bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and factor-dependent modulation of the interleukin-3 receptor subunits on human hematopoietic cells
Authors: Sato N , Caux C , Kitamura T , Watanabe Y , Arai K , Banchereau J , Miyajima A
Published: October 2019

Interleukin-3 (IL-3) regulates growth and differentiation of multipotential as well as lineage-committed progenitor cells. The human IL-3 receptor (IL-3R) consists of the alpha and common beta (beta c) subunits. The alpha subunit (IL-3R alpha) is specific for IL-3 and binds IL-3 with low affinity. In contrast, the beta c subunit does not bind any cytokine by itself, but forms a high-affinity receptor with IL- 3R alpha. As the same beta c subunit also forms high-affinity receptors for IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) with the respective cytokine-specific alpha subunit, the expression of the alpha subunits is responsible for specificity of cytokines. To examine the expression of IL-3R alpha, we have developed a monoclonal antibody (MoAb), N3A. N3A specifically bound to cells expressing IL-3R alpha and immunoprecipitated a 75 Kd glycoprotein, which became 43 Kd on N-glycosidase digestion. N3A and an anti-beta c antibody, CRS1, were used in double color fluorescence-activated cell sorter (FACS) staining with several lineage markers to see the IL-3R expression pattern in peripheral blood (PB), cord blood (CB), and bone marrow (BM) cells. Both IL-3R subunits were expressed on myeloid cell lineages (CD13+, CD14+, CD15Lo, or CD33+). To further study the IL-3R expression on hematopoietic progenitor cells, the CD34+ populations were isolated from both BM and CB cells. Those populations showed positive staining profiles with the N3A MoAb and were weakly stained with the CRS1 MoAb. Furthermore, anti c-kit antibody staining of the CD34+ fraction from CB, but not from BM, showed two intensities and the IL-3R alpha expression seemed to be higher in a fraction of low c-kit expression. Because IL-1, IL-6, G-CSF, stem cell factor (SCF), interferon (IFN)- gamma, and tumor necrosis factor (TNF)-alpha are known to enhance IL-3- dependent colony formation, we have examined whether this enhancement could be correlated with upregulation of the IL-3R expression. Incubation of CD34+ cells with TNF-alpha for 2 days significantly increased the level of beta c and G-CSF increased the number of cells with high level expression of alpha, while other factors did not affect the IL-3R expression. Thus, different cytokines appear to have different mechanisms for enhancement of IL-3-dependent proliferation.
In vivo administration of recombinant methionyl human stem cell factor expands the number of human marrow hematopoietic stem cells
Authors: Tong J , Gordon MS , Srour EF , Cooper RJ , Orazi A , McNiece I , Hoffman R
Published: October 2019

A growing number of in vitro studies suggest that recombinant human stem cell factor (SCF) is capable of augmenting the proliferative capacity of human hematopoietic progenitor cells (HPC) and stem cells (HSC). We further evaluated this biologic effect by analyzing the response of bone marrow (BM) HPCs and HSCs to the administration of SCF in eight patients with locally advanced or metastatic breast cancer who were enrolled in an ongoing phase I study. SCF was administered for 14 days by daily subcutaneous injection at dosages of 10, 25, or 50 micrograms/kg/d. BM CD34+ HLA-DR+ and CD34+ HLA-DR- CD15- cells, previously shown by our laboratory to be enriched for various classes of differentiated and primitive HPCs, respectively, were quantitated in BM samples on day 0 (pretreatment) and day 15 (posttreatment). These CD34+ HLA-DR+ and CD34+ HLA-DR- CD15- cells were then isolated by cell- sorting and assayed for several classes of HPCs, including the high-- proliferative potential colony-forming cell (HPP-CFC), the burst- forming unit--megakaryocyte (BFU-MK), and the long-term BM culture-- initiating cell (LTBMC-IC). SCF administration resulted in a 3.3-fold (range, 1.4- to 18.8-fold; P = .018) increase in the absolute numbers of CD34+ cells, a 3.7-fold (range, 1.2- to 8.2-fold; P = .028) increase in the absolute numbers of CD34+ HLA-DR+ cells, and a 2.4-fold (range, 1.1- to 29.3-fold; P = .010) increase in the absolute numbers of CD34+ HLA-DR- CD15- cells. Following the infusion of SCF, a statistically significant increase in the absolute numbers of HPP-CFC (P = .018), BFU- MK (P = .046), CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM: P = .043), BFU-erythrocyte (BFU-E; P = .043), CFU- granulocyte, macrophage (CFU-GM; P = .045), and CFU-megakaryocyte (CFU- MK; P = .028) per milliliter of marrow was observed. Stromal cell-free LTBMCs supplemented with SCF and interleukin-3 (IL-3), initiated with CD34+ HLA-DR- CD15- cells obtained on day 0, produced viable cells for 9.6 weeks, compared with 11.5 weeks for LTBMCs initiated with CD34+ HLA- DR- CD15- cells obtained on day 15. Cumulative cellular production by LTBMCs initiated with day 15 CD34+ HLA-DR- CD15- cells was statistically greater than that by day 0 LTBMCs (P = .031). These same cultures produced CFU-GM for 6.3 weeks (day 0) versus 9 weeks (day 15).(ABSTRACT TRUNCATED AT 400 WORDS)
Clinical significance of CD34 expression in childhood acute lymphoblastic leukemia
Authors: Pui CH , Hancock ML , Head DR , Rivera GK , Look AT , Sandlund JT , Behm FG
Published: October 2019

The CD34 antigen was detected on > or = 10% of the blast cells in 235 (70%) of 335 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL) treated in two consecutive chemotherapy trials. By immunophenotype, the distribution of positive cases favored early pre-B ALL (83%; n = 180) followed by pre-B ALL (61%; n = 89) and then T-cell ALL (46%; n = 61) (P < .001). Among the B-lineage cases, CD34 expression was significantly associated with favorable presenting features: age 1 to 10 years, white race, absence of central nervous system (CNS) leukemia, low serum lactate dehydrogenase level, CD10 expression, and leukemic cell hyperdiploidy (> 50 chromosomes or DNA index > or = 1.16). Event-free survival was clearly superior for patients with CD34+ leukemia (P = .01), with an estimated 83% +/- 6% (SE) of the cohort remaining free of adverse events at 5 years post diagnosis, as compared to 63% +/- 10% of the group without this feature. Multivariate analysis showed that the prognostic influence of the antigen was independent of age, leukocyte count, and other well- recognized factors, suggesting that it would add discriminatory power to current systems of risk assignment. Findings in T-cell ALL were the reverse: CD34 expression showed positive correlations with initial CNS leukemia and CD10 negativity but not with any good-risk presenting characteristics. Log-rank analysis indicated no adverse effect on treatment outcome by CD34 antigen expression, although additional patients with need to be studied to obtain a definitive answer. The opposed clinical associations of CD34 expression in B- and T-lineage ALL may reflect fundamental biologic differences between these leukemia species.
Involvement of the high-affinity receptor for IgG (Fc gamma RI; CD64) in enhanced tumor cell cytotoxicity of neutrophils during granulocyte colony-stimulating factor therapy
Authors: Valerius T , Repp R , de Wit TP , Berthold S , Platzer E , Kalden JR , Gramatzki M , van de Winkel JG
Published: October 2019

Three different classes of Fc receptors for IgG (Fc gamma R) are currently distinguished in humans, of which polymorphonuclear phagocytes (PMN) normally express both low-affinity receptor classes-- Fc gamma RII (CD32) and Fc gamma RIII (CD16). During therapy with granulocyte colony-stimulating factor (G-CSF), neutrophils from patients with various malignancies and different hematologic disorders were found to additionally express high levels of the receptor with high affinity for IgG (Fc gamma RI; CD64). For these patients, the relative fluorescence intensity (rFI) for Fc gamma RI was 5.3 (range, 1.7 to 10.3; n = 19), compared with 1.0 (range, 1.0 to 1.1; n = 8) for healthy donors. The expression of Fc gamma RI during G-CSF therapy could be confirmed by using a panel of six CD64-specific antibodies, and by showing mRNA for Fc gamma RI. So far, three genes for Fc gamma RI have been identified, encoding four distinct transcription products. By reverse transcriptase-polymerase chain reaction technology, transcripts for both membrane-associated isoforms (hFc gamma RIa and hFc gamma RIb2) could be detected. The functional activity of Fc gamma RI on PMN during G-CSF therapy was shown by measuring binding of monomeric human IgG and antibody-dependent cellular cytotoxicity (ADCC). Thus, Fc gamma RI-positive neutrophils displayed enhanced ADCC activity to glioma (A1207), squamous cell (A431), and ovarian (SK-ov3) carcinoma cell lines. The involvement of Fc gamma RI in this increased cytotoxic activity was shown by blocking Fc gamma receptors with monoclonal antibodies, and by using F(ab')2 x F(ab')2-bispecific antibodies with specificities against tumor-related antigens and Fc gamma RI, resulting in solely Fc gamma RI-mediated cytotoxicity. Therapeutically, this additional Fc receptor on PMN may increase the efficacy of experimental antibody therapy.
Autologous IgM, IgA, and complement binding to sickle erythrocytes in vivo. Evidence for the existence of dense sickle cell subsets
Authors: Green GA
Published: October 2019

We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities > 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high- density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high- density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.
Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells
Authors: Rosnet O , Schiff C , Pebusque MJ , Marchetto S , Tonnelle C , Toiron Y , Birg F , Birnbaum D
Published: October 2019

The human FLT3 cDNA was cloned from a pre-B cell line and characterized. The deduced amino acid sequence shows that FLT3 codes for a receptor-type tyrosine kinase of 993 residues, presenting a strong similarity with the corresponding mouse FLT3/FLK2 protein as well as with the receptors for colony-stimulating factor 1 (CSF1R/FMS) and steel locus factor (SLFR/KIT). An analysis of the expression of the gene using amplification of reverse transcribed FLT3 mRNA by polymerase chain reaction shows that FLT3 is expressed in various lymphohematopoietic cells and tissues, including a series of immature cell lines and leukemias of lymphocytic origin.
CD63 is a component of Weibel-Palade bodies of human endothelial cells
Authors: Vischer UM , Wagner DD
Published: October 2019

Weibel-Palade bodies are secretory granules of vascular endothelial cells specialized in the storage of von Willebrand factor (vWF) and P- selectin, two adhesion proteins that can be rapidly mobilized to the cell surface by exocytosis in response to thrombin or other agonists. In this study, we attempted to identify additional components of Weibel- Palade bodies by raising monoclonal antibodies to these granules, purified by cell fractionation. One antibody, 2C6, was found to be specific for CD63, a membrane glycoprotein previously described in the lysosomes of platelets and other cell types. The immunopurified 2C6 antigen was recognized by an anti-CD63 reference antibody, 2.28, by Western blotting. Also, the biosynthetic profile of the 2C6 antigen in endothelial cells showed a nascent molecular mass and a glycosylation pattern identical to that of CD63. Immunofluorescence staining with 2C6 showed the lysosomes, and also elongated structures identified as Weibel-Palade bodies by their shape, distribution, and positive staining with anti-vWF antibodies, CD63 was also found by Western blotting of subcellular fractions highly enriched in Weibel-Palade bodies. Our results indicate that CD63 colocalizes with vWF and P- selectin in the Weibel-Palade bodies of endothelial cells, and together with these adhesion proteins it could be rapidly expressed on the cell surface in areas of vascular injury and inflammation.
Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia
Authors: Rabinowe SN , Soiffer RJ , Gribben JG , Daley H , Freedman AS , Daley J , Pesek K , Neuberg D , Pinkus G , Leavitt PR
Published: October 2019

Twenty patients with poor prognosis B-cell chronic lymphocytic leukemia (B-CLL) underwent uniform high-dose chemoradiotherapy followed by rescue with multiple monoclonal antibody-purged autologous bone marrow (BM) (12 patients) or T-cell-depleted allogeneic BM from HLA-identical siblings (8 patients) in a pilot study to assess the feasibility of BM transplantation (BMT) in this disease. All had poor prognosis disease by either staging, BM pattern, tumor doubling time criteria, or cytogenetics. All patients achieved remission criteria (defined as < or = 2 adenopathy, absence of splenomegaly, < or = 20% of the intertrabecular space involved on BM biopsy) before BMT. Despite the use of fludarabine, a median of three treatment regimens were required to achieve BMT eligibility. After BMT, all patients achieved complete hematologic engraftment. Toxicities were not significantly different between autologous versus allogeneic BMT. Two toxic deaths were observed. Of 19 evaluable patients, 17 clinical complete clinical remissions (89%) were observed, with 2 patients (1 allogeneic and 1 autologous) exhibiting persistent BM disease. Complete clinical remissions were documented at the phenotypic and molecular level for the majority of patients in whom dual fluorescence for CD5 and CD20 (15 of 15; 100%) and Ig gene rearrangements (11 of 14; 79%) were performed. Although long-term follow-up is needed to assess any potential impact on the disease-free and overall survival of these patients, this study shows the feasibility of using high-dose chemoradiotherapy and BMT in patients with poor prognosis B-CLL.
Rescue of W-associated mast cell defects in W/Wv bone marrow cells by ectopic expression of normal and mutant epidermal growth factor receptors
Authors: von Ruden T , Stingl L , Ullrich A , Wagner EF
Published: October 2019

The normal human epidermal growth factor receptor (EGF-R) (HERc), a chimeric EGF-R/v-erbB (HERerbB) receptor, and the ligand-independent oncogenic EGF-R variant (v-erbB) were used to correct the mast cell defects in W/Wv bone marrow (BM) cells. In culture, all three receptor molecules transduced functional mitogenic signals in infected interleukin-3 (IL-3)-dependent bone marrow-derived mast cells (BMMCs) and enabled their differentiation into safranin-positive mast cells resembling connective tissue-type mast cells (CTMCs). Furthermore, expression of these receptors restored the capacity of W/Wv BMMCs to colonize the peritoneal cavity of mast cell-deficient W/Wv mice where they differentiated to safranin-positive cells with similar frequencies as wild-type BMMCs. These experiments show that expression of normal and mutant EGF-Rs in W/Wv BM cells is able to complement the function of the c-kit-encoded Steel factor receptor (SLF-R) in mast cell development. We conclude that signal transduction by normal and mutant EGF-Rs in murine hematopoietic cells apparently involves components also used by the SLF-R, which suggests that these receptors use overlapping pathways for signal transduction.
Erythropoietin structure-function relationships: high degree of sequence homology among mammals
Authors: Wen D , Boissel JP , Tracy TE , Gruninger RH , Mulcahy LS , Czelusniak J , Goodman M , Bunn HF
Published: October 2019

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5′ primers, we were able to successfully PCR- amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha- helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.
Severe chronic autoimmune thrombocytopenic purpura is associated with an expansion of CD56+ CD3- natural killer cells subset [see comments]
Authors: Garcia-Suarez J , Prieto A , Reyes E , Manzano L , Merino JL , Alvarez-Mon M
Published: October 2019

Recent studies have indicated that autoimmune thrombocytopenic purpura (ATP) patients show immune system alterations that are not restricted to the B-cell compartment, but that also affect T lymphocytes. This report studies the phenotypic characteristics of natural killer (NK) cells in the peripheral blood of ATP patients, as well as their clinical significance in 33 ATP patients with active disease. Ten patients had stable disease (sustained platelet counts > 50,000/microL without the need for treatment), whereas 23 patients had therapy- dependent disease (platelet counts < 50,000/microL). A significant increase in both CD56+ CD3- NK cells and CD56+ CD3+ cytotoxic T lymphocytes was observed in peripheral blood mononuclear cells and in purified CD2+ cells from therapy-dependent ATP patients as compared with ATP patients with stable disease and healthy controls. Moreover, there were more major histocompatibility complex (MHC) class II molecules in the CD56+ CD3- cells from the therapy-dependent patients' peripheral blood preparations than there were in the stable ATP patients' and healthy controls' peripheral blood preparations. This growth in the number of CD56+ CD3- NK cells was statistically higher in patients whose disease was refractory to conventional therapy (corticosteroids and splenectomy). In addition to the CD56+ CD3- NK cells, the percentage of CD3+ T lymphocytes and their proliferative response to phytohemagglutinin (PHA) stimulation were studied in fresh CD2+ preparations from nine patients with stable disease, 22 patients with therapy-dependent disease, and 26 healthy controls. The proliferative response of CD2+ lymphocytes from both groups was similar and significantly defective with respect to that found in healthy controls. In conclusion, clinically severe ATP (therapy-dependent disease) is associated with a significant increase of CD56+ CD3- NK cells, which is particularly marked in patients whose disease is refractory to therapy.
Synthesis of interleukin-5 by activated eosinophils in patients with eosinophilic heart diseases
Authors: Desreumaux P , Janin A , Dubucquoi S , Copin MC , Torpier G , Capron A , Capron M , Prin L
Published: October 2019

Eosinophilic endomyocardial disease represents a major evolutive risk in chronic eosinophilia-associated disorders. Eosinophil granule proteins appear to be involved in cardiac injury, but the mechanisms leading to eosinophil infiltration and degranulation are not clear. Interleukin-5 (IL-5) has been recently shown to be produced by eosinophils and might play a role in both chemoattraction and degranulation of eosinophils. In four cases of eosinophilic diseases with severe cardiac failure, we evaluated the proportion of eosinophil phenotypes and the serum levels of eosinophil cationic protein (ECP) and soluble IL-2 receptor (sIL-2R), markers of disease activity in the hypereosinophilic syndromes. All four patients showed a markedly increased proportion of hypodense eosinophils with elevated serum ECP and sIL-2R levels. In all four patients, extracellular deposition of eosinophil granule proteins and features of eosinophil activation were observed in cardiac tissues. The synthesis of IL-5 by eosinophils was detected in myocardial sections and blood cells by in situ hybridization and by immunostaining with a monoclonal antibody against human IL-5. Sixty percent to 90% of tissue eosinophils expressed IL-5 mRNA and IL-5 protein. These data suggest that IL-5 can be produced by eosinophils at the sites of myocardial tissue damage and might participate in local eosinophil activation.
Lysis of human monocytic leukemia cells by extracellular adenosine triphosphate: mechanism and characterization of the adenosine triphosphate receptor
Authors: Spranzi E , Djeu JY , Hoffman SL , Epling-Burnette PK , Blanchard DK
Published: October 2019

The present study shows that extracellular adenosine triphosphate (ATP) has the capacity to mediate dose-dependent lysis of the monocytic leukemia cell line THP-1. The lysis, assessed by 51Cr release, was found to be selective for ATP, because adenosine diphosphate (ADP) or other nucleotides were less effective in their ability to lyse the cells. The amount of 51Cr released was particularly enhanced by the stimulation of the cells with 1,000 U/mL of interferon gamma (IFN- gamma) for 3 days, and the sensitivity was time and dose dependent. Analysis of the mechanism of lysis indicated that the fully ionized form, ATP4-, mediated the lysis, because the addition of cation chelators or the absence of the divalent cations, Ca2+ and Mg2+, in the culture medium of a 6-hour 51Cr release assay increased the percent specific lysis. Therefore, the ATP receptors on THP-1 cells were classified as P2Z purinoceptors. Moreover, it is shown here that the Ca2+/calmodulin complex plays a role in the regulation of the lysis by extracellular ATP of THP-1 cells, because antagonists of this complex, such as trifluoperazine or KN-62, were found to inhibit the ATP- mediated cell lysis.
T-cell-rich B-cell lymphoma
Authors: Rodriguez J , Pugh WC , Cabanillas F
Published: October 2019

We analyzed 23 cases of T-cell-rich B-cell lymphomas (BCL) to determine if the clinical features are characteristic of a discrete entity. Cases encoded as T-cell-rich BCL in the hematopathology archives of the University of Texas M.D. Anderson Cancer Center between 1988 and 1991 formed the basis of this study. At least 50% of the total population of cells were required to be of T-cell phenotype. Actually, all but one patient had more than 70% T cells in the total population. Sixty-five percent of all cases were referred with other diagnosis such as Hodgkin's mixed cellularity, peripheral T-cell lymphoma (PTCL), or diffuse mixed lymphoma, and had received therapy accordingly. With the exception of splenomegaly, which occurred in 35% of cases, the other clinical characteristics and the response to therapy did not indicate that this entity represents a distinct type of lymphoma. Ann Arbor stage I-II presentations were seen in 10 of 23 (43%) T-cell-rich BCLs. Serum lactate dehydrogenase (LDH) was elevated in eight of 19 patients. Age, sex, and beta 2-microglobulin were not significantly different from classical B-cell large cell lymphoma. The clinical presentation and clinical outcome of T-cell-rich BCL did not differ from that of common B-cell large cell lymphoma, except for the higher proportion of splenomegaly seen in patients with T-cell-rich BCL. The presence of the T-cell-rich infiltrate varied: it frequently was not seen at relapse or at other sites of disease at presentation. It was thus considered an unstable parameter. The major importance of identifying this entity is to distinguish it pathologically from other disorders such as Hodgkin's disease and PTCL, which would be treated in a different manner.
E-selectin and vascular cell adhesion molecule-1 mediate adult T-cell leukemia cell adhesion to endothelial cells
Authors: Ishikawa T , Imura A , Tanaka K , Shirane H , Okuma M , Uchiyama T
Published: October 2019

We studied the adhesion properties of peripheral blood leukemic cells from 10 patients with adult T-cell leukemia (ATL) to endothelial cells to better understand the mechanism of leukemic cell infiltration. ATL cells expressed lymphocyte function-associated antigen-1 (LFA-1), but the expression of very late antigen-4 (VLA-4) and sialyl-Lewisx (SLex) was variable. They did not express sialyl-Lewisa (SLea). Cell adhesion assays, which were performed in nine patients, showed marked adhesion of ATL cells to interleukin [IL]-1-activated human umbilical vein endothelial cells (HUVEC). A monoclonal antibody (MoAb) against E- selectin consistently inhibited ATL cell adhesion, and an MoAb against vascular cell adhesion molecule-1 (VCAM-1) or an MoAb against VLA-4 sometimes diminished it. In contrast, an MoAb against LFA-1 had a minor effect on freshly isolated ATL cell adhesion to HUVEC. The percentage of SLex+ cells in the cell population adherent to IL-1-activated HUVEC was slightly higher than that in unseparated cells. These results, together with the detection of E-selectin expression on the endothelium at ATL skin lesions, indicate that E-selectin-mediated adhesion is the major pathway for the adherence of ATL cells to endothelial cells. In addition, the ligand for E-selectin on ATL cells appears to differ from that on neutrophils.
The interleukin-2 receptor: a target for monoclonal antibody treatment of human T-cell lymphotrophic virus I-induced adult T-cell leukemia
Authors: Waldmann TA , White JD , Goldman CK , Top L , Grant A , Bamford R , Roessler E , Horak ID , Zaknoen S , Kasten-Sportes C
Published: October 2019

Adult T-cell leukemia (ATL) is a malignancy of mature lymphocytes caused by the retrovirus human T-cell lymphotrophic virus-I (HTLV-I). It is an aggressive leukemia with an overall mortality rate of 50% within 5 months; no conventional chemotherapy regimen appears successful in inducing long-term disease-free survival in ATL patients. However, ATL cells constitutively express high-affinity interleukin-2 receptors (IL-2Rs) identified by the anti-Tac monoclonal antibody, whereas normal resting cells do not. To exploit this difference in receptor expression, we administered anti-Tac intravenously (IV) to 19 patients with ATL. In general the patients did not suffer untoward reactions, and in 18 of 19 cases did not have a reduction in normal formed elements of the blood. Seven patients developed remissions that were mixed (1 patient), partial (4 patients), or complete (2 patients), with partial and complete remissions lasting from 9 weeks to more than 3 years as assessed by routine hematologic tests, immunofluorescence analysis, and molecular genetic analysis of T-cell receptor gene rearrangements and of HTLV-I proviral integration. Furthermore, remission was associated with a return to normal serum calcium levels and an improvement of liver function tests. Remission was also associated in some cases with an amelioration of the profound immunodeficiency state that characterizes ATL. Thus the use of a monoclonal antibody that blocks the interaction of IL-2 with its receptor expressed on ATL cells provides a rational approach for treatment of this aggressive malignancy.
Recombinant rat stem cell factor synergizes with recombinant human granulocyte colony-stimulating factor in vivo in mice to mobilize peripheral blood progenitor cells that have enhanced repopulating potential
Authors: Briddell RA , Hartley CA , Smith KA , McNiece IK
Published: October 2019

Splenectomized mice treated for 7 days with pegylated recombinant rat stem cell factor (rrSCF-PEG) showed a dose-dependent increase in peripheral blood progenitor cells (PBPC) that have enhanced in vivo repopulating potential. A dose of rrSCF-PEG at 25 micrograms/kg/d for 7 days produced no significant increase in PBPC. However, when this dose of rrSCF-PEG was combined with an optimal dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 200 micrograms/kg/d), a synergistic increase in PBPC was observed. Compared with treatment with rhG-CSF alone, the combination of rrSCF-PEG plus rhG-CSF resulted in a synergistic increase in peripheral white blood cells, in the incidence and absolute numbers of PBPC, and in the incidence and absolute numbers of circulating cells with in vivo repopulating potential. These data suggest that low doses of SCF, which would have minimal, if any, effects in vivo, can synergize with optimal doses of rhG-CSF to enhance the mobilization of PBPC stimulated by rhG-CSF alone.
Activation of the contact system in insect-sting anaphylaxis: association with the development of angioedema and shock
Authors: van der Linden PW , Hack CE , Eerenberg AJ , Struyvenberg A , van der Zwan JK
Published: October 2019

A postulated role of the contact system in anaphylactic reactions to insect stings was investigated. During prospective, in-hospital sting challenge, we collected serial blood samples from five normal volunteers and 16 patients with a history of insect-sting anaphylaxis. Activation of the contact system was assessed by measuring plasma levels of factor XIIa-C1-inhibitor and kallikrein-C1-inhibitor complexes as well as those of cleaved high molecular weight kininogen (HK). In addition, antigenic levels of (pre)kallikrein, factor XII, and HK were measured. No significant changes in contact system parameters were observed in any of the five volunteers or the four patients who did not develop an anaphylactic reaction after sting challenge. In contrast, significant changes in contact system parameters occurred in 7 of the 12 patients with anaphylactic symptoms after challenge. Peak levels of either C1-inhibitor complex were found 5 minutes after the onset of anaphylactic symptoms. The increase in C1-inhibitor was most pronounced in the 4 patients with angioedema, 2 of which also developed shock. However, activation of HK was observed in all four patients with angioedema, the two patients with shock but no angioedema, as well as in 1 of the remaining 6 patients with anaphylactic symptoms other than angioedema or shock. Thus, activation products of the contact system may be involved in the pathogenesis of angioedema and shock in insect- sting anaphylaxis.
Controlled insect-sting challenge in 55 patients: correlation between activation of plasminogen and the development of anaphylactic shock
Authors: van der Linden PW , Hack CE , Struyvenberg A , Roem D , Brouwer MC , de Boer JP , van der Zwan JK
Published: October 2019

The pathogenesis of anaphylactic shock is not completely understood. Mast cell degranulation products may stimulate endothelial cells, leading to activation of fibrinolytic and coagulation systems. We investigated the activation of these systems in insect-sting anaphylaxis. Fifty-five patients with a previous insect-sting anaphylactic reaction and 8 volunteers were challenged with an in- hospital sting. Plasma levels of von Willebrand factor (vWF), coagulation, and fibrinolytic parameters were assessed. After the sting challenge, 20 patients developed anaphylactic symptoms, 7 of whom developed hypotension. In only these 7 patients, but not in the volunteers or in the other patients with no or mild anaphylactic symptoms, vWF levels increased from 107% +/- 33% (mean +/- SD) before, to 235% +/- 134% 60 minutes after the onset of clinical symptoms. This increase of vWF was accompanied by an increase of circulating tissue- type plasminogen-activator (tPA) levels from 5 +/- 3 micrograms/L to 50 +/- 59 micrograms/L and of plasminogen-alpha 2-antiplasmin complex (PAP- c) levels from 6 +/- 3 nmol/L to 297 +/- 225 nmol/L. Both tPA and PAP-c levels peaked 5 minutes after the onset of clinical symptoms. Such increases of tPA and PAP-c were not observed in the volunteers or in the patients who did not develop shock. The increase of tPA and PAP-c levels in the hypotensive patients correlated positively with the degree of mast cell degranulation and inversely with the mean arterial pressure. We conclude that activation of plasminogen may be involved in the pathogenesis of anaphylactic shock induced by insect venom.
The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues
Authors: Fujimoto T , McEver RP
Published: October 2019

P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.
A recombinant factor VIII A2 domain polypeptide quantitatively neutralizes human inhibitor antibodies that bind to A2
Authors: Scandella D , Mattingly M , Prescott R
Published: October 2019

Human antibodies that inactivate coagulation factor VIII (fVIII), known as inhibitors, have been shown by immunoblotting or immunoprecipitation assays to bind predominantly to epitopes within the A2 and/or C2 domains of the fVIII protein. Because these assays simply measure antibody binding, a soluble recombinant polypeptide containing the fVIII A2 domain was used to develop a quantitative inhibitor neutralization assay for antibodies that bound only to A2 by immunoblotting assay. Antibodies from six of eight inhibitor plasmas were fully neutralized by A2 (> or = 90%), whereas two were only partially neutralized. These results established the fVIII inhibitor properties of anti-A2 antibodies. In immunoprecipitation assays, five of the eight inhibitors also had significant levels of anti-light-chain antibody. In one case, this light-chain antibody was shown to have inhibitor activity. Because it did not bind to the C2 domain, this antibody appears to define a new inhibitor epitope within the fVIII light chain. Another inhibitor, which was partially neutralized by A2, was not neutralized by the light chain, even though it contained anti- light-chain antibodies by immunoprecipitation assay. Our results show additional complexities of the immune response to fVIII.
Isolation and characterization of the neutrophil-binding proteins for platelet-derived adherence-inhibiting factor
Authors: Iwabuchi K , Nagaoka I , Someya A , Yamashita T
Published: October 2019

Guinea pig neutrophils adhered to adherence-inhibiting factor (AIF)- coated plastic; the adherence was completely inhibited by the addition of AIF, but partly inhibited by type IV collagen. Binding of 125I- labeled AIF to neutrophils was inhibited by unlabeled AIF, but partly inhibited by type IV collagen. Scatchard analysis showed that neutrophils have two classes of binding sites for AIF, high-affinity binding sites (kd = 5.0 pmol/L) numbering 500 per cell and low-affinity binding sites (kd = 860 pmol/L) numbering 6,400 per cell. Type IV collagen increased the kd of low-affinity binding sites. We have isolated and characterized the AIF-binding sites. We have isolated and characterized the AIF-binding proteins. Using AIF affinity chromatography, the radioactive fraction containing six proteins of molecular mass 45, 63, 87, 90 to 105, 145, and 195 Kd was isolated from 125I surface-labeled neutrophil extracts. This radioactive fraction was further separated into two fractions using type IV collagen affinity chromatography, ie, one fraction was adsorbed on the type IV collagen column and contained the 45-, 63-, and 87-Kd proteins, whereas another fraction was not adsorbed on the column and contained the 45-, 63-, 90- to 105-, 145-, and 195-Kd proteins. To isolate the type IV collagen- binding proteins, 125I surface-labeled neutrophil extracts were applied to a type IV collagen-Sepharose column; the isolated radioactive fraction contained the 45-, 63-, and 87-Kd proteins and bound to an AIF- Sepharose column. Taken together, these results suggest that the AIF- binding proteins, which bind to type IV collagen, are the type IV collagen-binding proteins of neutrophils.
Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation
Authors: Cross NC , Feng L , Chase A , Bungey J , Hughes TP , Goldman JM
Published: October 2019

We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia- positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.
Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells
Authors: Bodine DM , Moritz T , Donahue RE , Luskey BD , Kessler SW , Martin DI , Orkin SH , Nienhuis AW , Williams DA
Published: October 2019

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.
Anticoagulant protein C pathway defective in majority of thrombophilic patients [see comments]
Authors: Griffin JH , Evatt B , Wideman C , Fernandez JA
Published: October 2019

A defect involving poor anticoagulant response to activated protein C (APC), an anticoagulant serine protease known to inactivate factors Va and VIIIa in plasma, was recently reported and the existence of a novel APC cofactor was suggested. To define the frequency of this defect among 25 venous thrombophilic patients with no identifiable laboratory test abnormality and among 22 patients previously identified with heterozygous protein C or protein S deficiency, the APC-induced prolongation of the activated partial thromboplastin time assay for these patients was compared with results for 35 normal subjects. The results show that this new defect in anticoagulant response to APC is surprisingly present in 52% to 64% of the 25 patients, ie, in the majority of previously undiagnosed thrombophilia cases, but is not present in 20 of 22 heterozygous protein C or protein S deficient patients, suggesting that the new factor is a risk factor independent of protein C or protein S deficiency. The results demonstrate that abnormalities in the anticoagulant protein C pathway are present in the majority of thrombophilic patients.
A randomized controlled trial of pentoxifylline for the prevention of regimen-related toxicities in patients undergoing allogeneic marrow transplantation
Authors: Clift RA , Bianco JA , Appelbaum FR , Buckner CD , Singer JW , Bakke L , Bensinger WI , Bowden RA , McDonald GB , Schubert M
Published: October 2019

This study evaluated the effect of pentoxifylline (PTX) on the incidence of regimen-related toxicity in patients receiving allogeneic marrow transplants from related donors. All patients received a regimen of methotrexate and cyclosporine as prophylaxis against acute graft- versus-host disease (GVHD). Patients were randomized to receive PTX or a placebo for 70 days and the outcome was examined in a blinded fashion. Forty-four patients were evaluate in each study arm. PTX had no significant effect on engraftment, the incidence of GVHD, venocclusive disease of the liver, infection, the need for oxygen, posttransplant survival, or the duration of hospitalization. Patients receiving PTX were significantly more likely to develop major elevations of serum creatinine levels. PTX was poorly tolerated and induced significantly more vomiting than the placebo. PTX as administered in this randomized study was associated with significant toxicity and offered no benefit in reducing transplant-related morbidity or mortality.
Cytokines in inflammatory malignant fibrous histiocytoma presenting with leukemoid reaction
Authors: Melhem MF , Meisler AI , Saito R , Finley GG , Hockman HR , Koski RA
Published: October 2019

Inflammatory malignant fibrous histiocytomas (IMFH) are rare tumors and are frequently associated with leukocytosis. In rare cases, leukemoid reactions were attributed to tumor production of unidentified hematopoietic factors. In this study, we used immunohistochemical techniques to show cytokine immunoreactivity in the malignant cells of two cases of IMFH presenting with leukemoid reactions and compared them with two malignant fibrous histocytomas, noninflammatory type. All four tumors stained positively for stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-2 (IL-2), IL-4, IL-5, interferon-alpha (IFN-alpha), and insulin-like growth factor-I. Other cytokines detected only in the two IMFH included IL-6, IL-7, IL-8, IFN- gamma, and keratinocyte growth factor. Granulocyte-macrophage-CSF, IL- 3, and transforming growth factor-beta staining was present in one of the two IMFH tumors and was not present in the noninflammatory tumors. The immunohistochemical staining was localized to the malignant cells, suggesting deregulated cytokine expression consistent with their monocytic/histocytic origin. Expression of certain cytokines in the IMFH may account for the local inflammatory infiltrate, tumor fibrosis, and the aggressive nature of the malignant cells. We also detected elevated serum levels of SCF, G-CSF, IL-6, and tumor necrosis factor in one or both of the IMFH patients. These latter observations may explain the bone marrow hypercellularity and other paraneoplastic symptoms, including fever, malaise, and weight loss, observed in both patients. Different cytokines present in the two IMFH tumors appear to be responsible for the eosinophilic leukemoid reaction observed in one case and for the granulocytic leukemoid reaction observed in the other patient. They may also be responsible for expansion of the tumor-cell population, fibroblast proliferation, and enhanced secretion of extracellular collagen.