bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Detection of FV Leiden Patients Using a Novel Snake-Venom-Based Activated Protein C Resistance Assay.
Authors: Wilmer Marianne , Stocker Christoph , Buehler Beatrice , Conell Brigitte , Calatzis Andreas
Published: October 2019

A new functional prothrombin-based activated protein C (APC) resistance (APC-R) test (Pefakit® APC-R Factor V Leiden, Pentapharm, Basel, Switzerland) is presented.

Methods: The plasma sample is mixed with a reagent containing APC and snake venom specifically activating FV (RVV-V, Daboia russelli) and plasma that has been depleted of FV. During an incubation period of 180 sec the activated FV is inactivated by APC. Subsequently a reagent that contains a FV dependent prothrombin activator (Noscarin, Notechis scutatus) and EDTA is added. The clotting time is recorded. A second determination is performed under identical conditions, with the exception that no APC is added to the first reagent. A ratio between the two measurements is calculated. 703 samples of patients undergoing thrombophilia screening were analysed. Results were correlated to PCR based FVL testing, aPTT, PT, and to levels of Protein C, Protein S, Fibrinogen, FVIII and lupus anticoagulant index.

Results: Using a predefined cut-off of a ratio of 2.5 a 100% sensitivity and specificity for the detection of a FVL mutation was found. Using a cut-off ratio of 1.2 a complete but narrow distinction of FVL heterozygous (n=192) and FVL homozygous samples (n=27) was determined. No interference by sample’s INR and aPTT, PS, fibrinogen and FVIII levels and lupus anticoagulant ratio was detected.

Conclusion: The new snake-venom-based APC-R assay provides an improved distinction of FV wild-type and FVL carriers compared to the data reported for the aPTT based methods. The use of a FV dependent prothrombin activator eliminates effects of FVIII concentration or lupus anticoagulants in the sample.
Transcriptional Response to IL-7 by Immature Human Thymocytes Demonstrates Specific Effects on Differentiation as Well as Survival Maintenance and Proliferation.
Authors: Senadheera S. , George A. , Barsky L. , Zielinska E. , Dudl E. , Schaub B. , Triche T. , Marjoram P. , Weinberg K.
Published: October 2019

Cytokine signaling has been thought to have either instructive effects on hematopoietic cell differentiation, or only survival and proliferation effects on cells whose differentiation is programmed by mechanisms other than cytokine signaling. Thymopoiesis is a useful model for examining the question of how cytokines affect lymphohematopoietic development because it is tightly regulated with well-defined developmental stages, and loss of thymopoiesis is not lethal. IL-7 is a cytokine that is known to be essential for normal thymocyte development as demonstrated by low thymocyte numbers in IL-7-deficient and IL-7R-deficient mice. To determine whether IL-7 has direct effects on human thymocyte differentiation or only acts a survival/proliferation factor, changes in gene expression in CD3-CD4-CD8-(”triple-negative”, TN) human thymocytes were analyzed after in vitro stimulation with IL-7. TN thymocytes were enriched by negative selection with anti-CD3, -CD4, -CD8 immunomagnetic columns, followed by FACS purification, factor starvation for 12–16 hours, and stimulation with IL-7. The RNA was extracted, amplified by in vitro transcription, labelled, and hybridized on Affymetrix U133 microarrays. Analyses of 10 paired samples of unstimulated and IL-7 stimulated cells revealed 56 genes which were up-regulated and 22 genes which were down-regulated greater than 1.25 fold (p<0.05). Confirmatory quantitative RT-PCR demonstrated significant data compression, e.g., signal changes of 1.25 fold by array corresponded to 8–20 fold differences by qRT-PCR. Many of the up-regulated genes were not lymphoid- or thymocyte-specific and were known previously, e.g., anti-apoptotic (Bcl-2) or cell cycle regulatory (pim-1 cyclin D2). There was a pattern of changes in gene expression which would be expected to down-modulate IL-7R signaling - decreased IL-7R itself, increased SOCS family genes, decreased PI-3K p85α. There were also alterations in genes specifically expressed during lymphocyte or thymocyte differentiation. Mal, which encodes a T lymphoid specific proteolipid tetraspannin that is thought to be a necessary component of lipid rafts containing the T cell receptor (TCR) for antigen, was up-regulated 4-fold. On the other hand, IL-7 down-regulated expression of terminal deoxynucleotidyl transferase (Tdt). IL-7 appeared to regulate cytokine production and responsiveness. There was altered expression of secreted protein and recptor genes that would alter the interactions of TN thymocytes with their microenvironment, e.g., increased P-selectin, IL-4R, frizzled 10, and decreased TGFβ 1, EGF. The complex changes in gene expression induced by IL-7 in TN cells suggest a direct instructive role for IL-7 signaling in the tight regulation of the different stages of thymic differentiation, rather than only regulation of survival, maintenance and/or proliferation.
Identification of a Novel Stromal Cell Membrane Protein that Binds to Megakaryocytes.
Authors: Tamashiro Kazuki , Fujimoto Tetsur-Takahiro , Fujimura Kingo
Published: October 2019

In bone marrow, hematopoiesis-supporting cells or stromal cells govern the growth, survival and differentiation of hematopoietic stem cells. We previously reported the method to produce the functional platelets from murine embryonic stem (ES) cells in vitro, using the coculture system with OP9 stromal cells. Also in this system, the cell contact between ES-derived cells and stromal cells is essential for megakaryocyte differentiation, and even after growth factors including TPO were supplied, ES-derived megakaryocytes cannot be maintained without stromal cells. In this study, we searched membrane proteins that were expressed in the stromal cells and were involved in the cell contact with megakaryocytes. We employed the retrovirus-based signal sequence trap (SST) method to identify such membrane proteins from murine stromal cells. 117 signal sequence-containing clones were identified, and the clones were further screened by the binding capacity to megakaryocytic cell lines. We finally identified a novel protein that contains two Ig domains and belongs to the transmembrane-type immunoglobulin superfamily. RT-PCR analysis showed that this protein is expressed in bone marrow stromal cell lines including OP9, and vascular endothelial cell lines, whereas no expression was detected in any hematopoietic cell lines. We next created a cDNA construct in which the extracellular domain of the protein was linked to IgG Fc domain, and the construct was transfected into 293 cells. The fusion protein was purified from the culture supernatant. By flow cytometer, the purified recombinant protein bound to megakaryocytic cell lines and CD41-positive bone marrow megakaryocytes. The binding to murine Sca-1-positive and human CD34-positive immature cells was also detected. The binding was dependent on calcium, but was not inhibited by RGD peptide. The effect on in vitro differentiation system of ES cells was further tested. The addition of the fusion protein showed no influences on the maintenance of undifferentiated state of ES cells. However, after the differentiation induction, the protein facilitated morphological change of the ES cells. Further analysis will contribute to understanding of the mechanisms by which the stromal cell contact regulates megakaryocyte differentiation and platelet production.
Determination of Soluble MICA in Serum as a Novel Marker for Tumor Stage and Metastasis.
Authors: Salih Helmut R. , Stieber Petra , Peterfi Andrea , Nagel Dorothea , Kanz Lothar , Steinle Alexander L. , Holdenrieder Stefan
Published: October 2019

The human NKG2D ligands (NKG2DL) MICA and MICB have been shown to be expressed on tumors of epithelial and hematopoietic origin in vivo. Recently we reported that MICA is shed from the cell surface of tumor cells and is present in sera of tumor patients (J Immunol169:4098 (2002), Blood102:1389 (2003)). Since the strength of an anti-tumor response by NK cells and CD8 T cells is critically depending on NKG2DL expression levels, down-regulation of MICA-expression on tumor cells represents an immune escape mechanism that diminishes anti-tumor reactivity of NKG2D-bearing lymphocytes. However, no data are yet available regarding the correlation of soluble MICA (sMICA) levels with specific tumor entities, aggressiveness of the disease, and hence the potential implementation of sMICA as novel marker in differential diagnosis and prognosis of cancer. In this study, we determined sMICA levels in sera of 512 individuals including 296 patients with various cancers, 154 patients with benign disorders and 62 healthy individuals. Healthy individuals revealed significantly lower sMICA values (median<30pg/mL) than patients with benign diseases (84pg/mL; p=0.005) and cancer patients (161pg/mL; p<0.0001). In addition, sMICA levels differed significantly between cancer patients and patients with benign disorders (p<0.0001) that represent the most relevant control group for differential diagnosis. In cancer patients, while there was no association between sMICA levels and tumor size (p=0.456), cell differentiation (p=0.271), or lymph node involvement (p=0.674), sMICA correlated significantly with cancer stage and metastasis (p=0.015 and p=0.007, respectively). Our data indicate that release of MICA might play a role in late stages of tumor progression by overcoming the confining effect of NK cells and CD8 T cells. Thus, determination of sMICA levels provides valuable information for cancer staging, and sMICA in serum seems to be an indicator for systemic manifestation of malignancy rather than for local tumor extent.
Human Monoclonal Cryocrystalglobulinemia: First Complete Heavy and Light Chain Variable Region Nucleotide Sequences.
Authors: Perfetti Vittorio , Navazza Valentina , Giorgietti Sofia , Marchese Loredana , Palladini Giovanni , Caporali Roberto , Montecucco Claudio , Merlini Giampaolo
Published: October 2019

Cryocrystalglobulinemia is characterized by a monoclonal immunoglobulin that is both cryoprecipitable and crystalforming (cryocrystalglobulin). It occours in humans as a rare complication of lymphoproliferative disorders. The monoclonal immunoglobulins may crystallize in serum and synovial fluid and are responsible for both microcrystalline synovial inflammation and occlusive vasculopathy in kidneys and skin. At present, there are no reports on sequence analysis and tertiary structure of human cryocrystalglobulins. In this study, we established the first complete light and heavy chain variable region sequences of a human cryocrystalglobulin (BEL) from a patient with severe microcrystalline arthropathy, cutaneous purpura and sacroiliitis. Serum cryoprecipitate was composed of unbound monoclonal IgGk at immunofixation (type I cryoglobulinemia) and revealed homogeneous crystals under light microscopy. Extra and intracellular crystals were also found in synovial fluid. Analysis of bone marrow aspirate showed 4% monoclonal plasma cell infiltration. Total RNA was extracted from Ficoll-separated bone marrow mononuclear cells and complete heavy (VH) and light chain (Vk) variable domain sequences of monoclonal Ig were obtained by means of an unbiased inverse-PCR strategy. Sequences were then matched in databases to identify rearranged germline V, D and J segments. The heavy chain sequence belonged to the g2 class and its variable region was most closely related (94% identity) to the VH3-30 germline gene (VH3 family) rearranged to D2-2 and JH4b segments, whereas the Vk region was derived (97% identity) from the germline VkL6 (VkIII) rearranged to Jk5. Partial protein sequence of serum cryocrystalprecipitate confirmed the correct identification of the monoclonal sequences. A previously reported murine model of cryocrystalglobulinemia suggested that acquisition of specific amino acids at critical positions within the VH domain (positions 6 and 23) could influence cryoprecipitation (Rengers J-U et al, Blood2000;95:3467–72). Analysis of BEL VH domain did not confirm these observations. Protein modelling and site-directed mutagenesis studies are undergoing to clarify the role of amino acid substitutions and interactions in immunoglobulin crystal-like aggregation and cryoprecipitation.
A New Vaccine Paradigm To Break Tolerance with Potential Applications for the Immunotherapy of B Cell Lymphoma.
Authors: Taylor Ronald P. , Whipple Emily C. , Lindorfer Margaret A. , Ditto Andrew H. , Shanahan Ryan S.
Published: October 2019

Complement (C) plays a critical role in the immune response by opsonizing immune complexes (IC) and thymus-independent type 2 antigens with C3 breakdown product C3dg. We investigated the in vivo fate and handling in mice of anti-CR1/CR2 mAb 7G6. We used this rat IgG mAb as a surrogate for C3dg-opsonized IC; mAb 7G6 binds to CR1/CR2 with high affinity, blocks C3dg binding and saturates mouse B cell CR2 at inputs of only 2 ug. RIA, flow cytometry, and fluorescence immunohistochemistry were used to examine the disposition of 0.5–2 ug quantities of mAb 7G6 infused i.v. in mice. The mAb binds to circulating B cells and in the spleen binds preferentially to marginal zone (MZ) B cells. However, within 24 h MZ B cells relocate and transfer the mAb to regions rich in follicular dendritic cells (FDC). Localization of intact antigen to FDC should induce a substantial immune response, and therefore we immunized mice and monkeys i.v. with low doses (1–20 ug/kg) of prototype antigens constructed with anti-CR1/2 mAb 7G6 or anti-CR2 mAb HB135, respectively. We observed a strong immune response characterized by early development of IgG antibodies and long-lasting immunity extending out to at least one year. We applied our immunization paradigm to mouse IgG idiotypes, based on i.v. infusion of mouse IgG2a mAbs which were cross-linked with mAb 7G6. The purpose of these experiments was to determine if tolerance can be broken in order to develop a more powerful vaccine strategy to induce a cytotoxic humoral immune response to malignant B cells based on targeting the idiotype of immunoglobulin molecules expressed on their surfaces. I.V. immunization with the constructs indeed generated a mouse IgG1 immune response to two different mouse IgG2a mAbs, as demonstrated by ELISA. The immune response was idiotype specific, but some anti-isotype antibodies were also detected. Moreover, sera from immunized mice immunoprecipitated the specific radiolabeled mouse mAbs in the presence of 7.5% polyethylene glycol. This humoral immune response was also demonstrable in flow cytometry assays in which IgG1 in sera of immunized mice bound to erythrocytes opsonized with bispecific mAb constructs consisting of the IgG2a mAb crosslinked with an anti-CR1 mAb. The present approach, based on coupling the targeted immunoglobulin to an anti-CR2 mAb for delivery to FDC, may lead to a more effective immunotherapeutic vaccine compared to methods currently in clinical trials which require use of glutaraldehyde to effect crosslinking of the targeted immunoglobulin to KLH.
Clinical and Quality of Life Outcomes in Patients with Multiple Sclerosis (MS) after High Dose Chemotherapy(HDCT)+Autologous Stem Cell Transplantation (ASCT).
Authors: Shevchenko Yury L. , Novik Andri A. , Afanasiev Boris V. , Lisukov Igor A. , Fedotov Yury N. , Ionova Tatiana I. , Kulagin Alexandr D. , Malysheva Olga A. , Bisaga Gennadi N. , Melnichenko Vladimir Y. , Chelombit Lubov V. , Voloshin Sergei V. , Fedorenko Denis A. , Vereschagina Inna V. , Kishtovich Anton V.
Published: October 2019

Objectives: HDCT+ASCT is a new and promising therapy for MS patients. Among a number of questions are the terms of conducting HDCT+ASCT. According to our concept there are 3 strategies of HDCT+ASCT depending on terms of disease stage: early, conventional and salvage. The aim of the research was to study clinical and quality of life (QoL) outcomes in MS patients with different terms of HDCT +ASCT.

Methods: Twelve patients with MS were included in the study. All the patients previously underwent conventional treatment. Clinical and QoL evaluation was provided at baseline, at discharge, at 3, 6, 9, 12 months and later every half a year after HDCT+ ASCT. MRI was conducted at baseline, in 6, 12 months and at the end of follow-up. QoL was assessed by FACT-BMT and FAMS. Integral QoL index was assessed by the method of integral profiles. Median EDSS at base-line was 6.0 (range 2 –7.5). The median follow-up duration was 18 months (range 3–60 months).

Results: Clinical examination and MRI revealed disease stabilization (DS) in ten patients and progression (DP) in two patients. EDSS decreased in seven patients; increased from 6.5 to 7.0 and from 6.0 to 6.5 in patients with DP and remained the same in three patients. Distinct QoL improvement was observed in all the patients with DS at the end of follow-up.

Three case reports with different terms of ASCT were analyzed. Patient K. underwent early ASCT (male, 21 years old; secondary progredient type; base-line EDSS - 2.0; follow-up 18 months); patient F.- conventional ASCT (female, 35 years old; secondary progredient type; base-line -EDSS 5.0; follow-up 60 months); patient P.- salvage ASCT (female, 49 years old; secondary progredient type; base-line EDSS 7.5; follow-up 48 months). Clinical examination and MRI revealed DS. EDSS decreased from 2.0 to 1.5 in patient K.; from 5.0 to 4.5 in patient F. and from 7.5 to 6.0 in patient P. at the end of follow-up. Integral QoL index increased dramatically as compared to base-line: in patient K. - from 0.6 to 0.7; in patient F. - from 0.2 to 0.7; in patient P. - 0.4 to 0.6.

Conclusion: HDCT+ASCT in MS patients resulted in DS in ten out of twelve patients under observation. Along with clinical stabilization dramatic improvement of QoL took place. Analysis of three case reports demonstrated feasibility of early, conventional and salvage ASCT in MS patients.
Dose Adjusted IV Busulfan/Cyclophosphamide (BU/CY) and Autologous (AU) Stem Cell Transplantation (SCT) for Recurrent Lymphoma.
Authors: Spitzer Thomas R. , Cox Benjamin , Sepe Paul , McAfee Steven L. , Dey Bimalangshu R. , Ballan Karen , Scadden David
Published: October 2019

The optimal preparative therapy for AuSCT for lymphoma is unknown. High dose Bu/CY is a generally well tolerated, and an effective regimen for allogeneic SCT for hematologic malignancy (HM), and autologous SCT for myeloid malignancies. It has, however, been less well studied for lymphoid malignancies. IV busulfan has largely replaced oral busulfan because of more predictable pharmacokinetics, and ease of administration. We have treated 46 patients (pts) with recurrent, chemosensitive lymphoma or lymphoma in which an initial complete remission was not achieved with the Bu/CY regimen and AuSCT from 2001 – 2004. 4 pts had AIDS related NHL (ARL). Patients had received a median of two chemoradiotherapeutic regimens prior to SCT. Diagnoses were indolent NHL (n = 12), aggressive NHL (n = 28) and Hodgkin’s disease (n = 6). IV busulfan was given at a dose of 0.8 mg/kg (based on actual or ideal body weight (IBW) whichever was less) every six hours for 16 doses (for patients < 50 years of age), or 14 doses (for patients = 50 years of age) on days -7 through -4. IV cyclophosphamide 60 mg/kg (based on a wt half way between actual and IBW for patients > 20% IBW) was given on days −3 and −2. AuSCT was performed on day 0. Additional autologous bone marrow was infused in pts who had less than 2 x 106/ kg CD 34+ progenitor cells (n = 2). Median (range) patient age was 56 (24–78) years. Four pts were = 70 years old at the time of transplant. Median (range) times to ANC = 0.5 and platelets = 20k were 10 (8–12) and 12 (8–64) days, respectively. Regimen related toxicities (according to the Bearman toxicity scale) included grade II-III mucosal toxicity (n = 7), hepatic VOD (n = 1), and grade II GI toxicity (n = 7). Documented infection occurred in 16 (35%) pts. Two pts (4.4%) died before day 100 from non-relapse causes (progressive pulmonary toxicity (n = 1), recurrence of melanoma (n = 1). Late (= day 100) non-relapse mortality (NRM) occurred in two pts (respiratory failure, (n = 1); hepatic VOD (n = 1). Overall NRM was 8.7% (4/46 patients). No NRM occurred in pts with ARL or pts > 70 yrs. Fourteen of 40 pts with NHL have had relapse/progression. Three of six pts with HD have had relapse/progression. Actuarial progression free survival (PFS) and overall survival (OS) probabilities are 67% and 100% at 32 mos for pts with indolent NHL and 44% and 79% at 25 mos for pts with aggressive NHL, respectively. High dose IV Bu/CY as preparation for AuSCT, with dose reduction of IV busulfan to 14 mg/kg for patients = 50 years is well tolerated and is associated with favorable PFS and OS probabilities. The regimen has also been well tolerated in pts over the age of 70 with no life threatening or fatal toxicities in this subgroup.
Differential Gene Expression in Leukemic Cells from Bone Marrow (BM) and Peripheral Blood (PB) of Pediatric Patients with Acute Lymphoblastic Leukemia (ALL).
Authors: Rhein Peter , Scheid Stefanie , Hagemeier Christian , Seeger Karl , Kirschner Renate , Schrappe Martin , Spang Rainer , Ludwig Wolf-Dieter , Karawajew Leonid
Published: October 2019

Although the genetic mechanisms underlying the induction of ALL are similar, the molecular events leading to diverse clinical presentation and course are not well characterized. High PB white blood cell (WBC) counts reflect tumor burden in PB and have been used in the past as a clinical risk factor in ALL. We hypothesized that, given differential microenvironment signaling provided by BM and PB, ALLs with high or low WBC counts (WBChigh and WBClow) might differ with regard to their dependence on the microenvironment. To approach this issue, we investigated gene expression changes in PB blasts as compared to their BM counterparts in patients with precursor B-cell ALL (n=15). Blasts were isolated from BM and PB samples by flow sorting and investigated for mRNA levels using Affymetrix HG U133A microarrays. Gene expression data, normalized by variance stabilization on probe level, were analysed in matched PB-BM pairs using a SAM-like analysis of log ratios in a balanced permutation test. By these procedures, a limited number of 38 genes consistently changed in PB vs BM blasts was identified (false discovery rate < 0.01). Leukemic PB cells were characterized by upregulation of genes encoding for cell adhesion- and trafficking-related proteins (CD11b, CD73, S100A4, EMP3). Furthermore, downregulation of the cell cycle- and proliferation-associated genes (thymidylate synthetase TYMS, kinesin KIF11, topoisomerase TOP2A, ribonucleotide reductase RRM2, kinetochore protein ZWINT) indicated decreased proliferative activity of leukemic cells in PB microenvironment. Finally, the gene encoding for Bcl-2 protein, known to be a powerful downstream mediator of survival signaling from microenvironment in various experimental settings, was consistently downregulated in PB as compared to BM blasts. Within the group of WBClow patients (n=8, mean WBC=17x103/μl), most of these genes retained their statistical significance. By contrast, no consistent expression changes in PB vs BM blasts were found in WBChigh patients (n=7, mean WBC=201x103/μl). In summary, investigation of differential gene expression in leukemic cells from PB and BM indicated decreased levels of proliferative and survival signaling in the PB microenvironment and a higher dependence on this signaling of leukemic cells from WBClow as compared to WBChigh patients.
Fatal Human Metapneumovirus Infection in Stem Cell Transplant Recipients.
Authors: Englund Janet A. , Nichols W. G. , Kuypers Jane , Hackman Robert C. , Morrow Rhoda , Fredricks David N. , Corey Lawrence , Boeckh Michael J.
Published: October 2019

The importance of human metapneumovirus (hMPV), a recently discovered human respiratory virus, in hematopoietic cell transplant (HCT) recipients is not known. We retrospectively surveyed 211 bronchoalveolar lavage samples (BAL) from symptomatic adults with new or changing pulmonary infiltrates after HCT during five-year period using a quantitative real time RT-PCR assay. Medical records, radiographic studies, biopsy specimens, and autopsy material were reviewed. hMPV was detected in 8/211 (3.8%) BAL specimens from 5/162 (3%) patients. The median titer of hMPV was 9.0 x 106 copies/ml of BAL fluid (range 5.5 x 104 to 3.5 x 108). Persistent hMPV infection was noted in all 3 patients who had multiple samples tested. hMPV was also detected in lung tissue in 1 of 2 samples tested. Infected patients were between the ages of 24–54 years, and became symptomatic within the first 40 days post transplant. Initial symptoms included fever, cough, nasal congestion, and sore throat. Clinical findings near the time of diagnosis included respiratory failure, pulmonary hemorrhage, and culture-negative septic shock. Four patients died as a result of acute respiratory distress and pulmonary disease with clinical and autopsy diagnoses of “idiopathic pneumonia.” We conclude that hMPV infection is associated with respiratory failure in immunocompromised adults and may result in fulminant respiratory decompensation in the post transplant period. Prospective studies of the role of this virus in idiopathic pneumonia syndromes of immunocompromised patients are warranted.
In Vitro Growth Regulation and the Identification of Potential Signaling Pathways in T (4; 11), Pediatric Pre-B Acute Lymphoblastic Leukemia (ALL).
Authors: Narendran Aru , Tijan Bijan , Coppes Lucas , Rhemtulah Farrah , Anderson Ron
Published: October 2019

Among the various chromosomal translocations found in pediatric pre-B cell acute leukemias, the t (4; 11) group contains patients of all ages as well as a clustering of children at birth or in the first year of life. As with all 11q23 abnormalities, t (4; 11) fusion confers an extremely poor prognosis. Using cell lines, many recent studies have described important molecular and cellular growth properties of these cells. We have investigated in vitro growth regulation of freshly isolated infant pre-B ALL cells with t (4; 11).

Leukemic blasts were affinity purified with anti CD19 antibodies and cultured in the presence of various cytokines including IL-3, IL-1, FLT-3 ligand (FL), SCF, IL-6, IL-7, GM-CSF, PDGF, SDF-1, VEGF, EPO and GCSF. Cell proliferation after four days in culture was quantified by Alamar blue assay. At pg/ ml concentrations, significant growth stimulation was seen in the presence of FL and IL-7. The FLT-3 effect was much less prominent against leukemic cells without t (4; 11). Then we aimed to identify the possible signaling pathways utilized by the FLT-3 mediated stimulation of these cells. Purified blasts were cultured in serum free medium for 24 hours and stimulated for 0, 5 and 10 minutes with 10 pg/ml of FL. These cells were subsequently lysed and total cellular extracts were analyzed by an antibody array technique. The arrays used consisted of antibodies against a selection of fifty proteins involved in all known signaling pathways. Changes in the amount of phosphorylated proteins following FL treatment were identified by enzyme conjugated anti phospho specific antibodies. A number of reproducible FL specific changes were observed. These include increased Ets-1/2, JNK1, 2,3, p38MAPK and Src. We describe the detail analysis of the signaling pathways potentially utilized by these cells and illustrate the use of this experimental approach to identify possible targeted therapeutic agents against this particular form of leukemia.
ZAP-70 Tyrosine Kinase Is Constituvely Expressed and Phosphorylated in B-Lineage Acute Lymphoblastic Leukemia Cells.
Authors: Guillaume Nicolas , Alleaume Celine , Capiod Jean-Claude , Pautard Brigitte , Desablens Bernard , Claisse Jean-François , Lefrere Jean-Jacques , Gouilleux Fabrice , Lassoued Kaiss , Gouilleux-Gruart Valerie
Published: October 2019

Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases is normally expressed in T and NK cells. ZAP-70 plays a key role in the signaling pathway of the TCR. It has been recently shown that this protein is also expressed in mouse B lineage cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of chronic lymphocytic leukemia (CLL), with a poor prognosis, that have nonmutated immunoglobulin variable region genes.

In this study, we examined the expression and phosphorylation status of ZAP-70 and Syk in B-lineage acute lymphoblastic leukemia (B-ALL) cells. ZAP-70 and Syk were constitutively expressed and phosphorylated (tyr319 for ZAP and tyr352 for Syk) in human precursor B-ALL cell lines as well as in B leukemic cells from all examined B-ALL patients (n=18). The expression of ZAP-70 was specific to these B-ALL cells as it was not detected in the K562 cell line or malignant myeloid cells. This expression was independant of maturation state of leukemic cells (pro-B or pre-B) and caryotypic status.

The expression and phosphorylation of the T-cell “specific” ZAP-70 kinase in all B-ALL cells raises important questions concerning the contribution of this protein to the leukemogenesis process resulting in B-ALL.
Possible Autocrine Mechanism of Placenta Growth Factor / Vascular Endothelial Growth Factor Receptor Type 1 in Acute Lymphoblastic Leukemia with Philadelphia Chromosome.
Authors: Ikai Toshiko , Miwa Hiroshi , Shikami Masato , Hiramatsu Akihito , Imai Noriaki , Satoh Atsushi , Itoh Masato , Miura Kazuhisa , Imamura Akira , Mihara Hidetsugu , Nishii Kazuhiro , Nitta Masakazu
Published: October 2019

The growth and/or survival of leukemia cells are affected by endothelial cell associated factors such as vascular endothelial growth factor (VEGF). Here, we studied the mRNA expression of VEGF and placenta growth factor (PLGF), a VEGF-related molecule, of acute lymphoblastic leukemia (ALL) cells (10 Philadelphia chromosome (Ph1) positive cases and 10 Ph1 negative cases) by real-time PCR. The expression of PLGF mRNA was significantly higher in Ph1 positive cases than Ph1 negative cases (p<0.05). The expression of VEGF mRNA was not different between Ph1 positive and negative cases. Then, recombinant PLGF was added to the culture of ALL cell lines: the growth of CRL1929 (Ph1 positive) was augmented by PLGF in a dose-dependent manner, while the growth of Nalm6 (Ph1 negative) was not affected by the addition of PLGF. Here, the growth enhancement effect of PLGF on CRL1929 was cancelled by the simultaneous addition of VEGF receptor type 1-Fc (VEGFR1-Fc), which binds to PLGF and abrogates the function. The growth enhancement effect was not cancelled by the simultaneous addition of VEGFR2-Fc, which binds to VEGF, but not to PLGF. Recombinant VEGF did not affect the growth of either cell line. Our data demonstrated that PLGF has a growth promoting effect on Ph1 positive ALL cells, and autocrine mechanism of PLGF-VEGFR1 might work in Ph1 positive ALL.
CXCL-12 Induces Prolonged Erk-Phosphorylation in Zap-70 Positive B-CLL.
Authors: Lust Sofie , Boelens Jerina , Janssens Ann , Philippé Jan , Bracke Marc , Offner Fritz
Published: October 2019

Introduction

Zap70 is superior to mutation status as a prognostic indicator in B-CLL, but whether it is causally implicated or just associated with progressive disease is unclear. Zap-70 transduces the TCR signal through the CD3 zeta-unit in T cells, but although it is important in preB development, it has no known function in mature B-cells. Zap-70 positive B-CLL show enhanced B cell receptor mediated Syk-phosphorylation upon surface IgM (sIgM) crosslinking, simulating antigen driven B cell proliferation. This may increase the proliferative drive in Zap-70+ B-CLL compared to Zap-70- leading to a higher risk of secondary chromosomal damage. Zap-70 was recently implied in Erk activation via CXCL-12/CXCR4 in T-cells and this is enhanced by cell adhesion through beta-1 integrins that may influence P-Erk phosphatases. Since both CXCL12/CXCR-4 and integrins influence B-CLL survival in vitro, we investigated the duration of downstream Erk activation in Zap-70 + versus Zap-70 B-CLL after CXCL-12 stimulation in the presence and absence of integrin binding by fibronectin.

Materials/methods

Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-gradient. Zap-70 was measured by flow cytometry (Upstate Ab), and PCR.

B-CLL cells from 6 patients were stimulated with 100 ng/ml CXCL-12 after preincubation in serum free RPMI for 2 hrs at 37°in fibronectin or BSA coated plates and in suspension culture. After CXCL-12 addition supernatant and adherent cells were separately lysed at 0, 2 and 10 minutes and studied by Western blot for total Erk, Akt, P-Erk and P-Akt. Parallel cultures were extended for 24 hours and apoptosis of supernatant versus adherent cells was performed by AnnexinV-PI staining and flow cytometry.

Results

Erk-phosphorylation was markedly stimulated by CXCL-12 after 2 minutes, both in supernatant and adherent fractions of fibronectin and BSA coated plates compared to suspension cultures. This stimulation decreased in Zap-70- B-CLL after 10 minutes but remained high in Zap-70+ B-CLL particularly in the fibronectin adherent fraction. These cells were also protected from spontaneous apoptosis at 24 hours when compared to supernatant Zap70+ and Zap-70- cells.

Conclusion

CXCL-12/CXCR4 ligation leads to prolonged Erk-phosphorylation in Zap-70+ but not in Zap70- B-CLL cells. Prolonged Erk-activation is required for nuclear translocation and effective signal transduction along this way. This effect is enhanced by concomitant fibronectin engagement of integrins, which may give Zap-70+ B-CLL cells a proliferative advantage upon homing in lymphoid tissues.
Secretable Human Megakaryocyte Factor V Endocytosed from Plasma in Complex with Multimerin: The Origin of Human Megakaryocyte Factor V.
Authors: Suehiro Youko , Veljkovic Dragoslava K. , Fuller Nola , Motomura Yasuaki , Masse Jean , Cramer Elizabeth , Hayward Catherine P.M.
Published: October 2019

Factor V (FV) is an essential coagulation cofactor that circulates in distinct plasma and platelet pools. Unlike plasma FV, platelet FV is stored complexed to the megakaryocyte (MK) synthesized α-granule protein multimerin, which is not found in plasma. The origin of platelet FV has been controversial and recent studies have suggested human platelet factor V originates from plasma. To determine if human MK could endocytose FV and generate the FV-multimerin complexes, we investigated FV endocytosis and storage by human MK derived from cord blood CD34+ cells. Cells were cultured with or without 10% plasma in 15% BIT/ IMDM containing 5ng/ml SCF and 50ng/ml TPO. Human plasma or purified plasma FV was added on day 7 of culture. Cells were harvested to analyze α-granule proteins on day 11. Enzyme-linked immunoassays were used to evaluate MK content of FV and multimerin, and their complexes. Control α-granule proteins analyzed included albumin, IgG and fibrinogen, as they are known to originate from plasma. Multimerin was detected in all MK whereas FV, albumin, IgG, fibrinogen and FV-multimerin complexes were only detected in MK cultured with plasma. The amounts of plasma-derived proteins in MK were orders of magnitude lower than in platelets. MK cultured with purified FV had dose-dependent FV uptake. With SFLLRN stimulation, MK cultured with plasma released multimerin, FV, albumin, IgG and fibrinogen, consistent with their storage in secretion granules. Like platelet FV, MK FV showed a proteolyzed pattern. Double immunostaining showed partial colocalization of intracellular multimerin and FV in MK cultured with FV. Immunoelectron microscopy confirmed that like bone marrow MK, MK cultured with FV contained FV within demarcation membranes, small vesicles and α-granules. Analyses of FV expression by cultured MK, using semi-quantitative RT-PCR, indicated there were no significant differences in FV expression by MK cultured with or without plasma, at any of the time points evaluated. However, there was a trend to increased FV expression with MK maturation, and for MK cultured in plasma, FV expression was significantly higher at 15 compared to 7 days, raising the possibility that some FV was synthesized at late stages of megakaryopoiesis. Our study illustrates that cultured human MK can proteolyze and target endocytosed plasma FV into α-granules and that they can generate complexes of FV with multimerin.
Formation of Tissue Factor-Factor VIIa-Factor Xa Complex Promotes Survival of Human Breast Cancer Cells by Inhibition of Apoptosis.
Authors: Jiang Xiaofeng , Guo Yan-Lin , Bromberg Michael E.
Published: October 2019

Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa). TF is constitutively expressed by a variety of tumor cells. Recently, TF has been shown to induce cellular signaling and promote tumor growth, angiogenesis, and metastasis. We showed previously that formation of TF-FVIIa-Factor Xa (FXa) complex induces cellular signaling in the Adr-MCF-7 cell line, a multidrug-resistant subline of the human breast cancer cell line, MCF-7 (Jiang et al. J. Thromb Haemost2: 2004; 93–101). This cell line has high endogenous expression of TF. Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induces phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and protein kinase B (PKB), whereas no increase in the basal level of phosphorylation of these proteins occurs with treatment of the cells with FVIIa (10 nM) alone. In the present study, we investigated whether TF-FVIIa-induced signaling might alter apoptosis in human breast cancer cells. Apoptosis of the Adr-MCF-7 cells was induced by serum starvation for 5–7 days. Treatment of the cells with the combination of FVIIa (10 nM) and FX (150 nM), reduced apoptosis of the cells by nearly 50% compared with untreated, control cells using an ELISA that detects histone-DNA fragments. In contrast, FVIIa (10 nM) alone did not significantly prevent apoptosis. Pre-treatment of the Adr-MCF-7 cells with hirudin did not inhibit the anti-apoptotic effect of the combination of FVIIa and FX, whereas this effect could be completely blocked by either U0126 (10 μM), which inhibits p44/42 MAPK phosphorylation, or LY294002 (50 μM), which inhibits PKB phosphorylation. In addition, treatment of the Adr-MCF cells with the combination of FVIIa and FX led to a 50% increase in the level of the anti-apoptotic protein, survivin, compared with untreated cells by Western blot analysis. Results from this study indicate that formation of TF-FVIIa-FXa complex prevents apoptosis of breast cancer cells by a thrombin-independent pathway. Moreover, the anti-apoptotic effect of this signaling pathway involves both phosphorylation of p44/42 MAPK and PKB and might be mediated in part by an increase in cell survivin levels.
Long Term Survival Using Intensive Multiagent Chemotherapy for Relapses of Pediatric Acute Lymphoblastic Leukemia (ALL).
Authors: Rheingold Susan R. , Bunin Nancy J. , Aplenc Richard , Leahey Ann M. , Lange Beverly J.
Published: October 2019

Relapses of childhood ALL that occur on therapy are associated with a dismal survival. To improve the prognosis for these patients, we developed an intensive multiagent chemotherapy protocol consisting of an induction with idarubicin, vincristine (VCR), dexamethasone (DEX), and peg-asparaginase (PEG). Consolidation included high-dose cytarabine (ARA-C), etoposide (VP-16), and PEG followed by VCR and methotrexate (MTX). After an interim maintenance (IM), induction and consolidation were repeated followed by maintenance therapy lasting two years. Maintenance and IM consisted of alternating two week cycles of VP-16, ARA-C, and PEG, with MTX ,VCR, DEX, and PEG. Between 1992 and 2002, 53 pts (32M, 21F) received treatment according to this protocol, 21 of whom were treated as part of the original study (Leahey AM et al., Med Ped Onco34(5):313–8, 2000). Median time to relapse was 37 months from diagnosis (range 12–86 mos). Twenty-one pts were on therapy at relapse and 25 pts were <36 months from diagnosis. Relapses included isolated bone marrow (BM) (32 pts), BM and central nervous system (CNS) (9 pts), BM and testicular (3 pts), and extramedullary (EM) (9 pts). By present day criteria 26 pts were standard risk (SR), 23 were high risk (HR), and 4 were infants. Two patients died in induction, and 2 never achieved a second remission. All others achieved remission by the end of induction (92%). Five-year event-free survival (EFS) and overall survival (OS) are both 56% +/−7% (CI 41%–68%), at a mean of 47 months from relapse (range 0–141 mos). Patients with a first complete remission (CR1) duration <36 months have an EFS of 40% +/−10% (CI 21%–58%); >36 months CR1 is associated with an EFS of 70% +/−9% (CI 50%–84%). Of the events in all pts who initiated therapy,13 were from refractory/recurrent ALL (25%), and 10 pts died of toxicity (19%). Four pts died from chemotherapy induced toxicity (8%), and 6 died from transplant (BMT) related toxicities (11%). Fourteen pts in second remission proceeded to BMT at a mean of 5 months from relapse (range 4.5–8 mos), and 7 of these pts remain in CR2. Of the 39 pts who continued on chemotherapy, 6 pts (35%) with CR1<36 months remain in CR2 and 16 patients (57%) with CR1>36 months remain in CR2 or 3. The 19 pts who were treated on modern standard risk (SR) protocols (CCG-1881 to present) were more salvagable than their 20 high risk (HR) counterparts (CCG-1882 to present). Five year EFS for SR and HR pts is 67% +/−8% (CI 48%–80%) and 38% +/−12% (CI 16%–59%) respectively. Intensive rotating therapy with reinduction and reconsolidation improves EFS. Children with early EM relapses of ALL, CR1 > 36 months, and SR patients all have very good long term survival. Novel therapies need to be integrated into intensive relapse protocols for children with early BM relapse and children treated upfront on HR protocols.

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Imatinib Mesylate Does Not Inhibit BCR-ABL Kinase Activity in CML Stem Cells In Vitro.
Authors: Elrick Lucy J. , Hamilton Ashley , Deininger Michael W. , Holyoake Tessa L.
Published: October 2019

Imatinib mesylate (IM, Gleevec, Novartis), a specific inhibitor of the tyrosine kinase BCR-ABL, promotes an impressive complete cytogenetic remission rate in patients with chronic phase Chronic Myeloid Leukaemia (CML). In most cases however, molecular disease persists and may lead to eventual relapse. IM selectively interacts with BCR-ABL thereby inhibiting activation of downstream targets, in particular phosphorylation of CrkL. We have previously demonstrated the sensitivity of rare Ph+ CD34+ CML stem cells (<5% of total CD34+ cells) to the anti-proliferative but, not the pro-apoptotic effects of IM, hence their persistence in vitro. We hypothesised that these rare cells may also behave differently from total CD34+ cells in terms of their kinase response to IM. The effect of IM on the phosphorylation status of CrkL was therefore investigated, in total CD34+ and in primitive CD34+/38- cells from 6 untreated chronic phase CML patients. Ph+ CD34+ and CD34+/38- cells were cultured in serum free medium for up to 72h, in the presence and absence of IM (5mM). After 16 and 72h the phosphorylation status of CrkL was measured with an antibody specifically designed to recognise only the phosphorylated form of the protein. After 16h total CD34+ cells showed a significant dephosphorylation of CrkL in response to IM. However, after 72h the remaining viable BCR-ABL+ cells failed to show any de-phosphorylation of CrkL, consistent with an IM selected, resistant population of stem cells. Primitive CD34+/38- cells from the same patients exhibited no de-phosphorylation of CrkL after either 16 or 72h, confirming their innate/inherent resistance to the drug. These data show, for the first time, that very primitive CML cells exhibit BCR-ABL dependent resistance (i.e. BCR-ABL kinase remains active during drug exposure) and suggest that the drug is not reaching or binding its target at inhibitory concentrations. Novel approaches to eliminate these cells will be critical to the development of curative strategies for CML.
The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.
Authors: Donato Nicholas J. , Wu Ji , Kong Ling Y. , Lee Francis , Talpaz Moshe
Published: October 2019

BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.