bluuebunny/crossref_metadata_2025 huggingface dataset:
Requirement for CD4 Help for Optimal GVL Effects of Alloreactive T Cells Given to Established Mixed Chimeras but Not Freshly Irradiated Mice.
Authors: Eom Hyeon-Seok , Chakraverty Ronjon , Sachs Jessica , Zhao Guiling , Sykes Megan
Published: October 2019

Donor lymphocyte infusion (DLI) may lead to lymphohematopoietic graft-versus-host (GVH) reactions without graft-versus-host-disease (GVHD). The graft-versus-leukemia (GVL) effect of DLI is greater in mixed chimeras than in full chimeras following fully MHC-mismatched bone marrow transplantation (BMT). Some studies showed, following MHC-mismatched BMT, that only CD8+ T cells are involved in GVL effects of DLI against MHC class I+ class II− tumors, but others showed that the GVL effect of DLI is dependent on both CD8+ and CD4+ T cells. Previously, we have observed that GVL against EL4 is mediated by CD8+ T cells but not CD4+ T cells following DLI in freshly irradiated mice treated with cytokines to reduce GVHD mortality in fully MHC-mismatched BMT. The exact contribution of CD4+ and CD8+ T cells to the GVL effects of DLI in established mixed chimeras is not known. We hypothesized that lethal conditioning leads to the production of many proinflammatory cytokines which may help to generate a CD4 cell-independent, CD8 cell-mediated anti-tumor effect. However, in delayed DLI recipients, CD4+ helper T cells might be needed to induce CD8+ anti-tumor effector cells. We evaluated the role of T-cell subsets in GVL effects of DLI following fully major histocompatibility complex (MHC)-mismatched BMT in freshly irradiated mice and mixed chimeric delayed DLI recipients. While GVL effects of delayed DLI given to established mixed chimeras were dependent on both CD4+ and CD8+ T cells, CD4+ T cells did not contribute to GVL effects in freshly irradiated mice. Consistent with this, MHC class II expression on host antigen-presenting cells (APCs) was needed to optimize GVL effects of DLI in established mixed chimeras. To address whether cross-presentation of tumor antigens (Ag) via class I would permit GVL responses in full chimeras, we generated full chimeras in which donor and recipient shared a class I locus. However, there were no GVL effects of delayed DLI in full chimeras, providing no evidence that cross-presentation of tumor Ag through class I molecules shared by donor and the tumor led to anti-tumor effects in this model. We conclude that following delayed DLI to established mixed chimeras, the GVL response requires extensive MHC disparity, MHC class II expression on host APCs and the presence of CD4+ T cells. In contrast, the requirement for CD4+ T cells can be bypassed in freshly irradiated recipients, but this occurs at the expense of a greater risk of GVHD. Administration of delayed DLI to mixed chimeras permits achievement of optimal GVL effects because large numbers of GVH-reactive T cells can be administered without inducing GVHD.
The Role of Age at First Exposure and Therapy Regimen on the Development of Neutralizing FVIII Antibodies in Hemophilia A.
Authors: Ettingshausen Carmen Escuriola , Zyschka Alexandra , Saguer Inmaculada Martinez , Heller Christine , Klingebiel Thomas , Kreuz Wolfhart
Published: October 2019

In order to assess the impact of age at first exposure to F VIII and therapy regimen on neutralizing FVIII-antibodies in previously untreated patients (PUP) with hemophilia A a prospective study was performed.

Over a 23-years study period a total of 74 severely affected hemophilia A -PUPs have been consecutively recruited, treated with F VIII and investigated for inhibitor development. The patients were divided into two groups according to their treatment regimen: Group 1 (n=23) started prophylaxis at the age of 1 year (before or immediately after the first relevant bleed). Group 2 (n=43) was treated on-demand or prophylaxis was started after more than 2 bleeds. The following parameters were equally distributed among both groups: caucasian ethnicity, intron-22-inversion, age at 1st ED >0.5 years.

Out of 74 hemophilia A patients 23 developed inhibitors (31%). Inhibitor incidence was 0% (1 transient inhibitor out of 23 patients) in those patients who received early prophylaxis (group 1) and 42% (18/43 patients) in case of delayed prophylaxis or on-demand treatment (group 2) (p=0.002).

No linear correlation was found between the age at first exposure and inhibitor formation. However, patients treated before the age of 0.5 years showed a significantly higher inhibitor incidence (62%) than those treated at an more advanced age. Our data confirm that very early age at first exposure is a risk factor for inhibitor development in severe hemophilia A patients. Early prophylaxis might be protective against inhibitor development. To confirm the data a larger patient cohort has to be investigated.
F VIII Inhibitors in Haemophilia A Patients after Continuous Infusion of Factor VIII Concentrates.
Authors: von Auer Ch. , Oldenburg J. , von Depka M. , Escuriola-Ettinghausen C. , Kreuz W. , Kurnik K. , Lenk H. , Varvenne M. , Scharrer I.
Published: October 2019

Continuous infusion (CI) of coagulation factor concentrates has been used since the early 1990s. Recent reports of the occurrence of an inhibitor (inh) after CI have raised concerns about this method of treatment. We conduct a retrospective study to investigate the development of inh after CI of FVIII concentrates in Germany. 99 haemophilia treating physicians in Germany were contacted and asked to answer a questionnaire. So far data of 13 departments have been reported and analyzed.

Three of these 13 centers never conducted a CI, in 5 no inh were detected and in 5 haemophilia centers 10 patients with inh development after CI were registered.

5 of these patients were suffering from severe, 1 from moderate and 4 from mild haemophilia (age between 7 months and 57 years). Indications for treatment were major bleeds and surgical procedures. Plasma derived (6) and recombinant (4) factor concentrates were given in various infusion sets. Data concerning infused amount (4300 to >100000 IE), exposure days (1 to >100) and inh characteristic (alloantibodies, 3 LR, 7 HR) were collected.

Regarding the genotype, we found 4 missense-mutations, 2 intron-22-inversions, 1 small deletion, 3 were unknown. In our own center we found no inh in 81 patients with major orthopaedic surgery and bolus infusion of factor VIII concentrate compared to 2 inh in 8 patients with major orthopaedic surgery and CI of FVIII.

In conclusion we found only in 2 patients the typical gene mutation for inh development. Strikingly the inh developed very often in patients with mild haemophilia. These findings agree with published results.

There might be an uncommon inh-pathomechanism due to CI or patients with mild haemophilia might exhibit a much higher prevalence of inhibitor development when treated with an “intensive FVIII-treatment” such as CI.
HIV-1 Tat Mediated Effects on Focal Adhesion Assembly and Permeability in Brain Microvascular Endothelial Cells.
Authors: Avraham Shalom , Jiang Shuxian , Lee Tae-Hee , Prakash O. , Avraham Hava Karsenty
Published: October 2019

The blood-brain barrier (BBB) is a network formed mainly by brain microvascular endothelial cells. The integrity of the BBB is critical for brain function. Breakdown of the BBB is commonly seen in AIDS patients with HIV-1-associated dementia (HAD), despite the lack of productive HIV-infection of the brain endothelium. The processes by which HIV causes these pathological conditions are not well understood. Here, we characterized the molecular mechanisms by which Tat mediates its pathogenic effects in-vitro on primary human brain microvascular endothelial cells (HBMECs). Tat treatment of HBMECs stimulated cytoskeletal organization and increased focal adhesion sites as compared to control cells or cells treated with heat-inactivated Tat. Pretreatment with Tat antibodies or with the specific inhibitor SU-1498, which interferes with VEGFR-2 (Flk-1/KDR) receptor phosphorylation, blocked the ability of Tat to stimulate focal adhesion assembly and the migration of HBMECs. Focal adhesion kinase (FAK) was tyrosine-phosphorylated by Tat and found to be an important component of focal adhesion sites. Inhibition of FAK by the dominant-interfering mutant form FRNK (FAK-related non-kinase) significantly blocked HBMEC migration and disrupted focal adhesions upon Tat activation. Furthermore, HIV-Tat induced permeability changes in HBMECs in a time dependent manner. Tat also impaired BBB permeability as observed in HIV-1 Tat transgenic mice. These studies define a mechanism for HIV-1 Tat in focal adhesion complex assembly in HBMECs, via activation of FAK, leading to cytoskeletal reorganization and permeability changes.
Identical Clonal Origin in Three Cases of Hodgkin’s Lymphoma Variant of Richter’s Syndrome Associated with EBV Infection.
Authors: Feugier Pierre , Monhoven Nathalie , Legres Luc , Saout Louis , Martin Laurent , Patey Martine , Janin Anne , Labouyrie Eric
Published: October 2019

Occurrence of an aggressive lymphoma in patients with chronic lymphocytic leukemia (CLL), clinically referred to as Richter’s syndrome, occasionally manifests as a lymphoproliferation resembling Hodgkin’s lymphoma (HL) and often containing the Epstein-Barr virus (EBV). Only a limited number of HL variants have been subject to informative analysis regarding EBV status and their clonal relation-ship to the CLL, with evidence of an identical clonal origin in some cases and of clonally unrelated neoplasms in most cases. We reported a clinical, pathological and molecular analysis of three new cases of Richter’s syndrome with HL features. The three patients (two males, one female), aged of 67, 70 and 74, have 4, 8 and 15-year history of CLL, respectively. At HL, clinical stages (CS) were IIIAa, IIBb and IIIBb, at HL diagnosis. All the three patients responded to HL specific chemotherapy but two of them died from infectious and cardiac complications and the last one died from HL relapse. Histologically the lesions were composite comprising areas of CLL, areas of CLL with Hodgkin Reed-Sternberg (HRS) cells and areas of HL. The HRS cells were CD45RC−/CD20−/CD79a−/CD30+/CD15+/J-chain-(all cases), Oct-2+ (case 2) and Pax-5+ (cases1 and 3). In all cases, HRS cells were positive for EBV with a latence 2 profile. Using single cell laser microdissection and PEP-PCR amplification of the immunoglobulin heavy chain gene (IgH) FR3A region of each cell type, we demonstrated a same clonal origin for the CLL cells and the HRS cells in all cases. These data were confirmed in two cases by Genescan reamplification with fluorescent FR3A primer and sequenced with BigDye Terminator chemistry. Clinicopathological presentation was similar to classical HL but persistent EBV infection is probably related to an underlined immunosuppression due to CLL and chemotherapy. Seven other cases have been reported in the literature using similar single cell techniques, four of them, EBV +, were clonally unrelated while the three other one, EBV−, were of same clonal origin. At the opposite and although our three cases are EBV+, we show that the HRS cells and CLL cells belong to the same clonal population. Our data do not support the hypothesis that EBV+ HL in CLL patients occurs as unrelated secondary neoplasm in immunocompromised patients.
Pinkie, the First Viable Germline Hypomorph Allele of Retinoid X Receptor Alpha, Reveals an Important Role for RXRa in Th2 Development.
Authors: Du Xin , Bigby Timothy , Tabeta Koichi , Janssen Edith , Crozat Karine , Navjiwan Mann , Beutler Bruce
Published: October 2019

Pinkie is a recessive ENU-induced mutation, homozygotes develop premature graying and subsequently, progressive alopecia. Histologically, the skin is characterized by the destruction of hair follicle architecture, formation of dermal cysts and appearance of black papules throughout the ventral skin late in life. The Pinkie mutation was mapped to the retinoid X receptor alpha (Rxra) locus (chromosome 2) on 700 meioses and positionally cloned. As a component of the nuclear receptors for retinoic acid and other bioactive molecular ligands, RXRa plays an irreplaceable role during embryogenesis and a null allele of RXRa invariably causes embryonic lethality. Pinkie, which results from the amino acid substitution N273I in the ligand-binding domain, is the first viable germline hypomorphic mutation of RXRa. Transfection studies suggest that the abnormal protein retains only 10 to 20% of the activity of the wild type receptor. Pinkie homozygotes develop a progressive phenotype, part of which entails a severe, age-dependent imbalance of Th1/Th2 differentiation upon antigenic challenge. When immunized with ovalbumin (OVA), pinkie homozygotes displayed markedly decreased IgG1 levels in serum as compared with normal C57BL/6 mice; in vitro OVA re-stimulation of pinkie splenocytes causes greatly enhanced IFN-g production and decreased IL-4 production. Upon MCMV infection, pinkie homozygotes showed significantly higher type I interferon levels in serum than normal C57BL/6 mice. The in vitro differentiation of naïve CD4 T cells demonstrated skewed Th1 development in pinkie mouse compared to that of C57BL/6 control animal. However, real-time PCR didn’t reveal significant difference in mRNA level of GATA-3 and T-bet, which are believed to be critical transcription factors in Th1/Th2 development. These data collectively imply that RXRa is required for Th2 development. This requirement might reflect an influence of RXRa on the expression of pertinent transcription factors (e.g., GATA-3, T-bet), cytokines (IL-4, IFN-gamma) and/or cytokine receptors that are involved in Th1/Th2 differentiation. RXRa represents a likely interface between the nutritional environment and the immune status of the host. Consumption of retinoids and vitamin D may therefore be expected to influence the development of the allergic/atopic (Th2) phenotype, and provide a plausible explanation for the increasing frequency of atopic disease observed in developed countries.
Acute Myeloid Leukemia in the Elderly: Longitudinal Outcomes and Hospital Charges in Chemotherapy and Non-Chemotherapy Patients.
Authors: Katz Laura M. , Stern Lee S. , Fastenau John M. , Piech Catherine T. , Doyle John J.
Published: October 2019

Background: Patients with acute myeloid leukemia (AML) have an estimated median age of 65 to 70 years (J Clin Oncol1999;17:3569–76). The overall incidence of the disease increases with increasing age. The five-year survival rate for AML is 2% among elderly patients (Arch Intern Med2002;162:1597–1603). Treatment includes cytotoxic chemotherapy [e.g., the “7+3” regimen consisting of cytarabine and daunorubicin or idarubicin (Leukemia1996;10:389–95)], which can be associated with serious morbidity and is often not tolerated by older patients. Over 80% of Medicare payments for treating AML are attributable to hospitalization costs (Arch Intern Med2002;162:1597–1603).

Methods: A longitudinal cohort design was employed using an inpatient database containing information on over three million individuals from 28 hospitals in the US. The study population included patients aged 60 years and older with initial AML diagnosis between January 2000 and June 2003. Patients were followed across multiple admissions, from first inpatient encounter through last discharge from hospital. Descriptive statistics (demographics and hospital characteristics), comorbidities, chemotherapies, outcomes, resource utilization, and charges were examined; multivariate linear regressions were performed.

Results: 275 patients were identified and included in these analyses. The mean age upon first AML admission was 74.3 years (SD=8.1) and 46.6% of patients were female. On first admission, the mean length of stay (LOS) was 14.7 days (SD=14.3), 37.8% of patients received chemotherapy, and one quarter of all patients (25.3%) died. Patients incurred a mean total charge of $70,305 on first admission, while the median was $17,640; patients on chemotherapy had mean and median total charges of $113,118 and $74,689, respectively, versus $43,999 and $9,784, respectively, for non-chemotherapy patients. Chemotherapy patients also had a higher mean LOS than non-chemotherapy patients (23.0 vs. 6.7 days, respectively). 44.4% of patients had a least one re-admission with a mean of 36.4 days (SD=62.1) between the first and second admissions. For re-admissions, the mean LOS and charges were 8.9 days (SD=15.3) and $38,785 (median $13,936), respectively. Across all admissions, 41.4% of patients received chemotherapy and 40.7% of patients died in-hospital. Over the entire study period, patients incurred a mean total charge of $115,471 with a median charge of $49,168. In multivariate analyses, after adjustment for covariates, chemotherapy was found to be a significant predictor (p<0.0001) of admission charges, LOS, and total days of stay.

Conclusion: Elderly patients with AML incurred substantial charges and inpatient mortality. The highest charges and the majority of deaths occurred during first admission. Current chemotherapy regimens in these patients were associated with substantially increased charges.
Targeted Gene Therapy Vectors for Acute Myeloid Leukemia Cells Selected from Random AAV-Displayed Peptide Libraries.
Authors: Trepel Martin , Lee Mi-Kyung , Kaul Felix , Kleinschmidt Jurgen
Published: October 2019

For acute myeloid leukemia, gene therapy may be a valuable tool to treat patients refractory to conventional chemotherapy regimens. However, there are no vectors available that sufficiently and specifically transduce this cell type. Cell type specific receptors can be used to target gene therapy vectors to the cells of interest (Trepel et al., Hum. Gene Ther., 2001). We have developed a screening system based on random peptide libraries displayed on adeno-associated virus type 2 (AAV) (Muller et al., Nat. Biotechnology, 2003). Such libraries consist of up to 108 different viruses that display a targeting peptide with random sequence in a capsid region that mediates binding of the virus particle to cell surface receptors. Targeted AAV gene therapy vectors that specifically transduce the cell types of interest can be selected from such a library. Here we report the selection of a random AAV display peptide library on a panel of acute myeloid leukemia cell lines (Kasumi-1, U937, HL 60). An X7 (X = any amino acid) oligonucleotide insert was cloned in to the AAV-library plasmid followed by the production of AAV library transfer shuttle AAV. To produce the final library, AAV producer cells were infected with shuttle AAV at an MOI of 1 to ensure encoding of displayed peptides by the packaged DNA. The target cells were then infected with the AAV library. Bound particles were internalized and amplified either by superfinfection of the cells with wild type adenovirus type 5 or by a high-sensitivity PCR-system of the random oligonucleotide followed by cloning of the insert and making a preselected library as above. Amplified clones were recovered and subjected to two more rounds of selection. Enriched peptide inserts were analyzed indirectly by DNA sequencing of recovered clones. After three rounds of selection, enrichment of a peptide motif within the selected AAV capsids was observed. The selected viruses were used for production of recombinant AAV vectors harboring a GFP reporter gene. Such recombinant targeted vectors transduced the target leukemia cells they have been selected on (e.g., Kasumi-1 cells) up to 60 times more efficient compared to a random clone of the initial, unselected library. We conclude that our novel targeting strategy can be applied to a great variety of cell types. The clones selected here may be used as valuable tools to target therapeutic genes to acute myeloid leukemia cells.
Live Attenuated Salmonella Carrying Platelet Factor 4 cDNAs as Radioprotection and Anti-Tumor Angiogenesis Agents.
Authors: Han Zhong Chao , Liu Bin , Zhao Lihua
Published: October 2019

In cancer therapy, specific radioprotection of normal tissue and antiangiogenesis are the ways to increase the therapeutic gain. Here we describe a novel gene therapy, which uses attenuated salmonella SL3261 as oral vectors carrying with cDNA of platelet factor 4 (PF4) or that of a truncated PF4. After oral administrations of attenuated salmonella carrying with cDNA of PF4 or truncated PF4, the survival rate of mice which received sublethal total body irradiation was improved by 50%, In comparison with the control mice, the bone marrow cells obtained from the mice of experimental group increased (13.2±8.3, 15.7±1.5 vs 4.1 ± 2.0 P<0.05) at day 7 after TBI, and the number of HPP-CFC of bone marrow cells also increased significantly (15.7±9, 11.7±5 vs 4.3±4.1 P<0.05) at day 7, suggesting a stimulating effect of PF4 on hematopoietic recovery. This gene therapy also caused significant tumor regression. The microvessel density (MVD) of tumors was significantly decreased in the group of treated mice compared to controls (4.25±0.96, 4.08±0.56 vs 11±0.83 P<0.05). Analysis TUNEL kit revealed an increase in the number of apoptosis cells in tumors of mice treated by SL3261 carrying with cDNA of PF4 or a truncated PF4. GFP expression and gene integration were detected in the liver, kidney, spleens, intestine, peripheral blood, bone marrow and tumors samples of the SL3261 treated mice, and the expression of GFP was higher in tumors than that in other tissues. These data demonstrate for the first time a dual biological function of PF4 against tumor growth and radiation injury. These results also demonstrate that attenuated salmonella can be used in vivo as a DNA delivery vector
E2F4 Plays a Critical Role in Early B-Cell Development.
Authors: Smith Clayton , Glozak Michelle , Gasparetto Maura , Rempel Rachel , Domens Jos , Cress Doug , Nevins Joseph
Published: October 2019

The E2Fs are important mediators of cell cycle control, DNA synthesis and apoptosis in many cell types. Recently E2F4 has been shown to play a role in hematopoietic cell growth and development (Rempel et al. Mol Cell, 6 p293, 2000). Here we report the effects of loss of E2F4 specifically on B-cell development. E2F4−/− mice have a partial block in early B-cell development prior to immunoglobulin gene rearrangement. The block is intrinsic to B-cell progenitors rather than secondary to micro-environmental effects since it occurs following transplant of E2F4−/− marrow into wild type recipients. Increases in apoptosis and abnormalities in cell cycle progression were found in B220+CD43+ B-cells of E2F4−/− mice indicating that E2F4 plays an important role in these processes in early B-cells. Expression of a variety of genes important in B-cell development including E2A, RAG, IL-7, EBF and Pax-5 were decreased in early E2F4−/− B-cells. In contrast, Id1 and Id2, regulators of a variety of genes critical to B-cell development, were relatively over-expressed in early E2F4−/− B-cells while Id3 was relatively under-expressed in these cells. E2F binding sites were identified in the Id2 and Id3 promoters and E2F4 was found to directly bind to these promoters in splenic B-cells. These findings suggest that E2F4 may also regulate early B-cell development by directly and indirectly modulating expression of the genes critical to B-cell differentiation. Together, these observations indicate that E2F4 is a critical mediator of early B-cell development via its effects on multiple pathways including those involved with apoptosis, cell cycle progression and differentiation. These findings also suggest that the E2Fs may serve to link cell survival and proliferation pathways to differentiation pathways in early B-cells and perhaps other cells aswell.
Correction of a Murine Model of Von Willebrand Disease by Gene Transfer.
Authors: Pergolizzi Robert G. , Dragos Rachel , Chan Diane , Wagner Denisa D. , Lenting Peter J. , Crystal Ronald G.
Published: October 2019

Von Willebrand disease (VWD) is the most common inherited bleeding disorder affecting 1 to 3% of the U.S. population. It is caused by defects in expression and processing of Von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis, that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. Since it is established that 1–5% of normal levels of VWF is sufficient to ameliorate the bleeding phenotype, the possibility that corrective levels of circulating VWF could be produced using a gene transfer strategy was investigated. However, since the normal sites of VWF production, endothelial cells, megakaryocytes and platelets, are difficult to target with traditional gene transfer methodologies, we explored the strategy of using gene transfer to the liver, an easily transduced ectopic site for VWF production. The study employed a murine “knockout” model of VWD created by disrupting the naturally occurring VWF gene (VWF-KO mice) which exhibit defects in hemostasis with a highly prolonged bleeding time and spontaneous bleeding events closely mimicking severe human VWD. Transected-tail wounds in untreated VWF-KO mice will bleed to death unless cauterized, whereas similar wounds in WT mice stop bleeding in <3 min. To investigate the impact of VWF gene transfer on this phenotype, a plasmid containing the complete murine VWF cDNA under the control of a cytomegalovirus immediate/early promoter/enhancer, was delivered to the VWF-KO mice intravenously. An identical plasmid containing the cDNA for green fluorescent protein (GFP) and saline were used as controls. Bleeding time after wounding was determined 24, 48 or 72 hr after transfer of 10 to 250 μg of plasmid. Bleeding time was normalized by 48 hr after delivery of 50 μg or more of VWF cDNA, but not by GFP cDNA. Western analysis of plasma from animals receiving VWF cDNA, but not GFP cDNA, revealed high-molecular weight VWF multimers similar to the pattern observed in wild type mice. These data suggest that it may be possible to correct VWD using gene therapy delivered to tissues other than the naturally occurring sites of VWF manufacture, and that the liver is capable of expressing, processing and secreting VWF in a physiologically active form.
Eicosapentaenoic Acid (EPA) Attenuates the Anemia Due to Ribavirin/Interferon a Treatment in Patients with Chronic Hepatitis C.
Authors: Seki Ritsuko , Okamura Takashi , Ide Tatsuya , Maruyama Toru , Fujino Takehiko , Uyesaka Nobuhiro , Sata Michio
Published: October 2019

Ribavirin(RBV)is a water soluble synthetic guanosine analog that exerts antiviral activity against DNA and RNA viruses after intracellular phosphorylation. Current studies indicate that combination therapy with RBV and Interferon a (IFNa) is associated with higher rates of sustained virological and biochemical response compared to IFN a monotherapy for chronic hepatitis type C (CHC). Hemolytic anemia is the major side effect of the therapy, with 67% of treated patients developing anemia. The exact mechanism for RBV-related anemia is unknown, but it is thought to be decreasing deformability of eythrocytes resulting from accumulation of phosphorylated RBV in erythrocytes. Highly purified eicosapentaenoic acid (EPA) is widely used for the treatment of hyperlipidemia and atherosclerosis in Japan. We have investigated that EPA has a beneficial effect in patients with RBV-related anemia (Int J Mol Med11:729–732, 2003).

Our aim of this study is to establish the safer treatment of RBV/IFNa using EPA for CHC patients and elucidate the mechanism of EPA action for improving anemia.

[Patients and methods] Thirty three patients with CHC were randomized to RBV/IFNa therapy (800mg/6MU/day) with or without EPA (1800mg/day) for 12 weeks (14:+EPA; 19:-EPA). Erythrocyte filterability (Nickel mesh filtration method; Jpn J Physiol53: 481-486, 2003) was measured before and after 12 weeks of treatment in 8+EPA and 9 -EPA patients. Statistical analysis was performed by Wilcoxon and ANOVA test.

[Result] As shown in Fig. 1, hemoglobin (Hb) levels decreased in both groups by the treatment. However, decrease of Hb levels significantly attenuated by the addition of EPA at 4 and 8 weeks after initiation of the treatment. This effect continued for 12 weeks. Initial mean erythrocyte filterability (%) at study entry was 65.3% and 68.1% in the + and -EPA group, respectively. After 12 weeks treatment, improvement of erythrocyte filterability occurred in 6/8 patients in the +EPA group. In contrast, 7/9 patients resulted in the decrease of erythrocyte filterability in the -EPA group. As shown in Fig 2, the mean erythrocyte filterability (%) for the +EPA group was 71.0%, against 51.4% in the -EPA group (P=0.01) after 12 weeks.

[Discussion and Conclusion] EPA significantly attenuated the anemic effect of RBV/IFNa therapy for CHC patients. Erythrocyte filterability decreased in the -EPA group. On the contrary, decrease of erythrocyte filterability was not found in the +EPA group. Therefore, EPA improved the anemic side effect of RBV/IFNa via the stabilizing action of the membrane by amelioration of erythrocyte filterability. This study would be useful for clinical application.
Stimulation of Transferrin Receptor Expression by Enhanced Heme Biosynthesis in Murine Erythroleukemia Cells.
Authors: Lok Chun-Nam , Ponka Prem
Published: October 2019

In erythroid cells iron uptake from transferrin (Tf) is utilized largely for heme synthesis. Here we provide evidence that Tf receptor (TfR) expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) resulted in an increase in TfR expression accompanied by enhanced uptake of iron from Tf and incorporation of iron into heme. ALA-mediated enhancement of TfR mRNA expression was completely prevented by succinylacetone, an inhibitor of ALA dehydratase, and N-methylprotoporphyrin, an inhibitor of ferrochelatase, indicating that the effect of ALA required its metabolism to heme. Treatment of cells with ALA was associated with enhanced iron regulatory protein-2 (IRP-2) binding activity, which could be blocked by inhibitors of heme synthesis and supplementation of the culture medium with a permeable iron chelate or Tf. In all cases, IRP-2 activities were correlated exactly with TfR mRNA levels. Thus, in addition to the previously characterized transcriptional up-regulation of TfR expression in differentiating erythroid cells, increased TfR expression mediated by enhanced heme biosynthesis may ensure sufficient iron availability for optimal heme synthesis and prevent possible protoporphyrin accumulation under conditions of inadequate iron supply.
Novel Iron Chelators Alleviate Symptoms of Thalassemia.
Authors: Szuber Natasha , Buss Joan L. , Trudel Marie , Felfly Hady , Ponka Prem
Published: October 2019

Thalassemia is a blood disorder requiring lifelong transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction. We hypothesized that removing this proximate iron compartment as a primary treatment using novel iron chelators, could prevent cell death and alleviate the need for transfusion. Novel, highly cell permeable iron chelators, pyridoxal isonicotinoyl hydrazone and pyridoxal ortho-chlorobenzoyl hydrazone (o-108) were compared to the present mainstay, desferrioxamine and deferiprone, in vitro and in vivo. Treatment of human model b-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron (P < 0.002) and reduced oxidative stress, as indicated by methemoglobin levels (P < 0.01). When administered to β-thalassemic mice, iron chelators mobilized erythrocyte membrane iron (P < 0.05), reduced cellular oxidation (P < 0.03), and prolonged erythrocyte survival. Consistently, these mice showed substantial correction of hematological abnormalities (P < 0.05). A beneficial effect as early as the erythroid precursor stage, was also determined by normalized proportions of mature versus immature reticulocytes (P < 0.05). Remarkably, all four compounds reduced iron accumulation in target organs (P < 0.05). Most importantly, o-108 revealed superior activity compared to desferrioxamine, further decreasing iron in spleen and liver by ~2-fold and ~5-fold, respectively (P < 0.05). Our study demonstrates that iron chelators ameliorate thalassemia in a human and murine model, and validates their primary use as an alternative to transfusion therapy.
ABCG2 Expression as Prospective Marker for Hematopoeitic Stem Cell Purification.
Authors: Tadjali Mehrdad , Zhou Sheng , Rehg Jerold , Sorrentino Brian P.
Published: October 2019

ABCG2 (BCRP/MXR/ABCP) is an ABC transporter that effluxes Hoechst 33342 and is required for the side population (SP) phenotype of hematopoietic stem cells (HSCs). Although SP cells are enriched for HSC activity, they are not a pure stem cell population and contain a significant number of differentiated and committed cells. We hypothesized that expression of ABCG2 would provide a more specific marker for HSCs, given our prior results showing that other ABC transporters were expressed in committed SP cells. To test this hypothesis, we have generated a mouse strain in which an IRES-GFP element was inserted immediately after the stop codon of Abcg2, so that GFP and the endogenous Abcg2 gene were co-expressed from the same transcriptional regulatory elements. Immunohistochemical analysis using an anti-GFP antibody showed that Abcg2/GFP allele was strongly expressed in proximal tubule cells of kidney, in the epithelial cells of small intestine, and in vascular endothelium, recapitulating the normal expression pattern of Abcg2. Using flow cytometry to analyze GFP expression in bone marrow cells, we found that 48% of SP cells expressed GFP. In the distal tip of the SP region, previously shown to have the highest concentration of HSCs, 93% of cells expressed GFP. In gated Lin- GFP+ cells within the whole bone marrow population, 55 % of the cells expressed c-Kit and Sca1, a previously identified phenotype for HSCs. We sorted Lin- cells into GFP+ and GFP− subpopulations and determined repopulation frequencies in a limiting dilution assay. Fifteen percent of recipient mice were reconstituted with 10 Lin- GFP+ cells, and 50% mice with 60 Lin- GFP+ cells. In groups transplanted with 125 to 1000 Lin- GFP+ cells, 100% of the mice were reconstituted. In contrast, no reconstitution was seen in any mice transplanted with 60,000 Lin− GFP− cells, indicating that most if not all HSCs expressed the Abcg2/GFP allele. Secondary transplant experiments and individual lineage analysis of peripheral blood cells confirmed that the Lin- GFP+ cells were long-term HSCs. These results demonstrate that expression of Abcg2 in Lin- cells defines a highly enriched population of HSCs, and is a more specific HSC marker than the SP phenotype. We are now increasing the stringency of the GFP sorting gate and transplanting mice with single cells to determine if a pure HSC population can be isolated with this simple 2 marker system.
CD43 Defines Early Hematopoietic Progenitors Derived from Human Embryonic Stem Cells (hESC).
Authors: Vodyanik Maxim A. , Thomson James A. , Slukvin Igor I.
Published: October 2019

We have demonstrated that co-culture of hESCs with OP9 bone-marrow stromal cells induced efficient hESC differentiation into hematopoietic colony-forming cells (CFCs). During hESC/OP9 co-culture, the first CFCs were detectable 1–2 days after emergence of CD34+ cells (day 4–5 of co-culture) and 3–4 days before generation of CD45+ cells, indicating that early CFCs arose in the CD45- population. To define phenotype of early hematopoietic progenitors, we examined the expression of CD41a, CD43, CD61 and glycophorin A (CD235a) on the CD34+ cells in the course of hESC/OP9 co-culture. The first detectable CD34+ cells expressed a high level of VEGF-R2 (KDR) and were CD41a, CD43, CD61 and CD235a negative. Appearance of CFCs was accompanied with down-regulation of KDR and almost simultaneous expression of CD41a, CD43 and CD235a on the CD34+ cells. CD43+ cells gradually increased thereafter, whereas the expression of CD41a was retained on the subset of CD43+ cells that co-express CD235a and CD61. CD45+ cells emerged within CD43+ population and the most of CD45+ cells did not express CD235a, CD41a or CD61. The expression of CD34 was decreased progressively on CD43+CD41a+CD235a+CD61+ cells, but retained by CD43+CD45+ cells. To assay hematopoietic potential of defined subsets, CD34+ cells were isolated by magnetic sorting and further separated into CD34+ CD43+ and CD34+CD43− cell fractions. In addition, CD34−CD43+ cells were isolated from CD34− fraction. CD34+ selection markedly enriched all types CFCs except of E-CFCs. All GEMM-CFCs were found within CD34+ population and none within CD34−. However, CD34− cell fraction contained E-CFCs and minimal numbers of M-CFCs and GM-CFCs. CD34+ cells were also heterogenous in morphologic appearance and contained endothelial cells that were capable to endocytose Ac-LDL. Positive selection of CD43+ cells resulted in total recovery of all types of CFCs. E-CFCs, which consistently detected in the CD34- fraction, were also recovered by CD43 selection. Morphologically, CD43+ cells comprised a homogenous blast-like population and were lack of endothelial-like cells. Thus, expression of CD43 molecule (leukosialin/sialophorin) is detected on the earliest clonogenic hematopoietic progenitors before expression of CD45, persists on developing hematopoietic cells after their lineage specification and most likely defines the divergence of hematopoiesis from endothelial cells during early development in humans.
Mitochondrial DNA (mtDNA) Sequence Heterogeneity among and within Single Human CD34 Cells, T Cells, B Cells and Granulocytes.
Authors: Ogasawara Yoji , Nakayama Kazutaka , Tarnowka Magdalena , McCoy J. Philip , Molldrem Jeffrey J. , Levin Barbara C. , Kajigaya Sachiko , Young Neal S.
Published: October 2019

Abnormalities of mitochondrial DNA (mtDNA) are responsible for a variety of inherited syndromes and have been broadly implicated in aging, cancer, and autoimmunity diseases. Mutations in mtDNA have been reported in myelodysplasia and leukemia, although their pathogenic mechanism remains uncertain. We have described age-dependent accumulation of mtDNA mutations, leading to a high degree of mtDNA sequence heterogeneity among normal marrow and blood CD34 cells as well as in granulocytes (Shin M et al, Blood101:3118 [2003], 103:553 [2004], 103:4466 [2004]). In order to examine mtDNA heterogeneity in detail, we developed a method for analysis of the mtDNA control region from single cells that were sorted by flow cytometry. Highly purified populations of CD34 cells, T cells, B cells, and granulocytes were obtained from five healthy adult donors. The sequence of the individual cells’ mtDNA was compared to the aggregate mtDNA for the respective cell type and differences were expressed as a measure of mtDNA heterogeneity among cells. Overall, heterogeneity was high: for circulating CD34 cells, 38±3.4%; for T cells, 37±14%; B cells, 36±10.8%; and for granulocytes, 48±7.2% (the value for granulocytes statistically differed from CD34 cells; p = 0.03). Most intercellular heterogeneity was due to polyC tract length variability; however, mtDNA base substitution mutations were also prevalent: 15±5.5% in CD34 cells; 15±9.0% in T cells, 15±6.7% in B cells; and 33±2.4% for granulocytes (granulocytes were significantly higher than other cells; p < 0.01). The higher rate of base substitution in granulocytes may reflect their greater exposure to reactive oxygen species. Surprisingly, for both polyC tract length differences and point mutations, the specific mtDNA abnormalities and the proportion of circulating cells characterized by these changes were similar among different cell lineages and relatively constant over time (~2 years) in the same donors. One inference from these results is that mtDNA heterogeneity during development is fixed in the primitive lymphohematopoietic stem cell compartment. In contrast to normal adults, circulating CD34 cells from patients obtained even years after successful allogeneic stem cell transplantation showed a remarkable level of mtDNA homogeneity, similar to the uniformity we have previously observed in cord blood CD34 cells and consistent with limited numbers of stem cells active in these individuals. Leukemic blast cells (from patients with AML-M2, AML evolving from CMML, and T-PLL) also showed a high degree of homogeneity. We propose that mtDNA sequence of single cells may be utilized as a natural genetic marker of hematopoietic progenitors and stem cells; to detect minimal residual disease in leukemia; and as a measure of the accumulation of mutagenic events in mammalian cells in vivo and in vitro.
Tmtsp, a Novel Gene Encoding a Cell Surface Protein Highly Specific to Hematopoietic Stem and Endothelial Cells.
Authors: Takayanagi Shin-ichiro , Hiroyama Takashi , Yamazaki Satoshi , Iwama Atsushi , Nakauchi Hiromitsu
Published: October 2019

In the adult mouse bone marrow, hematopoietic stem cells (HSC) are highly enriched in the CD34 (−/low), c-Kit (+), Sca-1 (+), and lineage marker (−) (CD34 (−) KSL cell population. Using in silico data base search against NCBI EST database, we identified a novel cell surface molecules specific to CD34 (−) KSL HSCs. We cloned its full-length cDNAs of both mouse and human counterparts from total embryo (E15) and human spleen cDNAs, respectively. They were 2815-bp and 2953-bp long and the deduced amino acid sequences consisted of 851 and 852 amino acids, respectively. They contain a signal sequence, three Ig-like domains, a Thrombospondin type 1 (TSP1) domain, and a transmembrane region. Its domain composition shows high similarity to that of Unc5h3, a receptor for netrin, which plays a key role in neuronal axon guidance. Therefore, we designated this gene as Transmembrane Molecule with Thrombospondin Module (Tmtsp). BLAST search against the mouse genome database displayed that the mouse Tmtsp is composed of four exons and spanning over 26kbp on the chromosome 8qA2. Tmtsp was translated into a 170 kDa transmembrane protein. An alternative splicing which splices out the 3rd exon was detected in all major organs tested, leading to in-frame translation of a variant protein without TSP1 domain. RT-PCR analysis of hematopoietic cells demonstrated that Tmtsp is confined to KSL cells in expression and shows even higher expression in CD34 (−) KSL HSCs. On the other hand, Northern blot analysis detected its expression in all major organs tested and the highest expression in the lung. To specify the cells that express Tmtsp, we generated monoclonal antibodies against the extracellular domain of Tmtsp. Immunohistochemical analysis using anti-Tmtsp monoclonal antibodies revealed that its expression is tightly restricted to endothelial cells in tissues. These findings indicate that Tmtsp is a novel cell surface transmembrane molecule specific to both hematopoietic stem/progenitor cells and endothelial cells. Among a list of molecules shared by HSC and endothelial cells, Tmtsp is the first one that possesses the TSP1 domain implicated in cell adhesion. In this context, structure and expression of Tmtsp strongly suggest important roles in the development and maintenance of HSCs and endothelial cells including HSC homing and formation of vascular network.
A Unique Transcriptional Profile for Spontaneously Immunoglobulin Secreting B-1 Cells.
Authors: Tumang Joseph R. , Frances Ruben , Rothstein Thomas L.
Published: October 2019

Natural antibodies are spontaneously arising polyreactive Ig, usually IgM, that are encoded by V genes in germline configuration and are produced by B-1 cells. Because of their ability to promiscuously bind to a variety of antigens, especially those on bacteria and viruses, natural antibodies act as a primary line of defense against infection. A key shortfall in our understanding of these key effector molecules centers on the molecular mechanisms regulating natural antibody secretion by B-1 cells. Recent advances in our understanding of plasmacytic differentiation now provide us with a conceptual framework by which we can begin to explore this issue. In the studies described herein, we demonstrate that secreting B-1 cells utilize some aspects of the plasmacytic differentiation program and deviate away from it in others. Specifically, we show that key repressors of the plasmacytic program, BCL-6 and PAX-5a, are indeed reduced in secreting B-1 B cells. Surprisingly, we found that key promoters of the plasmacytic program, BLIMP-1 and XBP-1, are not upregulated in secreting B-1 B cells. These data demonstrate that B-1 B cells operate under a differentiation program unique from prototypic secreting B-2 cells and cast BCL-6 and PAX-5a but not BLIMP-1 and XBP-1 as central regulators of natural immunoglobulin secretion.
Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).
Authors: Multhoff Gabriele , Gross Catharina , Dickinson Anne , Holler Ernst
Published: October 2019

Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT).

Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells.

Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells.

One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals.

Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT.

Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.