bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Immune Reconstitution After Unrelated and Related T-Cell–Depleted Bone Marrow Transplantation: Effect of Patient Age and Donor Leukocyte Infusions
Authors: Small T.N. , Papadopoulos E.B. , Boulad F. , Black P. , Castro-Malaspina H. , Childs B.H. , Collins N. , Gillio A. , George D. , Jakubowski A. , Heller G. , Fazzari M. , Kernan N. , MacKinnon S. , Szabolcs P. , Young J.W. , O’Reilly R.J.
Published: October 2019

Unrelated bone marrow transplantation (BMT) is often complicated by fatal opportunistic infections. To evaluate features unique to immune reconstitution after unrelated BMT, the lymphoid phenotype, in vitro function, and life-threatening opportunistic infections after unrelated and related T-cell–depleted (TCD) BMT were analyzed longitudinally and compared. The effects of posttransplant donor leukocyte infusions to treat or prevent cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infections on immune reconstitution were also analyzed. This study demonstrates that adult recipients of TCD unrelated BMTs experience prolonged and profound deficiencies of CD3+, CD4+, and CD8+ T-cell populations when compared with pediatric recipients of unrelated BMT and adults after related BMT (P < .01), that these adults have a significantly increased risk of life-threatening opportunistic infections, and that the rate of recovery of CD4 T cells correlates with the risk of developing these infections. Recovery of normal numbers of CD3+, CD8+, and CD4+ T-cell populations is similar in children after related or unrelated BMT. This study also demonstrates that adoptive immunotherapy with small numbers of unirradiated donor leukocytes can be associated with rapid restoration of CD3+, CD4+, and CD8+T-cell numbers, antigen-specific T-cell responses, and resolution of CMV- and EBV-associated disease after unrelated TCD BMT.
Selective Requirements for Leukocyte Adhesion Molecules in Models of Acute and Chronic Cutaneous Inflammation: Participation of E- and P- But Not L-Selectin
Authors: Catalina Michelle D. , Estess Pila , Siegelman Mark H.
Published: October 2019

Adhesion molecules borne by both endothelial cells and circulating leukocytes are in large measure responsible for guiding the process of extravasation. The selectin family has been primarily associated with the early stages of adhesion involving initial contact and rolling. A significant body of evidence has accumulated indicating a fundamental role for the endothelial members of this family, E- and P-selectin, in a variety of inflammatory states and models. Although originally identified as the lymph node-specific lymphocyte homing receptor, L-selectin has also been suggested to play an important role in leukocyte recruitment to sites of inflammation. We have recently demonstrated, using L-selectin–deficient mice, that defects in contact hypersensitivity (CHS) responses are in essence due to the inability of T cells to home to and be sensitized within peripheral lymph nodes, whereas nonspecific effector cells are fully capable of entry into sites of cutaneous inflammation (Catalina et al, J Exp Med184:2341, 1996). In the present study, we perform an analysis of adhesion molecule usage in two models of skin inflammation and show in both L-selectin–deficient as well as wild-type mice that a combination of P- and E-selectin is crucial for the development of both acute (croton oil) and chronic (contact hypersensitivity) inflammation at sites of the skin, whereas L-selectin does not appear to play a significant role. Moreover, 4 integrins are shown to be integral to a CHS but not an acute irritant response, whereas CD44 does not significantly contribute to either. These results provide a systematic examination in one study of major adhesion molecules that are critical in acute and chronic skin inflammation. They reinforce the essential role of the collaboration of E- and P-selectin in both specific and nonspecific skin inflammatory responses and the importance of 4 in the specific response only. In addition, they substantiate only a limited role, if any, for L-selectin in these cutaneous effector mechanisms and demonstrate the essential equivalence in this analysis of L-selectin–deficient mice compared with normal mice treated with blocking antibodies.
Detection of Normal and Chimeric Nucleophosmin in Human Cells
Authors: Cordell Jacqueline L. , Pulford Karen A.F. , Bigerna Barbara , Roncador Giovanna , Banham Alison , Colombo Emanuela , Pelicci Pier-Giuseppe , Mason David Y. , Falini Brunangelo
Published: October 2019

In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RAR and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.
Eradication of Pre-Established Lymphoma Using Herpes Simplex Virus Amplicon Vectors
Authors: Kutubuddin Mahmood , Federoff Howard J. , Challita-Eid Pia M. , Halterman Marc , Day Brian , Atkinson Meredith , Planelles Vicente , Rosenblatt Joseph D.
Published: October 2019

Herpes simplex virus amplicon vectors expressing RANTES (HSVrantes) and the T-cell costimulatory ligand B7.1 (HSVB7.1) were studied for their ability to elicit a tumor-specific T-cell response in a murine lymphoma model. HSVB7.1- and HSVrantes-transduced EL4 cells expressed high levels of B7.1 and RANTES as analyzed by flow cytometry and enzyme-linked immunosorbent assay, respectively. Inoculation of ex vivo HSVB7.1 transduced cells in syngeneic mice resulted in regression of both transduced cells and nontransduced cells inoculated contralaterally. Direct intratumoral injection of HSVB7.1 and/or HSVrantes alone or in combination into established EL4 tumors led to complete tumor regression in injected tumors as well as in nontransduced contralaterally implanted tumor, whereas control tumors or tumors injected with HSVlac expressing β-galactosidase did not regress. Maximal protection was achieved with combined injection of HSVB7.1 and HSVrantes; mice showing tumor regression were resistant to rechallenge with parental EL4 cells, and tumor cell-specific cytolytic T-cell activity was observed in mice demonstrating regression. HSV amplicon-mediated delivery of immune effector molecules may represent a useful strategy for immunotherapy in the setting of pre-existing tumor.
The Biologic Role of Interleukin-8: Functional Analysis and Expression of CXCR1 and CXCR2 on Human Eosinophils
Authors: Petering Holger , Götze Otto , Kimmig Daniela , Smolarski Regina , Kapp Alexander , Elsner Jörn
Published: October 2019

Chemokines play an important role in attracting granulocytes into sites of inflammation. Two chemokine subfamilies differ in their biologic activity for different granulocyte subsets. Whereas CXC chemokines such as interleukin-8 (IL-8) activate predominantly neutrophils, CC chemokines such as RANTES and eotaxin activate predominantly eosinophils. However, controversial results have been published in the past regarding the biologic role of IL-8 in eosinophil activation, particularly in allergic diseases. In this study, we investigated the functional evidence and expression of both IL-8 receptors, CXCR1 and CXCR2, on highly purified human eosinophils. In the first set of experiments, a chemotaxis assay was performed showing that IL-8 did not induce chemotaxis of eosinophils. In addition, and in contrast to neutrophils and lymphocytes, IL-8 did not induce a rapid and transient release of cytosolic free Ca2+([Ca2+]i) in eosinophils, even after preincubation with TH1- and TH2-like cytokines. To investigate whether neutrophil contamination might be responsible for the reported IL-8 effects on eosinophils, neutrophils were added to highly purified eosinophils from the same donor in different concentrations. Interestingly, as little as 5% of neutrophil contamination was sufficient to induce an increase of [Ca2+]iafter stimulation with IL-8. Flow cytometry experiments with monoclonal antibodies against both IL-8 receptors demonstrated no expression of CXCR1 and CXCR2 on eosinophils before or after cytokine activation. Reverse transcriptase-polymerase chain reaction experiments showed that eosinophils, in contrast to neutrophils and lymphocytes, did not express mRNA for CXCR1 and CXCR2. In summary, this study clearly demonstrates that CXCR1 and CXCR2 are not expressed on human eosinophils, even after priming with different bioactive cytokines. Because the CXC chemokine IL-8 did not induce in vitro effects on human eosinophils, IL-8 may also not contribute in vivo to the influx of eosinophil granulocytes into sites of allergic inflammation. Our results suggest that CC chemokines such as eotaxin, eotaxin-2, and MCP-4 are predominant for the activation of eosinophils.
Extreme Leukoreduction of Major Histocompatibility Complex Class II Positive B Cells Enhances Allogeneic Platelet Immunity
Authors: Semple John W. , Speck Edwin R. , Cosgrave Donna , Lazarus Alan H. , Blanchette Victor S. , Freedman John
Published: October 2019

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2d) SCID or BALB/c (H-2d) mouse platelets were transfused weekly into fully allogeneic CBA (H-2k) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 ± 0.6/μL and SCID mouse platelets could be prepared to have significantly lower (<0.05/μL) MC numbers. Transfusions with 108 BALB/c platelets (containing ≈100 MC/transfusion) stimulated IgG antidonor antibodies in 100% of the recipients by the fifth transfusion, whereas 108 SCID mouse platelets (containing ≈5 MC/transfusion) stimulated higher-titered IgG alloantibodies by the second transfusion. When titrations of BALB/c peripheral blood MC were added to the SCID mouse platelets, levels approaching 1 MC/μL reduced SCID platelet immunity to levels similar to BALB/c platelets. Characterization of the alloantibodies showed that the low levels of MC significantly influenced the isotype of the antidonor IgG; the presence of 1 MC/μL was associated with induction of noncomplement fixing IgG1 antidonor antibodies, whereas platelet transfusions, devoid of MC (<0.05/μL), were responsible for complement-fixing IgG2a production. When magnetically sorted defined subpopulations of MC were added to the SCID platelets, major histocompatability complex (MHC) class II positive populations, particularly B cells, were found to be primarily responsible for the reduced SCID mouse platelet immunity. The presence of low numbers of MC within the platelets was also associated with an age-dependent reduction in platelet immunogenicity; this relationship however, was not observed with SCID mouse platelets devoid of MC. The results suggest that a residual number of MHC class II positive B cells within allogeneic platelets are required for maximally reducing alloimmunization.
Marrow Ablative and Immunosuppressive Effects of131I-Anti-CD45 Antibody in Congenic and H2-Mismatched Murine Transplant Models
Authors: Matthews Dana C. , Martin Paul J. , Nourigat Cynthia , Appelbaum Frederick R. , Fisher Darrell R. , Bernstein Irwin D.
Published: October 2019

Targeted hematopoietic irradiation delivered by131I-anti-CD45 antibody has been combined with conventional marrow transplant preparative regimens in an effort to decrease relapse. Before increasing the proportion of therapy delivered by radiolabeled antibody, the myeloablative and immunosuppressive effects of such low dose rate irradiation must be quantitated. We have examined the ability of 131I-anti-CD45 antibody to facilitate engraftment in Ly5-congenic and H2-mismatched murine marrow transplant models. Recipient B6-Ly5a mice were treated with 30F11 antibody labeled with 0.1 to 1.5 mCi 131I and/or total body irradiation (TBI), followed by T-cell–depleted marrow from Ly5b-congenic (C57BL/6) or H2-mismatched (BALB/c) donors. Engraftment was achieved readily in the Ly5-congenic setting, with greater than 80% donor granulocytes and T cells after 0.5 mCi 131I (estimated 17 Gy to marrow) or 8 Gy TBI. A higher TBI dose (14 Gy) was required to achieve engraftment of H2-mismatched marrow, and engraftment occurred in only 3 of 11 mice receiving 1.5 mCi131I delivered by anti-CD45 antibody. Engraftment of H2-mismatched marrow was achieved in 22 of 23 animals receiving 0.75 mCi 131I delivered by anti-CD45 antibody combined with 8 Gy TBI. Thus, targeted radiation delivered via131I-anti-CD45 antibody can enable engraftment of congenic marrow and can partially replace TBI when transplanting T-cell–depleted H2-mismatched marrow.
P-Glycoprotein Protects Leukemia Cells Against Caspase-Dependent, but not Caspase-Independent, Cell Death
Authors: Johnstone Ricky W. , Cretney Erika , Smyth Mark J.
Published: October 2019

A major problem with treating patients with cancer by traditional chemotherapeutic regimes is that their tumors often develop a multidrug resistant (MDR) phenotype and subsequently become insensitive to a range of different chemotoxic drugs. One cause of MDR is overexpression of the drug-effluxing protein, P-glycoprotein.It is now apparent that P-glycoprotein may also possess a more generic antiapoptotic function that protects P-glycoprotein–expressing cancer cells and normal cells from cell death. Herein we show that cells induced to express P-glycoprotein either by drug selection or by retroviral gene transduction with MDR1 cDNA are resistant to cell death induced by a wide range of death stimuli, such as FasL, tumor necrosis factor (TNF), and ultraviolet (UV) irradiation, that activate the caspase apoptotic cascade.However, P-glycoprotein–expressing cells were not resistant to caspase-independent cell death mediated by pore-forming proteins and granzyme B.MDR P-glycoprotein–expressing cells were made sensitive to caspase-dependent apoptosis by the addition of anti–P-glycoprotein antibodies or verapamil, a pharmacological inhibitor of P-glycoprotein function. Clonogenic assays showed that P-glycoprotein confers long-term resistance to caspase-dependent apoptotic stimuli but not to caspase-independent cell death stimuli. This study has confirmed a potential novel physiological function for P-glycoprotein and it now remains to dissect the molecular mechanisms involved in the inhibition of capsase-dependent cell death by P-glycoprotein.
Biological Inactivation of 5-oxo-6,8,11,14-Eicosatetraenoic Acid by Human Platelets
Authors: Powell William S. , Gravel Sylvie , Khanapure Subhash P. , Rokach Joshua
Published: October 2019

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 μmol/L, but both blocked 5-oxo-ETE–induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 μmol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.
Dendritic Cells Derived In Vitro From Acute Myelogenous Leukemia Cells Stimulate Autologous, Antileukemic T-Cell Responses
Authors: Choudhury A. , Liang J.C. , Thomas E.K. , Flores-Romo L. , Xie Q.S. , Agusala K. , Sutaria S. , Sinha I. , Champlin R.E. , Claxton D.F.
Published: October 2019

We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor- or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.
Prolonged Expression of c-fos Suppresses Cell Cycle Entry of Dormant Hematopoietic Stem Cells
Authors: Okada Seiji , Fukuda Tetsuya , Inada Kunimasa , Tokuhisa Takeshi
Published: October 2019

The proto-oncogene c-fos was transiently upregulated in primitive hematopoietic stem (Lin−Sca-1+) cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-/β–inducible Mx-promoter (Mx–c-fos), and fetal liver cells from c-fos–deficient mice. Prolonged expression of the c-fos in Lin−Sca-1+ BM cells inhibited factor-dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin−Sca-1+ BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was downregulated. However, ectopic c-fos did not perturb colony formation by Lin−Sca-1+ BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells because the factor-dependent and the stroma-dependent hematopoiesis by Lin−Sca-1+ fetal liver cells from c-fos–deficient mice was not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state.
Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule
Authors: Evangelista Virgilio , Manarini Stefano , Sideri Rita , Rotondo Serenella , Martelli Nicola , Piccoli Antonio , Totani Licia , Piccardoni Paola , Vestweber Dietmar , de Gaetano Giovanni , Cerletti Chiara
Published: October 2019

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.
Genotype/Phenotype Correlations for Coagulation Factor XIII: Specific Normal Polymorphisms Are Associated With High or Low Factor XIII Specific Activity
Authors: Anwar Rashida , Gallivan Louise , Edmonds Stuart D. , Markham Alexander F.
Published: October 2019

Factor XIII is a transglutaminase essential for normal hemostasis. We have studied the plasma FXIII levels and FXIII activity in 71 individuals and found these to be normally distributed. FXIII specific activity is also normally distributed. However, we show that FXIII activity is not directly dependent on FXIII levels, and individuals with low FXIII levels may have high FXIII activity and vice versa. We have determined the FXIIIA genotype in these individuals to assess whether the variation observed in FXIII specific activity is dependent on specific polymorphisms in the FXIIIA gene. Our data show that the Leu34 and Leu564 variants give rise to increased FXIII specific activity, while the Phe204 variant results in lower FXIII specific activity. We also report preliminary evidence that the Phe204 polymorphism may be associated with recurrent miscarriage. Overall, we have identified 23 unique FXIIIA genotypes. Certain specific FXIIIA genotypes consistently give rise to high, low, or median FXIII specific activity levels, while others appear to have little or no consistent influence on the FXIII phenotype. These genotype to phenotype relationships are discussed in light of the growing interest in the role of FXIII in clinical problems involving an increased thrombotic tendency.
Association of a Common Polymorphism in the Factor XIII Gene With Venous Thrombosis
Authors: Catto Andrew J. , Kohler Hans P. , Coore Julie , Mansfield Michael W. , Stickland Max H. , Grant Peter J.
Published: October 2019

We have shown an association between a common mutation in the factor XIII a-subunit gene, coding for an amino acid change, 3 amino acids from the thrombin activation site (factor XIII Val34Leu) that may protect against myocardial infarction and predisposes to intracranial hemorrhage. To investigate the possible role of factor XIII Val34Leu in the pathogenesis of venous thromboembolism (VTE) and potential interactions with factor V Leiden (FV:Q506) and prothrombin G → A 20210, we studied 221 patients with a history of VTE and 254 healthy controls. Patients with VTE showed an increased frequency of the FXIII Val/Val genotype (63% v 49%) and a lower frequency of the Val/Leu genotype (31% v 42%) than controls (P = .007). FV:Q506 heterozygotes were more frequent in VTE patients (11%) than controls (5%; P = .04). The prothrombin G → A 20210 mutation was present in only 3 patients and no controls (P = .10). In a logistic regression model for a history of VTE, the odds ratio (95% confidence interval) for FXIII Val/Leu or Leu/Leu genotype was 0.63 (0.38 to 0.82) and for possession of FV:Q506 2.40 (1.17 to 4.90). There was no evidence for an interaction between factor XIII Val34Leu genotype and FV:Q506, prothrombin G → A 20210, sex, or age. It is concluded that possession of the Leu allele at factor XIII Val34Leu is protective against deep venous thrombosis.
Shaping the Repertoire of Cytotoxic T-Lymphocyte Responses: Explanation for the Immunodominance Effect Whereby Cytotoxic T Lymphocytes Specific for Immunodominant Antigens Prevent Recognition of Nondominant Antigens
Authors: Pion Stéphane , Christianson Gregory J. , Fontaine Pierre , Roopenian Derry C. , Perreault Claude
Published: October 2019

The immunodominance effect, whereby the presence of immunodominant epitopes prevents recognition of nondominant determinants presented on the same antigen-presenting cell (APC) considerably restricts the repertoire of cytotoxic T lymphocyte (CTL) responses. To elucidate the molecular basis of the immunodominance effect, we compared the interactions of a dominant (B6dom1) and a nondominant epitope (H-Y) with their restricting class I molecule (H2-Db), and their ability to trigger cognate CTLs. We found that B6dom1/Db complexes behaved as optimal T-cell receptor (TCR) ligands and triggered a more rapid in vivo expansion of cognate CTLs than H-Y/Db complexes. The superiority of the dominant epitope was explained by its high cell surface density (1,012 copies/cell for B6dom1v 10 copies/cell for H-Y) and its optimal affinity for cognate TCRs. Based on these results, we conclude that dominant class I–associated epitopes are those that have optimal ability to trigger TCR signals in CTLs. We propose that the rapid expansion of CTLs specific for dominant antigens should enable them to compete more successfully than other CTLs for occupancy of the APC surface.
Upregulated Expression of Fibronectin Receptors Underlines the Adhesive Capability of Thymocytes to Thymic Epithelial Cells During the Early Stages of Differentiation: Lessons From Sublethally Irradiated Mice
Authors: Dalmau Sergio R. , Freitas Claudia S. , Savino Wilson
Published: October 2019

A 250-cGy whole-body γ-radiation dose was used to induce thymus regression in mice, and to study the expression and function of extracellular matrix (ECM) receptors in distinct thymocyte subsets emerging during repopulation of the organ. The onset of regeneration was detected from day 2 to 3 postirradiation (P-Ir), when a remarkable increase in the absolute counts of CD3−CD25hiCD44+ and CD3−CD25in/hiCD44−cells occurred. Enhanced expression of L-selectin, 4, and 5 integrin chains (L-selhi 4hi5hi) was also exhibited by these cells. This pattern of expression was maintained until the CD4+CD8+ (DP) young stage was achieved. Afterward, there was a general downregulation of these ECM receptors in DP as well as in CD4+ or CD8+ single positive (SP) thymocytes (L-selin 4in5in). In some recently generated SP cells, 4 expression was downregulated before the 5 chain, and L-selectin was upregulated in half of more mature cells. The expression of the 6 integrin chain was downregulated only in maturing CD4+cells. Importantly, the increased expression of L-selectin and 4 and 5 chains in thymocytes was strongly correlated with their adhesiveness to thymic epithelial cells (TEC) in vitro. Blocking experiments with monoclonal antibody or peptides showed the following: (1) that the LDV rather than the REDV cell attachment motif in the IIIC segment of fibronectin is targeted by the 4 integrin during thymocyte/TEC adhesion; (2) that the RGD motif of the 120-kD fragment of fibronectin, a target for 5 integrin, has a secondary role in this adhesion; and (3) that the YIGSR cell attachment motif of the β1 chain of laminin/merosin recognized by a nonintegrin receptor is not used for thymocyte adherence. In conclusion, our results show that an upregulated set of receptors endows CD25+ precursors and cells up to the young DP stage with a high capability of interacting with thymic ECM components.
Cytogenetic Profile of Lymphoma of Follicle Mantle Lineage: Correlation With Clinicobiologic Features
Authors: Cuneo Antonio , Bigoni Renato , Rigolin Gian Matteo , Roberti Maria Grazia , Bardi Antonella , Piva Nadia , Milani Raffaella , Bullrich Florencia , Veronese Maria Luisa , Croce Carlo , Birg Françoise , Döhner Hartmut , Hagemeijer Anne , Castoldi Gianluigi
Published: October 2019

Conventional chromosome analysis (CCA) and interphase fluorescence in situ hybridization (FISH) was performed in 42 patients with mantle-cell lymphoma (MCL), with BCL1 rearrangement. The t(11;14)(q13;q32) or 11q abnormalities were detected by CCA in 34 cases, 20 of which had additional aberrations. A normal karyotype was observed in 8 cases. Probes detecting the chromosome aberrations that were observed in at least 3 cases by CCA, ie, +12, 13q14 deletion, and 17p deletion, were used for interphase FISH analysis. FISH detected total or partial +12, 13q14 deletion and 17p- in 28.5%, 52.4%, and 26% of the cases, respectively. The presence of these anomalies was not a function of karyotype complexity. Based on the results of CCA/FISH, three groups of increasing karyotype complexity were recognized: group 1, including 11 patients without detectable aberrations in addition to BCL1 rearrangement; group 2, including 14 patients with 1 to 2 additional anomalies; and group 3, including 17 patients with three or more additional anomalies. Clinical parameters associated with shorter survival were male sex (P= .006) and primary lymph-node involvement compared with primary bone marrow involvement (P = .015). Trisomy 12 was the only single cytogenetic parameter predictive of a poor prognosis (P = .006) and the best prognostic indicator was the derived measure of karyotype complexity (P < .0001), which maintained statistical significance in multivariate analysis (P< .0001). We arrived at the following conclusions: 13q14 deletion occurs at a high incidence in MCL; 17p deletion and total/partial +12 are relatively frequent events in MCL, the latter aberration being associated with a shorter survival; and the degree of karyotype complexity has a strong impact on prognosis in this neoplasia.
Activation of Nitric Oxide Release and Oxidative Metabolism by Leukotrienes B4, C4, and D4 in Human Polymorphonuclear Leukocytes
Authors: Lärfars Gerd , Lantoine Frédérique , Devynck Marie-Aude , Palmblad Jan , Gyllenhammar Hans
Published: October 2019

Because arachidonate metabolites are potent mediators of inflammation, we have studied the effects of leukotriene B4(LTB4) and the cysteinyl leukotrienes C4 and D4 (LTC4 and LTD4) on the release of nitric oxide (NO), in vitro, by human polymorphonuclear granulocytes (PMN). Two independent and highly sensitive real-time methods were used for these studies, ie, the NO-dependent oxidation of oxyhemoglobin (HbO2) to methemoglobin and a NO-sensitive microelectrode. When activated with LTB4, LTC4, or LTD4, but not with other lipoxygenase products such as 5S-HETE, 5-oxo-ETE or 5S,12S-diHETE, PMN produced NO in a stimulus- and concentration-dependent manner. The rank order of potency was LTB4 = LTC4 > LTD4, corresponding to 232 ± 50 pmol of NO/106 PMN for 100 nmol/L LTB4 after 30 minutes. The kinetic properties of the responses were similar for all three leukotrienes with a maximum response at 13 ± 3 minutes. Cysteinyl leukotriene and LTB4 antagonists inhibited the agonist-induced NO production by 70%, and treatment with Bordetella pertussis toxin, or chelation of cytosolic Ca2+, [Ca2+]i, also efficiently inhibited this response. In contrast, treatment of PMN with cytochalasin B (5 μg/mL) enhanced the LTB4-induced NO formation by 86%. Thus, this is the first demonstration that the cysteinyl leukotrienes LTC4 and LTD4, as well as LTB4, activate NO release from human PMN by surface receptor, G-protein and [Ca2+]i-dependent mechanisms. This effect differs from activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, for which only LTB4is an activator.
Constitutive Degradation of PML/RAR Through the Proteasome Pathway Mediates Retinoic Acid Resistance
Authors: Fanelli Mirco , Minucci Saverio , Gelmetti Vania , Nervi Clara , Gambacorti-Passerini Carlo , Pelicci Pier Giuseppe
Published: October 2019

PML/RAR is the leukemogenetic protein of acute promyelocytic leukemia (APL). Treatment with retinoic acid (RA) induces degradation of PML/RAR, differentiation of leukaemic blasts, and disease remission. However, RA resistance arises during RA treatment of APL patients. To investigate the phenomenon of RA resistance in APL, we generated RA-resistant sublines from APL-derived NB4 cells. The NB4.007/6 RA-resistant subline does not express the PML/RAR protein, although its mRNA is detectable at levels comparable to those of the parental cell line. In vitro degradation assays showed that the half-life of PML/RAR is less than 30 minutes in NB4.007/6 and longer than 3 hours in NB4. Treatment of NB4.007/6 cells with the proteasome inhibitors LLnL and lactacystin partially restored PML/RAR protein expression and resulted in a partial release of the RA-resistant phenotype. Similarly, forced expression of PML/RAR, but not RAR, into the NB4/007.6 cells restored sensitivity to RA treatment to levels comparable to those of the NB4 cells. These results indicate that constitutive degradation of PML/RAR protein may lead to RA resistance and that PML/RAR expression is crucial to convey RA sensitivity to APL cells.
Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12
Authors: Yao Lei , Sgadari Cecilia , Furuke Keizo , Bloom Eda T. , Teruya-Feldstein Julie , Tosato Giovanna
Published: October 2019

Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.
Randomized Phase II Study of Fludarabine + Cytosine Arabinoside + Idarubicin ± All-Trans Retinoic Acid ± Granulocyte Colony-Stimulating Factor in Poor Prognosis Newly Diagnosed Acute Myeloid Leukemia and Myelodysplastic Syndrome
Authors: Estey Elihu H. , Thall Peter F. , Pierce Sherry , Cortes Jorge , Beran Miloslav , Kantarjian Hagop , Keating Michael J. , Andreeff Michael , Freireich Emil
Published: October 2019

Preclinical data suggest that retinoids, eg, all-trans retinoic acid (ATRA), lower concentrations of antiapoptotic proteins such as bcl-2, possibly thereby improving the outcome of anti-acute myeloid leukemia (AML) chemotherapy. Granulocyte colony-stimulating factor (G-CSF) has been considered to be potentially synergistic with ATRA in this regard. Accordingly, we randomized 215 patients with newly diagnosed AML (153 patients) or high-risk myelodysplastic syndrome (MDS) (refractory anemia with excess blasts [RAEB] or RAEB-t, 62 patients) to receive fludarabine + ara-C + idarubicin (FAI) alone, FAI + ATRA, FAI + G-CSF, or FAI + ATRA + G-CSF. Eligibility required one of the following: age over 71 years, a history of abnormal blood counts before M.D. Anderson (MDA) presentation, secondary AML/MDS, failure to respond to one prior course of chemotherapy given outside MDA, or abnormal renal or hepatic function. For the two treatment arms containing ATRA, ATRA was given 2 days (day-2) before beginning and continued for 3 days after completion of FAI. For the two treatment arms including G-CSF, G-CSF began on day-1 and continued until neutrophil recovery. Patients with white blood cell (WBC) counts >50,000/μL began ATRA on day 1 and G-CSF on day 2. Events (death, failure to achieve complete remission [CR], or relapse from CR) have occurred in 77% of the 215 patients. Reflecting the poor prognosis of the patients entered, the CR rate was only 51%, median event-free survival (EFS) time once in CR was 36 weeks, and median survival time was 28 weeks. A Cox regression analysis indicated that, after accounting for patient prognostic variables, none of the three adjuvant treatment combinations (FAI + ATRA, FAI + G, FAI + ATRA + G) affected survival, EFS, or EFS once in CR compared with FAI. Similarly, there were no significant effects of either ATRA ignoring G-CSF, or of G-CSF ignoring ATRA. As previously found, a diagnosis of RAEB or RAEB-t rather than AML was insignificant. There were no indications that the effect of ATRA differed according to cytogenetic group, diagnosis (AML or MDS), or treatment schedule. Logistic regression analysis indicated that, after accounting for prognosis, addition of G-CSF ± ATRA to FAI improved CR rate versus either FAI or FAI + ATRA, but G-CSF had no effect on the other outcomes. We conclude that addition of ATRA ± G-CSF to FAI had no effect on CR rate, survival, EFS, or EFS in CR in poor prognosis, newly diagnosed AML or high-risk MDS.
Increased BAX Expression Is Associated With an Increased Risk of Relapse in Childhood Acute Lymphocytic Leukemia
Authors: Hogarth Linda A. , Hall Andrew G.
Published: October 2019

Studies in cell lines have indicated that expression of the BCL-2 family of proteins is an important determinant of chemotherapy-induced apoptosis; however, the level of expression of these proteins in childhood acute lymphoblastic leukemia (ALL) has not been extensively reported. Using quantitative Western blotting we have determined the level of expression of BCL-2, BAX, MCL-1, and BCL-X in lymphoblasts from 47 children with ALL (33 at presentation only, 4 at relapse only, and 10 at both presentation and on relapse). Results were determined as a ratio to actin as an internal control. BCL-2, BAX, and MCL-1 were detected in all samples. BCL-XL was only detected in 6 cases (4 at presentation and 2 at relapse) and BCL-XS in none. No correlation was found between expression and white blood cell count, age at diagnosis, gender, or blast karyotype. BCL-2 levels and the BCL/BAX and MCL-1/BAX ratios were found to be significantly higher in B-lineage as compared with T-lineage disease (P < .003, .02, and .02, respectively). No consistent pattern of change in expression was noted in the 10 cases studied at both presentation and relapse. Kaplan-Meier analysis showed a significant correlation between high BAX expression and an increased probability of relapse (P< .05 by the log rank test), suggesting that chemosensitivity in leukemic blasts may be regulated by factors that override the BCL-2 pathway.
Variant Genotypes of the Low-Affinity Fcγ Receptors in Two Control Populations and a Review of Low-Affinity Fcγ Receptor Polymorphisms in Control and Disease Populations
Authors: Lehrnbecher Thomas , Foster Charles B. , Zhu Shaoxian , Leitman Susan F. , Goldin Lynn R. , Huppi Konrad , Chanock Stephen J.
Published: October 2019

Fcγ-receptors (FcγR) provide a critical link between humoral and cellular immunity. The genes of the low-affinity receptors for IgG and their isoforms, namely, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and SH-FcγRIIIb, are located in close proximity on chromosome 1q22. Variant alleles may differ in biologic activity and a number of studies have reported the frequencies of variant FcγR alleles in both disease and control populations. No large study has evaluated the possibility of a nonrandom distribution of variant genotypes. We analyzed 395 normal individuals (172 African Americans [AA] and 223 Caucasians [CA]) at the following loci: FcγRIIa, FcγRIIIa, and FcγRIIIb, including the SH-FcγRIIIb. The genotypic distributions of FcγRIIa, FcγRIIIa, and FcγRIIIb conform to the Hardy-Weinberg law in each group. There was no strong evidence that combinations of 2-locus genotypes of the 3 loci deviated from random distributions in these healthy control populations. The distribution of SH-FcγRIIIb is underrepresented in CA compared with AA (P < .0001) controls. A previously reported variant FcγRIIb was not detected in 70 normal individuals, indicating that this allele, if it exists, is very rare (<1%). In conclusion, we present data that should serve as the foundation for the interpretation of association studies involving multiple variant alleles of the low-affinity FcγR.
The Risk of Residual Molecular and Cytogenetic Disease in Patients With Philadelphia-Chromosome Positive First Chronic Phase Chronic Myelogenous Leukemia Is Reduced After Transplantation of Allogeneic Peripheral Blood Stem Cells Compared With Bone Marrow
Authors: Elmaagacli Ahmet H. , Beelen Dietrich W. , Opalka Bertram , Seeber Siegfried , Schaefer Ulrich W.
Published: October 2019

The detection of residual molecular and cytogenetic disease was prospectively compared in patients with Philadelphia-chromosome (Ph1) positive first chronic phase chronic myelogenous leukemia (CML) who underwent allogeneic transplantation of unmanipulated peripheral blood stem cells (PBSCT) (n = 29) or bone marrow (BM) (n = 62) using genotypically HLA-identical sibling donors or partially HLA-matched extended family donors. A molecular relapse (MR), as defined by two consecutive positive polymerase chain reaction (PCR) assays for the detection of M-bcr-abl transcripts in a 4-week interval, was found in two of 29 (7%) patients after PBSCT compared with 20 of 62 (32%) patients after bone marrow transplantation (BMT). This corresponds to a 4-year molecular relapse estimate (± standard error) of 7% ± 5% after PBSCT and of 44% ± 8% after BMT (P < .009). With identical follow-up periods of survivors in both patient subsets between 6 and 55 months (median, 28 months), 14 of the 20 patients with MR after BMT progressed to an isolated cytogenetic (n = 10) or a hematologic (n = 4) disease recurrence, resulting in a 4-year cytogenetic relapse estimate of 47% ± 11%, while none of the patients after PBSCT has so far relapsed (P < .006). Multivariate analysis including all potential influencial factors of posttransplant disease recurrence identified the source of stem cells (P < .02) as the only independent predictor of molecular relapse. In conclusion, this prospective comparison of molecular and cytogenetic residual disease demonstrates that peripheral blood stem cell transplants have a more pronounced activity against residual CML cells than bone marrow transplants. Prospective randomized trials comparing PBSCT and BMT in patients with first chronic phase Ph1-positive CML are strictly required to further substantiate differences in the antileukemic activity of the two stem cell sources.
Dose-Intensive Melphalan With Stem Cell Support (MEL100) Is Superior to Standard Treatment in Elderly Myeloma Patients
Authors: Palumbo Antonio , Triolo Sabrina , Argentino Chiara , Bringhen Sara , Dominietto Alida , Rus Cecilia , Omedè Paola , Tarella Corrado , Pileri Alessandro , Boccadoro Mario
Published: October 2019

A clinical relationship between dose-intensity of melphalan and response rate has been demonstrated in multiple myeloma. Promising results have been reported after 200 mg/m2 melphalan, especially in younger patients. It is uncertain whether 100 mg/m2 melphalan (MEL100) can offer similar results in older patients. To address this issue, patients were treated with 2 or 3 MEL100 courses followed by stem cell support. Seventy-one patients (median age, 64 years) entered the protocol at diagnosis. Their clinical outcome was compared with that of 71 pair mates (median age, 64 years) selected from patients treated at diagnosis with oral melphalan and prednisone (MP) and matched for age and β2-microglobulin. Complete remission was 47% after MEL100 and 5% after MP. Median event-free survival was 34 months in the MEL100 group and 17.7 months in the MP group (P < .001). Median overall survival was 56+ months for MEL100 and 48 months for MP (P< .01). In a multivariate analysis, β2-microglobulin levels and MEL100 were independent risk factors associated with outcome: superior event-free and overall survival were observed in patients presenting low β2-microglobulin levels at diagnosis and receiving MEL100 as induction regimen. In conclusion, MEL100 was superior to MP in terms of complete remission rate, event-free survival, and overall survival.
Functional and Molecular Analysis of Hematopoietic Progenitors Derived From the Aorta-Gonad-Mesonephros Region of the Mouse Embryo
Authors: Delassus Sylvie , Titley Ian , Enver Tariq
Published: October 2019

Herein, we show that CD34, c-kit double-positive (CD34+c-kit+) cells from the aorta-gonad-mesonephros (AGM) region of the developing mouse are multipotent in vitro and can undergo both B-lymphoid and multimyeloid differentiation. Molecular analysis of individual CD34+c-kit+ cells by single-cell reverse transcriptase–polymerase chain reaction (RT-PCR) shows coactivation of erythroid (β-globin) and myeloid (myeloperoxidase [MPO]) but not lymphoid-affiliated (CD3, Thy-1, and λ5) genes. Additionally, most cells coexpress the stem cell–associated transcriptional regulators AML-1, PU.1, GATA-2 and Lmo2, as well as the granulocyte colony-stimulating factor receptor (G-CSF-R). These results show that the CD34+c-kit+ population from the AGM represents a highly enriched source of multipotent hematopoietic cells, and suggest that limited coactivation of distinct lineage-affiliated genes is an early event in the generation of hematopoietic stem and progenitor cells during ontogeny.