bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of p38 and ERK MAP kinase in proliferation of erythroid progenitors in response to stimulation by soluble and membrane isoforms of stem cell factor
Authors: Kapur Reuben , Chandra Saurabh , Cooper Ryan , McCarthy Jennifer , Williams David A.
Published: October 2019

Two alternatively spliced stem cell factor (SCF) transcripts encode protein products, which differ in the duration of membrane presentation. One form, soluble SCF (S-SCF) gets rapidly processed to yield predominantly secreted protein. The other form, membrane-associated SCF (MA-SCF) lacks the primary proteolytic cleavage site but is cleaved slowly from an alternate site, and thus represents a more stable membrane form of SCF. Mutants of SCF that lack the expression of MA-SCF (Steel-dickie) or possess a defect in its presentation (Steel17H) manifest deficiencies in erythroid cell development. In this study, we have compared the consequence(s) of activating Kit, the receptor for SCF by MA-SCF with S-SCF, and an obligate membrane-restricted (MR) form of SCF (MR-SCF) on erythroid cell survival, proliferation, cell cycle progression, and the activation of p38 and ERK MAP kinase pathways. Activation of Kit by MR-SCF was associated with a significantly lower incidence of apoptosis and cell death in erythroid cells compared to either other isoform. MR- or MA-SCF–induced stimulation of erythroid cells resulted in similar and significantly greater proliferation and cell cycle progression compared to soluble SCF. The increase in proliferation and cell cycle progression via MA- or MR-SCF stimulation correlated with sustained and enhanced activation of p38 and ERK MAP kinase pathways. In addition, MR- or MA-SCF–induced proliferation was more sensitive to the inhibitory effects of ERK inhibitor compared to S-SCF–induced proliferation. In contrast, soluble SCF-induced proliferation was more sensitive to the inhibitory effects of p38 inhibitor compared with MR- or MA-SCF. These results suggest that different isoforms of SCF may use different biochemical pathways in stimulation of survival and/or proliferation of erythroid cells.
Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor–dependent mechanism: relationship between the procoagulant and permeability effects of TNF
Authors: Friedl Josef , Puhlmann Markus , Bartlett David L. , Libutti Steven K. , Turner Ewa N. , Gnant Michael F. X. , Alexander H. Richard
Published: October 2019

Tumor necrosis factor (TNF) has marked effects on permeability and procoagulant activity on tumor-associated neovasculature when used in isolation perfusion, the latter effect primarily mediated via induction of cell surface expression of tissue factor (TF) on endothelial tissue. However, the cellular events that result in rapid alterations in endothelial cell (EC) permeability after intravascular TNF administration in isolation perfusion are not well characterized. We demonstrate that short exposure intervals to TNF induces TF expression on ECs but has no effect on permeability as assessed by flux of Evans blue–bound albumin across confluent EC monolayers using a 2-compartment model under basal culture conditions. However, a rapid and significant increase in EC permeability occurred with TNF in the presence of factor VIII–deficient plasma. Permeability was induced only with luminal versus abluminal TNF exposure and was blocked by antithrombin III, TF pathway inhibitor, or anti-TF antibody cotreatment. These data indicate that EC surface expression of TF and extrinsic clotting factors are critical in augmenting capillary leak following intravascular TNF administration. Alterations in permeability were associated with intercellular gap formation at sites of down-regulation of vascular endothelial (VE)–cadherin expression, the primary endothelial intercellular adhesion molecule, and intracellular contraction and alignment of F-actin cytoskeletal elements. Rapid induction of TF by TNF may be the primary EC response that results in alterations in permeability and procoagulant activity observed following intravascular TNF administration in isolation perfusion.
B cells immortalized by a mini–Epstein-Barr virus encoding a foreign antigen efficiently reactivate specific cytotoxic T cells
Authors: Moosmann Andreas , Khan Naeem , Cobbold Mark , Zentz Caroline , Delecluse Henri-Jacques , Hollweck Gabi , Hislop Andrew D. , Blake Neil W. , Croom-Carter Debbie , Wollenberg Barbara , Moss Paul A. H. , Zeidler Reinhard , Rickinson Alan B. , Hammerschmidt Wolfgang
Published: October 2019

Lymphoblastoid cell lines (LCLs) are human B cells latently infected and immortalized by Epstein-Barr virus (EBV). Presenting viral antigens, they efficiently induce EBV-specific T-cell responses in vitro. Analogous ways to generate T-cell cultures specific for other antigens of interest are highly desirable. Previously, we constructed a mini-EBV plasmid that consists of less than half the EBV genome, is unable to cause virus production, but still immortalizes B cells in vitro. Mini-EBV–immortalized B-cell lines (mini-LCLs) are efficiently produced by infection of B cells with viruslike particles carrying only mini-EBV DNA. Mini-EBV plasmids can be engineered to express an additional gene in immortalized B cells. Here we present a mini-EBV coding for a potent CD8+ T-cell antigen, the matrix phosphoprotein pp65 of human cytomegalovirus (CMV). By means of this pp65 mini-EBV, pp65-expressing mini-LCLs could be readily established from healthy donors in a one-step procedure. We used these pp65 mini-LCLs to reactivate and expand effector T cells from autologous peripheral blood cells in vitro. When generated from cytomegalovirus (CMV)–seropositive donors, these effector T-cell cultures displayed strong pp65-specific HLA-restricted cytotoxicity. A large fraction of CD8+ T cells with pp65 epitope specificity was present in such cultures, as demonstrated by direct staining with HLA/peptide tetramers. We conclude that the pp65 mini-EBV is an attractive tool for CMV-specific adoptive immunotherapy. Mini-EBVs could also facilitate the generation of T cells specific for various other antigens of interest.
Expression patterns of Th1 and Th2 cytokine genes in childhood idiopathic thrombocytopenic purpura (ITP) at presentation and their modulation by intravenous immunoglobulin G (IVIg) treatment: their role in prognosis
Authors: Mouzaki Athanasia , Theodoropoulou Maria , Gianakopoulos Ioannis , Vlaha Vassiliki , Kyrtsonis Maria-Christina , Maniatis Alice
Published: October 2019

Childhood idiopathic thrombocytopenic purpura (ITP) resolves usually after the first episode, although it may recur, and in 10% to 20% of patients develops into a chronic disorder. Evidence of the immunoregulatory role of Th1/Th2 responses in autoimmune diseases prompted us to perform a prospective study of Th1/Th2 gene expression profiles and transforming growth factor β (TGF-β) plasma levels in 18 children (median age, 6.4 years) with acute ITP, before and after intravenous immunoglobulin G (IVIg) infusion, and during a follow-up period (0.5-5 years). Initially, 12 of 18 patients had either low Th0/Th1 plus interleukin 10 (IL-10) or no in vivo cytokine gene expression (0). At 24 hours after IVIg infusion this pattern became 0 or Th2 (9 of 12) or remained low Th0/Th1 (3 of 12). During follow-up these patients did not relapse and maintained 0 or Th2 pattern without IL-10. Of the remaining 6 patients, 4 presented with a Th1 or Th0/Th1 pattern plus IL-10 that persisted after IVIg treatment (although interferon γ [IFN-γ] expression diminished) and stabilized to Th1 plus IL-10 at follow-up, which was marked by infrequent episodes of ITP. Two patients presenting with a strict Th1 pattern characterized by high expression of IFN-γ, which remained unchanged after IVIg and at follow-up, can be characterized as chronic ITP. TGF-β plasma levels were low in patients with active disease and increased in remission. Overall, acute ITP presents with Th1, Th0/Th1, or 0 in vivo cytokine gene expression. Stable remission is associated with a 0 or Th2 pattern. A 0 or Th2 pattern after IVIg gave the best prognosis, whereas sustained high expression of IFN-γ and refractoriness to IVIg were the main indicators of poor prognosis.
Gene Expression Profile of Acute Myeloid Leukemia (AML) with t(8;16)(p11;p13) and MYST3/CREBBP Rearrangement.
Authors: Camos Mireia , Esteve Jordi , Jares Pedro , Rozman Maria , Colomer Dolors , Villamor Neus , Costa Dolors , Gomis Federico , Nomdedeu Josep , Campo Elias , Montserrat Emili
Published: October 2019

Translocation t(8;16)(p11;p13) is an infrequent chromosomal abnormality in de novo and secondary AML cases, leading to the fusion of MYST3 (MOZ) and CREBBP (CBP) genes, both of them harboring histone lysine acetyl-transferase activity. This AML variety displays specific clinical and biological features, although its gene expression profile is currently unknown. In this study, the genetic signature of AML cases with MYST3/CREBBP rearrangement was compared with the genetic profile of other well-defined AML subtypes. Genotypic analyses using oligonucleotide U133A arrays (Affymetrix) were performed on RNA of 19 AML samples, including t(8;16)-AML (n=3), t(15;17) (n=3), t(8;21) (n=2), inv(16)/t(16;16) (n=3), t(9;11) with AF9/MLL rearrangement (n=2), 3 cases with normal karyotype and flt-3 internal tandem duplication (flt-3 ITD), the three remaining samples corresponding to monocytic cases (M4/M5) without MLL rearrangement nor flt-3 ITD. After unsupervised analysis, cases of AML with t(8;16) clustered together, displaying a differential expression profile. Supervised analysis allowed the identification of the top 53 up-regulated and 28 down-regulated genes. Among the set of genes overexpressed, genes involved in chromatin remodelling and transcription (HOXA9, HOXA10, MEIS1, CHD3, SATB1) and protooncogenes (RET, flt-3, LMO2) were identified. In contrast, CREBBP gene and several members of the JAK-STAT pathway (STAT3, STAT5B, JAK2) were underexpressed. Interestingly, overexpression of multiple homeobox genes was detected in flt-3 ITD cases, some of them as a distinctive finding (HOXA2, HOXA3, HOXB6), and others (HOXA9, HOXA10, MEIS1) were found to be highly expressed in MYST3/CREBBP and MLL-rearranged samples. In conclusion, AML with t(8;16) and MYST3/CREBBP rearrangement shows a distinctive gene expression profile, with some similarities with MLL rearranged leukemias and flt-3 ITD AML cases, thus suggesting a partially common leukemogenic pathway.
Expression of the Human Germinal Center-Associated Lymphoma (HGAL) Protein, a New Marker of Germinal Center B Cell Derivation.
Authors: Natkunam Yasodha , Lossos Izidore S. , Taidi Behnaz , Zhao Shuchun , Lu XiaoQing , Ding Feying , Hammer Anne S. , Marafioti Teresa , Byrne Gerald E. , Levy Shoshana , Warnke Roger A. , Levy Ronald
Published: October 2019

We identified the Human Germinal center-Associated Lymphoma (HGAL) gene in gene expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL was correlated with survival of patients with this diagnosis. The HGAL gene is the human homologue of M17, a mouse gene that had been previously identified to be expressed specifically in normal germinal center B cells. Here we generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC-lymphocytes and in lymphomas considered to be derived from the GC. Among 718 lymphomas tested by immunohistochemistry on tissue microarrays, staining for HGAL protein was present in 97% (103/107) of follicular lymphoma, 100% (40/40) of Burkitt lymphoma, 87% (7/8) of mediastinal large B-cell lymphoma and 70% (103/151) of DLBCL. Nodal and extranodal marginal zone lymphomas lacked HGAL expression. Lymphocyte predominant Hodgkin (70%, 12/17) and, surprisingly, a majority of cases of classical Hodgkin (73%, 78/107) lymphomas were found to be positive.

Double-immunohistologic labeling experiments show that HGAL protein expression overlaps with the expression of BCL6 and CD10 proteins within GCs. Hierarchical clustering of comparative immunohistologic results in 151 cases of DLBCL demonstrates that the expression of HGAL is similar to two other GC-associated proteins BCL6 and CD10, but different from two markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC-specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.
Quantitative Proteomic Profiling of Primary Cutaneous CD30+ T-Cell Lymphoma Progression.
Authors: Reddy Kalpana S. , Crockett David K. , Kadin Marshall E. , Elenitoba-Johnson Kojo S.J. , Lim Megan S.
Published: October 2019

Primary cutaneous CD30+ T-cell lymphoproliferative disorders (LPD) represent a complex spectrum of related conditions that are not very well understood. Many may progress to more aggressive or systemic lymphomas. The factors involved in the progression of these LPDs are largely unknown. Advances in robust high throughput proteomics techniques permit elucidation of the molecular mechanisms underlying disease development and progression. In this study we applied a quantitative proteomics methodology involving isotope coded affinity tag reagents (ICAT™), 3-dimensional liquid chromatography (3-D LC), and tandem mass spectrometry (MS/MS) to evaluate the differential protein expression patterns of two unique T-cell lymphoma derived cell lines. The Mac-1 and Mac-2A are clonally related cell lines derived from the same cutaneous T-cell lymphoma (CTCL) at relatively indolent versus aggressive stages of tumor progression, respectively. Equivalent quantities of total cell lysates obtained from the two cell lines were labeled with cleavable ICAT™ and dialyzed prior to digestion with proteinase K. The labeled peptides were then subjected to strong cation-exchange (SCX) chromatography followed by further purification of each offline fraction using avidin affinity chromatography. These cysteine enriched ICAT™ labeled peptides were analyzed by reverse phase nanospray LC-MS/MS. Differential expression of 490 unique proteins was determined. Of these, a total of 59 proteins showed a 1.5 fold or greater increase in abundance while 86 proteins exhibited a similar decrease in the clinically aggressive lymphoma cells as compared to the more indolent form. Functional classification of the differentially expressed proteins revealed increased expression of those involved in cell signaling (neurotrophic tyrosine kinase receptor, ATP binding cassette transporter 1, SH3 protein interacting with NCK), transcription (HFK1, general transcription factor IIIC), and cell adhesion (ICAM, protocadherin) in the advanced lymphoma. There was also decreased expression in the advanced lymphoma of proteins which were involved in apoptosis (caspase 8), cell signaling (STAT4, LIM kinase), cell cycle regulation (CDC10 homolog, ankyrin 3), cell adhesion (laminin, periostin), and ion transport (voltage-gated potassium channel). Overexpression of MALT1/paracaspase that leads to activation of the NF-kappaB pathway was observed in the advanced lymphoma. Proteins such as Smad4 which is involved in regulation of the TGF-beta signaling pathway and ubiquitin specific protease of the ubiquitin-proteasomal degradation pathway that in turn regulates Smads were differentially expressed. Some of the proteins identified by MS were selected for confirmation by Western blot analysis. This study implicates deregulation of multiple signal transduction pathways during progression of CTCL and provides novel insights into the underlying molecular pathogenetic mechanisms involved.
Identification of a Set of Seven Genes for the Monitoring of Minimal Residual Disease in Pediatric Acute Myeloid Leukemia.
Authors: Steinbach Daniel , Schramm Alexander , Eggert Angelika , Wittig Susann , Pfaffendorf Nadine , Voigt Astrid , Zintl Felix , Gruhn Bernd
Published: October 2019

A stepwise approach which combined genome wide expression profiling and a TaqMan realtime PCR based screening was used to identify new markers for the monitoring of minimal residual disease (MRD) in acute myeloid leukemia (AML). Leukemic cells from 52 children with AML were analyzed. Seven genes were identified which are vastly over-expressed in many patients with AML compared to healthy bone marrow: CCL23, GAGED2, MSLN, SPAG6, and ST18 as well as the previously described markers WT1 and PRAME. This set of genes was analyzed in 141 follow-up samples from 25 patients. The expression of all genes decreased to normal levels in patients who achieved a continuous complete remission. Elevated levels of MRD markers were found prior to relapse in 7 out of 10 patients who relapsed. This set of genes should allow a sensitive and specific monitoring of MRD in AML. Notably, some of these markers could also serve as therapeutic targets or might be involved in leukemogenesis. MSLN is already used as a target for immunotherapy in clinical trials in other malignancies. GAGED2 and SPAG6 belong to the family of cancer testis genes which are also studied intensively as targets for immunotherapy. ST18 is a recently discovered tumor suppressor which was not yet described in hematological malignancies. CCL23 is a chemokine that inhibits the proliferation of healthy hematological stem cells.

Names, symbols, and geneID of seven MRD markers Gene Symbol Gene Name GeneID CCL23 chemokine (C–C motif) ligand 23 6368 GAGED2 G antigen, family D, 2 9503 MSLN Mesothelin 10232 SPAG6 sperm associated antigen 6 9576 ST18 suppression of tumorigenicity 18 9705 WT1 Wilms tumor 1 7490 PRAME preferentially expressed antigen in melanoma 23532
Hypoxia-Inducible Factor 1 Mediated Expression of Hypoxia-Induced Vascular Endothelial Growth Factor in Human Bone Marrow Stromal Cells, Which Is Involved in MAP Kinase and Phosphatidylinositol 3-Kinase Signaling.
Authors: Tang Guangxiao , Liu Gexiu
Published: October 2019

INTRODUCTION: Recent studies indicate that transplantation of bone marrow stromal cells after rat traumatic brain injury provided functional recovery. Hypoxia is the major characteristic of ischemic microenvironment. Vascular endothelial growth factor (VEGF) has been demonstrated in vivo and in vitro to be the principal mediator of hypoxia-induced angiogenesis. Recent studies indicate that VEGF implicated in neuroprotection. We tested the hypothesis that hypoxia induces expression of vascular endothelial growth factor (VEGF) in human bone marrow stromal cells (hBMSC) by hypoxia-inducible factor 1 (HIF-1).

METHODS: Human bone marrow adherent cells were cultured, and were passaged in DMEM/F12 containing 10% FBS. The fifth passage cells were identified as stromal cells. Subconfluent cells were used, and were cultured with hypoxic DMEM/F12 in hypoxia condition (94% N2 + 1% O2 +5% CO2), and were treated with 50 μM PD98059, or 200 nM wortmannin, or SiRNA specific for HIF-1mRNA. Cells were harvested after 6 h for analysis of active phosphorylated kinases (Akt and MAPK) and the HIF-1-alpha protein or after 24 h for analysis of VEGF protein and mRNA. VEGF mRNA was detected by semi-quantitative RT-PCR, and VEGF protein by ELISA, and kinases and the HIF-1-alpha protein by Western blot.

RESULTS: Hypoxia increased significantly level of both VEGF mRNA and VEGF protein in hBMSC s, which were low level in normoxic cultured cells. 24 hours after treatment, ratio of RT-PCR product between VEGF and β-actin reach to (46.25±7.54)% from (19.61±4.57)% of control cells (P<0.01). VEGF protein increased to (142.77±22.33) pg/ml from (45.85±9.69) pg/ml (P<0.01). In addition, hypoxia induced active phosphorylated kinases (Akt and MAPK), and enhanced level of the HIF-1-alpha protein. Moreover, PD98059, or wortmannin, or SiRNA specific for the HIF-1-alpha mRNA inhibited induction of the VEGF gene by hypoxia.

CONCLUSION: The expression of VEGF mRNA and VEGF increased in human bone marrow stromal cells during hypoxia condition, which is involved in MAP kinase and phosphatidylinositol 3-kinase signaling. These results support that these cells play an important role in therapy of ischemic/hypoxic injury such as cerebral ischemia and myocardial ischemia. The hMSC capacity to increase expression of VEGF may be the key to the benefit provided by transplanted hMSCs in the ischemic injuries.
A Comparison of Outcome and Second Malignancies in Adolescents and Adult Patients with Hodgkin’s Lymphoma (HL). Results from the German Hodgkin Study Group.
Authors: Bredenfeld Henning , Gilman Elena , Pfistner Beate , Diehl Volker , Engert Andreas
Published: October 2019

Introduction: Whether adolescent (alc) patients with HL represent a distinct patient group, requiring a therapy strategy separated from adults, is currently focussed in international debates. Most study groups are treating adult (ad) patients (pts) (age > 18 years) apart from younger patients (age < 18 years), considering a difference in treatment related toxicity and therapy outcome in both patient groups. Between 1988 and 1998, the GHSG trial generations 2 (G2) and 3 (G3) included younger patients from the age of 15 (G2), and 16 (G3), in identically therapy regimen with adults up to 65, and 75 years.

The trials for early stages HD were comparing radiation therapy (RX) arms only (HD4, recruited 1988–1993), and, RX vs 2 cycles polychemotherapy plus RX (HD7, 1993–1998). Intermediate stages HD received 2 cycles of different chemotherapy regimen plus identically RX (HD5, 1988–1993), and, 2 identically cycles of chemotherapy plus different RX (HD8, 1993–1998). Advanced stages HL were treated with either 4 cycles conventional polychemotherapy plus EF, or, 4 cycles of a hybrid regimen plus EF RX (HD6, 1988–1993); the following trial (HD9, 1993–1998) compared 3 arms of different polychemotherapy followed by RX on bulky disease.

Methods: In G2 and G3, a total of 573 adolescents (15–21 yrs) in early, intermediate, and advanced stages HL were compared with 4917 pts (15–75 yrs) for complete remission rate (CR), 5 yrs survival rate (SV), 5 yrs freedom from treatment failure (FFTF), and, secondary neoplasias (2nd NPL). (table 1)

Conclusion: Based on this large analysis of HL patients treated in prospectively randomized trials, the prognosis of patients aged 15–21 years is rather similar to the prognosis of adult patients. A more detailed evaluation including age-adapted prognosis will be presented.

Results: Adolescents /Adults CR % 5 yrs FFTF % 5 yrs SV % Alc Ad Alc Ad Alc Ad Early stages HD4 97.7 97.3 81.6 80.5 97.6 95.1 HD7 93.3 94.3 82.0 83.3 100 94.1 Intermediate stages HD5 93.5 93.4 83.3 81.7 94.3 90.5 HD8 93.8 92.6 84.5 82.7 97.3 91.3 Advanced stages HD6 77.8 77.0 61.0 58.8 85.3 78.6 HD9 93.5 90.3 85.8 79.2 92.4 88.0
Hematopoietic Cell Transplantation-Specific Comorbidity Index To Predict Non-Relapse Mortality and Survival after Allografting.
Authors: Kato Ruri , Fukuda Takahiro , Usui Eiji , Yamasaki Satoshi , Maruyama Dai , Morita Yuriko , Kim Sung-Won , Mori Shin-ichiro , Tanosaki Ryuji , Tajima Kinuko , Heike Yuji , Makimoto Atsushi , Tobinai Kensei , Takaue Yoichi
Published: October 2019

Pre-transplant comorbidity can affect the outcome of allogeneic hematopoietic cell transplantation (HCT). The Seattle group recently proposed a new HCT-specific comorbidity index (HCT-CI) which was based on the Charlson Comorbidity Index (Blood, prepublished online, June 30, 2005).

To validate this scoring system, we retrospectively reviewed the medical records of 315 patients with hematologic malignancies who underwent allogeneic HCT after fludarabine-based reduced-intensity (n=160) or conventional (n=155) conditioning at our center between 2000 and 2004. The median age of the patients was 46 (range, 1–68) years. The diagnoses included acute myeloid leukemia or myelodysplastic syndrome (n=153), chronic myelogeneous leukemia (n=30), acute lymphoblastic leukemia (n=36), lymphoma (n=90), and other hematologic malignancies (n=6). Donors included HLA-matched (n=120) or mismatched (n=53) relatives and unrelated volunteers (n=142). Stem cell source was G-CSF-mobilized peripheral blood stem cell (n=169), bone marrow (n=117), or cord blood (n=29). We did not include the pulmonary function test results due to our inconsistency with the test.

The HCT-CI captured 45% of patients with scores >0 (score 1, n=71; score 2, n=22; score 3, n=27; score 4, n=15; score >4, n=6). The capture rate of HCT-CI in patients who received reduced-intensity conditioning was higher than that in those who received conventional conditioning (51% vs 38%). The involved organ systems included hepatic (n=68), recent infection (n=38), prior malignancies (n=17), cardiac (n=16), renal (n=11), metabolic (n=11), psychiatric (n=11), pulmonary (n=8), and gastrointestinal (n=4) abnormalities. The Kaplan-Meier estimate of overall survival was significantly different among risk groups stratified according to HCT-CI (Figure 1, p<0.0001). In Cox proportional hazard models, a higher HCT-CI score, disease risk, and transplant from donors other than HLA-matched relatives were associated with poor overall survival. A higher HCT-CI score, greater patient age, and transplant from donors other than HLA-matched relatives were associated with a significantly increased risk for non-relapse mortality.

In conclusion, the new HCT-CI using pre-transplant variables was the most significant predictor of non-relapse mortality and survival after allografting. Our validation study suggests that this index will be a useful tool for future use in clinical trials and standard practice.

Overall Survival according to HCT-specific Comorbidity Index Overall Survival according to HCT-specific Comorbidity Index
Fanconi Anemia D2 Protein Contributes to BCR/ABL-Mediated Transformation of Hematopoietic Cells.
Authors: Koptyra Mateusz , Houghtaling Scott , Grompe Marcus , Skorski Tomasz
Published: October 2019

Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DNA double-strand breaks (DSBs) induced by reactive oxygen species (ROS) or genotoxic treatment. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts. In addition, although the expression of FANCD2 was stimulated by BCR/ABL and growth factors, higher levels of FANCD2 monoubiquitination was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, monoubiquitination of FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. BCR/ABL kinase displayed an impaired transformation potential in FANCD2-/- murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2-/- human lymphoblast cell line. Growth ability of BCR/ABL-positive FANCD2-/- cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2-/- cells accumulate more DNA damage than +/+ counterparts as indicated by enzymatic assays converting oxidative DNA lesions into gaps detectable by comet assay. In addition, enhanced oxidative DNA damage in BCR/ABL-positive FANCD2-/- cells produced a variety of DNA lesions including abasic sites, and single- and double-strand breaks assessed by neutral comet assay. Moreover, BCR/ABL-positive FANCD2-/- cells accumulated higher numbers of DSBs detected by γ-H2AX immunostaining and displayed discrete apoptosis. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in the initial steps of BCR/ABL dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.
MDM2 Dependant Induction of Error Prone DNA Polymerase beta by EBV-LMP1 Oncoprotein.
Authors: Nathalie Faumont , Christophe Le Clorennec , Pierre Teira , Ibtissam Youlyouz-Marfak , Christine Mazeres , Sebastien Hoffmann Jean , Georges Delsol , Jean Feuillard , Fabienne Meggetto
Published: October 2019

Epstein Barr Virus (EBV) is able to immortalise B lymphocytes in vitro and is associated with several human tumours, such as Hodgkin’s disease (HD), suggesting that this virus could play a role in the initiation of the tumoural process. The LMP1 protein of EBV, coded by the LMP1-BNLF1 gene, was rapidly suspected to be involved in this process due to its oncogenic characteristics. LMP1 functions as a constitutively activated member of the tumour necrosis factor receptor superfamily, and activates trough its carboxyterminal cytoplasmic region several signalling pathways, including NF-kappa B. In order to identify new LMP1 target genes, we studied the transcriptome of the B lymphoid Ba/F3 cell line stably transfected with LMP1-BNLF1 genes from HD Reed-Sternberg tumour cells as well as from normal cells, using the cDNA microarray technique.

We show that the expression of several genes was modulated by the LMP1 protein, independently of the cellular origin of the LMP1 protein. Among overexpressed genes, we focused on the DNA polymerase-beta (beta-pol) gene. To confirm and extend this cDNA result, we demonstrated in different cell lines that the beta-pol protein expression was under the control of LMP1. In vivo and in vitro functional tests showed that LMP1 induced overexpression of beta-pol paralleled its increase in activity.

Regulatory sequences of the human beta-pol gene includes three motives that match the Sp1 factor binding site motive. LMP1 did not modulate Sp1 RNA neither protein expression. By contrast, we observed that MDM2 and beta-pol RNA expression were increased in parallel by LMP1, an effect trigerred by the carboxyterminal region of LMP1. Then, we demonstrated that MDM2 protein overexpression was associated with increased beta-pol protein expression.

Since MDM2 promoter is regulated by NF-kappaB, we investigated whether up-regulation of MDM2 and beta-pol were NF-kappaB pathway dependant. We show that inhibition of NF-kappaB by overexpression of an I-kappaB dominant negative down-regulated both MDM2 and beta-pol expression.

In conclusion, we demonstrated that the oncoprotein LMP1 regulates overexpression of beta-pol through NF-kappaB signaling pathway and the MDM2 protein, and that MDM2 was itself regulated by NF-kappaB. Beta-pol is the more error prone of all the known eucaryotic DNA polymerases. Elevated expression of beta-pol, an event found in many human tumours, has been shown to generate a mutator phenotype which is extensively associated with cancers. Our work leads to the hypothesis that LMP1 protein could play a role in the genetic instability in EBV associated malignant diseases such as HD through over-expression of beta-pol.
Differential Expression of the Homeobox Gene MIXL1 in Non Hodgkin (NHL) and Hodgkin Lymphomas (HL).
Authors: Drakos Elias , Rassidakis George Z. , Guo Wei , Medeiros L. Jeffrey , Nagarajan Lalitha
Published: October 2019

The gene MIXL1 (Mix1 homeobox-like) encodes a paired class homeobox transcription factor involved in early hematopoietic specification during embryogenesis. Previous studies have shown that MIXL1 gene is expressed in hematopoietic cells during adult life (Guo et al. Blood100;1;89–96, 2002). Furthermore 5′ MIXL1 sequences are a target of retroviral insertion in murine T-cell lymphoma (http:RTCGD.ncifcrf.gov), suggesting a selection advantage for aberrant expression of this gene. However, the status of MIXL1 expression in human lymphomas has not been examined. Using a highly specific antibody, we assessed for MIXL1 protein expression in 14 lymphoma cell lines (9 B-cell and 5 T-cell) by immunobloting. MIXL1 was detected predominantly in nuclear extracts of lysates of all cell lines tested, although at a variable level. We also assessed for MIXL1 protein expression in 126 B-cell and 21 T-cell NHLs of various types, as well as 14 Hodgkin lymphomas using immunohistochemical methods. The results of the immunohistochemical studies are summarized in table 1. Once again, MIXL1 immunoreactivity was primarily nuclear in the tumor cells. Based on distribution data (histogram), a 50% cutoff was selected for high versus low MIXL1 expression. Among B-cell tumors, high expression levels of MIXL1 protein were more frequently detected in high-grade NHL and HL compared with low/intermediate grade NHL (p<0.0001, chi-square test). As a continuous variable, the percentage of MIXL1-positive tumor cells was also significantly higher in high-grade B-cell NHL and HL compared with low/intermediate grade NHL (p<0.0001, Kruskal Wallis test). All Hodgkin lymphomas expressed high levels of MIXL1 with 60% to 100% of neoplastic cells being positive for MIXL1. Most T-cell NHLs also expressed high levels of MIXL1. In contrast, most low/intermediate-grade B-cell NHL and multiple myelomas expressed low levels of MIXL1. Frequent overexpression of MIXL1 gene product in most high-grade B-cell NHLs, HL and T-cell NHLs suggests that aberrant expression of MIXL1 may play a role in proliferation, block of differentiation or both.

Table 1. HL (n=14) B-NHL (n =126) T-NHL (n =21) N (%) Low/intermediate grade N (%) N (%) Classical HL 12/12(100%) Chronic lymphocytic leukemia /small lymphocytic lymphoma 0/8 (0% T-precursor lymphoblastic leukemia/lymphoma 2/2 (0%) Nodular lymphocyte predominance HL 2/2 (100%) MALT-lymphoma 0/8 (0%) Mycosis fungoides/Sezary syndrome 2/2 (0%) Follicular lymphoma 9/24 (38%) Extranodal NK/T-cell lymphoma, nasal type 3/3 (100%) Mantle cell lymphoma 5/34 (15%) Peripheral T-cell lymphoma, unspecified 6/9 (66% High grade Anaplastic large cell lymphoma 5/5 (100%) B-precursor lymphoblastic leukemia/lymphoma 1/3 (33%) Burkitt lymphoma/leukemia 2/2 (100%) Diffuse large B-cell lymphoma 30/31 (97%) Plasma cell myeloma/plasmacytoma 0/16 (0%)
Management of HIV Associated Thrombotic Thrombocytopenic Purpura.
Authors: Scully Marie , Miller Robert , Cohen Hannah , Roedling Sherie , Edwards Simon , Machin Samuel J.
Published: October 2019

Thrombotic thrombocytopenic purpura (TTP) is a life threatening disorder characterised by thrombocytopenia and microangiopathic haemolytic anaemia associated with a deficiency in ADAMTS 13. Human Immunodeficiency Virus (HIV) remains an important infectious cause of TTP. We report the characteristics and treatment of patients presenting over a 6.5 year period to a tertiary referral centre. Cases: 9 patients, 8 female, 1 male, all heterosexual Africans. In 5 of the patients, TTP presentation was associated with the initial diagnosis of HIV. Only 1 patient was on highly active anti-retroviral therapy (HAART) at diagnosis. Median haemoglobin on admission was 6.4 (range 4.6–7.2) g/dl, platelet count-12 (range 5–53) X 109/L, lactate dehydrogenase (LDH) 1401 (range 914-2532) IU/L. In 6 patients there were neurological features at presentation, from headache, to transient ischaemic attacks and coma;6 patients had renal impairment, median creatinine 127 (range 63–418) μmol/L. Notably, HIV associated TTP was not a late onset disorder associated with poor CD 4 counts and poor prognosis. Median CD 4 counts were 170 (range 70–400) cells/μl, with a median viral load 74,400 copies/ml. ADAMTS 13 activity ranged from <5%–50% (normal range 66–126%) with no evidence of inhibitor by mixing studies using collagen binding assay. All patients received single volume plasma exchange (PEX) with cryosupernatant, median 8 (range 3-16) until remission and started HAART as soon as the diagnosis was made. Two patients received fresh frozen plasma (FFP) replacement for PEX for refractory TTP: one had coma and suboptimal treatment pre-referral, the second patient had an initial speedy recovery, but developed septicaemia. Two patients stopped their anti-retroviral therapy and had a TTP relapse within 3 months. However, recommencement of HAART plus 3 and 6 PEX’s respectively was associated with remission. Six of the patients remain in remission from TTP at 6–74 months; 2 were lost to follow-up and 1 died 18 months later from renal failure, unrelated to TTP. These data suggest HIV status should be checked in all patients presenting with acute TTP as it accounted for 12.5% of all acute TTP admissions in our cohort. All HIV associated TTP patients were black African heterosexuals. Treatment with PEX and HAART was associated with speedy remission of TTP. In patients who stopped HAART, TTP relapsed within 3 months. The abscence of antibody to ADAMTS 13 suggests a possible direct effect of the retrovirus on endothelium. With HAART and a decrease in viral load, ADAMTS 13 activity increases as patients achieve remission.
Adoptive Transfer of Nf1+/− Bone Marrow Is Sufficient for Neurofibroma Progression in Krox20;Nf1flox/flox Mice.
Authors: Yang Fengchun
Published: October 2019

Mutations in the NF1 tumor suppressor gene cause neurofibromatosis type 1 (NF1), a GTPase activating protein for Ras called neurofibromin. NF1 is a genetic disorder that affects approximately 250,000 individuals in the US, Europe, and Japan alone. Neurofibromas, the hallmark of NF1, are complex tumors characterized by tumorigenic Schwann cells, neoangiogenesis, fibrosis, and degranulating mast cells. Studies in experimental models have emphasized the role of inflammatory cells in altering the microenvironment and facilitating malignant outgrowth. Similarly, Parada (Science, 2002) found that nullizygosity of Nf1 in Schwann cells of conditional knockout mice (Krox20;Nf1flox/flox) was necessary but not sufficient for neurofibroma formation and haploinsufficiency of Nf1 in lineages within the tumor microenvironment was required for neurofibroma progression. We previously provided the first genetic, cellular, and biochemical evidence that haploinsufficiency of Nf1 alters Ras activity and cell fates in mast cells (JEM, 2000, 2001) and identified a mechanism underlying the recruitment of mast cells to tumorigenic Schwann cells (JCI 2003). However, it remains unclear whether Nf1 +/− bone marrow derived hematopoietic cells can directly contribute to neurofibroma formation in vivo. To address this question, Nf1+/− or wildtype (WT) EGFP+ bone marrow (BM) was adoptively transferred into lethally irradiated Krox20;Nf1flox/flox mice and cohorts were followed prospectively for tumor formation using positron emission tomography and computerized axial tomography. Mice transplanted with Nf1+/− but not WT BM developed progressive enlargement of the trigeminal nerve, dorsal root ganglia, peripheral nerves, and motor paralysis similar to Krox20;Nf1flox/− mice that are haploinsufficient at Nf1 in all lineages of the tumor microenvironment. Postmortem analysis revealed that Krox20;Nf1flox/flox mice transplanted with Nf1+/− BM had cellular neurofibromas containing Schwann cells, fibroblasts, blood vessels and mast cells, which closely resembled the cellular architecture of human neurofibromas. Mice transplanted with WT BM did not develop neurofibromas. These studies establish that recruitment of Nf1 +/− BM derived cells to the neurofibroma microenvironment is directly linked to neurofibroma formation and progression. Given our observations, therapies which prevent both the recruitment and the tumor promoting functions of Nf1 +/− hematopoietic cells in neurofibroma formation are currently being tested in vivo as pre-clinical trials.
A Model of Severe Congenital Neutropenia Reveals Signaling Pathways Mediating Mutant Elastase-Triggered Agranulocytosis.
Authors: Aprikyan Andrew A. , Vaisar Tomas , Makaryan Vahagn , Heinecke Jay
Published: October 2019

Severe congenital neutropenia (SCN; Kostmann’s syndrome or infantile genetic agranulocytosis) defines an inheritable hematopoietic disorder of impaired neutrophil production due to a “maturation arrest” at the promyelocytic stage of differentiation in the bone marrow. SCN patients have recurring severe infections and often develop acute myelogenous leukemia. We and others reported accelerated apoptosis and cell cycle arrest of bone marrow-derived myeloid progenitor cells in SCN patients with autosomal dominant and autosomal recessive inheritance. Heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease, are present in a majority of SCN patients, but not in healthy members of the family, thus indicating a key role of mutant NE in pathogenesis of this disorder. To date, there are no animal or cellular models of SCN as both the knock-in of mutant NE as well as the knock-out of normal NE failed to result in neutropenia phenotype in mice. The molecular mechanisms of mutant NE-mediated severe neutropenia remain largely unknown.

We hypothesized that mutations in NE expose the protease to a new range of substrates. To explore this proposal, we established a cellular model of SCN based on tetracycline-regulated expression of mutant NE in human promyelocytic tet-off HL-60 cells that very closely recapitulated the human phenotype. Mutant NE expression resulted in a characteristic block of myeloid differentiation - the cellular hallmark of SCN. Expression of the mutant product was associated with a significant reduction in phosphatidylinosytol-3-kinase and phosphorylated PKB/Akt levels and an imbalance of anti-apoptotic Bcl-2 and pro-apoptotic Bax. These alterations contributed to observed dissipation of mitochondrial membrane potential as determined by FACS analysis, aberrant release of cytochrome C, and accelerated apoptosis. Marked changes in actin cytoskeleton that made the cells more rigid appeared to stem from a reduced level of alpha-actinin and elevated level of Rho GTPase.

Immunoprecipitation of cell lysates with elastase-specific monoclonal antibodies followed by mass spectrometric analysis revealed that NE interacted with histone H2B, one of the key components of the nucleosome core of the chromatin. Interestingly, the expression level of histone H2B was substantially reduced in cells expressing mutant NE, therefore supporting the notion of altered substrate specificity of mutant NE. Thus, these observations provide the first evidence that mutant NE affects specific signaling pathways that lead to alterations in cytoskeleton and chromatin reorganization, subsequent apoptosis, and a block of myeloid differentiation in SCN. This cellular model of SCN should provide an invaluable tool for screening potential therapeutic agents capable of preventing maturation arrest and leukemogenesis in subjects suffering from severe congenital neutropenia.
F8 Gene Mutations and ITT Response in the Italian Cohort of Haemophilia A Patients with Inhibitors.
Authors: Tagariello Giuseppe , Salviato Roberta , Belvini Donata , Radossi Paolo , Zanon Ezio , Gandini Giorgio , Castaman Giancarlo , Sartori Roberto
Published: October 2019

Introduction: Immune tolerance therapy (ITT), consists of administration of daily high doses of factor VIII concentrates by intravenous infusions. It can induce tolerance to the exogenous protein and eradicate the antibody response to FVIII, which still represents the main complication of replacement therapy in haemophilia A (HA) severe cases.

Methods: We have investigated 71 HA patients with inhibitors (sixty-nine severe, one moderate and one mild cases). We screened the patients for the causative mutations in the F8 gene using Long Range PCR for the intron22 inversion, multiplex-PCR for intron1 inversion or conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing for other mutation types.

Results: Diverse genetic defects were detected in the severe cases, with a predominance of gross mutations: F8 gene inversions, large deletions and nonsense mutations account for 68% of the mutations, whereas in the two non severe patients specific missense mutations were identified. ITT has been attempted in 16 HR patients of this cohort but failed in 5 cases.

Conclusion: We confirmed that the presence of inhibitors correlates well with the presence of null mutations, as reported by Schwaab et al (Thromb Haemost 1995). Several predictive factors for ITT outcome have been so far taken into consideration but no correlation has been made between F8 gene defect and ITT response. In our cohort 2 large deletions, 2 intron22 inversions and 1 nonsense mutations failed to respond. For the remaining 2 large deletions no ITT were attempted so no information are available. Large deletions appear to be a high risk genetic factor both for inhibitor development and for long term inhibitor persistence and ITT unresponsiveness. The identification of these patients at very high risk of inhibitor development and ITT unresponsiveness by mutation analysis should therefore be strongly recommended soon after the diagnosis, even because of the high cost of the therapy.
Concomitant Induction of CMVpp65-Specific CD4+ and CD8+ T Cells Using Dendritic Cells Transfected with mRNA Encoding an Invariant Chain-pp65 Fusion Protein.
Authors: Le Ngocdiep , Dannull Jens , Chao Nelson , Vieweg Johannes
Published: October 2019

Human cytomegalovirus (CMV) disease is increasingly recognized as a major cause of morbidity and mortality in post-stem cell transplant (SCT) recipients due to a lack of cellular immunity. Thus, novel therapies that offer the restoration of cellular immunity in SCT patients are highly desirable for clinical use. Cytotoxic T lymphocyte (CTL) responses against CMV represent a major effector arm of the immune system to control viremia. However, it is well established that the efficient induction and persistence of CTL responses in vivo requires the concomitant induction of antigen-specific CD4+ T helper cells. In this study, we sought to determine whether human dendritic cells (DC) transfected with mRNA encoding an invariant chain-CMVpp65 fusion protein (Ii-pp65) were capable of inducing concomitant CMV-specific CTL and CD4+ responses, thereby constituting a useful strategy for immunotherapy of CMV disease. We show that transfection of DC with Ii-pp65 mRNA leads to enhanced stimulation of CMV-specific CTL in vitro (Figure 1). Furthermore, DC expressing Ii-pp65 are potent inducers of primary CMV-specific CD4+ T cell responses as evidenced by ELISPOT analyses of primed CD4+CD45RA+ T cells (Figure 2). Lastly, efficient routing of Ii-pp65 into the MHC class II presentation pathway is demonstrated by confocal microscopy (data not shown). Based on these preclinical findings, we propose a clinical trial to administer DC, transfected with mRNA encoding a chimeric Ii-pp65, to post-SCT patients. Our primary goal will be to prevent CMV infection and reactivation by inducing strong immune reactivities against CMV.

Figure Figure