bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of CXCR1, CXCR2 and CXCR4 in Human Neutrophils: Potential Role in the Release from the Bone Marrow, Clearance of Senescent Cells, and Cell Function at Sites of Inflammation.
Authors: Yildirim Sedat , Bautz Frank , Boehmler Andreas M. , Kanz Lothar , Möhle Robert
Published: April 2020

In the mouse model, it has been shown that the interleukin-8 (IL-8) receptor CXCR2 is involved in the release of mature neutrophils from the bone marrow into the circulation. When neutrophils age, upregulation of CXCR4 and downmodulation of CXCR2 result in homing and subsequent sequestration of senescent cells in the bone marrow. In our study, we observed a similar time-dependent (starting at 3 hrs., maximum at 12–18 hrs.) downregulation of CXCR2 in human neutrophils during aging in ex vivo culture, while expression of the second IL-8 receptor CXCR1, which is mainly responsible for the IL-8-induced chemotaxis, was unchanged. Furthermore, strong upregulation of CXCR4 was noted on the cell surface which could not solely be attributed to re-expression of internalized, intracellular receptors, as an increased amount of CXCR4 mRNA was detected by Northern blot analysis in these cells. The increase in CXCR4 expression was not influenced by inflammatory cytokines such as TNF and IL-1, as well as by IL-8 or G-CSF. Accordingly, SDF-1-induced transendothelial migration of aged neutrophils was 6-fold increased and even exceeded migration in response to IL-8. We conclude that also in human neutrophils, loss of CXCR2 and gain of CXCR4 expression on the cell surface may favor homing and sequestration of senescent cells in the bone marrow. At sites of inflammation however, retained expression of CXCR1 and increased expression of CXCR4 still allow a response of aged, pre-apoptotic neutrophils to the chemotactic mediators IL-8 and SDF-1, as the latter is not only released in the bone marrow, but also at sites of tissue damage and necrosis.
Rb Plays an Essential Role in Erythroid Differentiation through Inhibition of Apoptosis Mediated by NFKB.
Authors: Matsuoka Sahoko , Hirao Atsushi , Arai Fumio , Takubo Keiyo , Miyamoto Kana , Hosokawa Kentaro , Suda Toshio
Published: April 2020

Inactivation of the retinoblastoma (Rb) gene results in embryonic lethality due to severe anemia and increased nucleated erythrocytes by day14. However, molecular mechanisms of the function of Rb in erythroid differentiation have been unclear. Recent studies have suggested that Rb has both intrinsic and extrinsic roles on erythroid differentiation. Using Rb-deficient (Rb−/−) embryos(E12), we showed that Rb regulates terminal erythroid differentiation through inhibition of apoptosis mediated by NFKB. Enucleation of erythroblasts was impaired in semisolid culture of Rb−/− hematopoietic progenitors in fetal liver. The lethally-irradiated recipient mice transplanted with Rb−/− hematopoietic stem cells (HSCs) showed severe anemia with splenomegaly, whereas the number of leukocytes and platelets were normal. In Rb−/− recipient mice, the nucleated erythrocytes and reticulocytes were significantly increased in the peripheral blood. We analyzed cell surface markers for erythroid lineage (TER119 and CD71) in the enlarged spleen. A block of erythroid differentiation at the early erythroblast stage (TER119high CD71high), accompanied with increased apoptosis, was observed in the recipient mice with Rb−/− HSCs. We speculated that the defect in the erythroid differentiation of Rb−/− HSCs might be caused by inappropriate cell death. Thus, we examined expression of apoptosis-related genes in early erythroblasts (CD71high Ter119high) and observed decrease of Bcl-XL expression. To clarify the function of Bcl-XL, we introduced exogenous cDNA of mouse Bcl-XL with GFP (Bcl-XL ires GFP) or GFP alone as control into HSCs and then transplanted them to lethally irradiated mice. From the point of CD71 and Ter119 expression pattern in GFP positive cells, Rb−/− erythoblasts still showed the block in differentiation. In contrast, overexpression of Bcl-XL in Rb−/− erythoblasts inhibited inappropriated apoptosis and restore the differentiation capacity. Further, we found that inactivation of NFKB, but not STAT5 in Rb−/− erythroblasts. Treatment of NFKB inhibitor suppressed erythroid differentiation, accompanied by enucleation, and also inhibited upregulation of Bcl-XL. These data demonstrates that Rb is essential for erythroid differentiation through inhibition of apoptosis mediated by NFKB.
The Role of the Small GTPase Rab27a in the Regulated Secretion of Granulocytes.
Authors: Catz Sergio D. , Munafo Daniela B. , Ellis Beverly A. , Johnson Jennifer L.
Published: April 2020

Neutrophils kill microorganisms using microbicidal products that they release into the phagosome or the extracellular space. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules that contain myeloperoxidase, specific granules, gelatinase granules and secretory vesicles. Since the uncontrolled release of the contents of these organelles is potentially harmful, granule exocytosis must be tightly regulated. However, the secretory machinery utilized by neutrophils is poorly characterized. Here, we evaluate the role of Rab27a and its effector JFC1 in the regulated secretion of granulocytes. First, we show that JFC1 and Rab27a are highly expressed in human neutrophils. Furthermore, in HL-60 promyelocytic cells, the expression of JFC1 and Rab27a dramatically increased when they are differentiated to neutrophil-like granulocytes by DMSO treatment, supporting a role for these proteins in the secretory machinery of granulocytes. We found that while Rab27a distribution is restricted to the membrane fraction after cell fractionation, JFC1 is distributed between the membrane fraction and the cytosol. The localization of Rab27a in neutrophils was confirmed by immuno-electron microscopy. Colocalization of endogenous JFC1 and Rab27a was observed by immunofluorescence analysis. After cell fractionation, JFC1 and Rab27a were mainly associated with the tertiary granules (γ fraction) which are enriched in MMP-9 and cytochrome b558. A small proportion of the myeloperoxidase-positive granules were also detected in this fraction. Both Rab27a and JFC1 colocalized with VAMP2, a marker of secretory vesicles. The introduction of the plasma membrane binding domain (C2A domain) of JFC1 into permeabilized neutrophils significantly decreased exocytosis, however, neither Rab27a nor JFC1 integrated to the phagolysosome when neutrophils were exposed to opsonized particles, suggesting that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.
Prospective Evaluation of Graft-Versus-Host Disease-Specific Proteomics Pattern in Patients after Allogeneic Stem Cell Transplantation.
Authors: Weissinger Eva M. , Hillmann Meike , Kolb Hans-Jochem , Zander Axel R. , Holler Ernst , Mischak Harald , Ganser Arnold , Hertenstein Bernd
Published: April 2020

We have recently described and published a proteomic pattern specific for the early diagnosis of acute graft versus host disease (aGvHD), based on the application of capillary electrophoresis (CE) and mass spectrometry (MS). Here we report the application of proteome based GvHD-patterns to prospectively collected samples from 86 patients (45 AML, 14 ALL, 8 MM, 5 CLL, 5 SAA, 3 MDS, 3CML, 3 NHL). Fifty three patients were transplanted from matched unrelated donors (MUD), 29 received stem cells from matched related donors (MRD), 3 from haplo-identical donors and 1 was transplanted from a syngeneic sibling. GvHD prophylaxis was methotrexat or mycophenolate and cyclosporin A in the majority of the patients. The follow up is currently between 60 and 549 days after allogeneic HSCT. Urine samples were collected on ice prior conditioning, once a week and monthly after day +60. To avoid degradation of the proteins/peptides the samples were frozen as soon as possible. After thawing and removal of confounding substances, like salts, lipids and of all molecules larger than 30 kDa, the samples were loaded onto the CE, separated according to their charge and, after ionization, directly analyzed in an electrospray ionization time of flight (ESI-TOF) -MS. Between 500 and 2500 peptides and proteins were detected in individual samples. All data generated are stored in a Microsoft MS database. Reproducibility of these analyses is greater 98%. The polypeptide patterns specific for the early detection of acute GvHD were applied to the data generated prospectively and the outcome of these analyses was compared to the clinical diagnosis of aGvHD, sepsis and CMV-reactivation. Forty patients were diagnosed with aGvHD in the current evaluation period, 18 had aGvHD ≥ II. From 461 samples, 68 gave a score with the aGvHD pattern, 15 were false positive, whereas 5 scored false negative. The sensitivity of the aGvHD pattern is more than 90%, the specificity is about 96%. Thus the application of the aGvHD pattern for early recognition of acute GvHD is very useful for predicting the development of aGvHD. In addition, we are currently evaluating samples of patients with chronic GvHD (cGvHD). Seven patients in the prospective cohort have developed cGvHD so far. First results show that in the majority of the patients the cGVHD markers are different from those forming the aGvHD pattern. The polypeptide patterns for aGvHD and cGvHD will be discussed together with those of other complications, like CMV-reactivation or sepsis.

Taken together our results demonstrate that the proteome analysis of body fluids collected from patients after HSCT maybe extremely useful for diagnosis of complications.
Suicide Gene Therapy of Graft-Versus-Host Disease Induced by Central Memory Human T Lymphocytes.
Authors: Bondanza Attilio , Valtolina Veronica , Magnani Zulma , Kaneko Shin , Ponzoni Maurilio , Sanvito Francesca , Bonyhadi Mark , Traversari Catia , Toma Salvatore , Radrizzani Marina , La Seta-Catamancio Simona , Fleischhauer Katharina , Ciceri Fabio , Bordignon Claudio , Bonini Chiara
Published: April 2020

Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.
Nucleophosmin Regulates Differentiation, Cell Cycle Progression, and Stress Response in Hematopoietic Progenitor Cells.
Authors: Li June , Sejas Daniel P. , Pang Qishen
Published: April 2020

Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Strong evidence indicates that NPM is involved in hematopoiesis and leukemic development. Here we report that NPM enhances the proliferative potential of hematopoietic stem/progenitor cells and increases cell survival upon stress challenge. Specifically, lin-Sca1+c-kit+ bone marrow cells transduced with retroviral vector expressing NPM exhibited higher proliferative rates in both short-term liquid culture and clonogenic progenitor cell assays, compared to the cells transduced with empty vector. Interestingly, NPM overexpression appears to inhibit differentiation of myeloid progenitors. Hematopoietic stem/progenitor cells infected with the NPM retrovirus expressed significantly lower levels of mature cell markers Gr-1 and Mac-1 compared to empty vector transduced cells, and majority of the NPM-overexpressing cells remained Sca1+C-Kit+ during the 5-day culture. Bone marrow transplantation experiments demonstrated that NPM overexpression increases long-term multi-lineage repopulating capacity of hematopoietic progenitors. We have not observed any evidence of proliferative disorders or leukemia in recipients transplanted with NPM-expressing progenitors thus far (4 months posttransplantation). Through cell-cycle profile analysis and single-cell division experiments, we showed that NPM overexpression induces rapid entry of hematopoietic progenitors into the cell cycle, probably via promoting G0/G1 to S transition. Furthermore, immunocytochemical and Western-blot analyses demonstrated that NPM-transduced cells expressed higher level of cyclin A compared to vector-transduced cells. Finally, overexpression of NPM significantly increased the survival of hematopoietic progenitors exposed to mitomycin C or hydrogen peroxide, suggesting that NPM can protect cells from DNA damage and oxidative stress. Together, these results indicate that NPM plays an important role in hematopoiesis via mechanisms involving modulation of progenitor differentiation, cell cycle progression, and stress response.
Daptomycin (DAP) Use in Patients with Hematologic (HEM) Cancer History; Post-Marketing Experience in a Registry.
Authors: Akpek Gorgun , Forrest Graeme , Lamp Kenneth C. , Donovan Brian J. , Friedrich Lawrence V.
Published: April 2020

Background: DAP is a novel lipopeptide antibiotic, approved for complicated skin and skin structure infections, with potent in vitro activity against many resistant Gram-positive organisms. Registration studies often limit or exclude patients with severe illness. Antibiotic resistant Gram-positive pathogens are frequently encountered in compromised patient populations; however, minimal information is available pre-approval in these patients.

Methods: Cubicin® Outcomes Registry and Experience (CORE) is a Phase IV retrospective observational chart review (45 institutions) to quantitate characteristics and clinical outcomes of patients (pts) receiving DAP. A standard case report form including demographic, disease state, clinical, and microbiologic data was completed by each investigator. Cure, failure, and improvement were assessed using standard definitions. Nonevaluable pts had insufficient data available to determine clinical outcome. Of the 1160 pts in CORE, 56 were reported with a history of HEM cancer. Eighteen (32%) were nonevaluable; 6 of those expired. This report describes the 38 pts with an assigned outcome.

Results: Fifty-five percent of pts were female, 63% were 31 to 65 years of age, and 76% of infections developed in a hospital setting. Common comorbidities included cardiovascular disease (24%), immunosuppression (18%), and transplant (11%). The most common infections were; bacteremia catheter-related (53%), skin and skin structure (34%), and bacteremia noncatheter-related (18%). Seventy-one percent (27/38) of pts were culture positive before DAP with the most common pathogens being Enterococcus sp. (52%, of which 71% were VRE), coagulase-negative staphylococci (41%), and S. aureus (19%, of which 80% were MRSA). A high percentage of pts (92%) received antibiotics (abx) prior to DAP. The most frequent initial dosing regimens were 4mg/kg QD (50%), and 6mg/kg QD (29%). The mean duration of DAP therapy was 13.6 days (range 2–44). Dose (P=0.3) and duration (P=0.58) did not vary by primary pathogen. Twenty-nine (76%) pts received concomitant abx therapy, most commonly with fluoroquinolones (31%), metronidazole (28%) or cephalosporins (24%). The mean time to clinical response was 2.4 days (n=28) and unaffected by primary pathogen (P=0.43) or the influence of concomitant abx therapy (P=0.55). All pts were reported as cured (66%) or improved (34%).

Conclusion: The data from the CORE registry demonstrates that DAP shows early clinical response and clinical cure for HEM patients where catheter associated infections and VRE are prevalent. These data provide preliminary data on the use of DAP in this compromised patient population. This information should lead to further prospective studies in cancer patients with DAP.
Cost-Effectiveness of Itraconazole for the Prophylaxis of Invasive Fungal Infections for Neutropenic Patients; a Study for 3 European Countries.
Authors: Postma Maarten J. , de Vries Robin , Christopherson Harriet , Howells Sarah , Tolley Keith , Daenen Simon , Glasmacher Axel , Prentice Archibald , Christie Angela
Published: April 2020

Background: Invasive fungal infections present a leading cause of mortality and morbidity in neutropenic patients treated for hematologic malignancies. As the diagnosis and management of invasive fungal infections is difficult, effective antifungal prophylaxis is desirable. Itraconazole is a broad spectrum antifungal agent with activity against Candida species as well as Aspergillus species, whereas fluconazole primarily targets Candida. There is no published data comparing cost-effectiveness of both antifungal agents.

Objective: To asses the cost-effectiveness of itraconazole compared with (i) prophylactic fluconazole and with (ii) no prophylaxis in the prevention of invasive fungal infections in Germany, the Netherlands and the UK.

Methods: A probabilistic decision model was designed to evaluate the incremental cost-effectiveness of itraconazole versus fluconazole and versus no prophylaxis (see Figure). Baseline risks for invasive fungal infections and risk reductions for itraconazole (>200mg/day) and fluconazole (400mg/day PO or 200mg/day IV) were taken from studies recently published in two meta-analyses. Costs were evaluated from the health care perspective. Drug acquisition costs for the two prophylactic regimens were calculated using 2003 prices. We estimated the increase in the length of stay as a result of an invasive infection using local or national databases and costed this using reference prices.

Results: The meta-analysis revealed that itraconazole is effective in averting invasive fungal disease, in particular aspergillosis. The mean increase in the length of stay for invasive fungal infections was estimated at 9.3 days (rather similar for all 3 countries). As an illustration for our findings: in the Netherlands, itraconazole prophylaxis was expected to be cost-saving compared to fluconazole and provided limited extra costs compared to no prophylaxis at all. Cost-effectiveness was €470 per invasive infection averted for the itraconazole compared to no prophylaxis (95%-CI ranging from cost-saving to €5926 per infection averted). Other country-specific results will be presented at the conference.

Discussion & Conclusions: Our study shows that itraconazole prophylaxis is effective and is clinically likely to result in cost-savings or provide an acceptable cost-effectiveness. Itraconazole should be the first choice in the prophylaxis of invasive fungal infections in neutropenic patients with hematologic malignancies, from the clinical and economic point of view.

Figure Figure
Constitutive Activation of STAT5 and Bcl-XL Overexpression Can Induce Endogenous Erythroid Colony Formation in Human Primary Cells.
Authors: Garçon Loïc , James Chloe , Lacout Catherine , Camara-Clayette Valérie , Ugo Valérie , Raphael Martine , Vainchenker William
Published: April 2020

In contrast with secondary erythrocytosis, progenitor cells from polycythemia vera (PV) patients can undergo in vitro erythroid differentiation despite absence of erythropoietin (EPO) and presence of such endogenous erythroid colonies (EEC) is routinely used as a diagnostic assay. Recent focus on the JAK2 mutation V617F in PV patients argue for a direct implication of JAK2 dependent signaling pathways in EEC formation. Because STAT5 is the principal target of JAK2 in erythroid cells, we investigated whether EEC formation was only dependent on STAT5 activation or required other signaling pathways that would be activated by JAK2. For this purpose, we transduced a retroviral vector coding for a constitutively active form of STAT5 (MIGR-STAT5CA) in UT7 cells, a leukemic cell line with erythroid properties. We observed in cells transduced with the MIGR-STAT5CA vector a spontaneous induction of erythroid differentiation in comparison with cells infected with the empty vector MIGR, as assessed by GPA staining. We next investigated effects of STAT5CA on erythroid differentiation of human primary progenitors. Purified CD34+ cells obtained from peripheral blood (PB) of patients treated with G-CSF were transduced with the STA5CA vector, the CD36+/GPA− erythroid progenitor cells were sorted and cultured in presence of SCF alone. When expressing STAT5CA, they both proliferate and undergo erythroid terminal differentiation despite the absence of EPO. We concluded that a phosphorylated form of STAT5 was sufficient to support in vitro erythroid differentiation of human primary cells. Because STAT5 has been shown to play a crucial role in erythropoiesis via induction of the antiapoptotic protein Bcl-xL, we next investigated whether effects of STAT5CA on erythroid maturation was dependent on Bcl-xL induction. Tansduction of human CD36+/GPA− cells with a retrovirus containing the coding sequence of human Bcl-xL progenitors allowed survival, proliferation and GPA acquisition despite the absence of EPO. We next investigated whether STAT5CA or Bcl-xL overexpression in normal primary cells could reproduce the malignant phenotype observed in PV patients, i.e. induction of EEC formation. CD36+/GPA− transduced with either the STAT5 CA or the Bcl-XL vectors were plated in methylcellulose in the absence of EPO. Bcl-xL as well as STAT5CA vectors could both induce endogenous erythroid colony formation. Regardless to these results, we hypothesized that the EEC formation observed in myeloproliferative disorders could be at least partially due to the JAK2 dependent activation of the STAT5/Bcl-XL pathway. Thus, both constitutive activation of STAT5 and Bcl-xL overexpression could substitute to EPO to induce terminal differentiation of human primary erythroid progenitors.
The Role of K-ras Signaling in Erythropoiesis In Vivo.
Authors: Zhang Jing , Liu Yangang , Beard Caroline , Jaenisch Rudolf , Jacks Tyler , Lodish Harvey F.
Published: April 2020

K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.
Essential Role and Regulatory Mechanism of the Erythropoietin Gene in the Liver.
Authors: Suzuki Norio , Obara Naoshi , Kim Ki-bom , Pan Xiaoqing , Jishage Ko-ichi , Imagawa Shigehiko , Yamamoto Masayuki
Published: April 2020

Erythropoietin (Epo) is produced from the kidney and liver under anemic and/or hypoxic conditions. This inducible and tissue-specific production of Epo is regulated at the Epo gene transcription level. To elucidate the mechanisms how hypoxia inducible gene expression is regulated, a number of studies have been carried out and the function of the hypoxia-responsible enhancer sequence located in the 3′ flanking region of the Epo gene (3′HRE) were identified using the Epo-producing human hepatoma cell lines, Hep3B and HepG2. In order to clarify in vivo function of 3′HRE, in this study we deleted 500-bp mouse genomic region (referred to as BS region) containing 3′HRE by the gene targeting strategy. The homozygous BS-null mutant neonates suffered from severe anemia (hematocrit 17±2%) compared with wild-type neonate (hematocrit 28±2%). This anemic phenotype was observed from embryonic day 15 (E15). To our surprise, however, erythropoiesis in the adult BS-null mice was within the normal range. We then examined the expression level of Epo gene in the kidney and liver of BS-null mouse line in the embryonic stage, and found that the Epo gene expression was completely silenced in the BS-null embryonic liver after E15 to adulthood. The BS deletion did not affect the Epo gene expression in the liver up to midgestation stage (E10 to E13) and in the kidney throughout life. These results thus demonstrate that the BS region including 3′HRE is the enhancer for Epo gene that is functional after the late embryonic stage and in the hepatocytes. BS-null mice also showed that the hepatic erythropoiesis in the embryonic and neonatal stages requires the paracrine Epo secretion from the hepatocytes, and the Epo production in the kidney is insufficient to compensate the loss of hepatic Epo production.
Ex Vivo Expansion of Human SCID-Repopulating Cells Using Recombinant TAT-HOXB4 Protein.
Authors: Krosl Gorazd , Giard Marie-Pier , Krosl Jana , Beauchemin Stephanie , Humphries Keith , Sauvageau Guy , Roy Denis Claude
Published: April 2020

One of the major obstacles to the successful clinical application of hematopoietic stem cell (HSC) transplantation, particularly in the context of related haplotype-mismatched transplantation, unrelated cord blood transplants for adults, and grafts that are processed ex vivo to remove malignant, or alloreactive T cells, is the number of available long-term repopulating HSCs. The addition of soluble recombinant TAT-HOXB4 protein was recently reported to enable rapid in vitro expansion of murine HSCs that retain their in vivo proliferation and differentiation capacity. However, the ability of this recombinant protein to effectively expand human hematopoietic stem cells remains hypothetical. In addition, limited information is available on underlying mechanisms of HOXB4 HSC expansion. First, to determine the capacity of recombinant TAT-HOXB4 protein to promote human HSC expansion, we treated human CD34+ cells for 4 and 8 days with 40 nM, or 80 nM TAT-HOXB4 protein in X-Vivo 15 medium supplemented with Stem Cell Factor, TPO, IL-6 and Flt3-ligand. Cultures exposed to TAT-HOXB4 treatment for 8 days had no pronounced effect on the total cell yield. During this period, a 2-fold net loss of CFU-GEMM was observed for controls, in comparison to ~8-fold and ~5-fold expansions in response to 40 nM and 80 nM TAT-HOXB4 (p<0.05), respectively. Recombinant TAT-HOXB4 also induced ~10–15-fold expansion of large CFU-GM, compared to only ~2.5-fold increase for controls (p<0.05). HSC numbers were enumerated at the beginning and at the end of the treatment using the principle of limiting dilution in a 4-month NOD/SCID repopulation assay. Culture for 8 days in cytokines devoid of TAT-HOXB4 resulted in ~2-fold loss of SCID Repopulating Cells (SRCs), while cultures supplemented with 40 and 80 nM TAT-HOXB4 protein showed a 2.5-fold (95% CI 1.7 – 3.3 fold) and 5.5-fold (95% CI 3.6 – 7.4 fold) increase, respectively. Then, to determine whether the increase in HSC numbers resulted from HOXB4-enhanced proliferation of HSCs, we examined the cell cycle profile of control and HOXB4-treated cell populations using Hoechst 33342 and pyronin Y dyes. After 4-day treatment with 80 nM TAT-HOXB4, 44%±12% of CD34+CD38+ cells were in Go, compared to only 19%±6% of the controls (p<0.05). In contrast, similar proportions (89–91%) of quiescent CD34+CD38− cells were observed for both conditions. Tracking cell divisions using CFSE also showed that during this period, HOXB4-treated CD34+CD38+ cells underwent ~2 population doublings less than controls (p<0.05). In conclusion, short-term exposure of human CD34+ populations to recombinant TAT-HOXB4 protein has the potential to achieve clinically relevant HSC expansion levels. At the HSC level, these observations suggest that TAT-HOXB4 preferentially affects cell fate (self-renewal?) rather than cell proliferation.
Morphine Induces Kidney Injury in Transgenic Sickle Cell Mice.
Authors: Weber Marc L. , Hebbel Robert P. , Gupta Kalpna
Published: April 2020

Sickle cell nephropathy is poorly understood and currently lacks any specific therapeutic options. Opioids are used to treat the pain associated with sickle cell disease. Interestingly, heroin (a metabolic precursor of morphine) is associated with glomerulosclerosis. Glomerulomegaly, an early sign of kidney damage, predicts the eventual severity of glomerulosclerosis. Using transgenic sickle mice as a disease model, we evaluated the effects of chronic opioid exposure on kidney pathology in the setting of sickle cell disease.

Transgenic sickle cell mice (carry a single copy of linked transgenes for human alpha and beta-S globins and are homozygous for deletion of murine beta major globin) were treated for three weeks with PBS, morphine (50–150mg/70kg human equivalent), naloxone (equimolar to morphine), or morphine and naloxone (n=6 per group). We evaluated the effects of morphine on kidney pathology by assessing kidney weight, glomerular volume (by the Weibel-Gomez method), and mesangial cell counts per glomerulus. Morphine stimulated kidney growth in transgenic sickle mice. Treatment with morphine significantly increased glomerular volume and mesangial cell counts per glomerulus compared to PBS. These effects were reversed with the addition of naloxone to morphine. (see Table)

Morphine-stimulated cultured mouse mesangial cells underwent proliferation, which was inhibited with the addition of naloxone (202% ±14.3 vs. 108% ±12.9 increase over control, p<0.01). Morphine-stimulated mesangial cells had increased expression of phosphorylated STAT-3 by western blot analysis. Blockade of STAT-3 with PpYLKTK-mts (0.2mM) resulted in inhibition of morphine-stimulated mesangial cell proliferation (202% ±14.3 vs.110±29, p<0.01).

In summary, morphine exposure markedly induced kidney growth in transgenic sickle mice, as evidenced by increased kidney size, glomerular volume, and glomerular cell counts. Furthermore, morphine stimulates a proliferative response in mesangial cells via STAT-3 signaling. These results suggest morphine may accelerate renal injury in sickle cell disease.

Group Kidney Weight (g) Glomerular Volume (mm3) #MC/glomerulus Results (mean ± SD): λ p<0.01 vs. Sickle PBS; π p=0.01 vs. Sickle MS; γ p<0.01 vs. Sickle MS. Sickle PBS 0.190 ± 0.02 133 ± 7 27 ± 0.5 Sickle Naloxone 0.176 ± 0.01 124 ± 13 26 ± 1.2 Sickle Morphine 0.251 ± 0.04 λ 163 ± 14 λ 37 ± 1.9 λ Sickle MS+ Nal 0.193 ± 0.01 π 127 ± 12 π 30 ± 1.5 γ
Myocardial Ischemia without Coronary Artery Obstruction in Patients with Sickle Cell Disease.
Authors: Raman Subha V. , Simonetti Orlando P. , Cataland Spero R. , Hicks William J. , Kraut Eric H.
Published: April 2020

Objectives. Sickle cell disease (SCD) is the most common single gene disorder in black Americans, with poorly described cardiovascular sequelae partly due to insensitive techniques to identify microvascular perfusion abnormalities. We employed cardiac magnetic resonance (CMR) and computed tomography (CTA) to assess myocardium, microvascular perfusion and coronary arteries in SCD patients.

Methods. Twelve adult patients with SCD (10 HbSS, 1 HbSC and 1 S-thal) age 20–45 years were enrolled. CMR protocol included: dark blood multiecho imaging, rest and stress myocardial perfusion, multiplane cines, and delayed post-gadolinium imaging (DME) for infarct imaging. Myocardial T2*, reflecting iron content, was calculated and verified using a T2* phantom. Left and right ventricular volumes and ejection fractions were quantified from short axis cines. Coronary CTA was performed in patients and five controls using a 64-slice scanner and 70cc of isotonic, isoosmolar contrast, with vessel review in multiple planes.

Results. Three subjects, all SS genotype, had striking subendocardial ischemia (top image, arrows) with vasodilator stress that was not present at rest (signal intensity-vs-time curves, solid line=abnormal subendocardium, dotted and dashed=normal myocardium, dot-dash=blood pool). Myocardial T2* was normal in all subjects (25 to 33ms) as were DME, LV mass and LVEF. One subject with pulmonary hypertension had marked RV dysfunction (RVEF 26%). No subject had obstructive epicardial coronary disease, though proximal coronary diameters were significantly larger for SCD patients compared to controls (2.7mm/m2 versus 1.9mm/m2, p<0.001).

Conclusions. A subset of SCD patients has quantifiable myocardial ischemia with coronary dilatation and absence of infarcted myocardium. Further studies are ongoing to correlate perfusion abnormalities with episodic chest pain and ischemia-based therapies.

Figure Figure Figure Figure
Lung Function and High Resolution Computed Tomography in Sickle Cell Disease.
Authors: Sylvester Karl P. , Desai Sujal R. , Wells Athol U. , Hansell David M. , Awogbade Moji , Thein Swee Lay , Greenough Anne
Published: April 2020

Adults with sickle cell disease (SCD) may develop sickle chronic lung disease which appears to be a complication of chronic hemolysis and repeated episodes of pulmonary vaso-occlusion. Pulmonary function tests have usually been used to document the severity of lung disease in SCD, but some patients cannot successfully undertake such examinations. Recently, high resolution computed tomography (HRCT) has been shown to be of value in the evaluation of patients with diffuse lung diseases. Assessment of lung disease in SCD by HRCT, however, has not been fully explored. The study sets out to investigate the relationship of any pulmonary function abnormalities in adults with SCD to a non-invasive measure of hemolysis and findings on HRCT. The study population consisted of 33 Hb SS subjects (12 males) with a median age of 36 (range 17–67) years, median height of 167 (range 53–188) cm, and median weight of 69 (range 52–92) kg.

Pulmonary function was assessed by measurements of lung volumes, spirometry, gas transfer and oxygen saturation, and hemolysis by measurement of end-tidal carbon monoxide (ETCO). The degree of a lobar volume loss and ground glass opacification and prominence of central vessels on HRCT were quantitatively assessed.

There was a wide variation in the lung function of the cohort. Nine patients had a restrictive lung function abnormality, five an obstructive abnormality and four a mixed restrictive/obstructive abnormality. Twenty-seven of the patients completed assessment of ETCO levels. ETCO levels correlated positively with bilirubin levels (rs=0.66, p=0.0002) and the absolute reticulocyte count (rs=0.70, p=0.0002), and negatively with hemoglobin (rs=−0.51, p=0.008). ETCO levels negatively correlated with FEV1 (p=0.006), VCpleth (p=0.006), sGaw (p=0.04) and SpO2 (p=0.007). A reticular pattern, lobar volume loss and prominent central vessels were the three most common abnormalities on HRCT. FEV1 (p<0.05), FVC (p<0.005) and TLC (p=0.008) correlated with HRCT findings, particularly lobar volume loss. Lobar volume loss, prominent central vessels and a reticular pattern/ground glass opacification were all present on HRCT in 12 of 18 subjects with a restrictive, obstructive or mixed defect.

Our results suggest that non-invasive assessment of hemolysis and/or HRCT examination might facilitate identification of SCD patients with respiratory function impairment. More of the patients had abnormalities on HRCT than on lung function testing, which suggests that HRCT is a more sensitive detector of respiratory abnormalities than lung function testing. This hypothesis merits testing by serially assessing SCD patients to determine if those with only HRCT abnormalities subsequently develop lung function abnormalities.
Vascular Abnormalities and Pulmonary Hypertension in the Berkeley Sickle Mouse.
Authors: Archer David R. , Elms Shawn , Boutwell Joshua , Perry Jennifer , Sutliff Roy
Published: April 2020

Clinically, pulmonary hypertension is a major risk factor for mortality in adults with sickle cell disease. Contributing factors probably include red cell hemolysis and vaso-occlusive injury with their associated oxidative and inflammatory stimuli. Previously, we have described RBC hemolysis and endothelial oxidative stress in the Berkeley sickle mouse model and extend those studies in this work to investigate cardiovascular and endothelial dysfunction. Eight to ten month old homozygous and hemizygous Berkeley sickle mice and C57BL/6 control mice were used for all aspects of these experiments. In vivo measurements of mean arterial pressure and right ventricular pressures were conducted in fully anesthetized mice using a pressure transducer inserted in the carotid and right ventricle respectively. Following in vivo readings hearts were excised for measurement of ventricular mass. The ascending aorta was removed and cut into 5 mm rings for in vitro studies of agonist- induced contractility and relaxation. The mean arterial pressure of the hemizygous sickle mice (70.6 ± 3.4) was significantly lower than the control mice (86.0 ± 3.1) and the mean arterial pressure of homozygous sickle mice (59.0 ± 2.2 mmHg) was significantly lower than the hemizygous and control mice (p≤0.05 and p≤0.001, respectively). The right ventricular pressure showed a trend that approached significance (p= 0.08) such that pressures in homozygous mice were ≥ than those in hemizygous which were ≥ than those in control mice. Increased basal cardiac output was suggested by significant left ventricular hypertrophy. In vitro examination of potassium chloride activation of voltage gated calcium channels showed no significant difference in sensitivity or maximal contraction. Similarly, there was no difference in sensitivity to the α1 agonist, phenylephrine. However, both hemi- and homozygous mice showed a significant reduction in maximal force of contraction (normalized to cross sectional area when compared to controls. Maximal acetylcholine induced relaxation of aortic rings was significantly reduced (p≤0.05) in homozygous sickle mice compared to controls. The same effect was not seen with sodium nitroprusside induced relaxation indicating that the acetylcholine effect was not due to effects on the smooth muscle but was endothelium-dependent. The Berkeley mouse model shows cardiac hypertrophy consistent with the increased cardiac output associated with chronic anemia and a reduced basal mean arterial blood pressure similar to that seen in humans. 8–10 month old mice have increased right ventricular pressure and RV mass indicative of pulmonary hypertension. Further endothelial dysfunction is characterized by a reduction in the maximal relaxation elicited by acetylcholine. Therefore, the Berkeley mouse is a good model for investigating sickle related endothelial dysfunction.
Lactate Dehydrogenase as a Biomarker of Hemolysis-Associated Nitric Oxide Resistance, Priapism, Leg Ulceration, Pulmonary Hypertension and Death in Patients with Sickle Cell Disease.
Authors: Kato Gregory J. , McGowan Vicki , Machado Roberto F. , Little Jane A. , Taylor James , Morris Claudia R. , Nichols James S. , Wang Xunde , Poljakovic Mirjana , Morris Sidney M. , Gladwin Mark T.
Published: April 2020

Pulmonary hypertension is prevalent in adult patients with sickle cell disease, and is strongly associated with markers of hemolysis, in particular serum lactate dehydrogenase (LDH), and early mortality. Intravascular hemolysis leads to a state of resistance to nitric oxide (NO), mediated by NO scavenging by plasma oxyhemoglobin and by arginine degradation by plasma arginase. We hypothesized that serum LDH may represent a convenient biomarker of intravascular hemolysis and NO bioavailability, characterizing a clinical subphenotype of hemolysis-associated vasculopathy. In a cohort of 213 patients with sickle cell disease, we found statistically significant associations of LDH with low levels of hemoglobin and haptoglobin and high levels of reticulocytes, bilirubin, plasma hemoglobin, aspartate aminotransferase, arginase and soluble adhesion molecules. LDH isoenzyme fractionation confirmed predominance of LD1 and LD2, the principal isoforms within erythrocytes. In a subgroup, LDH levels closely correlated with plasma cell free hemoglobin, accelerated nitric oxide consumption by plasma and impaired vasodilatory responses to an NO donor. Remarkably, this simple biomarker was statistically associated with a clinical subphenotype of pulmonary hypertension, leg ulceration, priapism, and risk of death in patients with sickle cell disease. We propose that LDH elevation identifies patients with a syndrome of hemolysis-associated NO-resistance, endothelial dysfunction and end-organ vasculopathy.

Figure Figure Figure Figure
Delayed Wound Healing in Hemophilia B Mice.
Authors: Hoffman Maureane , Lenkowski Angie , Brock Jacqueline , Hedner Ulla , Monroe Dougald M.
Published: April 2020

These studies use our previously reported wound healing model to establish the mechanisms behind the impaired wound healing we observed in hemophilia B animals. We hypothisized that thrombin mediated signals generated in response to the initial wound would be absent or reduced in hemophilia B mice, leading to an impaired course of wound healing and poor angiogenesis. All work was done in accord with the US Amended Animal Welfare Act and was monitored by the Institutional Animal Care and Use Committee of UNC. Three mm circular dermal wounds were made in normal and hemophilia B mice. All wounds were treated with an antibiotic gel at the time the wound was made. The mice were monitored for bleeding both at the time the wound was made and for delayed bleeding. Hemophilia B mice with excessive bleeding were treated. The wounds were monitored for regrowth of the epithelial layer. The size of the area not covered by epithelial tissue was measured and expressed as square millimeters (wound size). We have previously reported that there is a significant difference in the time to wound healing between untreated hemophilic (10 days) and normal animals (7 days). At the end of the time course, wound areas were excised and the skin was formalin fixed, paraffin embedded, and sectioned. Essentially all hemophilia but no normal animals showed unresolved subcutaneous hematoma. During the wound healing process, macrophage infiltration into the granulation tissue was significantly delayed. Unexpectedly, hemophilia B mice relative to wild type mice showed twice as many blood vessels in the healing wound. This difference persisted out to two weeks after the wound was made and well after reepithelialization. Pretreatment (30 minutes prior to wounding) of hemophilia B mice with a single bolus dose of human factor IX sufficient to give 1 U/mL corrected the wound healing time. Pretreated hemophilia B mice did not have obvious subcutaneous hematomoas. The observation that a single bolus dose of factor IX prior to wounding a hemophilia B animal normalized the wound healing time provides a starting point for determining how different doses or different administration times effect wound healing. It also provides a starting point from which to compare the effects of bypassing therapy on wound healing.
Epitope Mapping of Inhibitors in Acquired Hemophilia by Phage Display.
Authors: Königs Christoph , Kessel Christoph , Scholz Sabine , Stumpf Susanne , Krause Manuela , Klingebiel Thomas , Kreuz Wolfhart
Published: April 2020

In acquired hemophilia polyclonal autoantibodies (inhibitors) to coagulation factor VIII are generated. Inhibitors functionally interfere with interaction of molecules involved in the coagulation cascade for example the interaction of FVIII and the von Willebrand factor. Inhibitors in acquired hemophilia have been reported to bind either to the A2 or to C2 domain of FVIII.

Random peptide phage display libraries were screened with patient plasma to identify small peptides mimicking inhibitor epitopes. Peptide sequences were used to map the inhibitor epitope to the surface of FVIII. The phage libraries included libraries with linear or cyclic 7mer or 12mer inserts. Biopanning was performed including positive selections and also negative selections to deplete irrelevant binders. Phages bound to inhibitors were eluted by pH shift or by competition with FVIII. Single phage clones were tested for their specificity for inhibitor positive plasma by ELISA. For specific binders, the sequence of the inserts was identified by DNA sequencing. For one patient 33 phage clones were analysed from three libraries and 28 peptide sequences mimicking the inhibitor epitopemimotopes - were determined. The peptide sequences map to two conformational epitopes on the A2 and A1 domain of FVIII. Clusters of six and four amino acids are the proposed binding regions on the A2 and A1 domain respectively. The matching conformational motifs contain PPLRQ and PPLS. Synthetic peptides corresponding to the sequence displayed on phage were generated and binding of inhibitors to peptides was confirmed by different ELISA formats.

Currently, functional studies with synthetic peptides are being performed to further analyse the epitopes. Additionally, more patient samples are being screened to analyse further epitopes and understand immuno dominant regions of FVIII in acquired haemophilia and compare them to the immune response in hemophilia A. The mimotopes isolated could be a basis for the development of ligand effector conjugates to target inhibitor specific B cells.