bluuebunny/crossref_metadata_2025 huggingface dataset:
Prognostic variables and clinical staging in multiple myeloma
Authors: Cavo M , Galieni P , Zuffa E , Baccarani M , Gobbi M , Tura S
Published: October 2019

To evaluate the most important factors in the prognosis and staging of multiple myeloma (MM), the presenting clinical features of 163 previously untreated patients with MM were correlated with survival duration using univariate and multivariate regression analyses. The univariate proportional hazard analysis ranked the parameters in the following order of importance: platelet count, hemoglobin level (Hb), tumor cell mass stage, lytic bone lesions, creatinine, and age. When the individual contribution of each variable was assessed by multivariate regression analysis, platelet count was confirmed to be the dominant feature for prognosis and clinical stage provided additional information. The introduction of platelet count could then be used to improve the reliability of the Durie and Salmon staging, by allowing to separate the high-risk group (stages II and III) into a smaller subgroup (22%) of thrombocytopenic patients (less than 150 x 10(9) platelets/L) whose risk of death was actually very high (median survival, 9 months) and a larger subgroup (46%) of patients with normal platelet count and intermediate or standard risk (median survival, 48 months). This simple change in the prognostic system gave rise to markedly different survival curves also after the exclusion of patients with renal failure and applied successfully to both old and young patients (greater than and less than 50 years, respectively). Finally, platelet count, Hb, and lytic bone lesions could be combined simply to stratify patients with normal renal function into three risk groups: (1) low (39% of cases; median survival, 79 months), (2) intermediate (53% of cases; median survival, 48 months), and (3) high (8% of cases; median survival, 19 months).
Reticulocyte maturation and exosome release: transferrin receptor containing exosomes shows multiple plasma membrane functions
Authors: Johnstone RM , Bianchini A , Teng K
Published: October 2019

Vesicles (exosomes) released during sheep reticulocyte maturation contain a number of plasma membrane functions. Using an antibody coated, magnetic core bead, it has been shown unequivocally that vesicles that contain the transferrin receptor also contain other plasma membrane activities, such as the nucleoside transporter and acetylcholinesterase. Lysosomal activities, normally found in the same pellet, are excluded from the transferrin receptor-containing exosomes, suggesting that there is a common mechanism to segregate and externalize specific plasma membrane proteins. In addition to the sheep, electron micrographic studies show that exosomes can be retrieved from the circulation of anemic pigs, rats, and rabbits.
Wegener's granulomatosis autoantigen is a novel neutrophil serine proteinase [see comments]
Authors: Niles JL , McCluskey RT , Ahmad MF , Arnaout MA
Published: October 2019

Circulating IgG autoantibodies that produce cytoplasmic immunofluorescence staining of ethanol-fixed normal neutrophils have recently been found in a large percentage of patients with active Wegener's granulomatosis. Such autoantibodies are rarely found in other diseases and are therefore virtually diagnostic of Wegener's granulomatosis. The nature of the neutrophil antigen defined by these autoantibodies is controversial and the roles of the antigen and/or autoantibodies in the pathogenesis of Wegener's granulomatosis are unknown. We studied serum samples that produce the cytoplasmic pattern of staining from 10 patients with a diagnosis of Wegener's granulomatosis. By Western blot analysis, all 10 sera reacted with a 29- Kd neutrophil protein (p29). We generated a mouse monoclonal antibody directed against this antigen. The monoclonal antibody produced the same immunofluorescence staining pattern as the serum autoantibodies and was used to affinity-purify p29. The purified antigen had a novel N- terminal sequence homologous to members of the serine proteinase family and bound to radiolabeled diisopropyl fluorophosphate (DFP). We conclude that the neutrophil antigen responsible for the cytoplasmic staining pattern produced by autoantibodies in patients with active Wegener's granulomatosis is a distinctive serine proteinase.
Improvement of platelet counts in steroid-unresponsive idiopathic immune thrombocytopenic purpura after short-course therapy with recombinant alpha 2b interferon [see comments]
Authors: Proctor SJ , Jackson G , Carey P , Stark A , Finney R , Saunders P , Summerfield G , Maharaj D , Youart A
Published: October 2019

In 13 patients with severe steroid-refractory idiopathic immune thrombocytopenia (ITP), a short course of recombinant alpha 2b interferon (IFN), given at a dose of 3 MU for 12 doses, caused a significant increase in platelet count in 11 patients. The rise in platelet count occurred following completion of the short course of IFN in 10 patients and occurred during therapy in one patient. Three patients showed an increase to normal platelet counts within 14 days of discontinuing the drug, eight showed a partial response, with a platelet count increase from 30 to 100 x 10(9)/L, and two patients showed minimal response. One complete responder relapsed at 5 months from initial response, and a further course of alpha 2b IFN caused a second prompt response with a rise of platelet count to supranormal levels. Short-course alpha 2b IFN can be recommended as a therapy for severe ITP. Responses are seen in splenectomized and nonsplenectomized subjects, and thrombocytopenia is not exacerbated during treatment.
2'-Deoxycytidine protects normal human bone marrow progenitor cells in vitro against the cytotoxicity of 3'-azido-3'-deoxythymidine with preservation of antiretroviral activity
Authors: Bhalla K , Birkhofer M , Li GR , Grant S , MacLaughlin W , Cole J , Graham G , Volsky DJ
Published: October 2019

Bone marrow cytotoxicity of 3′-azido-3′-deoxythymidine (AZT), an anti- human immunodeficiency virus (anti-HIV) drug, has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined, in vitro, the effect of high, but clinically achievable and nontoxic, concentrations of 2′-deoxycytidine (dCyd) (greater than or equal to 100 mumol/L) on high-dose AZT mediated growth inhibition and intracellular biochemical perturbations in normal bone marrow progenitor cells. Colony formation by bone marrow progenitor cells in semisolid medium was significantly protected by dCyd against the inhibitory effects of co-administered, high concentrations of AZT (10 mumol/L). Also, dCyd significantly corrected AZT mediated depletion of intracellular thymidine triphosphate (dTTP) and dCyd triphosphate (dCTP) levels in normal bone marrow mononuclear cells (BMMC). Moreover, dCyd reduced the intracellular accumulation of AZT triphosphate (AZT-TP) and its DNA incorporation in BMMC. In contrast, co-administration of dCyd (100 mumol/L to 1 mmol/L) did not reverse AZT (10 mumol/L) mediated suppression of HIV infectivity in HUT-102 cells in culture, although a partial reduction in intracellular AZT-TP pools and its DNA incorporation as well as a correction of AZT mediated depletion of dTTP and dCTP pools was observed in these cells. These studies suggest that dCyd at high concentrations might ameliorate the bone marrow cytotoxicity of high-dose AZT without impairing its anti-HIV effect.
The fate of the open canalicular system in surface and suspension- activated platelets
Authors: Escolar G , Leistikow E , White JG
Published: October 2019

We have examined the movement of fibrinogen-gold (fgn-Au) complexes in platelets activated in suspension and by surface contact. Fgn-Au probes did not react with resting cells but were bound to the external membrane of platelets in suspension 5 seconds after addition of 1 U/mL of thrombin. At intervals over a period of 5 to 20 minutes, fgn-Au probes moved from the cell surface to peripheral and then deep channels of the open canalicular system (OCS). When platelets were surface activated by exposure to carbon-stabilized, formvar-coated grids for 5 to 20 minutes and then exposed to fgn-Au complexes for 5 minutes, probes were also observed in the OCS. At 5 minutes, over 40% of the platelets had concentrated fgn-Au in their OCS. Results after 10 minutes revealed 25% with gold-filled channels, 16% after 15 minutes, and 5% after 20 minutes. The decrease in frequency of OCS staining correlated with the increasing frequency of spread platelets, suggesting that tension produced by spreading may cause collapse of the OCS or that the OCS may evaginate onto the platelet during spreading. To evaluate the latter hypothesis, platelets were initially exposed to grids for 5 minutes and then incubated with fgn-Au for intervals of 5 to 20 minutes. The frequency of platelets with fgn-Au concentrated in the OCS was greatest at 5 minutes (44%) and decreased at the same rate as the frequency of spread platelets increased. Only 14.7% of the cells contained fgn-Au in the OCS after 20 minutes. These were primarily dendritic in form, while fully spread platelets rarely contained an OCS filled with the probe. The study indicates that fgn-Au particles are cleared to channels of the OCS independent of the mechanism of platelet activation. Fgn-Au that has been concentrated in the OCS at early stages of surface activation can be externalized during platelet spreading but remain internalized in suspension-activated cells. The OCS represents a membrane reservoir that can be evaginated onto the platelet surface during interaction with surfaces.
Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets
Authors: Parker RI , Gralnick HR
Published: October 2019

Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
Diffuse osteosclerosis in hairy cell leukemia
Authors: VanderMolen LA , Urba WJ , Longo DL , Lawrence J , Gralnick H , Steis RG
Published: October 2019

We describe two patients with a new clinical pathologic syndrome of diffuse osteosclerosis in association with hairy cell leukemia. In both patients bone marrow biopsies could not be obtained due to extremely hard bones and inability to insert the biopsy needle; neither patient had a history of bony pain or fracture. The osteosclerotic process in one patient stabilized after successful treatment of her hairy cell leukemia with interferon alpha and deoxycoformycin suggesting that the osteosclerosis observed was related to the underlying malignant disease. Possible etiologic mechanisms are discussed.
Interleukin-6 and interleukin-1 production in acute leukemia with monocytoid differentiation
Authors: van der Schoot CE , Jansen P , Poorter M , Wester MR , von dem Borne AE , Aarden LA , van Oers RH
Published: October 2019

Several authors have reported the in vitro production of colony- stimulating factors (CSF) and interleukin-1 (IL-1) by the neoplastic cells from patients with acute myeloid leukemia (AML). Using a sensitive bioassay for IL-6, the capacity of the leukemic cells of 30 patients with AML to produce IL-6 was examined. IL-6 production was found to be specific for cells from patients with an AML with monocytic differentiation (12 of 15 M4 and M5 patients, 0 of 15 M1 and M2 patients). Moreover, IL-6 production was paralleled by IL-1 production. The IL-6- and IL-1-producing cells were mainly found in the more mature monocytic cell fractions, defined as CD14-positive and CD34-negative adherent cells. By limiting dilution experiments, it could be excluded that the production of IL-1 or IL-6 was due to contamination with normal monocytes.
Human myeloperoxidase gene expression in acute leukemia
Authors: Zaki SR , Austin GE , Swan D , Srinivasan A , Ragab AH , Chan WC
Published: October 2019

To evaluate the relationship between myeloperoxidase (MPO) gene expression and specific lineages of hematopoietic differentiation, we analyzed expression of the MPO gene in a variety of normal and leukemic human cells. As a first step, we synthesized several oligonucleotide probes and isolated cDNA clones for the MPO gene. MPO expression was detected in most acute myeloid leukemias, and the level of expression correlated well with cytochemical enzymatic activity. No MPO message was detected in pure lymphoid leukemias, whereas very low basal levels of MPO transcripts were found in normal bone marrows. In four cases of acute undifferentiated leukemias cytochemically negative for MPO, significant levels of MPO transcripts were detected, suggesting a myeloid origin for these cases. These results indicate that MPO gene expression may serve as an additional marker for subclassification of acute leukemias and may be used to identify leukemic cells arrested at an early stage of the myeloid differentiation pathway.
Analysis of molecular events in leukemic cells arrested at an early stage of T-cell differentiation
Authors: Yumura-Yagi K , Hara J , Terada N , Ishihara S , Tawa A , Takihara Y , Champagne E , Minden MD , Mak TW , Kawa-Ha K
Published: October 2019

We analyzed the rearrangement and expression of T-cell receptor (TCR) genes, including the recently identified TCR delta gene, in 21 patients with T-lineage leukemia/lymphoma. Among 8 patients with CD3-, CD4-, and CD8- (group I), 2 patients showed germline configuration of the TCR delta, gamma, beta, and alpha genes and 1 patient demonstrated only TCR delta gene rearrangement. All nine patients with CD3-, CD4+, and/or CD8+ (group II) showed concomitant rearrangements of the TCR delta, gamma, and beta genes. TCR alpha gene rearrangement was also observed in two patients. Three of four patients with CD3+ (group III) showed rearrangement of the TCR alpha gene with deletion of both alleles or of a single allele of the TCR delta gene. With Northern blot analysis, full-length transcripts of the TCR delta gene were detected in 3 of 15 examined patients. All were restricted to group I or group II. In contrast, full-length transcripts of TCR beta and alpha were observed mainly in samples from groups II and III. Based on these findings, rearrangement of the TCR delta gene may be the earliest event in T-cell differentiation, preceding rearrangements of the other TCR genes.
Time-dependent loss of adenosine 5'-monophosphate deaminase activity may explain elevated adenosine 5'-triphosphate levels in senescent erythrocytes
Authors: Dale GL , Norenberg SL
Published: October 2019

Senescent erythrocytes from rabbits were previously shown to have elevated levels of adenine nucleotides. The present study documents that aged red blood cells have a normal synthetic capacity for adenine nucleotides, as indicated by normal levels of adenosine kinase. However, senescent erythrocytes do have decreased levels of adenosine 5′-monophosphate deaminase, the critical enzyme involved in degrading adenine nucleotides. These circumstances of a normal synthetic capacity in the presence of decreased catabolic ability were observed previously in a human genetic deficiency of adenosine 5′-monophosphate deaminase; the red blood cells in these patients accumulate adenosine 5′- triphosphate as do senescent erythrocytes in rabbits.
AMP deaminase as a cell-age marker in transient erythroblastopenia of childhood and its role in the adenylate economy of erythrocytes
Authors: Paglia DE , Valentine WN , Nakatani M , Brockway RA
Published: October 2019

Erythrocytes from 11 patients with presumptive diagnoses of transient erythroblastopenia of childhood were evaluated retrospectively (six) or prospectively (five) for a possible relationship between erythrocyte adenosine 5′-monophosphate aminohydrolase, adenylic acid deaminase (AMP deaminase) activity and intracellular concentrations of adenine nucleotides. Older red blood cell (RBC) cohorts in these patients consistently exhibited significantly decreased activities of AMP deaminase (approximately 5% to 70% of normal control mean) in association with increased concentrations (up to threefold) of adenosine triphosphate (ATP) and total adenine nucleotides. We postulate that the latter is a direct consequence of the former, since diminishing AMP deaminase activity in aging cells should reduce the drain on the adenine nucleotide pool imposed by irreversible deamination of AMP to inosine 5′-monophosphate. Consistent reductions in AMP deaminase activity indicate that this enzyme should also serve as a reliable marker of mean RBC age useful in diagnostic confirmation of transient erythroblastopenia. The observed increases in ATP and total adenine nucleotides in older RBCs require a reevaluation of the traditional view that age-related losses of these compounds mediate the ultimate demise of senescent erythrocytes. Similar alterations in the balance of degradative and salvage pathways in RBC nucleotide metabolism may also underlie certain cases of so-called “high ATP syndrome.”
Antimalarial effect of HBED and other phenolic and catecholic iron chelators
Authors: Yinnon AM , Theanacho EN , Grady RW , Spira DT , Hershko C
Published: October 2019

Previous studies showed that deferoxamine inhibits malaria by interacting with a labile iron pool within parasitized erythrocytes. Consequently, we studied the antimalarial properties of other iron- chelating drugs such as 2,3-dihydroxybenzoic acid (2,3-DHB) and its methyl ester as well as two polyanionic amines, N.N′-bis (o- hydroxybenzyl) ethylenediamine-N,N′-diacetic acid (HBED) and N,N′- ethylenebis(o-hydroxyphenylglycine) (EHPG) in rats infected with Plasmodium berghei. All drugs were delivered by subcutaneous injection at 8-hour intervals, 40 mg per animal per day. All animals receiving N,N′-ethylenebis(o-hydroxyphenylglycine) died of drug toxicity between days 6 and 11. Peak parasitemia on day 11 of infection was 32.8% in control animals; 25.3% with methyl 2,3-DHB, 15.5% with 2,3-DHB, 8.0% with deferoxamine, and 0.9% with HBED. Subsequent studies of HBED and deferoxamine in P falciparum cultures in human erythrocytes showed a marked suppression of parasite counts by both drugs at concentrations of greater than 5 mumol/L. At all concentrations tested, HBED was four to five times more effective than deferoxamine in suppressing parasite counts. The superior antimalarial activity of HBED is attributed to its increased lipophilicity and higher affinity to ferric iron. These findings indicate that selective iron deprivation by interaction with an intracellular chelator may represent a novel approach to antimalarial drug development, and that systematic screening of available iron-chelating drugs may result in identification of potentially useful antimalarial compounds.
Anti-B-cell monoclonal antibody-purged autologous bone marrow transplantation for B-cell non-Hodgkin's lymphoma: phenotypic reconstitution and B-cell function
Authors: Pedrazzini A , Freedman AS , Andersen J , Heflin L , Anderson K , Takvorian T , Canellos GP , Whitman J , Coral F , Ritz J
Published: October 2019

In the present report we have attempted to examine immunologic reconstitution following high-dose chemoradiotherapy and anti-B-cell monoclonal antibody (MoAb)-purged autologous bone marrow transplantation (ABMT). By cell-surface phenotypic analysis, the majority of patients had normal percentage of natural killer cells (NK), monocytes, and CD8+ T cells at one month post-ABMT. In contrast, the percentage of CD4+ T cells was reduced for at least 3 years, and the CD4:CD8 ratio reflected this imbalance. B-cell reconstitution was slightly prolonged, with normal percentage and absolute numbers of CD20+ B cells evident by 3 months. Although B cells returned by 3 months, in vitro assessment of B-cell function demonstrated impairment of proliferative responses to either anti-immunoglobulins bound to beads (anti-Ig), Epstein-Barr virus (EBV), or interleukin-2 (IL-2) for approximately 1 year and low molecular B-cell growth factor (BCGF) for approximately 2 or more years. Moreover, in vivo B-cell reconstitution demonstrated a more selective defect, with normal levels of immunoglobulin IgM returning at 6 months, IgG at 12 months, and IgA after 2 years. Despite normal numbers of B cells and relative normal levels of Ig early following ABMT, our in vitro data suggest an intrinsic defect in B-cell responsiveness. Moreover, these defects are similar to those observed following nonpurged autologous and allogeneic BMT, although the interval of immune impairment appears more prolonged.
Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridization assay
Authors: Durnam DM , Anders KR , Fisher L , O'Quigley J , Bryant EM , Thomas ED
Published: October 2019

A Y-chromosome-specific in situ hybridization assay was used to assess the frequency with which host bone marrow cells are retained after marrow grafting. The majority of patients (74%) showed the presence of both host and donor marrow cells when assayed 14 days after transplant. By 84 days posttransplant only 4% of the patients retained host marrow cells. Only 1 of 19 evaluable patients analyzed over 1 year posttransplant showed minimal retention of host cells. No statistical correlation was found between retention of host cells posttransplant and the development of relapse or acute or chronic graft-versus-host disease. Pretransplant conditioning regimen, HLA-matching, diagnosis, disease status at transplant, ABO-matching, and patient age also showed no correlation with the retention of host cells posttransplant.
T-cell ontogeny after autologous bone marrow transplantation: failure to synthesize interleukin-2 (IL-2) and lack of CD2- and CD3-mediated proliferation by both CD4- and CD8+ cells even in the presence of exogeneous IL-2
Authors: Cayeux S , Meuer S , Pezzutto A , Korbling M , Haas R , Schulz R , Dorken B
Published: October 2019

T cells generated during a second round of ontogeny after autologous bone marrow transplantation (ABMT) represent a unique model of early T- cell ontogeny in an autologous situation. Since grafted bone marrows were pretreated in vitro with the cyclophosphamide derivative ASTA Z 7557, circulating T cells had to be regenerated from reinfused hematopoietic progenitor cells. The T-cell population derived from 25 patients post-ABMT was phenotypically characterized: an increase in CD8+ cells, a low percentage of CD4+ cells, and a median of 12% CD56+ (NKH1+) cells were found. When the T cells were stimulated with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), defective interleukin-2 (IL-2) secretion was observed. In addition, proliferative responses of the T cells after activation through the antigen-receptor- dependent CD3 pathway, through the CD2 dependent alternative T-cell pathway, and by the lectin PHA were investigated. Despite the presence of CD2, CD3, alpha/beta chains of the T-cell receptor, and CD25+ IL-2 surface receptors, abnormal proliferative responses were obtained even in the presence of exogeneous IL-2. In experiments where the T-cell population was separated into CD4+ cells and CD8+ cells, both the CD4- and CD8+ subsets were unable to respond to activating and proliferating signals. Thus, T cells at early stages of ontogeny not only possess an intrinsic defect in IL-2 synthesis but, in addition, were unable to express functional IL-2 receptors in response to mitogenic stimuli.
Allogeneic mixed lymphocyte reactions during a second round of ontogeny: normal accessory cells did not restore defective interleukin- 2 (IL-2) synthesis in T cells but induced responsiveness to exogeneous IL-2
Authors: Cayeux S , Meuer S , Pezzutto A , Korbling M , Haas R , Schulz R , Dorken B
Published: October 2019

The T-cell-accessory-cell interaction in mixed lymphocyte cultures was investigated in 25 patients following autologous bone marrow transplantation (ABMT) using autologous bone marrow treated in vitro with the cyclophosphamide derivative ASTA Z 7557. In a previous study using the same group of patients, T cells failed to synthesize interleukin-2 (IL-2) and proliferate in response to CD3- and CD2- mediated stimuli even in the presence of exogenous IL-2. To investigate whether this defect in IL-2 synthesis and proliferation was caused by defective cell-to-cell interactions, we analyzed mixed lymphocyte reactions (MLR) using T cells and irradiated non-T cells. When normal T cells from 10 different healthy subjects were challenged with allogeneic normal non-T cells, IL-2 production and proliferation were observed. In contrast, when normal T cells were cultured with non-T cells derived from patients found between 20 and 330 days after ABMT, no IL-2 secretion and no proliferative responses could be seen. The addition of lymphokines such as interleukin-1 (IL-1), interleukin-3 (IL- 3), tumor necrosis factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), and interferon-gamma (IFN-y) did not improve the reactions. Furthermore, when patients' T cells were incubated with normal, irradiated non-T cells, defective IL-2 synthesis or proliferative response was obtained. However, when IL-2 was added to these cultures, an improvement in proliferative reactions was observed. Taken together, these new data provide additional evidence that T cells early in ontogeny possessed an intrinsic defect in IL-2 synthesis and that physical cell-to-cell contact between patients' T cells and allogeneic accessory cells induced functional responsiveness to exogeneous IL-2.
Detection of HIV-1-infected cells from patients using nonisotopic in situ hybridization
Authors: Singer RH , Byron KS , Lawrence JB , Sullivan JL
Published: October 2019

We have demonstrated that a sensitive, nonisotopic in situ hybridization (ISH) assay can be used to detect HIV-infected cells from seropositive, asymptomatic individuals. Our assay is based on the detection of a biotinated HIV DNA probe hybridized to human immunodeficiency virus (HIV)-infected peripheral blood lymphocytes (PBL) using streptavidin and alkaline phosphatase to identify positive cells. This assay is rapid in that it can be performed within a day and is sensitive enough to unambiguously identify a rare, single, positive cell. Patient samples derived from HIV-seropositive hemophiliacs and HIV-seropositive infants were analyzed before and after coculture with normal PBL. The same samples were investigated using a Dupont P24 antigen-capture kit. It was found that ISH always detected the same positive samples as antigen capture, often in shorter times of coculture. In situ hybridization detected over half of our HIV-infected hemophilia patient population as virus positive, whereas the antigen capture assay detected less than one fourth as virus positive. In situ hybridization detected positive cells directly, without coculture, in 12 out of 35 (34%) hemophiliacs and in three out of eight (37%) infants. The speed, sensitivity, and confidence of ISH and nonisotopic detection indicates that it will be useful as a tool for clinical research and diagnosis.
Therapy of chronic idiopathic thrombocytopenic purpura in adults [see comments]
Authors: Berchtold P , McMillan R
Published: October 2019

Chronic ITP is a common hematologic illness. Approximately three fourths of the patients respond to corticosteroids or splenectomy and need no further treatment. Patients refractory to these two therapeutic approaches are relatively resistant to present forms of treatment and are at much greater risk for morbidity and mortality. Future clinical studies evaluating therapy in this refractory group would be best performed in a cooperative group setting in which large numbers of patients could be treated in a prospective randomized manner.
Recombinant human interleukin-3 and granulocyte-macrophage colony- stimulating factor show common biological effects and binding characteristics on human monocytes
Authors: Elliott MJ , Vadas MA , Eglinton JM , Park LS , To LB , Cleland LG , Clark SC , Lopez AF
Published: October 2019

Two human hemopoietic growth factors involved in monocytopoiesis, interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied for their ability to stimulate blood monocytes and to bind to the monocyte membrane. Both cytokines maintained monocyte/macrophage numbers during long-term culture and increased cell size as compared with controls. Effects on cell numbers were present at low cytokine concentrations (6 to 20 pmol/L), whereas enhanced 3H-thymidine incorporation was observed only at higher concentrations (greater than or equal to 60 pmol/L). Autoradiographic studies showed only 1% to 3% of stimulated monocytes with nuclear grains. These results suggest that the primary mechanism for IL-3 and GM-CSF-induced maintenance of monocyte/macrophage numbers in humans is through an effect on cell survival. Surface receptors for both IL-3 and GM-CSF were studied by using 125I-labeled recombinant human (rh) cytokines and performing Scatchard analyses. Both cytokines showed curvilinear Scatchard plots, and computer analyses favored a two-site binding model. High-affinity binding data for 125I rhIL-3 (Kd 7.7 to 38.2 pmol/L; receptor number/cell 95 to 580) and for 125I rhGM-CSF (Kd 4.7 to 38.9 pmol/L; receptor number/cell 8 to 67) show similar binding affinities for the two cytokines but a lower receptor number/cell for 125I rhGM-CSF. Low-affinity binding characteristics for 125I rhIL-3 (Kd 513 to 939 pmol/L; receptor number/cell 179 to 5,274) and for 125I rhGM- CSF (Kd 576 to 1,120 pmol/L; receptor number/cell 130 to 657) show a similar pattern for the two cytokines. Specificity of 125I rhIL-3 and 125I rhGM-CSF binding to monocytes was established by the ability of the homologous cytokine to inhibit binding and the inability of a range of other cytokines to compete at 100-fold excess molar concentration. It is important, however, that binding of 125I rhIL-3 was partially inhibited by rhGM-CSF and that rhIL-3 partially inhibited binding of 125I rhGM-CSF to the monocyte membrane under conditions shown to prevent receptor internalization. The degree of inhibition varied between 25% and 80% in different experiments, and quantitative inhibition experiments showed that 1,000-fold excess concentrations of competitor failed to inhibit binding of the heterologous ligand completely. These results demonstrate that human IL-3 and GM-CSF have similar effects on growth and survival of human monocytes in vitro and suggest that these and other common biological effects may be mediated either through a common receptor or through distinct receptors associated on the monocyte membrane.
Heterogeneous phenotypes of platelet and plasma von Willebrand factor in obligatory heterozygotes for severe von Willebrand disease
Authors: Mannucci PM , Lattuada A , Castaman G , Lombardi R , Colibretti ML , Ciavarella N , Rodeghiero F
Published: October 2019

To characterize the heterogeneity of severe (type III) von Willebrand disease (vWD), plasma and platelet von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor activity (Ricof) were measured in 28 obligatory heterozygotes (ie, parents or children of probands from 15 different kindreds with severe vWD). On the average, heterozygotes had low levels of vWF in both platelets and plasma. There was, however, considerable heterogeneity, with four distinct patterns. Eleven heterozygotes had concordant reduction of vWF:Ag and Ricof in both plasma and platelets; five had low levels of vWF:Ag and Ricof in plasma contrasting with normal levels in platelets; eight had a peculiar pattern, the reverse of the above (ie, low levels in platelets and normal levels in plasma); and in one, both vWF measurements were normal in plasma and platelets. These patterns were genetically determined: they were consistent in four couples of consanguineous heterozygotes and in two couples carrying the same gene deletion. Only the remaining three heterozygotes had no clearly identifiable pattern. Other findings of this study were that although most of the heterozygotes had normal bleeding times, the 7 of 28 who had prolonged bleeding times had concordantly low levels of vWF measurements in both plasma and platelets. In conclusion, this large series of obligatory heterozygotes provides evidence for phenotypic and genotypic heterogeneity of severe vWD.
The C-terminal sequences of the gamma 57.5 chain of human fibrinogen constitute a plasmin sensitive epitope that is exposed in crosslinked fibrin
Authors: Haidaris PJ , Peerschke EI , Marder VJ , Francis CW
Published: October 2019

The gamma chain of human plasma fibrinogen is heterogeneous with three forms differing in length at the C-terminus. Alternative RNA splicing produces two gamma chain mRNAs encoding gamma 50 and gamma 57.5 polypeptides, while fibrinogen gamma 55 is produced by post- translational modification of the gamma 57.5 chain. The composition of purified variant gamma chain fibrinogens, which comprise 10% to 13% total plasma fibrinogen, is predominantly heterodimeric (A alpha, B beta, gamma 50/gamma 55 or A alpha, B beta, gamma 50/gamma 57.5), whereas the composition of purified fibrinogen with the major form of the gamma chain is homodimeric (A alpha, B beta, gamma 50/gamma 50). These gamma chain variations interrupt sequences that mediate platelet- fibrinogen interactions. Therefore, the structure and function of gamma 57.5 C-terminal sequences were investigated using synthetic peptides and a specific monoclonal antibody (MoAb), L2B. The L2B epitope was localized and included gamma 57.5 chain residues 409–412 (Arg-Pro-Glu- His), as determined by differential enzyme-linked immunosorbent assay (ELISA) reactivity with a His-412 deleted synthetic peptide and by Western blot analysis of plasmin cleaved fibrinogen gamma 57.5. L2B had no effect on adenosine diphosphate (ADP)-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. High concentrations (0.5 to 1 mmol/L) of synthetic peptide gamma 57.5 405– 416 only weakly inhibited ADP-induced platelet aggregation supported by either fibrinogen gamma 50 or gamma 57.5. Binding of fibrinogen gamma 50 (IC50 = 780 mumol/L) or gamma 57.5 (IC50 = 650 mumol/L) to ADP- stimulated platelets was weakly inhibited, and MoAb L2B failed to inhibit fibrinogen gamma 57.5 binding. Peptide gamma 57.5 408–416 failed to dissociate platelet-bound fibrinogens. These data indicate that the gamma 408–416 sequence of fibrinogen gamma 55 or gamma 57.5 alone is unlikely to bind to the platelet fibrinogen receptor, glycoprotein llb-llla (GPllb-llla), in support of platelet aggregation under physiologic conditions. The sequence recognized by L2B does not resemble known GPllb-llla binding site peptide sequences [Arg-Gly-Asp- Ser (RGDS) or gamma 50 400–411] as determined by competitive inhibition ELISA comparing these binding site synthetic peptides with gamma 57.5 408–416. This epitope is available for binding MoAb L2B in gamma 55 or gamma 57.5 chain dimers and binds to all gamma 57.5 408–416 epitopes equally in non-crosslinked and factor Xllla crosslinked fibrin clots.(ABSTRACT TRUNCATED AT 400 WORDS).
Increased density of ecto 5' nucleotidase antigen on leukemic T cells from patients with cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma
Authors: Fukunaga Y , Evans SS , Yamamoto M , Ueda Y , Tamura K , Takakuwa T , Gebhard D , Allopenna J , Demaria S , Clarkson B
Published: October 2019

Malignant CD4+ T cells in adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphoma (CTCL) express a number of cell surface molecules that are upregulated on normal T cells activated by foreign antigen. In this report we describe an interesting exception to the parallel phenotypic features of activated T cells and malignant CD4+ T cells. A monoclonal antibody (MoAb; termed 27.2) that was raised to HTLV-1+, CD4+25+ leukemic T cells stained weakly 25% of peripheral T cells, including approximately 50% of CD8+ T cells and 20% of CD4+ T cells. Flow cytometry analysis indicated that the surface density of the 27.2 antigen was unchanged or diminished when normal T cells were activated by antigen. However, 3/4 Sezary cases and 4/8 cases of ATL had relatively high densities of the 27.2 antigen. Immunoprecipitation and sodium dodecylsulfate polyacrylamide gel electrophoresis of the NP- 40-solubilized membranes of surface-iodinated ATL cells indicated that MoAb 27.2 reacted with a 75 Kd molecule. The size and distribution of the 27.2 antigen on T cell subsets suggested that it might be the enzyme ecto-5′ nucleotidase (NT), a phosphatidylinositol-linked enzyme that catalyzes dephosphorylation of monophosphate nucleotides to their respective nucleosides. This was confirmed by demonstrating that lymphocyte ecto-5′NT activity was blocked partially and inhibited completely by preincubating cells with MoAb 27.2 for 1 hour at 4 degrees C and 24 hours at 37 degrees C, respectively. When used with a second MoAb (27.1) to a novel T cell activation antigen found on all CTCL and ATL leukemias examined, 27.2 was found to discriminate between normal and leukemic T cells in two patients with ATL. These studies suggest that ecto-5′NT has diagnostic value in T cell malignancies and may be aberrantly expressed in some cases of ATL and CTCL.
Identification and characterization of a novel monocyte subpopulation in human peripheral blood
Authors: Passlick B , Flieger D , Ziegler-Heitbrock HW
Published: October 2019

With the aid of two-color immunofluorescence and flow cytometry, a new subset of cells coexpressing CD14 and CD16 antigens can be identified in human peripheral blood. Using the monoclonal antibody My4, these CD14+/CD16+ cells account for 2.2% of the mononuclear cells and form about 13% of all cells identified by the monocyte-specific CD14 monoclonal antibody. The CD14+/CD16+ cells can be assigned to the monocyte lineage based on typical morphology, on expression of additional monocyte-associated molecules, on the ability to form reactive oxygen intermediates and on the expression of monocyte- specific NaF-sensitive esterase. Light scatter analysis revealed lower forward angle and right angle light scatter for the CD14+/CD16+ cells compared with the regular monocytes, and the average cell size was determined to be 13.8 and 18.4 microns, respectively. Expression of class II antigens on these “small monocytes” was twofold higher compared with the regular monocytes. By contrast, the capacity to perform adherence to plastic surfaces, as well as the ability to phagocytize antibody-coated erythrocytes was clearly reduced in the CD14+/CD16+ monocyte subset as compared with the regular monocytes. Hence the CD14+/CD16+ cells appear to represent a new monocyte subset with a distinct functional repertoire. A survey of various tissues revealed that a large proportion of the alveolar macrophages, but not of the peritoneal macrophages, express the CD14+/CD16+ phenotype.
Post-transcriptional regulation of glutathione peroxidase gene expression by selenium in the HL-60 human myeloid cell line
Authors: Chada S , Whitney C , Newburger PE
Published: October 2019

We have used a cloned cDNA for the major human selenoprotein, glutathione peroxidase (GPx), to assess the mode of regulation of human GPx gene (GPX-1) expression by selenium. When the HL-60 human myeloid cell line is grown in a selenium-deficient medium, GPx enzymatic activity decreases 30-fold compared with selenium-replete cells. Upon return to a medium containing selenium in the form of selenite, GPx activity in the cells starts to increase within 48 hours and reaches maximal (selenium-replete) levels at 7 days. Steady-state immunoreactive protein levels correlate with enzymatic activity. Cycloheximide inhibits the rise in GPx activity that accompanies selenium replenishment, indicating that protein synthesis is required for the increase. However, GPx mRNA levels and the rate of transcription of the human GPx gene change very little and thus appear to be independent of the selenium supply. Thus the human GPx gene appears to be regulated post-transcriptionally, probably cotranslationally, in response to selenium availability.
Membrane deformability and the capacity for shape change in the erythrocyte
Authors: Chasis JA , Schrier SL
Published: October 2019

Erythrocytes must have the capacity to undergo marked membrane deformation and shape changes in order to circulate through capillaries and respond to a range of shear stresses. To study the interrelationships between membrane deformability and the capacity for shape transformation, we created rigid membranes using several agents and then examined the ability of these erythrocytes with rigid membranes to undergo amphipath-induced shape change. We have previously shown that wheat germ agglutinin (WGA) and a monoclonal antibody to glycophorin A (R-10) cause membrane rigidity as measured by ektacytometry. Experiments were therefore designed to produced comparably rigid membranes using WGA, R-10, and diamide, and then to test the ability of lysophosphatidylcholine to produce echinocytes, and primaquine to produce stomatocytes. We found that diamide treatment substantially blocked both types of shape change. In contrast, R-10 binding did not impair either primaquine- or lysophosphatidylcholine- induce shape change. Further, WGA blocked echinocyte transformation, as previously reported, but not stomatocytosis. Using reduced and unreduced gel electrophoresis and Triton extraction, we compared the biochemical changes associated with WGA-, diamide-, and R-10-induced rigidity, and found them to be different. We conclude that not all rigid cells are incapable of shape change, and therefore that decreased membrane deformability is not predictive of impaired capacity for shape change.
Autologous bone marrow transplantation in follicular non-Hodgkin's lymphoma before and after histologic transformation
Authors: Schouten HC , Bierman PJ , Vaughan WP , Kessinger A , Vose JM , Weisenburger DD , Armitage JO
Published: October 2019

Patients with disseminated follicular non-Hodgkin's lymphoma (NHL) are only occasionally cured with standard chemotherapy regimens. Although most of these tumors are initially responsive to chemotherapy, in 40% to 70% of patients the lymphoma will eventually transform to an NHL of higher grade malignancy and a poorer prognosis. We treated 18 patients having an original diagnosis of follicular NHL with high-dose therapy and autologous bone marrow transplantation. The lymphomas of 10 of the patients had already undergone histologic transformation and eight still had a follicular histologic pattern. The former group had been followed for a longer time from the diagnosis of NHL and had been more extensively treated with conventional chemotherapy regimens. All eight patients with follicular NHL at the time of transplantation are alive for 246+ to 1,804+ days and seven of the patients are in complete remission. In contrast, of the 10 patients in histologic transformation only 1 is alive and in CR. This reflects the inability of these patients to tolerate the high-dose chemotherapy and myelosuppression as well as resistance of their lymphoma to this therapy. This difference in survival between the two groups was highly significant (P = .002). We conclude that the outcome of patients with follicular NHL transplanted early before histologic transformation is better than for those who are transplanted later in the course of their illness. Because of the relapsing behavior of follicular NHL, a longer follow-up is necessary to prove any impact on the natural history of the disease.
Identification through chemical cross-linking of distinct granulocyte- macrophage colony-stimulating factor and interleukin-3 receptors on myeloid leukemic cells, KG-1
Authors: Gesner T , Mufson RA , Turner KJ , Clark SC
Published: October 2019

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high- affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM- CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.
Granulocyte-macrophage colony-stimulating factor reactivates fetal hemoglobin synthesis in erythroblast clones from normal adults
Authors: Gabbianelli M , Pelosi E , Bassano E , Labbaye C , Petti S , Rocca E , Tritarelli E , Miller BA , Valtieri M , Testa U
Published: October 2019

Reactivation of fetal hemoglobin (HbF, alpha 2 gamma 2) synthesis was previously reported in normal human adult erythroblast colonies (“bursts”) generated by erythroid progenitors (BFU-E) in fetal calf serum-supplemented (FCS+) semisolid cultures stimulated with erythropoietin (Ep). Our studies focused on the reactivation of HbF synthesis in normal adult erythroid bursts generated by peripheral blood mononuclear cells (PBMCs) seeded in FCS+ methylcellulose culture. Reactivation is almost totally suppressed when (a) PBMCs are grown in optimized FCS- culture, or (b) PBMCs are first stringently depleted of monocytes and then plated in FCS+ medium (ie, BFU-E growth in FCS+ Mo- culture). In both experimental conditions, the proliferation of lymphocytes and macrophages interspersed among colonies is drastically reduced, and the cloning efficiency of granulocyte-macrophage (GM) progenitors is sharply diminished. In either case, addition of biosynthetic GM colony-stimulating factor (GM-CSF) induces a dose- related increase of HbF synthesis up to the level in FCS+ culture, with even more elevated values on delayed addition of Ep. A dose-related increase was also observed in erythroblast clones generated by highly purified BFU-E. These results suggest that reactivation of HbF synthesis in normal adults is at least in part mediated by GM-CSF. Furthermore, they imply intriguing hypotheses on the mechanism(s) of perinatal Hb switching. Finally, they raise the possibility of reactivation of HbF synthesis in beta-thalassemia and sickle cell anemia by GM-CSF therapy.