bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential suppression of myelopoiesis in normal human bone marrow cells after in vitro challenge with human cytomegalovirus
Authors: Sing GK , Ruscetti FW
Published: October 2019

The pathogenic effects of human cytomegalovirus (CMV) infection in vitro on hematopoiesis were investigated. Normal human bone marrow cells from both seronegative and seropositive donors were challenged with CMV (Towne or wild-type strain) and tested for their responsiveness to the recombinant hematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF), respectively. Regardless of the serostatus of the donor, infection with CMV resulted in a significant decrease in the proliferation and colony formation of hematopoietic progenitor cells in response to both growth factors, with more pronounced suppression in response to G-CSF being observed. Evaluation of the colony composition revealed a profound decrease in colonies of the granulocytic (CFU-G), or granulocyte-macrophage (CFU-GM) lineages, while suppression of multipotential (CFU-GEMM) and erythroid (BFU-E) colony-forming cells occurred after infection with wild-type but not the laboratory strain of CMV. Although no evidence of productive virus infection could be seen in colony-forming cells, in situ hybridization studies and immunohistochemical staining revealed the presence of CMV-specific mRNA and immediate-early antigens, demonstrating that a small proportion of cells were abortively infected. These studies demonstrate that CMV can infect bone marrow progenitor cells and interfere with normal hematopoiesis in vitro, which may help to explain the hematologic defects seen during acute infections with CMV in vivo.
Expansion of a lymphocyte population co-expressing T4 (CD4) and T8 (CD8) antigens in the peripheral blood of a normal adult male
Authors: Kay NE , Bone N , Hupke M , Dalmasso AP
Published: October 2019

Approximately 2% to 3% of circulating human T cells co-express CD4 and CD8 (CD4+, CD8+) T-cell antigens. These CD4+, CD8+ cells may be immature precursors that function to replenish functional T-cell subsets. We detected a very high level of CD4+, CD8+ cells in the peripheral blood lymphocytes of a healthy white male, ranging from 21% to 36%. The morphology of his peripheral blood lymphocytes was normal, and he has maintained an elevated level of CD4+, CD8+ cells without clinical disease over a 19-month observation period. The CD4+, CD8+ cells did not possess thymocyte membrane antigen (CD6), nor did they have increased Tac (CD25) antigen. His intact, purified blood T cells had a normal proliferative response to phytohemagglutinin and interleukin-2 (IL-2), provided help for B-cell proliferation at control levels and exposure to IL-2 resulted in generation of cytotoxic cells. However, the purified blood CD4+, CD8+ cells were deficient in these latter functions except for help in B-cell function. Despite defective function, the isolated CD4+, CD8+ cells co-expressed CD2 and CD3. Prolonged in vitro culture of CD4+, CD8+ cells was possible in the presence of recombinant IL-2. The cultured CD4+, CD8+ cells retained the double antigens (CD4 and CD8) throughout a 4-week period. It is likely these cells are less mature than CD4+, CD8- or CD4-, CD8+ T cells as the CD4+, CD8+ have less in vitro function than the former cells, but it is not yet clear if they mature into either CD4+, CD8-, or CD4-, CD8+ cells. Finally, the presence of an expanded, hypofunctioning CD4+, CD8+ cell population in a normal adult male is apparently compatible with excellent clinical health.
Assignment of Sp alpha I/74 hereditary elliptocytosis to the alpha- or beta-chain of spectrin through in vitro dimer reconstitution
Authors: Pothier B , Alloisio N , Marechal J , Morle L , Ducluzeau MT , Caldani C , Philippe N , Delaunay J
Published: October 2019

Partial digestion of spectrin dimers in vitro has allowed the definition of domains. For example, the portions of the dimers that are involved in spectrin self-association are represented by the alpha I and the beta I domains. The alpha I domain (80 Kd) is further cleaved into a minor 78 Kd fragment and, more substantially, into a 74 Kd fragment. The intensity of the latter, which we expressed as the 74:(80 + 78 + 74) ratio, or the 74:alpha I ratio, is variable depending on the experimental conditions, eg, in fine, on the conformation of the alpha I domain. A number of cases of hereditary elliptocytosis (HE) are associated with an increase of the 74:alpha I ratio, also referred to as the Sp alpha I/74 abnormality. Several lines of evidence have suggested that the causal mutations may lie in the alpha- or the beta- chain, a point of importance before one undertakes studies at the gene level. In order to address this question, we reconstituted spectrin dimers in vitro, combining alpha- and beta-chains of various origins, and then carried out partial digestion and assayed the Sp alpha I/74 abnormality. The patterns obtained with reconstituted dimers were nearly identical to those of native dimers. We applied the assay to three spectrin variants that cause Sp alpha I/74 HE: (1) a variant that we previously designated spectrin Nice and whose beta-chain lacks a 4 Kd fragment in its C-terminal region; and two distinct variants that we found in two unrelated white families and that we provisionally designated spectrin Lyon and spectrin Culoz. The Sp alpha I/74 abnormality appeared in all kinds of dimers that harbored the beta- chain of spectrin Nice, or the alpha-chain of spectrin Lyon or spectrin Culoz, respectively. Therefore, we confirmed that spectrin Nice is a (alpha I/74) beta-variant, and established that both spectrin Lyon and spectrin Culoz are (alpha I/74) alpha-variants. The present assay may be extended to any spectrin variant displaying the Sp alpha I/74 abnormality.
Western blot identification of platelet proteins that bind normal serum immunoglobulins. Characteristics of a 95-Kd reactive protein
Authors: Reid DM , Jones CE , Vostal JG , Shulman NR
Published: October 2019

We have found that Western blots (WBs) of whole platelets exposed to normal autologous or homologous sera commonly have bands at 90 to 95 (95) Kd, and less often at 100 to 110, 80 to 85, 60 to 75, and 50 to 60 Kd when developed with antiglobulins. The percentages of normal sera producing a 95-Kd band with anti-immunoglobulin G (IgG), -IgA, and -IgM are 85, 50, and 30, respectively. Antiglobulin reagents alone also produce background bands on WBs that we have shown correspond to levels of platelet-associated Igs (PAIgs) or their derivatives. Titers of 95 Kd-reactive IgG in normal sera range from 10 to 1,280 (85% less than or equal to 50), and the reaction appears to be partially F(ab')2- mediated. The 95-Kd protein is internal and differs in many respects from surface glycoproteins IIIa, IV, and V of similar apparent molecular weight. In thrombocytopenic patients there was no correlation between severity of thrombocytopenia or PAIgG of platelet eluates and corresponding serum titers of 95 Kd-reactive IgG. Some WB reactions previously reported as evidence of autoimmunity may represent normal variations in reactions of Igs with internal platelet proteins. These reactions may be immunospecific or analogous to nonspecific, partially F(ab')2-dependent binding of Igs by certain bacterial proteins.
Hepatosplenic T-cell lymphoma: sinusal/sinusoidal localization of malignant cells expressing the T-cell receptor gamma delta
Authors: Farcet JP , Gaulard P , Marolleau JP , Le Couedic JP , Henni T , Gourdin MF , Divine M , Haioun C , Zafrani S , Goossens M
Published: October 2019

Peripheral T-cell lymphomas consist of a clinically heterogeneous group of malignant disorders whose immunophenotype usually corresponds to that of normal mature T cells. We describe and correlate the clinical, histopathologic, phenotypic, and genotypic findings in two patients with malignant lymphoma presenting with hepatosplenic disease. The morphologic pattern of lymphoma was that of a sinusal/sinusoidal infiltration in spleen, marrow, and liver. This morphologic characteristic was associated with the presence of a productive clonal rearrangement of the T-cell receptor (TCR) delta gene. Lymphoma cells expressed a CD3-TCR-gamma delta- phenotype. They were also double negative (ie, CD4-CD8-) and lacked the CD5 and CD7 antigens. In one patient, tumor progression was associated with phenotypic changes that resulted in a CD3-TCR-gamma delta- phenotype with the same delta-gene rearrangement as initially. These observations suggest the existence of a new type of peripheral T-cell lymphoma characterized by its hepatosplenic presentation, and by the sinusal/sinusoidal tropism and the TCR-gamma delta phenotype of the malignant cells.
BAVC regimen and autologous bone marrow transplantation in patients with acute myelogenous leukemia in second remission
Authors: Meloni G , De Fabritiis P , Petti MC , Mandelli F
Published: October 2019

Twenty-one acute myelogenous leukemia (AML) patients were submitted in second remission (II CR) to BAVC conditioning regimen followed by unpurged ABMT. Transplant was done after a median of 2 months from II CR (range, 1 to 13). Median first remission (I CR) duration was 16 months (range, 1–35). Conditioning regimen was well tolerated, with no major extra-medullary toxicity. One patient died during aplasia from fungal sepsis. Of the 20 evaluable patients, nine relapsed after a median time of 6 months (range, 2 to 18). Eleven patients are in continuous complete remission (CCR) after a median follow-up of 40 months (range, 24 to 63). The duration of II CR has exceeded the duration of I CR in all patients in CCR. Projected probability of disease-free survival is 52% at 63 months.
Interleukin-6 enhances murine megakaryocytopoiesis in serum-free culture
Authors: Koike K , Nakahata T , Kubo T , Kikuchi T , Takagi M , Ishiguro A , Tsuji K , Naganuma K , Okano A , Akiyama Y
Published: October 2019

We investigated the effect of interleukin-6 (IL-6) on murine megakaryocytopoiesis in a serum-free culture system. The addition of IL- 6 to a culture containing interleukin-3 (IL-3) resulted in a significant increase in the number of megakaryocyte colonies by bone marrow cells of normal mice. The megakaryocytic progenitors that survive exposure to 5-fluorouracil (5-FU) exhibited a more significant response to IL-6 and IL-3. Polyclonal anti-IL-6 antibody neutralized the stimulatory effect of IL-6 on megakaryocyte colony growth supported by IL-3. Delayed addition experiments and replating experiments of blast cell colonies showed that megakaryocytic progenitors are supported by IL-3 in the early stage of the development but require IL- 6 for their subsequent proliferation and differentiation. In addition, IL-6 increased the size of megakaryocytes in granulocyte-macrophage- megakaryocyte colonies. The combination of granulocyte colony- stimulating factor or granulocyte-macrophage colony stimulating factor with IL-3 resulted in an increase in the granulocyte-macrophage colony growth of bone marrow cells of 5-FU-treated mice or normal mice, respectively, but had little effect on the enhancement of pure and mixed megakaryocyte colony growth. These results suggest that IL-6 plays an important role in murine megakaryocytopoiesis.
Identification and characterization of a new surface membrane antigen found predominantly on malignant B lymphocytes
Authors: Gingrich RD , Dahle CE , Hoskins KF , Senneff MJ
Published: October 2019

A monoclonal antibody, 1D10, was derived that identifies a new antigenic epitope on the surface of malignant B lymphocytes. Normal resting and stimulated lymphocytes do not express the antigen. The majority of individuals with acute Epstein-Barr virus infection express the antigen on their lymphocytes, and in these patients, the T lymphocyte may also be antigen positive. The antigen was found on B- lymphoid neoplasia from the early pre-B cell stage through terminally differentiated plasma cells, a characteristic not reported for other B cell-associated antigens. Studies on homozygous typing cells and cells from individuals with known HLA phenotypes indicate that the antigen does not segregate in a pattern characteristic for major histocompatibility antigens. The molecule is a heterodimeric polypeptide with the molecular weight and isoelectric points of the alpha and beta chains being 32,000 d/4 and 28,000 d/6, respectively. Evidence is presented that the 1D10 molecule is not HLA-DR, -DP, or - DQ. By extrapolation, we suggest that this novel molecule may represent HLA D-region gene expression of a gene(s) not normally expressed. Potential candidates are D-region pseudogenes. We conclude that the antigenic epitope identified by the 1D10 monoclonal antibody is unique among previously described B-lymphocyte antigens. Further studies of the factors controlling the expression of this molecule, as well as studies designed to look at the possible cellular function, may provide insights for understanding crucial events in the malignant transformation of lymphocytes.
Expression of the CD34 gene in vascular endothelial cells
Authors: Fina L , Molgaard HV , Robertson D , Bradley NJ , Monaghan P , Delia D , Sutherland DR , Baker MA , Greaves MF
Published: October 2019

All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.
Induction of thymidine kinase activity and clonal growth of certain leukemic cell lines by a granulocyte-derived factor
Authors: Ho CK , Ou BR , Hsu ML , Su SN , Yung CH , Wang SY
Published: October 2019

Normal polymorphonuclear neutrophils (PMNs) constitutively secrete a mediator designated granulocyte-derived factor (GDF) that can enhance the uptake of 3H-thymidine (3- to 20-fold) by the molt-3, CTV-1, and K562 leukemic cell lines in a dose-dependent manner. GDF is heat labile (56 degrees C for 30 minutes) and acid labile (pH 2.0) and is sensitive to treatment with bacterial protease type IV. Our preliminary studies suggest that GDF is non-dialyzable (molecular weight cutoff, 12,000), binds to diethylaminoethyl (DEAE), and has an apparent molecular weight (mol wt) of about 40 Kd. Production of GDF is unaffected by treatment of PMN with activating agents (interferon gamma, OK432, phorbol ester, calcium ionophore, poly I:C) or metabolic inhibitors (actinomycin-D and cyclohexamide), suggesting that GDF is constitutively secreted. Despite the marked enhancement of 3H-thymidine uptake, cell number and the rate of DNA synthesis in GDF responsive cultures remain unchanged. In contrast, the clonogenic efficiency of the responsive cells is greatly increased in the presence of GDF. These phenomena occur in parallel to an amplification of the level of thymidine kinase activity in the sensitive cells. GDF is distinct from a panel of different lymphokines and monokines in antigenicity and biochemical and functional characteristics, and is possibly a novel cytokine that can alter the pattern of DNA synthesis and growth characteristics of certain hematopoietic cells. However, its biologic and physiologic significance remains to be determined.
Ca2+ permeability in deoxygenated sickle cells
Authors: Rhoda MD , Apovo M , Beuzard Y , Giraud F
Published: October 2019

Deoxygenation of sickle cells is known to increase cation permeabilities (Na+, K+, and Ca2+). The possible mechanisms involved in the increased uptake of Ca2+ were investigated: activation of Ca2+ channels, involvement of the anion channel, and the formation of endocytic vacuoles. The Ca2+ channel blocker nifedipine reduced the deoxy-stimulated Ca2+ uptake by about 30% to 40%. The anion channel inhibitor DIDS (4,4′ diisothiocyanate stilbene 2,2′ disulfonate) inhibited the deoxy-stimulated Ca2+ uptake by approximately 50%. Maximal possible endocytic uptake, measured by using an impermeant marker ([3H] inuline), accounted for 6% to 9% of the total Ca2+ uptake. These data indicate that the deoxygenation-induced increase in Ca2+ permeability could result from both the activation of a Ca2+ channel and of a transport system for cations involving interactions between polymerized hemoglobin S, band 3 and other membrane components. Endocytosis appears to play only a minor role in the Ca2+ uptake of deoxygenated sickle cells.
Randomized prospective trial comparing the native prothrombin antigen with the prothrombin time for monitoring oral anticoagulant therapy
Authors: Furie B , Diuguid CF , Jacobs M , Diuguid DL , Furie BC
Published: October 2019

The dosage of the anticoagulant warfarin sodium is based upon the prolongation of the prothrombin time into an optimal therapeutic range. We have developed a new assay for the native prothrombin antigen that measures the fully gamma-carboxylated prothrombin using a radioimmunoassay. Based on preliminary data that indicated that the native prothrombin antigen predicted both bleeding and thrombotic complications more accurately than the prothrombin time in patients anticoagulated with warfarin sodium, we have performed a randomized prospective trial comparing the complication rate in warfarin-treated patients monitored with the native prothrombin antigen or the prothrombin time. Patients with indications for anticoagulation were randomized to be monitored by the native prothrombin antigen (therapeutic range, 12 to 24 micrograms/mL) or the prothrombin time index (therapeutic range, 1.5 to 2.0). Of the prothrombin time group (N = 80), seven (8.8%) had bleeding or thrombotic complications, with a complication rate of 9.5%/patient-year. In the native prothrombin antigen group (N = 76), one subject (1.3%) had a bleeding complication. The complication rate per patient-year was 1.5%. These results indicate an 85% reduction in the complication rate of the native prothrombin antigen group compared with the complication rate of the prothrombin time group. This difference is statistically significant by the Fisher exact test (P = .037) and by Kaplan Meier survival analysis (P = .040). This study suggests that the use of the native prothrombin antigen assay has the potential to decrease the complications associated with anticoagulation therapy with warfarin sodium.
Alteration of N-ras gene mutation after relapse in acute lymphoblastic leukemia
Authors: Terada N , Miyoshi J , Kawa-Ha K , Sasai H , Orita S , Yumura-Yagi K , Hara J , Fujinami A , Kakunaga T
Published: October 2019

We investigated N-ras activation in childhood acute lymphoblastic leukemia (dALL) by the polymerase chain reaction (PCR) and the oligonucleotide hybridization method. The frequency of point-mutation of the N-ras gene was not high (2 of 15), and one positive case who relapsed was analyzed in detail. Although N-ras gene activation was detected at both onset and relapse, the mutation sites were different. At onset, Gly (GGT) was changed to Ser (AGT) at codon 12, and at relapse, Gly (GGT) to Asp (GAT) was observed at the same codon. In addition, the DNA at relapse showed a remarkably higher transforming activity than the DNA at onset on two independent recipient cell lines. The identical cell surface phenotype and the same rearrangement patterns of both the immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) gamma chain genes indicated that the leukemic cells at onset and those at relapse were derived from the same precursor cell. Therefore, this case supports the concept that ras activation is not the event initiating leukemogenesis, but may be involved in leukemic progression.
Occurrence of cytochrome b558 in B-cell lineage of human lymphocytes
Authors: Kobayashi S , Imajoh-Ohmi S , Nakamura M , Kanegasaki S
Published: October 2019

Cytochrome b558, involved in superoxide generation in phagocytes, was found to be expressed on the cell surface of most normal peripheral B lymphocytes. The cytochrome was not found on the surface of peripheral T lymphocytes, natural killer cells, or peripheral lymphocytes derived from patients with X-linked chronic granulomatous disease. On stimulation, at least half of peripheral B lymphocytes could generate superoxide anion as detected by superoxide dismutase-sensitive nitroblue tetrazorium reduction. Cytochrome b558 was not present on the surface of pre-pre B cells or pre-B cells, but did appear at the early B-cell stage. It disappeared from the B-cell surface during terminal differentiation to plasma cells. The transient expression of the cytochrome in B-cell lineage may indicate that superoxide generation is important for the function of these cells at certain stages.
Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury
Authors: La Celle P , Blumenstock FA , McKinley C , Saba TM , Vincent PA , Gray V
Published: October 2019

Plasma fibronectin augments the clearance of blood-borne foreign and effete complexes by mononuclear phagocytes. The release of a “gelatin- like” ligand into plasma after thermal injury has been reported. We quantified the release of this collagenous debris from thermally injured skin, and its potential interaction with soluble fibronectin in plasma using anesthetized rats. Collagen-like material debris in the plasma was detected by assay of hydroxyproline. Fibronectin was measured by a double antibody enzyme-linked immunosorbent assay (ELISA) technique. Over a 24-hour postburn interval, plasma hydroxyproline increased from 6.7 +/- 0.6 micrograms/mL to a maximum of 19.0 +/- 3.3 micrograms/mL at 60 minutes postburn, and normalized by 6 hours. A direct correlation existed between the magnitude of burn injury and the increase in plasma hydroxyproline. In parallel, plasma fibronectin declined over a 15-minute to 2-hour period postburn, and normalized by 3 to 4 hours with rebound hyperfibronectinemia observed at 24 hours. The elevation in total plasma hydroxyproline was not due to an increase in plasma Clq (zero time, 26.2 +/- 1.4 micrograms/mL; 60 minutes, 23.9 +/- 1.1 micrograms/mL). Tracer studies with 125I-fibronectin showed that the acute decline of plasma fibronectin was due to its uptake by the liver and binding to sites of tissue injury. Total hydroxyproline in extracts of burn skin, used as an index of soluble collagenous material, rose from 15 +/- 3.3 micrograms/g skin at zero time to 129.3 +/- 43.7 micrograms/g skin by 5 minutes postburn, with a decline to 38 +/- 22 micrograms/g skin by 24 hours. The formation of circulating fibronectin-gelatin complexes in vivo was documented by cross- immunoelectrophoresis coupled with autoradiography using 125I-gelatin as a model ligand. Thus, collagenous tissue debris from burned skin may enter the plasma after thermal injury and directly complexes with soluble fibronectin before hepatic phagocytic clearance.
Expression of major blood group antigens on human erythroid cells in a two phase liquid culture system
Authors: Wada H , Suda T , Miura Y , Kajii E , Ikemoto S , Yawata Y
Published: October 2019

In order to examine the sequential expression of major blood group antigens on human erythroblasts, a selective two phase liquid culture system for erythroid progenitors was established. After mononuclear cells obtained from peripheral blood were cultured in the presence of phytohemagglutinin stimulated-leukocyte conditioned medium (PHA-LCM) for 7 days (the first phase), nonphagocytic cells were recultured under hypoxic culture conditions containing 30% fetal calf serum, 1% bovine serum albumin, 300 micrograms/mL transferrin and 2 U/mL recombinant erythropoietin (the second phase). Mature (orthochromatic) erythroblasts were observed on day 4 of the second phase, and reached 57.1 +/- 3.1% of total cells on day 8, followed by the appearance of denucleated red cells, equivalent to mature red cells in peripheral blood. Hemoglobin contents reached the level of 16.8 +/- 0.7 micrograms/10(6) cells on day 8. Flow cytometric analyses revealed that, on day 3 of the second phase, cells became blood type M-positive, corresponding to the maturation of erythroid cells. Regarding the expression of ABH blood group antigens, a small number of blood type H- positive cells were initially detected on day 0 of the second phase, while blood type A-positive cells, which essentially were not observed on day 0, increased gradually corresponding to the extent of erythroid maturation. In the present system, Lewis and P1 blood group antigens were expressed at day 5 of the second phase, although autologous plasma was required to determine the expression of Lewis blood group antigens. This culture system is beneficial for studies on normal and abnormal human red cell membranes, because the erythroid progenitors in human peripheral blood were used, and a reasonable number of erythroid cells (0.5 to 1.5 x 10(7] was obtained with good maturation.
GMP-140 mediates adhesion of stimulated platelets to neutrophils
Authors: Hamburger SA , McEver RP
Published: October 2019

In vivo, platelets associate with neutrophils at sites of hemorrhage or inflammation. In vitro, stimulated platelets bind to neutrophils in a Ca2(+)-dependent manner. GMP-140, an integral membrane glycoprotein found in secretory granules of platelets and endothelium, is rapidly translocated to the cell surface after cellular activation. It shares sequence similarity with two leukocyte adhesion molecules, ELAM-1 and a lymphocyte homing receptor. We have recently shown that neutrophils bind to purified GMP-140 in a Ca2(+)-dependent fashion, and that GMP- 140 participates in adhesion of neutrophils to activated endothelium. In this study we demonstrate that GMP-140 also mediates adhesion of neutrophils to stimulated platelets. Fixed thrombin-activated human platelets, but not unstimulated platelets, formed rosettes around neutrophils in the presence of Ca2+. The binding of platelets to neutrophils was inhibited by a monoclonal antibody to GMP-140 and by purified GMP-140. By promoting close cell-cell contact, GMP-140 may recruit both platelets and neutrophils to sites of tissue injury as well as modulate the function of each cell type by the other.
A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor
Authors: Molineux G , Pojda Z , Dexter TM
Published: October 2019

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) induces leukocytosis in vivo in both intact and splenectomized mice. Full dose response data showed a plateau in this effect at doses over 500 micrograms rhG-CSF/kg body weight/d in intact mice. The effect is magnified in splenectomized mice, where leukocyte numbers reach 100 x 10(6) mL after 4 days' treatment at 250 micrograms/kg/d. Further hematopoietic precursor populations are also affected in both marrow and the spleen; in general, marrow parameters were depressed, while splenic populations were enlarged. In splenectomized mice, both blood- borne stem cells were enhanced, and foci of extramedullary hematopoiesis were enlarged in addition to the effects seen in intact mice. In the marrow of splenectomized and intact mice treated with a high dose of G-CSF, erythroid suppression in the marrow was confirmed with radioactive iron. Our studies confirm and extend previous work on the mode of action of G-CSF, and indicate that side effects of high dose G-CSF therapy might include erythroid suppression in the bone marrow.
Tumor cells are the site of erythropoietin synthesis in human renal cancers associated with polycythemia
Authors: Da Silva JL , Lacombe C , Bruneval P , Casadevall N , Leporrier M , Camilleri JP , Bariety J , Tambourin P , Varet B
Published: October 2019

One to five percent of human renal cell carcinomas are associated with polycythemia. It is generally assumed that polycythemia results from the secretion of erythropoietin (Epo) by the malignant cells. However, there is no direct proof supporting this hypothesis. Three patients with typical renal adenocarcinoma and polycythemia were studied. All three exhibited high Epo serum levels as measured by radioimmunoassay (RIA). A strong Epo signal was observed on Northern blot analysis of total RNA extracted from the renal tumors. The Epo message seemed to be of normal size and no Epo gene rearrangement was observed with the restriction enzymes tested. Using the in situ hybridization technique, a significant labeling was constantly observed on the tumor cells. Immunohistochemical studies showed that these tumor cells, known to be of tubular origin, were labeled by an anti-cytokeratin antibody and therefore were of epithelial nature. Thus, this study demonstrated that malignant cells of tubular origin were able to produce Epo constitutively, whereas in the mouse hypoxic kidney, peritubular cells (probably capillary endothelial cells) were the major site of Epo synthesis.
Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation
Authors: Keller JR , Mcniece IK , Sill KT , Ellingsworth LR , Quesenberry PJ , Sing GK , Ruscetti FW
Published: October 2019

We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1- negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5- fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.
Synergy between transforming growth factor-beta and tumor necrosis factor-alpha in the induction of monocytic differentiation of human leukemic cell lines
Authors: De Benedetti F , Falk LA , Ellingsworth LR , Ruscetti FW , Faltynek CR
Published: October 2019

We examined the effect of transforming growth factor-beta (TGF-beta) alone and in combinations with other factors on the growth and differentiation of the human promyelocytic cell line HL60 and the human monoblastic cell line U937. Treatment with TGF-beta alone did not significantly affect growth or differentiation of HL60 cells, while it significantly inhibited proliferation and induced monocytic differentiation of a small percentage of U937 cells. Combinations of TGF-beta and tumor necrosis factor-alpha (TNF-alpha) acted in synergy to inhibit cell proliferation and to induce monocytic differentiation of both HL60 and U937 cells. In contrast, no synergy was observed when HL60 cells were treated with TGF-beta in various combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and retinoic acid. Examination of TNF-alpha receptor expression on HL60 and U937 cells showed that these cell lines expressed comparable levels of high-affinity TNF-alpha binding sites. Treatment of HL60 and U937 cells with TGF-beta did not induce significant changes in TNF-alpha receptor expression in either cell line. In contrast, HL60 cells expressed much lower levels of TGF-beta receptors than did U937 cells. Treatment of both HL60 and U937 cells with TNF-alpha induced a dose-dependent increase in expression of TGF-beta receptors, suggesting that the synergy between TNF-alpha and TGF-beta may result, at least in part, from upregulation of TGF-beta receptor expression by TNF-alpha.
In vivo radioprotective effects of recombinant human granulocyte colony- stimulating factor in lethally irradiated mice
Authors: Uckun FM , Souza L , Waddick KG , Wick M , Song CW
Published: October 2019

The purpose of this study was to investigate the in vivo radioprotective effects of recombinant human granulocyte colony stimulating factor (rhG-CSF) in lethally irradiated BALB/c mice. We initially analyzed the effects of increasing doses of rhG-CSF on survival of mice receiving 700 cGy (LD100/30) single dose total body irradiation (TBI). While 1 microgram/kg to 100 micrograms/kg doses of rhG-CSF were not radioprotective, a dose-dependent radioprotection was observed at 200 micrograms/kg to 4,000 micrograms/kg rhG-CSF. We next compared four different rhG-CSF treatment regimens side by side for their radioprotective effects in LD100/30 irradiated mice. One hundred percent of control mice receiving phosphate buffered saline died within 21 days after TBI with a median survival of 14 days. The median survival was prolonged to 20 days and the actuarial 60-day survival rate was increased to 27% when mice received 2,000 micrograms/kg rhG- CSF 24 hours before TBI (P = .0002; Mantel-Peto-Cox). Similarly, the median survival time was prolonged to 24 days and the actuarial 60-day survival rate was increased to 33%, when mice were given 2,000 micrograms/kg rhG-CSF 30 minutes before TBI. Optimal radioprotection was achieved when 2,000 micrograms/kg rhG-CSF was administered in two divided doses of 1,000 micrograms/kg given 24 hours before and 1,000 micrograms/kg given 30 minutes before TBI. This regimen prolonged the median survival time of LD100/30 irradiated mice to more than 60 days and increased the actuarial 60-day survival rate to 62% (P = .0001; Mantel-Peto-Cox). By comparison, no survival advantage was observed when mice received rhG-CSF 24 hours post-TBI. Similar radioprotective effects were observed when mice were irradiated with 650 cGy (LD80/30). The presented findings provide conclusive evidence that rhG-CSF has significant in vivo radioprotective effects for mice receiving LD100/30 or LD80/30 TBI.
R68070, a combined thromboxane/endoperoxide receptor antagonist and thromboxane synthase inhibitor, inhibits human platelet activation in vitro and in vivo: a comparison with aspirin
Authors: Hoet B , Falcon C , De Reys S , Arnout J , Deckmyn H , Vermylen J
Published: October 2019

We have investigated the effects of R68070 on platelet function in vitro and in vivo. The drug inhibits U46619-induced aggregation (IC50 = 1.2 x 10(-6) mol/L), blocks serum thromboxane formation (IC50 = 1 x 10(- 7) mol/L), and increases serum prostaglandin (PG)E2 and 6-keto-PGF1 alpha levels, indicating that it combines thromboxane receptor blocking and thromboxane synthase inhibiting properties. The thromboxane- dependent aggregation of blood platelets is blocked by R68070, whereas no inhibition of thromboxane independent pathways occurs. A double- blind, randomized, cross-over study was performed on nine volunteers, comparing 400 mg placebo, 400 mg aspirin, and 400 mg R68070. Thromboxane-dependent aggregations were significantly inhibited by R68070 and by aspirin, the latter still having the most pronounced action. However, R68070 was clearly more powerful than aspirin (P less than .0005) in prolonging the bleeding time. Serum TxB2 formation was completely inhibited with both treatments, whereas serum 6-keto-PGF1 alpha and PGE2 and intralesional 6-keto-PGF1 alpha were inhibited after aspirin and stimulated after R68070. We conclude that R68070 inhibits platelet thromboxane synthase and its thromboxane receptor both in vitro and in vivo; local reorientation of cyclic endoperoxide metabolism toward prostacyclin induces a stronger inhibition of hemostasis than that produced by aspirin.
Complexing of tissue plasminogen activator with PAI-1, alpha 2- macroglobulin, and C1-inhibitor: studies in patients with defibrination and a fibrinolytic state after electroshock or complicated labor
Authors: Bennett B , Croll A , Ferguson K , Booth NA
Published: October 2019

Release of tissue plasminogen activator (t-PA) and its interaction with plasma protease inhibitors were studied in two patients with massive defibrination, one after electroshock and soft tissue injury and the other after complicated labor; both had very severe hemorrhage. Large quantities of free t-PA were present in the circulation for several hours. Complexes of t-PA with plasminogen activator inhibitor 1 (PAI- 1), alpha 2-macroglobulin and C1-inhibitor were also observed. PAI-1 antigen rose dramatically in both patients, and complexes of t-PA with PAI-1 rose rapidly during the period of observation. In contrast, the complexes of t-PA with alpha 2-macroglobulin and C1-inhibitor, present initially, persisted for short periods only and disappeared when free t- PA disappeared from the circulation. Plasmin was generated initially, as indicated by the presence of plasmin-alpha 2-antiplasmin complexes. Plasma concentrations of alpha 2-macroglobulin, C1-inhibitor, antithrombin III, and alpha 2-antiplasmin were severely depleted initially, but rapidly returned to normal. The observations demonstrate that there is a major release of t-PA in such defibrinating patients, that there is a role for protease inhibitors other than PAI-1 in the regulation of endogenous t-PA, and indicate the great rapidity with which such free t-PA is complexed and cleared.
A new human B-cell non-Hodgkin's lymphoma cell line (Karpas 422) exhibiting both t (14;18) and t(4;11) chromosomal translocations
Authors: Dyer MJ , Fischer P , Nacheva E , Labastide W , Karpas A
Published: October 2019

A unique B-cell non-Hodgkin's lymphoma (NHL) cell line (Karpas 422), bearing both t(14;18) and t(4;11) chromosomal translocations as well as several other chromosomal abnormalities, has been established from the pleural effusion of a patient with chemotherapy-resistant NHL. This cell line has the same karyotypic features as malignant cells from the patient. The major cell clone is characterized chromosomally by 46,XX t(2;10)(p23;q22.1), t(4;11)(q21.3; q23.1), t(14;18)(q32.1;q21.3), t(4;16)(q21.3;p13.1). Both phenotypically and genotypically, the cell line has features of a mature B-cell neoplasm with no evidence for commitment to other lineages. Rearrangements of the C-ETS-1 oncogene and N-CAM-1 and CD3 genes that map to 11q23 were not detected by conventional Southern analysis. BCL-2 was rearranged within the major breakpoint cluster. The K422 cell line has a unique karyotype; this is the first occasion that the t(4;11) translocation has been described in a t(14;18) lymphoma. The cell line will be of value in determining the molecular nature of the t(4;11) translocation.
Effect of glucocorticoids on the biologic activities of myeloma cells: inhibition of interleukin-1 beta osteoclast activating factor-induced bone resorption
Authors: Ishikawa H , Tanaka H , Iwato K , Tanabe O , Asaoku H , Nobuyoshi M , Yamamoto I , Kawano M , Kuramoto A
Published: October 2019

Regulatory effects of glucocorticoids (dexamethasone) on myeloma cells as well as bone resorption in multiple myeloma were investigated. Glucocorticoids significantly inhibited proliferation of myeloma cells, and decreased the messenger RNA (mRNA) expressions of interleukin-6 (IL- 6) and secretory type immunoglobulin G (IgG). The inhibitory effects of glucocorticoids on myeloma cell proliferation could be due to the decreased expression of IL-6 mRNA, decreased IL-6 production, and thus suppression of autocrine growth by IL-6, which is an autocrine growth factor for myeloma cells as reported previously (Nature 332:83, 1988). Glucocorticoids also inhibited M-protein secretion by decreasing the levels of secretory type Ig mRNA. On the other hand, because IL-1 beta rather than lymphotoxin is considered to be a major osteoclast activating factor (OAF) produced by myeloma cells, and glucocorticoids decreased the expression of IL-1 beta mRNA and markedly suppressed the bone resorbing activity induced by IL-1 beta OAF in 45Ca-release bone resorption assay, it is suggestive that glucocorticoids could inhibit bone resorption induced by IL-1 beta OAF in multiple myeloma. Therefore, from these data it is concluded that glucocorticoids could be more effective chemotherapeutic agents in multiple myeloma than we expected, especially with regards to the inhibitory effects on proliferation and M-protein secretion from myeloma cells, as well as bone resorption by myeloma cells.
Biochemical characterization of the neutrophil-specific antigen NB1
Authors: Stroncek DF , Skubitz KM , McCullough JJ
Published: October 2019

Neutrophil-specific alloantibodies and the antigens they recognize are important in clinical medicine, but little is known about the structure of these antigens. Alloimmunization to the antigen NB1 is a clinically important cause of neonatal neutropenia and febrile transfusion reactions. To study the immunochemistry of the NB1 antigen, we prepared neutrophil plasma membranes and granules by nitrogen cavitation and differential centrifugation and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with alloantibodies to several neutrophil-specific antigens. Two different antisera to the neutrophil-specific antigen NB1 identified an approximately 55-Kd protein by immunoblotting on neutrophil membranes from four NB1-positive donors but not on neutrophil membranes from five NB1-negative donors. Four anti-NB1 antisera immunoprecipitated a 58- to 64-Kd protein from extracts of NB1- positive neutrophils surface-labeled with 125I using lactoperoxidase, but not from similarly treated NB1-negative neutrophils. Normal human serum did not immunoprecipitate or immunoblot any proteins from these same neutrophil preparations. The NB1 antigen was detected by immunoblotting in secondary granules but was not found in primary granules. The electrophoretic mobility of the antigen was decreased slightly by reduction, suggesting that intrachain disulfide bonds were present. After reduction, the antigen could no longer be recognized by anti-NB1 antisera, but treatment of the antigen with periodate had no effect on the ability of anti-NB1 antisera to recognize the antigen, suggesting that it is not a carbohydrate. The data suggest that the neutrophil-specific antigen NB1 is present on a 58- to 64-Kd surface glycoprotein that is also present in secondary granules, and that the NB1 epitope is not a carbohydrate but probably resides in the tertiary structure of the protein backbone.
Clinical toxicity of cryopreserved bone marrow graft infusion
Authors: Davis JM , Rowley SD , Braine HG , Piantadosi S , Santos GW
Published: October 2019

We prospectively evaluated infusion-related toxicities in 82 recipients of autologous bone marrow grafts. The grafts were cryopreserved in 10% dimethylsulfoxide and stored in liquid nitrogen. All grafts were concentrated and buffy-coat cells were collected. Forty-seven grafts were treated ex vivo with 4-hydroperoxycyclophosphamide (4-HC) at 100 micrograms/mL; 26 grafts were further processed using density-gradient separation and treated with 4-HC at 60 micrograms/mL. Nine buffy-coat concentrates were frozen without drug treatment. Before infusion, patients were medicated with mannitol, hydrocortisone, and diphenhydramine. Grafts were rapidly thawed and immediately infused without further manipulation. During the infusions, 33 (70%) recipients of treated buffy-coat, 5 (56%) recipients of untreated buffy-coat, and 6 (23%) recipients of density-gradient separated grafts experienced varying symptoms including nausea, abdominal cramping, and flushing. Forced vital capacities for 83% of the recipients of treated buffy-coat concentrates decreased after the graft infusion; six of these patients complained of dyspnea and one patient experienced an acute episode of respiratory decompensation. Decreased heart rates were observed in 98% of the recipients of treated buffy-coat cells with asymptomatic bradycardia occurring in 45%. Forty-five patients (96%) in this group experienced transient hypertension, with 18 (38%) requiring additional medications within 6 hours after the infusion for control of blood pressure. Similar cardiovascular changes were observed in the recipients of untreated buffy-coat concentrates. One recipient of an untreated buffy-coat concentrate had 2 degrees heart block after the graft infusion. Twenty-three (88%) recipients of density-gradient separated grafts had decreased heart rates and 21 (81%) had increased blood pressure. However, the degrees of change were less than those experienced by the recipients of treated buffy-coat cells (P less than .01). Forced vital capacities were not affected by the infusion of the density-gradient separated grafts. No renal failure or obvious hemolytic episodes occurred for any patient group. Minor to moderate toxicities were associated with cryopreserved graft infusions. Recipients of buffy-coat separated grafts, both treated and untreated, experienced more complications than the recipients of density-gradient separated grafts. These toxicities may relate to the volumes of cryoprotectant and cell lysis products infused, which were less for the more highly purified density-gradient separated grafts.