bluuebunny/crossref_metadata_2025_split huggingface dataset:
siRNA-Mediated Silencing of αII Spectrin Gene Expression in Cells Containing DNA Interstrand Cross-Links and Its Relationship to Fanconi Anemia.
Authors: Lambert Muriel W. , McMahon Laura W. , Lefferts Joel A. , Sridharan Deepa M. 
Published: October 2019

Abstract
Fanconi anemia (FA) is characterized by bone marrow failure, a predisposition to cancer and, at the cellular level, a hypersensitivity to DNA interstrand cross-linking agents that correlates with a defect in ability to repair cross-links produced by these agents. We have previously shown that the structural protein, nonerythroid a spectrin (αSpII∑*), binds to DNA containing interstrand cross-links and plays an important role in the repair of this type of damage and that there is a deficiency in this protein in cells from FA-A, FA-B, FA-C, FA-D1, FA-F and FA-G cells. We have also shown that after damage to normal cells, αSpII∑* co-localizes in nuclear foci with FANCA, FANCF and the DNA interstrand cross-link repair protein XPF. In FA-A cells, where there is a deficiency in αSpII∑*, damage-induced nuclear foci formation is significantly reduced. In order to further assess the functional importance of αSpII∑* in the repair process and the DNA repair defect in FA cells, studies were carried out on siRNA-mediated silencing of αII spectrin gene expression. Three siRNA oligonucleotide templates were synthesized, which were targeted for different αII spectrin gene sequences. The 21-mer siRNAs produced were purified and normal human lymphoblastoid cells were transfected with these siRNAs. αII spectrin mRNA and protein levels in transfected cells were determined by RT-PCR and Western blot analysis, respectively. By 48 hours after transfection, levels of αII spectrin mRNA and protein were approximately 34% and 30% of mock transfected cells, respectively. Cells were then damaged with 8-MOP plus UVA light so as to produce DNA interstrand cross-links and localization of αII spectrin and FANCA in the nuclei was examined by immunofluorescence microscopy. The results showed that, in cells transfected with αII spectrin siRNA, there was a marked reduction in the number of αII spectrin and FANCA foci in the nuclei of the siRNA transfected cells compared to the mock transfected cells. There was also markedly reduced survival of the siRNA transfected cells after damage compared to mock transfected cells. These results show that there is a correlation between a reduction of αII spectrin levels in these cells and decreased formation of FANCA as well as αII spectrin nuclear foci after damage with a DNA interstrand cross-linking agent and that this in turn correlates with decreased survival and DNA repair in these cells after DNA damage. These results support our model that αII spectrin is needed in the DNA repair process where it acts as a scaffold in the recruitment and alignment of FANC and repair proteins at sites of DNA damage and that, in FA cells, where there is a deficiency in αII spectrin, this recruitment and repair are defective.
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Prospective Evaluation of a GvHD-Specific Proteom Pattern.
Authors: Weissinger Eva M. , Kaiser Thorsten , Kamal Haytham , Kolb Hans-Jochem , Holler Ernst , Ganser Arnold , Hertenstein Bernd 
Published: October 2019

Abstract
We have recently described and published a proteom pattern specific for the early diagnosis of acute GvHD, based on the application of capillary electrophoresis (CE) and mass spectrometry (MS). Here we report the application of the previously described GvHD-pattern, consisting of 29 polypeptides discriminatory for differential diagnosis of GvHD, towards the analysis of urine of a new group of 35 patients in the follow up phase after allogeneic HSCT. The samples were collected prospectively and analysed in a blinded fashion.
Fifteen patients were transplanted from matched unrelated donors, while 20 received stem cells from family donors. GvHD prophylaxis was metotrexat and cyclosporin A. Urine samples were collected prior to conditioning and once a week during the follow up phase. Screening of the patients’ urine with CE-MS yielded between 500 and 2500 polypeptides defined via their mass, charge, and retention time in the CE-MS. These polypeptides were depicted as a three dimensional picture (contour plot, Diapat) and the data for each individual patient were stored in a Microsoft Access data base. The evaluation of urine of 40 patients after HSCT (5 autologous, 35 allogeneic) led to the establishment of a GvHD-specific pattern, consisting of 16-GvHD-specific and 13- sepsis-specific polypeptides (Kaiser et al., Blood 2004). Sixteen differentially excreted polypeptides formed a pattern of early GvHD markers, allowing discrimination of GvHD from patients without complications with 82% specificity and 100% sensitivity, cross validated. Inclusion of 13 sepsis-specific polypeptides allowed to distinguish sepsis from GvHD with a specificity of 97% and a sensitivity of 100%. Sequencing two prominent GvHD-indicative polypeptides led to the identification of a peptide from leukotriene A4 hydrolase and a peptide from serum albumin. The comparison of the newly collected data to the GvHD-specific patterns will be presented.
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Dysregulation of MMSET Is Tumorigenic In-Vivo.
Authors: Chesi Marta , Naik Kruti , Robbiani Davide F. , Affer Maurizio , Nickerson Helen D. , Bergsagel Peter L. 
Published: October 2019

Abstract
Approximately 15% of multiple myeloma (MM) is characterized by a t(4;14) translocation that causes the simultaneous dysregulation of MMSET on der(4) and fibroblast growth factor receptor 3 gene (FGFR3) on der(14). We reported several lines of evidence indicating a role for FGFR3 in myeloma tumorigenesis. First, activated FGFR3 is an oncogene capable of transforming fibroblasts. Second, FGFR3 activating mutations are acquired by MM cells during tumor progression. Third, targeted inhibition of FGFR3 leads to terminal differentiation and apoptosis in two t(4;14) MM cell lines. However, expression of FGFR3, but never of MMSET, is lost in about 25% of t(4;14) MM. Therefore, the overexpression of MMSET in all MM tumors with a t(4;14) translocation, and its homology to MLL, the oncogene on 11q23 translocated in acute leukemia suggest a critical role for MMSET in MM.
To determine whether MMSET is an oncogene in vivo, we have generated transgenic mice in which MMSET expression in driven in lymphocytes by the lck proximal promoter juxtaposed to the Emu enhancer. Using the same expression vector we and others have obtained specific, high levels of transgene expression in B and T cells from spleen, bone marrow and thymus. Four transgenic lines were generated and although we detected MMSET expression in T cells in each of them, unexpectedly no expression in B cells was seen. This is consistent with our inability to ectopically express MMSET in B cell lines. Nevertheless B lymphoid tumors expressing MMSET developed at 23 month of age in each line (18/51 mice). Only 1/19 wild type matching control mice developed a splenomegaly. By Southern blot, monoclonal rearrangements of IgH, IgL and TCR β were detected within the same tumor population.
In conclusion, this is the first report that MMSET is an oncogene capable of transforming lymphoid cells in an animal model. We are currently crossing these mice with FGFR3 transgenic mice to assess cooperation between these two oncogenes in tumorigenesis. Obviously a more restricted expression of MMSET in germinal center cells is required to investigate the role of MMSET in MM. Therefore, as we have done for c-myc, we are generating new transgenic mice in which MMSET expression will be activated sporadically in germinal center B cells by somatic hypermutation.
Bexarotene-Induced T-Cell Immunomodulation and Response in CTCL.
Authors: Porcu Pierluigi , Baiocchi Robert , Buckner Maureen , Byrd John C. , Magro Cynthia M. 
Published: October 2019

Abstract
Cutaneous T-cell lymphoma (CTCL) is a group of chronic lymphoproliferative disorders mostly of skin-homing CD4+ T-cells associated with profound suppression of cell-mediated immunity and loss of T-cell reportoire. The immunological effects of current CTCL therapies and their impact on response have not been studied in large samples of patients. Bexarotene is a synthetic retinoic X receptor (RXR) agonist that induces apoptosis in malignant T-cells and has significant clinical activity in CTCL. Bexarotene also exerts multiple effects on normal T-cells. We investigated the in-vivo immunomodulatory effects of bexarotene in patients with CTCL and correlated them with response. 37 patients (pts) with stage IB-III CTCL (33 Mycosis Fungoides, 1 ALCL, 3 pleomorphic small cell) received oral bexarotene (150–300 mg/m2/day) for a median duration of 13 months (range 4–18). Peripheral blood (PB) T-cell subpopulations were measured by multicolor flow cytometry at baseline and during therapy. Circulating CTCL cells were defined as CD4+ CD7− T-cells. 32/37 patients had an elevated PB CD4/CD8 ratio at diagnosis, regardless of the presence of circulating CTCL cells (3/37 pts) and 33/37 pts had a low absolute CD8+ T-cell count (median 98 cells/mm3, normal 150–1000/mm3). After a median time of 6.5 weeks on bexarotene (range 3.5–12) the CD8+ T-cell count had returned within normal range in 26/33 pts and the CD4/CD8 ratio had decreased in 27/32 pts. Responses (defined as Pysician Global Assessment [PGA] of clinical condition) were observed in 24/37 pts (64.8%). Responders had significantly higher peak CD8+ T-cell counts compared to non-responders (median 975/mm3 vs 221/mm3, P=0.002) and lower CD4/CD8 ratios (median 0.8 vs 2.4, P=0.005). At this time 21 pts have relapsed, with median duration of response 9.5 months. A ≥50% decrease in the PB CD8+ T-cell count preceded cutaneous relapse in 17/21 pts (81%) by a median time of 4.5 weeks (range 3–6.5 weeks). Functional analysis (mitogenic response, cytokine secretion, antigenic repertoire) of PB T-cell subpopulations from these pts at baseline and during therapy with bexarotene is in progress. Bexarotene appears to have a profound in vivo T-cell immunomodulatory effect in CTCL pts. The importance of these immune effects for clinical response vis-a-vis direct induction of apoptosis in CTCL needs to be further studied. If these results are confirmed in larger samples, monitoring of PB T-cell subpopulations may provide clinically valuable information in predicting response and relapse.
Mutations in the Gene of Neutrophil Elastase Associated with Congenital Neutropenia Lead to Alteration of Intracellular Trafficking and Proteolytic Function of NE Protein.
Authors: Beger Carmela , Koellner Inga , Zeidler Cornelia , Germeshausen Manuela , von Neuhoff Nils , Schlegelberger Brigitte , Welte Karl 
Published: October 2019

Abstract
Severe congenital (SCN) and cyclic neutropenia (CyN) are characterized by early-stage maturation arrest of myelopoiesis and often are found associated with different heterozygous mutations in the gene encoding neutrophil elastase (ELA2). Neutrophil elastase (NE) is a serin protease stored mainly in azurophilic granules. However, the consequences of the mutations of this protein up to date are not clear. Recent studies suggest a correlation with incorrect intracellular transport leading to mis-localization of the mutant protein. In the present study, we have generated an ELA2-inducible expression system based on the monoblast-like cell line U937. This system allows controlled expression of the transgene to study functional correlations with specific mutations of ELA2. Applying this system, we have studied intracellular processing and proteolytic activities of ten different mutations of ELA2 that are typically detected in patients suffering from SCN or CyN. We detected two clusters within the neutrophil elastase protein, in which mutations caused alteration of protein glycosylation, associated with prevention of membrane localization of the mutant protein. Instead, the mutant protein was incorrectly directed to the nuclear region. Proteolytic activities of NE protein were measured using different substrates. NE activities were suppressed in most but not all mutations tested. To extend our localization studies, we analyzed the intracellular distribution of NE in granulocytes of patients suffering from CyN or SCN. Again, NE protein of patients was mis-localized to the nucleus correlated with loss of detection in granules and membranes as compared with healthy donors without or with rh-G-CSF treatment. In summary, our data suggest that intracellular processing and localization is altered due to specific mutations of ELA2, which may be crucial for the molecular mechanisms resulting in the neutropenic phenotype.
Characterization of Waldenström Macroglobulinemia Subtypes using Global Gene Expression Profiling.
Authors: Chung Tae-Hoon , Kim Seungchan , Baker Angela S. , Price-Troska Tammy , Xu Ruifang , Ghobrial Irene , Greipp Philip R. , Rajkumar S. Vincent , Gertz Morie A. , Van Wier Scott A. , Ahmann Gregory J. , Carpten John , Fonseca Rafael 
Published: October 2019

Abstract
Waldenström macroglobulinemia (WM) is a lymphoid neoplasm associated with accumulation of lymphoplasmacytic cells in the bone marrow and an elevated monoclonal IgM in the serum. Although the genetic and molecular mechanisms behind the pathogenesis of WM are not well understood, losses of the long arm of chromosome 6 have been found as the only recurrent genomic aberration. These deletions, mostly starting at 6q21, are observed in upwards of 50% of patients and involve the majority of the clonal cells. To better characterize pathologic gene expression signatures in different types of WM at the molecular level (6q deleted versus not), we examined gene expression level changes of more than 20000 human gene probes in bone marrow tissues of clinically proven WM patients. We used Affymetrix HG-U133A v2 chips (n = 24). Clonal cells were purified using a combination of anti-CD138 and anti-CD19 Miltenyi™ magnetic microbeads. Based on cIgM-FISH results, we classified samples into two groups - 6q normal (ND) group and 6q deletion (D) group. We then proceeded to identify genes that showed differential expression between the two groups. We selected 165 differentially expressed genes (DEG) using SAM, and underwent further investigation of biological annotations of them using Gene Ontology™ (GO) terms. Among165 DEG, only 32 showed high level of expression in ND group compared to D group while 133 showed high level of expression in D group. As expected, all genes that reside in 6q region showed under-expression in D group, constituting 53% (17 genes) of genes under-expressed in D group (32 genes). According to GO analysis, the 6q-deletion-induced under-expression affects many biological processes, and is associated with a transcriptional upregulation of downstream genetic cascades. This likely indicates release of regulatory control by a tumor suppressor gene. Genes involved in cell growth and maintenance, cell communication and morphogenesis are also over-expressed in D group. Interestingly, a gene that is involved in innate immune response and inflammatory response shows under-expression in D group but is not accounted for by other mechanisms. In similar vein, a gene that is involved in negative regulation of cytokine biosynthesis showed over-expression in D group. We think these transcriptional characteristics can provide basic framework for differentiating subtypes of WM and search for genes associated with WM pathogenesis
The Antithrombotic Activity of Kininogen Is Mediated by Inhibitory Effects of Domain 3 on Thrombin-Induced Platelet Activation, In Vivo.
Authors: Hassan Sarmina , Khan Mohammad M. , Isordia-Salas Irma , Sainz Irma , Kashem Sakeen W. , Liu Bo , Sartor R. Balfour , Colman Robert W. 
Published: October 2019

Abstract
Background: High molecular weight kininogen (HK) exhibits anticoagulant and profibrinolytic properties and can inhibit platelet activation at low thrombin concentrations. HK acting through domain 3 (D3) is antithrombotic by interfering with the ability of thrombin to bind to the platelet GPIb complex in vitro. We hypothesize that rapid occlusive thrombosis due to de-endothelization of the aorta is due to enhanced platelet activation by thrombin and will be reversed by infusion of D3 or D3-derived peptides.
Methods: F6HKd (deficient in HK) and F6WT (wild-type) strains were produced from offspring of kininogen-deficient Brown-Norway rats by backcrossing to Lewis rats for 6 generations to ensure homogenous genetic background. In the present study, we have expressed kininogen domain 3 (D3) and a fragment of D3, coded for by exon 7, E7P (aaG235-Q292), in E. Coli as glutathione S-transferase (GST) fusion proteins. GST-D3 and either GST-E7P or GST alone was infused intravenously 10 min before inducing the endothelial injury to the abdominal aorta. (Each group n = 5)
Results: Despite an equivalent vascular injury, the mean time (± SEM) for a 90% decrease in flow measured by an ultrasonic probe was 28±5.2 minutes in GST-treated F6HKd rats compared with >240 minutes in the GST-D3 and 233±6.7 in GST-E7P-treated animals. Differences between GST and either GST-D3 or GST-E7P pretreatments were highly significant (p<0.0002). F6WT showed no occlusion at 240 min after injury. In F6HKd rats, TAT concentrations were increased from 32±5.1 to 52±4.1 ng/ml (p<0.007). Similarly D-dimer concentrations were increased from 94±16.3 to 145±9.6 ng/ml (p<0.01) after endothelial injury with only GST pretreatment. With GST-E7P pretreatment, TAT and D-dimer levels were significantly decreased from the GST-treated group (TAT from 52±4.1 to 36±4 ng/ml, p<0.01 and D-dimer from 145±9.6 to 69±11.9 ng/ml, p<0.0007). No significant difference was observed between the GST-E7P pretreated injured and non-injured F6HKd rats or between the F6WT rats which were injured and non-injured.
Conclusions: The results suggest that F6HKd rats are prothrombotic. After endothelial injury thrombotic occlusion occurs and is characterized by thrombin formation with secondary fibrinolysis. Moreover, thrombin-induced platelet activation is inhibited by D3 or the polypeptide coded for by exon 7 product in vivo. These results indicate the importance of HK in modulating arterial thrombosis and the possibility of therapy with HK fragments.
Activated Peripheral Mononuclear Cells Plus Soluble Plasma Tissue Factor Activate Factors VII and X in Cardiac Surgical Wounds.
Authors: Colman Robert W. , Khan Mohammad M.H. , Hattori Takashi , Edmunds4 L. Henry 
Published: October 2019

Abstract
Objectives: This study examines the role of pericardial wound monocytes in thrombin generation during clinical cardiac surgery with cardiopulmonary bypass (CPB).
Background: The mechanism by which wound mononuclear cells rapidly express procoagulant activity is unexplained.
Methods: Factor VII activation (FVIIa) was measured using recombinant, truncated, soluble tissue factor (rsTF) and various blood cells in vitro. FVIIa was also measured with monocytes and soluble plasma tissue factor taken before CPB and simultaneously from the pericardial wound and perfusion circuit during CPB in thirteen patients.
Results: RsTF in combination with monocytes, but not platelets, neutrophils or red cells, accelerates activation of FVII beginning at 1 pmole/L rsTF. Less than 1% rsTF is bound, yet catalytic activity peaks at 7 minutes and decays afterwards. In wound plasma, monocytes are activated (MCP-1 = 29.5 ± 2.1 pmoles/L) and wound plasma tissue factor (wpTF) is substantially elevated (3.64 ± 0.45 pmoles/L) with 81.7% in the supernatant and 18.3% in microparticles. By Western blot all forms of plasma TF migrate at Mr 65 kDa [TF/FVII(FVIIa) complex]. Wound monocytes and C5a activated prebypass or perfusate monocytes plus wpTF convert all available FVII to FVIIa. Activated monocytes plus supernatant TF/FVII(VIIa) more efficiently activate factor X than microparticle TF/FVII(FVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wpTF is 0.944 (p = 0.0004).
Conclusions: During clinical cardiac surgery with CPB wound monocytes plus wpTF or microparticle-free, protein fragments of wound tissue factor preferentially accelerate activation of FVII and FX. This system represents a new mechanism of thrombin generation.
CALGB 90003: Adoptive Immunotherapy by Allogeneic Stem Cell Transplantation for Metastatic Renal Cell Carcinoma: An Intergroup Phase II Study.
Authors: Rini Brian I. , Halabi Susan , Barrier Robert , Margolin Kim A. , Avigan David , Logan Theodore , McCarthy Philip L. , Small Eric S. , Linker Charles A. 
Published: October 2019

Abstract
A graft-versus-tumor effect mediated by non-myeloablative allogeneic stem cell transplantation has been reported for patients with cytokine-refractory, metastatic renal cell carcinoma (RCC). An Intergroup phase II trial was undertaken to further define the feasibility, toxicity and efficacy of this approach in a multi-institutional setting. The conditioning regimen was fludarabine 30mg/m2/d, days −7 to −3, and cyclophosphamide 60mg/kg/d, −4 and −3. Patients received 2 to 8 x 106 G-CSF-mobilized CD34+ cells/kg from a 6/6 HLA-matched sibling donor. Graft-versus-host disease (GVHD) prophylaxis included tacrolimus (target level 5–10 ng/ml) starting day −1 and methotrexate 5 mg/m2 days +1, +3, +6. Tacrolimus was tapered to zero between days +60 and +90. Twenty-two patients were enrolled at 14 different institutions. Patient characteristics included median age of 53, (range, 39–60) and a performance status of 0 in 14 patients and 1 in 8 patients. Prior systemic therapies included 1 regimen in 13 patients, 2 regimens in 5 patients and 3 regimens in 1 patient. One patient failed to engraft and had autologous recovery. Greater than 95% donor T cell chimerism was seen in 10 of 13 patients (77%) by 120 days after BMT. The day 100 regimen-related mortality was 0 of 22 (0%). Acute GVHD was observed in 7 patients (41%). Chronic GVHD was observed in 11 patients (65%) with one patient death from liver failure secondary to chronic GVHD. Eleven patients have died: 7 from disease progression; 1, renal failure (not treatment related); 1, sepsis (pseudomonas); 1, liver failure secondary to chronic GVHD (treatment related); 1, unknown. No objective response has been reported in the 17 patients with follow-up data available. Progressive disease was reported for 14 patients. Median progression-free survival is 4.1 months (95% C.I.: 3.9 – 4.4 months). Median overall survival is 5.6 months (95% C.I.: 4.3 – 12.3 months). Non-myeloablative allogeneic stem cell transplant for metastatic RCC is feasible in a multi-institutional setting. Significant morbidity limits the eligible patient population. Lack of anti-tumor effect highlights the need for stringent patient selection and for further manipulation of the current approach. Non-myeloablative allogeneic stem cell transplantation in metastatic RCC remains investigational.