bluuebunny/crossref_metadata_2025 huggingface dataset:
Near-normal circulatory survival of rabbit red cells exposed to high levels of Ca and ionophore in vitro
Authors: Bookchin RM , Roth EF Jr , Lew VL
Published: October 2019

The belief is widely held, on the basis of indirect evidence, that a substantial, even brief elevation of red cell Ca content must result in a marked shortening of circulatory survival. To test this notion directly, we exposed rabbit red cells in vitro to the ionophore A23187 and Ca so as to produce sustained uniform cell Ca levels of 40 to 360 mumol/L cells for one to 60 minutes, and compared the survival of the Ca-loaded cells in vivo with that of ionophore-treated controls, simultaneously, in the same rabbits. Despite marked reductions in cell adenosine triphosphate and dehydration of the Ca-exposed cells prior to reinfusion, the majority of cells, all of which had experienced these high cytoplasmic Ca levels, showed normal or near-normal survival in the circulation.
Fibronectin binding to thrombin-stimulated platelets: evidence for fibrin(ogen) independent and dependent pathways
Authors: Plow EF , Marguerie GA , Ginsberg MH
Published: October 2019

Plasma fibronectin binds in a specific and saturable manner to thrombin- stimulated platelets. gamma-Thrombin stimulated 80% as much fibronectin binding to platelets as alpha-thrombin with conversion of less than or equal to 1% of platelet fibrinogen to fibrin. Afibrinogenemic and normal platelets bound similar quantities of fibronectin in the presence of calcium or magnesium-ethylene glycol tetra-acetic acid (EGTA). These observations indicate that fibronectin can interact with platelets without involvement of fibrin or fibrinogen. Nevertheless, two different effects of fibrin(ogen) on fibronectin binding were observed. First, exogenous fibrinogen inhibited fibronectin binding to thrombin-stimulated platelets. This inhibition was unidirectional, as fibronectin did not inhibit fibrinogen binding to ADP or thrombin- stimulated cells. Second, formaldehyde-fixed cells with surface- associated fibrin bound significant quantities of fibronectin. This interaction required calcium and did not occur on fixed cells with or without surface-bound fibrinogen. A portion of the ligand bound to fixed cells with surface-associated fibrin was modified to form a derivative with a molecular weight identical to that of the fibronectin subunit cross-linked to the alpha-chain of fibrin. This high mol wt derivative was also observed to a variable extent with living cells in the presence of magnesium or calcium but not in the presence of magnesium-EGTA. Thus, fibronectin binds to platelets by at least two mechanisms: (1) a fibrin(ogen)-independent pathway that requires divalent ions and is inhibited by exogenous fibrinogen; and (2) a fibrin-dependent pathway with an absolute calcium requirement. With nonaggregated, thrombin-stimulated platelets, the former pathway appears to predominate.
Modulation of thrombin-mediated activation of factor VIII:C by calcium ions, phospholipid, and platelets
Authors: Hultin MB
Published: October 2019

The activation of factor VIII:C by thrombin appears to be an important prerequisite for the function of factor VIII:C as a cofactor in factor X activation in coagulation. The possible modulation of factor VIII:C activation by potential cofactors such as calcium ions, phospholipid, and platelets was studied systematically. Factor VIII:C activation could not be studied in the complete absence of Ca2+, since factor VIII:C activity decayed rapidly in calcium-free buffers, EDTA, or ethylene glycol tetra-acetic acid (EGTA), with only partial or no recovery of activity after readdition of Ca2+, Mn2+, or Mg2+. Added calcium chloride at 1.25, 2.5, 4, 10, 50, and 200 mmol/L produced progressive inhibition of factor VIII:C activation, with complete inhibition achieved by 50 mmol/L. Crude phospholipid preparations gave varying results, while purified phospholipids either had no effect or inhibited activation. This paper reports the new finding that fresh washed human platelets markedly potentiated factor VIII:C activation by a low concentration of thrombin (0.02 U/mL), even with prostaglandin E1 (PGE1) or dibutyryl cyclic AMP (cAMP) added to the washed platelets. However, the activity of platelets in factor VIII:C activation was inhibited by inclusion of PGE1 or dibutyryl cAMP during platelet washing, and ionophore A23187 increased this platelet activity; these data suggest that platelet stimulation is involved in the development of this activity. When platelets were maximally stimulated by thrombin (0.5 U/mL), the external calcium concentration increased 55 to 160 mumol/L, as measured with murexide, supporting the possible modulation of factor VIII:C activation by a transient increase in Ca2+ at the platelet surface.
The transport of 125I-labeled human high molecular weight urokinase across the intestinal tract in a dog model with stimulation of synthesis and/or release of plasminogen activators
Authors: Sasaki K , Moriyama S , Tanaka Y , Sumi H , Toki N , Robbins KC
Published: October 2019

In an attempt to elucidate the mechanism of fibrinolytic enhancement by orally administered urokinase, studies on the intestinal transport of urokinase were carried out, using 125I-labeled human high mol wt urokinase, administered intraduodenally in the experimental dog model with a saphenous vein thrombus. Using the plasma sample obtained from blood 45 minutes after intraduodenal administration of the urokinase, protein fractions were isolated by a sequential two-step affinity chromatography method, first with [N alpha-(epsilon-aminocaproyl)-DL- homoarginine hexylester]-Sepharose followed by a specific anti-human low mol wt urokinase rabbit IgG-Sepharose (adsorbed-eluted and unadsorbed). Each of the isolated protein fractions was further purified by gel filtration on Sephacryl S-300. The proteins isolated by the two-step affinity chromatography method were transported human urokinase with radioactivity in the adsorbed-eluted fraction, and newly synthesized and/or released dog plasminogen activators, probably urokinase-type and tissue-activator type, without radioactivity. In an antibody quenching assay, dog urokinase and the immuno-affinity unadsorbed fraction were not neutralized, but the immuno-affinity adsorbed-eluted fraction was completely neutralized by the specific anti-urokinase IgG antibody. Proteins isolated from control plasma (after administration of saline) by the two-step affinity chromatography method in the unadsorbed fraction had negligible amounts of activator activity. In these studies, we were able to show that synthesis of plasminogen activators was stimulated, with the activators being released, from either the liver or the vascular endothelium. Also we showed that urokinase is transported across the intestinal tract in the dog model.
Liquid preservation of human neutrophils stored in synthetic media at 22 degrees C: controlled observations on storage variables
Authors: Glasser L , Fiederlein RL , Huestis DW
Published: October 2019

The purpose of this study was to use a chemically defined medium to identify essential substances and optimal conditions for the liquid storage of neutrophils at 22 degrees C. Several commercially available synthetic media were evaluated: L-15, McCoy's 5a, M199, minimum essential medium, Dulbecco's MEM, NCTC135, and RPMI 1640. Proteins, glucose, pH, and neutrophil concentration were systematically studied. Neutrophils were harvested by centrifugal cell separators or phlebotomy, and their maintenance was evaluated by monitoring cell counts, dye exclusion, phagocytosis, bacterial killing, and chemotaxis. Neutrophils stored equally well in all synthetic media except L-15; however, chemotaxis was poorly maintained in synthetic media as compared with autologous plasma. RPMI 1640 was arbitrarily selected as a basal medium to evaluate storage variables. RPMI 1640 supplemented with albumin to a concentration of 1% improved chemotaxis and was equivalent to plasma as a storage medium with regard to the in vitro functions tested. Cohn fractions IV-1, IV-4, and gamma globulin were not effective substitutes for albumin. Glucose is essential for neutrophil storage; its absence from the medium correlated with poor cell function. Optimal glucose requirements depend on the cell concentration. High glucose concentrations were toxic to neutrophils; at 1,000 mg/dL, chemotaxis was depressed by 58%. Glucose utilization was dependent on the initial pH of the medium and on the cell concentration. A wide range of hydrogen ion concentrations was tolerated, and the optimum pH range was 7.2 to 7.8. Cell concentration is an important variable because it affects the pH of the medium as well as glucose utilization.
Type IIB Tampa: a variant of von Willebrand disease with chronic thrombocytopenia, circulating platelet aggregates, and spontaneous platelet aggregation
Authors: Saba HI , Saba SR , Dent J , Ruggeri ZM , Zimmerman TS
Published: October 2019

Type IIB von Willebrand disease is characterized by enhanced ristocetin- induced platelet aggregation and absence of large von Willebrand factor multimers from plasma. An alteration of the von Willebrand factor molecule resulting in increased reactivity with platelets appears to be the basis for these abnormalities. We have now identified a new variant of type IIB von Willebrand disease in a family in which the four affected members also have chronic thrombocytopenia, in vivo platelet aggregate formation, and spontaneous platelet aggregation in vitro. In spite of repeatedly prolonged bleeding times and persistent thrombocytopenia, their bleeding diathesis is only moderate.
The effect of hydroxyurea on hemoglobin F in patients with myeloproliferative syndromes
Authors: Alter BP , Gilbert HS
Published: October 2019

Fetal hemoglobin (Hb F) may increase in patients receiving chemotherapeutic drugs, a result of potential use in patients with symptomatic hemoglobinopathies. We examined Hb F in 13 patients with myeloproliferative disease (six polycythemia vera, five polycythemia vera with myeloid metaplasia, one agnogenic myeloid metaplasia, and one chronic myelogenous leukemia) who were treated with hydroxyurea. Four patients showed an increase in Hb F from less than 1% to between 5% and greater than 8% while on hydroxyurea, and a decline to less than 1% when the drug was discontinued. This group of “responders” received a higher average daily dose of hydroxyurea, which was administered continuously rather than intermittently, when compared to the “nonresponders.” Mean corpuscular volumes (MCVs) rose in most patients, and i antigen remained elevated or decreased; neither parameter correlated with Hb F levels. Both responders and nonresponders had therapeutically desirable suppression of WBCs and platelets, and almost all had no depression of reticulocytes or Hb.
Treatment of baboon with vinblastine: insights into the mechanisms of pharmacologic stimulation of Hb F in the adult
Authors: Veith R , Papayannopoulou T , Kurachi S , Stamatoyannopoulos G
Published: October 2019

Vinblastine was administered to anemic baboons to test whether stimulation of Hb F takes place following distortion of erythropoiesis by an M-stage-specific compound. The treatments elicited erythroid cell cytoreduction followed by regeneration. During the phase of reticulocyte reduction, gamma/gamma + beta biosynthetic ratios increased without increment in F reticulocytes, suggesting that there was increased production of Hb F per F cell. The phase of reticulocyte regeneration was associated with sharp increments in relative (percentage) and absolute F reticulocytes. These data suggest that perturbations of erythropoiesis underlie the stimulation of Hb F synthesis by vinblastine. Accelerated or abnormal precursor maturation may account for the release of shift F reticulocytes with higher Hb F content, during the reduction phase. Accelerated total erythroid differentiation/maturation may account for the increment in F reticulocyte numbers during the phase of regeneneration.
Surface phenotypes of human hemopoietic progenitor cells defined by monoclonal antibodies
Authors: Ferrero D , Gabbianelli M , Peschle C , Lange B , Rovera G
Published: October 2019

A panel of ten monoclonal antibodies which react with antigens present on the surface of myeloid leukemic cells was used to investigate the distribution of these antigens on normal hemopoietic stem cells and progenitor cells at various stages of maturity. A population of immature cells, possibly stem cells, that are capable of regenerating CFU-GM in long-term marrow cultures reacts with four antibodies recognizing antigens abundantly expressed in leukemic cells, but does not react with antibodies against Ia-like molecules or against carbohydrate determinants specific for myeloid cells. Progenitor cells that form mixed colonies in semisolid medium (CFU-GEMM), early erythroid (BFU-E) and early myelomonocytic (type 1 CFU-GM) progenitors retain the antigens present on the hypothetical stem cell population and begin to express Ia-like antigens. As they differentiate, myeloid and erythroid progenitors undergo a series of quantitative and qualitative shifts in surface phenotype. They begin to express stage- related, lineage-specific antigens and cease expressing antigens common to early cells of different lineages. The identification of antigens present on very immature normal progenitor cells should be valuable in future studies aimed at the detailed characterization of this relatively little-known hemopoietic cell population.
SFL 23.6: a monoclonal antibody reactive with CFU-E, erythroblasts, and erythrocytes
Authors: Gupta AD , Samoszuk MK , Papayannopoulou T , Stamatoyannopoulos G
Published: October 2019

A cytotoxic (IgG2b) monoclonal antibody (McAb) for a novel erythroid differentiation antigen was generated by hyperimmunizing young mice with mononuclear cells obtained from livers of 20- to 22-week-old fetuses. This McAb, designated SFL 23.6, shows an extremely well- defined reactivity with the cells of the erythroid lineage at all stages of maturation as evident from the labeling of morphologically identifiable erythroid precursors and of erythrocytes present in peripheral blood, bone marrow, and fetal liver, and from its reactivity with culture-derived erythroblasts. The nonerythroid cells present in these and other tissue preparations were not labeled by SFL 23.6. The erythroid lineage specificity of McAb SFL 23.6 was confirmed by a cell- sorting experiment in which 97% of the cells in the fluorescent fraction sorted from SFL 23.6-treated bone marrow cells were erythroid precursors at various stages of maturation. Complement-mediated cytotoxicity and progenitor cell-sorting experiments showed that most (greater than 90%) of the late erythroid progenitors (CFU-E) and only a small proportion of the early erythroid progenitors (BFU-E) express the antigenic determinant identified by SFL 23.6. The myeloid progenitors (CFU-GM) and multilineage progenitors (CFU-GEMM) were negative for the SFL 23.6 antigenic determinant. The antigen recognized by SFL 23.6 has not been determined as yet. Because of the pattern of its reactivity and its dependence on sialic acid residues, the possibility of its relationship to glycophoria A was entertained. However, previous work using antiglycophorin McAbs (R-10) has shown that this determinant is not expressed in CFU-E. Therefore, among the erythroid lineage-specific McAbs described thus far, SFL 23.6 is unique in its reactivity with CFU- E and the mature erythron. Reagents with such specificity may be useful in studies of erythroid differentiation and commitment.
Comparison of inhibitory and binding characteristics of an antibody causing acquired von Willebrand syndrome: an assay for von Willebrand factor binding by antibody
Authors: Fricke WA , Brinkhous KM , Garris JB , Roberts HR
Published: October 2019

An acquired inhibitor of von Willebrand factor (vWF) activity occurring in a patient with benign gammopathy and von Willebrand syndrome (vWS) has been partially characterized. The inhibitor-induced syndrome resulted in low to undetectable plasma levels of vWF/ristocetin, vWF/botrocetin, FVIIIR:Ag, and FVIII:C with a normal to slightly prolonged bleeding time. Platelet vWF was normal. Intensive and continuous infusion of a heat-treated factor VIII concentrate (Hemofil- T, Hyland, Glendale, Calif) elevated the FVIII:C plasma levels to about 100%, with an increase in FVIIIR:Ag levels to about 340% and vWF/ristocetin levels to about 40%, much lower than expected based on the dose of Hemofil-T and its content of vWF and FVIII:C activities. The inhibitor bound to staphylococcal protein A (SpA) with high affinity, indicating an IgG antibody (Ab). An assay for the vWF-binding capacity was developed on the basis of absorption of the Ab from serially diluted plasma by SpA and removal of vWF and FVIII:C activities from normal plasma by the SpA-Ab complex. The Ab-binding site was on the vWF component of the factor VIII complex. The Ab was unable to bind isolated FVIII:C. The combined use of the new vWF- binding assay and a battery of tests for inhibition of vWF-dependent platelet aggregation with ristocetin (which detects high molecular weight vWF), with botrocetin (which detects high and low molecular weight vWF), and with platelet-aggregating factor (which detects high molecular weight vWF) provided a means of analysis of Ab effect on in vitro vWF function. Using these tests, a comparison was made of the effects of the vWS Ab with those of an Ab inhibitor occurring in homozygous von Willebrand's disease. The Ab of the vWS patient had weak inhibitory action on vWF/ristocetin without having an effect on vWF/botrocetin and platelet-aggregating factor, a high titer vWF- binding capacity, and no anamnestic response following concentrate therapy. These findings contrasted with those of the Ab occurring in inhibitor von Willebrand's disease in which vWF inhibitor and binding values were similar, with a strong anamnestic response. The findings indicate that the vWS Ab binds to an epitope on the molecular vWF in such a way that causes only limited inhibition of vWF/ristocetin function and no inhibition of vWF/botrocetin function, suggesting that these two functional domains are at separate sites.
Effects of in vitro depletion of T cells in HLA-identical allogeneic marrow grafts
Authors: Martin PJ , Hansen JA , Buckner CD , Sanders JE , Deeg HJ , Stewart P , Appelbaum FR , Clift R , Fefer A , Witherspoon RP
Published: October 2019

We report results of a pilot study designed to evaluate the effects of in vitro depletion of T lymphocytes from donor marrow in patients receiving HLA-identical marrow grafts for treatment of hematologic malignancies. Twenty patients aged 31 to 50 years were prepared for transplantation with cyclophosphamide (120 mg/kg) and fractionated total body irradiation (12.0 or 15.75 Gy). All received cyclosporine after grafting. The donor marrows were treated with a mixture of eight murine monoclonal antibodies and rabbit serum complement in a manner that achieved a 2- to 3-log depletion of T cells in most patients. Initial engraftment occurred promptly in 19 of the patients, and only three had clinically significant acute graft-versus-host disease. Depletion of donor T cells, however, was associated with an increased incidence of graft failure, which occurred as late as 244 days after transplantation. Graft failure was transient in one patient but apparently was irreversible in seven others. Three of the seven patients had cytogenetic but not morphological evidence of leukemic relapse at the time of graft failure. All seven patients with irreversible graft failure have died, six after receiving second bone marrow transplants. Seven of the eight cases of graft failure occurred among the 11 patients prepared for transplantation with 12.0 Gy of total-body irradiation, and only one occurred among the nine patients with advanced malignancies who received 15.75 Gy of total-body irradiation. This association with irradiation dose suggests that host factors were partly responsible for the graft failures. Because graft failure seldom occurs in irradiated recipients of unmodified HLA- identical allogeneic marrow transplants, it appears that T cells in the donor marrow may serve a beneficial function in helping to maintain sustained engraftment possibly by eliminating host cells that can cause graft failure. Optimal application of in vitro manipulation of donor marrow as a method for preventing graft-versus-host disease will require more effective immunosuppression of the recipient in order to assure sustained engraftment and function of donor stem cells.
Eccentric localization of von Willebrand factor in an internal structure of platelet alpha-granule resembling that of Weibel-Palade bodies
Authors: Cramer EM , Meyer D , le Menn R , Breton-Gorius J
Published: October 2019

Immunogold staining was used to study the ultrastructural distribution of von Willebrand factor (vWF) in unstimulated platelets. vWF was detected in the alpha-granules with a specific eccentric distribution pattern opposite the nucleoids. Similar findings were obtained with a polyclonal antibody or a pool of monoclonal antibodies to human vWF. This labeling coincided with the presence of tubular structures located at the periphery of the alpha-granules. These structures were better visualized on platelets treated for standard electron microscopy: they formed a group of one to four tubules ranging from 200 A to 250 A in diameter. They closely resembled the internal tubular structures found in Weibel-Palade bodies, which are the storage organelles of vWF in endothelial cells.
Glycoprotein IIb-IIIa complex and Ca2+ influx into stimulated platelets
Authors: Powling MJ , Hardisty RM
Published: October 2019

Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.Changes in intracellular Ca2+ concentrations [( Ca2+]i) in platelets stimulated with aggregating agents were measured with the fluorescent indicator dye quin 2. Ca2+ influx, but not intracellular mobilization, in response to adenosine diphosphate (ADP), platelet aggregating factor (PAF-acether), and sodium arachidonate was significantly inhibited by monoclonal antibodies against the glycoprotein (GP) IIb-IIIa complex; inhibition of thrombin-stimulated influx was inhibited to a lesser extent and reached statistical significance only at thrombin concentrations of 0.1 U/mL and below. Anti-GP Ib and HLA-ABC monoclonal antibodies had no effect on Ca2+ influx in response to any agonist. Thrombasthenic platelets gave normal [Ca2+]i responses to ADP and thrombin, which were not inhibited by an anti-GP IIb-IIIa antibody. It is suggested that Ca2+ influx in response to weak agonists occurs predominantly via a channel closely adjacent to the GP IIb-IIIa complex, but that higher concentrations of thrombin and A23187 also stimulate influx via another pathway.
Diadenosine 5',5'''-p1,p4-tetraphosphate deficiency in blood platelets of the Chediak-Higashi syndrome
Authors: Kim BK , Chao FC , Leavitt R , Fauci AS , Meyers KM , Zamecnik PC
Published: October 2019

Diadenosine tetraphosphate (AP4A) is an unusual nucleotide found in a variety of cells, including platelets. It has been suggested that platelet AP4A is stored in the dense granules and is metabolically inactive. We have studied the AP4A content of blood platelets in two patients and three cattle with Chediak-Higashi syndrome (CHS), a hereditary platelet defect with dense granule deficiency. Acid-soluble extractions of whole blood and platelets were neutralized. The adenosine triphosphate (ATP) level was measured by luminescence technique. To measure the AP4A content, the neutralized extract was treated with phosphomonoesterase for removal of ATP. The AP4A content was then measured by coupling the phosphodiesterase and luciferase reaction. The AP4A content was 0.43 nmol/mg protein for normal human platelets and 0.004 nmol/mg protein for CHS platelets. The ATP/AP4A ratio was 67 for normal and 3,023 for CHS platelets. The whole blood AP4A was reduced by 89% in CHS patients who had only a slight decrease in ATP level (26% reduction). Similarly, bovine platelets with CHS showed a marked decrease of AP4A content and a moderate reduction of the ATP level. The platelet ATP/AP4A ratio was 351 and 3,133 for normal and CHS cattle, respectively. Results demonstrate a marked reduction of AP4A in CHS platelets and suggest that AP4A may be a useful marker for the measurement of dense granule content in platelets.
Natural antibodies in sera from Japanese individuals infected with HTLV- I do not recognize HTLV-III
Authors: Hattori T , Robert-Guroff M , Chosa T , Matsuoka M , Yamaguchi K , Ishii T , Gallo RC , Takatsuki K
Published: October 2019

Seventy-one sera from Japanese individuals infected with human T cell leukemia virus type I (HTLV-I) were examined for the presence of antibodies to HTLV-III by an enzyme-linked immunosorbent assay (ELISA) and by a strip radioimmunoassay based on the Western blot technique. The sera were from 23 healthy carriers and from 48 patients, including 18 with smoldering adult T cell leukemia (ATL), 13 with chronic ATL, and 17 with acute ATL. All people tested lived in the southwestern part of Japan, a known endemic area for HTLV-I infection. Antibodies against HTLV-I were detected in all sera both by indirect immunofluorescent methods and strip radioimmunoassay using cell lysates. Six sera were reactive in the ELISA assay for HTLV-III. But these sera did not react specifically to HTLV-III-related proteins (p15, p24, gp41) when analyzed by strip radioimmunoassay. Our data suggest that coincidental infection of HTLV-I and HTLV-III is quite rare in Japan.
Application of molecular genetics to prenatal diagnosis and carrier detection in the hemophilias: some limitations
Authors: Graham JB , Green PP , McGraw RA , Davis LM
Published: October 2019

Prenatal diagnosis and carrier detection in the hemophilias have received much attention in recent years. The error rate in prenatal diagnosis by fetoscopy is less than 1%; fetoscopy is not possible, however, until the second trimester of pregnancy. Carrier detection based on bioassays of plasma has an irreducible error rate (approximately 5%?), because of the “lyonization” phenomenon in heterozygous women, and the final results are always probabilistic. New DNA methods promise to alleviate these difficulties. Prenatal diagnosis can be accomplished in the first trimester. “Lyonization” is bypassed in carrier detection, and the results may sometimes be essentially nonprobabilistic. But the DNA methods have certain limitations of their own which are not widely appreciated. Aside from cost and the necessity to adopt a new technology, there are inherent genetic problems: mothers must be heterozygous for both a disease gene and a marker gene, final results are probabilistic if the marker gene lies outside the disease gene, and multiple marker genes are often in linkage disequilibrium. We have concluded that a clinical unit planning to use the DNA methods must also maintain the conventional methods at a high level of performance.
Heparin cofactor II activity in patients with disseminated intravascular coagulation and hepatic failure
Authors: Tollefsen DM , Pestka CA
Published: October 2019

Heparin cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of heparin or dermatan sulfate. We have developed a functional assay for HCII in which inhibition of thrombin by plasma is determined in the presence of dermatan sulfate. The assay is specific for HCII by the following criteria: (a) under the conditions of the assay, 125I-thrombin forms complexes in plasma which comigrate with the thrombin-HCII complex during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE); (b) activity detected by the assay is decreased in plasma absorbed with monospecific antibodies against HCII; and (c) purified antithrombin III (ATIII) is unreactive in the assay system. Addition of Polybrene to the assay permits determination of HCII activity in samples containing less than or equal to 12 U/mL of heparin. The range of HCII concentrations in normal individuals is 1.2 +/- 0.4 mumol/L (mean +/- 2 SD, n = 34). HCII activity was determined in 54 consecutive patients undergoing evaluation for the possibility of disseminated intravascular coagulation (DIC). Ten of the 11 patients with documented DIC had decreased HCII activity as compared with only 7 of the 43 patients without DIC (chi 2 = 19.3, P less than .0001). The concentrations of HCII and ATIII varied in parallel in most of the patients tested. A significant correlation between decreased HCII activity and decreased serum albumin concentration was also observed in these patients and in eight additional patients with hepatic failure in the absence of DIC. We conclude that HCII activity is decreased in many patients with DIC and hepatic failure.
Serum lactic dehydrogenase level has prognostic value in childhood acute lymphoblastic leukemia
Authors: Pui CH , Dodge RK , Dahl GV , Rivera G , Look AT , Kalwinsky D , Bowman WP , Ochs J , Abromowitch M , Mirro J
Published: October 2019

Serum lactic dehydrogenase (LDH) levels were measured at diagnosis in 293 children with “standard-risk” acute lymphoblastic leukemia (ALL) to determine the prognostic value of this biologic feature. Standard risk assignment was based on an initial leukocyte count of less than 100 X 10(9)/L, the absence of a mediastinal mass, the absence of meningeal involvement, and the presence of lymphoblasts lacking sheep erythrocyte receptors or surface immunoglobulin. Serum LDH levels ranged from 97 to 6,595 U/L, with a mean of 547 U/L. Higher LDH levels were associated with higher leukocyte counts, lower blast cell DNA indices, lower platelet counts, a larger spleen size, and nonwhite race. LDH levels were not related to the percentage of marrow S-phase cells, liver size, French-American-British (FAB) classification, hemoglobin levels, age, sex, or the presence of the common ALL antigen on marrow blasts. Patients with the highest LDH levels (greater than 1,000 U/L) were most likely to fail treatment, whereas those with the lowest levels (less than 300 U/L) had the lowest risk of failure (P less than .0001). The prognostic significance of serum LDH level was retained in a subset of patients that included only those with leukocyte counts less than 25 X 10(9)/L (P = .0018). When 11 presenting characteristics were subjected to multivariate analysis, serum LDH level was found to have independent prognostic strength, contributing clinically important information to that gained from leukocyte count. Early measurement of serum LDH could be useful in identifying a group of standard-risk ALL patients with a high relapse hazard.
Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro
Authors: Wilson BS , Platt JL , Kay NE
Published: October 2019

Several mouse monoclonal IgG antibodies (AB1, AB2, AB3, and AB5) were developed that reacted with a 140,000 mol wt glycoprotein on the surface of cultured RAJI B lymphoid cells. The antibodies reacted with purified normal human peripheral blood B cells and CLL Ig+ B cells and showed specific germinal center and mantle zone staining in tissue sections of secondary lymphoid organs. Immunodepletion studies using 125I surface-labeled Raji cell membrane antigens demonstrated that the antigen identified by AB5 is the same 140,000 mol wt glycoprotein detected by anti-B2 that has recently been shown to react with the C3d fragment or CR2 receptor. (Iida et al: J Exp Med 158:1021, 1983). Addition of the AB series and anti-B2 monoclonal antibodies to cultures of purified human peripheral blood B cells resulted in the uptake of 3H- thymidine at two to six times background control levels provided that irradiated autologous T cells were added to the culture. Stimulation was not evoked by other monoclonal antibodies to B cell surface molecules (ie, B1, BA-1, BA-2, and HLA-DR). Pepsin-generated F(ab')2 fragments of anti-CR2 antibodies were essentially as effective as the intact IgG molecule in stimulating B cells. Induction of B cell proliferation by antibody binding to CR2 suggests that the C3d receptor may have an integral role in regulation of humoral immune response.
Stimulation of bone marrow-derived and peritoneal macrophages by a T lymphocyte-derived hemopoietic growth factor, persisting cell- stimulating factor
Authors: Crapper RM , Vairo G , Hamilton JA , Clark-Lewis I , Schrader JW
Published: October 2019

Several lines of evidence indicated that P cell-stimulating factor (PSF), a T lymphocyte-derived lymphokine known to stimulate the growth of hemopoietic stem and progenitor cells, also acted on macrophages. PSF was absorbed from medium that had been mixed for two hours at 0 degrees C with either resident or thioglycollate-elicited peritoneal cells, suggesting the presence of receptors for PSF on cells in the population. The addition of pure PSF to populations highly enriched in either resident or elicited adherent peritoneal macrophages resulted in stimulation of macrophages with morphological changes, including increases in size, spreading, vacuolation, and the number of cytoplasmic processes, together with stimulation of proliferation and the phagocytosis of opsonized yeast. PSF also stimulated the incorporation of [3H]thymidine by bone marrow-derived adherent macrophages. Addition of pure PSF to cultures that contained only a single macrophage resulted in enhanced survival and proliferation of these isolated cells, demonstrating that the effect of PSF on macrophages was direct. These results indicate that PSF can stimulate well-differentiated functional macrophages and raise the possibility that the effects of PSF on macrophages may play a regulatory role in immune responses.
Altered glycosaminoglycan production by HL-60 cells treated with 4- methylumbelliferyl-beta-D-xyloside
Authors: Luikart SD , Sackrison JL , Thomas CV
Published: October 2019

Glycosaminoglycans, mainly chondroitin 4-sulfate, are located in the primary granules of human myeloid cells. These polyanionic carbohydrates are believed to play an important role in leukocyte maturation and function. To study the effect of altered chondroitin sulfate metabolism on human promyelocytic leukemia cells, we have treated HL-60 cells with 4-methylumbelliferyl-beta-D-xyloside. beta-D- Xylosides initiate the synthesis of free chondroitin sulfate chains. Cytochemical studies of treated cells demonstrated a marked increase in cytoplasmic granules stained with cationic dyes. This was confirmed by radiolabeled precursor incorporation studies that demonstrated a 344% increase in 35S-sulfate uptake into glycosaminoglycans associated with the cells and a 39% increase in incorporation into glycosaminoglycans released into the media. Chromatographic analyses of these glycosaminoglycans from treated cells demonstrated that the newly formed chondroitin sulfate chains were not attached to protein core and were of shorter length, but of greater charge density than chondroitin sulfate produced by control cells. Thus, beta-D-xyloside appears to alter the protein linkage, chain length, and sulfation of chondroitin sulfate produced by HL-60 cells, and these changes are morphologically evident. These biochemically altered cells may provide important information concerning the role of these macromolecules in myeloid development.
Transferrin saturation, plasma iron turnover, and transferrin uptake in normal humans
Authors: Cazzola M , Huebers HA , Sayers MH , MacPhail AP , Eng M , Finch CA
Published: October 2019

The relationship between plasma iron, transferrin saturation, and plasma iron turnover was studied in 53 normal subjects whose transferrin saturation varied between 17% and 57%, in 25 normal subjects whose transferrin saturation was increased by iron infusion to between 67% and 100%, and in five subjects with early untreated idiopathic hemochromatosis whose transferrin saturation was continually elevated to between 61% and 86%. The plasma iron turnover of all of these subjects ranged from 0.45 to 1.22 mg/dL whole blood/d. The mean values for the above-mentioned three groups were 0.71 +/- 0.17, 1.01 +/- 0.11, and 1.01 +/- 0.13 mg/dL whole blood/d, respectively. Most of this variation, estimated at 72% by regression analysis, was due to a direct relationship between transferrin saturation and plasma iron turnover. This effect was attributed to a competitive advantage of diferric over monoferric transferrin in delivering iron to tissues. This was confirmed by the demonstration of a more rapid clearance of diferric as compared to monoferric transferrin in an additional group of eight normal subjects. Calculations were made of the amount of transferrin reacting with membrane receptors per unit time. Allowance was made for the noncellular (extravascular) exchange and for the 4.2:1 preference of diferric over monoferric transferrin demonstrated in vitro. The amount of iron-bearing transferrin leaving the plasma to bind to tissue receptors for 53 subjects with a transferrin saturation between 17% and 57% was 71 +/- 13; for 25 subjects with a saturation from 67% to 100%, 72 +/- 12; and for five subjects with early idiopathic hemochromatosis, 82 +/- 11 mumol/L whole blood/d. There were no significant differences among these groups. These studies indicate that while the number of iron atoms delivered to the tissues increases with increasing plasma iron and transferrin saturation, the number of iron-bearing transferrin molecules that leave the plasma per unit time to bind to tissue receptors is relatively constant and within the limits studied, independent of transferrin saturation.
Homozygous transcobalamin II deficiency maintained on oral hydroxocobalamin
Authors: Zeitlin HC , Sheppard K , Baum JD , Bolton FG , Hall CA
Published: October 2019

A case of transcobalamin II (TCII) deficiency in which a total absence of TCII was demonstrated both functionally and immunologically is reported. Unlike previously described patients, this child has been maintained on oral hydroxocobalamin, 2 mg daily, without any parenteral supplementation for the last five years. At the age of six years her development is normal and her health is good. Plasma cobalamin levels are in the range of 3,000 ng/L and most of this appears to be bound to a molecule, which on gel filtration, elutes with albumin. In an extended family study, a clear separation of heterozygotes from both the propositus and from normal subjects suggests that the underlying defect in this condition is confined to a single gene.
The immunohistology of follicle lysis in lymph node biopsies from homosexual men
Authors: Wood GS , Garcia CF , Dorfman RF , Warnke RA
Published: October 2019

Follicle lysis is a characteristic alteration of B cell follicles described recently in lymph node biopsies from homosexual men. It consists of disruption of germinal centers by aggregates of small mature lymphocytes variably associated with erythrocyte extravasation. We studied the immunohistology of follicle lysis identified in lymph node biopsies from 11 homosexual men. The results indicate that follicle lysis has two principal immunohistologic features: (1) intrafollicular aggregates of small lymphocytes predominantly of polytypic mantle B cell phenotype (T015+/Leu-8+/mu+/delta+/k+ or lambda+), and (2) disruption of the normal, unified follicular meshwork of R4/23+ dendritic reticulum cells by these B cell aggregates. These structural alterations may affect the functional integrity of the germinal center as it pertains to the abnormal B cell effector function and the increased prevalence of B cell lymphoma recently documented in the acquired immunodeficiency syndrome and related disorders. Because dendritic reticulum cells weakly express the Leu-3 (T4) antigen, which is known to be an essential component of the receptor for human T- lymphotropic virus type III/lymphadenopathy-associated virus (HTLV- III/LAV) retrovirus infection, it is possible that retroviral infection of dendritic reticulum cells may play a role in the pathogenesis of follicle lysis.
Quantitation of adenosine-5'-triphosphate used for phosphoinositide metabolism in human erythrocytes
Authors: Dale GL
Published: October 2019

The human erythrocyte actively phosphorylates and dephosphorylates phosphatidylinositol present in the membrane in an apparent “futile cycle.” Recent reports have proposed that this phosphorylation/dephosphorylation cycle is a significant consumer of adenosine-5′-triphosphate (ATP) in the erythrocyte. This study details two independent techniques for quantitating the ATP consumed by this phosphoinositide futile cycle. With the first technique a quasi-steady- state labeling of erythrocyte ATP with 32P-phosphate was obtained, and the rate of synthesis of 32P-phosphoinositides was then monitored. The second technique used a novel labeling strategy that allowed only ATP to be labeled with 32P; the transfer of 32P from ATP to phosphoinositides was then an independent measure of the ATP consumed for phosphoinositide synthesis. These two techniques documented that 0.5% to 1.0% of net ATP produced by the erythrocyte is used for phosphoinositide synthesis.
Graded responses of human neutrophils induced by serum-treated zymosan
Authors: Whitin JC , Ryan DH , Cohen HJ
Published: October 2019

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3′- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.
The kinetics of inhibition of thrombin by antithrombin in the presence of components of the hemostatic system
Authors: Jesty J
Published: October 2019

The inhibition of human thrombin by antithrombin has been measured in pure systems in the presence of other components of the hemostatic system that might affect the kinetics of the reaction. These included fibrinogen, calcium ions, phospholipid, prothrombin, platelets (both adenosine diphosphate [ADP]-stimulated and -unstimulated), and platelet extracts. Inhibition rates were measured in each case by a discontinuous amidolytic assay over a range of antithrombin concentrations, from 0 to 4.5 mumol/L. Under all conditions, rates of inhibition were proportional to antithrombin concentration. Calcium ions at 5 mmol/L caused a small (20%) reduction in rate, but phospholipid and prothrombin had no additional effect. In contrast, both fibrinogen and platelets significantly changed the rate of inhibition. In the presence of calcium, fibrinogen at concentrations from 0 to 12 mumol/L reduced the rate of inhibition in a competitive manner, giving an apparent Kd for fibrinogen of 6.0 mumol/L. As the plasma fibrinogen level is about 8 mumol/L, one may therefore predict that variations in fibrinogen level will have a significant effect on the rate of thrombin inhibition in plasma. More unexpected was the observation that platelets increase the rate of inhibition: unstimulated platelets increased the rate constant by 40%, and ADP- stimulated platelets increased it by 55%. However, this acceleratory effect could not be mimicked with either a KCI extract or a Triton extract of platelets, and its cause remains unknown. In sum, it has been shown that the rate of inhibition of thrombin can be modulated in at least three ways-antithrombin concentration, fibrinogen concentration, and platelets; each of which can vary independently in vivo. It is well known that defects of the first lead to an increased risk of thrombosis, and it is proposed that this may be substantially caused by changes in the kinetics of inhibition such as those described. Additionally, it is suggested that changes in inhibition rate caused by other components may also be significant, for the same reason, in modulating the clotting system in vivo.
Correction of symptoms of platelet storage pool deficiency in animal models for Chediak-Higashi syndrome and Hermansky-Pudlak syndrome
Authors: Novak EK , McGarry MP , Swank RT
Published: October 2019

Two human diseases of platelet storage pool deficiency (SPD), Hermansky- Pudlak syndrome and Chediak-Higashi syndrome, are recessively inherited disorders characterized by hypopigmentation, prolonged bleeding, and normal platelet counts accompanied by a reduction in dense granule number. We have recently described seven independent recessive mutations in the mouse regulated by separate genes which are likely animal models for human SPD. Reciprocal bone marrow transplants were carried out between normal C57BL/6J mice and two of these mutants, beige and pallid, in order to test whether the platelet defects are due to a defect in platelet progenitor cells or to humoral factors. Normal and congenic mutant mice were transplanted with marrow after 950 rad whole body radiation. The long bleeding times and low serotonin concentrations of the two mutants were converted to normal values after transplantation with normal marrow. Likewise, normal mice displayed symptoms of SPD when transplanted with mutant marrow. These studies demonstrate that with each of the two mutations, platelet SPD results from a defect in bone marrow precursor cells. Also, the studies suggest that in severe cases, platelet SPD may be successfully treated by bone marrow transplantation.