bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of T-cell receptor Dβ1 promoter by KLF5 through reiterated GC-rich motifs
Authors: Yang Xuexian O. , Doty Raymond T. , Hicks Justin S. , Willerford Dennis M.
Published: May 2003

Rearrangement of T-cell receptor (TCR) and immunoglobulin genes by a common V(D)J recombination machinery is regulated by developmentally specific chromatin changes at the target locus, a process associated with transcription. At the TCRβ locus, the Eβ enhancer and the Dβ1 promoter regulate germline transcription originating near the TCR Dβ1 gene segment. The Dβ1 promoter contains 3 GC-rich motifs that bind a common set of nuclear proteins from pro–T-cell lines. Mutations that diminish the binding of nuclear proteins also diminish the activity of the Dβ1 promoter in transcriptional reporter assays. Using a yeast one-hybrid approach, 3 Krüppel-like factors—KLF3, KLF5, and KLF6—and a novel zinc finger protein were identified in a thymus library, all of which bound the GC-rich motif in a sequence-specific manner. Of these genes, KLF5 mRNA was expressed in a restricted manner in lymphoid cells and tissues, with highest expression in pro–T-cell lines and Rag-deficient thymocytes. Antibody supershift studies and chromatin immunoprecipitation assay confirmed that KLF5 bound the Dβ1 promoter. In reporter gene assays, KLF5 but not KLF6 efficiently transactivated the Dβ1 promoter, whereas a dominant-negative KLF5 construct inhibited reporter expression. These data suggest that reiterated GC motifs contribute to germline TCRβ transcription through binding of KLF5 and other Krüppel family members and that restricted expression of KLF5 may contribute to lineage-specific regulation of germline TCRβ transcription.
The low-frequency allele of the platelet collagen signaling receptor glycoprotein VI is associated with reduced functional responses and expression
Authors: Joutsi-Korhonen Lotta , Smethurst Peter A. , Rankin Angela , Gray Elaine , IJsseldijk Martin , Onley Catherine M. , Watkins Nicholas A. , Williamson Lorna M. , Goodall Alison H. , de Groot Philip G. , Farndale Richard W. , Ouwehand Willem H.
Published: May 2003

Interaction of platelets with collagen under conditions of blood flow is a multi-step process with tethering via glycoprotein IbIXV (GPIbIXV) over von Willebrand factor, adhesion by direct interaction with the integrin GPIaIIa, and signaling via GPVI. GPVI can be specifically agonized by cross-linked collagen-related peptide (CRP-XL), which results in a signaling cascade very similar to that evoked by native collagen. The GPVI gene has 2 common alleles that differ by 3 replacements in the glycosylated stem and 2 in the cytoplasmic domain. We used CRP-XL to elucidate the variation in responses observed in platelet function in different individuals. We observed a 3-fold difference in the response to CRP-XL in platelet aggregation when comparing platelets from 10 high-frequency allele homozygotes with 8 low-frequency ones (2-way analysis of variance [ANOVA], P < .0001). The difference in functional responses was reflected in fibrinogen binding and in downstream signaling events as measured by tyrosine phosphorylation, the expression of P-selectin, and the binding of annexin V and the generation of thrombin on the platelet surface (2-way ANOVA, P < .001). Platelets homozygous for the low-frequency allele tended to be less able to form a thrombus on a collagen surface in flowing whole blood or in the platelet function analyzer–100 (t test, P = .065 and P = .061, respectively). The functional difference was correlated to a difference in total and membrane-expressed GPVI measured by monoclonal and polyclonal antibodies. This study demonstrates for the first time that platelet function may be altered by allelic differences in GPVI.
KRN5500: a novel therapeutic agent with in vitro activity against human B-cell chronic lymphocytic leukemia cells mediates cytotoxicity via the intrinsic pathway of apoptosis
Authors: Byrd John C. , Lucas David M. , Mone Andrew P. , Kitner Joshua B. , Drabick Joseph J. , Grever Michael R.
Published: May 2003

Therapy of B-cell chronic lymphocytic leukemia (CLL) is currently palliative, emphasizing the need for identification of new therapies for this disease. KRN5500 is a novel agent that has a unique sensitivity pattern in the National Cancer Institute cell line screening panel, suggesting a unique mechanism of action. To assess its in vitro activity in CLL, we exposed peripheral mononuclear cells from CLL patients (n = 11) to varying concentrations of this agent. Viability of the CLL cells was reduced by 50% (LC50) at 4 hours, 24 hours, and 4 days at KRN5500 concentrations of 2.50 μM, 0.276 μM, and 0.139 μM, respectively. KRN5500 induced cellular injury via caspase-dependent apoptosis involving the intrinsic mitochondrial (caspase-9) initiating caspase and caspase-3 effector caspase; however, expression of the antiapoptotic mitochondrial membrane protein Bcl-2 was unaffected. These data demonstrate KRN5500 has significant in vitro activity against human CLL cells, thus providing support for introduction of this agent into clinical trials for patients with CLL.
Rituximab in lymphocyte-predominant Hodgkin disease: results of a phase 2 trial
Authors: Ekstrand Bradley C. , Lucas Jennifer B. , Horwitz Steven M. , Fan Zhen , Breslin Sheila , Hoppe Richard T. , Natkunam Yasodha , Bartlett Nancy L. , Horning Sandra J.
Published: May 2003

Lymphocyte-predominant Hodgkin disease (LPHD) is a unique clinical entity characterized by indolent nodal disease that tends to relapse after standard radiotherapy or chemotherapy. The malignant cells of LPHD are CD20+ and therefore rituximab may have activity with fewer late effects than standard therapy. In this phase 2 trial, 22 patients with CD20+ LPHD received 4 weekly doses of rituximab at 375 mg/m2. Ten patients had previously been treated for Hodgkin disease, while 12 patients had untreated disease. All 22 patients responded to rituximab (overall response rate, 100%) with complete response (CR) in 9 (41%), unconfirmed complete response in 1 (5%), and partial response in 12 (54%). Acute treatment-related adverse events were minimal. With a median follow-up of 13 months, 9 patients had relapsed, and estimated median freedom from progression was 10.2 months. Progressive disease was biopsied in 5 patients: 3 had recurrent LPHD, while 2 patients had transformation to large-cell non-Hodgkin lymphoma (LCL). All 3 patients with recurrent LPHD were retreated with rituximab, with a second CR seen in 1 patient and stable disease in 2. Rituximab induced prompt tumor reduction in each of 22 LPHD patients with minimal acute toxicity; however, based on the relatively short response duration seen in our trial and the concerns about transformation, rituximab should be considered investigational treatment for LPHD. Further clinical trials are warranted to determine the optimal dosing schedule of rituximab, the potential for combination treatment, and the possible relationship of rituximab treatment to the development of LCL.
RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation
Authors: Elagib Kamaleldin E. , Racke Frederick K. , Mogass Michael , Khetawat Rina , Delehanty Lorrie L. , Goldfarb Adam N.
Published: May 2003

Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor β (CBFβ), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFβ up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFβ, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1–mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins αIIb and α2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).
Central venous line–related thrombosis in children: association with central venous line location and insertion technique
Authors: Male Christoph , Chait Peter , Andrew Maureen , Hanna Kim , Julian Jim , Mitchell Lesley ,   the PARKAA Investigators
Published: May 2003

Venous thromboembolic events (VTEs) in children are associated with central venous lines (CVLs). The study objective was to assess whether CVL location and insertion technique are associated with the incidence of VTE in children. We hypothesized that VTE would be more frequent with (1) CVL location on the left body side, (2) CVL location in the subclavian vein rather than the jugular vein, and (3) CVL insertion by percutaneous technique rather than venous cut-down. This was a prospective, multicenter cohort study in children with acute lymphoblastic leukemia who had a CVL placed in the upper venous system during induction chemotherapy. Characteristics of CVL were documented prospectively. All children had outcome assessment for VTE by objective radiographic tests, including bilateral venography, ultrasound, echocardiography, and cranial magnetic resonance imaging. Among 85 children, 29 (34%) had VTE; 28 VTEs appeared in the upper venous system, and 1 was sinovenous thrombosis. Left-sided CVL (odds ratio [OR], 2.5; 95% confidence interval, 1.0-6.4; P = .048), subclavian CVL (OR, 3.1; 95% CI, 1.2-8.5; P = .025), and percutaneous CVL insertion (OR, 3.5; 95% CI, 1.3-9.2; P = .011) were associated with an increased incidence of VTE. Interaction occurred between CVL vein location and insertion technique. Subclavian vein CVL inserted percutaneously had an increased incidence (54%) of VTE compared with any other combination (P = .07). For CVL in the upper venous system, CVL placement on the right side and in the jugular vein may reduce the risk for CVL-related VTE. If subclavian vein placement is necessary, CVL insertion by venous cut-down appears preferable over percutaneous insertion.
Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool
Authors: Jensen Peter D. , Jensen Finn T. , Christensen Thorkil , Eiskjær Hans , Baandrup Ulrik , Nielsen Johan L.
Published: May 2003

Evaluation of myocardial iron during iron chelation therapy is not feasible by repeated endomyocardial biopsies owing to the heterogeneity of iron distribution and the risk of complications. Recently, we described a noninvasive method based on magnetic resonance imaging. Here, the method was used for repeated estimation of the myocardial iron content during iron chelation with deferrioxamine in 14 adult nonthalassemic patients with transfusional iron overload. We investigated the repeatability of the method and the relationship between the myocardial iron estimates and iron status. The repeatability coefficient (2sD) was 2.8 μmol/g in the controls (day-to-day) and 4.0 μmol/g in the patients (within-day). Myocardial iron estimates were elevated in 10 of all 14 patients at first examination, but normalized in 6 patients after 6 to 18 months of treatment. If liver iron declined below 350 μmol/g all but one of the myocardial iron estimates were normal or nearly normal. At start (R2 = 0.69, P = .0014) and still after 6 months of iron chelation (R2 = 0.76, P = .001), the estimates were significantly and more closely related to the urinary iron excretion than to liver iron or serum ferritin levels. In conclusion, our preliminary data, which may only pertain to patients with acquired anemias, suggest the existence of a critical liver iron concentration, above which elevated myocardial iron is present, but its extent seems related to the size of the chelatable iron pool, as reflected by the urinary iron excretion. This further supports the concept of the labile iron pool as the compartment directly involved in transfusional iron toxicity.
Phase 2 study of alemtuzumab (anti-CD52 monoclonal antibody) in patients with advanced mycosis fungoides/Sézary syndrome
Authors: Lundin Jeanette , Hagberg Hans , Repp Roland , Cavallin-Ståhl Eva , Fredén Susanne , Juliusson Gunnar , Rosenblad Eija , Tjønnfjord Geir , Wiklund Tom , Österborg Anders
Published: May 2003

This phase 2 study evaluated the safety and efficacy of alemtuzumab in 22 patients with advanced mycosis fungoides/Sézary syndrome (MF/SS). Most patients had stage III or IV disease, reduced performance status, and severe itching. The overall response (OR) rate was 55%, with 32% of patients in complete remission (CR) and 23% in partial remission (PR). Sézary cells were cleared from the blood in 6 of 7 (86%) patients, and CR in lymph nodes was observed in 6 of 11 (55%) patients. The effect was better on erythroderma (OR, 69%) than on plaque or skin tumors (OR, 40%) and in patients who had received 1 to 2 previous regimens (OR, 80%) than in those who had received 3 or more prior regimens (OR, 33%). Itching, self-assessed on a 0 to 10 visual analog scale, was reduced from a median of 8 before treatment to 2 at end of therapy. Median time to treatment failure was 12 months (range, 5-32+ months). Cytomegalovirus (CMV) reactivation (causing fever without pneumonitis and responding to ganciclovir) occurred in 4 (18%) patients. Six additional patients had suspect or manifest infection (fever of unknown origin, 3; generalized herpes simplex, 1; fatal aspergillosis, 1). One patient had fatal Mycobacterium pneumonia at 10+ months. All serious infectious adverse events (except CMV) occurred in patients who had received 3 or more prior regimens. Progression of squamous cell skin carcinoma was noted in 1 patient. Alemtuzumab shows promising clinical activity and an acceptable safety profile in patients with advanced MF/SS, particularly in patients with erythroderma and severe itching and those who were not heavily pretreated.
Impaired recovery of Epstein-Barr virus (EBV)—specific CD8+ T lymphocytes after partially T-depleted allogeneic stem cell transplantation may identify patients at very high risk for progressive EBV reactivation and lymphoproliferative disease
Authors: Meij Pauline , van Esser Joost W. J. , Niesters Hubert G. M. , van Baarle Debbie , Miedema Frank , Blake Neil , Rickinson Alan B. , Leiner Ingrid , Pamer Eric , Löwenberg Bob , Cornelissen Jan J. , Gratama Jan W.
Published: May 2003

Epstein-Barr virus (EBV)—specific cytotoxic T lymphocytes are considered pivotal to prevent lymphoproliferative disease (LPD) in allogeneic stem cell transplantation (SCT) recipients. We evaluated the recovery of EBV-specific CD8+ T cells after partially T-cell—depleted SCT and studied the interaction between EBV-specific CD8+ T cells, EBV reactivation, and EBV-LPD. EBV-specific CD8+ T cells were enumerated using 12 class I HLA tetramers presenting peptides derived from 7 EBV proteins. Blood samples were taken at regular intervals after SCT in 61 patients, and EBV DNA levels were assessed by real-time polymerase chain reaction. Forty-five patients showed EBV reactivation, including 25 with high-level reactivation (ie, more than 1000 genome equivalents [geq] per milliliter). Nine of these 25 patients progressed to EBV-LPD. CD8+ T cells specific for latent or lytic EBV epitopes repopulated the peripheral blood at largely similar rates. In most patients, EBV-specific CD8+ T-cell counts had returned to normal levels within 6 months after SCT. Concurrently, the incidence of EBV reactivations clearly decreased. Patients with insufficient EBV-specific CD8+ T-cell recovery were at high risk for EBV reactivation in the first 6 months after SCT. Failure to detect EBV-specific CD8+ T cells in patients with high-level reactivation was associated with the subsequent development of EBV-LPD (P = .048). Consequently, the earlier defined positive predictive value of approximately 40%, based on high-level EBV reactivation only, increased to 100% in patients without detectable EBV-specific CD8+ T cells. Thus, impaired recovery of EBV-specific CD8+ T cells in patients with high-level EBV reactivation may identify a subgroup at very high risk for EBV-LPD and supports that EBV-specific CD8+ T cells protect SCT recipients from progressive EBV reactivation and EBV-LPD.
Down-regulation of BRCA1 in BCR-ABL–expressing hematopoietic cells
Authors: Deutsch Eric , Jarrousse Sylvie , Buet Dorothée , Dugray Aymeric , Bonnet Marie-Laure , Vozenin-Brotons Marie-Catherine , Guilhot François , Turhan Ali G. , Feunteun Jean , Bourhis Jean
Published: May 2003

BCR-ABL fusion oncogene is the molecular hallmark of chronic myelogenous leukemia (CML), a condition characterized by a progression from a chronic to acute phase leukemia because of secondary genetic events, the nature of which remains largely unknown. Here, we report that the expression of the p210 BCR-ABL fusion protein leads to a down-regulation of BRCA1 protein, a gene product involved in the maintenance of genome integrity. BRCA1 protein is nearly undetectable in leukemia cells from patients with CML, both during the chronic phase and in blast crisis. Similarly, stable transfection-enforced expression of p210 protein in established hematopoietic cell lines leads to severe BRCA1 depletion. The lack of significant change in BRCA1 mRNA level in cells expressing p210 supports the hypothesis that the regulation of BRCA1 protein level occurs after transcription. It is abolished on exposure of the cells to STI571 and by mutation in the adenosine triphosphate (ATP) pocket of p210 and thus seems to require the tyrosine kinase activity of BCR-ABL. Cell lines expressing high levels of BCR-ABL display an increased rate of sister chromatid exchange and chromosome aberrations after ionizing radiation. These findings reveal a novel link between the oncoprotein BCR-ABL and the tumor-suppressor protein BRCA1.
Clinical and biologic implications of recurrent genomic aberrations in myeloma
Authors: Fonseca Rafael , Blood Emily , Rue Montserrat , Harrington David , Oken Martin M. , Kyle Robert A. , Dewald Gordon W. , Van Ness Brian , Van Wier Scott A. , Henderson Kimberly J. , Bailey Richard J. , Greipp Philip R.
Published: May 2003

Nonrandom recurrent chromosomal abnormalities are ubiquitous in multiple myeloma (MM) and include, among others, translocations of the immunoglobulin heavy chain locus (IgH). IgH translocations in MM result in the up-regulation of oncogenes, and include more commonly t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23). Based on the recurrent nature of these translocations and their finding since the early stages of the plasma cell (PC) disorders, we hypothesized that they would confer biologic and clinical variability. In addition, deletions of 13q14 and 17p13 have also been associated with a shortened survival. We used cytoplasmic Ig—enhanced interphase fluorescent in situ hybridization to detect deletions (13q14 and 17p13.1), and translocations involving IgH in 351 patients treated with conventional chemotherapy entered into the Eastern Cooperative Oncology Group clinical trial E9486/9487. Translocations were frequently unbalanced with loss of one of the derivative chromosomes. The presence of t(4; 14)(p16;q32) (n = 42; 26 vs 45 months, P < .001), t(14;16)(q32;q23) (n = 15; 16 vs 41 months, P = .003), – 17p13 (n = 37; 23 vs 44 months, P = .005), and – 13q14 (n = 176; 35 vs 51 months, P = .028) were associated with shorter survival. A stratification of patients into 3 distinct categories allowed for prognostication: poor prognosis group (t(4;14)(p16;q32), t(14; 16)(q32;q23), and – 17p13), intermediate prognosis (– 13q14), and good prognosis group (all others), with median survivals of 24.7, 42.3, and 50.5 months, respectively (P < .001). This molecular cytogenetic classification identifies patients into poor, intermediate, and good risk categories. More importantly it provides further compelling evidence that MM is composed of subgroups of patients categorized according to their underlying genomic aberrations.
Tumor and CD4 T-cell interactions: tumor escape as result of reciprocal inactivation
Authors: Flynn Sarah , Stockinger Brigitta
Published: May 2003

This paper addresses the capacity of naive, effector, and memory CD4 T cells to control growth of a major histocompatibility complex (MHC) class II—positive B-cell lymphoma in vivo. To assess the role of T cells on their own without contributions by B cells, antibodies, or natural killer (NK) cells, we generated pure effector or memory CD4 T cells in Rag–/–gc–/– mice deficient in endogenous lymphocytes and NK cells. Lymphoma cells expressing a model antigen were injected into mice with T cells of cognate specificity that were either naive or in effector or resting memory state. Naive T cells were unable to prevent tumor growth, probably due to delay of efficient cross-presentation by dendritic cells. However, both effector and memory T cells, dependent on the amount of antigen available, controlled the tumor for a considerable period of time without the need for dendritic cell stimulation. Nevertheless, the tumor eventually grew uncontrolled in all cases. This was not because of a defect in T-cell homing to the tumor site or loss of MHC class II or costimulatory molecules by the tumor, but reflected mutual paralysis of T-cell responsiveness and antigen processing by tumor cells.
Expression of CD4 on human peripheral blood neutrophils
Authors: Biswas Priscilla , Mantelli Barbara , Sica Antonio , Malnati Mauro , Panzeri Carla , Saccani Alessandra , Hasson Hamid , Vecchi Andrea , Saniabadi Abby , Lusso Paolo , Lazzarin Adriano , Beretta Alberto
Published: May 2003

CD4, the primary receptor for entry of HIV, is known to be expressed on T cells and monocytes/macrophages; healthy natural killer (NK) lymphocytes; in vitro human herpesvirus 6 (HHV6)–infected CD8+, NK, and γδ T lymphocytes; CD34+ progenitor cells; and a subset of eosinophils and basophils. We here report the unconventional expression of CD4 at the surface of peripheral blood neutrophils derived from 4 of 51 (7.8%) HIV-1–infected and 3 of 25 (12%) uninfected donors, with similar frequency within the 2 groups. The percentage of CD4+ neutrophils ranged from 39% to 97% of the total neutrophil population. Both surface and cytoplasmic forms of CD4 were present in neutrophils. Quantitative RNA polymerase chain reaction (PCR) revealed that neutrophils contain levels of CD4 mRNA comparable to those of peripheral blood mononuclear cells derived from the same donor. The conformation of CD4 expressed at the surface of neutrophils was similar to that of CD4 expressed on T lymphocytes as determined by the binding of monoclonal antibodies specific for conformational epitopes and the binding of recombinant HIV-1 gp120. Thus, our data provide evidence that neutrophils express endogenous CD4 and bind HIV. Owing to their abundance in peripheral blood, CD4+ neutrophils may influence significantly the biodistribution of HIV delivering it to sites of inflammation or to additional tissue reservoirs.
Glycophorin C is the receptor for the Plasmodium falciparum erythrocyte binding ligand PfEBP-2 (baebl)
Authors: Lobo Cheryl-Ann , Rodriguez Marilis , Reid Marion , Lustigman Sara
Published: May 2003

We report in this paper that glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. PfEBP-2 is a member of the Duffy binding–like erythrocyte binding protein (DBL-EBP) family. Although several reports have been published characterizing PfEBP-2, the identity of its erythrocytic receptor was still unknown. Using a combination of enzymatically treated red blood cells (RBCs) and rare, variant RBCs lacking different surface proteins, we have shown that PfEBP-2 does not bind to cells lacking GPC. Additionally, we found that PfEBP-2 binds differentially to variants of GPC lacking exon 2 or exon 3, and determined that the binding domain on GPC is potentially restricted to amino acid residues 14 through 22 within exon 2. Thus PfEBP-2 is involved in a sialic acid–dependent pathway of invasion, which does not involve glycophorin A or glycophorin B and represents a novel route of entry into the RBCs.
Tumor-derived, chaperone-rich cell lysate activates dendritic cells and elicits potent antitumor immunity
Authors: Zeng Yi , Feng Hanping , Graner Michael W. , Katsanis Emmanuel
Published: May 2003

We have utilized a free-solution isoelectric focusing technique (FS-IEF) to obtain chaperone-rich cell lysate (CRCL) fractions from clarified tumor homogenates and have previously reported on their vaccinating potential. To better understand the underlying mechanisms as well as to improve on the immunizing efficacy of tumor-derived chaperone complexes, in the present study we examined the effects of CRCL-loaded dendritic cells (DCs) against 12B1, an aggressive bcr-abl+ murine leukemia tumor. We found that DCs incubated with 12B1-derived CRCL had higher expression of CD40 and major histocompatibility complex class II (MHC-II) on their cell surface, produced more interleukin-12 (IL-12), and had superior immunostimulatory capacity in a mixed leukocyte reaction (MLR) when compared with DCs exposed to unfractionated tumor lysate or purified heat-shock protein 70 (HSP70). Vaccination of mice with 12B1 CRCL–pulsed DCs significantly prolonged their survival, with more than 80% of mice rejecting their tumors following a lethal challenge with live 12B1 compared with those immunized with tumor lysate or HSP70-loaded DCs. The protective immunity generated was tumor specific, long lasting, and both CD4+ and CD8+ T-cell dependent. Moreover, immunization with CRCL-loaded DCs resulted in a 75% cure rate in mice with pre-existing 12B1 tumors. Our findings indicate that CRCL has prominent adjuvant effects and is a very effective source of tumor antigen for pulsing DCs. FS-IEF–derived CRCL-pulsed DCs are a promising anticancer vaccine that warrants clinical research and development.
Macrophage- and dendritic cell—dependent regulation of human B-cell proliferation requires the TNF family ligand BAFF
Authors: Craxton Andrew , Magaletti Dario , Ryan Elizabeth J. , Clark Edward A.
Published: May 2003

Macrophages and dendritic cells play an important role in regulating B-cell responses, including proliferation to antigens such as trinitrophenyl (TNP)—Ficoll and TNP-Brucella abortus. However, the mechanisms and molecule(s) that regulate these processes are relatively undefined. In this report, we show that human macrophages generated in vitro strongly costimulate proliferation of dense human tonsillar B cells ligated via their B-cell antigen receptor (BCR) but not proliferation via CD40. Similarly, dendritic cells also markedly enhance BCR-activated B-cell proliferation. Soluble molecule(s) are required for human macrophages to costimulate proliferation of B cells triggered via their BCR. Importantly, a TACI (trans-membrane activator and CAML interactor)—Fc fusion protein inhibits both macrophage- and dendritic cell (DC)—dependent BCR-activated B-cell proliferation, indicating a requirement for at least one of the known TACI ligands, BAFF and/or APRIL. Consistent with a major role for BAFF, macrophages release BAFF at levels sufficient to potently costimulate BCR-induced B-cell proliferation. In addition, BAFF is more than 100-fold more potent than APRIL in enhancing BCR-mediated human B-cell proliferation. Furthermore, immunodepletion of APRIL under conditions that prevent APRIL-mediated B-cell costimulation does not block macrophage enhancement of B-cell proliferation. Finally, there is no correlation between the high levels of a proliferation-inducing ligand (APRIL) expressed by macrophages compared with DCs and the similar abilities of macrophages and DCs to enhance BCR-stimulated B-cell proliferation. In summary, our results suggest that macrophage- and DC-derived B-cell—activating factor belonging to the TNF family (BAFF) represents a key molecule by which macrophages and DCs directly regulate human B-cell proliferative responses to T-cell—independent stimuli.
Modulation of the folate receptor type β gene by coordinate actions of retinoic acid receptors at activator Sp1/ets and repressor AP-1 sites
Authors: Hao Hong , Qi Huiling , Ratnam Manohar
Published: May 2003

Folate receptor (FR) type β is a promising target for therapeutic intervention in acute myelogenous leukemia (AML) owing particularly to its specific up-regulation in AML cells by all-trans retinoic acid (ATRA). Here we identify functional elements in the FR-β gene and examine the molecular mechanism of transcriptional induction of FR-β by ATRA. The basal promoter activity of FR-β resulted from synergistic interaction between Sp1 and ets binding sites (EBSs) and repression by upstream AP-1–like elements, whose action required EBSs. A minimal promoter containing the Sp1 and ets elements was ATRA-responsive. The repressor elements bound Fos family proteins; association of the proteins with the repressor elements correlated negatively with FR-β expression in peripheral blood neutrophils and monocytes and also in KG-1 (AML) cells grown in the absence or in the presence of ATRA. Furthermore, down-regulation of FR-β in KG-1 cells treated with O-tetradecanoylphorbol 13-acetate (TPA) was accompanied by increased AP-1 binding to the repressor elements. From chromatin immunoprecipitation (ChIP) assays, the nuclear retinoic acid receptor α (RARα) associated with the Sp1 region, and RARs β and γ associated with the AP-1 and Sp1 regions; treatment of KG-1 cells with ATRA did not alter Sp1 binding but increased the association of RARα and decreased the association of RARs β and γ. ATRA also decreased RAR expression levels. The results suggest that the FR-β gene is a target for multiple coordinate actions of nuclear receptors for ATRA directly and indirectly acting on a transcriptional complex containing activating Sp1/ets and inhibitory AP-1 proteins. The multiple mechanisms favor the prediction that ATRA will induce FR-β expression in a broad spectrum of AML cells. Further, optimal FR-β induction may be expected when all 3 RAR subtypes bind agonist.
Low levels of tissue factor pathway inhibitor (TFPI) increase the risk of venous thrombosis
Authors: Dahm Anders , van Hylckama Vlieg Astrid , Bendz Bjorn , Rosendaal Frits , Bertina Rogier M. , Sandset Per Morten
Published: May 2003

There is now strong experimental evidence that tissue factor pathway inhibitor (TFPI) is a critical inhibitor to modulate tissue factor–induced coagulation, but the role of TFPI as a risk factor for thrombosis is yet to be to be determined. This study investigated the role of low TFPI levels for the development of deep-vein thrombosis (DVT). We determined TFPI activity and TFPI-free and total antigen levels in the subjects enrolled in the Leiden Thrombophilia Study, which is a large population-based case-control study of 474 patients and 474 controls. The odds ratio (OR) for DVT in subjects who had TFPI-free antigen levels below the 10th percentile, as compared with those who had TFPI-free antigen levels above this cutoff, was 1.7 (95% confidence interval [CI], 1.1-2.6). The ORs for low TFPI activity and low total antigen were also mildly increased. When the 5th percentile was used as a cutoff, the ORs were 2.1 (95% CI, 1.1-4.1) for both TFPI-free antigen and TFPI total antigen. Exogenous female hormones had a profound lowering effect on TFPI levels, with lower levels in oral contraceptive users than in premenopausal nonusers, who had lower levels than men and postmenopausal women. These results indicate that low levels of TFPI, especially low TFPI-free and total antigen in plasma, constitute a risk factor for DVT.
Dysfunction and infection of freshly isolated blood myeloid and plasmacytoid dendritic cells in patients infected with HIV-1
Authors: Donaghy Heather , Gazzard Brian , Gotch Frances , Patterson Steven
Published: May 2003

Recently it has been shown that the 2 populations of blood dendritic cells (DCs), termed plasmacytoid (pcDCs) and myeloid (myDCs), are reduced in HIV-1 infection. This study aimed to determine whether these 2 populations are targets for HIV-1 infection and whether their ability to stimulate T-lymphocyte proliferation is affected. Highly purified populations of myDCs and pcDCs were isolated from the blood of antiretroviral treatment–naive patients and assessed for the level of HIV provirus by polymerase chain reaction (PCR). We show that both populations are targets for HIV-1 infection as indicated by the presence of provirus in 12 of 14 pcDC and 13 of 14 myDC samples tested. A proportion of this provirus is integrated in myDCs. The ability of both myDCs and pcDCs from HIV-1–infected patients to stimulate allogeneic T-lymphocyte proliferation in a 6-day mixed leukocyte reaction was severely impaired, but was not mediated by secondary infection of T lymphocytes. Thus, in addition to depletion, both myeloid and plasmacytoid DCs are infected and show impaired functional capacity. These findings suggest that infection, depletion, and dysfunction of dendritic cells may contribute to the immunosuppression associated with HIV-1 disease.
A predominant role for cAMP-dependent protein kinase in the cGMP-induced phosphorylation of vasodilator-stimulated phosphoprotein and platelet inhibition in humans
Authors: Li Zhenyu , Ajdic Jasna , Eigenthaler Martin , Du Xiaoping
Published: May 2003

The vasodilator-stimulated phosphoprotein (VASP) plays an important role in cGMP-induced platelet inhibition. Since VASP is an in vitro substrate for cGMP-dependent protein kinase (PKG), it has been presumed that VASP phosphorylation induced by cGMP is mediated by PKG. Here we show that, in human platelets, phosphorylation of VASP at Ser239 induced by either cGMP analogs or nitric oxide (NO) donor glyco-SNAP1 is inhibited by PKA inhibitors KT5720, PKI, Rp-Br-cAMPS, and H89, but not by PKG inhibitors KT5823 or Rp-pCPT-cGMPS. Unlike human platelets, cGMP analog–induced phosphorylation of VASP in mouse platelets is inhibited by both PKG and PKA inhibitors. Ineffectiveness of PKG inhibitors in inhibiting VASP phosphorylation in human platelets is not due to an inability to inhibit PKG, as these PKG inhibitors but not PKA inhibitors inhibit a different cGMP-induced intracellular signaling event: phosphorylation of extracellular signal–responsive kinase. Furthermore, PKA inhibitors reverse cGMP-induced inhibition of thrombin-induced platelet aggregation, whereas PKG inhibitors further enhance the inhibitory effect of cGMP analogs. Thus, PKA plays a predominant role in the cGMP-induced phosphorylation of VASP and platelet inhibition in human platelets.
Myosin: a noncovalent stabilizer of fibrin in the process of clot dissolution
Authors: Kolev Krasimir , Tenekedjiev Kiril , Ajtai Katalin , Kovalszky Ilona , Gombás Judit , Váradi Balázs , Machovich Raymund
Published: May 2003

Myosin modulates the fibrinolytic process as a cofactor of the tissue plasminogen activator and as a substrate of plasmin. We report now that myosin is present in arterial thrombi and it forms reversible noncovalent complexes with fibrinogen and fibrin with equilibrium dissociation constants in the micromolar range (1.70 and 0.94 μM, respectively). Competition studies using a peptide inhibitor of fibrin polymerization (glycl-prolyl-arginyl-proline [GPRP]) indicate that myosin interacts with domains common in fibrinogen and fibrin and this interaction is independent of the GPRP-binding polymerization site in the fibrinogen molecule. An association rate constant of 1.81 × 102 M–1 · s–1 and a dissociation rate constant of 3.07 × 10–4 s–1 are determined for the fibrinogen-myosin interaction. Surface plasmon resonance studies indicate that fibrin serves as a matrix core for myosin aggregation. The fibrin clots equilibrated with myosin are stabilized against dissolution initiated by plasminogen and tissue-type plasminogen activator (tPA) or urokinase (at fibrin monomer-myosin molar ratio as high as 30) and by plasmin under static and flow conditions (at fibrin monomer-myosin molar ratio lower than 15). Myosin exerts similar effects on the tPA-induced dissolution of blood plasma clots. Covalent modification involving factor XIIIa does not contribute to this stabilizing effect; myosin is not covalently attached to the clot by the time of complete cross-linking of fibrin. Thus, our in vitro data suggest that myosin detected in arterial thrombi binds to the polymerized fibrin, in the bound form its tPA-cofactor properties are masked, and the myosinfibrin clot is relatively resistant to plasmin.
Signaling through MHC in transgenic mice generates a population of memory phenotype cytolytic cells that lack TCR
Authors: McFarland Hugh I. , Hansal Susan A. , Morris Diane I. , McVicar Daniel W. , Love Paul E. , Rosenberg Amy S.
Published: May 2003

We constructed a chimeric molecule, composed of the T-cell receptor (TCR)–ζ chain fused to the extracellular domains of a prototypical allogeneic major histocompatibility complex (MHC) class I molecule, Dd, to assess whether such a construct could affect Dd allospecific responses in vitro and in vivo. To generate cytotoxic T lymphocytes (CTLs) expressing the construct, Dd-ζ was targeted to lymphocyte populations in transgenic mice by placing its expression under control of the CD2 promoter. In response to ligation of Dd, lymphocytes from transgenic mice expressing high levels of Dd-ζ are activated to proliferate and kill cells binding to Dd, despite the near total loss of CD8+ T cells in these mice. Thus, the Dd-ζ cytolytic cell was found not to be a conventional CD8+ CTL, but rather an unusual T lineage cell (CD3-CD5+Thy1.1+) that lacked αβ or γδ TCRs, as well as CD4 and CD8 coreceptors, but expressed surface markers strikingly similar to memory CTLs, including CD44, Ly-6C, and CD122. These cells originate in the thymus and potently veto responses to Dd in vitro. Lacking TCRs, these veto cells are unlikely to mediate graft-versus-host disease (GVHD) and thus may be useful as a cellular therapy for therapeutic deletion of alloreactive T cells in the settings of graft rejection and GVHD.
Characterization of molecular defects of Fitzgerald trait and another novel high-molecular-weight kininogen-deficient patient: insights into structural requirements for kininogen expression
Authors: Krijanovski Yelena , Proulle Valerie , Mahdi Fakhri , Dreyfus Marie , Müller-Esterl Werner , Schmaier Alvin H.
Published: May 2003

A 6-year-old male with vertebral-basilar artery thrombosis was recognized to have high-molecular-weight kininogen (HK) deficiency. The propositus had no HK procoagulant activity and antigen (< 1%). Using monoclonal antibodies (Mabs) to kininogen domain 3, the propositus, family members, and Fitzgerald plasma were determined to have detectable low-molecular-weight kininogen. Mabs to HK domains 5 and 6 do not detect HK antigen in the propositus' plasma. The propositus has a single base pair (bp) deletion in cDNA position 1492 of exon 10 affecting amino acid 480 of the mature protein and resulting in a frameshift and a premature stop codon at position 1597 (amino acid 532). Unexpectedly, Mabs to the heavy chain and domain 5 of HK detect a 92-kDa form of HK in Fitzgerald plasma, the first HK-deficient plasma. The 92-kDa Fitzgerald HK has amino acid residues through 502, corresponding to domains 1 through 5, but lacks epitopes of domain 6 (positions 543 to 595). Fitzgerald DNA has a normal exon 10, but a 17-bp mutation in intron 9. These combined results indicate that mutations in the kininogen gene may differentially affect biosynthesis, processing, and/or secretion of HK.
Inhibition of coagulation, fibrinolysis, and endothelial cell activation by a p38 mitogen-activated protein kinase inhibitor during human endotoxemia
Authors: Branger Judith , van den Blink Bernt , Weijer Sebastiaan , Gupta Abhya , van Deventer Sander J.H. , Hack C. Erik , Peppelenbosch Maikel P. , van der Poll Tom
Published: May 2003

P38 mitogen-activated protein kinase (MAPK) is an important component of intracellular signaling cascades that initiate various inflammatory cellular responses. To determine the role of p38 MAPK in the procoagulant response to lipopolysaccharide (LPS), 24 healthy subjects were exposed to an intravenous dose of LPS (4 ng/kg), preceded 3 hours earlier by orally administered 600 or 50 mg BIRB 796 BS (a specific p38 MAPK inhibitor), or placebo. The 600-mg dose of BIRB 796 BS strongly inhibited LPS-induced coagulation activation, as measured by plasma concentrations of the prothrombin fragment F1 + 2. BIRB 796 BS also dose dependently attenuated the activation and subsequent inhibition of the fibrinolytic system (plasma tissue-type plasminogen activator, plasmin-α2-antiplasmin complexes, and plasminogen activator inhibitor type 1) and endothelial cell activation (plasma soluble E-selectin and von Willebrand factor). Activation of p38 MAPK plays an important role in the procoagulant and endothelial cell response after in vivo exposure to LPS.
Rapamycin inhibits IL-4—induced dendritic cell maturation in vitro and dendritic cell mobilization and function in vivo
Authors: Hackstein Holger , Taner Timucin , Zahorchak Alan F. , Morelli Adrian E. , Logar Alison J. , Gessner Andre , Thomson Angus W.
Published: May 2003

Rapamycin (RAPA) is a potent immunosuppressive macrolide hitherto believed to mediate its action primarily via suppression of lymphocyte responses to interleukin 2 (IL-2) and other growth factors. We show here that this view is incomplete and provide evidence that RAPA suppresses the functional activation of dendritic cells (DCs) both in vitro and in vivo. In vitro, RAPA inhibits IL-4—dependent maturation and T-cell stimulatory activity of murine bone marrow—derived DCs. These effects are associated with posttranscriptional down-regulation of both subunits of the IL-4 receptor complex (CD124, CD132) and are mediated via binding of RAPA to its intracellular receptor FK506-binding protein 12 (FKBP12). In vivo, RAPA impairs steady-state DC generation and fms-like tyrosine 3 kinase ligand (Flt3L)—induced DC mobilization. In addition, in vivo administration of RAPA impairs DC costimulatory molecule up-regulation, production of proinflammatory cytokines, and T-cell allostimulatory capacity. These novel findings have implications for RAPA-based therapy of chronic DC-triggered autoimmune diseases, transplant rejection, and hematologic malignancies with activating Flt3 mutations.
A novel genetic leukocyte adhesion deficiency in subsecond triggering of integrin avidity by endothelial chemokines results in impaired leukocyte arrest on vascular endothelium under shear flow
Authors: Alon Ronen , Aker Memet , Feigelson Sara , Sokolovsky-Eisenberg Maya , Staunton Donald E. , Cinamon Guy , Grabovsky Valentin , Shamri Revital , Etzioni Amos
Published: May 2003

Leukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective β2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte β1 and β2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein–coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of β2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fail to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow.
A role for Toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells
Authors: Bernasconi Nadia L. , Onai Nobuyuki , Lanzavecchia Antonio
Published: May 2003

Toll-like receptors (TLRs) are pattern recognition receptors that trigger innate immunity. In this study we investigated the expression of 10 TLRs in human naive and memory B-cell subsets. We report that in human naive B cells most TLRs are expressed at low to undetectable levels, but the expression of TLR9 and TLR10 is rapidly induced following B-cell-receptor (BCR) triggering. In contrast, memory B cells express several TLRs at constitutively high levels. The differential expression of TLR9 correlates with responsiveness to its agonist, CpG DNA. Thus, human memory B cells proliferate and differentiate to immunoglobulin (Ig)–secreting cells in response to CpG, while naive B do so only if simultaneously triggered through the BCR. The BCR-induced expression of TLRs in human naive B cells prevents polyclonal activation in a primary response, because it restricts stimulation to antigen-specific B cells. In contrast, the constitutive expression of TLRs in memory B cells allows polyclonal activation of the entire memory pool. Thus, in human B cells TLRs are downstream of BCR and play a role both in the primary response and in the memory phase.