bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Efficent Gene Therapy for the Treatment of Erythrocyte Pyruvate Kinase Deficiency.
Authors: Meza Nestor W. , Alonso Maria Eugenia , Navarro Susana , Guenechea Guillermo , Quintana-Bustamante Oscar , Garcia-Gomez Maria , Bueren Juan A. , Bautista Jose M. , Segovia Jose C.
Published: October 2019

Human erythrocyte R-type pyruvate kinase deficiency (PKD) is an autosomal recessive disorder produced by mutations in the PKLR gene. Haemolytic anaemia is the major symptom of the disease. A severe deficiency in PKD causes ATP depletion in the RBC metabolism, which ultimately leads to haemolysis that may require periodical blood transfusion, splenectomy, and in some cases bone marrow transplantation. These clinical features make this disease a good candidate for gene therapy. With this aim, we have developed different gammaretroviral and lentiviral vectors expressing the human RPK and have characterized the functionality of the erythroid specific expression regulatory sequences of the human RPK gene in a lentiviral backbone. The transduction of mouse hematopoietic stem cells from a mouse strain deficient in the pklr gene [AcB55: pklr269A/269A mice], which mainly resembles the human RPK deficiency, with a retroviral vector expressing the human RPK, followed by the transplantation into irradiated syngenic recipients completely recovered the red cell parameters in peripheral blood, spleen and bone marrow. Also, intracellular values of ATP, plasmatic iron and circulating erythropoietin levels were recovered to normal values. After 100 days of transplantation, treated mice did not show any clinical symptom of the disease. Secondary pklr269A/269A recipients were also transplanted and their hematological symptoms were also reverted, demonstrating the stable therapeutic efficacy of the vector. To specifically express the RPK in the erythroid lineage, a lentiviral vector expressing the EGFP marker gene under the above RPK promoter (LVpRPKEG) was constructed to test its specificity. EGFP expression was detected in erythroid, but not in non-erythroid cell lines, transduced with this vector. Human CD34+ cells were also transduced with the LVpRPKEG vector and transplanted into NOD/SCID mice. Forty days post-transplantation human bone marrow cells were obtained and seeded in semisolid media. Significantly, only human erythroid colonies expressed the EGFP protein, demonstrating the efficacy of the RPK promoter to specifically express proteins in the erythroid lineage. Experiments using a lentiviral vector expressing the human RPK gene under the control of the human RPK promoter are now in progress. Overall, the transfer of lentiviral vectors harbouring the hRPK cDNA driven by its own promoter in PKLR mutated hematopoietic stem cells could represent an efficient therapeutic treatment of severe clinical cases of human erythrocyte PKD.
Cause-Specific Mortality and Second Cancer Incidence after Non-Hodgkin Lymphoma: A Report from the Childhood Cancer Survivor Study.
Authors: Bluhm Elizabeth C. , Ronckers Cecile , Hayashi Robert J. , Neglia Joseph P. , Mertens Ann C. , Meadows Anna T. , Mitby Pauline A. , Stovall Marilyn , Robison Leslie L. , Inskip Peter D.
Published: October 2019

Background: Second primary malignancies and premature death are a concern for patients surviving treatment for childhood lymphomas. Despite in-depth reports on second cancer risk after Hodgkin lymphoma, there is limited published information on clinical, demographic, and treatment risk factors for second malignant neoplasms (SMNs) or deaths after non-Hodgkin lymphoma (NHL).

Methods: We assessed mortality and SMNs among 1,082 five-year survivors of NHL in the Childhood Cancer Survivor Study, a multi-institutional North American retrospective cohort study of pediatric cancer survivors diagnosed between 1970 and 1986 before age 21. We searched the National Death Index for dates and causes of death and inquired about SMNs in periodic questionnaires, with confirmation of all reported SMNs by a study pathologist. We calculated standardized mortality ratios (SMRs) and standardized incidence ratios (SIRs) based on United States population rates. We calculated relative risks (RRs) for death and SMNs by demographic, clinical, and treatment characteristics using proportional hazards modeling. Medical physicists determined organ exposure to radiation therapy (RT) based on clinical RT records.

Results: There were 87 deaths among 5-year survivors (SMR=4.2, 95% confidence interval (CI): 1.8, 4.1). Leading causes of death were NHL (n=21), solid tumors (n=17), circulatory diseases (n=12), and leukemia (n=10). There were elevated rates of mortality due to NHL, solid tumor SMNs, leukemia, cardiac disease, and pneumonia. The mortality rate remained elevated beyond 20 years after NHL. Risk factors for death included NHL relapse (RR=8.5), doxorubicin (RR=1.8), bleomycin (RR=4.1), platinum agents (RR=4.0), and cardiac RT exposure (RR=1.8). There were 31 SMNs, including 27 solid tumor SMNs (SIR=3.9, 95%.CI: 2.6, 5.7), specifically SMNs of the breast (n=6, SIR=9.1), thyroid (n=6, SIR=9.4), oral cavity and pharynx (n=3), brain and nervous system (n=3), bone (n=2) and urinary bladder (n=2). Risk factors for solid tumor SMNs were female sex (RR=3.1), mediastinal NHL disease (RR=5.2), and breast RT exposure (RR=4.3). Overall cumulative incidence of solid tumor SMNs from 5 to 20 years after NHL diagnosis was 0.03 (95% CI: 0.02, 0.05).

Conclusions: We identified a four-fold risk of mortality and three-fold risk of SMNs in a large cohort of pediatric NHL survivors relative to the general U.S. population. Survivors of childhood NHL, particularly those treated with chest RT, may be at continued increased risk of early mortality and solid tumor SMNs. Health care providers responsible for this population should remain alert to second cancers and other severe late health outcomes.
Comparison between Intravenous Iron and Oral Iron Preparations for the Treatment of Anemia of Chronic Kidney Disease - A Systematic Review and Meta-Analysis
Authors: Gafter-Gvili Anat , Rozen-Zvi Benaya , Paul Mical , Leibovici Leonard , Uzi Gafter , Shpilberg Ofer
Published: October 2019

Background: There is confounding data regarding the best method of iron supplementation in chronic kidney disease (CKD), without a consistent approach in clinical practice. Objectives: To evaluate the efficacy and safety of intravenous (IV) iron versus oral iron in patients treated for anemia of CKD.

Methods: Systematic review and meta-analysis of randomized controlled trials comparing IV iron preparation with oral iron preparation for the treatment of anemia in patients with CKD (stage III, IV and V). The Cochrane Library, MEDLINE, conference proceedings and references were searched until 2007. Primary outcomes: absolute hemoglobin (Hb) level or change in Hb level from baseline at two months or at end of study; all-cause mortality. Secondary outcomes: need for renal replacement therapy (RRT) in predialysis patient and adverse events. Weighted mean differences (WMD) for outcomes with continuous variables and relative risks (RR) for dichotomous outcomes with 95% confidence intervals (CI) were estimated and pooled.

Results: Our search yielded 11 trials which compared IV iron preparations (iron sucrose, iron gluconate or iron dextran) to oral iron. Compared to oral iron, there was a significant rise in Hb level in the IV iron treated hemodialysis patients (WMD 1.17; 95%CI 0.19–2.15, fig). Significant heterogeneity was observed due to different baseline Hb values and baseline iron status, different dosages of oral iron, and different dosages of erythropoiesis stimulating agents (ESA). For predialysis patients, there was a small but significant difference in the Hb level favoring the IV iron group (WMD 0.28; 95% CI 0.15–0.4, fig). For both groups effect estimates were not influenced by ESA administration. In predialysis patients, there was no significant difference in the risk for requiring RRT during the trial between the different groups (RR 0.63; 95%CI 0.25–1.65). Data on all-cause mortality were sparse (RR 0.54; 95%CI 0.09–3.13, 3 trials) and there was no difference in adverse events (RR 0.9; 95%CI 0.65–1.24) between the IV and oral treated patients. However, discontinuations of treatment were more common (RR 3.27; 95%CI 1.15–9.26) for the IV iron treated patients.

Conclusions: Our review demonstrates that dialysis patients treated with IV iron have better Hb response than patients treated with oral iron. For predialysis patients, this effect is very small. IV iron should be preferred in the treatment of anemia in dialysis patients. In predialysis patients the slight advantage in Hb response should be weighed against the inconvenience and cost of IV iron treatment.
Proteomics Analysis of Naturally Occurring CD4+/CD25+ Regulatory T Cells.
Authors: Garin Marina I. , Chu Cheri C. , Wait Robin , Bueren Juan A. , Lechler Robert I.
Published: October 2019

Recently, a naturally occurring regulatory T cell population (Tregs) has been identified and shown to actively suppress immune responses of self-reactive T cells. The molecular basis for the function of Tregs remains unclear. We are interested in identifying molecules specifically expressed in human Tregs that are candidates for mediating suppression by comparative proteomic and transcriptomic analysis of human CD4+/CD25+ and CD4+/CD25- T cells using two-dimensional electrophoresis (2-DE) followed by electrospray tandem MS on the Micromass Q-Tof (HPLC-MS/MS) and high-density cDNA microarray technology, respectively. Thus far, the most revealing results of the 2-DE analysis are the detection of an upregulation in the expression of a protein kinase inhibitor and FUSE binding protein (FBP), a regulator of c-Myc expression. The gene expression pattern of the Tregs was overall very similar compared to CD4+/CD25- T cells despite their major functional differences. We have identified several sets of genes differentially expressed in the Tregs. Of special interest are adhesion molecules and chemokine receptors that could reveal distinct trafficking patterns. We are in the process of validating and screening at the protein level several of these candidate genes that could lead to the identification of specific molecular events in the Tregs. The upregulation of the PKC inhibitor is especially interesting, as the reduction of IL-2 production may explain the hypoproliferative status of Tregs. The increase in FBP and a resulting increase in c-Myc transcription may promote the survival of Tregs, thereby contributing to the prevention of autoimmunity.
Von Willebrand Disease in Taiwan: A Clinical and Laboratory Studies of 52 Patients.
Authors: Chen Yeu-Chin , Chao Tsu-Yi , Cheng Shin-Nan
Published: October 2019

Von Willebrand disease (VWD) has been found to be the most common inherited bleeding disorder in Caucasian with a prevalence of around 1%, yet it has not been well recognized in Taiwan and Asian countries. Only a small number of patients have been identified and reported. The aim of this retrospective study was to identify patients with VWD by clinical manifestation and laboratory tests in Taiwan. VWD was detected by a panel of laboratory tests, including bleeding time, aPTT, factor VIII activity assay, Von Willebrand antigen (VWF: Ag) and ristocetin cofactor activity (VWF: RCo), and platelet function analyzer (PFA) test using both Collagen/ADP and Collagen/Epinephrine (CEPI). VWF multimer analysis was performed by western blot to confirm the disease subtype. From October, 2003 to April, 2007, 52 of 494 (9.5%) patients, from 44 families, were identified to have VWD, including 16 men and 36 women. Their median age was 29 with a range of 4∼69 years of age. The most frequently encountered reasons for VWD detection was menorrhagia accounting for 19.2%. The others included inherited detection because of affected family (11.5%), prolonged aPTT (11.5%), excessive bleeding after dental procedures (9.6%) and excessive bleeding after surgery or invasive procedure (9.6%). By laboratory tests, the mean value of VWF: Ag and VWF: RCo was 46.9 ± 18.4% and 32.3 ± 17.4%, respectively. The most sensitive test was VWF: RCo with 90.3%, followed by CEPI with 83.8%. Of 37 patients with VWF multimer analysis, 34(92%) were revealed to have type I VWD, 1 had type IIA, and 2 from the same family had type III VWD. In conclusion, our study demonstrated that VWD was not an uncommon inherited bleeding disease in Taiwan. Menorrhagia was the most common reason for VWD determination, and VWF: RCo and PFA test CEPI were the more sensitive tests for the VWD identification.
D-DImer Dosing for Risk Prediction of Central Venous Catheter Thrombosis (The DIReCT Trial).
Authors: Doucet Stephane , Miron Marie-Jose , Denis Soulieres , Karl Belanger , Marie Florescu , Jean-Pierre Ayoub , Louise Yelle , Olney Harold J. , Blais Normand
Published: October 2019

Cancer patients receiving chemotherapy often need central venous catheters (CVC). There is no laboratory marker to identify patients at high risk of thrombosis who may benefit from thromboprophylaxis. D-Dimer dosing is a well studied test that may correlate with thrombosis risk. The aim of this study is to determine if d-dimer levels are a predictor of CVC thrombosis. Adult cancer patients needing a new CVC for more than 1 month and without an ongoing indication for anticoagulation were included. D-dimer dosing was done before (0–3 days), 24–48h after and 7 days after insertion of CVC. Duplex ultrasonography (US) was performed approximately 30 days after installation. We planned to look at quantitative d-dimer variation in patients to establish its prognostic value in relationship with eventual thrombosis occurrence. Thirty five patients are included in this analysis. There were 63% males and 37% females and the median age was 57. Eighty nine percent had solid tumors (64% gastrointestinal, 23% head and neck, 13% breast) and 11% had an hematologic malignancy. Fifty one percent received concomitant radiotherapy. Seventy nine percent had continuous infusion chemotherapy. Twenty two patients underwent US and 27 patients had at least 2 out of three planned d-dimer values. The incidence of venous thrombosis in the whole cohort was 31% (5 symptomatic, 6 asymptomatic) whereas 24 patients were considered free of thrombosis (11 confirmed by duplex, 13 clinically asymptomatic). In patients with available US results, baseline d-dimer level was similar for those with or without thrombosis (924 and 902). For these patients, the absolute increase of d-dimer was not significantly different in the two groups (194 vs 115 at day 1–2; 421 vs 63 at day 7) as well as relative increase (51% vs 31% at day 1–2; 145% vs 40% at day 7). Baseline d-dimer level was similar for the five patients with symptomatic thrombosis compared to the other 30 patients (1038 and 859), but higher at day 7 (1616 vs 905 p=0,048). Different strategies were analysed to try to define a high and a low risk group and two were selected. First, the population with a cutoff value of d-dimer levels at day 7 of > 1100 had a 40% risk of symptomatic thrombosis vs 0% for those below 1100. Second, an absolute increase in d-dimer values at day 1–2 of more than 500 was associated with a 66,7% risk of symptomatic thrombosis vs 0% for the others. Although numbers are small, patients with symptomatic thrombosis were more frequently receiving G-CSF, red cell transfusions, platelets transfusions and were less likely to be receiving continuous infusion chemotherapy. Our pilot study suggests that d-dimer testing after CVC insertion might permit risk stratification for thrombotic complications. If confirmed by larger trials, this strategy might be useful to plan more effective prophylaxis studies in these populations.
Non Hodgkin’s Lymphoma (NHL) and Deep Vein Thrombosis (DVT): A Dangerous Liaison Still Unexplored.
Authors: Giordano Giulio , Piano Silvia , Marra Roberto , De Stefano Valerio , Hohaus Stefan , Iacoviello Licia , Leone Giuseppe , Donati Maria Benedetta , Storti Sergio
Published: October 2019

Introduction DVT incidence ranges from 3 to 15% in cancer patient. Nevertheless there are only few data about DVT in lymphoma. Aim of our study is to define the real DVT risk and incidence in lymphoma patients.

Patients and Methods Our study is a retrospective study including patients of two haematology centers. We considered the age of the patients, sex, histological type of lymphoma (indolent -IL- vs aggressive -AL), localization over or under diaphragm, extranodal localizations, vascular compression, stage of disease, IPI, LDH level, chemotherapy type and timing of administration (weekly vs every 2wks or 3 weeks), the use of chemotherapy regimen containing methotrexate as potential risk factors in DVT onset. Data regarding 567 NHL patients, observed from 2001 to 2006, were collected. 400 patients had indolent NHL (IL) and 167 Aggressive NHL (AL). Median age was 59 years (R 13–94), M/F ratio was 300/267. DVT was diagnosed by ultrasound or CT scan. The statistical analysis was conducted with Yates corrected chi square test, Odds Ratio (OR), Log-rank test (to compare Kaplan-Meier curves).

Results 87 patients (15%) showed DVT. Of these, 37(43%) were localized at legs and 19(22%) involved abdominal veins (especially iliac veins, 10% of total). DVT onset median time was 3 months from NHL diagnosis (Range 0–156 months). Sex, histological type of lymphoma (IL vs AL), localization over or under diaphragm, extranodal localization, stage of disease, IPI, LDH level, and use of chemotherapy regimen containing methotrexate were not related to an increased risk to develop DVT. Vascular compression was the most significant risk factor for DVT development with OR 3.1 (CI95%:1.9–5), Chi Square22.7(p< 0.0001). Patients receiving weekly chemotherapy showed an increased risk to develop DVT (OR 1.8; CI95%:1.1–2.8), Chi Square5.1, p0.024. Patients aged ≥60 presented increased risk of DVT with OR 1.7(CI95%: 1–2.7), Chi Square 4.28 (p 0.04). Patients with DVT within 3 months from NHL diagnosis had an higher risk of disease relapse or non response at first line chemotherapy: OR 3.7 (CI95%: 1.8–7.3), Chi Square 13.4 (p < 0.0001), positive predictive value 0.71(CI95%: 0.57–0.82), specificity 0.95(CI95%: 0.93–0.97). However DVT had no impact on mortality and survival, also in the subgroup of patients with DLBCL. Discussion In our study vascular compression by enlarged lymphonodes, patient age ≥60 y.o., and weekly administration of chemotherapy seem to be the main risk factors to develop DVT in NHL patients. Further studies, concerning genetic conditions and other disease-related parameters, are ongoing to individuate other risk factors for thrombosis in NHL patients. The DVT development within 3 months from NHL diagnosis seems to be a risk factor for non response/relapse. An antithrombotic prophylaxis could be considered in the patients with higher thrombotic risk.
Massive Transfusion in Wartime: Experience from Northern Israel, Summer 2006.
Authors: Dann Eldad J. , Dally Najib , Chezar Judith , Michaelson Moshe , Barzelay Mirit , Braester Andrei , Hoffman Ron , Bonstein Lilach
Published: October 2019

In July 2006 hostilities erupted in Israel/Lebanon. Reported here is the experience of three medical centers in Northern Israel during 33 days of the warfare; the Rambam Health Care Campus in Haifa - a level I trauma center, the Rebecca Sieff Hospital in Safed and the Western Galilee Hospital in Nahariah - both secondary trauma centers. 504, 1138 and 868 wounded were presented to the three medical centers and 281, 415 and 195, respectively, required hospitalization. Sixty, 32 and 15 hospitalized patients were concomitantly transfused in each corresponding center, representing 20%, 7% and 7%, respectively, of admitted patients. Patients with an injury severity score of ≥16 had a higher need for blood products than those less severely injured, with a mean packed red blood cell (PRBC) transfusion of 7 versus 4 units (p=0.03) and FFP transfusion of 13 versus 1.5 units (p=0.002), respectively. Twenty four soldiers and one civilian had massive transfusions and twenty three of these patients survived. The median ratio between transfused fresh frozen plasma (FFP) and packed red blood cells (PRBC) was 0.8, ranging from a ratio of 0.25 to 1.3. Among 25 massively transfused patients 21 received cryoprecipitate and 19 - platelets. The median prothrombin time (INR) and partial thromboplastin time (PTT) increased during the first 2 hours after admission from 1.29 to 1.51 and from 33.6 seconds to 39 seconds, respectively. In the cohort of massively transfused patients 3 individuals additionally received 3 g of tranexamic acid, while another 2 patients were treated with recombinant factor VII. In conclusion, massively transfused patients with wartime penetrating injuries have an ongoing coagulopathy despite vigorous replacement therapy, which needs to be continued until the patients are stabilized. Early intervention and consultation in the Emergency Room by transfusion-service specialists is essential to the overall management of critically and massively wounded patients in wartime.

Wounded (hospitalized) Transfused patients Packed RBC units FFP units Cryo units Platelet units Massive transfusion (patients) Rambam 504 (281) 60 463 413 266 258 21 Rebecca Sieff 1138 (415) 32 134 34 50 30 4 Western Galilee 868 (195) 15 71 68 51 10 1
Polyphosphate Enhances Fibrin Clot Structure.
Authors: Smith Stephanie A. , Morrissey James H.
Published: October 2019

Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V. Our previous studies found that polyP did not affect the time to clot formation when plasma was clotted with thrombin, however, suggesting that polyP exerts its procoagulant actions upstream of thrombin. We now report that polyP enhances fibrin clot structure.

Methods: Purified fibrinogen and polyP were preincubated for 15 min in multiwell plates in buffer containing CaCl2, after which clotting was initiated by adding 0.1 to 8 nM thrombin and fibrin clot formation was evaluated by quantifying the change in turbidity (A405). Mass-length ratios were calculated from scans of A400 to A800. The effect of polyP on fibrinolysis was examined by adding 8 nM plasmin to the reaction mixtures immediately prior to thrombin. Scanning electron microscopy (SEM) was employed to visualize clot structure, and time courses of covalent fibrin cross-linking were assessed by SDS-PAGE.

Results: PolyP had no effect on time to clot formation, but clots formed in the presence of polyP had markedly (up to threefold) higher turbidity than clots formed in the absence of polyP (see figure), irrespective of thrombin concentration. The increased turbidity in the presence of polyP was calcium-dependent and was enhanced when fibrinogen, CaCl2, and polyP were preincubated for up to 15 min prior to initiation of clotting with thrombin. PolyP increased the mass-length ratio of fibrin, and SEM confirmed that fibers formed with polyP were thicker than those formed without polyP. The ability of polyP to enhance fibrin clot turbidity was independent of factor XIIIa activity, and polyP did not alter the rate or extent of covalent fibrin cross-linking by factor XIIIa. When plasmin was included in clotting reactions containing polyP, mean times to 50% clot lysis were 28.5 ± 0.8 min for clots without polyP but 120.4 ± 5.6 min for clots with polyP.

Conclusions: PolyP alters polymerization of fibrin, resulting in fibers of higher mass-length ratio that are lysed more slowly. This effect is calcium-dependent and is enhanced by preincubation of fibrinogen with calcium and polyP. Release of polyP from activated platelets or infectious microorganisms may therefore enhance fibrin clot structure.

Figure Figure
Evidence for a Metabolic Stem Cell Niche.
Authors: Cross Michael , Alt Rudiger , Schnapke-Hille Lydia , Riemer Thomas , Niederwieser Dietger
Published: October 2019

The hematopoietic stem cell niche presents a localised environment supporting the balanced maintenance, self-renewal and occasional expansion of the stem cell pool. These options are widely assumed to be regulated exclusively by signalling from specific combinations of stroma-bound or soluble ligands. However, a consideration of the rare conditions under which absolute numbers of stem cells increase in vivo as well as the selective pressures acting on regenerative systems during evolution has led us to propose a metabolic component to the stem cell niche which serves to limit cumulative damage, to avoid the selection of potentially oncogenic mutations and to tie symmetric division to slow proliferation. This would mean that traditional cell culture media based on “systemic” substrates such as glucose and glutamine may actively prevent the symmetric amplification of high quality stem cells, offering a possible explanation for the limited success in this area to date. To investigate this possibility, we have examined the effects of range of carbon and energy sources on the proliferation and maintenance of stem and progenitor cells. Our strategy is to screen a wide variety of culture conditions using murine FDCPmix cells, which are non-tumorigenic but have an innate tendency to amplify symmetrically in the presence of IL-3, and then to test key observations in human UCB CD133+ cells provided with SCF, TPO and FLT-3L. In both cell systems, we do indeed find an unusually low requirement for the systemic substrates glucose and glutamine normally included as major energy and carbon sources in cell culture media. Reducing glucose reduces the yield of committed cells from CD133+ cultures without affecting the accumulation of CD133+CD34+cKit+ progenitors. When provided with alternative substrates more likely to reflect a “niche” type environment, FDCPmix cells can be maintained for long periods in media containing only the trace levels of glucose or glutamine derived from dialysed serum, and show improved self-renewal under these conditions. We have also found that raising osmolarity reduces glucose dependence and simultaneously favours the maintenance both of self-renewing CFU (FDCPmix culture) and of CAFCweek13 (CD133+ culture). In parallel, the use of NMR and mass spectrometry techniques to profile intracellular metabolites in self-renewing and differentiating FDCPmix cells reveals a shift in the metabolite balance indicating reduced glycolysis in the early cells. Taken together, these results suggest that hematopoietic stem cells do indeed have remarkable metabolic characteristics consistent with adaptation to a metabolically limiting niche environment. It may therefore be necessary to identify niche substrates and to combine these with the relevant signalling environment in vitro in order to effectively increase stem cell numbers for research, stem cell transplantation and tissue engineering applications.
Supportive Role of TNF-Family Death Receptors in Hematopoietic Cell Engraftment.
Authors: Pearl-Yafe Michal , Askenasy Nadir , Stein Jerry , Yaniv Isaac
Published: October 2019

The activity of death receptors of the tumor necrosis factor (TNF) superfamily as negative regulators of distal differentiation in the immuno-hematopoietic system is well characterized. The role of these receptors, classically associated with transduction of apoptotic signals, is unknown. A series of studies have attributed these receptors also negative regulatory functions in ex vivo expanded hematopoietic progenitors, showing variable supporting and suppressive interactions with growth factors. We observed robust upregulation of Fas, TNF-R1, TNF-R2 and TRAIL-R2 in bone marrow homed donor cells, reaching levels of 60–75% expression after 6 days. All putative SCA-1+c-kit+lin− progenitors ubiquitously expressed all four receptors. These receptor-positive cells were largely resistant to apoptotic signals delivered by the corresponding soluble and membranous ligands. Furthermore, there was no evidence of receptor cross-talk in sensitization of the cells to apoptotic death. The engraftment potential of cells was markedly diminished by ex vivo incubation, without any relationship to the viability of the putative progenitors. Taken together these data suggest a supportive role of the “death receptors” in hematopoietic cell engraftment and dissociate the deficient engraftment of cultured cells from apoptosis of progenitors.
Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells.
Authors: Ramakrishnan Aravind , Hayes Brian , Fagerlie Sara R. , Baran Szczepan , Harkey Michael , Graf Lynn , Bar Merav , Bendoraite Ausra , Tewari Muneesh , Torok-Storb Beverly
Published: October 2019

Embryonic stem (ES) cells have created considerable excitement in the last few years due to their unlimited potential to produce cells for tissue repair and replacement. However, a large animal pre-clinical model is necessary to establish the safety and efficacy of ES cell-derived tissue replacement therapy. The canine model has long been used in medical research, has been well established to study adult stem cell transplantation and has been highly predictive of clinical outcomes in humans, more so than rodent models. Given the documented record for extrapolating from dog to man, we hypothesize that the dog would serve as an ideal pre-clinical in vivo model for studying the clinical applications of ESC derived tissue. Eleven putative ES cell lines were initiated from canine blastocysts harvested from natural matings. One line described here, FHDO-7, has been maintained through 34 passages and has many characteristics of ES cells from other species. FHDO-7 cells are alkaline phosphatase positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and do not express message for Cdx2 which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miR-302b, miR-302c and miR-367) that have been found to be characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts (MEF) as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders the cells form embryoid bodies (EB). Under various culture conditions the EBs give rise to ectoderm-derived neuronal cells expressing β3-tubulin, mesoderm-derived osteocytes producing bone, and endoderm-derived cells expressing alpha feto protein or Clara cell specific protein. These results indicate that FHDO-7 is a pluripotent embryonic stem cell line.
Autologous Bone Marrow Transplant for Human Diabetes Mellitus.
Authors: Novoa Jose E. , Medina Maria A. , Gordillo Felipe , Ravera Jorge , Olivet Carlos
Published: October 2019

Background: stem cells have tha capacity of both self-renewal and multilineage differentiation. They have the ability to proliferate and differentiate into any type of cell. Bernat Soria and coworkers showed how to guide stem cells in animals to differentiate in specific cell lineages as neurons,cardiomyocytes an insulin-secreting cells (Diabetologia2001;44:407–415).Therapeutic angiogenesis has recently been developed as a new method of treatment for several ischaemic diseases performing stem cell transplantation. Ernesto Novoa et al. reported in march 2007 the Conzi’s Effect inhuman diabetes mellitus (Archivos de Medicina Interna2007;XXIX suppl1:S24.25). After therapeutic angiogenesis the patients discontinued antidiabetic treatment as a consequence of normalization in glucose metabolism.

Aims: to study the bahaviour in glucose metabolism in a group of patients receiving autologous bone marrow transplantation for arterial ischaemic limb disease and prior mobilization with G-CSF. To evaluate the safety of the procedure.

Methods: from july 2004 to july 2007, 71 patients developing severe arterial limb ischaemia (candidates to amputation) were included in this study. 40 men and 31 women. Median age 66 years old (44–86). Mobilized with filgrastim (Neupogen) 5 ug/kg weight daily (5 days). Bone marrow harvest at 6th day. Local anaesthesia was performed in all the patients. Unmanipulated cells, concentrated by gradient of density, were injected in the affected limb in 2 ml aliquots into the gastrocnemius muscle. Mean volume 40 ml. Follop up of each patient was done regularly evaluating: amputation, rest pain, frequency of analgesics intake, healing of the ulcers, peak walking time, insulin or oral antidiabetic drugs required. Doppler and angiographic test were also performed. The mean number of injected mononuclear cells was 1,9 x109/kg. All the patients received low molecular weight heparin (nadroparin, Fraxiparine)3800–5600 IU anti-Xa subcutaneously, aspirin 81 mg and pentoxifiline 400 mg daily, as medical treatment after the procedure for at least 60 to 90 days. A control population of 67 patients affected by severe arterial limb ischaemia was considered. They didn’t receive angiogenic treatment.

Results: there was no treatment related mortality. In the control group 65 patients were amputated. In the “ABMT Group” 3 patients received amputation of the lower limb. The statistical difference between the two groups was highly significant in favor of the angiogenic group. They were evaluated by the chi square test and log rank test with a p value < 0,05. In the ABMT Group, 33% of type 1 diabetes and 74% of type 2 diabetes, discontinued their antidiabetic treatment for more than 3 months after ABMT.

Conclusions: autologous bone marrow transplantatioan can be performed safely and appears to be a benefical therapy for selected patients with severe peripheral arterial occlusive disease and diabetes mellitus. novoa.je@gmail.com
Characterization of BioSafe SEPAX Manufactured Stem Cell Intended for Cardiac Cell Therapy.
Authors: O’Connor Sean L. , Sepulveda Carmen A. , Kaur Indreshpal , Sumari Robert D. , McMannis John D.
Published: October 2019

Numerous cardiology groups are actively pursuing the use of hematopoietic stem cells for myocardial delivery. Most published studies have used a manual ficoll density separation procedure to enrich the Mononuclear Cell (MNC) population thus enriching the stem cell content in the final therapeutic cell product. Manual ficoll procedures have certain intrinsic limitations including technologist variability, training time, and risk of contamination due to its open nature. Most open and manual cell processing procedures have been supplanted by recent technology developments in automated and closed devices. The BioSafe SEPAX is a closed system-automated ficoll density separation device that currently has FDA 510k clearance for the processing of umbilical cord blood products. In support of multi-center clinical studies being conducted by the NHLBI Cardiovascular Cell Therapy Research Network (CCTRN), the MD Anderson Cell Therapy Laboratory has evaluated bone marrow derived, MNC enriched, stem cells for their cardiovascular therapeutic potential. Traditional transplantation immunophenotyping was performed on these cells to evaluate hematopoietic stem cells (CD34+ and CD133+), T-Cell subsets, B-Cells, and NK-Cells. Colony Forming Units (CFU) as wells as surrogates used in cardiac regeneration studies including endothelial invasion assays and CXCR4 immunophenotyping (a chemokine receptor involved in cell migration) were also performed. Our data suggests the cells produced using the automated ficoll BioSafe SEPAX device are equivalent to cells produced using a manual ficoll procedure with respect to TNC, MNC, viability, and CD34+ recoveries. Cell subsets and function including invasion and CXCR4 analysis were also similar between the two procedures. The automated method produced a product with statistically higher CFU potential than the manual method suggesting the SEPAX isolates a more potent subset of the mononuclear fraction. These studies support the use of the SEPAX device as for enriching BM-MNCs for the CCTRN. Additional benefits might include the use of a closed system, ease of training, speed of cell manufacturing, and product consistency.
GlycoPEGylated Filgrastim XM22 Fixed Dose Demonstrates Similar Activity in Comparison to Pegfilgrastim in Healthy Volunteers after Single Administration.
Authors: Lubenau Heinz , Pokorny Rolf , Lopez-Lazaro Luis , Bias Peter , Wallin Bruce , Hinderer Walter , Maly Anne-Katrin , Seiberling Michael
Published: October 2019

XM22 is a glycoPEGylated recombinant human G-CSF that interacts with the G-CSF receptor by binding and activation. In pilot studies in non-neutropenic animals, XM22 demonstrated a similar PK/PD profile compared to pegfilgrastim. By its longer duration of action compared to filgrastim a once per cycle dosing as fixed dose in chemotherapy treated cancer patients for the reduction of duration of severe neutropenia and incidence of febrile neutropenia is intended.

METHODS: A single center, randomized, single-blind study of XM22, administered as single fixed dose given by the s.c. route, was conducted to assess safety, pharmacokinetic profile and pharmacodynamic properties based on absolute neutrophil count (ANC) and CD34+ cell count. Male or female healthy volunteers were assigned to either XM22 6 mg or pegfilgrastim 6 mg fixed dose treatment.

RESULTS: 36 healthy volunteers were enrolled in two treatment groups and completed 21 day follow-up. Injections were generally well-tolerated with no discontinuations due to adverse events or serious adverse events. Pharmacodynamic data were compared between the fixed doses of 6 mg XM22 and pegfilgrastim and in body weight strata within the treatment groups. Pharmacokinetic and antibody data will be presented.

CONCLUSIONS: Single doses of 6 mg fixed dose of XM22 administered to healthy volunteers were generally well-tolerated with regard to the expected side effect profile and demonstrated increases in absolute neutrophil count and CD34+ cell count. These results supplement data from another study in healthy volunteers in which XM22 was administered in ascending single doses adjusted according to body-weight.
NF-kappa B Plays a Key Role in the Hematopoietic Niche Function of Osteoblast after Ionizing Radiation.
Authors: Xiao Mang , Inal Cynthia E. , Whitnall Mark H.
Published: October 2019

Recent studies demonstrated that osteoblasts in the endosteal region of bone are a key cellular component of the hematopoietic niche that directly regulates hematopoietic stem cell (HSC) survival, proliferation and differentiation. In experiments with the human fetal osteoblast cell line hFOB1.19, we found survival of gamma-irradiated primary human hematopoietic CD34+ cells was significantly enhanced by co-culture with hFOB cells or by conditioned medium (CM) from hFOB cells, as shown by increased colony efficiency and colony size. In clonogenic assays of CD34+ cells starting with 5 × 103 cells/dish, co-culture with hFOB cells increased colony number from 428±53 (control) to 988±65 (co-culture) after 2 Gy (p<0.01), 301±44 to 739±56 after 4 Gy (p<0.01), and 45±15 to 72±10 after 6 Gy (p<0.05). Surprisingly, although radiation induces osteoblast cell damage, CM from 6 Gy-irradiated hFOB cells had a greater effect than CM from unirradiated hFOB cells in supporting CD34+ clonogenicity and resulted in colony number increase from 430±40 (CM from unirradiated hFOB cells) to 773±48 (CM from 6 Gy-irradiated hFOB cells) after 2 Gy IR (p<0.01). Using a cytokine array for 120 cytokines and chemokines, we detected 11 factors released from hFOB cells, and 4 of them (IL-6, G-CSF, GRO, and IL-8) were enhanced by ionizing radiation (IR). IR (2–8 Gy) caused hFOB cell cycle arrest in G2 phase after 24 h, and irreversible cell cycle block and apoptotic cell death were observed 72 h after IR in a radiation dose-dependent manner. IR induced NF-kappaB (NFkB) p65 phosphorylation and NFkB activity in hFOB cells. Inhibition of NFkB expression with siRNA blocked the ability of hFOB CM to support clonogenic survival of CD34+ cells, and abrogated the effect of IR on release of IL-6, G-CSF, GRO, and IL-8. Radiation upregulated p53 (ser15) phosphorylation within 4 h, and p21 expression after 24 h in hFOB cells. However, NFkB gene silencing accelerated p21 expression and induced a massive apoptosis of hFOB cells. We propose that IR induces NFkB and p53 activation simultaneously in hFOB cells. The fate of osteoblasts after irradiation depends on the amount and severity of injury, and on repair processes regulated by NFkB and p53. Silencing the NFkB gene disrupted this balance and resulted in hFOB cell apoptosis.
Ciprofloxacin-Induced G2 Arrest and Apoptosis in TK6 Lymphoblastoid Cells Is Not Dependent on Double-Strand Break Formation.
Authors: Halicka Dorota , Smart Daniel J. , Seiter Karen , Traganos Frank , Darzynkiewicz Zbigniew , Williams Gary M.
Published: October 2019

Drugs indicated for conditions other than tumors have shown promise in the potential treatment of leukemia and lymphoma. The fluoroquinolone antibiotic ciprofloxacin (CPFX) is known to modulate cycle cell progression and apoptosis in cancer cells and thought to induce DNA double-strand breaks (DSBs) via topoisomerase II (topo II) inhibition. DSBs trigger the phosphorylation of histone H2AX on serine 139 (γH2AX) by PI-3-like kinases including ATM; γH2AX can serve as a marker of DNA damage when measured in situ using immunocytochemistry and flow cytometry. The aim of the present study was to investigate the relationship between CPFX-mediated DNA damage and the induction of apoptosis in human lymphoblastoid cells and phytohaemagglutinin (PHA)-stimulated normal lymphocytes using γH2AX and multiparameter flow cytometry. Treatment of TK6 cells (wild-type p53) with 100 mg/ml CPFX for 2–10 hours produced no increase in γH2AX compared to controls; levels were in fact reduced in S phase cells at 6 and 10 hours. Phosphorylated ATM and G2 arrest was also observed in TK6 cells exposed to CPFX for 4 hours. γH2AX was dramatically increased after 24 hours treatment, indicating the onset of apoptosis (confirmed by presence of activated caspase-3). CPFX had a similar lack of effect on γH2AX at early time points in WTK-1 and NH32 lymphoblastoid cells (devoid of functional p53) and proliferating lymphocytes, however, induction of apoptosis was not as pronounced in these cells compared to TK6. The data suggest that, at early time points, CPFX may act a radical scavenger to reduce the level of endogenous oxidant-induced DSBs. However, phosphorylation of ATM (but lack of γH2AX induction) does indicate the presence of a non-DSB DNA modification. At later time points, induction of apoptosis appears to be facilitated by a functional p53 signaling pathway. CPFX may therefore have a potential use as a novel chemotherapeutic agent in the treatment of lymphoblast-derived cancer.
Bone Marrow Small Molecule Radioprotectors.
Authors: Epperly Michael W. , Franicola Darcy , Dixon Tracy , Zhang Xichen , Komanduri Paavani , Greenberger Benjamin , Wipf Peter , Koide Kazunori , Greenberger Joel S.
Published: October 2019

Development of small molecule radioprotectors is a major national priority. Two groups of compounds have particular promise. The first group targets the mitochondria based upon previous data with transgene MnSOD which when expressed in the mitochondria prevents apoptosis and increases radioprotection. These agents contain the antioxidant tempol or nitric oxide synthetase inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) attached to a hemi-gramicidin linker which targets the mitochondria. The second group consists of the dietary agent resveratrol and acetylated variants. Mouse hematopoietic progenitor 32Dcl3 cells were incubated for 1 hr in 10 μM tempol, AMT, or gramicidin linked tempol XJB-5-125 (tempol), XJB-7-75 (tempol) or JP4-039 (AMT). In separate experiments, 32Dcl3 cells were incubated for 1 hr in resveratrol or acetylated resveratrol. The cells were then irradiated to doses ranging from 0 to 8 Gy, plated in 0.8% methylcellulose, and incubated in a 5% CO2 incubator for 7 days. Colonies of greater than 50 cells were counted with the data analyzed using linear quadratic or single-hit, multi-target models. 32Dcl3 cells incubated in 10 μm tempol before irradiation resulted in no change in radiation sensitivity while incubation in XJB-5-125 or XJB-7-75 had decreased radiosensitivity. XJB-5-125 had an increased Do of 1.91 ± 0.67 Gy compared to 1.32 ± 0.09 Gy for 32Dcl3 cells incubated in tempol and 1.35 ± 0.27 Gy for control 32Dcl3 cells (p = 0.045 or 0.040, respectively). Incubation in XJB-5-75 resulted in an increased shoulder on the survival curve with an ñ of 19.4 ± 2.6 compared to 8.7 + 1.6 for cells incubated in tempol or 6.9 +1.8 for control 32Dcl3 cells (p = 0.025 or 0.022). Incubation in JP4-039 resulted in an increased Do of 2.2 ± 0.1 Gy compared to 1.24 ± 0.15 or 1.13 ± 0.06 for cells incubated in AMT or control 32Dcl3 cells only, respectively (p = 0.0115 or 0.0098, respectively). Incubation of 32Dcl3 cells in resveratrol or acetylated resveratrol before irradiation resulted in an increased shoulder on the survival curve of 33.2 ± 5.7 or 57.5 ± 9.9, respectively, compared to 6.9 ± 1.8 for 32Dcl3 cells (p = 0.0122 or 0.0072, respectively). These compounds were tested in mice receiving an LD50/30 irradiation dose. C57BL/6NHsd mice were injected intraperitoneally with 10 mg/kg of XJB-5-125, XJB-7-75or JP4-039 or 25 mg/kg of resveratrol or acetylated resveratrol and irradiated 10 mins later along with control mice to 9.5 Gy whole body irradiation. The mice injected with XJB-5-125, XJB-7-75, JP4-039 or acetylated-resveratrol had increased survival compared to control irradiated mice (p ≤ 0.0004). Therefore, four new small molecules have been identified which demonstrate significant radioprotective properties both in vitro and in vivo.
Mobilised Peripheral Blood Could Be a Suitable Source of Mesenchymal Stem Cells?.
Authors: Almici Camillo , Verardi Rosanna , Braga Simona , Neva Arabella , Russo Domenico , Rossi Giuseppe , Marini Mirella
Published: October 2019

Mesenchymal stem cells (MSC) are multipotent cells that are considered one of the most promising product for cellular therapy in regenerative medicine. MSC have been obtained and expanded from bone marrow and umbilical cord blood in adequate amounts for clinical applications. Under the right conditions, MSC could migrate from bone marrow into the peripheral circulation; however MSC have not been routinely isolated from peripheral blood, and studies are rare and not conclusive. The aim of the present study was to evaluate mobilised peripheral blood (MPB), obtained from patients undergoing apheresis collection of circulating hematopoietic progenitor cells, as a potential source of MSC for clinical applications. MPB samples (500–900 × 106 cells, N = 17) were separated by negative lineage-depletion immunoselection (RosetteSep). Selected cells were seeded in multi-well plates at low density in MesenCult Basal Medium without and with different combinations of growth factors (EGF, PDGF-BB, b-FGF). On reaching confluence, adherent cells were detached by 0.25% trypsin-EDTA treatment and replated for at least two passages. At each passage, surface antigen expression was analyzed by flowcytometry (CD45, CD34, CD105, CD44, CD73, CD166, CD31, HLA-DR and VE-caderine). Following immunoselection 9.5–17.1 × 106 cells were recovered from MPB samples. Cultured cells reached confluency in 3–4 weeks on first passage and in two weeks thereafter. Immunophenotyping showed negativity for CD45 antigen. The absence of growth factors in culture medium conditioned MSC growth capability, while the addition of PDGF-BB+EGF or b-FGF was able to boost the number of CD45−/CD73+/CD90+ cells in culture (see figure). However expansion remains still sub-optimal, having been reached in 8/17 samples. In conclusion, we demonstrate that MSC can be obtained from MPB, but expansion requires longer time period and appears more difficult compared to bone marrow. Therefore, further studies need to be conducted to find better culture conditions and optimal growth factor combinations to support MPB-derived MSC expansion.

Figure Figure