bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the protein kinase C inhibitor staurosporine on chemosensitivity to daunorubicin of normal and leukemic fresh myeloid cells
Authors: Laredo J , Huynh A , Muller C , Jaffrezou JP , Bailly JD , Cassar G , Laurent G , Demur C
Published: October 2019

The effect of the protein kinase C (PKC) inhibitor staurosporine (ST) on the chemosensitivity of normal (colony-forming unit granulocyte- macrophage [CFU-GM]) and leukemic (acute myeloid leukemia-CFU [AML- CFU]) myeloid progenitors to daunorubicin (DNR) was evaluated. Primary colony inhibition assays allowed us to characterize two distinct groups of AML, a DNR-resistant group (patients no. 1 through 6), which displayed significantly lower DNR sensitivity than normal CFU-GM (D50 = 11.3 +/- 1.4 ng/mL v 1.8 +/- 0.5 ng/mL, after 7 days of exposure, respectively; P < 0.01) and a DNR-sensitive group (patients no. 7 through 12) with D50 = 2.7 +/- 0.4 ng/mL. This classification remained unaltered when assessed by secondary colony inhibition assay (evaluating the self-renewal fraction of AML-CFU) or by viability assay (evaluating the ultimately differentiated blast cell population), suggesting that the DNR sensitivity profile in maintained throughout AML-CFU differentiation. DNR resistance of the differentiated blast cell population was not correlated with the level of P-glycoprotein (P- gp) expression but rather with the ability to extrude rhodamine 123 (Rh123). ST used at subtoxic concentrations induced a twofold to threefold enhancement of DNR cytotoxicity, increased Rh123 accumulation, and decreased Rh123 efflux kinetics in resistant AML cells. These effects were observed for ST concentrations much lower than those required to displace the P-gp-binding probe azidoprazosin, suggesting that ST might act through its PKC inhibitory effect and not through P-gp binding. Finally, this study provides evidence that DNR resistance in AML cells is, at least in part, related to the multidrug- resistance (MDR) phenotype. Because P-gp function can be downregulated by ST, it seems likely that the MDR pheno-type can be functionally regulated by cellular signalization in AML cells.
HLA-DR-, CD33+, CD56+, CD16- myeloid/natural killer cell acute leukemia: a previously unrecognized form of acute leukemia potentially misdiagnosed as French-American-British acute myeloid leukemia-M3 [see comments]
Authors: Scott AA , Head DR , Kopecky KJ , Appelbaum FR , Theil KS , Grever MR , Chen IM , Whittaker MH , Griffith BB , Licht JD
Published: October 2019

We have identified and characterized a previously unrecognized form of acute leukemia that shares features of both myeloid and natural killer (NK) cells. From a consecutive series of 350 cases of adult de novo acute myeloid leukemia (AML), we identified 20 cases (6%) with a unique immunophenotype: CD33+, CD56+, CD11a+, CD13lo, CD15lo, CD34+/-, HLA-DR- , CD16-. Multicolor flow cytometric assays confirmed the coexpression of myeloid (CD33, CD13, CD15) and NK cell-associated (CD56) antigens in each case, whereas reverse transcription polymerase chain reaction (RT- PCR) assays confirmed the identity of CD56 (neural cell adhesion molecule) in leukemic blasts. Although two cases expressed CD4, no case expressed CD2, CD3, or CD8 and no case showed clonal rearrangement of genes encoding the T-cell receptor (TCR beta, gamma, delta). Leukemic blasts in the majority of cases shared unique morphologic features (deeply invaginated nuclear membranes, scant cytoplasm with fine azurophilic granularity, and finely granular Sudan black B and myeloperoxidase cytochemical reactivity) that were remarkably similar to those of acute promyelocytic leukemia (APL); particularly the microgranular variant (FAB AML-M3v). However, all 20 cases lacked the t(15;17) and 17 cases tested lacked the promyelocytic/retinoic acid receptor alpha (RAR alpha) fusion transcript in RT-PCR assays; 12 cases had 46,XX or 46,XY karyotypes, whereas 2 cases had abnormalities of chromosome 17q: 1 with del(17)(q25) and the other with t(11;17)(q23;q21) and the promyelocytic leukemia zinc finger/RAR alpha fusion transcript. All cases tested (6/20), including the case with t(11;17), failed to differentiate in vitro in response to all-trans retinoic acid (ATRA), suggesting that these cases may account for some APLs that have not shown a clinical response to ATRA. Four of 6 cases tested showed functional NK cell-mediated cytotoxicity, suggesting a relationship between these unique CD33+, CD56+, CD16- acute leukemias and normal CD56+, CD16- NK precursor cells. Using a combination of panning and multiparameter flow cytometric sorting, we identified a normal CD56+, CD33+, CD16- counterpart cell at a frequency of 1% to 2% in the peripheral blood of healthy individuals. Our studies suggest that this form of acute leukemia may arise from transformation of a precursor cell common to both the myeloid and NK cell lineages; thus we propose the designation myeloid/NK acute leukemia. Recognition of this new leukemic entity will be important in distinguishing these ATRA- nonresponsive cases from ATRA-responsive true APL.
Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor
Authors: Menegazzi R , Cramer R , Patriarca P , Scheurich P , Dri P
Published: October 2019

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF- induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF- induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr- 9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin- coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.
Growth enhancement of established tumors by allogeneic blood transfusion in experimental animals and its amelioration by leukodepletion: the importance of the timing of the leukodepletion
Authors: Bordin JO , Bardossy L , Blajchman MA
Published: October 2019

We had reported previously (Blood 81:1880, 1993) that allogeneic blood transfusions (ABT) administered before the infusion of tumor cells in both inbred and outbred experimental animals promote tumor growth and that this effect can be ameliorated by leukodepletion. To better reproduce the human situation, we evaluated, in this present study, the effect of ABT in animals with established tumors using enumeration of pulmonary metastatic nodules as the end point. The role of allogeneic blood component transfusions in promoting tumor growth and the relative efficacy of prestorage versus poststorage leukodepletion of the ABT in preventing tumor growth enhancement were also evaluated. In an inbred murine animal model, C57Bl/6J mice were administered nonleukodepleted allogeneic (ABT), leukodepleted allogeneic (LD-ABT), or syngeneic (SBT) blood transfusions after the intravenous infusion of syngeneic methylcholanthrene-induced fibrosarcoma cells using two different protocols. A significant increase in the number of pulmonary nodules was observed in those mice that received ABT, in both protocols, compared to animals transfused with SBT or LD-ABT. Significantly higher numbers of pulmonary nodules were also seen in mice transfused with allogeneic buffy-coat leukocytes compared with mice that received either nonleukodepleted allogeneic plasma or LD-ABT. In an outbred animal (rabbit) model, recipient rabbits were administered either nonleukodepleted ABT, prestorage LD-ABT, poststorage LD-ABT, or SBT on days +4 and +9 after the infusion of syngeneic epithelial tumor cells. A significant increase in the number of pulmonary nodules was seen in rabbits that received nonleukodepleted ABT compared to animals transfused with SBT. Significantly lower numbers of pulmonary nodules were observed in rabbits that received prestorage LD-ABT compared to animals transfused with poststorage LD-ABT, but no significant difference was seen in rabbits that received poststorage LD-ABT compared with animals transfused with nonleukodepleted ABT. These studies show that ABT promote tumor growth of established animal tumors, that the ABT-induced tumor growth effect is related to the presence of donor allogeneic leukocytes, and that this effect can be ameliorated by prestorage leukodepletion. The present results also provide evidence for the lack of efficacy of poststorage leukodepletion in preventing ABT tumor growth promotion.(ABSTRACT TRUNCATED AT 400 WORDS).
Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells
Authors: Szilvassy SJ , Cory S
Published: October 2019

Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.
Lymphocyte predominance Hodgkin's disease: lineage and clonality determination using a single-cell assay
Authors: Delabie J , Tierens A , Wu G , Weisenburger DD , Chan WC
Published: October 2019

Lymphocyte predominance Hodgkin's disease (LPHD) is a clinically indolent condition. Although there is evidence that the putative neoplastic cell in this disease, the “L&H” cell, is of B-cell lineage, there is conflicting data concerning the clonality of these cells. Our study was aimed at clarifying the issue of lineage and clonality of the L&H cells of LPHD using a single-cell assay. Four cases of LPHD were studied. To circumvent the difficulties of obtaining fresh tissue and to be able to study representative cases, a new method was developed to obtain single-cell suspensions of L&H cells from archival formalin- fixed paraffin-embedded tissue. Single L&H cells were identified by morphology and immunostaining for epithelial membrane antigen, isolated using a micropipette, and subjected to polymerase chain reaction (PCR) amplification of the complematarity determining region 3 (CDR3) of the Ig heavy chain (IgH) gene, which is B-cell clone-specific. The PCR products were size-fractionated by polyacrylamide gel electrophoresis and representative products were directly sequenced. Single T cells and small B cells were also isolated from the tissues and used as negative and positive controls, respectively. In all four cases of LPHD, the IgH CDR3 of single L&H cells could be amplified. Within each case, the IgH CDR3 of single L&H cells was found to be of different length or of different sequence. Therefore, our results provide strong evidence for the B-cell origin of the L&H cells and the polyclonal nature of LPHD.
Human herpesvirus 6 (HHV-6) isolation from bone marrow: HHV-6- associated bone marrow suppression in bone marrow transplant patients [see comments]
Authors: Carrigan DR , Knox KK
Published: October 2019

These studies tested the hypothesis that human herpesvirus 6 (HHV-6) can cause posttransplant bone marrow (BM) suppression in BM transplant (BMT) patients. Fifteen adult patients who received T-lymphocyte- depleted, allogeneic BM transplants and who developed posttransplant BM suppression were studied. Detailed chart reviews were used to divide the patients into two groups: (1) those with diagnosed BM suppression (DBMS) and (2) those with idiopathic BM suppression (IBMS). BM aspirates obtained from the patients at the onset of BM suppression were subjected to an HHV-6 isolation procedure using mitogen-stimulated blood mononuclear cells. BM specimens obtained from another population of BMT patients solely to document engraftment irrespective of their BM function were also subjected to the HHV-6 isolation procedure as controls. HHV-6 was isolated from 6 of 15 BM samples from BMT patients with BM suppression. BM samples from patients with IBMS were more likely to be positive for HHV-6 than those from patients with DBMS (P < .01). Also, HHV-6-positive BM were significantly more likely (P < .05) to come from patients with suppression of more than one BM lineage than HHV-6-negative BM. Finally, samples of BM from an unselected series of BMT patients studied without regard to their BM function were less likely (P < .01) to be positive for HHV-6 than patients with IBMS.
Natural interferon-alpha versus its combination with 6-methyl- prednisolone in the therapy of type II mixed cryoglobulinemia: a long- term, randomized, controlled study
Authors: Dammacco F , Sansonno D , Han JH , Shyamala V , Cornacchiulo V , Iacobelli AR , Lauletta G , Rizzi R
Published: October 2019

Type II mixed cryoglobulinemia (MC) is an often progressive vasculitis characterized by circulating cold-precipitable proteins that usually consists of polyclonal IgG and monoclonal IgM kappa with rheumatoid factor (RF) activity. Its etiology is unknown, although recent evidence strongly suggests that hepatitis C virus (HCV) plays a major role. Plasmapheresis, corticosteroids, and cytotoxic drugs have been used in the therapy of MC patients. Recently, favorable results with recombinant interferon-alpha (rIFN alpha) have been reported. To further assess its effectiveness, we studied the effects of natural human interferon-alpha (nIFN alpha), alone and in combination with 6- methyl-prednisolone (PDN), in a prospective, randomized, controlled trial in patients with symptomatic MC. Sixty-five patients were enrolled onto the trial, 52 (80%) of whom presented serum anti-HCV antibodies and specific genomic RNA sequences. Fifteen patients received nIFN alpha (3 MU) intramuscularly (IM) three times weekly, whereas 17 patients also received 16 mg/d of PDN orally on non-IFN days. Moreover, 18 patients received 16 mg/d of PDN only, and 15 were untreated. Treatment was discontinued after 1 year and patients were monitored for 8 to 17 months (mean, 13). A complete response was achieved in eight of 15 patients (53.3%) treated with nIFN alpha and nine of 17 (52.9%) treated with nIFN alpha plus PDN, as compared with three of 18 patients (16.7%) who received PDN only (P < .05) and one of 15 (6.7%) untreated controls (P < .01). Partial response occurred in two of 15 (13.3%) patients treated with nIFN alpha, three of 17 (17.6%) who received nIFN alpha plus PDN, one of 18 (5.5%) who received PDN only, and one of 15 (6.7%) controls. A complete response in six patients (66.7%) was achieved within 3 months in the group that received nIFN alpha plus PDN, as compared with two patients (25%) of those who received nIFN alpha alone (P < .02). In anti-HCV-positive patients, the clinical response occurred in step with reduced or undetectable levels of HCV RNA and transaminase normalization. Quantification of circulating HCV RNA represented a good predictive response marker. The probability of relapse within 3 months after treatment was 100% (three of three patients) and 75% (six of eight patients), respectively, in patients who received PDN alone or nIFN alpha alone as compared with none of those who received nIFN alpha plus PDN (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)
B-cell non-Hodgkin's lymphoma cell line (Karpas 1106) with complex translocation involving 18q21.3 but lacking BCL2 rearrangement and expression
Authors: Nacheva E , Dyer MJ , Metivier C , Jadayel D , Stranks G , Morilla R , Heward JM , Holloway T , O'Connor S , Bevan PC
Published: October 2019

A B-cell non-Hodgkin's lymphoma (B-NHL) cell line (Karpas 1106) with an unusual three-way translocation involving 18q21.3 has been derived from a patient with mediastinal lymphoblastic B-NHL. Although conventional cytogenetics showed a derivative 18q-identical to that seen in cases with t(14;18)(q32.3;q21.3), no translocations of either chromosome 14 could be detected. Instead fluorescent in situ hybridization analysis using a chromosome-18 paint showed that the segment 18q21.3–18qter had become sandwiched on a derivative chromosome X between segments Xqter-c- Xq28 and 13q12-qter, with the centrometric site of 18q21.3 subband juxtaposed to the X sequences. Pulsed-field DNA blots failed to detect rearrangement of the BCL2 gene. Conventional DNA blots using a variety of restriction digests and both 52 and 32 BCL2 and FVT 1 probes also failed to detect rearrangement in Karpas 1106. A rearranged fragment seen only in HindIII digests with 52 BLC2 probes may represent a local microalteration, which is either a mutation or small deletion involving the HindIII site as seen in other cases of B-NHL. Neither BCL2 RNA nor BCL2 protein expression were detected. These and other data suggest that genes at 18q21.3, other than BCL2 and FVT1, may be targets for translocation in certain subgroups of B-NHL.
Fluorescence in situ hybridization mapping of translocations and deletions involving the short arm of human chromosome 12 in malignant hematologic diseases
Authors: Kobayashi H , Montgomery KT , Bohlander SK , Adra CN , Lim BL , Kucherlapati RS , Donis-Keller H , Holt MS , Le Beau MM , Rowley JD
Published: October 2019

Translocations and deletions of the short arm of chromosome 12 [t(12p) and del(12p)] are common recurring abnormalities in a broad spectrum of hematologic malignant diseases. We studied 20 patients and one cell line whose cells contained 12p13 translocations and/or 12p deletions using fluorescence in situ hybridization (FISH) with phage, plasmid, and cosmid probes that we previously mapped and ordered on 12p12–13. FISH analysis showed that the 12p13 translocation breakpoints were clustered between two cosmids, D12S133 and D12S142, in 11 of 12 patients and in one cell line. FISH analysis of 11 patients with deletions demonstrated that the deletions were interstitial rather than terminal and that the distal part of 12p12, including the GDI-D4 gene and D12S54 marker, was deleted in all 11 patients. Moreover, FISH analysis showed that cells from 3 of these patients contained both a del(12p) and a 12p13 translocation and that the affected regions of these rearrangements appeared to overlap. We identified three yeast artificial chromosome (YAC) clones that span all the 12p13 translocation breakpoints mapped between D12S133 and D12S142. They have inserts of human DNA between 1.39 and 1.67 Mb. Because the region between D12S133 and D12S142 also represents the telomeric border of the smallest commonly deleted region of 12p, we also studied patients with a del(12p) using these YACs. The smallest YAC, 964c10, was deleted in 8 of 9 patients studied. In the other patient, the YAC labeled the del(12p) chromosome more weakly than the normal chromosome 12, suggesting that a part of the YAC was deleted. Thus, most 12p13 translocation breakpoints were clustered within the sequences contained in the 1.39 Mb YAC and this YAC appears to include the telomeric border of the smallest commonly deleted region. Whether the same gene is involved in both the translocations and deletions is presently unknown.
The association of erythroblasts with macrophages promotes erythroid proliferation and maturation: a 30-kD heparin-binding protein is involved in this contact
Authors: Hanspal M , Hanspal JS
Published: October 2019

Although the association of erythroblasts with macrophages has been well documented in the human bone marrow, the function and identification of the intimate contacts occurring between the membranes of these two cell types in the physiology of erythropoiesis is not known. Using in vitro cultures of human peripheral blood derived erythroid progenitors, we have shown the presence of erythroblastic islands consisting of a central macrophage surrounded by a ring of erythroblasts that undergo terminal maturation leading to enucleation. However, when cultures were carried in the absence of intact macrophages, erythroid cells matured to the late erythroblast stage but failed to enucleate. Furthermore, the number of erythroid cells was markedly reduced in macrophage-depleted cultures, suggesting that the erythroblast-macrophage contact promotes proliferation and terminal maturation of erythroid cells leading to their enucleation. To examine the molecule(s) involved in the interaction between erythroblasts and macrophages, we have used a cell attachment assay involving incubation of solubilized surface-labeled erythroblasts with macrophage membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Erythroblast surface proteins specifically attached to a 30-kD protein from macrophage membranes, whereas no adhesion was seen to the protein standards. An apparently similar protein of 30 kD was also detected on erythroblasts and was shown to mediate erythroblast-erythroblast contact in addition to the erythroblast-macrophage contact. The extraction of plasma membranes with Triton X-100 showed that the 30-kD protein is linked to the membrane skeleton via an integral membrane protein both in erythroblasts and macrophages. Furthermore, our results show that the cell:cell interactions mediated by the 30-kD protein are calcium-independent and could be specifically inhibited by heparin. We conclude that the association of erythroblasts with macrophages promotes erythroid proliferation and maturation leading to erythroblast enucleation and that a 30-kD heparin-binding protein present on the surface of macrophages and erythroblasts is involved in this contact. This protein is capable of binding homotypic and heterotypic cells.
Hereditary hemolytic anemia caused by diverse point mutations of pyruvate kinase gene found in Japan and Hong Kong
Authors: Kanno H , Wei DC , Chan LC , Mizoguchi H , Ando M , Nakahata T , Narisawa K , Fujii H , Miwa S
Published: October 2019

We identified four distinct point mutations in homozygous pyruvate kinase (PK) variants in Japanese and Chinese patients with chronic nonspherocytic hemolytic anemia. All gene abnormalities were missense mutations that caused single amino acid substitutions. 1261A (Q421K) and 1436A (R436H), which were identified in PK Sendai and PK Shinshu, had been found in unrelated Japanese and Amish PK variants, respectively. The clinical severity and extremely low residual erythrocyte PK activity of PK Shinshu as well as of the Amish PK might be caused partly by aberrant splicing, because the 1436A mutation changes a nucleotide at the last nucleotide in the exon 10. Recently, we diagnosed a 42-year-old Japanese woman with chronic nonspherocytic hemolytic anemia as having a homozygous PK deficiency. DNA sequencing of the variant PK gene showed a homozygous missense mutation at 1403GCT- ->GTT, resulting in a single amino acid substitution from 468la-->Val. The gene mutation is likely to impair the allostericity of this enzyme, speculated from the tertiary structure. A homozygous missense mutation in PK Hong Kong, a boy of a non-Han southern Chinese minority group, was identified in exon 7 of the human L-PK gene, 941ATT-->ACT, resulting in a single amino acid substitution from 314lle-->Thr. The R- PK activity is expected to be severely affected, because the mutated amino acid residue is located between the 313 Lys and the 315 Glu, which are very important for acid-base catalysis and magnesium binding, respectively. Both the R- and M2-type PK were shown by polyacrylamide gel electrophoresis of the PK Hong Kong erythrocyte lysate, and this is the first report of a homozygous individual whose erythrocytes contain the immature (M2)-type isozyme.
Donor CD4-enriched cells of Th2 cytokine phenotype regulate graft- versus-host disease without impairing allogeneic engraftment in sublethally irradiated mice
Authors: Fowler DH , Kurasawa K , Smith R , Eckhaus MA , Gress RE
Published: October 2019

We have recently shown that donor CD4-enriched cells of Th2 cytokine phenotype, generated by treating mice in vivo with a combination of interleukin-2 (IL-2) and IL-4, prevent lipopolysaccharide-induced, tumor necrosis factor-alpha-mediated lethality during graft-versus-host reaction. To assess the potential regulatory role of such Th2-type cells in lethal graft-versus-host disease (GVHD) and graft rejection, we used a fully allogeneic murine transplant model using sublethally irradiated hosts (B6-->C3H, 500 cGy). Such recipients generated a strong host-versus-graft response, as reflected by their ability to reject T-cell-depleted inocula. The administration of T-cell-containing donor whole spleen inocula resulted in alloengraftment, but such recipients developed lethal GVHD. However, mice receiving sequential donor whole spleen (day 0) and CD4-enriched, Th2-type (day 1) populations engrafted, and had prolonged survival with protection from histologically defined tissue injury associated with GVHD. The findings in this fully allogeneic model thus extend our previous observations and indicate that the transfer of donor Th2-type cells may be an important strategy for regulating GVHD. Furthermore, the sequential “Th1(-)-->Th2-type” donor cell transfer described in this report represents a novel approach for abrogating graft rejection with concomitant control of GVHD and illustrates the importance of kinetics in the interaction of functionally distinct donor T-cell populations.
Evidence for engraftment of donor-type multipotent CD34+ cells in a patient with selective T-lymphocyte reconstitution after bone marrow transplantation for B-SCID
Authors: Tjonnfjord GE , Steen R , Veiby OP , Friedrich W , Egeland T
Published: October 2019

Severe combined immunodeficiencies (SCID), a heterogeneous group of disorders of infancy, are fatal without treatment directed at immunologic reconstitution. Allogeneic bone marrow transplantation (BMT), which is such a treatment presents some unique features in SCID, especially when T-lymphocyte-depleted HLA haploidentical allografts are used. Donor-type T lymphopoiesis, less often B lymphopoiesis, develops, whereas myelopoiesis remains the recipient-type. Little is known about the engrafting cells in this peculiar lymphohematopoietic chimerism and the pathophysiology of the frequent failure of B-lymphocyte reconstitution. To address these issues, we purified CD34+ BM cells from a patient with selective T-lymphocyte reconstitution after HLA haploidentical BMT for B-SCID. Phenotypic analysis of CD34+ cells was performed by flow cytometry, and functional studies of donor- and recipient-type CD34+ cells were performed in vitro. Donor-type CD34+ cells, constituting approximately 2% of the CD34+ cells, were detected; both CD34+ HLA-DR- cells and CD34+ cells coexpressing B-(CD10 and CD19) and T-(CD2 and CD7) lymphocyte-associated cell surface molecules. Donor- type CD34+ cells coexpressing myeloid-associated molecules (CD13, CD14, CD15, and CD33) were undetectable. However, donor-type CD34+ myeloid progenitors could be shown in functional assays. Recipient-type CD34+ cells coexpressing B- and T-lymphocyte- as well as myeloid-associated molecules were detected, but recipient-type CD34+ cells could not be driven into T-lymphocyte differentiation in vitro. These findings provide evidence for engraftment of multipotent stem cells in our patient with B-SCID. Furthermore, the failure of B-lymphocyte reconstitution cannot be explained by lack of donor-type B-lymphocyte progenitors. Donor-type B lymphopoiesis and myelopoiesis are prevented by an unidentified mechanism.
Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells
Authors: Li CL , Johnson GR
Published: October 2019

The effects of stem cell factor (SCF) have been tested on a murine bone marrow subpopulation (RH123lo, Lin-, Ly6A/E+) that is highly enriched for long-term hematopoietic repopulating cells. SCF maintained cells from this population with long-term repopulating ability for up to 10 days in vitro. However, compared with freshly isolated cells, the level of engraftment in vivo by the cultured cells declined during the in vitro culture period, suggesting that SCF alone was unable to stimulate the self-renewal of long-term repopulating cells. By direct visualization of cultures, only small numbers of cells survived and rarely underwent cell division. However, SCF did directly stimulate proliferation of a population (Rh123med/hi,Lin-,Ly6A/E+) enriched for short-term repopulating cells. These data suggest that stem cell differentiation is associated with the development of mitogenic activity by SCF at least in some progenitor cell populations.
Myeloid differentiation mediated through retinoic acid receptor/retinoic X receptor (RXR) not RXR/RXR pathway
Authors: Dawson MI , Elstner E , Kizaki M , Chen DL , Pakkala S , Kerner B , Koeffler HP
Published: October 2019

Retinoids, such as all-trans-retinoic acid and 9-cis-retinoic acid, are naturally occurring ligands of the nuclear retinoic acid receptors (RARs). In concert with binding of ligand, these receptors from heterodimers with the retinoic X receptor (RXR) and transactivate RAR/RXR-responsive genes. Retinoids can differentiate leukemic cell lines in vitro and induce clinically complete remissions in patients with acute promyelocytic leukemia. Synthetic ligands to the RAR and RXR receptors have been developed that selectively bind and activate RAR/RXR (TTAB) and RXR/RXR dimers (SR11217). We investigated the affect of these ligands, either alone or in combination, on in vitro growth and differentiation of cells from the HL-60, KG-1, THP-1, and WEHI-3 myeloid cell lines as well as on clonal growth of fresh myeloid leukemic blasts from patients. Clonal inhibition of proliferation of these cells was studied in soft agar cultures. Cells were plated in the presence of either one or a combination of retinoids at concentrations of 10(-5) to 10(-10) mol/L. TTAB inhibited 50% clonal growth at an effective dose (ED50) that was about 1,000-fold lower than the concentration of SR11217 required to achieve an ED50 for the same leukemic cells. Combination of both ligands at a variety of concentrations showed no synergistic effects. Superoxide production (nitroblue tetrazolium reduction) and CD11b expression as parameters of differentiation of HL-60 cells were also examined. Results paralleled those of clonal growth, with SR11217 being markedly less potent than TTAB. These results show that the ligand selective for RXR-homodimers has little effect on either inducing differentiation or inhibiting clonal growth of leukemic cells. The differentiating and antiproliferative effects of retinoids are mainly induced through RAR/RXR heterodimers, and development of therapeutic analogs should focus on this category of retinoids.
A functionally active retrovirus vector for gene therapy in Fanconi anemia group C
Authors: Walsh CE , Grompe M , Vanin E , Buchwald M , Young NS , Nienhuis AW , Liu JM
Published: October 2019

Fanconi anemia (FA) is a rare genetic disorder characterized by progressive pancytopenia, congenital abnormalities, and a predisposition to malignancy. Recently, mutation in a novel gene named FACC (Fanconi anemia C complementing) has been identified as causing one type of FA. Here, we report successful functional complementation of four FA(C) cell lines using a retroviral vector to transfer a copy of the normal FACC gene. The hallmark of the FA cell phenotype is extreme sensitivity to cross-linking agents such as mitomycin C (MMC). Cell lines transduced by FACC viral vectors were distinguished by their ability to grow at concentrations of MMC several orders of magnitude higher than those concentrations inhibitory of parental controls. The genetically corrected cell lines were analyzed for susceptibility to MMC-induced chromosomal breakage and were found to have been normalized. These two different assays confirmed that our retroviral vectors were capable of transferring a functional FACC gene to lymphoid cell lines established from FA(C) patients. We next analyzed the ability of our viral vectors to functionally correct hematopoietic progenitor cells from a patient bearing a splice donor mutation. Progenitor cells were purified by an immunoaffinity column to enrich for cells with high CD34 expression. Similar to FA lymphoid cell lines, this patient's CD34-enriched cells were extremely sensitive to MMC. After infection of these progenitor cells with viral vectors bearing normal FACC, increased numbers of colonies formed both in the absence and presence of < or = 5 nmol/L MMC, but no colonies formed from uninfected cells, even in the absence of MMC. Polymerase chain amplification was used to confirm proviral DNA integration. Thus, retroviral vectors can be engineered to transfer a normal FACC gene to lymphoid cell lines and primary hematopoietic cells bearing four different FACC mutations. FA stem cells rescued by gene transduction should have a selective growth advantage within the hypoplastic FA marrow environment in vivo. These experiments suggest that gene therapy may be an effective treatment strategy for FA.
Epstein-Barr viral nuclear antigen 1 antisense oligodeoxynucleotide inhibits proliferation of Epstein-Barr virus-immortalized B cells
Authors: Roth G , Curiel T , Lacy J
Published: October 2019

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a latent viral protein that is expressed in all EBV-immortalized lymphocytes and plays an essential role in immortalization y EBV. EBNA-1 protein is required for replication and maintenance of the episomal viral genome in latently infected, immortalized cells. Given the essential function of EBNA-1 in immortalization, we have examined the effect of EBNA-1 antisense oligodeoxynucleotides on expression of EBNA-1 protein and proliferation in EBV-immortalized lymphoblastoid cells. We have shown that exposure to unmodified antisense oligodeoxynucleotide of codons 6 through 10 of EBNA-1 partially suppressed EBNA-1 protein expression in EBV-immortalized lymphoblastoid cells relative to untreated cells or cells exposed to two scrambled sequences of the EBNA-1 antisense. Furthermore, EBNA-1 antisense inhibited proliferation of EBV- immortalized cells by at least 50% compared with the scrambled antisense sequences. There was no difference in the effect of antisense and scrambled antisense oligodeoxynucleotides on the proliferation of EBV-negative cells, indicating that the antiproliferative effect of EBNA-1 antisense was EBV-specific. These findings underscore the essential role of EBNA-1 in immortalization and, furthermore, have potential therapeutic implications for EBV-associated neoplastic diseases.
Evidence for nonclonal hematopoietic progenitor cell populations in bone marrow of patients with myelodysplastic syndromes
Authors: Asano H , Ohashi H , Ichihara M , Kinoshita T , Murate T , Kobayashi M , Saito H , Hotta T
Published: October 2019

Clonality of marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS) was analyzed by X-chromosome inactivation pattern using polymerase chain reaction (PCR). Five female patients were included in this study; two with refractory anemia (RA) and three with RA with excess blasts (RAEB). They were heterozygous for BstXI restriction fragment length polymorphisms (RFLP) of the X-chromosome-linked phosphoglycerate kinase (PGK) gene. In each patient, erythroid and nonerythroid colonies, grown in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibited no remarkable difference in clonal constitution. Two patients showed only one methylation pattern, suggesting the monoclonal origin of hematopoietic progenitor cells. Colonies of two other patients exhibited predominant and minor methylation patterns in PGK gene, indicating that nonclonal progenitor cells remain a minor population. The bone marrow of one patient appeared to contain a greater proportion of nonclonal progenitors. Stem cell factor (SCF), a potent colony- stimulating factor, enhanced both erythroid and nonerythroid colony formation. However, it did not notably alter the clonal constitutions. We conclude that nonclonal hematopoietic progenitor cells can persist in a substantial number of MDS patients.
Antibodies to myeloid precursor cells in autoimmune neutropenia
Authors: Hartman KR , LaRussa VF , Rothwell SW , Atolagbe TO , Ward FT , Klipple G
Published: October 2019

Antibodies to mature blood neutrophils and to bone marrow myeloid cells have been described in the sera of some patients with apparent autoimmune neutropenia. To further explore the prevalence and specificities of antibodies to myeloid precursor cells, we evaluated sera from 148 patients with suspected autoimmune neutropenia for the presence of antibodies to neutrophils, to cultured myeloid cell lines, and to highly purified bone marrow myeloid progenitor cells. Using an immunofluorescence flow cytometric assay, we identified IgG antibodies in 42 (28%) of these sera that bound specifically to K562 cells, a multilineage cell line originally derived from a patient with chronic myelogenous leukemia. Twenty-two (15%) of the sera also contained IgG antibodies that bound specifically to the primitive myelomonocytic leukemia cell line KG1a. Twenty-five (17%) of the sera had IgG antibodies to myeloid cell lines in the absence of antibodies to mature neutrophils. There was a trend toward more severe neutropenia in patients with antibodies to K562 cells, without antineutrophil antibodies. In further studies, antibodies from 12 sera bound to mononuclear CD34+ cells that had been purified from normal human bone marrow by an immunomagnetic separation procedure. Moreover, two of these sera suppressed the growth of granulocyte-macrophage colony- forming units (CFU-GM) in methylcellulose cultures. The presence of antibodies to primitive hematopoietic cells in the sera of some patients with suspected immune neutropenia suggests that these antibodies may have a role in the pathogenesis of the neutropenia observed.
Long-term immunity to measles, mumps, and rubella after allogeneic bone marrow transplantation
Authors: Ljungman P , Lewensohn-Fuchs I , Hammarstrom V , Aschan J , Brandt L , Bolme P , Lonnqvist B , Johansson N , Ringden O , Gahrton G
Published: October 2019

A total of 124 patients who had survived at least 2 years after allogeneic bone marrow transplantation (BMT) were studied. Serum was collected at least once yearly. IgG antibodies were determined by enzyme-linked immunosorbent assay for measles and mumps. Rubella antibodies were analyzed by radial hemolysis. Antibody levels were interpreted as representing immunity, seronegativity, or seropositivity, but with uncertain immunity. The median follow-up of the patients was 6.5 years (range, 2 to 13.5 years). The calculated probabilities of being immune to measles at 3, 5, and 7 years from BMT were 47%, 27%, and 20%, respectively. The corresponding probabilities for mumps were 37%, 12%, and 6%, respectively; and for rubella, 47%, 33%, and 28%, respectively. The probabilities for being seronegative for measles, mumps, and rubella at 5 years after BMT were 60%, 73%, and 52%, respectively. When compared with those patients who had experienced previous natural measles disease (P < .05), the only factor that was important for immunity to measles after BMT was whether the patient had been immunized before BMT. There was no influence of donor seropositivity on the probability of becoming seronegative to mumps during follow-up. We conclude that most allogeneic patients will become seronegative to measles, mumps, and rubella during follow-up. Therefore, long-term B-cell memory function is not maintained, regardless of the immune status of the donor.
2-Chlorodeoxyadenosine therapy in patients with T-cell lymphoproliferative disorders
Authors: O'Brien S , Kurzrock R , Duvic M , Kantarjian H , Stass S , Robertson LE , Estey E , Pierce S , Keating MJ
Published: October 2019

Mature T-cell lymphoproliferative disorders comprise a heterogenous group of diseases for which there is no standard therapy. These disorders are uncommon, and are usually treated similarly to their B- cell counterparts, but with less success. Nucleoside analogues have proven effective in indolent B-cell disorders but have been less well explored in T-cell malignancies. We treated 22 patients with mature T- cell lymphoproliferative diseases with 2-chlorodeoxyadenosine (2-CDA) administered as a continuous infusion at a daily dose of 4 mg/m2 over 7 days. Nineteen of the patients had received prior therapy with a median number of prior regimens of three. Eleven patients had leukemia or large granular lymphocytosis, eight patients had mycosis fungoides, and three had T-cell lymphoma. Nine patients (41%) responded to 2-CDA. Four of the patients had responses that were complete remissions, and three of these four patients remain in remission at 23, 24, and 23 months. The only important toxic effects were fever or infection, seen during 38% of courses. In conclusion, 2-CDA appears to be an effective therapy in T-cell lymphoproliferative disorders and deserves wider evaluation in this subset of patients.
Characterization and purification of osteogenic cells from murine bone marrow by two-color cell sorting using anti-Sca-1 monoclonal antibody and wheat germ agglutinin
Authors: Van Vlasselaer P , Falla N , Snoeck H , Mathieu E
Published: October 2019

Osteogenic cells were sorted from bone marrow of 5-fluorouracil (5-FU)- treated mice based on light scatter characteristics, Sca-1 expression, and their binding to wheat germ agglutinin (WGA). Four sort gates were established using forward (FSC) and perpendicular (SSC) light scatter and were denominated as FSChigh SSClow, FSClow SSChigh, FSClow SSClow, and FSChigh SSChigh cell. Cells from the FSChigh SSChigh gate, but not from the other gates, synthesized alkaline phosphatase, collagen, and osteocalcin and formed a mineralized matrix in culture. The number of osteoprogenitor cells was significantly enriched after depleting the 5- FU bone marrow from cells of the lymphoid and myeloid lineage, eg, T cells, B cells, natural killer cells, granulocytes, macrophages, and erythrocytes. Approximately 95% of the FSChigh SSChigh cell population of this “lineage-negative” (Lin-) marrow expressed the Sca-1 antigen (Sca-1+) and bound WGA. Three additional sort windows were established based on WGA binding intensity and were denominated as Sca-1+ WGAdull, Sca-1+ WGAmedium, and Sca-1+ WGAbright. Cells from the Sca-1+ WGAbright gate, but not from the other gates, synthesized bone proteins and formed a mineralized matrix. However, they lost this capacity upon subcultivation. Further immunophenotypic characterization showed that FSChigh SSChigh Lin- Sca-1+ WGAbright cells expressed stromal (KM16) and endothelial (Sab-1 and Sab-2) markers, but not hematopoietic surface markers such as c-kit and Thy1.2. Sorted FSChigh SSChigh Lin- Sca-1+ WGAbright cells form three-dimensional nodules that stain with the von Kossa technique and contain osteoblast and osteocyte-like cells.
Coxsackievirus B3 infection in human leukocytes and lymphoid cell lines
Authors: Vuorinen T , Vainionpaa R , Kettinen H , Hyypia T
Published: October 2019

Although coxsackie B viruses (CBVs) are known to cause viremia during acute infection, the role of the blood cells as a target for virus replication is poorly understood. We have analyzed the susceptibility of human peripheral blood mononuclear cells (PBMCs), granulocytes, bone marrow (BM) cells, and lymphoid cell lines to coxsackievirus B3 infection. Lymphoid cell lines with B- and T-cell characteristics (Raji and Molt-4, respectively) supported virus replication to high titers and virus protein synthesis was detected by metabolic labeling and immunoprecipitation. CBV3 synthesis in the U937 cell line with mononuclear phagocytic characteristics was very limited. The virus was able to infect a small proportion of leukocytes and BM cells, and intracellular virus antigens were detected by immunofluorescent staining. However, only a diminutive amount of infectious virus was produced in isolated PBMCs and granulocytes, and no virus protein synthesis was detected by metabolic labeling and immunoprecipitation in these cells.