bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased levels of total and reduced glutathione in CD4+ lymphocytes in common variable immunodeficiency are associated with activation of the tumor necrosis factor system: possible immunopathogenic role of oxidative stress
Authors: Aukrust P , Svardal AM , Muller F , Lunden B , Berge RK , Froland SS
Published: October 2019

We have previously shown chronic immune activation and enhanced generation of reactive oxygen species in common variable immunodeficiency (CVI). In the present study, we examined levels of glutathione, the dominant intracellular thiol, that play an important protective role against oxidative and inflammatory stress in plasma and in monocytes and lymphocyte subsets in 20 CVI patients and in 16 healthy controls. CD4+ lymphocytes from CVI patients had significantly lower levels of both total and reduced glutathione as well as a lower ratio of reduced to total glutathione compared with healthy controls. This decrease in glutathione levels in CD4+ lymphocytes was most pronounced in the CD45RA+ subset. Plasma levels of total glutathione were also significantly decreased in CVI. In contrast, monocytes from CVI patients exhibited increased levels of both total and reduced glutathione compared with blood donor monocytes. CVI patients had significantly raised serum levels of tumor necrosis factor alpha (TNF alpha) and TNF alpha concentration was strongly associated with glutathione depletion in CD4+ lymphocytes. Furthermore, the lowest levels of both total and reduced glutathione were found in a subgroup of CVI patients characterized by persistent immune activation in vivo, decreased numbers of CD4+ lymphocytes in peripheral blood, and splenomegaly. Finally, supplementation of cell cultures with glutathione-monoethyl ester did significantly enhance interleukin-2 production from peripheral blood mononuclear cells in CVI patients. These glutathione abnormalities in CVI indicate increased oxidative stress, particularly in CD4+ lymphocytes, and intracellular depletion of reduced glutathione of the demonstrated magnitude may have profound implications for CD4+ lymphocyte function and the immunodeficiency in CVI.
Delineation of an immunodominant and human T-cell lymphotropic virus (HTLV)-specific epitope within the HTLV-I transmembrane glycoprotein
Authors: Hadlock KG , Goh CJ , Bradshaw PA , Perkins S , Lo J , Kaplan JE , Khabbaz R , Foung SK
Published: October 2019

Antibody reactivity to the transmembrane region of human T-cell lymphotropic virus type I (HTLV-I) envelope, gp21, is observed in virtually all individuals infected with HTLV-I or HTLV-II. Recombinant proteins encoding selected portions of gp21 are described and used to define two immunogenic regions. The first epitope (designated GD21-I) contains amino acids 361 to 404 of the HTLV-I envelope and reacted with all of 54 sera from HTLV-I- and HTLV-II-infected individuals. The second epitope (designated BA21) expresses amino acids 397 to 430 of the HTLV-I envelope and was recognized by 33 of 54 HTLV antisera. To determine the specificity of GD21-I and BA21, sera from 17 HTLV- negative individuals with nonspecific reactivity to p21E were tested. None of these sera reacted with GD21-I, but 16 of 17 sera reacted with BA21. With virtually complete reactivity to sera from HTLV-infected individuals and no reactivity to sera from p21E-reactive uninfected individuals, GD21-I will be useful in immunoassays for the detection of HTLV infection.
Consolidation treatment of adult acute lymphoblastic leukemia: a prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. BGMT Group
Authors: Attal M , Blaise D , Marit G , Payen C , Michallet M , Vernant JP , Sauvage C , Troussard X , Nedellec G , Pico J
Published: October 2019

A prospective, randomized trial was initiated in adult acute lymphoblastic leukemia (ALL) to compare (1) disease-free survival (DFS) after allogeneic or autologous bone marrow transplantation (BMT) and (2) the relapse rate of patients treated with or without interleukin-2 (IL-2) after autologous BMT. A total of 135 previously untreated patients, aged under 55 years, received the Berlin-Frankfurt-Muster (BFM) induction regimen: 126 patients (93%), of which 120 were HLA- typed, achieved complete remission (CR). According to this genetic randomization, patients with (n = 43) or without an HLA-identical sibling (n = 77) were to receive allogeneic or autologous BMT, respectively. The 3-year post-CR probability of DFS was significantly higher in the HLA-identical sibling group than in the non-HLA-identical sibling group (68% v 26%; P < .001). Eligible patients were randomized to receive (n = 30) or not to receive (n = 30) IL-2 after autologous BMT: the 3-year post-BMT probability of continuous CR was similar in both groups (29% v 27%, respectively). We conclude that, in ALL, early allogeneic BMT after the BFM induction regimen is an effective consolidation treatment and that IL-2 does not decrease the high relapse rate observed after autologous BMT.
Human Rh D monoclonal antibodies (BRAD-3 and BRAD-5) cause accelerated clearance of Rh D+ red blood cells and suppression of Rh D immunization in Rh D- volunteers
Authors: Kumpel BM , Goodrick MJ , Pamphilon DH , Fraser ID , Poole GD , Morse C , Standen GR , Chapman GE , Thomas DP , Anstee DJ
Published: October 2019

The use of prophylactic anti-D to prevent Rh D immunization in Rh D- women and subsequent hemolytic disease in Rh D+ infants is widespread, but has led to shortages of the anti-D Ig. With the aim of substituting monoclonal anti-D for Rh D prophylaxis, we have compared the abilities of monoclonal and polyclonal anti-D to clear Rh D+ red blood cells (RBCs) infused into Rh D- male volunteers and to suppress Rh D immunization. Two human monoclonal antibodies (MoAbs), BRAD-3 (IgG3) and BRAD-5 (IgG1), produced from stable Epstein-Barr virus-transformed B-lymphoblastoid cell lines, were selected because of their proven in vitro activity in promoting RBC lysis in antibody-dependent cell- mediated cytotoxicity assays. RBC clearance was assessed by intravenous injection of 3 mL of 51chromium-labeled D+ RBCs into 27 volunteers 48 hours after intramuscular injection of monoclonal or polyclonal anti-D. Further 3-mL injections of unlabeled D+ cells were administered at 6 and 9 months to induce immunization. Blood samples were taken throughout the 12-month period of study for the serologic detection of anti-D. The mean half-life (t50%) of RBCs in 7 recipients of 300 micrograms BRAD-5 (5.9 hours) was similar to that in 8 recipients of 500 IU polyclonal anti-D (5.0 hours), whereas D+ cells were cleared more slowly in some of the 8 subjects injected with 300 micrograms BRAD- 3 (mean t50% 12.7 hours) and in 1 individual administered 100 micrograms BRAD-3 (t50% 41.0 hours). The rate of RBC clearance in both groups administered 300 micrograms monoclonal anti-D correlated with the amount of antibody bound per cell, determined by flow cytometry. There was no evidence of primary immunization having occurred in any subject after 6 months of follow-up. Five of 24 subjects produced anti- D after one or two further injections of RBCs, confirming that they were responders who had been protected by the monoclonal or polyclonal anti-D administered initially. Four of these responders were recipients of monoclonal anti-D (3 BRAD-3, 1 BRAD-5). One individual who received BRAD-5 produced accelerated clearance of D+ RBCs at the third unprotected RBC challenge but did not seroconvert. This study shows that the human MoAbs BRAD-3 and BRAD-5 can prevent Rh D immunization, and indicates that they may be suitable replacements for the polyclonal anti-D presently used in prophylaxis of Rh D hemolytic disease of the newborn.(ABSTRACT TRUNCATED AT 400 WORDS)
Cell proliferation on fibrin: modulation by fibrinopeptide cleavage
Authors: Sporn LA , Bunce LA , Francis CW
Published: October 2019

Fibrin forms the cohesive network of hemostatic plugs and thrombi, and it also provides the temporary matrix for initial support of healing and revascularization. Because cell proliferation is needed for revascularization after vessel injury, we have characterized structural requirements of fibrin needed to support cell proliferation on fibrin in vitro. Proliferation of cultured human endothelial cells and fibroblasts was measured by 3H-thymidine incorporation on fibrin surfaces varying in structure. Fibrin prepared with thrombin and lacking both fibrinopeptides A and B (desAB fibrin) supported proliferation of both endothelial cells and fibroblasts. In contrast, fibrin prepared with reptilase, which cleaves only fibrinopeptide A, supported significantly less proliferation. Also, fibrin prepared by thrombin treatment of fibrinogen lacking residues beta 1–42 supported only a low level of proliferation. Therefore, fibrinopeptide B cleavage and exposure of beta 15–42 enhanced proliferation of cells on fibrin. Specific proteolytic inhibitors were used to eliminate the potential mitogenic effects of residual fibrin-bound thrombin. Additional controls showed that neither catalytically inactive thrombin nor addition of the thrombin receptor-activating peptide (SFLLRNPNDKYEPF [SFLL]) stimulated proliferation on desA fibrin. The results indicate that cell proliferation on fibrin is enhanced by fibrinopeptide B cleavage and exposure of the amino terminus of the fibrin beta chain. They also show that specific structural features of the temporary fibrin matrix formed at sites of injury may modulate the proliferative response of vascular cells.
Factor VNew Brunswick: Ala221-to-Val substitution results in reduced cofactor activity
Authors: Murray JM , Rand MD , Egan JO , Murphy S , Kim HC , Mann KG
Published: October 2019

We have characterized the factor V protein and cDNA of a patient displaying factor V deficiency (parahemophilia) and correlated the reduced activity with a missense mutation of Ala221-to-Val. Plasma from the subject individual (C1) presented reduced factor V antigen (39% of normal) that displayed reduced activity (approximately 26% of normal). Factor V purified from this individual by standard techniques shows normal migration on sodium dodecyl sulfate gels and a normal pattern of activation by thrombin. Purified antigen from sibling C2 gives a much reduced specific activity of 263 U/mg (17% of normal). Sibling C3, the mother, and the father have antigen within the normal range (57% to 200%) that has approximately normal specific activity. The cDNA encoding the factor Va heavy and light chains of the subject individual was polymerase chain reaction-amplified and sequenced and revealed an A- to-G substitution at position 3 of codon 51 (silent mutation), a C-to-T substitution in position 2 of codon 221 (Ala221-Val), a T-to-C substitution at position 3 of codon 708 (silent mutation), and a G-to-A substitution at position 1 of codon 2185 (Thr2185-Ala). The latter mutation was also observed in control individuals and is proposed to be a possible polymorphism. Restriction analyses demonstrated the presence of one mutant and one normal allele in the father. The subject individual (C1) and sibling C2 carry only the mutant allele. The mother and sibling C3 carry only the normal allele. The inheritance pattern suggests the presence of a missing or nonexpressed allele in the mother that is passed on to all the siblings. Expression of only the mutant allele by the subject individual (C1) and sibling C2 is consistent with reduced factor V antigen and activity in these patients. We have designated this mutant as Factor VNew Brunswick.
Endothelial-specific gene expression directed by the tie gene promoter in vivo
Authors: Korhonen J , Lahtinen I , Halmekyto M , Alhonen L , Janne J , Dumont D , Alitalo K
Published: October 2019

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.
Hepatitis C virus within a malignant lymphoma lesion in the course of type II mixed cryoglobulinemia
Authors: De Vita S , Sansonno D , Dolcetti R , Ferraccioli G , Carbone A , Cornacchiulo V , Santini G , Crovatto M , Gloghini A , Dammacco F
Published: October 2019

Hepatitis C virus (HCV) has been implicated as the major etiologic factor sustaining B-cell clonal expansion in type II mixed cryoglobulinemia (MC). A putative pathogenetic role of HCV in the development of MC-associated B-cell malignancies has also been speculated. We report for the first time the localization of HCV within a parotid non-Hodgkin's lymphoma (NHL) lesion in the course of HCV- related type II essential MC, an important step to implicate any infectious agent in the lymphomagenesis. Plus and minus strand HCV RNA was first demonstrated by polymerase chain reaction on the whole RNA from the lesion. Further immunohistochemical studies localized HCV c22 proteins in the residual ductal or acinar parotid structures, which also abnormally expressed HLA-DR antigens. Weak c22 signals were inconstantly detected in cells strictly confined around the residual epithelium, while all the remaining infiltrating cells in the parotid lesion stained c-22-negative. Staining for c33 and c100 HCV antigens was negative. In situ hybridization (ISH) studies again identified the residual parotid epithelial cells as the site of HCV infection and replication in the NHL lesion. Sialotropic viruses previously involved in lymphoproliferation, ie, Epstein-Barr virus and human herpesvirus-6, were absent in the same tissue lesion. According to the current models of B-cell lymphomagenesis, a role of HCV as an exogenous antigenic stimulus should be considered for NHL development in the present case, whereas malignant B cells do not appear permissive of active HCV replication. Further efforts would be worthwhile to clarify a role of HCV infection in the development of some B-cell malignancies.
Consecutive appearance of coagulation factor XIII subunit A in macrophages, megakaryocytes, and liver cells during early human development
Authors: Kappelmayer J , Bacsko G , Birinyi L , Zakany R , Kelemen E , Adany R
Published: October 2019

Thirty embryonic and fetal samples were investigated to study the appearance and characteristics of factor XIII subunit A (FXIIIA)- containing cells in the course of human development. Samples were either vacuum-embedded in paraffin for staining FXIIIA by a sensitive biotin-streptavidin system or snap-frozen for double-labeling studies to characterize FXIIIA-containing cells. FXIIIA appeared as early as the fifth gestational week in yolk sac samples in stellate-shaped cells. Nonparenchymal cells in liver samples showed intense labeling for FXIIIA from the sixth week of gestation. The relative amount of FXIIIA-containing cells rapidly diminished up to the 13th gestational week. When characterized, the majority of these cells proved to be KiM7- positive macrophages, while GPIb (CD42b)-labeled cells accounted for less than 10% of FXIIIA-positive cells. Liver cells did not show any staining for FXIIIA in first trimester samples. The earliest liver specimen showing FXIIIA was at the 20th week, when FXIIIA appeared in some liver cells, particularly in those surrounding the central veins. In bone marrow smears, FXIIIA-positive cells started to appear at week 10 in the clavicles and increased in number in subsequent stages of development. Intracellular FXIIIA was distributed between GPIb-, RFD7-, and KiM7-positive cells. The results indicate that, apart from liver cells, at least three different cell populations (KiM7+ RFD7+ GPIb-, KiM7- RFD7- GPIb-, and KiM7- RFD7- GPIb+) contain FXIIIA in the early phase of human development. We conclude that FXIIIA appears very early during human development and is detectable in both extra- and intraembryonic hematopoietic organs. Intracellular FXIIIA in early human development is distributed between different macrophages and megakaryocytes, and by week 20, it appears in liver cells as well.
TCL1 oncogene activation in preleukemic T cells from a case of ataxia- telangiectasia
Authors: Narducci MG , Virgilio L , Isobe M , Stoppacciaro A , Elli R , Fiorilli M , Carbonari M , Antonelli A , Chessa L , Croce CM , Russo G
Published: October 2019

The TCL1 oncogene on human chromosome 14q32.1 is involved in chromosome translocations [t(14;14)(q11;q32.1) and t(7;14)(q35;q32.1)] and inversions [inv14(q11;q32.1)] with TCR alpha/beta loci in T-cell leukemias, such as T-prolymphocytic (T-PLL). It is also involved in T- acute and -chronic leukemias arising in cases of ataxia-telangiectasia (AT), an immunodeficiency syndrome. Similar chromosomal rearrangements occur also in the clonally expanded T cells in AT patients before the appearance of the overt leukemia. We have analyzed the expression of TCL1 mRNA and protein in peripheral blood lymphocytes (PBLs) from four AT cases and from healthy controls. We found that the TCL1 gene was overexpressed in the PBLs of an AT patient with a large clonal T-cell population exhibiting the t(14;14) translocation but not in the lymphocytes of the other cases. Fluorescence in situ hybridization of the TCL1 genomic locus to lymphocyte metaphases from the AT patient with the T-cell clonal expansion showed that the breakpoint of the t(14;14) translocation lies within the TCL1 locus and is accompanied by an inverted duplication of the distal part of chromosome 14. These data indicate that TCL1 is activated in preleukemic clonal cells as a consequence of chromosome translocation involving sequences from the TCR locus at 14q11. Deregulation of TCL1 is the first event in the initiation of malignancy in these types of leukemias and represents a potential tool for clinical evaluation.
Hematopoietic inhibition by interferon-gamma is partially mediated through interferon regulatory factor-1
Authors: Sato T , Selleri C , Young NS , Maciejewski JP
Published: October 2019

Biologic responses to cytokines are mediated by intracellular pathways involving induction of signaling and metabolic cascades. Interferon (IFN) regulatory factor-1 (IRF-1) is a major transcription factor induced not only by IFN-gamma but also by other cytokines including tumor necrosis factor-alpha (TNF-alpha). Possible IRF-1 binding sequence elements have been located in the promoter regions of several genes, including p53, inducible nitric oxide synthase, and cyclin D1. IFN-gamma and TNF-alpha can inhibit hematopoiesis in vitro and have been implicated in the pathophysiology of bone marrow (BM) failure. We investigated whether the inhibitory effects of these cytokines were intracellularly mediated through the expression of IRF-1 or -2 in target cells. In total BM cells, IRF-1 mRNA expression increased after stimulation with IFN-gamma and TNF-alpha; the stronger effect was observed with IFN-gamma. In contrast, IRF-2 mRNA expression was constitutive and not altered by cytokine stimulation. By gene amplification, low levels of IRF-1 mRNA were present in unstimulated, highly purified CD34+ cells; on exposure to IFN-gamma and TNF-alpha, amplified IRF-1 mRNA showed a much stronger signal than control. When CD34+ cells were treated with IFN-gamma and TNF-alpha, IRF-1 antisense oligodeoxynucleotide (ODN) partially reversed the suppressive effects on CD34+ cell-derived colony formation by IFN-gamma but not those by TNF-alpha. In parallel experiments, IRF-1 antisense ODN decreased both IRF-1 protein and mRNA expression. The effects of ODN were sequence- specific and concentration-dependent. These results suggest that the inhibitory hematopoietic effects of IFN-gamma and TNF-alpha are mediated by different pathways. For IFN-gamma, IRF-1 is involved in the activation of cellular genes responsible for IFN-gamma suppressive effects.
Oligodeoxynucleotides antisense to c-abl specifically inhibit entry into S-phase of CD34+ hematopoietic cells and their differentiation to granulocyte-macrophage progenitors
Authors: Rosti V , Bergamaschi G , Lucotti C , Danova M , Carlo-Stella C , Locatelli F , Tonon L , Mazzini G , Cazzola M
Published: October 2019

A number of experimental observations suggest that the proto-oncogene c- abl participates in the regulation of hematopoietic cell growth. We used an antisense strategy to study the relationship between c-abl expression and hematopoietic cell proliferation and differentiation. Purified normal human bone marrow-derived CD34+ cells were obtained by immunomagnetic selection and incubated with 18-base-unmodified antisense oligodeoxynucleotides complementary to the first six codons of the two alternative first exons of c-abl, la and lb. At the end of incubation, an aliquot of cells was assayed for clonogenic growth and the remainder was used for flow cytometric analyses. Cell kinetics were evaluated by means of both single parameter DNA and bivariate DNA/bromodeoxyuridine (BrdU) flow cytometry. Apoptosis was routinely studied by DNA flow cytometric analysis and, in some cases, also through DNA agarose gel electrophoresis for detection of oligonucleosomal DNA fragments. Expression of differentiation markers was studied by flow cytometry. Exposure to antisense oligonucleotides specifically inhibited the accumulation of c-abl mRNA in CD34+ cells. Preincubation with the c-abl antisense oligomers reduced the proportion of cells in S-phase from 19% +/- 5% (mean +/- SD) to 7% +/- 4% (P < .05), and BrdU labeling from 13% +/- 6% to 6% +/- 3% (P < .05). Flow cytometry and DNA agarose gel electrophoresis showed that treated CD34+ cells accumulated in the G0/G1 region of the DNA histogram with no evidence of either differentiation or apoptosis. By contrast, both growth factor deprivation and exposure of CD34+ cells to the tyrosine kinase inhibitor tyrphostin AG82 clearly induced apoptosis. When cells were preincubated with antisense oligonucleotides and then plated for evaluation of colony formation, this resulted in a significant inhibition of colony forming unit granulocyte-macrophage growth (from 44 +/- 15 to 22 +/- 9; P < .01) but had no effect on burst-forming unit erythroid growth (24 +/- 11 v 21 +/- 11; P < .05). These results suggest that c-abl expression is critical for entry of human CD34+ hematopoietic cells into S-phase and for their differentiation to granulocyte-macrophage progenitors. They also indicate that other tyrosine kinases besides p145c-alb are active in the prevention of apoptosis, so that inhibition of c-abl RNA accumulation arrests CD34+ cells in G0/G1 without activating programmed death.
Terminal differentiation of murine resident peritoneal macrophages is characterized by expression of the STK protein tyrosine kinase, a receptor for macrophage-stimulating protein
Authors: Iwama A , Wang MH , Yamaguchi N , Ohno N , Okano K , Sudo T , Takeya M , Gervais F , Morissette C , Leonard EJ
Published: October 2019

STK, a new member of the hepatocyte growth factor receptor family, is the receptor for macrophage-stimulating protein (MSP), which acts on murine resident peritoneal macrophages. We established polyclonal and monoclonal antibodies against STK and characterized the structure of STK protein and STK expression on cells of the mononuclear phagocyte system. Western blotting showed that the STK transcript is translated into a single-chain precursor and then cleaved into a 165-kD disulfide- linked heterodimer composed of a 35-kD alpha-chain and a 144-kD beta- chain. Western blotting detected STK protein on resident peritoneal macrophages, a target of MSP, and showed that it was autophosphorylated in cells stimulated by MSP. By flow cytometric analysis using a monoclonal anti-STK antibody, we showed that STK protein is expressed on restricted macrophage populations such as resident peritoneal macrophages, but not on exudate peritoneal macrophages or mononuclear phagocytes of the bone marrow, peripheral blood, spleen, or alveoli. Resident peritoneal macrophages were classified into two fractions according to their reactivity with an anti-STK antibody and a marker antibody for macrophages: STKhigh-F4/80high cells and STKnegative- F4/80low cells. Acute exudative macrophages were all STKnegative- F4/80low, but they gradually became predominantly STKhigh-F4/80high several days after entrance into the peritoneal cavity. These results showed that after monocytes migrate into the peritoneal cavity, they undergo terminal differentiation in the peritoneal microenvironment. This is the first evidence of tissue-specific terminal differentiation of peritoneal macrophages, and this terminal differentiation can be characterized by the expression of STK receptor tyrosine kinase.
The Ser 460 to Pro substitution of the protein S alpha (PROS1) gene is a frequent mutation associated with free protein S (type IIa) deficiency
Authors: Duchemin J , Gandrille S , Borgel D , Feurgard P , Alhenc-Gelas M , Matheron C , Dreyfus M , Dupuy E , Juhan-Vague I , Aiach M
Published: October 2019

A Ser 460 to Pro mutation of protein S (PS), involving a T to C transition in exon XIII of the protein S alpha (PROS1) gene and known as the Heerlen polymorphism, was found in 16 of 85 symptomatic patients with PS deficiency (18.8%) and only 1 of 113 healthy subjects (0.8%). Another frequent polymorphism was described in exon XV of the PROS1 gene, in the codon for Pro 626 (CCA/CCG). We found that Heerlen polymorphism was associated with allele CCA and not with allele CCG, suggesting a probable transmission by a common ancestor. Most subjects bearing the Ser 460 to Pro mutation were deficient in free PS, but had normal total PS levels. Normal levels of the C4b-binding protein (C4b- BP) isoform containing a beta chain (C4b-BP beta +) ruled out increased C4b-BP beta + as a cause of the free-PS deficiency. The binding curves of the mutated (Heerlen) PS on C4b-BP immobilized on microplates were biphasic, suggesting that one molecule of C4b-BP can bind two molecules of Heerlen PS. Because normal PS binds to C4b-BP with 1:1 stoichiometry, this may explain the free-PS deficiency observed in patients carrying the Ser 460 to Pro mutation.
Histamine distribution in human basophil secretory granules undergoing FMLP-stimulated secretion and recovery
Authors: Dvorak AM , MacGlashan DW Jr , Morgan ES , Lichtenstein LM
Published: October 2019

We examined subcellular histamine localizations in purified human basophils that were stimulated to degranulate with FMLP using an ultrastructural enzyme-affinity technique. Basophils were collected at early (0, 20 seconds, 1 minute) and late (10 minutes to 6 hours) time points poststimulation and were prepared for routine ultrastructural and diamine oxidase-gold (DAO-gold) cytochemical analysis. Histamine was present in unaltered cytoplasmic secretory granules (30.77 gold particles per square micrometer; P < .001 compared with background); specificity controls (histamine absorption, diamine oxidase digestion) abrogated granule labeling for histamine. Altered granules in stimulated cells were not significantly labeled for histamine, as compared with background (P = not significant); unaltered granules in the same cells contained more histamine than altered granules (P < .05). During recovery times, spanning 10 minutes to 6 hours, granules again appeared to be electron-dense and contained histamine (33.49/microns2; P = not significant as compared with unaltered granules in 1-minute FMLP-stimulated cells, and P < .05 as compared with altered granules in 1-minute FMLP-stimulated samples). Other structures devoid of histamine in actively secreting cells included extruded granules and intragranular and extruded Charcot-Leyden crystals. Recovering basophils displayed morphologic evidence of material and membrane conservation, granule content condensation, and biosynthesis. Subcellular histamine-rich sites in actively recovering basophils included condensing granules and collections of cytoplasmic vesicles in three locations: beneath the plasma membrane, adjacent to granules, and in the Golgi region. These studies show that unaltered granules of actively releasing human basophils, as well as similar granules that are reconstituted after FMLP-stimulated degranulation, contain histamine, but that altered granules in stimulated cells undergoing degranulation are devoid of histamine. Reconstitution of histamine-rich granules is associated with DAO-gold-positive cytoplasmic vesicles, suggesting transport of histamine derived from either new synthesis, re-uptake of released histamine, or both, to reconstituted granules.
In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells
Authors: Rosenzweig M , Marks DF , Zhu H , Hempel D , Mansfield KG , Sehgal PK , Kalams S , Scadden DT , Johnson RP
Published: October 2019

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.
Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor
Authors: MacVittie TJ , Farese AM , Herodin F , Grab LB , Baum CM , McKearn JP
Published: October 2019

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine- SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.
CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells
Authors: Kawashima I , Zanjani ED , Almaida-Porada G , Flake AW , Zeng H , Ogawa M
Published: October 2019

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.
Differences in serum cytokine levels in acute and chronic autoimmune thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity
Authors: Semple JW , Milev Y , Cosgrave D , Mody M , Hornstein A , Blanchette V , Freedman J
Published: October 2019

Patients with both acute and chronic autoimmune thrombocytopenic purpura (AITP) have in vitro lymphocyte defects in the form of platelet- stimulated proliferation and cytokine secretion. A blinded study was performed to determine if these defects are related to serum cytokine levels and/or platelet antigen expression. Compared with controls, 53% of children with chronic AITP, but only 9% of those with acute AITP, had increased serum interleukin-2 (IL-2), interferon-gamma, and/or IL- 10; however, none of the patients had detectible serum levels of IL-4 or IL-6, cytokine patterns suggesting and early CD4+ Th0 and Th1 cell activation. In children with chronic AITP, the levels of serum IL-2 correlated with in vitro platelet-stimulated IL-2 production. Few (17%) patients with AITP showed platelet activation, as measured by CD62 expression, or abnormal expression levels of platelet membrane glycoprotein (GP) IIbIIIa, but abnormal GPIb levels were observed in one-third of children with AITP. In contrast to normal controls and patients with nonimmune thrombocytopenia, a significant number of children with acute (80%), chronic (71%), or chronic-complex (55%) AITP and GPIb+ peripheral blood cells expressing HLA-DR. HLA-DR was variably coexpressed on distinct smaller and larger-sized GPIb+ cell populations with CD41, CD45, CD14, CD80, and/or glycophorin molecules. GPIb+ cells isolated from spleens of patients with chronic AITP had high expression (49% +/- 30%) of HLA-DR and splenic T cells had a high level of in vitro platelet-stimulated IL-2 secretion compared with controls. Platelet HLA-DR expression correlated inversely with platelet count, but not with therapy, serum cytokines, or in vitro lymphocyte antiplatelet reactivity. The results indicate that platelet HLA-DR expression is a common occurrence in patients with immune thrombocytopenia, whereas a large subpopulation of children with chronic AITP can be identified by increased serum cytokine levels and in vitro platelet-stimulated IL-2 secretion by lymphocytes, suggesting that differences exist in the immune pathogenesis of acute and chronic AITP, particularly at the level of platelet reactive T cells.
Altered expression of Pax-5 gene in human myeloma cells
Authors: Mahmoud MS , Huang N , Nobuyoshi M , Lisukov IA , Tanaka H , Kawano MM
Published: October 2019

Recent phenotypic analysis of plasma cells showed that normal plasma cells do express the B-cell lineage-specific molecule CD19, but their malignant counterpart (myeloma cells) are CD19-. To clarify the meaning of loss of CD19 antigen on myeloma cells, we first compared the expression of CD19 and Pax-5 genes among B cells, normal plasma cells, myeloma cell lines, and primary myeloma cells, because the Pax-5 gene was reported to encode the transcriptional factor, B-cell-specific activating protein (BSAP), necessary for CD19 gene expression. Neither CD19 nor Pax-5 mRNA could be detected in those primary myeloma cells and cell lines, whereas normal plasma cells did express both CD19 and Pax-5 mRNA. Furthermore, we could confirm that BSAP-binding activity was not detected in the nuclear extract from CD19- myeloma cell line (KMS-5) but was detected in CD19+ B-cell line (Raji) by gel-shift assay. We further examined the expression of E2A and Id genes, because E2A and Id are considered to be positive and negative regulators in the expression of Pax-5 gene, respectively. However, no significant differences in the expression of these E2A and Id-2 genes could be observed between myeloma cells and normal plasma cells. Therefore, these data suggest that the altered expression of Pax-5, but not E2A or Id, is responsible for the loss of CD19 expression in human myeloma cells, although the underlying mechanism of the altered Pax-5 gene expression remains to be clarified.
The p53 gene in pediatric therapy-related leukemia and myelodysplasia
Authors: Felix CA , Hosler MR , Provisor D , Salhany K , Sexsmith EA , Slater DJ , Cheung NK , Winick NJ , Strauss EA , Heyn R , Lange BJ , Malkin D
Published: October 2019

We investigated the frequency of p53 mutations in 19 pediatric cases of therapy-related leukemia or myelodysplastic syndrome. Eleven children presented with acute myeloid leukemia, one with mixed-lineage leukemia, two with acute lymphoblastic leukemia, and five with myelodysplasia at times ranging from 11 months to 9 years after a primary cancer diagnosis. The primary cancers, which included 11 solid tumors and eight leukemias, were treated with various combinations of DNA topoisomerase II inhibitors, alkylating agents, or irradiation. Leukemic or myelodysplastic marrows were screened for possible mutations by single-strand conformation polymorphism (SSCP) analysis of p53 exons 4 to 8. The only observed mutation was an inherited 2- basepair deletion at codon 209 in exon 6 that would shift the open reading frame, create a premature termination codon, and foreshorten the resultant protein. Prior therapy in this patient included DNA topoisomerase II inhibitors, alkylating agents, and irradiation. The secondary leukemia presented as myelodysplasia with monosomies of chromosomes 5 and 7 and abnormalities of chromosome 17. Although the primary cancer was an embryonal rhabdomyosarcoma and there was a family history of cancer, the case did not fulfill the clinical criteria for Li-Fraumeni syndrome. This study suggests that germline p53 mutations may predispose some children to therapy-related leukemia and myelodysplasia, but that p53 mutations otherwise are infrequent in this setting.
Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion
Authors: Francia di Celle P , Mariani S , Riera L , Stacchini A , Reato G , Foa R
Published: October 2019

Several cytokines have been suggested to play a regulatory action on the neoplastic clone of patients with B-cell chronic lymphocytic leukemia (B-CLL) by interfering in the differentiation, proliferation, or death/survival pathways. Interleukin-8 (IL-8) is a chemoattractant protein constitutively expressed at the mRNA level and released by B- CLL cells. In view of the presence of the IL-8 receptor mRNA and of specific IL-8 binding, confirmed also by Scatchard analysis using 125I- IL-8, the study was extended to evaluate the possible regulatory role of this cytokine on B-CLL cells. IL-8 failed to show any in vitro proliferative effect on leukemic B-CLL cells. By contrast, the propidium iodide (PI) staining of the DNA content showed that IL-8 could prolong the survival of resting B-CLL cells in 11 of 16 cases studied. In the remaining 5 cases, 90.6% +/- 4.39% SD of the cells after culture remained viable and IL-8 could exert a significant death protection action after pretreatment with 10(-4) mol/L hydrocortisone, which reduced the percentage of viable B-CLL cells. The dose range of IL-8 capable of inducing the prolonging survival effect is comparable with the levels of IL-8 released constitutively by B-CLL cells, indicating that the death protection action is exerted at physiologic doses. The in vitro rescue from death induced by IL-8 is reflected by an increased expression of bcl-2 mRNA in B-CLL cases incubated in the presence of IL-8. These findings were further confirmed at the protein level, because in B-CLL cells that displayed a bimodal bcl-2 intracytoplasmatic protein expression IL-8 was capable of upmodulating the bcl-2high expression peak. The potential autocrine regulatory action exerted by IL-8 is supported by the evidence that exogenous IL-8 can upregulate IL-8 mRNA in B-CLL cells. These results, together with the demonstration that antibody-mediated neutralization of endogenous IL-8 could induce a significant in vitro reduction in the number of living cells, further support the hypothesis that, in B-CLL, the physiologic doses of IL-8 released constitutively by the leukemic clone may play an autocrine role in the process of cell accumulation characteristic of this disease.
Association of lymphomatoid granulomatosis with Epstein-Barr viral infection of B lymphocytes and response to interferon-alpha 2b
Authors: Wilson WH , Kingma DW , Raffeld M , Wittes RE , Jaffe ES
Published: October 2019

Lymphomatoid granulomatosis (LyG) is an angiodestructive lymphoproliferative disorder (LPD) often involving the lungs. Its etiology is uncertain, but a number of previous studies had suggested it is a T-cell LPD associated with Epstein-Barr virus (EBV). Because of the similarity between LYG and nasal angiocentric lymphoma, the term angiocentric immunoproliferative lesion was proposed for both entities. Optimal therapy is unknown, but chemotherapy is often used. We studied four patients with LYG over a 5-year period. Biopsy samples were analyzed by immunohistochemistry, EBV in situ hybridization, and for Ig heavy-chain (IgH) gene rearrangements, Clinically, we assessed EBV serology, lymphocyte subsets, and the efficacy of interferon-alpha2b (IFN-alpha2b), All biopsy samples showed an exuberant T-cell infiltrate with scattered atypical large B cells. Double labeling showed EBV in the B cells but not T cells. Clonal IgH gene rearrangements were detected in 2 of 3 patients studied, 1 of whom had three distinct clones, and light-chain restriction showed two clones in an additional patient. All patients had positive EBV serologies. and markedly abnormal lymphocyte subsets. With IFN, 3 patients are alive and disease free at 36, 43, and 60 months; 1 patient achieved a partial response for 16 months but discontinued therapy and died with lymphoma. These results indicate that LYG is a T-cell-rich EBV-associated B-cell LPD in which the infiltrating T cells are numerous but reactive. IgH gene rearrangements may be polyclonal, monoclonal, or oligoclonal. Its association with immune defects suggests it is related to posttransplant LPD. However, LYG and nasal angiocentric lymphoma are distinct entities and should no longer be included together under the term angiocentric immunoproliferative lesion. IFN is effective therapy and should be studied further.
Thrombopoietin primes human platelet aggregation induced by shear stress and by multiple agonists
Authors: Oda A , Miyakawa Y , Druker BJ , Ozaki K , Yabusaki K , Shirasawa Y , Handa M , Kato T , Miyazaki H , Shimosaka A , Ikeda Y
Published: October 2019

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine- diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.