bluuebunny/crossref_metadata_2025 huggingface dataset:
Human interleukin-3 receptor modulates bcl-2 mRNA and protein levels through protein kinase C in TF-1 cells
Authors: Rinaudo MS , Su K , Falk LA , Halder S , Mufson RA
Published: October 2019

Upon withdrawal of interleukin-3 (IL-3) from human factor-dependent erythroleukemic cell line TF-1, bcl-2 mRNA and protein levels decrease within 8 to 24 hours. Accompanying this decrease is the onset of apoptosis as determined by flow cytometric analysis of DNA degradation. By 8 to 18 hours of deprivation approximately 70% to 80% of the cells have entered apoptosis. Downregulation of protein kinase (PK) by a 24- hour incubation in 100 nmol/L 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of IL-3 dramatically reduced bcl-2 mRNA levels, and induced apoptosis in the presence of IL-3. We have also found that even in the presence of IL-3, two inhibitors of PKC, light-activated calphostin and H-7, substantially reduced the levels of bcl-2 mRNA between 8 and 24 hours as measured by a semi-quantitative reverse transcriptase/polymerase chain reaction assay method; however, the cyclic nucleotide-dependent PK inhibitor HA 1004, that is a structural analog of H-7 but a poor inhibitor of PKC, did not reduce bcl-2 levels in the presence of IL-3. This decrease in bcl-2 mRNA was accompanied by a decline in bcl-2 protein levels by 8 to 24 hours after addition of light-activated calphostin. In addition to interfering with the maintenance of bcl-2 mRNA levels, inhibition of PKC with H-7 inhibited the induction of bcl-2 mRNA in factor-deprived TF-1 cells restimulated with IL-3. The cyclic nucleotide-dependent PK inhibitor HA 1004 did not inhibit IL-3-induced bcl-2 mRNA. Studies with actinomycin D showed that transcription plays a major role in maintaining bcl-2 levels in TF-1 cells, and it is therefore likely that IL-3 plays a role in maintaining bcl-2 transcription through activation of PKC in these cells.
Role of light chain variable region in myeloma with light chain deposition disease: evidence from an experimental model
Authors: Khamlichi AA , Rocca A , Touchard G , Aucouturier P , Preud'homme JL , Cogne M
Published: October 2019

Light chain deposition disease (LCDD) results from a propensity of some human monoclonal L chains to form tissue deposits. We designed an experimental model for in vivo expression of human kappa L chain sequences in mice and compared a somatically mutated LCDD chain with a closely related control kappa chain, both encoded by the unique V kappa IV gene. Mice secreting the LCDD chain but not those producing the control chain showed deposits with a distribution similar to that observed in patients. These data show that discrete changes in V region sequences can play a major role in tissue deposition of human L chains.
Polyclonal primitive hematopoietic progenitors can be detected in mobilized peripheral blood from patients with high-risk myelodysplastic syndromes
Authors: Delforge M , Demuynck H , Vandenberghe P , Verhoef G , Zachee P , van Duppen V , Marijnen P , Van den Berghe H , Boogaerts MA
Published: October 2019

Myelodysplastic syndromes (MDS) form a heterogeneous group of clonal hematopoietic disorders with unfavourable prognosis. Allogeneic bone marrow transplantation is the only potentially curative treatment, but remains limited to a small subgroup of younger patients with HLA- compatible donors. As autologous stem cell transplantation is currently being explored as an alternative treatment strategy for MDS, more information needs to be acquired regarding the clonal nature of the progenitor cells in these autografts. Therefore, we have analyzed the clonal patterns of highly purified hematopoietic progenitors and their mature daughter cells in mobilized peripheral blood collections produced from five female patients with high-risk MDS in complete hematologic remission. X-chromosome activation patterns of flow-sorted immature (CD34 + 38low, CD34 + 33low) and committed (CD34 + 38high, CD34 + 33high) progenitors were studied with the polymerase chain reaction-based HUMARA assay. In four patients, a polyclonal remission was shown in all stem cell subpopulations and their mature daughter cells whereas one patient was found to remain skewed in all fractions, except T lymphocytes. This study provides strong evidence that polyclonal immature hematopoietic progenitors can be mobilized and harvested in patients high-risk MDS after treatment with high-dose chemotherapy.
A functional comparison of CD34 + CD38- cells in cord blood and bone marrow
Authors: Hao QL , Shah AJ , Thiemann FT , Smogorzewska EM , Crooks GM
Published: October 2019

We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7- aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as = 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.
The human immunodeficiency virus type-1 Tat protein upregulates Bcl-2 gene expression in Jurkat T-cell lines and primary peripheral blood mononuclear cells
Authors: Zauli G , Gibellini D , Caputo A , Bassini A , Negrini M , Monne M , Mazzoni M , Capitani S
Published: October 2019

The regulatory Tat protein of human immunodeficiency virus type-1 (HIV- 1) exerts a pleyotropic activity on the survival and proliferation of different cell types in culture. In this report, we investigated the effect of either endogenous or exogenous Tat on Bcl-2 proto-oncogene expression and cell survival in Jurkat T-cell lines and primary peripheral blood mononuclear cells. Stable and transient transfections of Jurkat cells with the cDNA of tat and a plasmid containing Bcl-2 promoter in front of CAT (Bcl-2 Pr/CAT) stimulated CAT activity and showed an increase of Bcl-2 mRNA and protein expression. This effect was specifically related to tat, because Jurkat cells transfected with the cDNA of tat in antisense orientation, tat carrying a mutation in the amino acid cys22-gly22, or the control vector alone (pRPneo-SL3) did not show any significant difference in Bcl-2 promoter activity with respect to parental Jurkat cells. We also observed a specific correlation between tat-induced Bcl-2 gene expression and inhibition of apoptosis induced by serum withdrawal. Our results suggest that the structural integrity of the activation domain of Tat was required for the promotion of the Bcl-2 promoter and Jurkat cell survival, because a single mutation in the aminoacid cys22 was sufficient to completely block the upregulation of Bcl-2 and inhibition of apoptosis. Moreover, picomolar concentrations of native or recombinant Tat were able to upregulate Bcl-2 expression both in Jurkat and primary peripheral blood mononuclear cells, suggesting that extracellular Tat, actively released by infected cells, may also play a significant role in suppressing apoptosis. An aberrant cell survival of lymphoid cells consequent to the upregulation of Bcl-2 may represent an additional pathogenetic mechanism that could help explain both the dysregulated immune response and the frequent occurrence of hyperplastic/neoplastic disorders in HIV- 1-seropositive individuals.
Analysis of VH gene expression in CD5+ and CD5- B-cell chronic lymphocytic leukemia
Authors: Maloum K , Davi F , Magnac C , Pritsch O , McIntyre E , Valensi F , Binet JL , Merle- Beral H , Dighiero G
Published: October 2019

In contrast to highly mutated follicular lymphomas and multiple myelomas, chronic lymphocytic leukemias (CLLs) frequently express VH genes in germline configuration. It is currently unclear whether this difference is related to the expression of CD5 or to the differentiation stage of the B cell when malignant transformation occurs. We have studied the VH sequence of 11 cases of CD5- B-CLL to address the question whether CD5- B-CLL are derived from naive pregerminal B cells (low mutation pattern) or from germinal center- derived memory B cells (high mutation pattern). Among the 12 detected rearrangements (2 distinct rearrangements in 1 case) VH1 family was found in 2, VH2 in 2, VH3 in 4, and VH4 in 4. Nine different VH genes were detected among the 12 rearrangements, including 2 cases expressing V1–69 (51p1) and 1 case expressing V4–39 (VH4.18), previously reported to be overexpressed in CD5+ B-CLL. A higher mutation pattern, following a random distribution, was observed when compared with classical CD5+ B- CLL. However, as reported in normal B cells, these results appeared to be related to membrane Ig phenotype (less mutations in membrane mu delta-expressing forms in leukemias expressing exclusively membrane mu). Overall, the differences found when comparing the mutational profile with classical CD5+ B-CLL were not clearcut and might be explained more by the membrane isotype (mu v mu delta) than by CD5 expression.
Autologous progenitor cell transplantation: prior exposure to stem cell- toxic drugs determines yield and engraftment of peripheral blood progenitor cell but not of bone marrow grafts
Authors: Dreger P , Kloss M , Petersen B , Haferlach T , Loffler H , Loeffler M , Schmitz N
Published: October 2019

Agents with stem cell-toxic potential are frequently used for salvage therapy of Hodgkin's disease (HD) and high-grade non-Hodgkin's lymphoma (NHL). Because many patients with relapsed or refractory lymphoma are candidates for autologous progenitor cell transplantation, possible toxic effects of salvage chemotherapy on progenitor cells must be taken into account. In a retrospective study, we have analyzed the influence of a salvage regimen containing the stem cell-toxic drugs BCNU and melphalan (Dexa-BEAM) on subsequently harvested bone marrow (BM)- and peripheral blood-derived progenitor cell grafts (PBPC) and compared it with other factors. Progenitor cells were collected from 96 patients with HD or high-grade NHL. Seventy-nine grafts were reinfused (35 PBPC and 44 BM) after high-dose chemotherapy. Compared with patients autografted with BM, hematopoietic recovery was significantly accelerated in recipients of PBPC. For PBPC, the number of Dexa-BEAM cycles ( > or = v > 1) was the predominate prognostic factor affecting colony-forming unit-granulocyte-macrophage (CFU-GM) yield (66 v 6.8 x 10(4)/kg, P = .0001), CD34+ cell yield (6.6 v 1.6 x 10(6)/kg, P = .0001), neutrophil recovery to > 0.5 x 10(9)/L (9 v. 11 days, P = .0086), platelet recovery to > 20 x 10(9)/L (10 v 15.5 days, P = .0002), and platelet count on day +100 after transplantation (190 v 107 x 10(9)/L, P = .031) using univariate analysis. Previous radiotherapy was associated with significantly lower CFU-GM and CD34+ cell yields but had no influence on engraftment. Patient age, patient sex, disease activity, or chemotherapy other than Dexa-BEAM did not have any prognostic impact. Multivariate analysis confirmed that Dexa-BEAM chemotherapy was the overriding factor adversely influencing CFU-GM yield (P < .0001), CD34+ cell yield (P < .0001), and platelet engraftment (P < .0001). BM grafts were not significantly affected by previous Dexa-BEAM chemotherapy or any other variable tested. However, prognostic factors favoring the use of BM instead of PBPC were not identified using joint regression models involving interaction terms between the graft type (PBPC or BM) and the explanatory variables investigated. We conclude that, in contrast to previous radiotherapy or other chemotherapy, exposure to salvage regimens containing stem cell- toxic drugs, such as BCNU and melphalan, is a critical factor adversely affecting yields and performance of PBPC grafts. Marrow progenitor cells appear to be less sensitive to stem cell-toxic chemotherapy. PBPC should be harvested before repeated courses of salvage chemotherapy involving stem cell-toxic drugs to preserve the favorable repopulation kinetics of PBPC in comparison with BM.
IgE-independent interleukin-4 expression and induction of a late phase of leukotriene C4 formation in human blood basophils
Authors: Ochensberger B , Rihs S , Brunner T , Dahinden CA
Published: October 2019

T-helper cells can differentiate into at least two subtypes secreting distinct profiles of cytokines, Th1 and Th2, regulating immunoprotection and different immunopathologies. Interleukin-4 (IL-4) is both the product and the inducer of Th2 cells, raising the question whether IL-4 can be produced in response to antigen-independent stimuli. Here we show that human basophils produce IL-4 on stimulation with IL-3 and C5a or C5adesarg in similar amounts as induced by IgE- receptor-cross-linking. C5a-induced IL-4 production requires the presence of IL-3, with little effect of the sequence of stimuli addition. No “Th1-cytokines” (interferon-gamma and IL-2) and even no “Th2-cytokines” (IL-3, IL-5, IL-10, and granulocyte-macrophage colony- stimulating factor) are produced by basophils in response to either IgE- dependent or IgE-independent activation. The generation of leukotriene C4 (LTC4) is regulated in a similar manner. However, C5a induces a rapid, transient burst of leukotriene formation only if added after IL- 3. Interestingly, upon prolonged culture, a late phase of continuous LTC4 production is observed, which also requires two signals (IL-3 and C5a), but rather depends on their continuous presence than on their sequence of action. These data describe an antigen-independent pathway of very restricted IL-4 expression. Thus, basophils must be considered as central immunoregulatory cells of the innate immune system. Furthermore, the results show that LTC4 can also be generated more continuously for many hours, a phenomenon that may be of particular importance in chornic allergic inflammation, such as asthma.
Monoclonal antibody F1 binds to the kringle domain of factor XII and induces enhanced susceptibility for cleavage by kallikrein
Authors: Ravon DM , Citarella F , Lubbers YT , Pascucci B , Hack CE
Published: October 2019

In a previous study we have shown that monoclonal antibody F1 (MoAb F1), directed against an epitope on the heavy chain of factor XII distinct from the binding site for anionic surfaces, is able to activate factor XII in plasma (Nuijens JH, et al: J Biol Chem 264; 12941, 1989). Here, we studied in detail the mechanism underlying the activation of factor XII by MoAb F1 using purified proteins. Formation of factor XIIa was assessed by measuring its amidolytic activity towards the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302) in the presence of soybean trypsin inhibitor and by assessing cleavage on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Upon incubation with MoAb F1 alone, factor XII was auto-activated in a time-dependent fashion, activation being maximal after 30 hours. Factor XII incubated in the absence of MoAb F1 was hardly activated by kallikrein, whereas in the presence of MoAb F1, but not in that of a control MoAb, the rate of factor XII activation by kallikrein was promoted at least 60-fold. Maximal activation of factor XII with kallikrein in the presence of MoAb F1 was reached within 1 hour. This effect of kallikrein on the cleavage of factor XII bound to MoAb F1 was specific because the fibrinolytic enzymes plasmin, urokinase, and tissue-type plasminogen activator could not substitute for kallikrein. Also, trypsin could easily activate factor XII, but in contrast to kallikrein, this activation was independent of MoAb F1. SDS-PAGE analysis showed that the appearance of amidolytic activity correlated well with cleavage of factor XII. MoAb F1-induced activation of factor XII in this purified system was not dependent on the presence of high- molecular-weight kininogen (HK), in contrast to the activation of the contact system in plasma by MoAb F1. Experiments with deletion mutants revealed that the epitopic region for MoAb F1 on factor XII is located on the kringle domain. Thus, this study shows that binding of ligands to the kringle domain, which does not contribute to the proposed binding site for negatively charged surfaces, may induce activation of factor XII. Therefore, these findings point to the existence of multiple mechanisms of activation of factor XII.
Procoagulant activity of reversibly acylated human factor Xa
Authors: Wolf DL , Lin PH , Hollenbach S , Wong A , Phillips DR , Sinha U
Published: October 2019

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p′-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the “tenase” complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.
An essential role for lysophosphatidylcholine in the inhibition of platelet aggregation by secretory phospholipase A2
Authors: Yuan Y , Jackson SP , Newnham HH , Mitchell CA , Salem HH
Published: October 2019

The release of secretory phospholipase A2 (sPLA2) into the mammalian circulation may contribute to the development of hemorrhagic and inflammatory diseases. sPLA2 has previously been shown to alter the behavior of platelets, leukocytes, and endothelial cells, although the molecular basis for these cellular effects has not been established. Our studies indicate that the inhibition of platelet aggregation by snake, bee venom, and pancreatic sPLA2 is dependent on a plasma cofactor. This cofactor resides within the lipoprotein fraction of plasma, with 54%, 31%, and 11% of the activity present in the high- density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions, respectively. Delipidation of HDL and LDL was associated with the complete loss of platelet-inhibitory activity. Incubation of purified sPLA2 with the HDL fraction of plasma resulted in the time-dependent generation of lysophosphatidylcholine (lysoPC). The formation of lysoPC correlated with the inhibition of platelet aggregation. Purified lysoPC (10 to 100 micrograms/mL) inhibited platelet aggregation and dense granule release induced by thrombin (0.05 U/mL), collagen (1 micrograms/mL), ionophore A23187 (2 mumol/L), ADP (12.5 mumol/L), and adrenaline (3.2 mumol/L). The inhibition of platelet aggregation by lysoPC was dose-dependent and correlated with decreased fibrinogen binding to glycoprotein IIb-IIIa. Our studies indicate that the enzymatic generation of lysoPC from plasma lipoproteins is essential for the sPLA2-mediated inhibition of platelet activation in the presence of albumin. These results raise the possibility that the toxic effects of circulating sPLA2 may be due in part to the generation of the bioactive lysophospholipid, lysoPC.
CD43 regulates tyrosine phosphorylation of a 93-kD protein in T lymphocytes
Authors: Manjunath N , Ardman B
Published: October 2019

The leukocyte sialyloglycoprotein CD43 exhibits features of a signal transducing molecule and is thought to be important for T-cell activation and adhesion. However, cellular biochemical events in which CD43 participates remain poorly understood. Here we provide evidence that CD43 regulates tyrosine phosphorylation of a specific substrate in T cells. A 93-kD tyrosine phosphoprotein was identified specifically in the CD43+ T-cell line CEM, but not in their CD43-deficient counterparts derived by gene targeting. The 93-kD phosphoprotein was detected in the CD43-deficient CEM cells after transfection with CD43 cDNA, and it could be specifically phosphorylated in lysates from the CD43-deficient cells by incubation with a CD43 immunoprecipitate obtained from the CD43+ cells. Expression of CD43 in HeLa cell transfectants was associated with the appearance of novel phosphoproteins including one with a molecular weight of approximately 93 kD, confirming that tyrosine phosphorylation of cellular substrates results specifically from CD43 expression. We conclude that CD43 regulates tyrosine phosphorylation of a 93-kD T-cell substrate.
Characterization of T cells immortalized by Tax1 of human T-cell leukemia virus type 1
Authors: Akagi T , Ono H , Shimotohno K
Published: October 2019

Peripheral blood T cells were immortalized in vitro by introduction of the Tax1 gene of human T-cell leukemia virus type 1 (HTLV-1) with a retroviral vector and were characterized for transformation-associated markers. Long-term observation showed that these Tax1-immortalized T cells eventually exhibited very similar features that were characteristic of HTLV-1-immortalized T cells, ie, increased expression of egr-1, c-fos, IL-2R alpha, and Lyn and decreased expression of Lck and cell-surface CD3 antigen. Among these changes, an increase in the expression of Lyn and a decrease in the expression of Lck and cell- surface CD3 antigen were observed only in Tax1-immortalized T cells after long-term culture. The expression level of Tax1 protein did not differ significantly between early and late passage of cells, and the cellular clonality was found to be the same by the analysis of the retroviral vector integration site and the T-cell receptor beta-chain gene rearrangement pattern. These changes in the expression of Lyn, Lck, and cell-surface CD3 antigen probably resulted from indirect effects of Tax1 that appeared after extended culture.
Macrophage inflammatory protein-1 alpha receptors are present on cells enriched for CD34 expression from patients with chronic myeloid leukemia
Authors: Chasty RC , Lucas GS , Owen-Lynch PJ , Pierce A , Whetton AD
Published: October 2019

The response of normal and chronic myeloid leukemia (CML), CD34+ cells to human macrophage inflammatory protein-1 alpha (MIP-1 alpha or LD78) was assessed. In tritiated thymidine incorporation assays, stem cell factor plus granulocyte-macrophage colony-stimulating factor stimulated thymidine incorporation in normal CD34+ cells was reduced to 72% of control values in the presence of MIP-1 alpha, whereas incorporation by CML CD34+ cells exposed to the same factors was not altered. In clonogenic assays, the presence of MIP-1 alpha gave a level of colony formation that was 71% of control values for normal progenitor cells, whereas for CML CD34+ cells colony formation was enhanced by 25%. These results suggest that, in vitro, CML progenitor cells are relatively refractory to the growth inhibitory effects of MIP-1 alpha. Using flow cytometry, the specific binding of a biotinylated human MIP-1 alpha/avidin fluorescein (FITC) conjugate to normal and CML mononuclear and CD34+ cell populations was quantified. The data indicate that (for both normal and CML CD34+ cells) there was a single population of cells that express cell surface receptors for MIP-1 alpha and this receptor expression was independent of cell cycle status. CML progenitor cells may be refractory to the effects of MIP-1 alpha as a result of events downstream from receptor expression.
Anti-B4-blocked ricin synergizes with doxorubicin and etoposide on multidrug-resistant and drug-sensitive tumors
Authors: O'Connor R , Liu C , Ferris CA , Guild BC , Teicher BA , Corvi C , Liu Y , Arceci RJ , Goldmacher VS , Lambert JM
Published: October 2019

Anti-B4-blocked ricin (anti-B4-bR) is an immunotoxin directed against CD19-positive cells that is currently being tested in several B-cell leukemia/lymphoma clinical trials. To explore the possibility of using anti-B4-bR in combination with chemotherapy protocols, we investigated the in vitro and in vivo cytotoxic effects of combining it with doxorubicin or etoposide using the lymphoma cell line Namalwa and a P- glycoprotein-expressing cell line, Namalwa/mdr-1, obtained by retroviral infection of Namalwa cells with the mdr-1 gene. Namalwa/mdr- 1 cells were slightly more sensitive to anti-B4-bR than Namalwa cells; IC37 values were approximately 4 pmol/L and 8 pmol/L, respectively. When anti-B4-bR was combined simultaneously with doxorubicin or etoposide, additive to supra-additive killing of Namalwa and Namalwa/mdr-1 cells was observed. In xenografts of Na-malwa/mdr-1 cells in severe combined immunodeficiency (SCID) mice, doxorubicin and etoposide at their maximum tolerated doses (3 mg/kg x 3 or 15 mg/kg x 3) showed no therapeutic effect. However, treatment with 5 daily bolus injections of anti-B4-bR (50 micrograms/kg) followed by treatment with doxorubicin or etoposide significantly increased the life span of the mice by 129% and 115%, respectively. After treatment with anti-B4-bR, the Namalwa/mdr-1 population expressed lower levels of P-glycoprotein, and this decrease may account for the synergistic action of the drug combinations. These results suggest that anti-B4-bR could be used to good effect in combination with current treatment regimens and further hint at a promising role for this immunotoxin in treatment of disease at the minimal residual disease stage, where cells may be resistant to chemotherapy.
Increased cation permeability in mutant mouse red blood cells with defective membrane skeletons
Authors: Joiner CH , Franco RS , Jiang M , Franco MS , Barker JE , Lux SE
Published: October 2019

Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Nac+) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 +/- 0.9, 28.9 +/- 0.3, and 26.9 +/- 1.5 mmol/L cell, respectively, whereas cellular potassium (Kc+) was 102 +/- 2.6, 101 +/- 7.8, and 97.4 +/- 3.0. Nac+ and Kc+ in normal reticulocyte preparations were 11.3 +/- 0.7 and 123 +/- 10, respectively. Net Na+ and K+ fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 +/- 1.6, 15.4 +/- 3.3, and 14.7 +/- 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+ loss was 17.0 +/- 4.0, 15.0 +/- 3.8, and 14.1 +/- 2.6. Normal mouse reticulocytes gained Na+ at a rate of 3.9 +/- 1.0 mmol/L cell/h and lost K+ at 6.0 +/- 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphha and nb/nb cells was not dependent on the presence of a Na+ gradient, and net cation movements were insensitive to bumetanide (sphha/sphha and nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.
Pyruvate kinase deficiency of mice associated with nonspherocytic hemolytic anemia and cure of the anemia by marrow transplantation without host irradiation
Authors: Morimoto M , Kanno H , Asai H , Tsujimura T , Fujii H , Moriyama Y , Kasugai T , Hirono A , Ohba Y , Miwa S
Published: October 2019

Mutant mice with splenomegaly and nonspherocytic hemolytic anemia were found in an inbred colony of the CBA/N (hereafter CBA) strain maintained in the Japan SLC Haruno farm (Shuchi-gun, Shizuoka, Japan). The activity of pyruvate kinase (PK) in red blood cells (RBCs) of the anemic mutants decreased to 16.2% of normal (+/+) CBA mice. Because the mutant CBA mice showed a remarkable reticulocytosis (41.6%) and because the PK activity of reticulocytes is much higher than that of mature RBCs, the PK activity in mature RBCs of the mutant CBA mice was calculated to be 2.8% that of mature RBCs of CBA-(+/+) mice. Because RBC type PK is encoded by the Pk-1 locus of the mouse (chromosome 3), we designated the mutant locus as Pk-1slc. The anemia and PK deficiency of CBA-Pk-1slc/Pk-1slc mice were cured by bone marrow transplantation (BMT) from CBA-(+/+) mice. Prior irradiation was not necessary for the curative BMT. On the other hand, the BMT from CBA-Pk-1slc/Pk-1slc mice to nonirradiated CBA-(+/+) mice did not result in the decrease of RBCs and the reduction of PK activity. The present results indicate that CBA- Pk-1slc/Pk-1slc mice are a potentially useful animal model for studying pathophysiology of PK deficiency and for developing new therapeutic methods to correct PK deficiency.
BB-10010: an active variant of human macrophage inflammatory protein-1 alpha with improved pharmaceutical properties
Authors: Hunter MG , Bawden L , Brotherton D , Craig S , Cribbes S , Czaplewski LG , Dexter TM , Drummond AH , Gearing AH , Heyworth CM
Published: October 2019

The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.
Corticosteroid alteration of carboplatin-induced hematopoietic toxicity in a murine model
Authors: Rinehart J , Keville L , Measel J , Spiekerman AM , Burke K
Published: October 2019

Corticosteroids exhibit extensive hematopoietic effects both in vitro and in vivo. Some of the previously studied effects suggested that corticosteroids may alter hematopoietic toxicity of chemotherapeutic agents. In this study, we examined (1) the optimum dose and schedule of cortisone acetate (CA) to reduce hematopoietic toxicity of carboplatin (CB) and (2) possible mechanisms involved in this protective effect. CA given subcutaneously at 0.5 mg/d per mouse for 7 days before CB reduced CB-induced mortality due to neutropenia from 88% in controls to 14% in CA-treated mice (P < .05). Lower CA doses were not effective. Three days of pretreatment (but not 1 day) was as effective as 7 days. CA given after CB had no effect on mortality. Pharmacokinetic studies of CA at 0.5 mg per mouse demonstrated blood levels of cortisol achievable in patients (peak level, 82 micrograms/dL). CA treatment markedly reduced spleen cell number and colony-forming units- granulocyte/macrophage (CFU-GM) as well as bone marrow CFU-GM. Bone marrow CFU-GM removed from CA-treated mice demonstrated increased resistance to platinum and increased resistance to high specific activity 3H-thymidine. These findings suggest that treatment of mice with CA induces cellular resistance of hematopoietic precursors to platinum and, thus, reduces CB hematotoxicity. CA or other corticosteroids may be useful in reducing clinical toxicity of CB.
The expression of CD26 and CD40 ligand is mutually exclusive in human T- cell non-Hodgkin's lymphomas/leukemias
Authors: Carbone A , Gloghini A , Zagonel V , Aldinucci D , Gattei V , Degan M , Improta S , Sorio R , Monfardini S , Pinto A
Published: October 2019

CD26 and CD40 ligand (CD40L) are surface molecules on human activated T lymphocytes that play a critical role in the regulation of lymphopoiesis. Both molecules are expressed on a restricted fraction of human T-cell non-Hodgkin's lymphomas (NHL)/leukemias; however, little is known about their functional and/or clinical significance in these disorders. In this study, the pattern of expression of CD40L was compared with that of the CD26 molecule. A series of 67 human T-cell NHL/leukemias and a panel of leukemia/lymphoma T-cell lines were evaluated by immunohistochemistry, flow cytometry, and RNA studies. The overall frequency of CD26+ and CD40L+ samples was rather similar (25/67 [37%] v 18/67 [27%]). However, the majority of CD26-expressing cases clustered in the lymphoblastic lymphomas (LBL)/T-acute lymphoblastic leukemias (ALL; 12/23) and CD30+ anaplastic large-cell (ALC) lymphomas (5/8), whereas CD40L+ lymphomas included a large fraction of mycosis fungoides (11/21 [52%]). CD26 and CD40L coexpression was found only in 2 myocosis fungoides cases and 1 small lymphocytic lymphoma. Thus, the expression of the two antigens was mutually exclusive in almost all T- cell lymphomas/leukemias. Accordingly, lymphoma cell lines expressed either one of the molecules or the relative amounts of CD26 and CD40L were inversely proportional. In contrast, reactive T lymphocytes from patients with non-neoplastic T-cell expansions and in vitro activated CD3+ or CD4+ normal T cells were found to coexpress CD40L and CD26. Results of a multivariate analysis showed that the expression of CD26 in T-cell LBL/ALL patients was associated to a worse outcome in terms of survival, as compared with patients with CD26- tumors (P < or = .0001). Based on our results, it can be concluded that, (1) as opposed to activated or reactive normal T cells, the expression of CD26 and of CD40L is mutually exclusive in human T-cell lymphomas/leukemias; (2) expression of CD26 is restricted to aggressive pathologic entities, such as T-cell LBL/ALL and T-cell CD30+ ALC lymphomas, whereas CD40L is expressed on slow progressing diseases such as mycosis fungoides; and (3) within the T-cell LBL/ALL group of tumors, CD26 may identify a subset of poor prognosis patients.
Hematopoietic stem cells in the blood after stem cell factor and interleukin-11 administration: evidence for different mechanisms of mobilization
Authors: Mauch P , Lamont C , Neben TY , Quinto C , Goldman SJ , Witsell A
Published: October 2019

Peripheral blood stem cells and progenitor cells, collected during recovery from exposure to cytotoxic agents or after cytokine administration, are being increasingly used in clinical bone marrow transplantation. To determine factors important for mobilization of both primitive stem cells and progenitor cells to the blood, we studied the blood and splenic and marrow compartments of intact and splenectomized mice after administration of recombinant human interleukin-11 (rhlL-11), recombinant rat stem cell factor (rrSCF), and IL-11 + SCF. IL-11 administration increased the number of spleen colony- forming units (CFU-S) in both the spleen and blood, but did not increase blood long-term marrow-repopulating ability (LTRA) in intact or splenectomized mice. SCF administration increased the number of CFU- S in both the spleen and blood and did not increase the blood or splenic LTRA of intact mice, but did increase blood LTRA to normal marrow levels in splenectomized mice. The combination of lL-11 + SCF syngeristically enhanced mobilization of long-term marrow-repopulating cells from the marrow to the spleen of intact mice and from the marrow to the blood of splenectomized mice. These data, combined with those of prior studies showing granulocyte colony-stimulating factor mobilization of long-term marrow repopulating cells from the marrow to the blood of mice with intact spleens, suggest different cytokine- induced pathways for mobilization of primitive stem cells.
Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells
Authors: Carrier E , Lee TH , Busch MP , Cowan MJ
Published: October 2019

Significant morbidity and mortality are associated with the conditioning therapy needed for postnatal bone marrow transplantation (BMT) for inherited diseases. This could be eliminated with hematopoietic stem cell (HSC) transplantation in utero, when the immunoincompetence of the fetus permits engraftment without the need for immunosuppressive therapy. We have established an in utero (day 11 to day 13) model of HSC transplantation in nondefective, allogeneic major histocompatibility complex (MHC)-mismatched mice. Donor cells wre from pooled fetal livers of C57BL/6 (H-2b, GPI-1b) mice. Engraftment was tested by quantitative polymerase chain reaction (PCR) for the Y chromosome in female recipients (with 0.00001% sensitivity). Eight percent (3 of 36) of allogeneic mismatched (Balb-c, H-2d) recipients and 25% (3 of 12) of congenic (C57B1/6, GPI-1a) recipients showed durable engraftment (male donor cells detected beyond 20 weeks of age) based on analysis of peripheral blood leukocytes (P > .08). When spleen and liver were analyzed, 51% (17 of 33) of allogeneic recipients and 50% (6 of 12) of congenic recipients showed durable engraftment (P > .3). The percent donor cells that durably engrafted varied from as low as 0.0001% in spleen and liver to as high as 0.6% in peripheral blood. Postnatal boosting with a single dose of allogeneic MHC-mismatched donor cells in a tolerant, engrafted mouse resulted in a significant increase in donor cells in the peripheral blood from 0.2% pre-boost to 5% 6 months after the boost. There was no evidence of engraftment in nontolerant mice after the postnatal boost with a similar dose of donor cells. Twenty-two allogeneic recipients were evaluated for donor skin graft acceptance at 6 to 12 months of age. Three mice with engraftment in blood and/or tissue permanently accepted donor skin grafts, one of them with donor cells detectable only in the liver. Six additional mice that showed prolonged skin graft acceptance had no evidence of durable engraftment in the blood but were engrafted in the liver and/or spleen. The degree of engraftment in tolerant mice was low (< or = 0.1% donor cells). We conclude that at an early gestational age in nondefective mice (1) high rates of durable engraftment are achievable, although the degree of engraftment is usually low (less than 1%); (2) the percent of donor cells in the peripheral blood may be increased by a postnatal boost of donor cells in tolerant animals without conditioning therapy; (3) MHC appears to have little influence on engraftment efficiency at an early gestational age; (4) a very small number of circulating donor cells in the blood or the tissues is sufficient for the induction and maintenance of tolerance, and (5) the presence of donor cells in the circulating blood is not necessary for prolonged skin graft acceptance or maintenance of permanent skin graft acceptance.
Detection of clonal CD34+19+ progenitors in bone marrow of BCL2-IgH- positive follicular lymphoma patients
Authors: Macintyre EA , Belanger C , Debert C , Canioni D , Turhan AG , Azagury M , Hermine O , Varet B , Flandrin G , Schmitt C
Published: October 2019

The frequent occurrence of BCL2-IgH rearrangements in follicular lymphoma (FL) makes detection of low numbers of tumor cells possible by polymerase chain reaction (PCR). The presence of BCL2-IgH in the bone marrow (BM) and peripheral blood of many FL patients at the time of autografting has led to the suggestion that selection of the CD34- enriched fraction may lead to reinfusion of lower numbers of tumor cells. To address this issue, we PCR-amplified BCL2-IgH from fluorescence-activated cell sorting (FACS)-purified BM CD34+ and CD34- fractions in seven FL patients showing a PCR-detectable translocation in the major breakpoint region of BCL2, five of which showed morphological BM involvement. The total CD34+ fraction showed diminished but residual positivity in the first two cases tested. Therefore, BM cells from the remaining five patients were sorted for the CD34+19- immature population, the CD34+19+ B-cell precursors, and the CD34–19+ mature B-cell fraction. The CD34+19- subpopulation was negative in four of five, despite evident BM infiltration in three cases. In contrast, the CD34+19+ fraction was positive in all three cases tested. These cells represented 0% to 50% (mean, 18%) of the total CD34+ population, suggesting that, if reinfusion of BCL2-IgH- positive cells plays a role in postautograft relapse in FL, therapeutic CD34 selection procedures should include additional purging of the CD34+19+ B-cell precursors or, at least, assessment of the proportion of CD19+ cells in the CD34+ fraction and its correlation with clinical outcome postreinfusion.
Concomitant granulocyte colony-stimulating factor and induction chemoradiotherapy in adult acute lymphoblastic leukemia: a randomized phase III trial
Authors: Ottmann OG , Hoelzer D , Gracien E , Ganser A , Kelly K , Reutzel R , Lipp T , Busch FW , Schwonzen M , Heil G
Published: October 2019

This prospective multicenter study examined whether simultaneous administration of granulocyte colony-stimulating factor (G-CSF; Filgrastim) and induction chemotherapy for adult acute lymphoblastic leukemia (ALL) could prevent treatment-related neutropenia, infections, and resulting treatment delays. Seventy-six patients were randomly assigned to receive either G-CSF (n = 37) or no growth factor (n = 39) in conjunction with a uniform chemotherapy consisting of cyclophosphamide, cytarabine, mercaptopurine, intrathecal methotrexate, and cranial irradiation. The median duration of neutropenia (absolute neutrophil count < 1 x 10(9)/L) during chemotherapy was 8 days in patients receiving C-CSF, compared with 12.5 days in the control group (P < .002). A similar reduction from 11.5 to 7 days was observed in patients with T-ALL receiving additional mediastinal irradiation (P = .13). Infections occurred in 43% and 56% of patients in the G-CSF and control arm, respectively (P = .25); the incidence of nonviral infections was reduced by 50%, from 32 episodes in the control arm to 16 episodes in the G-CSF arm. Prolonged interruptions of chemotherapy administration were less frequent, with delays of 2 weeks or more occurring in only 24% of patients receiving G-CSF as opposed to 46% in the control arm (P = .01). Accordingly, chemotherapy was completed significantly earlier with the use of G-CSF (39 v 44 days, P = .008). With a median follow-up of 20 months, the probability of disease-free survival was 0.45 in the G-CSF group and 0.43 in the control group (P = .34). In conclusion, adult ALL patients appear to benefit by the simultaneous administration of G-CSF with induction chemotherapy because of a significant reduction in the duration of neutropenia, a trend to fewer infections, and a more rapid completion of chemotherapy.
Signal transduction by the receptors for thrombopoietin (c-mpL) and interleukin-3 in hematopoietic and nonhematopoietic cells
Authors: Morella KK , Bruno E , Kumaki S , Lai CF , Fu J , Wang HM , Murray L , Hoffman R , Timour M , Benit L
Published: October 2019

Antisense oligonucleotide to the translation initiation sequence of human c-mpI reduced the proliferation of human CD34+ bone marrow cells in response to interleukin-3 (IL-3) alone or to the combination of IL-3 and thrombopoietin (TPO). To investigate the molecular basis for these cytokine interactions, we analyzed the relationship between the receptor subunits for IL-3 and TPO and determined whether both receptors activate identical signal transduction pathways. The function of the receptor subunits was characterized in transiently transfected hepatoma cells and fibroblasts by the activation of gene expression via specific regulatory elements and by the stimulation of DNA-binding activity of STAT proteins. Although c-mpl and IL-3 receptor (IL-3R) reconstituted a qualitatively comparable gene regulatory response, there was no detectable functional interaction between their respective receptor subunits. By comparing the receptor action in different cell lines, we observed that in human hepatoma cells the signaling of c-mpI was 100-fold less sensitive to TPO than in rat hepatoma cells. However, IL-3R signaling was comparable between the two cell types, suggesting that c-mpI and IL-3R do not use identical signal transducing mechanisms. The cytoplasmic domains necessary for c-mpI signaling were determined by testing deletion mutants. The membrane-proximal box 1 sequence motif was critical for gene regulation and for STAT protein activation that seemed to involve the Janus kinase 2 (JAK2). Because IL- 3R was less dependent on JAK2 than c-mpI, different levels of JAK2 expression may account, in part, for the quantitative difference in IL- 3 and TPO response among various cell lines.
The mouse alpha-globin locus regulatory element
Authors: Gourdon G , Sharpe JA , Higgs DR , Wood WG
Published: October 2019

We have identified and cloned the major alpha globin locus regulatory element in the mouse (m alpha RE). This element shows a high level of sequence homology to its human counterpart (HS -40) and lies between the same two exons of an upstream, widely expressed gene in both species. Footprinting and band shift studies of the core element show conservation of many (but not all) of the protein binding sites identified as functionally important in HS -40. The functional equivalence of the mouse element was shown by attaching it to a human alpha globin gene and examining expression in transgenic mice. Readily detectable levels of human alpha mRNA were produced in these mice but they were lower than the endogenous gene expression and did not show copy number dependence. These results suggest that sequences additional to this major regulatory element may be necessary to obtain complete regulation of the alpha globin genes in both species.