- bactericidal activity were studied before and 2, 6, 9, and 12 months after BMT. Skin window migration was quantitatively assessed 2 months after BMT. Infectious complications were recorded prospectively with preset criteria during 1 year. Six of the 20 leukemic patients had defective neutrophil function before BMT. Two months after BMT all 10 patients with greater than stage II graft-versus-host disease (GVHD), and 6 of 10 patients with less than or equal to stage II GVHD had at least one decreased function. At this time, patients with subsequent pyogenic infections had lower chemotaxis (P less than .05), phagocytic- bactericidal activity (P less than .005), and superoxide production (P less than .025) than those without. Defective skin window migration and combined defects were predictive for late pyogenic infections. At 9 months all tests were normal in seven patients surviving without GVHD. In contrast, at 9 months three of three patients, and at 1 year two of three with chronic GVHD had still decreased neutrophil function. In conclusion, neutrophil function is frequently impaired during the first months after BMT. Combined neutrophil defects predispose to pyogenic infections and indicate the patient at risk.
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

Abstract
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.
Administration of interleukin-6 stimulates multilineage hematopoiesis and accelerates recovery from radiation-induced hematopoietic depression
Authors: Patchen ML , MacVittie TJ , Williams JL , Schwartz GN , Souza LM 
Published: October 2019

Abstract
Hematopoietic depression and subsequent susceptibility to potentially lethal opportunistic infections are well-documented phenomena following radiotherapy. Methods to therapeutically mitigate radiation-induced myelosuppression could offer great clinical value. In vivo studies in our laboratory have demonstrated that interleukin-6 (IL-6) stimulates pluripotent hematopoietic stem cell (CFU-s), granulocyte-macrophage progenitor cell (GM-CFC), and erythroid progenitor cell (CFU-e) proliferation in normal mice. Based on these results, the ability of IL- 6 to stimulate hematopoietic regeneration following radiation-induced hematopoietic injury was also evaluated. C3H/HeN female mice were exposed to 6.5 Gy 60Co radiation and subcutaneously administered either saline or IL-6 (1,000 micrograms/kg) on days 1 through 3 or 1 through 6 postexposure. On days 7, 10, 14, 17, and 22, femoral and splenic CFU-s, GM-CFC, and CFU-e contents and peripheral blood white cell, red cell, and platelet counts were determined. Compared with saline treatment, both 3-day and 6-day IL-6 treatments accelerated hematopoietic recovery; 6-day treatment produced the greater effects. For example, compared with normal control values (N), femoral and splenic CFU-s numbers in IL-6-treated mice 17 days postirradiation were 27% N and 136% N versus 2% N and 10% N in saline-treated mice. At the same time, bone marrow and splenic GM-CFC values were 58% N and 473% N versus 6% N and 196% N in saline-treated mice; bone marrow and splenic CFU-e numbers were 91% N and 250% N versus 31% N and 130% N in saline-treated mice; and peripheral blood white cell, red cell, and platelet values were 210% N, 60% N, and 24% N versus 18% N, 39% N, and 7% N in saline- treated mice. These studies demonstrate that therapeutically administered IL-6 can effectively accelerate multilineage hematopoietic recovery following radiation-induced hematopoietic injury.