bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of Retroviral Vector Insertion Sites after T-Cell Directed Gene Therapy.
Authors: Konno Akihiro , Jagadeesh G. Jayashree , Moratto Daniele , Bosticardo Marita , Holt Ingeborg , Muul Linda M. , Schurman Shepherd H. , Wolfsberg Tyra , Candotti Fabio
Published: October 2019

Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking.

We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p<0.002) and CpG islands (p<0.001).

These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.
Mechanisms of Erythropoietin-Mediated Cardioprotection during Ischemia-Reperfusion Injury: Role of Protein Kinase C Signaling.
Authors: Arcasoy Murat O. , Hanlon Paul , Fu Ping , Steenbergen Charles , Murphy Elizabeth
Published: October 2019

The biologic effects of erythropoietin (EPO) are mediated by its cellular receptor EPOR, a member of the cytokine receptor superfamily. EPOR expression in non-hematopoietic cells is associated with novel biologic effects for EPO in diverse organ systems. We recently demonstrated functional EPOR expression in adult rat cardiac myocytes and found that recombinant EPO exerts a rapid cardioprotective effect during ischemia-reperfusion injury of the isolated, perfused heart. Here we investigated the mechanisms of the cardioprotective effect of EPO using Langendorff-perfused rat hearts while left-ventricular-developed pressure (LVDP) was measured continuously to assess contractile function. Hearts were treated directly with EPO in the presence or absence of inhibitors of specific signal transduction pathways prior to normothermic global ischemia followed by reperfusion. Post-ischemic recovery of contractile function was determined by measuring LVDP at the end of reperfusion and expressed as a percentage of the baseline pre-treatment measurement. We investigated EPO-mediated activation of signal transduction pathways in the isolated, perfused heart and observed phosphorylation of p44/p42 MAP kinases ERK 1/2 (Thr202/Tyr204) and protein kinase B/Akt (Ser473), a downstream target of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Furthermore, EPO treatment of the isolated, perfused heart was associated with translocation of protein kinase C (PKC) ε and δ isoforms to the membrane fraction. We investigated the role of specific signaling pathways in EPO-mediated cardioprotection by employing inhibitors targeting PI3K, PKC and MAP kinase kinase (MEK1). PI3K inhibitors LY294002 and wortmannin attenuated EPO-induced phosphorylation of Akt but had no effect on EPO-mediated cardioprotection. MEK1 inhibitor U0126 had no effect on EPO-mediated cardioprotection. The PKC catalytic inhibitor chelerythrine (chel) significantly inhibited EPO-mediated improvement in post-ischemic recovery of LVDP (figure 1). Hearts pre-treated with EPO exhibited significantly improved post-ischemic recovery of LVDP compared to control hearts (mean±SE: 72±3 in EPO-treated versus 35±3% in control hearts, P<0.05 by ANOVA and Bonferroni post-hoc test, n=10 experiments each group) and the protective effect of EPO was significantly inhibited in chel-treated hearts (52±4% in EPO+chel versus 72±3% in EPO-treated hearts, P<0.05, n=10). As a control, treatment of the hearts with chelerythrine alone had no significant effect on LVDP (49±4%) compared to control hearts. These data demonstrate that EPO-mediated activation of the PKC signaling pathway is required for the cardioprotective effect of EPO during ischemia-reperfusion injury.

Figure Figure
Autologous Stem Cell Transplantation (ASCT) as Consolidation Therapy for Patients with Low-Intermediate (LI) Risk Diffuse Large B-Cell Lymphoma (DLBCL) and Overexpression of Bcl2 Protein. Results of the First Interim Analysis of the GELA Trial LNH98-B2.
Authors: Morel Pierre , Mounier Nicolas , Briere Josette , Ferme Christophe , Coiffier Bertrand , Tilly Herve , Gaulard Philippe , Lederlin Pierre , Reyes Felix , Gisselbrecht Christian
Published: October 2019

The anti-apoptotic protein bcl2 expression is associated with treatment failure in elderly patients (pts) with DLBCL (Blood 101:4279,2003). The ACVBP regimen with conventional consolidation therapy (JCO 18:3025,2000) yield a 2-year (yr) EFS at 71% in DLBCL pts aged 60 yrs or less with only 1 adverse age-adjusted International-Prognostic-Index factor (LI risk pts). To assess if ASCT reduces treatment failure in these pts, we initiated a trial stratified by bcl2 expression. Centralized staining for bcl2 was prospectively performed within 6 weeks after inclusion. Induction regimen consisted of 4 courses of ACVBP given every 2 weeks. In case of response, bcl2 negative pts received the conventional consolidation regimen, whereas bcl2 positive pts underwent peripheral blood stem cell collection after the 4th cycle, and ASCT (mitoxantrone 45 mg/m2, cyclophosphamide 1.5 g/m2 x 4d, etoposide 250 mg/m2 x 4d and carmustine 300 mg/m2) between d80 and d90. From 1999 to 2002, 272 LI-risk pts aged 18 to 59 (median 46, M/F=1.4) presented with bcl2 overexpression (151) or without (121). There was no difference in initial characteristics, toxicity of induction and complete response (CR+CRu) rates between bcl2 positive and bcl2 negative pts (78% and 82%, respectively, p = 0.38). ASCT was not performed in 35 pts, mainly because of early failure (37%). Although bcl2 negative pts retained a better 2-yr survival than bcl2 positive pts (95% vs. 86%, p=0.01) with a median follow-up of 21 months, 2-yr EFS and 2-yr DFS did not differ significantly (84% vs. 79%, p=0.33, and 89% vs. 87%, p=0.94 respectively). This analysis demonstrates the feasibility of taking bcl2 expression into account for the design of multicentre protocol. The lack of difference in EFS and DFS between bcl2 positive pts who received ASCT and bcl2 negative pts treated with conventional consolidation suggested that ASCT may overcome the adverse prognostic value of bcl2 expression in LI-risk pts who achieve CR or Cru.
Identification of Genes Potentially Involved in Disease Transformation of CML.
Authors: Janssen Jeroen J. , Klaver S. Marjolein , Waisfisz Quinten , Pasterkamp Gerard , de Kleijn Dominique P. , Schuurhuis Gerrit J. , Ossenkoppele Gerrit J.
Published: October 2019

In patients with chronic myeloid leukemia who do not reach a (near) complete cytogenetic response upon treatment, the disease progresses over months to several years from an indolent, chronic phase into a rapidly fatal blast crisis. Events that are responsible for this transformation process are largely unknown. To identify changes occurring in the course of the disease, we performed cDNA subtraction on sequentially stored peripheral blood mononuclear cell pellets, collected from a single CML patient throughout the course of disease. Thirty-three differentially expressed sequences were identified, of which 28 corresponded to known genes. Quantitative PCR confirmed cDNA subtraction results for 24 of 25 tested genes. Differential expression levels ranged from <2- to 106-fold. The sequences that were identified include genes encoding for several apoptosis related proteins, cell cycle proteins, interleukins/chemokines, heat shock proteins and a DNA repair enzyme (see table). Thus far, expression levels of eight genes have been tested in sequential samples of three to six additional CML patients, gathered throughout their disease course. PBCEF, IL8, IL6, CCL4 and YWHAZ show comparable patterns as the index patient; expression levels of APEX, NXF1 and PRG1 were consistent with those of the index patient in only a part of the additional patients. More patients are being tested on these and remaining genes. We conclude that this set of genes can be considered as a valuable starting point for further research on the causes of disease transformation in CML.

Name of gene Accession No. Fold difference (Q-PCR) * Genes hypothetically encoding proteins, ** Reverse to subtraction result, ***orf: open reading frame diagnosis > blast crisis: PRG1 NM_002727 14 IVNS1ABP NM_006469 <2 MPO NM_000250 ND blast crisis > diagnosis: EMR1 NM_001974 <2 CD96 NM_198196 3 GAS2 NM_005256 12 CHI3L2 NM_004000 11 late chronic phase > blast crisis: IL8 NM_000584 2 S100A8 NM_002964 16 FPR1 NM_002029 51 LYZ NM_000239 ND MEN1 AF001893 ** blast crisis > late chronic phase: MMRN NM_007351 6 NACSIN NM_015252 24 LARS NM_020117 7 FLJ25286* NM_152546 ND C5orf13*** NM_004772 ND C15orf15*** NM_016304 ND FLJ10849* NM_018243 ND diagnosis > late chronic phase: DNAJB1 NM_006145 35 HSPA8 NM_006597 5 NXF1 NM_006362 2 DC2 NM_021227 <2 APEX1 NM_080648 4 RPS6 NM_001010 <2 EEF1A1 NM_001402 3 FLJ25286* NM_152546 ND late chronic phase > diagnosis: SAT NM_002970 7 CCL4 NM_002984 106 YWHAZ NM_145690 3 PBEF1 NM_182790 7 IL6 NM_000600 42 IL8 NM_000584 9
STAT5 and STAT5A Signaling Play an Important Role in the Pathophysiology of the CML-Like Myeloproliferative Disorder in Mice.
Authors: Ye Dan , Li Li , Ilaria Robert L.
Published: October 2019

Although numerous studies have implicated signal transducers and activators of transcription (STAT)5 in Bcr/Abl-dependent transformation and survival, the role of STAT5 in chronic myelogenous leukemia (CML) has not been fully elucidated. In mice, the CML-like myeloproliferative disorder can occur in the absence of STAT5A/5B, but the repopulating and colony-formation defects of STAT5A/5B double knockout (−/−) bone marrow (BM) cells have made it difficult to separate Bcr/Abl-specific and Bcr/Abl-non-specific effects. To clarify the role of STAT5 signaling in P210BCR/ABL leukemogenesis, we have expressed Bcr/Abl in primary murine STAT5A-deficient (−/−) BM cells, which have no demonstrable intrinsic hematopoietic defects. Interestingly, the absence of STAT5A profoundly decreased the incidence of the pure CML-like disease. Only 24% of wild-type mice reconstituted with P210-transduced STAT5A(−/−) BM cells developed CML, compared to 93% of P210-STAT5A(+/+)- and 83% of P210(+/−)-reconstituted mice. The remainder of P210-STAT5A(−/−)-reconstituted mice developed an acute lymphoblastic leukemia (ALL)-like disease (20%) or a CML/ALL mix (56%), underscoring the relative importance of STAT5 signaling in P210-induced myeloid leukemia compared to P210-induced lymphoid leukemia. There was also a trend for increased latency of the CML-like disease in P210-STAT5A(−/−)-reconstituted mice, with P210-STAT5A(−/−)-reconstituted mice having a median survival of 38 days, compared to only 22 days for P210-STAT5A(+/+) animals. Compared to P210-control cells, primary leukemia cells from P210-STAT5A(−/−) mice had decreased constitutive expression of the STAT5 target genes Bcl-XL and oncostatin M, but preserved expression of pim-1 and cis. Of mice that developed CML, P210-STAT5A(−/−)-reconstituted animals had greater splenomegaly and tended to have higher Bcr/Abl expression than P210-control animals, suggesting that enhanced expression of Bcr/Abl might compensate for the absence of STAT5A in these mice. Importantly, there was no difference in the number of unique P210-STAT5A(−/−) or P210-STAT5A(+/+) leukemic clones by Southern blot, indicating that the reduced incidence of CML in STAT5A (−/−) mice was not due to the decreased availability of CML progenitors. Altogether, these findings indicate that STAT5, particularly STAT5A, plays an important role in the efficient induction of the CML-like disease in mice. A greater understanding of the STAT5 target genes important in CML will provide important insights into the pathophysiology of CML, and may provide additional molecular targets for CML therapy.
Single Nucleotide Polymorphisms Influence the Phenotype of Mutations P618A and R1336W found in the ADAMTS13 Gene of Congenital TTP Patients.
Authors: Plaimauer Barbara , Mohr Gabriele , Wernhart Waltraud , Bruno Katharina , Antoine Gerhard , Rieger Manfred , Scheiflinger Friedrich
Published: October 2019

ADAMTS13 cleaves plasmatic von Willebrand factor (VWF) between Tyr1605 and Met1606 and regulates thereby the hemostatic activity of VWF. Mutations in the ADAMTS13 gene leading to severe ADAMTS13 deficiency have been found in patients with congenital thrombotic thrombocytopenic purpura (TTP). We have analyzed the ADAMTS13 gene defects in two brothers with hereditary TTP [Antoine et al, Brit. J. Hematol., 2003] where we detected a total of six nucleotide exchanges causing point mutations. On the maternal allele we found an accumulation of five amino acid substitutions (R7W, Q448E, P618A, A732V, R1336W) and on the paternal allele a stop mutation (Q44X) leading to premature protein termination in the propeptide region. Both brothers were double heterozygotes with < 3% of ADAMTS13 activity, whereas their asymptomatic parents have ~ 50% activity. Four (R7W, Q448E, P618A, A732V) of the five maternal mutations constitute single nucleotide polymorphisms (SNP) but R1336W was identified as novel rare mutation in the second cub domain. To evaluate the biologic phenotype of a given haplotype, e.g. the functional significance of the presence of the various SNPs, we analyzed the functional impact of the individual mutations on ADAMTS13 antigen levels and ADAMTS13 activity. A series of mutant ADAMTS13 molecules was expressed which contained either single amino acid substitutions or combinations of mutations with each other. We found that the common SNPs R7W, Q448E and A732V, as single mutations, had either no or only a minor impact on ADAMTS13 secretion and ADAMTS13 activity, whereas P618A and R1336W conferred a dominant adverse effect on ADAMTS13 secretion levels. Co-expression of SNPs R7W or Q448E with SNP P618A lead to improved ADAMTS13 secretion levels and could therefore partly attenuate the detrimental effect of P618A. Concomitant expression of all four SNPs reconstituted secretion levels similar to wild-type implicating a complex synergistically interaction of SNPs located in different ADAMTS13 domain regions, however, functional activity was impaired to 50%. Mutation R1336W was shown to be, as a single amino acid exchange, responsible for reduced ADAMTS13 antigen levels, but in contrast to P618A, the negative effect of R1336W was rather enhanced by the co-expression of R7W and Q448E, than rescued, leading to the total absence of ADAMTS13 secretion from the maternal allele. Our findings provide for the first time evidence that fairly common SNPs, dependent on the presence or absence of other mutations, may differently modulate functional ADAMTS13 protease levels.
The Stress-Induced PAI-1 Expression Is Enhanced in Obese Mice: A Possible Mechanism of Stress-Associated Thrombosis in Obese Subjects.
Authors: Yamamoto Koji , Kojima Tetsuhito , Adachi Tatsuya , Hayashi Mutsuharu , Matsushita Tadashi , Takamatsu Junki , Loskutoff David J. , Saito Hidehiko
Published: October 2019

Cardiovascular/thrombotic diseases are frequently induced by a variety of stressors. Obese patients are susceptible to thrombotic diseases associated with stress, but the underlying mechanisms are unknown. We previously reported that restraint stress to mice caused a significant induction of a primary inhibitor of fibrinolysis, plasminogen activator inhibitor-1 (PAI-1), in association with tissue microthrombosis. We have investigated the effect of obesity on the stress-induced PAI-1 expression using genetically obese mice. Male obese mice (C57BL/6J ob/ob) of 6 weeks old and their lean counterparts (C57BL/6J +/?) were placed into 50-100 ml conical centrifuge tubes fitted with multiple punctures so as to allow ventilation. The tubes were placed in horizontal holders and the animals thus maintained for a continuous period of restraint. After 2 or 20 hours of restraint, the mice (n=8) were sacrificed, and the plasma and tissues were harvested. PAI-1 antigen in plasma was measured by ELISA and PAI-1 mRNA in tissues was quantitated by competitive RT-PCR assay. Obese mice were hyperresponsive to short- and long-duration of restraint stress in the induction of PAI-1 antigen in plasma and PAI-1 mRNA in tissues, especially in their hearts and adipose tissues (Fig. 1).

Figure Figure

In situ hybridization analysis of the stressed mice revealed that strong signals for PAI-1 mRNA were localized to adipocytes, cardiovascular endothelial cells, and renal glomerular cells in obese animals. Immunohistochemical analysis for fibrin revealed that renal glomerular fibrin deposition was induced by 2 hour-restraint stress in obese mice (arrow in Fig. 2B), but rare in lean mice (Fig. 2A). Quantitative evaluation of fibrin was also achieved by counting the number of fibrin-positive glomeruli in each kidney section in a blinded fashion (Fig. 2C).

Figure Figure

Elevation of tumor necrosis factor-alpha (TNF-alpha) level in plasma after stress was pronounced in obese mice and pre-treatment of mice with anti-TNF-alpha antibody partially attenuated the stress-mediated induction of PAI-1 gene in adipose tissues. These results suggest that larger induction of PAI-1 may be relevant to the increased risk of stress-induced thrombosis in obese subjects and that TNF-alpha may be involved. This study presents a novel finding regarding the molecular process of the stress-induced thrombosis in obesity, and suggests that PAI-1, stress, and obesity, may be closely associated with the increased risk for cardiovascular and thrombotic diseases.
Monitoring of FVIII Replacement Therapy with Cell-Based Monitoring Tests.
Authors: Bassus Steffen , Wegert Wolfgang , Krause Manuela , Scharrer Inge , Scholz Thomas , Escuriola-Ettinghausen Carmen , Siegemund Annelie , Kreuz Wolfhart , Engelmann Lothar , Kirchmaier Carl M.
Published: October 2019

Although FVIII replacement therapy is clinically safe and effective, there is still a debate about the individually sufficient efficacy of treatment. With routinely performed one-stage and chromogenic assays individual haemostatic effects of FVIII therapy are poorly detectable due to artificial test conditions, e.g. strong dilution and adding contact activators, phospholipids or activated coagulation factors to patients plasma. Moreover, the crucial relevance of platelets for coagulation characteristics can not be investigated. For our study cell-based coagulation assays (thrombin generation in platelet rich plasma and thrombelastography in whole blood) as well as an analytic strategy were adapted to make them suitable for measuring individual haemostatic effects of FVIII therapy. 40 patients (age 7–79) with haemophilia A replacements therapies were investigated. We measured FVIII activity resp. efficacy before and after substitution with one-stage, chromogenic, thrombin generation and thrombelastography assay. 2/3 of patients received a recombinant, 1/3 of patients a plasma-derived concentrate. The results showed an average FVIII activity, measured with chromogenic and one-stage assays, of 8% before and 76% after substitution of an average of 27,3 U/kg body weight. Thrombin generation and clot firmness showed almost maximum values for relatively low FVIIIactivities (20 to 30%). Moreover, the results of the cell-based tests showed large interindividual variations in pre-substitution residual activities and in haemostatic effects after substitution for same FVIII plasma activities. This indicates the need for different interindividual FVIII activities for achieving sufficient thrombin activity and clot firmness. Therefore it seems to be advantageous to measure patients FVIII efficacy profiles before treatment and use them as their own standard to assess FVIII efficacy after treatment instead of using standard curves from pooled plasma. According to this we measured thrombin generation and clot firmness before treatment. Than we added in vitro one unit FVIII to the same sample to determine patients individual maximum of thrombin generation and clot firmness (100%). After FVIII replacement therapy we determined the same parameters and calculated the relative haemostatic effect of FVIII therapy compared to in vitro results in relative per cent of the maximum FVIII efficacy. With thrombin generation and thrombelastography assays and our revised strategy of analysis we are able to determine the individual haemostatic effect of FVIII replacement therapy and, moreover, to predict the required FVIII-amount for successful treatment with pre-substitutional in vitro spiking of patients’ own blood.
HIV-1 Nef Alleles Show Differential Activation of Hematopoietic Cell Kinase in a Yeast Model System.
Authors: Trible Ronald P. , Smithgall Thomas E.
Published: October 2019

Hematopoietic cell kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed strongly in macrophages, an important target cell type for human immunodeficiency virus (HIV). The HIV Nef protein, a critical AIDS progression factor, binds Hck with unusually high affinity and induces Hck activation. Nef-mediated Hck activation has been proposed to trigger signaling pathways important for the establishment of HIV infection and subsequent progression to AIDS. Previous studies have utilized sequence alignments and binding assays to predict the functionality of Hck-Nef interactions, but these studies do not directly investigate the ability of Nef alleles to activate Hck. To better address this issue, we have created a model system in Saccharomyces cerevisiae based on previous findings that ectopic expression of c-Src arrests yeast growth in a kinase-dependent fashion. To establish the utility of this system for Hck and Nef, we first created a Hck variant containing a C-terminal sequence modified to encode the high-affinity SH2-domain binding sequence Tyr-Glu-Glu-Ile (HckYEEI). This modified sequence allows for autophosphorylation of the new tail and the subsequent intramolecular binding to the Hck SH2 domain. The resulting HckYEEI molecule models the physiologically down-regulated (inactive) form of Hck in structure and function. Unlike wild-type Hck, HckYEEI showed little kinase activity and failed to induce growth suppression in yeast. Importantly, co-expression with the Nef-SF2 allele activated HckYEEI, resulting in growth arrest. This effect was dependent on the conserved Nef proline-rich motif essential for Hck binding through its SH3 domain. However, Nef-SF2 did not activate FynYEEI, consistent with our previous data that the Fyn tyrosine kinase is not activated by Nef in mammalian cells. We then tested several additional laboratory HIV-1 Nef alleles for their ability to activate HckYEEI. Consensus, Lai, NL4.3, SF2, and YU-2 Nef alleles activated HckYEEI to varying degrees, as reflected by yeast growth inhibition. In contrast, Nef-Eli failed to suppress growth in the presence of HckYEEI, consistent with its inability to bind the Hck SH3 domain in vitro. Growth inhibition paralleled tyrosine phosphorylation of yeast proteins on anti-phosphotyrosine immunoblots. The differential effects of these Nef alleles on Hck activation correlated closely with previous results in mammalian cells. Taken together, these studies validate the use of yeast to reconstitute Nef-Hck interactions in a system amenable to screens of clinical Nef isolates, which may have predictive value in terms of AIDS progression.
High Frequency CD4+CD25+ Treg Cells in Nasopharyngeal Carcinoma Is Associated with Suppression of CD8 T Cell Mediated Response to Latency II EBV Antigens.
Authors: Chen Frederick E. , Wen Wensheng , Huang Guangwu , Travers Paul , Dodi I. Anthony , Madrigal J. Alejandro
Published: October 2019

Nasopharyngeal Carcinoma (NPC) is associated with latent Epstein Barr Virus (EBV) infection and expression of EBV latent antigen LMP2. Because of the possibility of targeting viral antigens, there is interest in developing EBV-LMP2-specific Cytotoxic T lymphocyte (CTL) immunotherapy for NPC. However, evidence suggests that CD8+ T cell responses to EBV latency II antigens are rarely detectable in these patients. Regulatory T cells have been shown to inhibit stimulation of CD8+ T cells by Antigen Presenting Cells (APC) in vitro, and may play an important role in immune tolerance to tumours. Thirteen newly diagnosed untreated HLA A2 NPC patients were investigated for CTL responses to EBV latency II antigens by flow cytometry using HLA A2 restricted tetramers specific for LMP2a derived peptides (CLG, LTA). No LMP2-specific CD8+ T cells were detected amongst peripheral blood CD8+ T cells either ex vivo or in vitro following short stimulation in ELIspot assays, although strong responses to CMV and flu peptides and PHA were elicited. To investigate the antigen presenting capability of professional APC in NPC, dendritic cells (DC) were generated from ex vivo peripheral blood monocytes and shown to express a stimulatory mature phenotype with expression of CD83 and markers of costimulation CD80 and CD86. Despite this, mature DC pulsed with LMP2 derived peptides failed to stimulate and expand autologous LMP2-specific CTL, suggesting either absence or tolerance of LMP2-specific CTL. CD4+CD25+ regulatory cells have been implicated in peripheral tolerance and inhibition of antigen-specific T cell responses, and analysis of ex vivo peripheral blood T cells from NPC patients showed increased CD25 expression constituting a mean of 22.23 % of total CD4+ T cells compared to normal control mean of 5.35% (student t-test p<0.001). CD25 was not expressed by non-CD4+ T cells including CD8+ and NK cells, indicating that CD25 expression was unlikely to have represented activation. The findings suggest that CD4+CD25+ regulatory cells may play an important role in inhibiting antigen-specific anti-tumour responses in patients with established disease.
Leukemia during Pregnancy: 32 Cases from a Single Institution.
Authors: Alhejazi Ayman , Aljurf Mahmoud , Sahovic Entezam , Almhareb Fahed , Maghfoor Irfan , Alsharif Fahad , Alzahrani Hazzaa , Alomar Hamad , Alshanqeeti Ali , Albeihani Amal , Zaidi Syed , Chaudhri Naeem
Published: October 2019

In an attempt to evaluate the outcome of leukemia during pregnancy we performed a study of all such cases identified in our institution leukemia database. Thirty two cases were identified as cases of leukemia during pregnancy among the cohort of patients treated between January 1991 and July 2003. The cases were AML (15), ALL (6) and CML (11).

At diagnosis the median age was 24.5 years and the median gestational age was 16 weeks. Twenty patients were treated for their leukemia during pregnancy at a median gestational age of 20 weeks (4 in the first trimester, 8 in the second trimester and 8 patients in the third trimester).

Among the acute leukemia patients (n=21), 10 patients (47.6 %) received chemotherapy during pregnancy, 9 (90%) of them achieved CR and one patient needed salvage chemotherapy. Of these 10 patients, 7 had normal live births, one spontaneously aborted at 15 weeks of gestation and 2 had therapeutic abortion at 16 and 19 weeks of gestation. The remaining 11 (52.4%) were not given chemotherapy while pregnant as 3 patients (27.3%) presented late in their pregnancy after 34 weeks of gestation ending in normal live births and then received induction chemotherapy and 8 patients (72.7%) presented early in their pregnancy and they either spontaneously aborted or had a therapeutic termination of their pregnancy before starting chemotherapy. Among the CML patients (n=11), 9 patients received Hydroxyurea (HU) treatment during pregnancy, one patient received α-Interferon and one patient was treated with leukapheresis. Eight of the 11 patients (63.6%) had normal live births. Three patients (18.1%) aborted, 2 at 11 weeks of gestation while on HU treatment and one shortly after diagnosis before treatment. Of the total 32 patients 13 (40.6%) subsequently underwent allogeneic stem cell transplant, 8 for AML, 2 for ALL and 3 for CML. At a median follow up of 19.1 months, the disease free survival (DFS) and overall survival (OS) were 19% and 33.3% respectively for the acute leukemia patients. For CML patients, the OS was 90.9% at a median follow up of 38 months.

The above data suggest that a high CR rate and satisfactory OS can be achieved in leukemia during pregnancy. Furthermore, chemotherapy did not lead to any teratogenic effects among live births. All spontaneous and therapeutic abortions were for disease related complications and not chemotherapy induced.

This series of cases, which is the largest from a single institution, clearly supports early treatment of leukemia during pregnancy based on the underlying disease rather than concomitant pregnancy.
Activation of Monocytes by Aspergillus Fumigatus Hyphae and Conidia.
Authors: Haddad Ziad , Loeffler Juergen , Bonin Michael , Schumacher Ulrike , Markulin Marija , Frank Friederike , Kapp Markus , Einsele Hermann , Hebart Holger
Published: October 2019

Invasive aspergillosis is a life-threatening infection in immunocompromised patients. Activation of innate immune cells is crucial for the successful control of invasive aspergillosis. Monocytes play an important role in the cellular defense against Aspergillus fumigatus by attacking defined differentiation states of the fungus and by the release of pro-inflammatory cytokines and chemokines. In order to get a better understanding on pathways involved in these defense mechanism, genome-wide expression profiling was performed after incubation of CD14+ selected monocytes with A. fumigatus conidia and hyphae for 3, 6 and 9 hours using the Affymetrix HG-U133A array. Moreover, phagocytosis of conidia was analyzed by immunofluorescence. Expression of defined cytokines and chemokines was confirmed by Real-time PCR and on protein level by ELISA. Phagocytosis of conidia was observed during the first 3 hours (up to 2–3 conidia/monocyte). The conidia became swollen after 6 hours. By genome-wide expression profiling, ~600 genes were differentially regulated after 3 hours of incubation with hyphae and ~200 after incubation with conidia. At 6 and 9 hours of incubation, the number of differentially regulated genes showed no difference (~400 genes by both stimuli). Hyphae but not conidia strongly induced the expression of chemokines attracting neutrophils and T-lymphocytes as well as their receptors already after 3 hours, indicating that phagocytosis of conidia does not immediately induce expression of inflammatory genes. Pentraxin-3 was upregulated after 3 but not after 6 hours by both conidia and hyphae. PTX-3 binds to A. fumigatus and plays a role in initiating phagocytosis. Genes encoding for inflammatory cytokines and chemokines as well as their receptors were found to be up-regulated at 6 and 9 hours by both stimuli. Urokinase plasminogen activator (uPA), which plays an essential role in regulating fibrinolysis and thereby monocyte/macrophage recruitment to sites of inflammation, was also found to be upregulated after stimulation with both stimuli. ELISA experiments confirmed these results on protein level, demonstrating a steady increase of chemokine and cytokine concentrations in culture supernatants from 3 to 9 hours.

In conclusion, A. fumigatus differentially regulates a wide number of genes expressed by human monocytes, which play a crucial role in immune response during infection. The expression of these genes is dependent on the differentiation and germination state of the fungus and the incubation period.
Iron Acquisition in Reticulocytes: Evidence for a Kiss and Run Mechanism.
Authors: Sheftel Alex D. , Zhang An-sheng , Shirihai Orian , Ponka Premysl
Published: October 2019

During differentiation, immature erythroid cells acquire vast amounts of iron at a breakneck rate. Proper coordination of iron delivery and utilization in heme synthesis is essential and disruption of this process likely underlies iron loading disorders such as sideroblastic anemia and myelodysplastic syndrome with ringed sideroblasts. Iron is taken up by the cells via receptor mediated endocytosis, a process whereby diferric transferrin (Tf) binds to its cognate receptor (TfR) on the erythroid cell plasma membrane, followed by internalization of the Tf-TfR complex. Subsequent to endocytosis, the endosome is acidified by a H+-ATPase, allowing the release of iron from Tf. Through an unknown mechanism, iron is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe into protoporphyrin IX, ferrochelatase, resides. Although it has been demonstrated that the divalent metal transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the iron atoms after their transport across the vesicular membrane remains unknown. Because reduced iron is a strong pro-oxidant, contributing to free radical formation through Fenton chemistry, it has been predicted that an iron binding molecule shuttles Fe from the endosome to mitochondria. However, this much sought iron binding intermediate, that would constitute the labile iron pool (LIP), has yet to be identified. Thus, we hypothesize that, in Hb-producing cells, there is a direct relaying of Fe from the endosomal machinery to that of the mitochondria. By loading the cytoplasm of reticulocytes with an impermeant form of the iron chelator, desferrioxamine, (hDFO) which would intercept Fe that traversed the cytosol bound to the putative LIP intermediate, we show that 59Fe, delivered via Tf, can bypass the cytosol. Importantly, iron delivered to these cells in a form that freely diffuses across the membrane, iron-salicylaldehyde isonicotinoyl hydrazone (59FeSIH2), was significantly prevented from being used for heme synthesis in hDFO-laden reticulocytes. We have also used time-lapse confocal microscopy to track iron-loaded endosomes and mitochondria with high spatial and temporal resolutions. Using this approach, we have found that endosomes are very mobile organelles that continuously traverse the cytosol and touch a number of mitochondria multiple times. Addition of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), a calmodulin antagonist that is known to block the myosin motor, to cells that had been treated with fluorescent holo-Tf, inhibited endosomal movement. Interestingly, W-7 treatment of cells whose endosomes were loaded with 59Fe-Tf blocked both incorporation of that iron into heme or mobilization of the iron by chelators. Together, these data are congruent with our hypothesis that iron is directly delivered to mitochondria by endosomes in a “kiss and run” paradigm.
Consequences of DMT1 Mutation on Proliferation and Hemoglobinization of Erythroid Progenitors In Vitro.
Authors: Priwitzerova Monika , Pospisilova Dagmar , Indrak Karel , Ponka Prem , Divoky Vladimir
Published: October 2019

Disorders of iron metabolism and homeostatis belong to the most common diseases in humans; however, neither the regulation of iron metabolism in erythroid cells nor the pathophysiology of these diseases are completely understood. Last year we reported at this meeting a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 (DMT1) gene (DMT1 1285G>C). The defective growth of this patient’s erythroid progenitors in vitro was rescued by the addition of 10 μM iron-salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), an iron chelate that has been shown to deliver iron for heme synthesis without involving the transferrin receptor/DMT1 pathway. To further characterize the impact of the DMT1 mutation on the in vitro growth of erythroid progenitors, 10 μM Fe-SIH, 25 μM hemin or 10 μM zinc chloride (ZnCl2) were added to the erythropoietin (Epo) containing cultures. All substances increased the plating efficiency in normal control as well as mutant BFU-Es. Moreover, all substances led to a 1.4 to 1.6 fold increase in hemoglobin (Hb) content in normal BFU-Es. Although hemin did not significantly increase Hb content in the patient’s BFU-E, the addition of Fe-SIH to the Epo/hemin-containing cultures increased Hb content 2.2 fold. Since Hb synthesis in heme-deficient erythroid cells is inhibited by heme-regulated inhibitor (HRI) kinase, which specifically phosphorylates the α subunit of the eukaryotic initiation factor 2 (eIF2α), we have analyzed the level of eIF2α phosphorylation in cytospine-harvested BFU-Es. Immunofluorescence analysis of erythroid cells with phospo-eIF2α (Ser51) antibody revealed increased phosphorylation of eIF2α in mutant BFU-Es compared to control BFU-Es grown under all aforementioned conditions. The most striking differences were observed in Epo and Epo/hemin containing cultures, in which the fractions of phospho-eIF2α-positive cells were significantly higher in mutant BFU-Es. These data suggest that the block of terminal differentiation/hemoglobinization in the DMT1-mutant erythroid cells is only partially caused by translational arrest of globin chain synthesis; the delivery of iron may be required for other cellular functions apart from Hb synthesis. Interestingly, the most prominent positive effect on the proliferation (i.e., BFU-E cellularity) as well as hemoglobinization of mutant BFU-Es had a combination of Epo/Fe-SIH and ZnCl2; this combination led to the highest increase in Hb content that was associated with almost complete lack of eIF2α phosphorylation. This suggests that the addition of Zn2+ to Fe-SIH containing cultures further stimulates processes that are initially rescued in DMT1-mutant erythroid cells by the addition of iron. We conclude, that the DMT1 defect in erythroid cells is complex, probably involving the combination of transcriptional and translational defects that cannot be rescued by hemin alone.

Figure Figure
Aberrant Ferroportin 1 Regulation and Iron Homeostasis Interferes with Development of the Spleen Stroma during Murine Embryogenesis.
Authors: Mok Henry , Mendoza Miriam , Prchal Josef T. , Balogh Peter , Schumacher Armin
Published: October 2019

We have previously identified a regulatory mutation in the ferroportin 1 (Fpn1) locus in polycythaemia (Pcm) mutant mice, leading to transient iron overload and polycythemia during postnatal development. A 58-base pair deletion within the Fpn1 promoter region caused aberrant transcription initiation and resulted in the absence of the iron-responsive element in the 5′ untranslated region of the vast majority of Fpn1 transcripts, thereby disrupting translational regulation of Fpn1 expression. Interestingly, at birth Pcm mutant pups were remarkable for severe iron deficiency and a hypochromic, microcytic anemia. Here, we demonstrate that decreased placental syncytiotrophoblast expression of Fpn1 protein governs decreased embryonic iron levels during late gestation, implicating Fpn1 in maternal-to-fetal iron transport in the placenta. The decreased placental expression of Fpn1 protein appeared to be regulated at the transcriptional level. In striking contrast, increased Fpn1 protein was observed in the embryonic spleen during late gestation, mediated by a post-transcriptional mechanism. This correlated with a significant increase in apoptotic cell death of spleen stromal cells, culminating in a progressive regression of the spleen during late embryogenesis, more severe in Pcm homozygotes. Importantly, Pcm describes a phase in spleen development subsequent to that affected in mutant alleles of the transcription factors Wt1, Hox11, Bapx1, and capsulin. During postnatal development, whereas Pcm heterozygotes retain a significant amount of spleen tissue, Pcm homozygotes display only a spleen rudiment, and are likely to be functionally asplenic throughout postnatal life. Phenylhydrazine treatment of Pcm heterozygotes during early postnatal development demonstrated a reduced functional capacity for erythroid hyperplasia in the spleen. Despite the significantly reduced organ size, splenic white pulp follicles appear to form normally in both Pcm heterozygotes and homozygotes. However, severe abnormalities of the red pulp sinusoidal endothelial are observed in mutant spleens using markers specific for endothelial cell subpopulations. Therefore, the Pcm mutation represents a novel murine model for disrupted splenogenesis that is due to aberrant tissue-specific regulation of the iron transporter Fpn1 during embryonic development.
Hepcidin Contributes to Anemia of Malignancy by Causing Sequestration of Iron in Hepatic Stores.
Authors: Rivera Seth , Liu Lide , Nemeth Elizabeta , Gabayan Victoria , Ganz Tomas
Published: October 2019

The iron regulatory hormone hepcidin is presumed to be the central mediator of anemia of inflammation (AI, anemia of chronic disease). Hepcidin is an acute phase protein which restricts iron absorption and release of iron from stores. Patients with hepatic adenomas producing large amounts of hepcidin had severe AI that resolved when the tumors were resected (Weinstein et al, 2002). In order to study the effects of hepcidin in anemia of malignancy, we created tumor xenografts in SCID mice, some of which were engineered to secrete large amounts of hepcidin (33 mice with control tumors, 30 with hepcidin producing tumors). Hepcidin producing tumors expressed 2000-times more hepcidin mRNA which also correlated with increased levels of hepcidin peptide detected in urine of these mice. Mice with hepcidin producing tumors were significantly more anemic (median hemoglobin = 10.4 vs. 8.4 g/dL, p=0.02) and had significantly lower serum iron values (median serum iron = 242 vs. 96 mg/dL, p<0.001). Liver iron was significantly higher in mice with hepcidin producing tumors (mean liver iron 199 vs. 142 mg/100 mg dry weight, p<0.001) even in the face of anemia and hypoferremia. Splenic and bone marrow iron levels were unchanged. Hepcidin contributes to the anemia of malignancy by sequestering iron predominately in hepatic stores, similar to the pattern seen in AI.
Lack of Anemia Despite Marked Elevation of Serum Methylmalonic Acid and Total Homocysteine in a Multiethnic Cohort.
Authors: Stabler Sally P. , Anand Kishlay , Huang Sam , Boden-Albala Bernadette , Allen Robert H. , Sacco Ralph L.
Published: October 2019

It is controversial whether asymptomatic patients with metabolic evidence of cobalamin (Cbl) deficiency need vitamin treatment. Patients with clinical abnormalities due to Cbl deficiency which are proven to correct with treatment invariably have elevated serum methylmalonic acid(MMA) and total homocysteine(tHcy). However, many subjects with elevated MMA are found in screening studies of seniors and they often have no classical clinical abnormalities. We correlated hematologic and neurologic findings with elevated MMA and tHcy in subjects enrolled in the Northern Manhattan Study. Eligible subjects were over 40 yr, had not had a stroke and lived in Manhattan. Approximately 3000 subjects had serum MMA, tHcy and other relevant data available. The samples were collected from 1993 to 2001 thus spanning food folate fortification in the United States. We analyzed those in the top 1% MMA and/or tHcy since these subjects had values seen also in clinical Cbl deficiency. There were 30 subjects with MMA 975 to 179,900 nM (normal<271 nM), 15 female, age 56 to 90 yr, and 19% white, 15% black, 60% other including Hispanic. Only 7of 27 had mild anemia (lowest value, 33.6%) and 6 of 27 had elevated MCV>100fL. The male subject with the highest MCV (109.8 fL), had a hematocrit of 42.7 %, MMA 179,900 nM, tHcy 86.3 uM and Cbl 34 pg/mL with no neurologic findings. Neurologic abnormalities were present in 5 of 30. One subject had high grade spastic paraparesis, 3 had peripheral nervous system deficits, and one had central nervous system defects.Serum tHcy and MMA were not higher in those with neurologic abnormalities. The serum creatinine was < 1.3 mg/dL in the 27 with values thus the elevated MMA was not attributable to renal failure. The serum Cbl was < 200 pg/mL in only 6 of 22 and < 350 pg/mL in 16 of 22. The top 1% of tHcy value ranged from 24.9 to 102 uM (normal<14uM) and only 7 of 31 of these subjects had elevated tHcy without associated elevated MMA. Eight of those in the top 1 percentile for tHcy were also in the top 1 percentile for MMA. We conclude 1.Serum MMA is elevated > 975 nM in 1% of a randomly selected urban multiethnic cohort. 2. Hematologic abnormalities were absent or mild in this group with high MMA. 3. Neurologic abnormalities were found consistent with Cbl deficiency in 17 % of those with high MMA. 4. Both MMA and tHcy are usually elevated. 5. Even though the elevations of MMA and tHcy overlapped those found in clinical studies of Cbl deficiciency these randomly discovered subjects were generally asymptomatic, suggesting that the pathophysiology of megaloblastic anemia and combined systems disease still needs to be investigated.