bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B
Authors: Palmer TD , Thompson AR , Miller AD
Published: October 2019

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.
Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation
Authors: Heeb MJ , Mosher D , Griffin JH
Published: October 2019

Activated protein C (APC) is inhibited by two major plasma inhibitors (PCIs). To find evidence for in vivo complexation of APC, immunoblotting studies were performed on plasmas of 85 patients with suspected disseminated intravascular coagulation (DIC). Samples from 62 of these patients contained 5% to 35% of protein C antigen in APC:inhibitor complexes, indicating that protein C activation and inhibition had occurred. In 24 normal plasmas, no detectable APC:PCI complexes were observed (less than 5%). Patients with higher levels of complexes had more abnormal coagulation test data for DIC. The major band of APC complexes detected by anti-protein C antibodies did not react with antibodies to the heparin-dependent protein C inhibitor (PCI- 1) previously described. Rather, APC was complexed with another recently described plasma protein C inhibitor, PCI-2. Immunoblotting studies for protein S, the cofactor for APC, revealed that the majority of the DIC patient plasmas contained a higher than normal proportion of protein S in cleaved form, suggesting that protein S may have been proteolytically inactivated. Protein S total antigen levels were also found to be low in DIC patients, excluding those with malignancy. These studies support the hypothesis that the protein C pathway is activated during DIC.
Identification of a leukocyte alloantigen with a high-frequency expression in leukemia patients
Authors: O'Connor R , Bradley JG , O'Meara A , Cotter TG
Published: October 2019

In this report we describe the production and characterization of a monoclonal antibody to the human promyelocytic leukemia cell line HL- 60. The antibody, NC-2, is of the IgG1 subclass and precipitates a 50- Kd protein from 125I-labeled HL-60 cells. The antigen is insensitive to treatment with trypsin, papain, or neuraminidase. NC-2 did not react with a number of established human cell lines, including Daudi, Molt-4, K562, U937, KG-1, CEM, Raji, and Gash-P. Neutrophils and monocytelike cells derived from HL-60 cells that were induced to differentiate continued to express the antigen. NC-2 reacted with all peripheral- blood cells except erythrocytes from eight (5%) of 150 normal individuals tested. Bone marrow samples from patients with myelogenous leukemias were more frequently reactive with NC-2 than were those from normal individuals (12/33 v 1/10). Family studies indicated that the antigen was inherited in an autosomal-dominant manner. These findings suggest that the expression of the above alloantigen is associated with an increased incidence of leukemia.
Characterization of the serum In(Lu)-related antigen: identification of a serum protein related to erythrocyte p80
Authors: Lucas MG , Green AM , Telen MJ
Published: October 2019

The In(Lu) gene has been shown previously to downregulate expression by erythrocytes and by a subset of leukocytes of an 80-Kd protein antigen defined by monoclonal antibody (MoAb) A3D8. A3D8 antibody has also been shown by inhibition studies to recognize a serum antigen; this serum antigen is present in reduced amount in serum from In(Lu) donors. The present study demonstrates that the serum antigen recognized by A3D8 antibody also resides on a protein similar in size to the protein present in erythrocyte membranes. Studies using chromatographically purified protein have further shown that this antigen shares many epitopes with that present in RBCs and is therefore likely to be extremely homologous or identical to the erythrocyte In(Lu)-related p80.
Systemic monoclonal antibody therapy for eliminating minimal residual leukemia in a rat bone marrow transplant model
Authors: Wagner JE , Johnson RJ , Santos GW , Kim BK , Shin HS
Published: October 2019

In an animal bone marrow transplant (BMT) model that mimics the human clinical condition, we evaluated the effectiveness of monoclonal antibody (MoAb) therapy in eliminating minimal residual disease (MRD) in a leukemic host. Leukemic rats were prepared with marrow ablative but noncurative doses of busulfan (BU) and cyclophosphamide (CY). Two days after syngeneic BMT, rats were treated with MoAb. Although all control rats died of leukemia relapse, 58% of those treated with MoAb were cured without any demonstrable effect on the rate of peripheral blood leukocyte recovery. Furthermore, the level of complement, an important effector in suppressing leukemia proliferation in the normal rat, was not adversely affected by BU/CY, BMT and MoAb. Thus, we demonstrated in an animal model that MoAb therapy may be a useful, nontoxic adjunct to high-dose chemotherapy and BMT in eliminating MRD.
Recombinant human granulocyte/macrophage colony-stimulating factor enhances monocyte cytotoxicity and secretion of tumor necrosis factor alpha and interferon in cancer patients
Authors: Wing EJ , Magee DM , Whiteside TL , Kaplan SS , Shadduck RK
Published: October 2019

The colony-stimulating factors (CSFs) promote the proliferation and differentiation of hematopoietic precursors and more recently have been shown to amplify the functions of mature phagocytes in vitro. In this study recombinant human granulocyte/macrophage colony-stimulating factor (rGM-CSF) was administered to cancer patients to determine whether the cytotoxic and secretory activity of their blood monocytes could be enhanced. Patients with refractory neoplastic disease were treated with rGM-CSF either as a single bolus or as a constant infusion for 14 days at either 100 or 500 micrograms/m2 per day. As has been reported by others, the number of peripheral blood monocytes and granulocytes rose markedly in a dose-response fashion during infusion with rGM-CSF. The functional capacity of monocytes was increased by rGM- CSF, since the cytotoxicity of monocytes against antibody-coated xenogeneic cells was increased during the constant infusion compared to baseline. In addition, monocytes harvested during the constant infusion and stimulated with lipopolysaccharide (LPS) in vitro secreted increased quantities of tumor necrosis factor alpha (TNF-alpha) and interferon (IFN). These data indicate that rGM-CSF can enhance both the number and the function of peripheral blood monocytes in vivo.
Treatment of B-cell lymphomas with anti-idiotype antibodies alone and in combination with alpha interferon
Authors: Brown SL , Miller RA , Horning SJ , Czerwinski D , Hart SM , McElderry R , Basham T , Warnke RA , Merigan TC , Levy R
Published: October 2019

Idiotypes are distinct clonal markers for B-cell lymphomas. Previously we reported the use of anti-idiotype antibodies in the therapy of patients with B-cell malignancies. Because synergy was demonstrated with the addition of alpha interferon to anti-idiotype antibodies in a murine lymphoma model, we performed a clinical trial combining these two agents. Here we provide an update of the original trial of anti- idiotype antibodies alone and report the outcome of the new combination trial. In 16 treatment courses of anti-idiotype antibodies alone there were seven partial responses and one complete response. In 12 courses of combination anti-idiotype antibody and alpha interferon there were two complete responses and seven partial responses. Substantial tumor regressions occurred with minimal toxicity in both trials even in patients refractory to conventional chemotherapy. Tumor specimens obtained at the time of disease progression often contained a preponderance of idiotype-negative lymphoma cells, suggesting that anti- idiotype antibody treatment exerted a strong antitumor effect against antigen-positive cells. Anti-idiotype antibodies have reproducible objective antitumor activity in B-cell lymphoma. The addition of alpha interferon may improve the initial rate of response to this treatment. Strategies that deal effectively with idiotype-negative lymphoma cells should improve the extent and duration of these responses.
Effects of normal mouse serum on the IL-3-induced proliferation of bone marrow cells
Authors: Dahl CA , Lindqvist C
Published: October 2019

Normal mouse serum (NMS) devoid of colony-stimulating factor (CSF) was found to enhance the interleukin 3 (IL-3)-driven colony formation of bone marrow in vitro. Inclusion of NMS in bone marrow colony-forming assays resulted in greatly increased numbers of colonies and clusters following seven days incubation; however, incubation of bone marrow with NMS before the colony-forming assay had no effect on resultant colony number. The levels of serum-enhancing activity (SEA) did not appear to vary significantly with age and in part was species restricted, in that human and guinea pig serum did not enhance mouse bone marrow colony formation. Conversely, NMS had no effect on human bone marrow colony formation. Levels of SEA were found to vary between strains, as did the degree to which bone marrow from various strains was enhanced by the serum. Serum fractionation studies indicated three active fractions with molecular weights of 800–900 Kd, 60–70 Kd, and 20- 30 Kd. The fraction at 800–900 Kd inhibited colony formation at high concentrations and enhanced colony formation on dilution, whereas the two other active fractions contained enhancing activity at all concentrations tested. These results would indicate that normal serum can play a greater role in colony-forming assays than nutritional supplements. The relationship of the SEA factors to other factors that have been reported to modulate bone marrow colony formation is discussed.
Immunohistochemical detection and quantitation of P-glycoprotein in multiple drug-resistant human myeloma cells: association with level of drug resistance and drug accumulation
Authors: Dalton WS , Grogan TM , Rybski JA , Scheper RJ , Richter L , Kailey J , Broxterman HJ , Pinedo HM , Salmon SE
Published: October 2019

Using several multiple drug-resistant human myeloma cell lines as standards, we developed an immunohistochemical staining technique and means of quantitating P-glycoprotein in individual myeloma cells. The level of staining intensity for P-glycoprotein in individual myeloma cells was quantitated by measuring the average optical density of each cell with a microscopic computerized cell analysis system. Using this system, we observed that the level of P-glycoprotein for individual cells within a cell population of known drug sensitivity was very homogeneous (coefficient of variation less than or equal to 13%). Analysis of cell lines with gradually increasing levels of multidrug resistance (8226/S, 8226/Dox6 and 8226/Dox40) demonstrated a close association between the level of resistance to doxorubicin, defined by the mean lethal dose (D0) and the amount of P-glycoprotein on individual cells determined by the optical density (r = 0.82, P less than 0.0005). Intracellular doxorubicin (DOX) accumulation in the individual cell lines was inversely related to the level of drug resistance expressed as D0. P-glycoprotein was also detected in the marrow-derived myeloma cells of patients with drug refractory disease using immunohistochemical staining. The amount of P-glycoprotein in the cells of one patient was directly compared to the amount found in the simultaneously stained standard cell lines (8226/Dox6 and 8226/Dox40) by comparing the optical densities for individual cells. Using this immunohistochemical technique to detect and quantitate P-glycoprotein in patient myeloma cells and comparing it to standard multidrug resistant myeloma cell lines may be of value in determining the contribution of P-glycoprotein to clinical drug resistance in patients with multiple myeloma.
c-myb but not c-myc suppresses the hemin-induced nonterminal expression of hemoglobin by murine erythroleukemia cells
Authors: Prochownik EV
Published: October 2019

Clonal lines of Friend murine erythroleukemia (F-MEL) cells have been generated following transfection with c-myc or c-myb expression plasmids. These clones produce high levels of abnormally regulated proto-oncogene transcripts and fail to terminally differentiate in the presence of dimethyl sulfoxide. To determine the relative levels at which the two proto-oncogenes might exert their inhibitory effects, we asked whether these clones could express differentiated functions in the absence of terminal differentiation. It was found that exposure of c-myc-transfected cells to hemin allows for the induction of hemoglobin, whereas c-myb-transfected cells were refractory to hemin induction. It thus appears that c-myb exerts a more globally suppressive effect on F-MEL-differentiated functions than does c-myc and may prevent the expression of those events that can otherwise be dissociated from the terminally differentiated state.
Combined procoagulant activity and proteolytic activity of acute promyelocytic leukemic cells: reversal of the bleeding disorder by cell differentiation
Authors: Wijermans PW , Rebel VI , Ossenkoppele GJ , Huijgens PC , Langenhuijsen MM
Published: October 2019

In the human promyelocytic cell line HL60, we observed both a strong procoagulant activity (PCA) on the cell membrane and proteolytic activity in the lysate of these cells. Because these cell-line cells are susceptible to differentiation to either a more mature granulocytic or monocytic form, we were able to study the hypothesis that the combination of PCA and proteolytic activity is confined to the promyelocyte. This may explain the severe coagulopathy seen in patients with acute promyelocytic leukemia. Cell differentiation in a myeloid direction induced by retinoic acid or DMSO led to a diminished PCA, while not affecting the fibrinolytic activity. On the other hand, monocytic differentiation obtained by culturing the cells in the presence of 1; 25 dihydroxy vitamin D3 led to the complete disappearance of the proteolytic activity of the cell lysate, although the procoagulant activity was still present. Furthermore, we found that the elastase activity almost disappeared after monocytic differentiation. We also studied the PCA, proteolytic activity, and elastase activity of blast cells of patients with acute myeloid leukemia. Only in patients with acute promyelocytic leukemia did we observe both a strong PCA and fibrinolytic activity. This supports our hypothesis that the combination of these activities is unique to the promyelocyte and may explain the observed bleeding complications in patients with acute promyelocytic leukemia.
Biphenotypic leukemic lymphocyte precursors in CD2+CD19+ acute lymphoblastic leukemia and their putative normal counterparts in human fetal hematopoietic tissues
Authors: Uckun FM , Muraguchi A , Ledbetter JA , Kishimoto T , O'Brien RT , Roloff JS , Gajl- Peczalska K , Provisor A , Koller B
Published: October 2019

During detailed immunophenotypic analyses of marrow blasts from 336 acute lymphoblastic leukemia (ALL) patients, a very small percentage of cases reactive with B-cell-directed as well as T-cell-directed monoclonal antibodies (MoAbs) were identified. Five ALL cases were biphenotypic since they coexpressed CD2 (Tp50) and CD19 (Bp95) antigens at the single-cell level. The composite immunophenotype of these biphenotypic ALL cases was [TdT+HLA-ABC+CD2+CD3-CD10+CD13-CD14-CD16- CD19+CD20+ ++-CD21-CD33-CD34+Bgp95-C mu- slg-]. Low-molecular-weight B- cell growth factor (LMW-BCGF), recombinant interleukin-2 (rIL-2), and rIL-3 stimulated the proliferative activity of biphenotypic leukemic lymphocyte precursors without inducing differentiation. In the presence of the phorbol ester TPA, leukemic blasts from two cases differentiated along the B precursor pathway to the [CD2-CD10+CD19+CD20+C mu+slg-] pre- B cell stage. Biphenotypic ALL cases did not share a common configuration and gene rearrangement pattern of the immunoglobulin heavy chain genes or T-cell receptor (TCR) genes. Three cases had rearranged C mu genes but germline TCR genes, one case showed rearrangement of both C mu and TCR genes, and the remaining case had rearranged TCR genes but germline C mu genes. All five patients attained prompt remission after standard induction chemotherapy. Three to four years after initial diagnosis, four patients are now off chemotherapy and remain alive in their first remission. One patient relapsed at 3 years, 7 months, but promptly achieved complete remission after reinduction chemotherapy and remains in second remission off chemotherapy greater than 3 years after her reinduction therapy. With two-color immunofluorescence staining techniques and multiparameter flow cytometric analyses, we identified a small population of CD2+CD19+ lymphoid cells in fetal livers (FLs) and fetal bone marrows (FBMs), which may represent the putative normal counterparts of biphenotypic ALL blasts. A CD2+CD19+ normal biphenotypic lymphoid precursor cell line, designated FL 8.2 CD2+, was established from an FL of 8-weeks of gestational age by Epstein-Barr virus (EBV)-induced blastoid transformation. The composite immunophenotype of FL 8.2 CD2+ cell line was [TdT+HLA-ABC+HLA-DR+ CD2+CD5-CD7-CD10+/-CD13-CD19+CD20-CD21+ CD22+CD33-CD34+/-Bgp95-CDw40+C mu-slgD-slgM-]. FL 8.2 CD2+ cells showed germline patterns of immunoglobulin heavy-chain joining region, heavy- chain constant region, kappa light-chain constant region genes, and TCR beta-chain genes. Cross-linking of CD2 as well as CD19 antigens on FL 8.2 CD2+ cells caused an increase of intracellular ionized calcium.(ABSTRACT TRUNCATED AT 400 WORDS)
Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging
Authors: Gulliya KS , Pervaiz S
Published: October 2019

Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin’s lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray- irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested. These results may have therapeutic relevance in patients undergoing interferon treatment who require bone marrow transplantation, as the complete elimination of tumor cells by marrow-purging procedures may be hampered by this increased survival in the presence of interferon.
Plasma cell myeloma--new biological insights and advances in therapy
Authors: Barlogie B , Epstein J , Selvanayagam P , Alexanian R
Published: October 2019

Plasma cell myeloma is a more complex neoplasm than suggested by the relative uniformity of its dominant plasma cells, which represent the terminal stage of normal B-cell differentiation. Phenotypic, molecular, and cellular genetic data favor the presence of a myeloma stem cell early in hematopoietic development so that, as in chronic myelogenous leukemia (CML), a far distance exists between the primordial malignant cell that was the target of malignant transformation and the dominant clinical phenotype. Traces of pre-B, myeloid, and T cells are coexpressed with the mature B-cell phenotype, an occurrence unknown in normal B-cell differentiation. Analogous to CML, disease progression is marked by disease dedifferentiation, occasionally with cessation of myeloma protein production and development instead of extramedullary lymphomalike features with high LDH or myelodysplasia/acute myelogenous leukemia (AML) syndromes. The prognostic importance of serum LDH levels even in newly diagnosed myeloma suggests the early presence of tumor cells with “LDH phenotype,” which, as a result of drug resistance and proliferative advantage, expand preferentially during disease progression. Further characterization of these cells may provide important clues about the ontogeny of multiple myeloma. Myeloma cells express many receptors for different biological signals that might be exploitable for therapy with immunotoxins or radioisotopes. Plasma cells and their precursors also produce a variety of cytokines, some of which have putatively autostimulatory functions (eg, IL-1, IL-5, IL-6) and/or are related to disease manifestations (eg, IL-1 and TNF-beta as OAF). The wealth of cellular expression by plasma cells provides clues for understanding the mechanisms of gene activation and the nature of abnormal growth and differentiation. The accuracy of prognostically relevant staging systems has been refined with the use of new quantitative parameters that reflect tumor mass (ie, serum B2M levels) and biology. Further studies of cellular and molecular biology (ie, CAL- LA, H-ras) may reveal those tumor cell features that define clinical entities, response to therapy, and long-term prognosis. The lack of a major advance in prognosis despite the use of more drugs and more intensive regimens justifies the continued use of standard melphalan- prednisone for patients with a highly favorable prognosis, for the very aged, and for those with a short life expectancy due to other major medical problems. However, a radical departure from standard practice is required to improve the prognosis for younger patients with poor risk features.(ABSTRACT TRUNCATED AT 400 WORDS)
Expression of lymphocyte homing receptor as a mechanism of dissemination in non-Hodgkin's lymphoma
Authors: Pals ST , Horst E , Ossekoppele GJ , Figdor CG , Scheper RJ , Meijer CJ
Published: October 2019

To investigate whether the lymphocyte homing receptor (LHR), an adhesion molecule believed to play an important role in the control of normal lymphocyte circulation, influences the spread of non-Hodgkin's lymphoma (NHL), expression of LHR was examined in 107 NHL of various histologic and immunophenotypic subclasses. This analysis revealed that whereas NHL with a putative derivation from recirculating mature T and B lymphocytes almost invariably express high levels of LHR, those akin to sessile mature or immature lymphocytes tend to express lower levels of LHR. Furthermore, in a survey among diffuse large-cell lymphomas of the B-lineage, the tumors of 11 of 13 patients with stage III/IV disease expressed moderate to high levels of LHR, whereas only two of 17 patients with stage I/II disease had tumors that did so. These findings suggest that LHR is involved in the dissemination of NHL.
Differential expression of MHC class II antigens in myelomonocytic leukemia cell lines
Authors: Yunis JJ , Band H , Bonneville F , Yunis EJ
Published: October 2019

Major histocompatibility complex (MHC) class II antigens are discordantly expressed on hematopoietic progenitor cells. Their expression is linked to differential responsiveness of the cells to growth factors and inhibitors. We examined the expression of different MHC class II antigens in a panel of human myelomonocytic cell lines representing different stages of differentiation, by cytofluorographic analysis with monoclonal antibody (MoAb) and Northern blot analysis with specific cDNA probes. These analyses revealed discordant expression of different MHC class II antigens both in basal state and after gamma-IFN induction. Thus KG-1 myeloblast cells express all class II antigens (DR greater than DP greater than DQ) constitutively and their expression increased after gamma-IFN treatment. KG-1a, an immature blast variant of KG-1, does not express class II antigens, even after gamma-IFN treatment. THP-1, a monocytic cell line expresses DR but not DP or DQ under basal conditions. DP and DQ are, however, gamma-IFN inducible. The class II negative HL-60 promyelocytic cell line, expresses DR and DP but not DQ after gamma-IFN induction. In all the above cell lines, surface expression of class II antigens correlated with the levels of mRNA expression as determined with specific cDNA probes. In U-937, a monocytic cell line, no surface expression of class II MHC antigens was observed either with or without gamma-IFN, however, specific mRNA message was observed under basal conditions and was further increased with gamma-IFN, indicating a possible defect in assembly or transport of class II antigens. The patterns of class II MHC antigens in these leukemic cell lines may be a useful model to delineate molecular basis of discordant MHC class II expression during myelomonocytic differentiation.
Human fibroblast tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apolipoprotein A-II
Authors: Gramzinski RA , Broze GJ Jr , Carson SD
Published: October 2019

Studies of proteins that inhibit tissue factor activity have generally been conducted using either an extracted tissue homogenate (“thromboplastin”) or tissue factor protein reconstituted into phospholipid vesicles rather than with tissue factor expressed in cell membranes (its physiological environment). In the present study, a human fibroblast cell strain was used to evaluate the effects of lipoprotein associated coagulation inhibitor (LACI), placental anticoagulant protein (PAP), and apolipoprotein A-II (apo A-II) on human tissue factor in cell membranes. LACI was tested from 7.8 to 500 pmol/L on fibroblasts cultured at cell densities ranging from 3,500 to 9,925 cells/well, and caused a progressive inhibition of tissue factor activity. PAP was tested from 3.9 nmol/L to 1 mumol/L at cell densities ranging from 4,500 to 15,400 cells/well and caused up to 83% inhibition of tissue factor activity. Inhibition by these proteins appeared to be influenced by cell density as well as whether the cells were intact or disrupted. Apo A-II, up to 1 mumol/L, did not inhibit the tissue factor activity of intact or disrupted fibroblasts at any cell density examined even though it did inhibit the activity of tissue factor in phospholipid vesicles. Of these inhibitors of tissue factor-dependent activation of factor X, LACI was the most effective in suppressing the generation of factor Xa activity. The effects obtained with apo A-II are clearly dependent on the nature of the tissue factor preparation with which it is tested. The disparity between the inhibitory effect of apo A-II on the activity of tissue factor reconstituted into lipid vesicles and the absence of effect on the activity of tissue factor remaining in cell membranes serves to reemphasize the necessity of reexamining results obtained with model systems using as nearly physiological reagents as possible.
Acute promyelocytic leukemia: treatment results during a decade at Memorial Hospital
Authors: Cunningham I , Gee TS , Reich LM , Kempin SJ , Naval AN , Clarkson BD
Published: October 2019

Fifty-seven adult patients with acute promyelocytic leukemia (APL) were treated between 1974 and 1984 with daunorubicin (DNR) or 4-(9- acridinylamino)methanesulfan-m-anisidide (AMSA) in combination with arabinosylcytosine (Ara-C) and 6-thioguanine (TG); they also received prophylactic heparin. Forty-one patients (72%) achieved complete remission (CR), including 11 of 12 patients who received the AMSA- containing regimen. The incidence of early fatal hemorrhage was 14%, lower than that of earlier studies or other published reports. Elevated WBC and serum lactate dehydrogenase levels at diagnosis were associated with an increased incidence of life-threatening hemorrhage and shorter remission duration. Advanced age was an unfavorable prognostic factor for male patients. Both DNR and AMSA in combination protocols are effective treatments for APL. The incidence of CR is similar to that achieved in other types of acute nonlymphoblastic leukemia (ANLL) with the same protocols, but the median duration of remission is significantly longer in APL (24 v 9 months) and the percentage of remissions longer than 60 months is also higher in APL (35% v 5%).
Tumor necrosis factor induces proliferation of neoplastic B cells from chronic lymphocytic leukemia
Authors: Digel W , Stefanic M , Schoniger W , Buck C , Raghavachar A , Frickhofen N , Heimpel H , Porzsolt F
Published: October 2019

The biologic effects of recombinant tumor necrosis factor-alpha (rTNF- alpha) and the expression of specific TNF membrane receptors on isolated neoplastic B cells from previously untreated patients with chronic lymphocytic leukemia (CLL) were investigated in vitro. Isolated B cells were incubated up to six days with various concentrations of rTNF-alpha (0.1 to 100 ng/mL). B cells from most patients proliferated ranged from two to 104 times that of unstimulated cells from the same patients. An optimal proliferative effect was achieved at 25 ng/mL rTNF- alpha and an incubation time between 96 and 120 hours, whereas a low concentration of rTNF-alpha (1 ng/mL) reduced [3H]TdR incorporation in four cases. Metaphase cells were detected in the rTNF-alpha-stimulated cultures that proliferated in response to rTNF-alpha. B cells from three of ten patients proliferated spontaneously and proliferation was further enhanced in two patients by rTNF-alpha. TNF binding assays gave a value of approximately 390 to 1,400 binding sites/cell for TNF and a dissociation constant (kd) of approximately 60 pmol/L. These data indicate that rTNF-alpha, in contrast to its cytotoxic/cytostatic effects, can also induce proliferation of tumor cells.
The bcr gene in Philadelphia chromosome positive acute lymphoblastic leukemia
Authors: Heisterkamp N , Jenkins R , Thibodeau S , Testa JR , Weinberg K , Groffen J
Published: October 2019

In chronic myelogenous leukemia (CML) and in a percentage of childhood and adult acute lymphoblastic leukemia (ALL) the Philadelphia (Ph′) chromosome is present in the leukemic cells of patients. This chromosome is the result of a reciprocal translocation between chromosomes 9 and 22. In CML the break on chromosome 22 occurs within the major breakpoint cluster region (Mbcr) of the bcr gene. In this study, we report on the examination of DNAs from nine Ph′-chromosome positive ALL patients for rearrangements within the bcr gene using Southern blot analysis. Of nine patients having a karyotypically identifiable Ph′-chromosome, only five exhibited rearrangements of the bcr gene. This could indicate that in ALL, chromosome 22 sequences other than the bcr gene are involved in the Ph′-translocation. Within the group of Ph′-positive ALL patients having a bcr gene breakpoint, a correlation appears to exist between the age of the patient and the location of the breakpoint within the gene: all or the vast majority of pediatric patients analyzed to date do not have a Mbcr breakpoint as found in CML and in adult ALL.
Ph1-positive, bcr-negative acute leukemias: clustering of breakpoints on chromosome 22 in the 3′ end of the BCR gene first intron
Authors: Chen SJ , Chen Z , Hillion J , Grausz D , Loiseau P , Flandrin G , Berger R
Published: October 2019

About 50% of the Philadelphia-positive acute leukemias undergo molecular rearrangements outside the now classical bcr sequence (or M- BCR-1) rearranged in chronic myeloid leukemia (CML). Most of the breakpoints on chromosome 22 have been shown to be clustered in a 10.8- kb region of the first intron of the BCR gene (called bcr2 or m-BCR-1). In this report we examined two cases of Ph1 acute lymphoblastic leukemia in adult patients that exhibited breakpoints in a 5-kb segment of the BCR gene first intron, 16 kb upstream of the previously described cluster, suggesting the possibility of a second minor breakpoint cluster. In addition, the breakpoints on chromosome 9 were located in a region just 5′ of the c-abl exon la.
Extracellular adenosine triphosphate increases cation permeability of chronic lymphocytic leukemic lymphocytes
Authors: Wiley JS , Dubyak GR
Published: October 2019

Extracellular adenosine triphosphate (ATP) is known to reversibly increase the cation permeability of a variety of freshly isolated and cultured cell types. In this study the effects of extracellular ATP were studied using peripheral blood lymphocytes (PBL) isolated from both normal subjects and from patients with chronic lymphocytic leukemia (CLL). Changes in the permeability to Na+, Rb+, and Li+ ions were measured using conventional isotope and flame photometry techniques. In addition, changes in cytosolic (Ca2+) were fluorimetrically monitored to assess possible changes in net Ca2+ influx. ATP produced a 12-fold increase in 22Na+ influx into CLL cells but only a 3.5-fold increase in this flux in PBL cells. A maximal response was produced by 0.1 mmol/L ATP in the absence of Mg2+, while a twofold molar excess of Mg2+ over ATP abolished the response. ATP had no effect on the passive (ouabain-insensitive) 86Rb+ influx into PBL cells but stimulated this flux by fivefold in the CLL cells. Li+ influx into CLL cells was also stimulated threefold by ATP. Under these same conditions ATP also produced a net increase in total cell Na and a decrease in total cell K in the CLL cells. Exclusion of two normally impermeable dyes, trypan blue and ethidium bromide, was not altered in the ATP-treated CLL cells. Finally, extracellular ATP (3 mmol/L) produced no significant change in the cytosolic (Ca2+) of normal, monocyte-depleted populations of PBL. Conversely, this same concentration of ATP produced a very rapid (complete within 30 seconds) and a significant (an average threefold peak change) increase in the cytosolic (Ca2+) of cell preparations derived from five out of nine CLL patients. In these latter CLL cells, the ATP-induced elevation in cytosolic (Ca2+) appeared to be due to a net increase in Ca2+ influx, since no elevations were observed when the extracellular (Ca2+) was reduced to less than 0.1 mmol/L. These actions of ATP were specific in that equimolar concentrations of other nucleotides were without effect. These data indicate that treatment of CLL lymphocytes with extracellular ATP4 produces large increases in cation permeability. In contrast, there is less or no ATP-induced permeabilization of normal PBL.
Expression of protooncogene c-myb in normal human hematopoietic cells
Authors: Kastan MB , Slamon DJ , Civin CI
Published: October 2019

Expression of the protooncogene, c-myb, in various subpopulations of normal human hematopoietic cells was characterized. Cells expressing the immature cell surface marker, CD34 (My10), were isolated by immune adherence with the “panning” technique or immunomagnetic microspheres and were shown to be strongly positive for c-myb protein expression in an immunoperoxidase assay. The CD34+ progenitor cell population was further separated into myeloid plus erythroid progenitors (CD34+, CD10- ) v B-lymphoid precursors (coexpressing CD34 and CD10) by two-color FACS. Both CD34+ progenitor cell subsets strongly expressed c-myb protein by the immunoperoxidase assay. A flow cytometric assay was then developed which permitted simultaneous detection of a cell-surface antigen (to characterize lineage and stage of maturation) and the nuclear oncoprotein. This assay confirmed that CD34+ cells were strongly positive for c-myb expression and also allowed quantitative comparisons of c-myb expression in selected populations of other normal hematopoietic cells. Most human bone marrow cells appear to express some level of c-myb protein, although the CD34+ progenitor cell population expresses the highest amount.
Myelostimulatory activity of recombinant human interleukin-2 in mice
Authors: Talmadge JE , Schneider M , Keller J , Ruscetti F , Longo D , Pennington R , Bowersox O , Tribble H
Published: October 2019

In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.
Myeloid growth factor(s) regulation of ornithine decarboxylase: effects of antiproliferative signals interferon-gamma and cAMP
Authors: Farrar WL , Harel-Bellan A
Published: October 2019

Colony-stimulating factors (CSFs) stimulate the activation and steady- state mRNA accumulation of an important regulatory enzyme for macromolecular synthesis, ornithine decarboxylase (ODC). Cloned murine CSF-dependent cell lines exhibited a rapid activation of ODC enzyme activity, detectable within ten minutes of stimulations with either interleukin-3 (IL-3), GM-CSF, or G-CSF. This early phase of enzyme activation did not require early protein or mRNA synthesis. The subsequent protracted rise in ODC activity occurring four to six hours after CSF treatment was dependent on increases in steady-state ODC mRNA accumulation and de novo protein synthesis. CSF, therefore, modulates both posttranslational activation of preexisting ODC and stabilization and accumulation of ODC mRNA. Antiproliferative signals, such as cAMP or interferon-gamma (IFN-gamma), effectively inhibited the CSF-directed increase in steady-state ODC mRNA. Cotreatment of the murine NSF 60.8 cell line with IFN-gamma and GM-CSF decreased steady-state ODC mRNA greater than 80% as compared with GM-CSF-treated cells alone. IFN treatment did not cause any appreciable destabilization of mature ODC mRNA, suggesting that its major effect may be at the level of ODC mRNA transcription or posttranscriptional processing. These data indicate that the ODC gene-protein system is an important molecular locus of the effects of myeloid proliferative and antiproliferation signals.
Biogenesis of the vitronectin receptor in human endothelial cell: evidence that the vitronectin receptor and GPIIb-IIIa are synthesized by a common mechanism
Authors: Polack B , Duperray A , Troesch A , Berthier R , Marguerie G
Published: October 2019

Human endothelial cells express a membrane glycoprotein alpha beta heterodimer similar to the human platelet glycoprotein IIb-IIIa complex (GPIIb-IIIa). This noncovalently associated complex is the vitronectin receptor (VNR). These two receptors belong to the cytoadhesin family and share the same beta subunit. They express different recognition specificities: platelet GPIIb-IIIa is a receptor for fibrinogen, fibronectin, and von Willebrand factor (vWF), whereas VNR is a receptor for vitronectin, and is possibly a receptor for fibrinogen and vWF. We analyzed the biosynthesis of the endothelial cell VNR. Our data show that VNR alpha is a two-chain protein which is biosynthesized as a single-chain precursor: the pro-VNR alpha. Pro-VNR alpha forms a complex with VNR beta, and this association occurs prior to the Golgi- mediated processing of the oligosaccharide side chains. Mature VNR beta is glycosylated by not fully processed oligosaccharide side chains because it remains endoglycosidase H (endo H) sensitive, even when the complex is expressed on the cell surface. This characteristic appears as a common feature for the members of the cytoadhesin family. These results indicate that although VNR and GPIIb-IIIa are biosynthesized in different cells, their expression is controlled by similar mechanisms, providing further support for the concept that the cytoadhesin family constitutes a distinct group of adhesion receptors.
The activation of the contact phase of coagulation by physiologic surfaces in plasma: the effect of large negatively charged liposomal vesicles
Authors: Mitropoulos KA , Martin JC , Reeves BE , Esnouf MP
Published: October 2019

The endogenous, negatively charged surface that induces activation of the contact coagulation factors was investigated in plasmas taken from women in late pregnancy and control subjects of child-bearing age. The plasmas from the two groups of subjects were incubated at 4 degrees C for 24 hours either in plastic or in glass tubes and the factor VII coagulant activity (VIIc) was assayed in the treated plasmas. The activation of factor VII under these conditions involves the generation of enzymes derived from factor XII (XIIa). The contact surface is rate- limiting for the activation of factor VII in the plasmas in both groups of subjects and can be supplemented by large multilamellar liposomal vesicles carrying the appropriate density of negative charge. The size of these vesicles is within the range of sizes of the large lipoprotein particles (chylomicrons, very low and intermediate-density lipoproteins). The relationship between the density of negative charge on the liposomal vesicles and VIIc was similar in the late pregnancy and the control plasmas incubated in plastic tubes. At a saturating density of negative charge the observed relative VIIc was similar in both sets of plasmas. The incubation of late pregnancy or control plasma in plastic tubes in the presence of sodium stearate caused VIIc to increase with increasing concentration of the added fatty acid. These results suggest that large lipoprotein particles carrying the appropriate free fatty acid at a sufficient density of negative charge could provide the contact surface that induces the generation of factor XIIa and the subsequent activation of factor VII. Moreover, plasmas from women in late pregnancy have a higher concentration of potential surface and a higher density of negative charge than the plasmas from nonpregnant women.
High pp60c-src level in human platelet dense bodies
Authors: Rendu F , Lebret M , Danielian S , Fagard R , Levy-Toledano S , Fischer S
Published: October 2019

Phosphoproteins phosphorylated in vivo were examined in resting and thrombin-activated human blood platelets. Thrombin-stimulation resulted in an overall increase in labeled proteins containing phosphotyrosine. The most prominent was a protein of 60 Kd. By electroblotting, the 60 Kd protein was identified as the pp60c-src, the normal cellular homolog of the transforming protein of Rous sarcoma virus. We have examined the intracellular distribution of the pp60c-src within platelets. Use of immunoprecipitation and electrotransfer to study isolated membranes, alpha-granules, lysosomes, and dense granules (also termed dense bodies) revealed that pp60c-src was highly enriched in dense bodies. In view of the prominent role of these granules in platelet function, We postulate that protein phosphorylation by activated pp60c-src is involved in early steps of platelet activation.
Interleukin-1 beta rather than lymphotoxin as the major bone resorbing activity in human multiple myeloma
Authors: Kawano M , Yamamoto I , Iwato K , Tanaka H , Asaoku H , Tanabe O , Ishikawa H , Nobuyoshi M , Ohmoto Y , Hirai Y
Published: October 2019

Human myeloma cells were purified from bone marrow aspirates from four patients having advanced myeloma, including one with common acute lymphoblastic leukemia antigen-positive myeloma. All of these myelomas had marked bone lytic lesions. From the culture supernatants of these purified myeloma cells, bone-resorbing activities were significantly revealed by 45Ca-release bone resorption assay, and IL-1 activities were also detected by IL-1 bioassay (mouse thymocyte comitogenic assay). Sandwich enzyme immunoassay for IL-1 alpha or IL-1 beta revealed that IL-1 beta was responsible for IL-1 activity of these culture supernatants. Furthermore, the bone resorbing activities of these culture supernatants were completely neutralized by pretreatment of anti-IL-1 beta, but not anti-IL-1 alpha antibody. By Northern blot analysis, IL-1 beta mRNA was identified from these myeloma cells. Therefore, it is concluded that myeloma cells produce IL-1 beta, which acts as bone-resorbing activity in multiple myeloma.