bluuebunny/crossref_metadata_2025 huggingface dataset:
The regulation of platelet-dense granules by Rab27a in the ashen mouse, a model of Hermansky-Pudlak and Griscelli syndromes, is granule-specific and dependent on genetic background
Authors: Novak Edward K. , Gautam Rashi , Reddington Madonna , Collinson Lucy M. , Copeland Neal G. , Jenkins Nancy A. , McGarry Michael P. , Swank Richard T.
Published: October 2002

The ashen (ash) mouse, a model for Hermansky-Pudlak syndrome (HPS) and for a subset of patients with Griscelli syndrome, presents with hypopigmentation, prolonged bleeding times, and platelet storage pool deficiency due to a mutation which abrogates expression of the Rab27a protein. Platelets of mice with the ashen mutation on the C3H/HeSnJ inbred strain background have greatly reduced amounts of dense granule components such as serotonin and adenine nucleotides though near-normal numbers of dense granules as enumerated by the dense granule-specific fluorescent dye mepacrine. Thus, essentially normal numbers of platelet dense granules are produced but the granule interiors are abnormal. Collagen-mediated aggregation of mutant platelets is significantly depressed. No abnormalities in the concentrations or secretory rates of 2 other major platelet granules, lysosomes and alpha granules, were apparent. Similarly, no platelet ultrastructural alterations other than those involving dense granules were detected. Therefore, Rab27a regulates the synthesis and secretion of only one major platelet organelle, the dense granule. There were likewise no mutant effects on levels or secretion of lysosomal enzymes of several other tissues. Together with other recent analyses of the ashen mouse, these results suggest a close relationship between platelet dense granules, melanosomes of melanocytes and secretory lysosomes of cytotoxic T lymphocytes, all mediated by Rab27a. Surprisingly, the effects of the ashen mutation on platelet-dense granule components, platelet aggregation, and bleeding times were highly dependent on genetic background. This suggests that bleeding tendencies may likewise vary among patients with Griscelli syndrome and HPS with Rab27a mutations.
Reconstitution of adhesive properties of human platelets in liposomes carrying both recombinant glycoproteins Ia/IIa and Ibα under flow conditions: specific synergy of receptor–ligand interactions
Authors: Nishiya Takako , Kainoh Mie , Murata Mitsuru , Handa Makoto , Ikeda Yasuo
Published: October 2002

Liposomes carrying both recombinant glycoprotein Ia/IIa (rGPIa/IIa) and Ibα (rGPIbα) (rGPIa/IIa-Ibα-liposomes) instantaneously and irreversibly adhered to the collagen surface in the presence of soluble von Willebrand factor (VWF) at high shear rates, in marked contrast with translocation of liposomes carrying rGPIbα alone on the VWF surface. In the absence of soluble VWF, the adhesion of rGPIa/IIa-Ibα-liposomes to the collagen surface decreased with increasing shear rates, similar to liposomes carrying rGPIa/IIa alone. While adhesion of liposomes with exofacial rGPIa/IIa and rGPIbα densities of 2.17 × 103 and 1.00 × 104molecules per particle, respectively, was efficient at high shear rates, reduction in rGPIbα density to 5.27 × 103molecules per particle resulted in decreased adhesion even in the presence of soluble VWF. A 50% reduction in the exofacial rGPIa/IIa density resulted in a marked decrease in the adhesive ability of the liposomes at all shear rates tested. The inhibitory effect of antibody against GPIbα (GUR83-35) on liposome adhesion was greater at higher shear rates. Further, the anti-GPIa antibody (Gi9) inhibited liposome adhesion more than GUR83-35 at all shear rates tested. These results suggest that the rGPIa/IIa–collagen interaction dominates the adhesion of rGPIa/IIa-Ibα-liposomes to the collagen surface at low shear rates, while the rGPIa/IIa–collagen and rGPIbα–VWF interaction complements each other, and they synergistically provide the needed functional integration required for liposome adhesion at high shear rates. This study thus has confirmed for the first time the proposed mechanisms of platelet adhesion to the collagen surface under flow conditions using the liposome system.
Paraneoplastic myasthenia gravis correlates with generation of mature naive CD4+ T cells in thymomas
Authors: Ströbel Philipp , Helmreich Markus , Menioudakis Georgios , Lewin Sharon R. , Rüdiger Thomas , Bauer Andrea , Hoffacker Viola , Gold Ralf , Nix Wilfred , Schalke Berthold , Elert Olaf , Semik Michael , Müller-Hermelink Hans Konrad , Marx Alexander
Published: October 2002

Myasthenia gravis (MG) is the leading paraneoplastic manifestation of thymomas and is probably related to the capacity of thymomas to mature and export potentially autoreactive T cells. Why some thymomas are MG associated (MG+) and others are not (MG−) has been unclear. We addressed this question by comparing the percentages of intratumorous naive mature CD45RA+ thymocytes in 9 MG(+) and in 13 MG(−) thymomas by fluorescence-activated cell sorting analysis. Our results show that intratumorous naive CD4 T cells were present in all MG(+) thymomas and in one MG(−) thymoma with the development of MG only 2 months after surgery. By contrast, the percentage of naive CD4+ T cells was significantly reduced in all 13 MG(−) thymomas (P < .0001). Alterations in intratumorous thymopoiesis were reflected by corresponding alterations of naive T-cell subset composition in the blood, in that only MG(−) patients had significantly decreased levels (P = .02) of naive CD4+ T cells compared with age- and sex-matched control persons. We conclude that paraneoplastic MG is highly associated with the efficiency of thymomas to produce and export naive CD4+T cells. The acquisition of the CD45RA+ phenotype on CD4+ T cells during terminal intratumorous thymopoiesis is associated with the presence of MG in most thymoma patients.
Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation
Authors: Angelucci Emanuele , Muretto Pietro , Nicolucci Antonio , Baronciani Donatella , Erer Buket , Gaziev Javid , Ripalti Marta , Sodani Pietro , Tomassoni Silvia , Visani Giuseppe , Lucarelli Guido
Published: October 2002

To identify the role of iron overload in the natural history of liver fibrosis, we reviewed serial hepatic biopsy specimens taken annually from patients cured of thalassemia major by bone marrow transplantation. The patients underwent transplantation between 1983 and 1989 and did not receive any chelation or antiviral therapy. Two hundred eleven patients (mean age, 8.7 ± 4 years) were evaluated for a median follow-up of 64 months (interquartile range, 43-98 months) by a median number of 5 (interquartile range, 3-6) biopsy samples per patient. Hepatic iron concentration was stratified by tertiles (lower, 0.5-5.6 mg/g; medium, 5.7-12.7 mg/g; upper, 12.8-40.6 mg/g dry weight). Forty-six (22%) patients showed signs of liver fibrosis progression; the median time to progression was 51 months (interquartile range, 36-83 months). In a multivariate Cox proportional hazard model, the risk for fibrosis progression correlated to medium hepatic iron content (hazard rate, 1.9; 95% confidence interval [CI], 0.74-5.0), high hepatic iron content (hazard rate, 8.7; 95% CI, 3.6-21.0) and hepatitis C virus (HCV) infection (hazard rate, 3.1; 95% CI, 1.5-6.5). A striking increase in the risk for progression was found in the presence of both risk factors. None of the HCV-negative patients with hepatic iron content lower than 16 mg/g dry weight showed fibrosis progression, whereas all the HCV-positive patients with hepatic iron concentration greater than 22 mg/g dry weight had fibrosis progression in a minimum follow-up of 4 years. Thus, iron overload and HCV infection are independent risk factors for liver fibrosis progression, and their concomitant presence results in a striking increase in risk.
HLA class II haplotype and quantitation of WT1 RNA in Japanese patients with paroxysmal nocturnal hemoglobinuria
Authors: Shichishima Tsutomu , Okamoto Masatoshi , Ikeda Kazuhiko , Kaneshige Toshihiko , Sugiyama Haruo , Terasawa Takashi , Osumi Kazuoki , Maruyama Yukio
Published: October 2002

It is unclear how a paroxysmal nocturnal hemoglobinuria (PNH) clone expands in bone marrow, although immune mechanisms involving cytotoxic T lymphocytes, autosomal proliferation, and apoptosis resistance have been hypothesized. To clarify aspects of immune mechanisms and proliferation of PNH cells, we investigated HLA-DRB1, -DQA1, and -DQB1 alleles by polymerase chain reaction (PCR)–based genotyping and expression of the Wilms' tumor gene, WT1, by real-time reverse transcriptase–PCR (RT-PCR) in 21 PNH and 21 aplastic anemia (AA) patients. HLA genotyping indicated that the frequency of DRB1*1501, DQA1*0102, and DQB1*0602 alleles in PNH patients and of DQB1*0602 allele in AA patients was significantly higher than in 916 Japanese controls, and that the HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype, found in 13 of 21 PNH patients, 5 of 7 AA-PNH syndrome patients, and 7 of 21 AA patients showed significant differences compared with healthy individuals. RT-PCR analysis showed that the mean values of WT1 RNA were 3413, 712, and 334 copies/μg RNA in PNH, AA, and healthy individuals, respectively. The values for PNH patients were significantly higher than for AA patients and healthy volunteers and were correlated with the proportion of CD16b−granulocytes. The high frequency of HLA-DRB1*1501-DQA1*0102-DQB1*0602 haplotype in PNH, including AA-PNH syndrome, and AA patients suggests that linkage exists between the disorders and that immune mechanisms in an HLA-restricted manner play an important role in the pathogenesis of these disorders. In addition, high expression of WT1 RNA in PNH patients is related to a PNH clone, but it remains unclear whether this causes expansion of a PNH clone.
Repair of DNA interstrand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma
Authors: Spanswick Victoria J. , Craddock Charles , Sekhar Mallika , Mahendra Prem , Shankaranarayana Paneesha , Hughes R. George , Hochhauser Daniel , Hartley John A.
Published: October 2002

Melphalan is widely used as a preparative agent in patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (SCT). Although disease relapse is the major cause of death after a melphalan-conditioned autograft, the mechanism remains unclear. Melphalan produces a number of DNA adducts with the DNA interstrand crosslink (ICL) considered to be the critical cytotoxic lesion. By using a modification of the single-cell gel electrophoresis (Comet) assay, we have measured formation and repair of DNA ICL in plasma cells from melphalan- naive and melphalan-treated patients (ie, those who have relapsed after a melphalan-conditioned autologous SCT or oral melphalan therapy). Similar levels of dose-dependent DNA interstand crosslinking were observed in cells from both melphalan-naive and -treated patients. However, marked differences in ICL repair were observed: cells from naive patients showed no repair, whereas those from treated patients exhibited between 42% and 100% repair at 40 hours. In vitro sensitivity to melphalan in plasma cells was found to correlate with ICL repair. These findings suggest that ICL repair may be an important mechanism by which melphalan resistance emerges after autologous SCT or oral therapy. This mechanism may have implications for MM patients undergoing melphalan therapy.
Intersubunit circular permutation of human hemoglobin
Authors: Sanders Kevin E. , Lo John , Sligar Stephen G.
Published: October 2002

For many years, human hemoglobin (Hb) isolated from erythrocytes has been investigated as a potential oxygen delivery therapeutic. Advantages with respect to the need for blood typing were balanced with various undesirable properties of cell-free Hb, including cost, overall oxygen affinity, alterations in cooperativity, and ready dissociation into toxic dimeric species. The use of total gene synthesis has resulted in very high levels of functional human Hb expression inEscherichia coli, but there remains a desire for effecting the crosslinking of the hemoglobin tetramer and providing for ready means for increasing the globular molecular weight. In this communication, we report a novel method for linking alpha chains. By circularly permuting one alpha sequence, the second alpha chain in the Hb tetramer can be linked with glycine residues to form 2 bridges across the central cavity. The second alpha chain thus presents its amino and carboxyl termini on a solvent exposed surface, providing for additional polymerization of oxygen-carrying subunits or attachment of any other peptide-based therapeutic.
Persistence of lymphoblasts in bone marrow on day 15 and days 22 to 25 of remission induction predicts a dismal treatment outcome in children with acute lymphoblastic leukemia
Authors: Sandlund John T. , Harrison Patricia L. , Rivera Gaston , Behm Frederick G. , Head David , Boyett James , Rubnitz Jeffrey E. , Gajjar Amar , Raimondi Susana , Ribeiro Raul , Hudson Melissa , Relling Mary , Evans William , Pui Ching-Hon
Published: October 2002

We determined the prognostic importance of morphologically identifiable persistent disease at day 15 and days 22 to 25 of remission induction in childhood acute lymphoblastic leukemia (ALL). Among 546 patients entered on 2 consecutive protocols, 397 patients had evaluable bone marrow (BM) examinations on day 15 (± 1 day) and 218 on days 22 to 25 (± 1 day). Fifty-seven patients (14%) had persistent lymphoblasts (≥ 1%) in the BM on day 15 and 27 patients (5.5%) had persistent lymphoblasts on days 22 to 25. The 5-year event-free survival (EFS) was significantly worse for patients with lymphoblasts on day 15 (40% ± 6%) or on days 22 to 25 (4% ± 3%) as compared to those without lymphoblasts on these dates (78% ± 2% and 76% ± 2%, respectively, P < .001 for both comparisons). A worse prognosis was observed even for patients with a low percentage of lymphoblasts (ie, 1%-4%) at either day 15 (5-year EFS = 56% ± 8%) or days 22 to 25 (5-year EFS = 0%) compared to those without morphologically identifiable persistent lymphoblasts at these times (P < .001 for both comparisons). The prognostic impact of persistent lymphoblasts on both dates remained significant after adjusting for other known risk factors, including treatment protocol, age, white blood cell count, DNA index, cell lineage, and central nervous system status, and National Cancer Institute/Rome criteria simultaneously. Hence, persistence of lymphoblasts (even 1%-4%) on day 15 of remission induction was associated with a poor prognosis and on days 22 to 25 signified a particularly dismal outcome; these very high-risk patients require novel or more intensive therapy to improve outcome.
Characterization of a new subpopulation of mouse CD8α+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential
Authors: Martı́n Pilar , del Hoyo Gloria Martı́nez , Anjuère Fabienne , Arias Cristina Fernández , Vargas Héctor Hernández , Fernández-L Africa , Parrillas Verónica , Ardavı́n Carlos
Published: October 2002

We describe a new B220+ subpopulation of immaturelike dendritic cells (B220+ DCs) with low levels of expression of major histocompatibility complex (MHC) and costimulatory molecules and markedly reduced T-cell stimulatory potential, located in the thymus, bone marrow, spleen, and lymph nodes. B220+ DCs display ultrastructural characteristics resembling those of human plasmacytoid cells and accordingly produce interferon-α after virus stimulation. B220+ DCs acquired a strong antigen-presenting cell capacity on incubation with CpG oligodeoxynucleotides, concomitant with a remarkable up-regulation of MHC and costimulatory molecules and the production of interleukin-12 (IL-12) and IL-10. Importantly, our data suggest that nonstimulated B220+ DCs represent a subset of physiological tolerogenic DCs endowed with the capacity to induce a nonanergic state of T-cell unresponsiveness, involving the differentiation of T regulatory cells capable of suppressing antigen-specific T-cell proliferation. In conclusion, our data support the hypothesis that B220+ DCs represent a lymphoid organ subset of immature DCs with a dual role in the immune system—exerting a tolerogenic function in steady state but differentiating on microbial stimulation into potent antigen-presenting cells with type 1 interferon production capacity.
The N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated endothelium via P-selectin in vivo
Authors: Burch Erin E. , Patil Vivek R. Shinde , Camphausen Raymond T. , Kiani Mohammad F. , Goetz Douglas J.
Published: October 2002

P-selectin glycoprotein ligand-1 (PSGL-1) is present on leukocytes and is the major ligand for endothelial expressed P-selectin. A variety of studies strongly suggests that the N-terminal region of PSGL-1 contains the binding site for P-selectin. We hypothesized that this relatively small N-terminal peptide of PSGL-1 is sufficient to support adhesion to P-selectin in vivo. To test this hypothesis, we coated 2 μm–diameter microspheres with a recombinant PSGL-1 construct, termed 19.ek.Fc. The 19.ek.Fc construct consists of the first 19 N-terminal amino acids of mature PSGL-1 linked to an enterokinase cleavage site that, in turn, is linked to human immunoglobulin G Fc. The 19.ek.Fc-coated microspheres were injected into the jugular vein of mice. Intravital microscopy of postcapillary venules within the cremaster muscle of mice revealed that a significantly greater number of 19.ek.Fc microspheres rolled compared with control microspheres. The number of rolling 19.ek.Fc microspheres was significantly diminished by pretreatment of the mice with a monoclonal antibody to P-selectin or by pretreatment of the 19.ek.Fc microspheres with a monoclonal antibody to PSGL-1. Combined, the results indicate that the N-terminal peptide of PSGL-1 can mediate adhesion to trauma-activated microvascular endothelium via P-selectin in vivo.
Induced disruption of the transforming growth factor beta type II receptor gene in mice causes a lethal inflammatory disorder that is transplantable
Authors: Levéen Per , Larsson Jonas , Ehinger Mats , Cilio Corrado M. , Sundler Martin , Sjöstrand Lottie Jansson , Holmdahl Rikard , Karlsson Stefan
Published: October 2002

Recent studies in mouse models deficient in transforming growth factor beta (TGF-β) signaling have documented TGF-β as one of the major regulators of immune function. TGF-β1–null animals demonstrated massive autoimmune inflammation affecting multiple organs, but attempts to transfer the phenotype to normal animals by bone marrow transplantation only resulted in minor inflammatory lesions. We wanted to ask whether a lethal inflammatory phenotype would develop following transplantation of bone marrow deficient for the TGF-β type II receptor (TβRII) gene to normal recipient animals. The TβRII-null mutation would generate a cell autonomous phenotype that cannot be reverted by the influence of endocrine or paracrine TGF-β derived from the recipient animal. We have generated conditional knockout mice in which the TβRII gene is disrupted upon induction with interferon-αβ or polyI:polyC. We show that induction of TβRII gene disruption in these mice by polyI:polyC results in a lethal inflammatory disease. Importantly, bone marrow from conditional knockout mice transferred to normal recipent mice caused a similar lethal inflammation, regardless of whether induction of TGF-β receptor deficiency occurred in donor animals before, or in recipient animals after transplantation. These results show that TGF-β signaling deficiency within cells of hematopoietic origin is sufficient to cause a lethal inflammatory disorder in mice. This animal model provides an important tool to further clarify the pathogenic mechanisms in animals deficient for TGF-β signaling and the importance of TGF-β to regulate immune functions.
MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+precursors
Authors: Masterson Allan J. , Sombroek Claudia C. , de Gruijl Tanja D. , Graus Yvo M. F. , van der Vliet Hans J. J. , Lougheed Sinéad M. , van den Eertwegh Alfons J. M. , Pinedo Herbert M. , Scheper Rik J.
Published: October 2002

Many human myeloid leukemia–derived cell lines possess the ability to acquire a dendritic cell (DC) phenotype. However, cytokine responsiveness is generally poor, requiring direct manipulation of intracellular signaling mechanisms for differentiation. In contrast, the CD34+ human acute myeloid leukemia cell line MUTZ-3 responds to granulocyte macrophage– colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNFα), cytokines known to be pivotal both in vivo and in vitro for DC generation from monocytes and CD34+ stem cells. In all respects, MUTZ-3 cells behave as the immortalized equivalent of CD34+ DC precursors. Upon stimulation with specific cytokine cocktails, they acquire a phenotype consistent with either interstitial- or Langerhans-like DCs and upon maturation (mDC), express CD83. MUTZ-3 DC display the full range of functional antigen processing and presentation pathways. These findings demonstrate the unique suitability of MUTZ-3 cells as an unlimited source of CD34+DC progenitors for the study of cytokine-induced DC differentiation.
Analysis of the hematopoietic potential of muscle-derived cells in mice
Authors: Geiger Hartmut , True Jarrod M. , Grimes Barry , Carroll Elizabeth J. , Fleischman Roger A. , Van Zant Gary
Published: October 2002

Cells in murine muscle have been reported to differentiate into hematopoietic stem and progenitor cells and thus repopulate the hematopoietic system of an irradiated animal. This activity was attributed to muscle stem cells. We used an in vitro and in vivo approach to identify the hematopoietic repopulating activity found in muscle tissue of mice by antibody staining and cell sorting. We confirmed existence of a hematopoietic repopulating cell in muscle tissue, but the data strongly suggest that repopulation is due not to muscle stem cells but to hematopoietic cells present in muscle tissue. Unexpectedly, the blood-forming cells were enriched in muscle relative to their frequency in peripheral blood.
Mannose-binding lectin gene polymorphisms are associated with major infection following allogeneic hemopoietic stem cell transplantation
Authors: Mullighan Charles G. , Heatley Sue , Doherty Kathleen , Szabo Ferenc , Grigg Andrew , Hughes Timothy P. , Schwarer Anthony P. , Szer Jeff , Tait Brian D. , Bik To L. , Bardy Peter G.
Published: October 2002

Life-threatening complications such as graft versus host disease and infection remain major barriers to the success of allogeneic hemopoietic stem cell transplantation (SCT). While pretransplantation conditioning and posttransplantation immunosuppression are important risk factors for infection, the reasons that similarly immunosuppressed transplant recipients show marked variation in frequency of infection after allogeneic SCT are unclear. Mannose-binding lectin (MBL) deficiency is a risk factor for infection in other situations where immunity is compromised. We investigated associations betweenMBL2 gene polymorphisms and risk of major infection following allogeneic SCT. Ninety-seven related allogeneic donor-recipient pairs were studied. Clinical data including survival, days of fever, graft versus host disease incidence and severity, and infection were collected by case note review. Five single-nucleotide polymorphisms in the MBL2 gene were genotyped using the polymerase chain reaction and sequence-specific primers.MBL2 coding mutations were associated with an increased risk of major infection following transplantation. This association was seen for donor (P = .002, odds ratio [OR] 4.1) and recipient (P = .04, OR 2.6) MBL2 genotype.MBL2 promoter variants were also associated with major infection. The high-producing haplotype HYA was associated with a markedly reduced risk of infection (recipient HYA P = .0001, OR 0.16; donor HYA P = .001, OR 0.23). Donor MBL2 coding mutations and recipientHYA haplotype were independently associated with infection in multivariate analysis. These results suggest that MBL2genotype influences the risk of infection following allogeneic SCT and that both donor and recipient MBL2 genotype are important. These findings raise the possibility that MBL replacement therapy may be useful following transplantation.
In vivo depletion of hematopoietic stem cells in the rat by an anti-CD45 (RT7) antibody
Authors: Dahlke Marc H. , Lauth Oliver S. , Jäger Mark D. , Roeseler Till , Timrott Kai , Jackobs Stefan , Neipp Michael , Wonigeit Kurt , Schlitt Hans J.
Published: October 2002

Anti-CD45 monoclonal antibodies (mAbs) are potentially powerful tools for the depletion of mature leukocytes. As their application for immunotherapy also depends on their effects on bone marrow (BM) progeny, the in vivo effects of an anti-CD45 mAb (anti-RT7a mAb) on BM precursor cells were analyzed in a rat model. Anti-RT7a mAb treatment was performed in LEW.1W (RT1u RT7a) rats with the use of different dosages. In addition, major histocompatibility complex (MHC)–congenic BM transplantation making use of a diallelic polymorphism (RT7a/RT7b) of rat CD45 was applied. Following injection of anti-RT7a mAb into normal LEW.1W rats, T cells were profoundly depleted in blood, lymph nodes, and spleen, whereas B cells were coated only by the antibody. Single injection of anti-RT7a mAb in a high dose induced a lethal aplastic syndrome with severe thrombocytopenia. Rescue of antibody-treated animals with BM from congenic LEW.1W-7B rats (RT1u RT7b) and transplantation of BM from LEW.1W rats pretreated with anti-RT7a mAb into sublethally irradiated LEW.1W-7B recipients revealed a profound effect of the mAb on progeny of myeloid and T-cell lineage. Following repeated antibody treatment of stable mixed chimeras (RT7b/RT7a), very few RT7a-positive B cells were still detectable after 6 months and their number declined during the subsequent year. These observations show that this anti-RT7a mAb effectively depletes mature T cells as well as BM precursor cells of myeloid, T-cell, and thrombocytic lineage after in vivo application. In contrast, mature B cells are not depleted, but precursors also appear to be eliminated. Overall, the findings suggest that the anti-RT7a mAb efficiently depletes early rat hematopoietic stem cells.
Factor XII interacts with the multiprotein assembly of urokinase plasminogen activator receptor, gC1qR, and cytokeratin 1 on endothelial cell membranes
Authors: Mahdi Fakhri , Madar Zia Shariat , Figueroa Carlos D. , Schmaier Alvin H.
Published: October 2002

Investigations were performed to define the factor XII (FXII) binding site(s) on cultured endothelial cells (HUVECs). Biotin- or fluorescein isothiocyanate (FITC)–FXII in the presence of 10 μM Zn2+ specifically binds to HUVEC monolayers or cells in suspension. Collagen-stimulated platelets release sufficient Zn2+ to support FXII binding. On laser scanning confocal microscopy or electron microscopy, FITC-FXII or Nanogold-labeled FXII, respectively, specifically bind to HUVECs. Antibodies to gC1qR, urokinase plasminogen activator receptor (uPAR) and, to a lesser extent, cytokeratin 1 (CK1) block FXII binding to HUVECs as determined by flow cytometry and soluble or solid phase binding assays. FITC-FXII on endothelial cells colocalizes with gC1qR, uPAR and, to a lesser extent, CK1 antigen. Combined recombinant soluble uPAR and CK1 inhibit 80% FITC-FXII binding to HUVECs. Peptide Y(39)HKCTHKGR(47) (YHK9) from the N-terminal region of FXII and peptide H(479)KHGHGHGKHKNKGKKNGKH(498) from HK's domain 5 cell-binding site block FITC-FXII binding to HUVECs. Peptide YHK9 also inhibits FXIIa's activation of prekallikrein and FXI on HUVECs. These combined investigations indicate that FXII through a region on its fibronectin type II domain binds to the same multiprotein receptor complex that comprises the HK binding site of HUVECs. However, plasma concentrations of HK and vitronectin inhibit FXII binding to HUVECs 100% and 50%, respectively, and plasma albumin and other proteins prevent a sufficient level of free Zn2+ to be available to support FXII binding to HUVECs. Thus, physiologic FXII expression on HUVECs is secondary to HK binding and highly restricted in its ability to initiate prekallikrein or FXI activation.
T-cell repopulation and thymic volume in HIV-1–infected adult patients after highly active antiretroviral therapy
Authors: Franco Jaime M. , Rubio Amalia , Martı́nez-Moya Manuel , Leal Manuel , Merchante Elena , Sánchez-Quijano Armando , Lissen Eduardo
Published: October 2002

The origin of T cells after highly active antiretroviral therapy (HAART) in patients infected with human immunodeficiency virus 1 (HIV-1) is now under discussion. The possibility of renewed lymphopoiesis in aged thymuses is still controversial. In this work we combine the analysis of naı̈ve T cells, T-cell receptor excision circles (TRECs), and computed tomography scanning of thymic tissue to further assess whether the thymus is involved in immune reconstitution. Fifteen antiretroviral-naı̈ve HIV-1–infected patients were evaluated during 48 weeks of HAART. At baseline, significant correlation was present among age and both thymic volume and TRECs, and between naı̈ve T cells and TRECs. After starting HAART, there was a significant increase at week 12 in naı̈ve CD4+and CD8+ T cells, TRECs, and thymic volume. The initial net increases in naı̈ve T cells and TREC counts were significantly correlated. Changes in thymic volume and TRECs were also indirectly related; splitting the population into 2 groups of high and low baseline TREC levels, only the group with low TREC levels had significant increases in both TRECs and thymic volume. Thus, the increase in thymic volume might be functional, in response to depleted TREC levels. Taken together, our data strongly suggest a thymic role in immune reconstitution, at least in patients with depleted baseline TREC levels.
Vaccination with CD20 peptides induces a biologically active, specific immune response in mice
Authors: Roberts Wendy K. , Livingston Philip O. , Agus David B. , Pinilla-Ibarz Javier , Zelenetz Andrew , Scheinberg David A.
Published: October 2002

CD20 is a 33-kD B-cell antigen that is expressed from the early pre–B-cell stage of development and is lost on differentiation of B cells into plasma cells. Because CD20 is expressed strictly by B cells, it is an attractive target for B-cell lymphoma therapy. Monoclonal antibodies to CD20 have been used successfully in the treatment of B-cell lymphomas. We hypothesized that a vaccine consisting of CD20 peptide sequences might be capable of inducing an active, specific, humoral immune response to the protein. Vaccine therapy would have the advantage of generating a polyclonal response to the antigen in contrast to the monoclonal response of an infused antibody. Balb/c mice were vaccinated with prototype vaccine constructs that consisted of peptides representing the human or mouse CD20 extracellular sequences conjugated to carrier proteins and mixed with QS21 adjuvant. Sera from the vaccinated mice demonstrated high-titer, specific antibodies to various epitopes on the immunizing peptides in enzyme-linked immunosorbent assay, weaker antibody binding to native CD20 on cells by flow cytometry, and antibody-mediated complement killing of CD20+ cells in some cases. Specific proliferation and secretion of interleukin 4 and interferon γ by mouse spleen cells in response to the immunizing peptides were also demonstrated. Mice vaccinated with the CD20 peptide keyhole limpet hemocyanin conjugates had a 25% decrease in CD19+ splenic B cells relative to control mice. These data indicate that a biologically active, specific immune response to CD20 can be elicited in mice vaccinated with CD20 peptide conjugates.
The AF10 leucine zipper is required for leukemic transformation of myeloid progenitors by MLL-AF10
Authors: DiMartino Jorge F. , Ayton Paul M. , Chen Everett H. , Naftzger Clarissa C. , Young Bryan D. , Cleary Michael L.
Published: October 2002

The t(10;11)(p12;q23) chromosomal translocation in human acute myeloid leukemia results in the fusion of theMLL and AF10 genes. The latter codes for a novel leucine zipper protein, one of many MLL fusion partners of unknown function. In this report, we demonstrate that retroviral-mediated transduction of an MLL-AF10complementary DNA into primary murine myeloid progenitors enhanced their clonogenic potential in serial replating assays and led to their efficient immortalization at a primitive stage of myeloid differentiation. Furthermore, MLL-AF10–transduced cells rapidly induced acute myeloid leukemia in syngeneic or severe combined immunodeficiency recipient mice. Structure/function analysis showed that a highly conserved 82–amino acid portion of AF10, comprising 2 adjacent α-helical domains, was sufficient for immortalizing activity when fused to MLL. Neither helical domain alone mediated immortalization, and deletion of the 29–amino acid leucine zipper within this region completely abrogated transforming activity. Similarly, the minimal oncogenic domain of AF10 exhibited transcriptional activation properties when fused to the MLL or GAL4 DNA-binding domains, while neither helical domain alone did. However, transcriptional activation per se was not sufficient because a second activation domain of AF10 was neither required nor competent for transformation. The requirement for α-helical transcriptional effector domains is similar to the oncogenic contributions of unrelated MLL partners ENL and ELL, suggesting a general mechanism of myeloid leukemogenesis by a subset of MLL fusion proteins, possibly through specific recruitment of the transcriptional machinery.
Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3
Authors: Ott German , Katzenberger Tiemo , Lohr Andreas , Kindelberger Steffi , Rüdiger Thomas , Wilhelm Martin , Kalla Jörg , Rosenwald Andreas , Müller Justus G. , Ott M. Michaela , Müller-Hermelink Hans Konrad
Published: October 2002

Follicular lymphoma (FL) grades 1 and 2 are regarded as a distinct disease entity, whereas data suggest that FL grade 3 might be an inhomogeneous tumor category. To define the biologic spectrum of FL, 89 follicular lymphomas were studied for their cytologic composition, antigen expression, mitotic and proliferation indices, cytogenetics, and clinical data. In contrast to the homogeneous appearance of FL grades 1 and 2 (29 and 33 cases, respectively), 2 types of FL grade 3 were recognized. Eleven cases of FL 3a displayed structural features similar to those of FL 1 and 2 and were composed of centroblasts and centrocytes, whereas 16 cases of FL 3b, with (n = 4) or without (n = 12) a diffuse large B-cell lymphoma component (DLBL) (FL 3b ± DLBL), consisted exclusively of blasts. In contrast to FL 3a, FL 3b ± DLBL were CD10+ in only 50% of cases and displayed plasmacytoid differentiation in 44% of cases.  Although FL3a was t(14;18)+ in 8 of 11 (73%) cases, only 2 of 16 (13%) FL3b ± DLBLs harbored this translocation. In contrast, chromosomal breaks at 3q27 were encountered in 7 of 16 (44%) FL 3b ± DLBL in contrast to only 2 of 11 (18%) FL 3a, and the spectrum of secondary aberrations in FL 3b ± DLBL was similar to that of diffuse large B-cell lymphoma. We conclude, therefore, that FL grade 3 is a heterogeneous disease group and that the distinction proposed in the new World Health Organization classification between FL 3a (with centrocytes) and FL3b (without centrocytes) is of biologic, and possibly clinical, importance.
Maintenance of elevated fetal hemoglobin levels by decitabine during dose interval treatment of sickle cell anemia
Authors: DeSimone Joseph , Koshy Mabel , Dorn Louise , Lavelle Donald , Bressler Linda , Molokie Robert , Talischy Nasrin
Published: October 2002

We have previously demonstrated that 5-aza-2′-deoxycytidine (decitabine) augments fetal hemoglobin (HbF) levels in patients with sickle cell anemia (SS) who did not respond to hydroxyurea (HU). The present study was designed to determine the effect of repeated decitabine dosing on HbF levels and hematologic toxicity over a 9-month treatment period. Seven patients (5 HU nonresponders) were entered. One patient had α-thalassemia sickle cell anemia. Decitabine was administered by intravenous infusion at a starting dose of 0.3 mg/kg per day, 5 days a week for 2 weeks, followed by a 4-week observation period. If the absolute neutrophil count dropped below 1000, the dose was reduced by 0.05 mg/kg per day in the next cycle. A drug dose was obtained for each patient, and it resulted in an elevated HbF without neutropenia (absolute neutrophil count nadir greater than 1500) or evidence of cumulative toxicity. Average HbF and average maximal HbF levels attained during the last 20 weeks of treatment for the 6 SS patients increased to 13.93% ± 2.75% and 18.35% ± 4.46%, respectively, from a pretreatment mean of 3.12% ± 2.75%. Mean and mean maximal hemoglobin (Hb) levels increased from 7.23 ± 2.35 g/dL to 8.81 ± 0.42 g/dL and 9.73 ± 0.53 g/dL, respectively. Individual maximal F-cell number observed during the trial was 69% ± 10.12%. The absence of cumulative toxicity may allow shorter intervals between drug treatments, which may lead to higher hemoglobin and HbF levels after several treatment cycles and, therefore, to greater clinical improvement.
Platelet-erythrocyte interactions enhance αIIbβ3 integrin receptor activation and P-selectin expression during platelet recruitment: down-regulation by aspirin ex vivo
Authors: Vallés Juana , Santos M. Teresa , Aznar Justo , Martı́nez Marcial , Moscardó Antonio , Piñón Marta , Broekman M. Johan , Marcus Aaron J.
Published: October 2002

Activated platelets release biologically active compounds, which then recruit additional platelets into an evolving thrombus. We studied activation of αIIbβ3 and exposure of P-selectin on platelets recruited by releasates obtained from collagen-treated platelets and evaluated modifications in prothrombotic effects of releasates induced by platelet-erythrocyte interactions and aspirin treatment. Releasates from collagen-stimulated platelets induced αIIbβ3 activation and P-selectin exposure (monitored by flow cytometry using fluorescein isothiocyanate–PAC-1 and phycoerythrin-CD62 antibodies). These responses were markedly amplified by releasates from combined platelet-erythrocyte suspensions. This finding demonstrates a novel mechanism(s) by which erythrocytes intensify platelet aggregability and mediate increased platelet recruitment. Because P-selectin and αIIbβ3 are potential sites for platelet-leukocyte interactions, erythrocytes may also modulate leukocyte recruitment. Following aspirin ingestion both the recruiting capacity of platelet releasates and erythrocyte-induced amplification of platelet recruitment were down-regulated. These events represent an additional antithrombotic property of aspirin. We also examined the possibility that arachidonic acid, or eicosanoids derived therefrom, can induce a prothrombotic activity of erythrocytes. The TXA2-analog U46 619 and free arachidonate, but not PGI2 or 12-HETE, induced increases in cytosolic Ca++ and promoted phosphatidylserine (PS) exposure on a subpopulation of erythrocytes. PS exposure and increases in erythrocyte [Ca++]i are associated with enhanced procoagulant activity, increased endothelial adhesion, and reduced erythrocyte deformability. Our findings, therefore, suggest that TXA2 and arachidonic acid, derived from activated platelets, induce a prothrombotic phenotype on erythrocytes in proximity. We conclude that by these mechanisms, erythrocytes can actively contribute to platelet-driven thrombogenesis and microvascular occlusion.
Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2
Authors: Feng Dian , Crane Katharine , Rozenvayn Nataliya , Dvorak Ann M. , Flaumenhaft Robert
Published: October 2002

Morphologic studies have demonstrated a process by which α-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for α-granule secretion. These observations raise the possibility that morphologic features of α-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins—human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca++-induced α-granule secretion by approximately 50%. Inhibition of α-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of α-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of α-granule secretion that include homotypic α-granule fusion and the fusion of α-granules with the open canalicular system and plasma membrane.
Biologic sequelae of nuclear factor–κB blockade in multiple myeloma: therapeutic applications
Authors: Mitsiades Nicholas , Mitsiades Constantine S. , Poulaki Vassiliki , Chauhan Dharminder , Richardson Paul G. , Hideshima Teru , Munshi Nikhil , Treon Steven P. , Anderson Kenneth C.
Published: October 2002

The transcription factor nuclear factor–κB (NF-κB) confers significant survival potential in a variety of tumors. Several established or novel anti–multiple myeloma (anti-MM) agents, such as dexamethasone, thalidomide, and proteasome inhibitors (PS-341), inhibit NF-κB activity as part of their diverse actions. However, studies to date have not delineated the effects of specific inhibition of NF-κB activity in MM. We therefore investigated the effect of SN50, a cell-permeable specific inhibitor of NF-κB nuclear translocation and activity, on MM cells. SN50 induced apoptosis in MM cell lines and patient cells; down-regulated expression of Bcl-2, A1, X-chromosome–linked inhibitor-of-apoptosis protein (XIAP), cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2, and survivin; up-regulated Bax; increased mitochondrial cytochromec release into the cytoplasm; and activated caspase-9 and caspase-3, but not caspase-8. We have previously demonstrated that tumor necrosis factor–α (TNF-α) is present locally in the bone marrow microenvironment and induces NF-κB–dependent up-regulation of adhesion molecules on both MM cells and bone marrow stromal cells, with resultant increased adhesion. In this study, TNF-α alone induced NF-κB nuclear translocation, cIAP-1 and cIAP-2 up-regulation, and MM cell proliferation; in contrast, SN50 pretreatment sensitized MM cells to TNF-α–induced apoptosis and cleavage of caspase-8 and caspase-3, similar to our previous finding of SN50-induced sensitization to apoptosis induced by the TNF-α family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L. Moreover, SN50 inhibited TNF-α–induced expression of another NF-κB target gene, intercellular adhesion molecule–1. Although the p38 inhibitor PD169316 did not directly kill MM cells, it potentiated the apoptotic effect of SN50, suggesting an interaction between the p38 and NF-κB pathways. Our results therefore demonstrate that NF-κB activity in MM cells promotes tumor-cell survival and protects against apoptotic stimuli. These studies provide the framework for targeting NF-κB activity in novel biologically based therapies for MM.
Inactivating mutations of CASP10 gene in non-Hodgkin lymphomas
Authors: Shin Min Sun , Kim Hong Sug , Kang Chang Suk , Park Won Sang , Kim Su Young , Lee Shi Nae , Lee Jong Heun , Park Jik Young , Jang Ja June , Kim Chul Woo , Kim Sang Ho , Lee Jung Young , Yoo Nam Jin , Lee Sug Hyung
Published: October 2002

Caspase 10 (Mch4/FLICE2) is a caspase homologous to caspase 8. A recent report described that inherited CASP10 gene mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome (ALPS). In this study, to explore the possibility that mutation of this gene might be involved in the development of non-Hodgkin lymphoma (NHL), we have analyzed the entire coding region and all splice sites of the CASP10gene for the detection of somatic mutations in 117 human NHLs. Overall, 17 NHLs (14.5%) were found to have CASP10mutations, which were identified in the coding regions of the prodomain (n = 3), the p17 large protease subunit (n = 11), and the p12 small protease subunit (n = 3). We expressed the tumor-derived caspase 10 mutants in 293 cells and found that apoptosis was suppressed. These data suggest that the inactivating mutations of theCASP10 gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human NHLs.
BCR-ABL–induced adhesion defects are tyrosine kinase–independent
Authors: Wertheim Jason A. , Forsythe Kevin , Druker Brian J. , Hammer Daniel , Boettiger David , Pear Warren S.
Published: October 2002

The t(9;22) chromosomal translocation results in expression of P210BCR-ABL, a fusion protein necessary for the development of chronic myelogenous leukemia (CML). The constitutive activation of the P210BCR-ABL tyrosine kinase results in phosphorylation of multiple signaling pathways leading to the transformed phenotype. Additionally, extracellular interactions between P210BCR-ABL–expressing progenitor cells and bone marrow stroma may provide external signals that facilitate CML development. In contrast to the intracellular signaling pathways involved in CML, little is known about how P210BCR-ABLexpression modifies cell-cell and cell-substratum interactions. To investigate the role of P210BCR-ABL in modulating cellular adhesion, we used a highly sensitive and quantitative cell detachment apparatus that measures the strength of association between a population of cells and an adhesive matrix. Our findings show that P210BCR-ABL expression increased adhesion nearly 2-fold between the myeloblastic cell line, 32D, and fibronectin compared to a control vector. We then investigated whether abnormal adhesion due to P210BCR-ABL expression was caused by its tyrosine kinase activity. A quantitative analysis of cell-fibronectin adhesion found that neither expression of a kinase-inactive P210BCR-ABL mutant in 32D cells or attenuation of kinase activity by STI571 (imatinib mesylate) in 32D cells transduced with wild-type P210BCR-ABL could correct the nearly 2-fold increase in cell-fibronectin adhesion. Similarly, STI571 treatment of Meg-01 cells, a P210BCR-ABL–expressing cell line derived from a patient in blast crisis, failed to inhibit adhesion to fibronectin. Together, our results indicate that changes in adhesion induced by P210BCR-ABL are independent of its tyrosine kinase activity.