bluuebunny/crossref_metadata_2025 huggingface dataset:
The 47-kD fragment of talin is a substrate for protein kinase P
Authors: Simons PC , Elias L
Published: October 2019

This laboratory has been characterizing protein serine/threonine kinase reactions of hematopoietic tissues, whose most distinguishing characteristics in vitro are stimulation with vesicular phosphatidyl glycerol, and the ability to function using Mn2+ as the sole divalent cation. The major protein substrates are a 73-kD protein and a protein migrating near ovalbumin on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 47-kD protein was partially purified from cells harvested by leukapheresis from a patient with acute myelogenous leukemia, using ammonium sulfate precipitation and ion exchange chromatography. This partially purified ion-exchange fraction contained an endogenous kinase activity with characteristics similar to those we previously described of protein kinase P (protein kinase, phospholipid- stimulable: PK-P), but not typical of any form of protein kinase C (PK- C). With longer phosphorylation, the 47-kD band showed increasingly lower mobility demonstrable both by Coomassie blue staining and autoradiography, suggesting both that it was multiply phosphorylated, and that the excisable band was pure. The protein was thus eluted from preparative gel slices and digested with endoproteinase lys C. Sequence data from the fragments identified the protein as the 47-kD calpain fragment of talin, a protein found in focal adhesion plaques and some cell-cell contacts. PK-C phosphorylated the 47-kD protein, as has been reported previously, and phosphopeptide mapping disclosed a similar pattern of phosphorylation using either PK-C or the endogenous activity. The 47-kD protein labeled with the endogenous kinase contained predominantly phosphoserine, with some phosphothreonine and a trace of phosphotyrosine. Intact, purified talin was also phosphorylated by PK-P in a phospholipid-stimulable manner, but at 1/20 the rate of the 47-kD fragment.
Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis
Authors: Delorme MA , Saeed N , Sevcik A , Mitchell L , Berry L , Johnston M , Andrew M
Published: October 2019

A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.
Amino acid 489 is encoded by a mutational "hot spot" on the beta 3 integrin chain: the CA/TU human platelet alloantigen system
Authors: Wang R , McFarland JG , Kekomaki R , Newman PJ
Published: October 2019

A new platelet alloantigen, termed CA, has recently been implicated in a case of neonatal alloimmune thrombocytopenia (NATP) in a Filipino family in Canada. Maternal anti-CA serum reacted with glycoprotein (GP) IIIa and maintained its reactivity after removal of high mannose carbohydrate residues from GPIIIa. The monoclonal antibody (MoAb) AP3 partially blocked binding of anti-CA to GPIIIa, suggesting that the CA polymorphism is proximal to the AP3 epitope. Platelet RNA polymerase chain reaction (PCR) was used to amplify the region of GPIIIa cDNA that encodes this region of the protein. DNA sequence analysis showed a G<==>A nucleotide substitution at base 1564 that results in an arginine (Arg) (CGG)<==>glutamine (Gln) (CAG) polymorphism in amino acid (AA) 489. Further analysis of PCR-amplified genomic DNA from 27 normal individuals showed that AA 489 is encoded by a mutational “hot spot” of the GPIIIa gene, as three different codons for the wild-type Arg489 of GPIIIa were also found. The codon usage for Arg489 was found to be: CGG (63%), CGA (37%), and CGC (< 1%). These frequency data were valuable in determining the relationship of the CA alloantigen to the serologically defined TU GPIIIa polymorphism that is present in low frequency in the Finish population. Analyses of PCR-amplified genomic DNA showed the CA and TU alloantigens to be identical at the molecular level. Definition of these new molecular variants of the beta 3 integrin chain should prove valuable in the diagnosis of NATP in these two geographically disparate populations, and it may also provide useful genetic markers for examining other pathologic variations of the GPIIb-IIIa complex.
Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals
Authors: Oyaizu N , McCloskey TW , Coronesi M , Chirmule N , Kalyanaraman VS , Pahwa S
Published: October 2019

This study investigates apoptosis as a mechanism for CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1) infection. Although several recent studies have shown that T cells of HIV-infected individuals show enhanced susceptibility to cell death by apoptosis, the mechanisms responsible for apoptosis are largely unknown. By using a flow cytometric technique and by morphology, we have quantitated the percentage of cells undergoing apoptosis in peripheral blood mononuclear cells (PBMCs) from HIV-seronegative donors and from HIV- infected asymptomatic patients. The PBMCs were cultured without any stimulus or with staphylococcus enterotoxin B, anti-T-cell receptor (TCR) alpha beta monoclonal antibody WT-31, or phytohemagglutinin for periods up to 6 days. In addition, we sought to determine whether cross- linking of CD4 followed by various modes of TCR stimulation in vitro could induce apoptosis in normal PBMCs. Here we show that (1) patient PMBCs undergo marked spontaneous apoptosis; (2) stimulation of T cells of patients as well as normal donors results in increased apoptosis; and (3) cross-linking of CD4 molecules is sufficient to induce apoptosis in CD4+ T cells if cross-linking is performed in unfractioned PBMCs, but not if CD4 molecules are cross-linked in purified T-cell preparations. These observations strongly suggest that accelerated cell death through apoptosis plays an important role in the pathogenesis of HIV-1 infection. At the same time, our observations implicate cross- linking of CD4 in vivo as a major contributor to this mechanism of accelerated cell death in HIV infection.
Subpopulations of bone marrow fibroblasts support VLA-4-mediated migration of B-cell precursors
Authors: Tang J , Scott G , Ryan DH
Published: October 2019

Proliferation of normal human lymphoid progenitors in culture is dependent on interaction with bone marrow-derived fibroblast-like cells (BM-FB). To investigate possible heterogeneity in this lymphoid- supportive microenvironment, we studied the interaction of a human B- precursor cell line (NALM-6) with BM-FB. NALM-6 cells associate with BM- FB by either adhesion or migration underneath the fibroblast. Individual fibroblasts in the BM-FB layer showed significant variation in the number of migrating NALM-6 cells. Migration of NALM-6 cells was primarily VLA-4-dependent, although residual migration observable after blocking with anti-VLA-alpha 4 antibody was inhibited by anti-VLA-alpha 5 antibody. Migration was not inhibited by blocking either of the known VLA-4 counterreceptors (VCAM-1 or fibronectin), although slight inhibition was observed using a combination of blocking antibodies to VCAM-1 and fibronectin. In contrast, NALM-6 adhesion without migration was significantly inhibitable by anti-VCAM-1 antibody. VCAM-1 or fibronectin expression on individual BM-FB did not correlate with NALM- 6 migration. These results indicate that the adhesion and migration of human B-lymphoid precursors in the bone marrow microenvironment are mechanistically separable events and suggest the possibility of novel VLA-4 ligand(s), which may be important in human lymphopoiesis. Subpopulations of cells in the bone marrow microenvironment may preferentially support important aspects of lymphoid progenitor development.
Analysis of the mechanism of adhesion of precursor-B acute lymphoblastic leukemia cells to bone marrow fibroblasts
Authors: Bradstock K , Makrynikola V , Bianchi A , Byth K
Published: October 2019

Normal B lymphopoiesis is dependent on a close relationship between B- cell precursors and the bone marrow (BM) microenvironment. To further understand the mechanisms regulating the proliferation of the malignant counterpart of B-cell precursors, namely precursor-B acute lymphoblastic leukemia (ALL), we examined the adhesion to BM fibroblasts (BMF) of 19 cases of precursor-B ALL using a chromium labeling assay. Eleven of 19 cases showed greater than 10% binding to BMF (range 2.3% to 54.8%, mean 19.1%). Binding was increased approximately twofold by preincubation of BMF with tumor necrosis factor and interleukin-4, which also resulted in upregulation of expression of vascular cell adhesion molecule-1 (VCAM-1) on BMF. The mechanism of attachment was investigated using murine monoclonal antibodies to leukocyte integrins, principally the beta, integrins VLA- 4 and VLA-5, which were demonstrated to be present on most cases by flow cytometry. Statistically significant inhibition of adhesion was observed with antibodies to the beta 1 common subunit, VLA-4, and VLA- 5, whereas little effect was seen with antibodies to VLA-6 or the beta 2 integrin subunit. Preincubation of fibroblasts with an antibody to VCAM-1 (a ligand of VLA-4) inhibited leukemic cell binding in the majority of cases, which was an effect also observed on cytokine- stimulated BMF. However, a minority of cases, as well as the pre-B lines NALM-6 and KM-3, showed no evidence of inhibition of adhesion with anti-VCAM-1 antibodies. Treatment of BMF with antifibronectin antibody alone had little effect on ALL adhesion and did not enhance the inhibitory effect of anti-VCAM-1. These data indicate that precursor-B ALL cells bind to BM stroma through the beta 1 integrins VLA-4 and VLA-5 and that this effect is partly mediated by VCAM-1 on stromal cells, although other undefined VLA ligands are also likely to be involved. Attachment of ALL cells to stroma is likely to play a key role in regulating the survival and growth of these cells through exposure to stromal cytokines.
p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression
Authors: el Rouby S , Thomas A , Costin D , Rosenberg CR , Potmesil M , Silber R , Newcomb EW
Published: October 2019

We studied 53 patients with B-cell chronic lymphocytic leukemia (B-CLL) and found mutations of the p53 gene in 15%. Patients with p53 gene mutations were found to have an aggressive form of B-CLL disease characterized by advanced Rai stage, rapid lymphocyte doubling time (LDT), and resistance to chemotherapy. While 27 of 29 treated patients (93%) without p53 mutations achieved a partial remission, only one of seven treated patients (14%) with p53 mutations achieved a partial remission (P = .00009). Adjusting for prognostic factors (age, sex, race, and Rai stage), patients with p53 gene mutations had a 13-fold greater risk of death than patients without p53 mutations (P = .013). In addition to examining the clinical relevance of p53 gene mutations in B-CLL, we investigated the possible role of p53 gene regulation in the expression of the multidrug resistance genes MDR1 and MDR3. We quantitated MDR1 and MDR3 mRNA expression by reverse transcription- polymerase chain reaction (RT-PCR). Expression of both the MDR1 and MDR3 genes was independent of p53 gene mutation or prior drug treatment, and did not predict for clinical response. Our findings indicate that p53 gene mutations in B-CLL are associated with a poor clinical outcome and may be a prognostic indicator for drug resistance.
Alternatively spliced CD44 transcripts in diffuse large-cell lymphomas: characterization and comparison with normal activated B cells and epithelial malignancies
Authors: Salles G , Zain M , Jiang WM , Boussiotis VA , Shipp MA
Published: October 2019

CD44 exists in a variety of alternatively spliced isoforms that include variable numbers of additional exons from the membrane proximal domain (exons 6–14). Lymphocytes express high levels of a “hematopoietic” isoform (CD44H) with no additional variable exons. CD44H binds lymphocytes to postcapillary venules and promotes lymphocyte extravasation into nodal areas. Epithelial carcinomas also express CD44 variants, including exon 10-containing isoforms associated with metastasis in a rat model. An exon 10-containing CD44 isoform is also transiently expressed by normal activated lymphocytes, suggesting that the protein may function in the trafficking of both normal lymphocytes and metastatic tumors. To identify the specific CD44 transcripts in tumors from patients with primary nodal, extranodal, and disseminated large-cell lymphomas (LCLs) and compare them with CD44 mRNAs in normal Ig-activated splenic B cells and epithelial cells, we used a semiquantitative RNA-based polymerase chain reaction. Specific CD44 variants were identified by size, hybridization with exon-specific probes, and sequence analysis. In comparison to primary nodal LCLs, extranodal and disseminated LCLs had increased levels of CD44H and additional isoforms, including a directly spliced (exon 5-->exon 10-- >exon 15) exon 10-containing CD44 variant. The CD44 transcripts in extranodal and advanced-stage LCLs were similar to those in normal Ig- activated splenic B cells, whereas epithelial malignancies contained decreased CD44H and increased amounts of larger CD44 variants with additional exons from the membrane proximal domain. The regulated expression of specific CD44 variants is likely to influence the trafficking of lymphoid and epithelial malignancies.
Effectiveness and pharmacokinetics of low-dose all-trans retinoic acid (25 mg/m2) in acute promyelocytic leukemia
Authors: Castaigne S , Lefebvre P , Chomienne C , Suc E , Rigal-Huguet F , Gardin C , Delmer A , Archimbaud E , Tilly H , Janvier M
Published: October 2019

It has been shown that all-trans retinoic acid (ATRA) at doses of 45 to 100 mg/m2/d induces complete remission (CR) of acute promyelocytic leukemia (APL) by a differentiation process. To date, ATRA dose-ranging studies have not yet been evaluated. Thus, we initiated in May 1990 a multicenter study with ATRA at a lower dose of 25 mg/m2/d until CR. Thirty patients with APL were treated with ATRA, of whom 12 were previously untreated, 14 were in first relapse, and 4 had failed after conventional first induction chemotherapy. Twenty-four of 30 achieved CR, 3 failed, and 3 died before day 30. Median time to CR was 45 days. Hyperleucocytosis (14 to 43 x 10(9) white blood cells per liter) was observed in 9 patients between days 10 and 23. Clinical complications that may have been related to the retinoic acid syndrome were observed in 8 patients, of whom 3 died. Pharmacokinetics studies were performed in 5 patients. Peak plasma concentrations and mean area under the concentration-time curve were not lower than previous levels obtained under the 45 mg/m2 dose. Overall, our study shows that there is no difference in terms of therapeutic efficacy, triggering of hyperleukocytosis, or retinoic acid syndrome and pharmacokinetic results with ATRA at 25 or 45 mg/m2/d.
Chemotherapy of intermediate- and high-grade non-Hodgkin's lymphomas with an intensive epirubicin-containing regimen
Authors: Zuckerman KS , Case DC Jr , Gams RA , Prasthofer EF
Published: October 2019

An intensive chemotherapy regimen (EVDAC), including high-dose epirubicin, vincristine, and dexamethasone followed by cyclophosphamide and high-dose cytarabine, was administered to 54 untreated adults with intermediate or high-grade non-Hodgkin's lymphomas (NHL). The median age was 59, 61% were Ann Arbor Stage IV, 57% had “B” symptoms, 50% had serum lactate dehydrogenase greater than 250 U/L, and 48% had masses greater than 7 cm (33% > 10 cm) in diameter. Seventy-six percent of patients attained complete or probable complete remissions. The Kaplan- Meier actuarial failure-free survival at 7 years is 50%, and 59% (32 of 54) of all patients started on therapy remain alive and in first remission at a median of 62+ (range, 49+ to 76+) months from completion of therapy. Nearly all patients developed severe neutropenia. Febrile episodes requiring hospitalization during neutropenia occurred after 56% of courses of epirubicin, vincristine, and dexamethasone and after 9% of courses of cyclophosphamide and cytarabine; 80% of patients were hospitalized at least once. Platelet count nadirs of less than 20,000/microL occurred after only 1 of 146 evaluable courses of epirubicin and after none of the cyclophosphamide/cytarabine courses. Although 8 patients had decreases of at least 0.12 in their left ventricular ejection fractions (5 to below normal levels), none have developed clinically evident congestive heart failure. Clinically significant mucositis occurred after only 8% of courses of high-dose epirubicin. Three deaths from infections and one from hyperkalemia with cardiac arrest occurred during therapy. These results confirm that high remission and sustained, failure-free survival rates can be achieved in patients with aggressive NHL, using high-dose anthracycline-containing chemotherapy regimens. Epirubicin appears to have an advantage over doxorubicin at high doses because of decreased toxicity at a therapeutically equivalent dose. These phase II study results need to be validated in a randomized phase III trial, and growth factors should be used to attempt to reduce the neutropenia-associated complications.
Additive effect of erythropoietin and heme on murine hematopoietic recovery after azidothymidine treatment [see comments]
Authors: Harrison J , Kappas A , Levere RD , Lutton JD , Chertkov JL , Jiang S , Abraham NG
Published: October 2019

The ability of combination treatment with erythropoietin (Epo) and heme to rescue hematopoietic activity in mice from the suppressive effect of azidothymidine (AZT) was determined. Exposure of mice to AZT for 5 weeks produced marked anemia, thrombocytopenia, neutropenia, and weight loss, whereas mice that received Epo and heme for 3 subsequent weeks showed significant alleviation of AZT cytotoxicity. Treatment with Epo (10 U for 5 times/week) stimulated hematopoietic recovery in the AZT- treated animals and reduced the severe anemia and thrombocytopenia by 3 weeks. Administration of a lower Epo dose (1 U Epo) resulted in only a modest retardation of AZT-induced anemia, although, when combined with heme, there was a great improvement in recovery of erythropoiesis. The combination of heme with Epo (10 U) produced the optimum response, resulting in almost normal recovery of bone marrow cellularity as well as recovery of burst-forming units-erythroid (BFU-E) and splenic hematopoietic progenitor content (colony-forming unit-spleen [CFU-S]) by the end of 3 weeks of post-AZT treatment. Treatment with heme alone markedly enhanced the recovery of BFU-E and CFU-S, as well as body weight post-AZT; however, this recovery was not to the extent seen in combination with Epo (10 U). Long-term bone marrow cultures (LTBMCs) established from mice exposed to AZT for 8 weeks showed a marked reduction in cellularity and this was completely alleviated when mice received heme and Epo (10 U) for 3 weeks after 5 weeks of AZT administration. The additive effect of heme and Epo was seen in BFU-E production, as well as in CFU-S production, in LTBMCs. Thus, heme exerts a significant protective effect on hematopoietic progenitors in vivo and may be of potential clinical use in combination with Epo to promote effective erythropoiesis in the setting of AZT therapy.
Desferrioxamine induces erythropoietin gene expression and hypoxia- inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction
Authors: Wang GL , Semenza GL
Published: October 2019

Erythropoietin (EPO) gene transcription is activated in kidney cells in vivo and in Hep3B cells exposed to hypoxia or cobalt chloride. Hypoxia- inducible factor 1 (HIF-1) is a nuclear factor that binds to the hypoxia-inducible enhancer of the EPO gene at a site that is required for transcriptional activation. HIF-1 DNA-binding activity is induced by hypoxia or cobalt chloride treatment of Hep3B cells. We report that treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of HIF-1 by hypoxia or cobalt chloride. Induction by each of these stimuli was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. DFX appears to induce HIF-1 by chelating iron as induction was inhibited by coadministration of ferrous ammonium sulfate. DFX administration to mice transiently increased EPO RNA levels in the kidney. As previously shown for hypoxia and cobalt treatment, DFX also induced HIF-1 activity in non-EPO-producing cells, suggesting the existence of a common hypoxia signal-transduction pathway leading to HIF-1 induction in different cell types.
Solid-phase von Willebrand factor contains a conformationally active RGD motif that mediates endothelial cell adhesion through the alpha v beta 3 receptor
Authors: Denis C , Williams JA , Lu X , Meyer D , Baruch D
Published: October 2019

The interaction of von Willebrand factor (vWF) with the alpha v beta 3 integrin of human umbilical vein endothelial cells is dependent on the RGD sequence present at residues 1744–1746 of the mature vWF subunit. We compared vWF and its two dimeric fragments, SpIII (residues 1–1365) and SpII (residues 1366–2050), as adhesion substrates. Solid-phase vWF and SpII supported endothelial cell adhesion, whereas SpIII, which contains the glycoprotein (GP) Ib binding domain, did not. Soluble SpII inhibited adhesion to immobilized ligands, whereas soluble vWF did not, suggesting that exposure of the cell attachment domain involves a conformational modification of vWF. Dendroaspin and albolabrin, two RGD- containing peptides of the disintegrin family, were potent inhibitors of cell adhesion to vWF (IC50 approximately 15 nmol/L). Complete inhibition of endothelial cell adhesion to vWF was obtained in the presence of F(ab')2 of monoclonal antibody 9 to vWF, which blocks vWF binding to platelet GPIIb/IIIa. In contrast, monoclonal antibody 713 to vWF, which blocks its binding to platelet GPIb, did not inhibit cell adhesion to vWF. These results indicate that endothelial cell adhesion to vWF is mediated by an RGD-dependent interaction with alpha v beta 3, but does not seem to involve a GPIb-like receptor, and show the importance of the conformation of the RGD sequence.
Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein
Authors: Vasconcellos CA , Lind SE
Published: October 2019

Actin is an abundant intracellular protein that is released into the blood during tissue injury and its injection into rats causes microthrombi to form in the vasculature. This report and others have shown that actin filaments are able to aggregate platelets in an adenosine diphosphate (ADP)-dependent manner. The effects on this process of two plasma actin-binding proteins, vitamin D-binding protein (DBP) and gelsolin, were examined separately and together. The addition of DBP, a monomer-binding protein, to actin filaments did not affect their ability to induce platelet aggregation. However, severing of actin filaments with gelsolin resulted in an increased degree of platelet aggregation. Preincubation of F-actin with both gelsolin and DBP resulted in a significant inhibition of aggregation. The effects of DBP and gelsolin on actin-induced aggregation paralleled their effects on exchange of actin-bound adenine nucleotides. DBP inhibited 1, N6- ethenoadenosine 5′ triphosphate (epsilon-ATP) exchange with G-actin but not with F-actin. Gelsolin increased epsilon-ATP exchange with F-actin, which was largely abrogated by the addition of DBP. These results suggest that gelsolin's severing (and subsequent capping) of actin filaments not only results in an increase in the number of pointed filament ends but also in the dissociation of actin monomers containing ADP. Phalloidin, which stabilizes actin filaments while decreasing both monomer and nucleotide exchange, inhibited actin-induced aggregation, as well, indicating that depolymerization of actin filaments is not required to inhibit aggregation. Platelet activation by either G- or F- actin may thus be regulated by the local concentrations of the plasma actin-binding proteins gelsolin and DBP. Together, these proteins inhibit platelet aggregation in a manner that can be explained by their effects on actin's filament structure and the accessibility of its bound ADP. Depletion of DBP or gelsolin may allow actin released from injured tissues to stimulate purinergic receptors on platelets, and perhaps other cells, via its bound adenine nucleotides.
Interleukin-1 receptor antagonist blocks chemokine production in the mixed lymphocyte reaction
Authors: Lukacs NW , Kunkel SL , Burdick MD , Lincoln PM , Strieter RM
Published: October 2019

The mixed lymphocyte reaction (MLR) has previously been used to elucidate pathways of cytokine activation and T-lymphocyte proliferation and is regarded as a model that simulates responses in allograft rejection. Studies have indicated that interleukin-1 (IL-1), a potent inflammatory cytokine, may have an important activating role in the MLR response. The discovery of a naturally occurring IL-1 receptor antagonist protein (IRAP) has renewed interest in control of IL-1--dependent responses both in vitro and in vivo. MLR cultures were used to study the role of IL-1 and IRAP in the regulation of subsequent cytokines during a T-lymphocyte-mediated alloantigen response. The temporal expression of IL-1 and IRAP during 5-day one-way MLR assays suggested antagonistic production of the two cytokines. IL-1 was produced early in the response, peaking at 4 hours through day 2, subsequently declining to near-background levels on day 5 of culture. In contrast, production of IRAP was delayed until day 2, steadily increased on days 3 and 4, and peaked on day 5 of culture, which correlated with the declining levels of IL-1. The addition of graded doses of IRAP (25 to 1,000 ng/mL) to MLR cultures decreased IL-1 production but had no effect on T-lymphocyte proliferative response. In addition, IRAP had little effect on the production of either IL-2 or tumor necrosis factor. The addition of 25 ng/mL of IRAP to MLR assays showed significantly decreased levels of two potent chemotactic cytokines, IL-8 and macrophage inflammatory protein-1 alpha (MIP-1 alpha), at peak chemokine production on day 5 of culture. The levels of IL-8 and MIP-1 alpha could be restored by the addition of IL-1 to the IRAP-treated cultures. IL-8 and MIP-1 alpha represent the two different families of chemotactic cytokines, C-X-C (IL-8) and C-C (MIP-1 alpha), and potentially play important roles in the recruitment of leukocytes to a site of immune allogeneic response. These studies indicate that regulation of IL-1 by IRAP does not significantly reduce T-lymphocyte activation but can regulate the production of chemokines involved in leukocyte recruitment.
Transduction of human melanoma cell lines with the human interleukin-7 gene using retroviral-mediated gene transfer: comparison of immunologic properties with interleukin-2
Authors: Miller AR , McBride WH , Dubinett SM , Dougherty GJ , Thacker JD , Shau H , Kohn DB , Moen RC , Walker MJ , Chiu R
Published: October 2019

Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.
Regulation of AP-1 expression and activity in antigen-stimulated mast cells: the role played by protein kinase C and the possible involvement of Fos interacting protein
Authors: Lewin I , Nechushtan H , Ke Q , Razin E
Published: October 2019

We have recently observed that protein kinase C (PKC) was involved in the regulation of the accumulation of mRNAs of the AP-1 components in cultured Abelson-transformed murine fetal-liver-derived mast cells stimulated by exocytotic stimuli. Here we analyzed the probable regulatory effect of PKC on the synthesis and DNA-binding activity of AP-1 complexes in immunologic stimulated mast cells. In this study we used the interleukin-3--dependent murine fetal-liver--derived mast cells that were not transformed by the Abelson oncogene. Study of PKC- depleted cells showed PKC dependency of c-fos mRNA accumulation and protein expression in IgE-Ag stimulated cells. In contrast, the c-jun mRNA accumulation was unaffected by PKC depletion, whereas its protein expression was dependent on this enzymatic activity. This suggests the involvement of PKC in the regulation of translation of c-Jun, a level of c-Jun regulation that was not previously described. The amount of AP- 1 DNA-bound complex was also lowered in PKC-depleted cells. Therefore, PKC plays an important regulatory role in different stages of the signal transduction pathway because of IgE-Ag stimulation. Surprisingly, we have observed that although the amount of total synthesized c-Fos began to increase 15 minutes after immunologic stimulation, the amount of c-Fos associated with Juns did not increase, even after 45 minutes. This association was not affected by PKC. Using a Fos-interacting protein (FIP)-cDNA probe, an expression of 2.9 kb mRNA was detected in these cells. Furthermore, immunologic stimulation caused an increase in the amount of a Fos-containing protein complex that bound to an FIP-binding DNA oligonucleotide. Therefore, we propose that this protein complex that contains most of the immunologically induced c-Fos has an important role in IgE-Ag-stimulated signal transduction.
Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact
Authors: Marijt WA , Veenhof WF , Goulmy E , Willemze R , van Rood JJ , Falkenburg JH
Published: October 2019

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA- A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY- specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units- granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.
Clinical and biologic relevance of immunologic marker studies in childhood acute lymphoblastic leukemia
Authors: Pui CH , Behm FG , Crist WM
Published: October 2019

Immunologic marker studies of the lymphoid leukemias have greatly improved the precision of diagnosis of these disorders by providing specific information regarding the lineage and stage of maturation of the malignant cells. Such studies have also enhanced our understanding of normal lymphocyte development, permitting reproducible identification of lymphoid cells in discrete developmental stages. By elucidating the functions of lymphoid cell differentiation antigens, it has been possible to gain insight into the signal transduction mechanisms by which these cells interact among themselves and with other cell types. Similar studies have shown that ALL is an immunophenotypically heterogeneous disease with clinically important subtypes representing clonal expansions of lymphoblasts at different stages of maturation. Furthermore, successful correlation of immunophenotype with certain karyotypic and molecular abnormalities, which appear to underlie most or all leukemias, were made possible by the inclusion of immunologic marker assessment. Interestingly, many of these phenotype-related abnormalities have involved either the Ig or TCR genes, thus providing additional clues to the mechanisms of leukemogenesis. Knowledge of the immunologic features of leukemic cells has been essential for the generation of phenotype-specific response data in the context of modern therapy for ALL. With wider use of intensive treatment, the traditional prognostic distinctions among immunophenotypes have begun to disappear; however, certain classes of agents have more favorable toxicity/efficacy ratios against some immunophenotypes than others, justifying continued efforts to target therapy by immunologic species of ALL. Antibody-toxin conjugates, or immunotoxins, have induced complete responses in preliminary trials and may prove clinically useful, perhaps in combination with chemotherapy, if their toxic side effects can be controlled. Finally, immunologic markers may serve as sensitive targets for the detection of minimal residual disease; the clinical usefulness of this approach will depend on prospective comparisons with molecular methods.
Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe beta-thalassemia (Cooley's anemia) [see comments]
Authors: Yuan J , Angelucci E , Lucarelli G , Aljurf M , Snyder LM , Kiefer CR , Ma L , Schrier SL
Published: October 2019

The profound and life-threatening anemia in patients with Cooley's anemia is ascribed primarily to intramedullary hemolysis (ineffective erythropoiesis), the cause of which is obscure. Based on prior morphologic data showing nuclear abnormalities, we hypothesized that accelerated apoptosis could occur in these erythroid precursors. The highly successful bone marrow (BM) transplantation program for patients with Cooley's anemia provided us with a unique opportunity to test this hypothesis. We obtained pretransplantation BM aspiration samples from patients undergoing BM transplantation in Pesaro, Italy and from their allogeneic donors. The erythroid precursors were isolated using ficoll sedimentation and then panning selecting fro CD45- cells. Cytospin and Giemsa staining showed that the separation provided greater than 90% erythroblasts. Five million of these erythroblasts were lysed and their DNA was isolated. There were obvious ladder patterns of DNA breakdown products in beta-thalassemia major samples, with less occurring in beta- thalassemia trait. Normal individuals showed only a slight smear of breakdown of DNA. These results indicate there is enhanced apoptosis in the erythroblasts in the BMs of Cooley's anemia patients. This finding might partially explain why most of these erythroblasts never survive to become mature erythrocytes.
Stem cell factor influences the proliferation and erythroid differentiation of the MB-02 human erythroleukemia cell line by binding to a high-affinity c-kit receptor
Authors: Broudy VC , Morgan DA , Lin N , Zsebo KM , Jacobsen FW , Papayannopoulou T
Published: October 2019

Stem cell factor (SCF) acts in synergy with other growth factors such as erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3), to stimulate the growth of primitive hematopoietic cells. Because of the prominent role of CSF in the maintenance of normal erythropoiesis in vivo, we examined the effects of SCF on the Epo-inducible human erythroleukemia cell line MB- 02, and characterized the c-kit receptor in these cells. MB-02 cells cultured in serum-containing media do not survive in the absence of exogenous growth factors, but the addition of SCF, Epo, or IL-3 as a single factor enhanced MB-02 survival. Furthermore, in the presence of Epo, SCF (5 to 25 ng/mL) enhanced MB-02 proliferation in a dose- dependent manner, and increased the relative and absolute number of benzidine-positive cells generated. SCF also enhanced cell proliferation in the presence of either IL-3 or low concentrations of GM-CSF. A neutralizing anti-c-kit receptor monoclonal antibody (SR-1) blocked binding of 125I-SCF to MB-02 cells by 98%, and the effect of SCF on MB-02 growth, c-kit receptor-binding parameters were quantitated by equilibrium-binding experiments with 125I-SCF. MB-02 cells display a single class of high-affinity (50 pmol/L) c-kit receptors, with approximately 8,000 receptors per cell. The molecular weight of the c- kit receptor was determined by affinity cross-linking 125I-SCF to MB-02 cells. 125I-SCF-c-kit receptor complexes of approximately 155,000 and approximately 310,000 daltons were found, likely representing the monomeric and dimeric forms of the c-kit receptor. The binding affinity and molecular weight of the c-kit receptor on MB-02 cells are similar to those of normal human marrow cells. These results suggest that SCF synergizes with Epo to influence not only the proliferation but the erythroid differentiation of MB-02 cells. Thus, the MB-02 cell line may be a useful model in which to investigate the molecular mechanisms of SCF action.
Expression of platelet C1 inhibitor
Authors: Schmaier AH , Amenta S , Xiong T , Heda GD , Gewirtz AM
Published: October 2019

Human platelets contain a pool of C1 inhibitor (C1 INH) distinct from that in plasma. Twelve normal platelet samples washed by centrifugation had a mean platelet C1 INH antigen level of 19.3 +/- 2.8 ng (mean +/- SEM) per 10(8) platelets. These values contrast with the mean +/- SEM platelet C1 INH antigen level of 6.1 +/- 0.9 per 10(8) platelets from 12 C1 INH-deficient patients. The level of platelet C1 INH correlated (r = .7) with the level of plasma C1 INH in normal individuals and patients with classic hereditary angioedema. Platelet C1 INH, like plasma C1 INH, was a 105-Kd protein on immunoblots of solubilized platelets and in thrombin- or collagen-induced platelet releasates. On indirect immunofluorescence, morphologically and immunochemically identifiable elutriated human megakaryocytes had C1 INH antigen. Using nested primer polymerase chain reaction, C1 INH mRNA was detected in megakaryocytes. When activated, human platelets expressed a portion of their total pool of C1 INH antigen on their membrane. Using a competitive enzyme-linked immunosorbent assay for C1 INH as a quantitative, indirect antibody consumption assay, the surface of unstimulated platelets had 0.55 +/- 0.4 ng C1 INH/10(8) platelets (mean +/- SEM). When activated with thrombin, platelets secreted 7.37 +/- 2.2 ng C1 INH/10(8) platelets into the suspension buffer and simultaneously expressed 4.4 +/- 1.2 ng C1 INH/10(8) platelets on their external membrane. These studies showed that activated platelets secreted 38% of their C1 INH and externalized another 23% of the total platelet C1 INH on their membrane. Furthermore, in 125I-anti-C1 INH Fab' binding experiments to platelets, about 8 ng of the antibody fragment specifically bound to 10(8) activated platelets. These data suggest that level of platelet C1 INH packaged into platelet alpha-granules is modulated by the amount of protein produced in megakaryocytes. Platelet alpha-granule C1 INH can both be secreted from platelets and expressed on their activated membranes. The cell membrane expression of C1 INH may be important to modulate the activity of the proteases of the complement and contact systems of plasma proteolysis in the microenvironment of the inflammatory response.
Platelets lacking functional CD36 (glycoprotein IV) show reduced adhesion to collagen in flowing whole blood
Authors: Diaz-Ricart M , Tandon NN , Carretero M , Ordinas A , Bastida E , Jamieson GA
Published: October 2019

A parallel-plate perfusion chamber has been used to evaluate the contribution of the adhesive membrane glycoprotein CD36 (GPIV) to platelet adhesion on type I collagen in flowing whole blood at a shear rate of 800 s-1. In one series of experiments, reconstituted normal blood (hematocrit 0.4; platelet count 1.5 x 10(5)/microL) was prepared from washed red blood cells, plasma, and washed platelets that had been incubated with Fab fragments of a monospecific polyclonal anti-CD36 antibody (50 micrograms/mL, 30 minutes, 37 degrees C). Percent surface coverage of collagen-coated coverslips using reconstituted blood with antibody-blocked platelets, as compared with paired reconstituted controls (100%), was 50% at 2 minutes, 87% at 5 minutes, and 90% at 10 minutes. Further studies were performed by perfusion of whole blood from a healthy donor of the Naka-negative phenotype, whose platelets constitutively lack CD36, over collagen-coated coverslips. In this case, percent surface coverage was 55% of normal controls at 2 minutes, 76% of controls at 5 minutes, and 72% of controls at 10 minutes. In both preparations, platelets lacking functional CD36 had a statistically significant decrease (P < .005) in adhesion after 2 minutes and 10 minutes perfusion but not at 5 minutes. These results show that functional CD36 facilitates the rapid adhesion of platelets to collagen and that this effect is seen at the earliest time points of their interaction.
Four ricin chain A-based immunotoxins directed against the common chronic lymphocytic leukemia antigen: in vitro characterization
Authors: Faguet GB , Agee JF
Published: October 2019

The common B-chronic lymphocytic leukemia (B-CLL) antigen (cCLLa) appears to be ideal for targeted immunotherapy in that it is the most prevalent and disease-restricted marker in B-CLL. To assess this potential, we developed four immunotoxins (ITs) of anti-cCLLa monoclonal antibody CLL2m (an IgG2a kappa), using ricin chain A (RTA) or its deglycosylated derivative (dgA), each conjugated to either the whole IgG molecule or its Fab' fragment. Each IT was tested in vitro for specificity and cytotoxic activity (assessed by protein synthesis inhibition [PSI] and by cell kill [CK] in the clonogenic assay) against B-CLL cells. RTA-based anti-CD5 ITs and enriched normal B and T lymphocytes were used as controls. Each IT exhibited antigen-specific, dose-dependent activity. Thus, whereas B-CLL cells exhibited dose- dependent PSI and CK (whether the B-CLL clone was CD5+ or CD5-), normal B (cCLLa-/CD5-) and T lymphocytes (cCLLa-/CD5+) remained unaffected. IT potency was independent of toxin glycosylation, but was slightly influenced by antibody valence; divalent ITs were twice as potent as monovalent ITs (IC50, 2.3 v 7.1 x 10(-11) mol/L; CK, 2.6- v 2.0-log reached with 524 v 1,072 IT molecules bound/cell, respectively). In the presence of ammonium chloride or Verapamil, IT-induced CK was enhanced 10- to 80-fold. These data suggest that the cCLLa is a promising target for IT-based immunotherapy of B-CLL in vivo and ex vivo.