bluuebunny/crossref_metadata_2025 huggingface dataset:
Interactions of plasma kallikrein and C1-s with normal and dysfunctional C1(-)-inhibitor proteins from patients with hereditary angioneurotic edema: analytic gel studies
Authors: Donaldson VH , Wagner CJ , Tsuei B , Kindness G , Bing DH , Harrison RA , Rosen FS
Published: October 2019

Purified preparations of normal C1(-)-inhibitor (C1(-)-INH) formed high mol wt complexes with plasma kallikrein that were stable during sodium dodecyl sulfate (SDS)-gel electrophoresis, but most of the dysfunctional C1(-)-INH proteins isolated from plasma of patients with type II hereditary angioneurotic edema (HANE) did not. Two of eight dysfunctional C1(-)-INH proteins were cleaved to lower mol wt forms that were not seen following the reaction of normal C1(-)-INH with equimolar amounts, or less, of plasma kallikrein. Only the higher mol wt component of normal C1(-)-INH (106,000 mol wt) appeared to form a stable complex with the plasma kallikrein, whereas both the 106,000 and 96,000 mol wt forms made stable complexes with C1-s. When a preparation of normal C1(-)-INH containing a homogeneous single band of C1(-)-INH was exposed to C1-s or kallikrein, a “doublet” form evolved in which the heaviest band was in the original position of native C1(-)-INH; C1- s cleavage provided a second band of 96,000; and cleavage by kallikrein, a second band of 94,000 mol wt. We conclude that dysfunctional C1(-)-INH proteins from plasma of persons with type II hereditary angioneurotic edema have impaired interactions with plasma kallikrein and are heterogeneous with respect to these interactions. Moreover, the requirements for the formation of stable complexes between normal C1(-)-INH and plasma kallikrein differed from those for stable complex formation with C1-s. The doublet form of C1(-)-INH, which purified preparations frequently demonstrate, may be due to prior cleavage by C1-s or kallikrein.
Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures
Authors: Alberico TA , Ihle JN , Liang CM , McGrath HE , Quesenberry PJ
Published: October 2019

Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.
A component of factor VIII preparations which can be separated from factor VIII activity down modulates human monocyte functions
Authors: Eibl MM , Ahmad R , Wolf HM , Linnau Y , Gotz E , Mannhalter JW
Published: October 2019

In this study we investigated different aspects of monocyte functions following interaction of monocytes (Mo) with therapeutic concentrations of factor VIII (F VIII) concentrate. A short (one-hour) treatment of normal Mo with F VIII concentrates led to a significant (P less than 0.001) down modulation of Fc receptors expressed in the Mo plasma membrane. This down modulation was accompanied by a decrease of Mo effector functions that was expressed by a reduced capacity of F VIII- treated Mo to release O2 radicals (40% of controls) and to kill bacteria (% killing: control Mo, 65%; F VIII-treated Mo, 24% to 51%). Further studies showed that the modulating activity was due to a contaminant present in F VIII concentrates (immune complexes or IgG aggregates). Fractionation using molecular sieving revealed that the modulatory activity was confined to a high-molecular range fraction (Mr greater than 1,270,000 daltons), while the fraction containing monomeric IgG had no effect. Further fractionation by affinity chromatography on protein A-Sepharose separated the coagulation activity (effluent) from the Mo function-modulating activity (eluate). We conclude that treatment with F VIII concentrates might contribute to an immunocompromised state in some hemophiliacs and facilitate opportunistic infections in these patients.
Role of colony-stimulating factor in myelopoiesis in murine long-term bone marrow cultures
Authors: Lipschitz DA , Udupa KB , Taylor JM , Shadduck RK , Waheed A
Published: October 2019

Weekly medium change or midweek feeding of long-term bone marrow cultures (LTMBCs) results in a significant increase in total myeloid cell production. Proliferative myeloid cells peak 48 hours after feeding, and nonproliferative myeloid cells reach maximum levels at 72 hours. This increase in myelopoiesis is invariably preceded by a significant elevation in biologically and immunologically measurable colony-stimulating factor (CSF) in the supernatants of LTBMC. The level peaks 24 hours after medium change, then gradually returns to basal values. The decrease in CSF relates to its consumption by generating myeloid precursors because no fluctuation in the levels occur in cultures without active myelopoiesis. No significant inhibitors or promoters of CSF were detected. When highly purified L cell CSF, CSF in lung-conditioned medium, or CSF concentrated from LTBMC supernatant is added to cultures, an identical increase in myelopoiesis occurs. Anti- CSF antiserum, added to culture at the time of medium change, totally neutralizes supernatant CSF levels but does not affect myelopoiesis. These findings suggest a potential regulatory role for CSF in myelopoiesis in LTBMC. CSF appears to function within the microenvironment through a mechanism involving cell:cell interactions or by causing the production of other substances that stimulate myelopoiesis. Because exogenous CSF stimulates myelopoiesis, it is likely that it too can react either directly or through microenvironmental cells to stimulate primitive myeloid cells to divide.
In vitro tests that predict tumor-associated idiotype levels in the serum of patients with B cell lymphomas and leukemias
Authors: Miller RA , Lowder JN , Gralow J , Meeker T , Levy R
Published: October 2019

The presence of circulating tumor idiotype interferes with the in vivo effectiveness of anti-idiotype antibodies. We developed two assays that permit identification of patients with high levels of serum idiotype without the need for first producing an anti-idiotype antibody. A cell suspension made from the tumor was cultured for seven days with or without phytohemagglutin (PHA) and/or phorbol myristic acetate (PMA). Ig secretion in vitro by patients' tumor cells varied. In 4 patients, no secretion in vitro occurred, 5 patients had low levels, and 5 patients had high levels of Ig secretion. In three patients, Ig secretion occurred only after stimulation with PHA, PMA, or both. Spontaneous or induced immunoglobulin secretion in vitro is related to the levels of tumor idiotype secretion that exist in vivo. Eight patients with serum idiotype levels greater than 100 micrograms/mL (mean 265 micrograms/mL), had a minimum of 1.0 microgram/10(6) cells of idiotype secretion in vitro. Nine patients with serum idiotype levels less than 30 micrograms/mL (mean 3.7 micrograms/mL), had less than or equal to 0.5 microgram/10(6) cells of idiotype secretion in vitro. In another assay, the levels of IgM kappa and IgM lambda in patients' sera were compared with those in normal serum. An imbalance in the relative amounts of IgM kappa and IgM lambda indicated high levels of circulating idiotype in the serum, but this assay was less sensitive than the in vitro secretion assay and limited to IgM-secreting tumors. These assays will be useful for future clinical studies using anti- idiotype antibodies.
Expression of the macrophage-specific colony-stimulating factor in human monocytes treated with granulocyte-macrophage colony-stimulating factor
Authors: Horiguchi J , Warren MK , Kufe D
Published: October 2019

The macrophage-specific colony-stimulating factor (CSF-1, M-CSF) regulates the survival, growth and differentiation of monocytes. We have recently demonstrated that phorbol ester induces expression of CSF- 1 in human monocytes. These findings suggested that activated monocytes are capable of producing their own lineage-specific CSF. The present studies demonstrate that the granulocyte-macrophage colony-stimulating factor (GM-CSF) also induces CSF-1 transcripts in monocytes. Furthermore, we demonstrate that the detection of CSF-1 RNA in GM-CSF- treated monocytes is associated with synthesis of the CSF-1 gene product. The results thus suggest that GM-CSF may indirectly control specific monocyte functions through the regulation of CSF-1 production. These findings indicate another level of interaction between T cells and monocytes.
Bone marrow transplantation in multiple myeloma: report from the European Cooperative Group for Bone Marrow Transplantation
Authors: Gahrton G , Tura S , Flesch M , Gratwohl A , Gravett P , Lucarelli G , Michallet M , Reiffers J , Ringden O , van Lint MT
Published: October 2019

Of 14 patients who received an allogeneic bone marrow graft from HLA- compatible sibling donors, 10 have survived for 6 to 34 months posttransplantation (median, 12 months). Four patients have died, two of relapse at extramedullary sites, one of severe acute GVHD, and one from GI bleeding and pericardial effusion. One patient is alive in relapse and four patients have signs of minimal persistent disease. Five patients are well without signs of active disease. Minor improvement in osteolytic lesions on X-ray were seen in three patients, but the X-ray bone structure was mainly unchanged in most patients. Bone marrow transplantation appears promising for treatment of certain patients with multiple myeloma.
Measurement of platelet-associated IgG in animal models of immune and nonimmune thrombocytopenia
Authors: Arnott J , Horsewood P , Kelton JG
Published: October 2019

Platelet-associated IgG (PAIgG) is elevated in idiopathic thrombocytopenic purpura (ITP), but it also is elevated in other thrombocytopenic disorders traditionally considered to be nonimmune. Consequently it is possible that elevated PAIgG is a nonspecific finding secondary to thrombocytopenia. To study this issue we developed a rabbit model of immune and nonimmune mediated thrombocytopenia. The mechanism of the thrombocytopenia was validated by platelet survival studies. Immune thrombocytopenia was produced by injection of antirabbit platelet serum that was raised in guinea pigs. Nonimmune aregenerative thrombocytopenia was produced by irradiation of the animals; nonimmune consumptive thrombocytopenia was produced by injection of adenosine diphosphate (ADP). PAIgG was measured in a direct binding assay using 125I-labeled staphylococcal protein A (SpA). Washed platelets from normal, nonthrombocytopenic rabbits bound an average of 81 molecules of SpA per platelet (81 +/- 168, mean +/- 2 SD, n = 39). Infusion of the antiplatelet antiserum produced thrombocytopenia with a rise in PAIgG that was closely correlated with the level of PAIgG (r = 0.86, n = 12). The thrombocytopenia was consumptive, as shown by a very short platelet life span using 111In- labeled platelets. In contrast, both nonimmune thrombocytopenic states resulted in an equal or greater drop in the platelet count but no change in the level of PAIgG. The animals with aregenerative thrombocytopenia had normal or only moderately reduced platelet life spans; however, in every animal the level of PAIgG was not different from the nonthrombocytopenic controls, irrespective of the platelet count. Similarly, the level of PAIgG was unchanged in those rabbits with nonimmune consumptive thrombocytopenia following infusion of ADP (82 +/- 55 molecules of SpA per platelet, mean +/- SD, n = 6). These studies indicate that elevated PAIgG is a specific finding of immune thrombocytopenia and is not secondary to thrombocytopenia itself. Indirectly these results support our hypothesis that immune mechanisms contribute to more thrombocytopenic disorders than was once thought likely.
The pathogenesis of accelerated fibrinolysis in liver cirrhosis: a critical role for tissue plasminogen activator inhibitor
Authors: Hersch SL , Kunelis T , Francis RB Jr
Published: October 2019

The pathogenesis of accelerated fibrinolysis in liver cirrhosis was investigated by comparing the results of specific assays for tissue plasminogen activator (tpa) antigen, tpa activity, tpa inhibitor, and alpha-2 plasmin inhibitor (a2PI) in 12 patients with cirrhosis and markedly accelerated fibrinolysis (dilute whole blood clot lysis time (DWBCLT) less than two hours), in nine patients with cirrhosis and moderately accelerated fibrinolysis (DWBCLT two to four hours), and in nine patients with cirrhosis and normal fibrinolysis (DWBCLT greater than four hours). Mean tpa antigen was markedly increased in all three groups, but no correlation was observed between overall fibrinolytic activity as measured by the DWBCLT and the level of tpa antigen. In contrast, there was a significant correlation between overall fibrinolytic activity and tpa activity and an equally significant correlation between fibrinolytic activity and decreased tpa inhibition. Mean a2PI activity was significantly lower than normal in groups 1 and 2 but was normal in group 3. The pathogenesis of accelerated fibrinolysis in liver cirrhosis thus appears to depend critically on the capacity of plasma inhibitors to inhibit increased circulating tpa antigen. Reduced a2PI also appears to play a role.
Receptor-specific inhibition of bone marrow erythropoiesis by recombinant DNA-derived interleukin-2
Authors: Burdach SE , Levitt LJ
Published: October 2019

Interleukin-2 (IL-2) induces differential secretion of lymphokines by IL-2 receptor (IL-2R)-positive and IL-2R-negative T cells. We studied T cell IL-2R-specific modulation of adult bone marrow erythropoiesis by recombinant IL-2 (rIL-2). I3–2R were induced by CD3 T cell surface determinant-triggering and analyzed by cytofluorography. Bone marrow monocyte and T cell-depleted (NAB-T) target cells were assessed for early erythroid progenitor expression (BFU-E) in the presence of 0 to 10(3) U/mL of rIL-2, rIL-2 had no significant effect on BFU-E expression in the absence of T cells or in the presence of IL-2R- negative T cells. rIL-2 caused a dose-dependent inhibition (75% to 90%) of BFU-E in the presence of autologous IL-2R-positive T cells. The addition of anti-IL2-receptor antibody to cultures containing rIL-2 plus IL-2R-positive T cells entirely abrogated rIL-2-mediated inhibition of BFU-E. In the presence of rIL-2 (10(2) U/mL) production of interferon gamma (IF-gamma) by adult marrow CD3-triggered IL-2R- positive T cells was increased 37- to 125-fold compared to IL-2R- negative T cells. rIF-gamma caused a dose-dependent (88% +/- 17% at 10(3) U/mL) inhibition of adult BFU-E in the presence of CD3-triggered autologous T cells. rIL2-mediated inhibition of adult BFU-E in the presence of IL-2R-positive T cells was partially abrogated (52% +/- 16%) following addition of monospecific IF-gamma antibody. These results demonstrate (a) rIL-2 modulation of adult marrow erythropoiesis is selectively dependent upon both the presence or absence of autologous T cells and the IL-2R status of these T cells; and (b) rIL-2- induced inhibition of adult marrow erythropoiesis is mediated in part by release of IF-gamma from IL-2R-positive T cells.
Genetic expression of adenosine deaminase in human lymphoid malignancies
Authors: Gan TE , Dadonna PE , Mitchell BS
Published: October 2019

Adenosine deaminase (ADA) is an enzyme in the purine catabolic pathway that has been used as an enzymatic marker of T cell lymphoblastic malignancies due to its high specific activity in thymocytes and immature T cells. We have investigated whether the level of ADA activity in lymphoid leukemic cells correlates with the amount of ADA- specific RNA and/or immunoreactive protein in these cells as an initial step toward characterizing the nature of the genetic regulation of ADA expression during differentiation. We have found a good correlation between the steady state levels of ADA-specific RNA and ADA- immunoreactive protein in T lymphoblastic leukemic cell lines, mature T cell lines, a B lymphoblast cell line, and leukemic cells directly isolated from four patients with acute lymphoblastic leukemia and three patients with chronic lymphocytic leukemia. Southern blot analysis of DNA from these cells shows no evidence for differences in ADA gene copy number or gene rearrangement to account for the variability in ADA expression. We conclude that levels of ADA in lymphoid leukemic cells are directly related to the amount of ADA-specific mRNA present. These findings imply that ADA expression in leukemic cells reflects either the transcriptional activity of the ADA gene or the stability of ADA mRNA in these cells.
Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry
Authors: Johnston GI , Pickett EB , McEver RP , George JN
Published: October 2019

Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.
Monoclonal antibody 7D5 raised to cytochrome b558 of human neutrophils: immunocytochemical detection of the antigen in peripheral phagocytes of normal subjects, patients with chronic granulomatous disease, and their carrier mothers
Authors: Nakamura M , Murakami M , Koga T , Tanaka Y , Minakami S
Published: October 2019

We have established a monoclonal hybridoma clone that produces IgG1 against the cytochrome b558 of human neutrophils. The antibody 7D5, secreted by the hybridoma, bound to solubilized cytochrome b of the neutrophils but not to other proteins such as hemoglobin, myeloperoxidase, and pig cytochrome P-450. Immunocytochemical studies of normal human peripheral blood showed that 7D5 bound to neutrophils and monocytes but not to lymphocytes or erythrocytes. The neutrophils of male patients but not of a female patient with chronic granulomatous disease lacked the antigen of 7D5 as well as the absorption spectrum for cytochrome b558. A mosaic of the antigen-positive and -negative neutrophils was observed in mothers of the male patients. These biochemical and immunocytochemical results indicate that 7D5 is a specific antibody against cytochrome b558 of human phagocytes.
Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae
Authors: Edwards JE Jr , Rotrosen D , Fontaine JW , Haudenschild CC , Diamond RD
Published: October 2019

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida- endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51Cr release from radiolabeled monolayers. From these studies, we conclude that neutrophils are capable of killing Candida hyphae selectively within human vascular endothelial cell monolayers and may have protective rather than detrimental effects on endothelial cell integrity.
Iron deficiency and neutrophil function: different rates of correction of the depressions in oxidative burst and myeloperoxidase activity after iron treatment
Authors: Murakawa H , Bland CE , Willis WT , Dallman PR
Published: October 2019

The polymorphonuclear granulocyte (PMN) kills ingested bacteria by mechanisms that include myeloperoxidase (MPO) and a sudden increase in oxygen consumption (the oxidative burst), both of which are iron dependent. The magnitude of the oxidative burst and activity of MPO were determined in PMNs during the progression of iron deficiency (ID) and following its treatment in rats. As ID developed, the oxidative burst after zymosan activation was less depressed than the activity of MPO. There was no change in the oxidative burst after activation with phorbol myristate acetate (PMA) or in the generation of superoxide (O2- ) by NADPH oxidase-containing particles from PMNs. Following iron treatment, impairment of the oxidative burst after zymosan activation was corrected after 1 day. In contrast, the deficit in MPO activity was not corrected until 7 days after initiation of iron treatment. The pattern of recovery in MPO activity after iron treatment corresponded to the prolonged period of maturation of the PMN primary granule since the formation of primary granules, which contain MPO, takes place only in the early, mitotic stages of maturation. The tendency of the PMN to maintain the oxidative burst allows the cell to preserve its capacity for bacterial killing during the progression of iron deficiency.
Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells
Authors: Mufson RA , Gesner TG
Published: October 2019

Erythropoietin (EPO) biosynthetically labelled with [35S]cysteine was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant [35S]EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The [35S]-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 to 200 binding sites per cell at 37 degrees C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with 125I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation at 37 degrees C, most of the bound [35S]EPO was internalized, whereas most of the [125I]EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.
Synergistic inhibition of platelet activation by plasmin and prostaglandin I2
Authors: Schafer AI , Zavoico GB , Loscalzo J , Maas AK
Published: October 2019

Endothelial cell prostacyclin (PGI2) inhibits platelet activation by raising platelet cyclic AMP. Previously, platelet activation was also shown to be blocked by plasmin formed by endothelium-derived tissue plasminogen activator (TPA). We have now studied interactions between PGI2 and plasmin in the control of platelet function. PGI2 and plasmin cause synergistic inhibition of thrombin- and ADP-induced aggregation of washed platelets. Inhibition by PGI2 is similarly potentiated by TPA added to platelet-rich plasma to generate plasmin. Thrombin-stimulated rise in platelet cytosolic Ca2+, measured by fura2 fluorescence, and thromboxane A2 formation, measured by radioimmunoassay (RIA), are likewise synergistically inhibited by PGI2 and plasmin. Plasmin neither increases nor potentiates PGI2-stimulated increases in platelet cyclic AMP. Thus, PGI2 and plasmin cause synergistic inhibition of platelet activation by both cyclic AMP-dependent and independent mechanisms. This interaction between two different endothelium-derived products may play an important role in localizing the hemostatic plug to a site of vascular injury by preventing further thrombin-mediated accrual of platelets.
Vasoactive amines directly modify endothelial cells to affect polymorphonuclear leukocyte diapedesis in vitro
Authors: Doukas J , Shepro D , Hechtman HB
Published: October 2019

Bovine aortic endothelial cells were cultured on the basement membrane surface of amnionic membrane and used as a substrate for polymorphonuclear leukocyte (PMN) diapedesis in vitro. Norepinephrine (NE), serotonin (5HT), or phalloidin treatment of the endothelial cells (ECs) reduces, whereas histamine or cytochalasin B increases, the number of PMNs migrating across the ECs and amnionic membrane. In contrast, amine treatment of PMNs or acellular amnionic membrane does not alter PMN diapedesis or chemotaxis. The NE and histamine effects are blocked by appropriate receptor antagonists, but the 5HT effect is not. All the agents' effects are also reversible. Qualitatively similar effects on EC permeability to Evan's blue-labeled albumin occur with all agents; however, PMN adhesion to ECs is not affected. Previously, we reported that NE and 5HT increase stress fiber numbers and decrease EC permeability to macromolecules in vitro, whereas histamine has the opposite effects, and that NE and 5HT eliminate the erythrocyte extravasation associated with thrombocytopenia in vivo. In this study, we propose that these vasoactive amines also alter PMN diapedesis in vitro through a direct effect on the EC, in part due to alterations in the EC cytoskeleton.
Intermediate lymphocytic lymphoma: immunophenotypic and cytogenetic findings
Authors: Weisenburger DD , Sanger WG , Armitage JO , Purtilo DT
Published: October 2019

A detailed immunohistologic and cytogenetic analysis of 12 cases of intermediate lymphocytic lymphoma was performed. The characteristic immunophenotype of intermediate lymphocytic lymphoma was: surface IgM and IgD+, BA1+, B1+, BA2-, B2-, B4+, Leu 14+, Leu 1+, HLA-DR+, and common acute lymphocytic leukemia associated (CALLA) antigen negative. Clonal chromosome abnormalities were identified in ten cases, with structural or numerical abnormalities of chromosomes 11 or 12 in nine cases. Five cases had structural abnormalities involving the long arm of chromosome 11; three of these had translocations with chromosome 14 at band q32. Three cases had trisomy 12, and one case had a translocation involving the long arm of chromosome 12. The tenth case had a translocation involving the long arms of chromosomes 7 and 9. These characteristic immunophenotypic and cytogenetic findings suggest a close lineage relationship between intermediate lymphocytic lymphoma and small lymphocytic (well differentiated) lymphoma/chronic lymphocytic leukemia. Their differing clinical, cytologic, and architectural features suggest, however, that intermediate lymphocytic lymphoma should be considered a separate category of lymphocytic lymphoma in the International Working Formulation.
A randomized prospective comparison of chemotherapy to total body irradiation as initial treatment for the indolent lymphoproliferative diseases
Authors: Jacobs P , King HS
Published: October 2019

One hundred eight consecutive patients with indolent lymphoproliferative diseases were stratified into chronic lymphocytic leukemia (CLL), stage III and IV well-differentiated lymphocytic lymphoma (WDLL), and stage III and IV follicular lymphoma (FL). Within each stratum, patients were prospectively and randomly assigned to receive chemotherapy with chlorambucil and prednisone (CP) or fractionated total body irradiation (TBI). Morbidity from both regimens was negligible. Complete response (CR) was defined as the resolution of organ enlargement and the return of blood count to normal. The CR rate for the entire CP group (n = 54) was 59% and that for the TBI group (n = 54), 52%; median survivals were 53 and 57 months respectively. In the 41 patients with CLL the CR rate for CP (n = 17) was 47% and that for TBI (n = 24), 50%; the median survival for CP was 48 months, and for TBI it was 51 months. In the 21 patients with WDLL the CR rate for CP (n = 15) was 53% and that for TBI (n = 6), 67%; the median survival for CP was 42 months and has not yet been reached for TBI. For the 46 patients with FL the CR rate for CP (n = 22) was 72% and that for TBI (n = 24), 50%; the median survival was 55 months, and for TBI it was 56 months. None of the differences in CR or survival are statistically significant (P greater than .05). In these indolent lymphoproliferative diseases, CP and TBI are equally effective forms of initial treatment irrespective of the end point being defined as CR or survival.
Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia
Authors: Raza A , Maheshwari Y , Preisler HD
Published: October 2019

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.
The proliferative response of B cell chronic lymphocytic leukemia to interleukin 2: functional characterization of the interleukin 2 membrane receptors
Authors: Touw I , Dorssers L , Lowenberg B
Published: October 2019

To determine the growth properties of B cell chronic lymphocytic leukemia (B CLL) and to identify possible abnormalities thereof, we examined the in vitro action of interleukin 2 (IL2) in four patients. Using radiolabeled IL2 and monoclonal antibodies reactive with IL2 membrane receptors we show that CLL cells, after their activation in vitro, express IL2 receptors of a high- as well as a low-affinity type, exactly as has been reported for normal T and B blasts. In three of the four reported cases, CLL proliferation (measured with 3H-thymidine incorporation) depended on the addition of phytohemagglutinin (PHA) to activate the cells and IL2 (optimal concentration, 10 to 100 U IL2/mL). In contrast, the cells of the fourth case of CLL (CLL-4) proliferated in an autonomous fashion, ie, without a need for PHA and IL2 in culture. Specific blocking of the IL2-binding sites with anti-IL2 receptor monoclonal antibodies almost completely inhibited the proliferation of these cells, which indicated that functional IL2 receptors were required for the autonomous proliferation. The demonstration of low concentrations of IL2 activity in the culture medium conditioned by the cells suggests that endogenous IL2 had been responsible for the spontaneous 3H-thymidine uptake by the CLL cells of patient 4. However, we were unable to extract IL2 mRNA from the cells (neither fresh nor after various in vitro incubations) in quantities detectable by Northern blot analysis that would prove that the CLL cells of patient 4 were actively synthesizing IL2 during culture. Thus, individual cases of B CLL are subject to variable growth regulation involving functional IL2 receptors on the cell surface: after activation with PHA the cells respond to exogenous IL2 in a fashion similar to normal B lymphocytes, or the cells are stimulated by endogenous IL2 (or an IL2-like activity) and do not require activation with PHA.
T lymphocytes lack rearrangement of the bcr gene in Philadelphia chromosome-positive chronic myelocytic leukemia
Authors: Bartram CR , Raghavachar A , Anger B , Stain C , Bettelheim P
Published: October 2019

To study the possible involvement of T lymphocytes in Philadelphia chromosome (Ph)-positive chronic myelocytic leukemia (CML) we analyzed the arrangement of the bcr gene in T cell and non-T cell samples of 12 CML patients. Although all the patients showed bcr rearrangements in non-T cell fractions, T cell populations lacked respective gene recombinations. Moreover, by Southern blot analyses using T cell receptor beta chain sequences our data indicate polyclonality of T cell samples from 11 of 12 cases; in one patient a clonal T cell population could be identified. These results suggest that T lineages of most Ph- positive CML patients are not derived from pluripotent stem cells involved in leukemogenesis and thus confirm previous investigations based on cytogenetic or glucose-6-phosphate dehydrogenase analyses. The demonstration of polyclonal T cell populations may reflect persistence of stem cells committed to differentiate only into T cells.
A killing defect of natural killer cells with the absence of natural killer cytotoxic factors in a child with Hodgkin's disease
Authors: Komiyama A , Kawai H , Yamada S , Kato M , Yanagisawa M , Miyagawa Y , Akabane T
Published: October 2019

A killing defect of natural killer (NK) cells in the absence of NK cytotoxic factors (NKCF) was first demonstrated in a child with Hodgkin's disease. The patient lacked detectable NK cell activity in every phase of the disease as measured by a four-hour 51Cr-release assay using K562 cells as a target. The percent lysis at a 40:1 effector:target ratio by the patient's lymphocytes was persistently below 0.3% as compared with the normal lymphocyte value of 46.2% +/- 5.8% (mean +/- SD). NK cell activity was not detectable at effector:target ratios of 10:1 to 80:1 and by prolongation of the incubation time, and the NK cell defect was not restored or improved by lymphocyte stimulation with polyinosinic-polycytidilic acid, interferon (IFN)-alpha, or interleukin 2 (IL 2). The numbers of Leu-7+ cells and Leu-11+ cells were normal as counted by flow cytometry. A single cell- in-agarose assay demonstrated normal numbers of target binding cells (TBCs), and they showed the morphology of “large granular lymphocytes.” However, there were no TBCs with dead targets. These results indicated that the patient's lymphocytes contained normal numbers of NK cells that were capable of recognizing and binding to a target but were incapable of killing the bound target cell. The patient's lymphocytes were then studied for their release of NKCF upon interaction with K562 cells. The patient's cells did not release NKCF, and the NK cell defect was not restored or improved by stimulation of the cells with IFN or IL 2. It is suggested that the deficient release of NKCF may have been related to the killing defect of the NK cells in this patient.
Type I Glanzmann thrombasthenia patients from the Iraqi-Jewish and Arab populations in Israel can be differentiated by platelet glycoprotein IIIa immunoblot analysis
Authors: Coller BS , Seligsohn U , Little PA
Published: October 2019

A sensitive immunoblot technique for platelet glycoprotein IIIa (GPIIIa) was used to analyze the platelets of patients living in Israel who meet the diagnostic criteria for type I Glanzmann thrombasthenia. When reacted with solubilized normal platelets, a rabbit antiserum to GPIIIa identified a major band at molecular weight (mol wt) 90,000 and three additional minor bands at Mr 110,000, 81,000, and 64,000. The major band could not be detected, and the minor bands were either markedly reduced or absent in the platelet samples from 14 of the 15 patients from the Iraqi-Jewish population. In contrast, in all four Arab patients tested, the major band was detectable, although at markedly reduced levels, and the minor bands were either markedly reduced or absent; an additional minor band at mol wt 47,000 was also present in the platelets from these patients. One Iraqi-Jewish patient had a unique pattern in which two of the bands were present but reduced and two were undetectable. We conclude that the protein defect, and thus presumably the genetic defect, causing Glanzmann thrombasthenia in the majority of patients in the Iraqi-Jewish population differs from that in the Arab population, and we confirm that there is considerable biochemical heterogeneity among the patients who meet the criteria for type I Glanzmann thrombasthenia.
Liver transplantation in hemophilia A
Authors: Bontempo FA , Lewis JH , Gorenc TJ , Spero JA , Ragni MV , Scott JP , Starzl TE
Published: October 2019

Four patients with hemophilia A have undergone liver transplantation in our institution, three successfully. The first was a 21-year-old man with chronic active hepatitis (CAH) in whom the effects of previous abdominal operations prevented the satisfactory technical insertion of the new liver. He died intraoperatively. The second patient was a 15- year-old boy with CAH who began to synthesize factor VIII coagulant activity (F VIII:C) within 18 hours of successful liver transplantation and has continued to do so for almost 2 years (F VIII:C range 0.89 to 3.20 U/mL). The first 2 months of his postoperative course were complicated by infections, but since that time he has done well and has returned to school. The third patient was a 48-year-old man with portal fibrosis and severe ascites. He synthesized F VIII:C (range 0.96 to 1.50 U/mL) within six hours after reestablishment of circulation through the new liver. His postoperative course was complicated by numerous infections, and he died with sepsis and an acquired immunodeficiency-like syndrome 4 months after transplantation. The fourth patient was a 47-year-old mild hemophiliac with CAH who produced adequate factor VIII:C levels following transplantation (range 0.79 to 2.80 U/mL). These patients demonstrate that liver transplantation in hemophiliacs with end-stage liver disease may be lifesaving and results in correction of the F VIII:C deficiency and associated hemorrhagic tendency.
Fetal hemoglobin levels and beta s globin haplotypes in an Indian populations with sickle cell disease
Authors: Kulozik AE , Kar BC , Satapathy RK , Serjeant BE , Serjeant GR , Weatherall DJ
Published: October 2019

To further explore the cause for variation in hemoglobin F (Hb F) levels in sickle cell disease, the beta globin restriction-fragment length polymorphism haplotypes were determined in a total of 303 (126 SS, 141 AS, 17 S beta degrees, 7 A beta, degrees and 12 AA) Indians from the state of Orissa. The beta s globin gene was found to be linked almost exclusively to a beta S haplotype ( -++-), which is also common in Saudi Arabian patients from the Eastern Province (referred to as the Asian beta s haplotype). By contrast, the majority of beta A and beta degree thalassemia globin genes are linked to haplotypes common in all European and Asian populations (+-----[+/-]; --++-++). Family studies showed that there is a genetic factor elevating Hb F levels dominantly in homozygotes (SS). This factor appears to be related to the Asian beta s globin haplotype, and a mechanism for its action is discussed. There is also a high prevalence of an independent Swiss type hereditary persistence of fetal hemoglobin (HPFH) determinant active in both the sickle cell trait and in sickle cell disease.
Transfusion-associated AIDS: donor-recipient human immunodeficiency virus exhibits genetic heterogeneity
Authors: Srinivasan A , York D , Ranganathan P , Ferguson R , Butler D Jr , Feorino P , Kalyanaraman V , Jaffe H , Curran J , Anand R
Published: October 2019

The genetic diversity of the human immunodeficiency virus (HIV) isolated from transfusion-associated AIDS patients has been examined. Restriction enzyme mapping studies of integrated proviral DNA of donor and recipient origin demonstrated genomic variation between isolates. Analysis of the molecularly cloned viral genomes of one donor-recipient pair showed that virus from the recipient had restriction enzyme site differences from the donor, noticeably clustered in the env and orf-2 regions, and also had a greater number of restriction sites in common with the donor as well. These results suggest that HIV may undergo genomic variation in vivo. Comparison of donor-recipient viruses may further the understanding of the molecular basis for AIDS pathogenesis.
Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium
Authors: Annamalai AE , Rao AK , Chiu HC , Wang D , Dutta-Roy AK , Walsh PN , Colman RW
Published: October 2019

We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.
A new mutation in IVS-1 of the human beta globin gene causing beta thalassemia due to abnormal splicing
Authors: Atweh GF , Wong C , Reed R , Antonarakis SE , Zhu D , Ghosh PK , Maniatis T , Forget BG , Kazazian HH Jr
Published: October 2019

A G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites. Only one of the two major cryptic sites was utilized in a cell-free splicing extract. The effects of this mutation on messenger RNA (mRNA) splicing are similar to that of another beta thalassemia gene with a G to C transition at the same position.
Erythrocyte membrane skeleton abnormalities in severe beta-thalassemia
Authors: Shinar E , Shalev O , Rachmilewitz EA , Schrier SL
Published: October 2019

The protein composition of ghosts, inside-out vesicles (IOV), and membrane skeletons (MS) of erythrocytes (RBC) from splenectomized (spx) and nonsplenectomized (non-spx) patients with beta-thalassemia major and beta-thalassemia intermedia was determined. Ghosts from spx thalassemia intermedia patients had a significant increase in their globin content (which was mostly heme reactive) and contained extra polypeptides in the protein 4.2 to 5 and 6-globin areas. The Triton- extracted MS from all of the thalassemic patients showed two major abnormalities: they retained up to twice the amount of protein 3 when compared with controls; they had a significant increase in their globin content, the concentration of which was independent of their protein 3 content. Analysis of the IOV revealed no differences between those prepared from normal controls and those of the patients. MS from spx thalassemia intermedia patients were grossly abnormal when examined by scanning electron microscopy and they exhibited aggregates of material that on transmission electron microscopy suggested the presence of globin precipitates. We propose that, although the integral protein composition, as reflected in the IOV, from severely affected beta- thalassemics is intact, their MS assembly is deranged. The altered skeletal structure of thalassemic RBC could result from attachment of denatured globin to the skeleton components. These abnormalities may contribute to the premature cell death seen in severe beta-thalassemia.