bluuebunny/crossref_metadata_2025 huggingface dataset:
Rates of Clinically Apparent Venous Thromboembolism and Death Resulting from Pulmonary Embolism among Unselected Hospitalized Acutely Ill Medical Patients Versus Rates Predicted from Clinical Studies.
Authors: Piovella Franco , Anderson Frederick A. , Decousus Hervé , FitzGerald Gordon , Bergmann Jean-François , Kakkar Ajay K. , Spyropoulos Alex C. , Zotz Rainer B. , Spencer Frederick A. , Turpie Alexander G.G. ,   for the IMPROVE Investigators
Published: April 2020

Background

Without evidence from autopsies, the majority of deaths resulting from pulmonary emboli (PE) are indistinguishable from deaths due to other cardiovascular diseases. This has led to a gap in perceptions between the benefits and risks of providing venous thromboembolism (VTE) prophylaxis. In this study, we estimated the incidence of clinically apparent VTE in hospitalized acutely ill medical patients in The International Medical Prevention Registry on Venous Thromboembolism (IMPROVE), and compared this with the expected incidence derived from clinical studies that used autopsy or prospective venographic confirmation of clinically important VTE.

Methods

Beginning in July 2002, a consecutive, unselected sample of patients who were aged ≥18 years and hospitalized for ≥3 days with an acute medical illness, were enrolled in this observational cohort from 49 hospitals in 12 countries. Up to 31 March 2005, 6946 patients were enrolled.

Results

Based on autopsy series of all-cause in-hospital deaths reported in the literature, PE is associated with 10% of deaths, is the primary cause in 5%, and is clinically recognized as the primary cause in 1.5% of deaths. A review of clinical studies with mandatory venography resulted in predicted rates of 10% for all VTE and 1% for clinically recognized (confirmed) VTE. In IMPROVE, there were 4/291 (1.4%) deaths due to clinically recognized PE (vs. 4 predicted). There were 79 (1.1%) treated VTE events (vs. 69 predicted).

Conclusions

Observed rates of death due to PE and clinically recognized VTE in a real-world setting are consistent with predictions from clinical study data. Physicians should be aware of the significant gap that exists between clinically important and clinically evident VTE events. Reliance on the low rates of clinically recognized events to assess the seriousness of this disease can lead to a significant under-estimation of its impact on public health and a consequent failure to realize the proven benefits of VTE prophylaxis in hospitalized acutely ill medical patients.
The Risk of Dialysis Access Thrombosis Is Related to Polymorphisms in the Transforming Growth Factor-β1 and Plasminogen Activator Inhibitor-Type 1 Genes.
Authors: Lazo-Langner Alejandro , Knoll Greg A. , Wells Philip S. , Pilkey Rachel M. , Carson Nancy , Rodger Marc A.
Published: April 2020

Background. Transforming growth factor-β1 (TGF-β1) is involved in cell growth and differentiation and it plays an important role in the genesis of fibrosis through the stimulation of neointima proliferation and accumulation of components of the extracellular matrix and it has been suggested that polymorphisms (polym) in its gene contribute to determining the patency of the vascular access (VA) in patients (pts) on hemodialysis (HD) by contributing to both atherogenesis and VA thrombosis. On the other hand it has been demonstrated that polym in the gene encoding the plasminogen activator inhibitor type-1 (PAI-1) are a risk factor for ischemic heart disease and possibly stroke although their role in other vascular territories is unknown. It is also known that TGF-β1 is an important up-regulator of the PAI-1 gene.

Methods. We conducted a case-control study to determine the relationship between TGF-β1 polym of the signal sequence (869 T>C; 915 G>C) and VA thrombosis in 416 HD pts. (107 with VA thrombosis, 309 controls had no thrombosis). We also explored for possible interactions with the 4G/5G polym of the PAI-1 gene. TGF-β1 and PAI-1 polym were amplified using PCR and genotyped using an ABI PRISM 3100 Genetic Analyzer. TGF-β1 producing haplotypes (haplo) were defined as low, intermediate or high as previously reported. All pts were also tested for thrombophilia. Statistical analysis was done using univariate and multivariate logistic regression adjusted for thrombophilia, age, access type, etc.

Results. Frequencies for low, intermediate and high TGF-β1 producing haplo were 9.3, 26.2 and 64.5% in cases and 2.6, 22.3 and 75.1% in controls.

Odds of thrombosis for TGF-β1 haplotypes Haplotype Crude OR (95% CI) p Adjusted OR (95% CI) p High producing haplotype is reference category Low 5.11 (1.93, 13.5) 0.001 7.31 (2.15, 24.88) 0.001 Intermediate 1.30 (0.74, 2.29) 0.36 1.39 (0.70, 2.75) 0.35

Figure Figure

Frequencies for 5G/5G, 4G/5G and 4G/4G PAI-1 polym. were 21.5, 56.1 and 22.4% in cases and 25.6, 50.2 and 24.3% in controls respectively. When we explored the interaction between both gene polym we found a highly significant result for the interaction between the low TGF-β1 producers and the 4G/4G PAI-1 polym (adjusted OR 19.3; 95% CI 2.82, 132.40; p=0.003).

Conclusions. Our results show that intermediate and high producing TGF-β1 haplo have a protective effect against VA thrombosis in HD patients that is not modified by PAI-1 polym and also suggest that the interaction between low TGF-β1 producing haplo and the 4G/4G PAI-1 polym might be an important contributor to thrombosis of the VA in HD pts. Further studies are ongoing to determine the relationship between TGF-β1 haplo, TGF-β1 level, and PAI-1 polym with thrombosis in this and other populations as well as to clarify the mechanisms underlying this apparently paradoxical effect.
Prevalence and Clinical Significance of Single and Combined Inherited Thrombophilias.
Authors: Losonczy Hajna , Nagy Agnes , Toth Orsolya , Balassa Katalin , David Marianna , Kecskes Marianna , Vidra Timea , Szomor Arpad
Published: April 2020

The prevalence of inherited single and combined thrombophilias were examined in 986 patients (330 males and 656 females) between 1995–2004 with venous thromboembolism (VTE).

Methods: Screening blood coagulation tests (PT, APTT, TT, FG), coagulation inhibitor activities and antigen levels (AT, PC, PS), APC-R, FV: Q506, FII 20210A, anti-phospholipoid antibodies, plasma homocystein level and circumstantial risk factors were examined. The age at first thromboembolic episode, family history for thrombosis, localization of VTE, therapies administered, recurrences, chronic venous insufficiency (CVI) and crural ulcer (CU) were registered.

Results: The mean age at the first thromboembolic episode was 34,4 +/− 11,4 y. VTE was present in the family in 416 cases (46%), the mean observation time was 9,24+/−8,81 years. We found AT deficiency in 4,3%, PC in 5,9 % (mutation in the PC gene was verified in 7 families), PS in 5,4%. APC resistence was 25,3%, FV:Q506 mutation 24%. Inherited defect was present in 459 p. (46,5%). Single inherited abnormality in 279/986 p. (28,2%), combined in 82/986 p. (8,3%), two abnormalities in 70, three in 11 and four in 1 patient. The distribution of circumstantial risk factors among patients with and without inherited coagulation abnormalities were equal, except OC usage, pregnancy and labor. These obstetrical risk factors occured significantly higher in patients having inherited abnormalities. VTE in the family, the ileofemoral localization of the VTE and the need for thrombolythic therapy was also significantly higher among patients with inherited abnormalities. We also examined and compared the occurence rate of recurrence, CVI and CU among patients with single and combined inherited abnormalities vs. those without such abnormalities. Recurrence rate was significantly higher in patients with coagulation defects vs. without defects, and between those having only one vs. more defects. It was not dependent on the type of the single coagulation defect. FVQ506+PC and FVQ506+AT defects increased, whereas the FVQ506+FII20210A and +PS defects did not increase singificantly the relapse rate. The occurance of CVI and CU in patients with coagulation defects was significantly increased independently of having one vs. more defects.

Conclusions: Inherited coagulation abnormalities predisposing to VTE are detectable in 46,5% of young Hungarian VTE patients. Among them 60,7% had single and 17,8% had combined abnormalities. Recurrence rate was significantly higher in patients with more then one vs. no inherited coagulation abnormality. CVI and CU developed significantly more frequently in patients having one or even more inherited coagulation defects. The detailed detection of coagulation abnormalities is of prognostic significance. (Supported by OTKA T026428 and ETT 124/96.)
Predictors of the Development of Post-Thrombotic Syndrome after Acute Symptomatic Deep Vein Thrombosis: Two-Year Results from a Canadian Multicenter Prospective Cohort Study (The VETO Study).
Authors: Kahn Susan R. , Ducruet Thierry , Arsenault Louise , Miron Marie Jose , Roussin Andre , Desmarais Sylvie , Joyal France , Kassis Jeannine , Solymoss Susan , Ginsberg Jeffrey S. , Desjardins Louis , Johri Mira , Lamping Donna L. , Shrier Ian
Published: April 2020

Background and Objectives: It is not understood why some patients develop post thrombotic syndrome (PTS) after DVT while others recover without sequelae. We performed a prospective, multicenter study of long-term outcomes after DVT (the Venous Thrombosis Outcomes [VETO] Study) whose principal aim was to identify predictors of PTS.

Methods: From 2001–2002, consecutive patients with objectively diagnosed DVT were recruited at 7 hospital centres. During study visits at Baseline, 1, 4, 8, 12 and 24 months, clinical data were collected, thrombophilia testing was performed and standardized assessments for PTS (using Villalta scale) were carried out by trained personnel. Patients were classified as having developed PTS if the ipsilateral Villalta score was ≥5 on at least 2 visits or at the final follow-up visit, and severe PTS if a score of >14 or a venous ulcer was documented on any one occasion. The influence of clinical, demographic, ergonomic and biological determinants on the risk of developing PTS was evaluated in bivariate and multivariate analyses.

Results: 359 patients were recruited; 49% were male, mean age was 56 years, 2/3 were outpatients and 55% had proximal DVT. The cumulative risk of PTS was 37%, of which 1 in 6 cases was severe. Patients who developed PTS, compared with those who did not, were more likely to be older (p=.005), have had previous episode(s) of DVT (p=.0003), have atherosclerosis risk factors [hypertension (p=.0002), high cholesterol (p=.02)], higher BMI (p=0.0007), longer duration of symptoms before DVT diagnosis (p=.049) and work in a job with high physical demands (p=.05), and they were less likely to have the Prothrombin gene mutation (p=0.03). Variables of interest that did not influence the risk of PTS included: sex, location of DVT (proximal vs. distal), side of DVT (left vs. right), type of DVT (cancer-related, temporary risk factors [e.g. surgery, trauma] or idiopathic), initial anticoagulation with LMWH vs. UFH, duration of warfarin, Factor V Leiden mutation or prior ipsilateral musculoskeletal condition. In adjusted multivariate analyses, BMI (odds ratio [OR] 2.1 for BMI >30 vs. < 30, 95% confidence interval [CI] 1.1, 4.2), previous DVT (OR 2.6, 95% CI 1.4, 4.9) and hypertension (OR 1.9, 95% CI 1.04, 3.6) were independently associated with PTS.

Conclusions: In our cohort, high BMI, previous history of DVT and hypertension were independent predictors of PTS, whereas presenting characteristics of the index DVT and its treatment did not influence the risk of developing PTS.
Regulation of Protein Kinase C by the Platelet P2Y12 ADP Receptor.
Authors: Lova Paolo , Guidetti Gianni F. , Campus Francesca , Bernardi Bruno , Balduini Cesare , Torti Mauro
Published: April 2020

The Gi-coupled P2Y12 receptor plays a crucial role in platelet activation induced by ADP, and is also required for the action of a number of agonists whose receptors are couple to Gq, such as thromboxane A2. Stimulation of P2Y12 leads to the inhibition of adenylyl cyclase through the Gai subunit, and to the activation of phosphatidylinositol 3-kinase (PI-3K) and Rap1b through the bg subunit. Pharmacological inhibition of P2Y12 receptor results in the prevention of platelet aggregation induced by a number of agonists, including ADP and the the thromboxane A2 analogue U46619. In this study, we report that P2Y12 receptor is absolutely necessary for the stimulation of protein kinase C (PKC) and for PKC-directed protein phosphorylation. By using 32P-labeled human platelets, as well as by immunoblotting with anti-phospho(Ser) PKC substrates antibody, we analyzed the phosphorylation of pleckstrin, the main platelet substrate for PKC. As expected, we found that ADP-induced pleckstrin phosphorylation was totally suppressed by antagonists of the Gq-coupled P2Y1 receptor, but, surprisingly, we found that it was also completely prevented upon blockade of the P2Y12 receptor by AR-C69931MX or by 2-MeSAMP. Although we failed to directly detect any production of IP3 in ADP-stimulated platelets, we observed that ADP-induced Ca2+ release from intracellular stores was not affected by P2Y12 receptor antagonists. Moreover, AR-C69931MX or 2-MeSAMP strongly impaired pleckstrin phosphorylation induced by U46619, without affecting intracellular Ca2+ mobilization. These results indicated that blockade of the P2Y12 receptor did not interfere with phospholipase C stimulation, and that the reduced activation of PKC is probably not the consequence of a reduced production of diacylglycerol. Experiments performed with specific cell permeable inhibitors demonstrated that, in ADP-treated platelets, neither inhibition of adenylyl cyclase nor activation of PI-3K downstream P2Y12 receptor stimulation was responsible for PKC regulation. Subthreshold concentrations of PMA were able to restore ADP- or U46619-induced pleckstrin phosphorylation in the presence of P2Y12 antagonists, but were unable to restore platelet aggregation. Moreover, direct inhibition of PKC by Ro31-8220 prevented ADP-induced pleckstrin phosphorylation, but aggregation occurred normally. These results indicate that although PKC-directed protein phosphorylation is targeted by the P2Y12 receptor, it is not responsible for the essential contribution of this receptor to platelet aggregation. By contrast, we found that inhibition of platelet diacylglycerol kinase by R59949 completely reverted the inhibitory effect of AR-C69931MX or 2-MeSAMP on ADP- and U46619-induced activation of PKC, and completely restored platelet aggregation. These results suggest that diacylglycerol kinase may be regulated downstream the Gi-coupled P2Y12 receptor, and may be an important new regulator of platelet aggregation.
Neither Free αIIb nor Free β3 Limits αIIbβ3 Biogenesis, and Pro-αIIbβ3 Adopts a Conformation Akin to Ligand-Bound Mature αIIbβ3.
Authors: Mitchell W. Beau , Newman Peter J. , Coller Barry S.
Published: April 2020

The first steps in the biogenesis of αIIbβ3 involve the individual synthesis of each subunit within the endoplasmic reticulum, followed by complex formation. Previous studies performed in HEL cells and in megakaryocyte-lineage cells cultured from cryopreserved progenitor cells of patients with CML have led to conflicting data with regard to whether αIIb or β3 is produced in limiting amounts that might then control complex formation. To study this question in greater detail, we conducted immunoprecipitation studies on HEK293 cells transfected with αIIb and β3 cDNAs and megakaryocyte-lineage cells derived from human umbilical cord blood grown in the presence of thrombopoietin and IL-11. Cells were labeled for 15 min with 35S-methionine/cysteine, and then lysed after 2 hr with a 1% triton X-100 buffer. The antibodies employed included murine monoclonal antibodies 10E5 (anti-αIIbβ3 complex, which, reacts with αIIb cap domain), 7E3 (anti-αIIbβ3 + αvβ3, which reacts with β3 specificity-determining loop and α1 helix), AP-5 (LIBS anti-β3, which reacts with β3 amino acids 1 - 6), 7H2 (anti-β3, which reacts with PSI domain near C13), 1990 (anti-αIIb, which reacts at unknown site), and LIBS2 (LIBS anti-β3, which reacts with a site within amino acids 602 – 690). The relative amounts of pro-αIIb, mature αIIb, and β3 immunoprecipitated by this panel of antibodies is shown in Table 1. Results were similar in both the HEK293 cells and in megakaryocyte-lineage cells derived from human umbilical cord blood. These data indicate that there are sizeable pools of both free pro-αIIb and free β3 in both cell types, and thus neither subunit controls complex formation by limited availability. Based on the selective precipitation of pro-αIIb + β3 by AP5, it appears that the pro-αIIbβ3 complex adopts a conformation similar to that of ligand-bound mature αIIbβ3. We conclude that αIIbβ3 complex formation is slow relative to the production of the individual subunits, and is probably controlled either by the ability of the subunits to adopt the proper fold and/or to interact with a chaperone(s) that facilitates complex formation. Assuming that AP5 selectively recognizes αIIbβ3 complexes in which the angle of the β3 βA (I-like) domain - hybrid domain interface results in separation of the αIIb and β3 leg domains, these data also suggest that αIIbβ3 head-head interactions precede αIIbβ3 leg-leg interactions during biogenesis, and that αIIb maturation occurs rapidly after αIIbβ3 leg-leg interactions. Such a model is consistent with our previous data demonstrating that pro-αIIb binds to the ER membrane-resident chaperone calnexin via the N-linked glycan at αIIb N15.

Table 1 Antibody Pro- αIIb Mature αIIb β3 1990 ++++ +++ +++ 10E5 ++ +++ ++ 7E3 ++ +++ ++ 7H2 ++ +++ ++++ LIBS2 + − ++++ AP5 ++ − ++++
Age-Decline in the Activity of the Plasma Membrane Ca2+ Pump (PMCA) of Normal Human Red Blood Cells (RBCs).
Authors: Bookchin Robert M. , Daw Nuala , Etzion Zipora , Tiffert Teresa , Lew Virgilio L.
Published: April 2020

We recently demonstrated that the activity of the PMCA varies greatly among the RBCs in a normal blood sample (Lew et al., Blood102:4206,2003). To test the possibility that these variations might be related to cell age, we designed a new experimental protocol to separate RBCs with different PMCA Vmax, which used glycosylated hemoglobin (Hb) A1c as an age marker for the normal RBCs, and avoided Co2+ (used in our usual PMCA activity assay) which we found to interfere with Hb A1c measurements: The ionophore A21837 was used to generate a high, rapid and uniform [CaT]i in RBCs, which were suspended in a 90mM-K+ buffer to prevent RBC dehydration by Gardos channel activation. These RBCs were then washed in high-K+ buffer (ice-cold to inhibit the Ca2+ pump) with 1% albumin to remove the ionophore. At t=0, the washed and packed ionophore-free cells were delivered into 20 volumes of high-K+ buffer at 37oC to initiate Ca2+ extrusion by the PMCA, and then sampled at 15 sec intervals into 25 vol of an ice-cold, K+-free, isotonic buffer containing 10 mM SCN−; these conditions were designed to trap un-extruded Ca2+ and to elicit rapid dehydration of those cells which had not yet pumped out all their Ca2+ load. After ~ 40 min at 0oC each sample was spun; the packed RBCs were resuspended in 10 vol of the same buffer and spun again through diethylphthalate oil (D=1.117 g/ml) to separate the dehydrated RBCs (pellets) from the non-dehydrated cells. The fraction of cells in pellets and on top of the oil was estimated in each sample from Hb measurements, and their Hb A1c content was measured by HPLC. Initially, almost all RBCs, except those with the most vigorous pumps, were recovered in the pellets, but with time, the fraction of cells which had fully extruded their Ca2+ load and were recovered on top of the oil approached 100%. The progressively smaller pellets contained RBCs with progressively weaker pumps; their Hb A1c fraction increased with decrease in the yield of RBCs in the pellet. This inverse correlation between yield and Hb A1c fraction in the pellets was observed in all six experiments performed with RBCs from four donors. Since the decline in PMCA activity correlated directly with an increase in Hb A1c, the observed population variation in PMCA activity reflects an age-related decline in PMCA activity. Whether this decline is due to progressive glycosylation of of a lysine residue near the catalytic ATP domain on the pump ATPase (Gonzalez Flecha et al., J Membr Biol171:25,1999) or to other mechanisms, remains to be determined. The age-decline in PMCA activity may be responsible for the increasing density of aging RBCs, by allowing intermittent episodes of [Ca2+]i elevations in the circulation, with transient Gardos channel activation and gradual dehydration by net KCl and water loss.
Alpha Hemoglobin Stabilizing Protein (AHSP) Is a Susceptibility Gene to Drug /Infection-Induced Hemolytic Anemia.
Authors: Kanno Hitoshi , Kamatani Naoyuki , Hamada Takako , Furihata Kaori , Hattori Yukio , Miwa Shiro , Fujii Hisaichi
Published: April 2020

Approximately 500 patients are diagnosed as having congenital hemolytic anemia every year in Japan, and the etiology in 75% of these patients involves red cell membrane disorders. The remaining cases are categorized as congenital non-spherocytic hemolytic anemia (CNSHA) and only about 25 % of CNSHA patients are diagnosed as either unstable hemoglobinopathies or erythroenzymopathies. As a result, up to 100 subjects remained diagnosed as having idiopathic hemolytic anemia. Recently, α-hemoglobin stabilizing protein (AHSP) has been identified, and found to play an important role as molecular chaperones in the stabilization of unstable free α-globin. Intact α-globin monomer generates reactive oxygen species, resulting in damage to red cell protein and lipids. AHSP knockout mice showed compensated hemolysis with Heinz bodies. Genetic defects in protection against oxidative stress such as glucose-6-phosphate dehydrogenase deficiency may account for acute hemolysis only when subjects are exposed to either oxidative drugs or infection, but chronic hemolysis is not evident in those cases. These observations led us to hypothesize that AHSP gene defects might account for either Heinz body-positive or drug/infection-induced hemolytic anemia.

We examined 25 CNSHA patients with unknown etiology: 6 cases showing Heinz body-positive red cells and 19 cases showing episodes of drug/infection-induced acute hemolysis. Erythroenzymopathies and unstable hemoglobinopathies were ruled out using red cell enzyme analysis, isopropanol stability tests and DNA sequencing of globin genes. Seven SNPs (single nucleotide polymorphisms) surrounding the AHSP gene (rs 4889672, rs 8046452, rs 8050390, rs 4296276, rs 17677, rs 10843, and a novel SNP at 92-bp upstream of rs 4889672) were examined, and compared with 100 control subjects. We found that a certain haplotype, 5′-GTCGA**-3′, was significantly prevalent in CNSHA cases (p=0.0086). Each SNP within the haplotype is located in the 5′-flanking region of AHSP gene, suggesting that the SNP(s) might account for decreased expression of AHSP gene. The reporter gene assay using K562 cells revealed that a construct containing 5′-GTCGA**-3′ had about 30% of transcriptional activity compared to a major haplotype 5′-ACCAG**-3′. Taken together, we conclude that the AHSP gene is correlated to disease-susceptibility in a group of hemolytic anemia with either Heinz body-positive or drug/infection-induced episodes.
A Pilot Study To Evaluate the Safety, Efficacy and Effects on Liver Function of Granulocyte Colony-Stimulating Factor To Mobilize Hematopoietic Stem Cells in Patients with Liver Cirrhosis.
Authors: Isidori Alessandro , Catani Lucia , Talarico Simona , Lorenzini Stefania , Loggi Elisabetta , Gramenzi Annagiulia , Bonifazi Francesca , Aluigi Michela , Bernardi Mauro , Andreone Pietro , Baccarani Michele , Lemoli Roberto M.
Published: April 2020

In animal models hematopoietic stem cells (HSCs) and the hematopoietic cytokine granulocyte colony-stimulating factor (G-CSF) contribute to tissue regeneration after acute or chronic liver damage. In this study, we assessed whether: 1) G-CSF can be safely administered to patients with liver cirrhosis to expand and mobilize HSCs into peripheral blood; 2) G-CSF treatment affects residual liver function. Eighteen patients with a Child-Turcotte-Pugh (CTP) score < 10 and a Mayo Model for End Stage Liver Disease (MELD) score < 20 were considered eligible for the study. Increasing doses of G-CSF were administered subcutaneously for 7 consecutive days to 5 cohorts of 3 patients each, starting from 2.2 μg/kg/daily, until the achievement of the maximum-tolerated dose and/or the successful mobilization of ≥10 CD34+ cells/μl in at least 2/3 patients. G-CSF mobilizing dose was found at 15 μg/kg/day. Four additional patients were treated with the mobilizing G-CSF dose. All of them showed a successful mobilization of HSCs with a median peak value of CD34+ and CD133+ cells of 27.1(±15.5) and 15.2 (±5.1)/μl, respectively. Circulating HSCs were then collected by single volume leukapheresis and a median of 1.4(±0.73)x106 CD34+ HSCs/kg body weight were cryopreserved. G-CSF treatment induced a significant increase of hepatocyte growth factor serum level. When we analyzed stem cell subsets, we found significant mobilization of early hematopoietic (CD34+/CD38−) and endothelial (CD34+/KDR+, CD133+/KDR+) progenitors as well as CD34+/CXCR4+ cells. No severe adverse events were observed at any dosage or during leukapheresis. According to CTP and MELD score, no significant modification of liver function was observed during the treatment period and follow up (median=198 days) in both mobilizing and non-mobilizing patients. Nevertheless, in all treated patients a significant reduction of alpha-fetoprotein values was observed (p<0.001). In conclusion, the administration of G-CSF to patients with liver cirrhosis is safe, feasible and capable of mobilizing HSCs at the dosage of 15 μg/kg/day. Our study represents the first step for evaluating the role of G-CSF and mobilized HSCs to improve liver function in patients with liver cirrhosis.
Characterization of ‘Early’ vs ‘Late’ Endothelial Progenitor Cells (EPCs) Which Are Derived from Human Umbilical Cord Blood (HCB) during Ex Vivo Expansion.
Authors: Yoo Eun-Sun , Ahn Jee-Young , Bae Yun-Kyung , Lee Seung-Eun , Lee Sang min , Mun Yeung-Chul , Lee Kyung-Eun , Seong Chu-Myong
Published: April 2020

EPCs have been isolated from adult peripheral blood and bone marrow. Recently, several groups reported that two types (‘early’ & ‘late’) of EPC could be isolated from peripheral blood and bone marrow when pertinent cocktails of cytokines were used. Interestingly, early and late EPCs are different in terms of expression of surface markers, the abilities of tube formation in vitro and the capabilities of re-vascularization on hind limb ischemia models in mice. We found EPC formation during ex vivo expansion of HCB and one EPC could be found from 314 CD34+ cells from HCB based on limiting dilutional assay (ref. Stem Cells; 2003, Yoo et al). However, little is known about the characteristics of ‘early’ and ‘late’ EPCs that are derived from HCB. In this study, our aims are to isolate the ‘early’ and ‘late’ EPCs from HCB during ex-vivo HCB expansion period and to characterize the biologic properties between ‘early’ and ‘late’ EPCs. 1 x 108 mononuclear cells were plated on a 100mm culture dish coated with 50ug/ml of human fibronectin (Calbiochem) and cultured in EGM-2 BulletKit system (Clonetics). Endothelial cells were assessed by colony counts, flow cytometry, proliferation assay, RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCs were also measured by in vitro transmigration assay in the presence of VEGF and SDF-1. In results, early spindle-shaped cells (‘early’ EPCs) which were grown at first week of culture were positive for CD31, CD14 and CXCR-4. Cobblestone shaped cells (‘late’ EPC) were in peak growth at second and third weeks of culture and were also positive using above antibodies except CD14. Early EPCs had not expressed mRNA of KDR, vWF and VE-Cadherin by RT-PCR. However, late EPCs expressed high level of mRNA of those endothelial marker genes. Both early and late EPCs expressed mRNA of eNOS. Late EPC produced more nitric oxide and formed more capillary tubes than those of early spindle-shaped cells. Early EPCs were readily migrated by VEGF and SDF-1 compared with those of late EPCs. In conclusions, we have found two different types of EPCs with different biologic properties during HCB ex vivo expansion. These findings may have potential clinical applications for “cell therapy” on vascular injuries (ie, hindlimb ischemia and myocardial infacrtion). Murine models for vascular injuries are being established to test the efficacy of different types of EPCs from HCB in our Lab.
Modulation of Aldehyde Dehydrogenase and Retinoid Signaling Induces the Expansion of Human Hematopoietic Stem Cells.
Authors: Chute John P. , Muramoto Garrett G. , Whitesides John , Colvin Michael , Saffi Rachid , Chao Nelson J. , McDonnell Donald
Published: April 2020

Aldehyde dehydrogenases are cytosolic enzymes that convert aldehydes into carboxylic acids. Human aldehyde dehydrogenase 1 (ALDH1) is highly expressed in the liver and in hematopoietic stem cells (HSCs). Although ALDH1 is a selectable marker of HSCs, its HSC-specific function is unknown. We hypothesized that ALDH might play a critical role in HSC fate determinations since it is required for the production of retinoic acids, which are broadly implicated in tissue differentiation, tissue patterning and embryonic development in vertebrates. In this study, highly purified human CD34+CD38−lin− HSCs were cultivated with early acting cytokines, thrombopoietin, stem cell factor and Flt-3 ligand (TSF) in the presence or absence of the competitive ALDH inhibitor, diethylaminobenzaldehyde (DEAB). Treatment of human BM and CB HSCs with TSF x 7 days caused a loss of CD34+CD38− cells in culture, morphologic differentiation, amplification of committed colony forming cells (CFCs) and a loss of primitive cells capable of repopulating non-obese diabetic/severe combined immunedeficient mice (SCID-Repopulating Cells, SRCs). Conversely, 7 day culture of BM and CB HSCs with TSF plus 100 μM DEAB blocked the morphologic differentiation and lineage commitment of HSCs in culture, expanded the CD34+CD38− population, and amplified SRCs 2-fold compared to input, indicating a fundamental role for ALDH in HSC differentiation. The effects of DEAB could be reversed by the co-administration of the retinoic acid receptor (RAR) agonist, all-trans retinoic acid (ATRA), suggesting that the ability of ALDH to produce retinoic acids is important in determining HSC fate. Via screening studies of direct ligands of RAR and RXR, we also identified a selective RXR modulator which functioned similarly to DEAB by impeding HSC differentiation and causing the 4-fold expansion of SRCs. Interestingly, treatment with either DEAB or the RXR modulator reversed the down-modulation of HOXB4 transcription that was otherwise observed in CD34+CD38−lin- cells during culture with cytokines, suggesting that inhibition of ALDH or RXR modulation may promote HSC self-renewal via discrete interactions with other regulatory pathways. Modulation of ALDH activity and retinoid signaling is a novel and effective strategy to amplify human HSCs.
Relationship between ABCG2 Expression and Side Population Stem Cell Phenotype in Murine Hematopoietic System Revealed by an Abcg2/GFP Knock-In Mouse Model.
Authors: Tadjali Mehrdad , Zhou Sheng , Rehg Jerold , Sorrentino Brian P.
Published: April 2020

Stem cells from a variety of tissues could be identified by a side population (SP) phenotype. We have identified an association between expression of the ABCG2 transporter and the SP phenotype, although the exact relationship between these two cellular characteristics is still not well understood. To further explore the relationship between Abcg2 expression and SP phenotype, we have generated a Abcg2/GFP reporter mouse model in which an IRES-GFP expression cassette was inserted down-stream of the stop codon of Abcg2 gene by homologous recombination, so that the expression of GFP is under the control of Abcg2 locus but endogenous Abcg2 expression was unperturbed. Immunohistochemistry of tissue sections using an anti-GFP antibody confirmed expression of GFP in tissues that normally express Abcg2, such as renal proximal tubules and intestinal epithelium. Flow cytometry analysis showed that about 10% of total bone marrow mononuclear cells expressed the Abcg2/GFP allele. Within this total GFP+ population, greater than 99% of the cells fall outside the SP region. Staining with lineage specific antibodies showed that 77% of the non-SP/GFP+ cells are Ter119+ erythroid cells. In contrast, only 0.5% of the total GFP+ population falls within the SP region. Approximately 90% of these bone marrow SP cells expressed the Abcg2/GFP allele. Transplantation studies with bone marrow cells that were depleted of lineage positive cells were then performed. All 5 mice transplanted with 100 Lin−, SP+, GFP+ cells were reconstituted in both myeloid and lymphoid lineages. In contrast, no repopulating activity was detected with up to 10,000 Lin−, non-SP, GFP− cells. When Lin−, non-SP, GFP+ cells were transplanted, only 2 of 5 animals were reconstituted with 5000 cells, and no repopulation was seen with lower cell doses. These results show a complex relationship between Abcg2 expression and the SP phenotype. Virtually all SP cells express the Abcg2/GFP allele, and these cells are highly enriched for repopulating activity in transplant assays. However, the majority of bone marrow cells that expressed the Abcg2/GFP allele were Ter119+ erythroid cells that do not bear the SP phenotype. In addition, there are non-SP, GFP+ cells that lack expression of Ter119 and other mature lineage markers. These Lin−, non-SP, GFP+ cells have relatively lower repopulating activity compared to SP cells.
Gata-Independent Regulation of Red Cell Specific Gene Expression.
Authors: Galloway Jenna L. , Wingert Rebecca A. , Thisse Christine , Thisse Bernard , Zon Leonard I.
Published: April 2020

During vertebrate embryonic hematopoiesis, the first blood cells can be identified by expression of the transcription factor genes scl and GATA2, followed by expression of GATA1, a gene required for the erythroid lineage. A high-throughput in situ hybridization screen in zebrafish analyzed the expression pattern of 3700 clones from a hematopoietic cDNA library and discovered 24 genes with expression in the blood. Examination of gene expression in Biklf, GATA1, GATA2, and GATA1/GATA2-deficient animals revealed that most blood genes are dependent upon GATA factors for expression rather than the Krüppel-like transcription factor Biklf. Three novel genes, expressed specifically in erythroid precursors, did not require GATA factors for their expression, demonstrating that some blood genes are regulated in a GATA-independent manner. These three genes were kelch-repeat protein (kelch repeats have been implicated in diverse cellular functions from actin binding to sequestering transcriptions factors), kiaa0650, which contains an SMC-hinge domain, and testhymin, which has no known structural motifs. By using combinations of antisense morpholinos to the known hematopoietic genes biklf , GATA1, GATA2, and scl, we were able to examine the regulation of these novel genes in double and triple knock-down embryos. While expression of kelch-repeat protein was lost in the absence of GATA1 and Biklf, expression of testhymin and kiaa0650 was maintained in GATA1/GATA2/Biklf-deficient embryos, suggesting that these similarly expressed genes are differentially regulated. As with GATA1, kiaa0650 and kelch-repeat protein required Scl for their expression in the early hematopoietic mesoderm while testhymin did not. Furthermore, loss of Scl and GATA2 did not completely ablate testhymin expression, suggesting that this gene is induced by factors upstream or parallel to Scl and GATA2. Taken together, our zebrafish studies establish a regulation of gene expression by a developmental hierarchy of specific transcription factors that act in combination during blood cell maturation.
Red Blood Cell (RBC) and Platelet (PLT) Transfusion after Allogeneic Transplant for Leukemia: Similar Utilization after Sibling (SIB) Peripheral Blood Stem Cell (PBSC) and Umbilical Cord Blood (UCB) Graft.
Authors: Brunstein Claudio G. , Larson Sarah , Rodgers Tyson , Eastlund Ted , Symons Katherine , Weisdorf Daniel
Published: April 2020

Transfusion independence is an important goal of hematopoietic stem cell transplantation (HSCT) for acute leukemia. We hypothesized that time to red blood cell (RBC) and platelet (PLT) transfusion independence would differ depending on the source of stem cells and the intensity of the preparative regimen. We retrospectively analyzed all transfusions administered to 146 acute leukemia patients who underwent a HSCT at our institution receiving a sibling (SIB) peripheral blood stem cell (PBSC) or an unrelated umbilical cord blood (UCB) graft, between 7/01 and 12/03. Median age was 35.6 yrs. (range 0.5–69.6) and 88 (60%) were male. Ninety-eight (67%) patients (pts) received an UCB and 48 (33%) a SIB graft. Thirty-four (71%) SIB and 56 (57%) UCB pts. received a myeloablative preparative regimen. Median time to RBC independence among all SIB PBSC recipients (n= 25) was 56 days [myeloablative conditioning (MA) (n= 18) 52.5 days; non-myeloablative conditioning (NMA) (n=7) 58 days], and the cumulative incidence of RBC independence at 6 months was 52% (95%CI: 39–69) [MA 53% (95%CI: 38–73); NMA 43% (95%CI: 28–89)]. For recipients of a UCB graft (n= 58) median time to RBC independence was 47 days [MA (n= 38) 56.5 days; NMA (n=20) 41.5 days], and the cumulative incidence of RBC independence at 6 months was 59% (95%CI: 50–70) [MA 68% (95%CI: 56–82); NMA 48% (95%CI: 34–66)]. Median time to platelet (PLT) independence among all SIB PBSC recipients (n= 27) was 25 days [MA (n= 19) 26 days; NMA (n=8) 12 days], and the cumulative incidence of PLT independence at 6 months was 56% (95%CI: 44–73) [MA 56% (95%CI: 41–76); NMA 57% (95%CI: 35–94)]. There was no statistically significant difference in the cumulative incidence of RBC independence between SIB PBSC vs. UCB (p=0.49), and MA vs. NMA (p=0.18). For recipients of an UCB graft (n= 68) median time to PLT independence was 43 days [MA (n= 44) 47.5 days; NMA (n=24) 39 days], and the cumulative incidence of PLT independence at 6 months was 69% (95%CI: 61–79) [MA 79% (95%CI: 68–90); NMA 57% (95%CI: 44–75)]. There was no statistically significant difference in the cumulative incidence of PLT independence between SIB PBSC vs. UCB (p=0.52), and MA vs. NMA (p=0.21). In multivariate analysis, the source of HSC and intensity of the preparative regimen were not predictors of RBC or PLT independence. This analysis shows similar RBC and PLT utilization in recipients of SIB PBSC and UCB following either MA or NMA conditioning. The substantial costs associated with transfusion support for allogeneic HSC transplant are similar in SIB PBSCT and UCB graft recipients.
A Randomized Trial of Heparin Plus Anti-Thrombin III (ATIII) vs. Heparin Alone To Prevent VOD after HSCT.
Authors: Jung Chul W. , Lee Jeeyun , Park Byeong Bae , Park Se H. , Kim Jung Han , Kwon Jung Mi , Oh Sung Yong , Kim Hyun Jung , Lee Hyorak , Kim Kihyun , Ahn Jin Seok , Kim Won Seog , Park Keunchil
Published: April 2020

Background: Hepatic veno-occlusive disease (VOD) is a devastating regimen-related toxicity after hematopoietic stem cell transplantation (HSCT). There is no safe and proven therapy for established VOD, and focus has been on its prevention. Previous studies have shown that ATIII level is markedly decreased in patients with VOD. We conducted a prospective, randomized trial to compare heparin plus ATIII versus heparin alone in HSCT recipients.

Methods: A clinical diagnosis of hepatic VOD was defined as satisfying two of the following criteria: bilirubin > 2.0 mg/dl, unexplained weight gain of > 2% from baseline, and hepatomegaly. The levels of PAI-1, TFG-β and TNF-α were measured at weekly basis for correlative analysis. Patients were randomized to receive or not receive prophylactic ATIII 1000U twice daily from day 1 until day +14 post-HSCT plus heparin 5U/kg/hr from day 1 until day +21 or discharge after HSCT.

Results: Thirty patients were enrolled for the study. Fourteen patients received heparin + ATIII and 16 patients received heparin alone for VOD prophylaxis during HSCT. There were 9 AML, 6 NHL, 4 ALL, 3 CML, 2 ABL, 2 MM, 1 MDS, 1 HD, 1 AA and 1 SLL patient. Of the 30 patients enrolled, 13 patients received sibling allogeneic HSCT, 13 autologous, 3 unrelated allogeneic and 1 cord blood HSCT. VOD occurred in 4 patients (25%) in the heparin alone group and none in the heparin + ATIII group (0%, P = 0.044). The incidence of bleeding complication was higher in the heparin alone group (4/16, 25%) than the heparin + ATIII group (1/14, 7%) without statistical significance (P = 0.190). The mean baseline level of PAI-1 was slightly higher in the heparin + ATIII group (30.9 ± 31.0 ng/ml) when compared to the heparin group (18.9 ± 13.0 ng/ml, P = 0.062). The PAI-1 level decreased markedly in the heparin + ATIII group (23.0 ±10.1 ng/ml) by the third week following VOD prophylaxis therapy (heparin group; PAI level 40.3 ± 40 ng/ml, P = 0.010). The mean baseline levels of TGF-β and TNF-α did not differ significantly between the two groups. There was no VOD-related death.

Conclusions: Although it was a preliminary analysis, the combination of ATIII and heparin for VOD prophylaxis in HSCT recipients seems to be more effective than heparin alone without increasing the hemorrhagic complication.
ZAP-70 Is Not Phosphorylated on the Activating Tyrosine Residue (Tyr319) in Chronic Lymphocytic Leukemia and B-Cell Lymphoma Cells.
Authors: Gobessi Stefania , Petlickovski Aleksandar , Laurenti Luca , Efremov Dimitar G.
Published: April 2020

The protein tyrosine kinase ZAP-70 is expressed at high levels in leukemic B-cells from chronic lymphocytic leukemia (CLL) patients with progressive disease and short survival. ZAP-70 is a key component of the proximal T-cell receptor signaling pathway and is highly homologous to Syk, an important B-cell receptor signaling (BCR) molecule. Recent studies indicate that ZAP-70 may participate in BCR signaling as well, but the mechanism of action is still not well understood. In T-cells, upon TCR stimulation ZAP-70 becomes phosphorylated on Tyr319 by the Src-like kinase Lck, which results in the release of the ZAP-70 kinase domain from an autoinhibited state to a fully active conformation. The Tyr319 site in ZAP-70 corresponds to the Tyr352 site in Syk, which is phosphorylated in B-cells following BCR stimulation. We therefore investigated the activation status of ZAP-70 and Syk in BCR stimulated CLL B-cells, using phosphorylation of Tyr319 and Tyr352 as markers of their activation. Analysis of 10 ZAP-70-positive CLL samples by immunoblotting with the phospho-ZAP70Tyr319/SykTyr352 antibody revealed that ZAP-70 is not phosphorylated at this site either before or after BCR stimulation, although in control experiments with Jurkat T-cells ZAP-70 became phosphorylated on Tyr319 upon TCR stimulation. Moreover, the Tyr352 site in Syk was phosphorylated following BCR stimulation in 6 of the 10 CLL B-cell samples. To further investigate the reasons for the unexpected lack of ZAP-70 activation in CLL B-cells, we produced stable transfectants of the BJAB lymphoma B-cell line that expressed ZAP-70 at levels similar to those found in CLL cases with progressive disease. In agreement with the CLL B-cell experiments, the Tyr319 site in ZAP-70 was not phosphorylated either before or after BCR stimulation. Since phosphorylation of Tyr319 is Lck-dependent in T-cells, and this kinase is expressed also in CLL B-cells, we ectopically expressed Lck in the ZAP-70-positive BJAB clones. Again, the Tyr319 site was not phosphorylated, indicating that ZAP-70 does not undergo activation of the kinase domain also in this cellular system. In contrast, BCR crosslinking in BJAB cells induced significant phosphorylation of Tyr352 in Syk, which was further enhanced in the clones that coexpressed ZAP-70. Furthermore, analysis of downstream signaling pathways following BCR stimulation showed stronger and prolonged activation of ERK and to a lesser extent Akt in the ZAP-70 positive clones, whereas no difference was observed in terms of activation of PLC-γ 2, JNK and degradation of the NF-kB inhibitor IkB. These data indicate that ZAP-70 does not undergo full activation in B-cells, but can still enhance activation of certain downstream BCR signaling pathways, possibly by affecting the activity of the related PTK Syk.
A G>A Transition in the Non-Coding Exon 1 of the Factor XI Gene as the Cause of Factor XI Deficiency in Three Afro-Caribbean Patients.
Authors: Mitchell Michael J. , Dai Letian , Alhaq Anwar , Savidge Geoffrey F.
Published: April 2020

Factor XI deficiency (MIM 264900) is an autosomal bleeding disorder of variable clinical severity. In contrast to haemophilia A or B the clinical symptoms do not correlate well with plasma levels of factor XI; it is therefore difficult to predict the bleeding tendency from either the factor level or the molecular defect. FXI deficiency is particularly common in the Ashkenazi Jews with a heterozygous frequency of 9%, associated with two common founder mutations E117X (Type II) and F283L (Type III). However, factor XI deficiency is found in all ethnic groups, with causative mutations being highly heterogeneous - mutations having been described in all exons with the exception of the non-coding exon 1.

In a study of >120 ethnically diverse factor XI deficient patients, three patients of Afro-Caribbean origin were found to be heterozygous for a G>A transition at nucleotide −53 within exon 1 of the factor XI gene. All three patients showed a low FXI:C on at least 3 different occasions (SM[female] 44.3–57.1, AB[female] 42.3–51.2 and GA[male] 70.3–72.9, Range 76–136u/dl). The 2 female patients were both reported to have a lupus anticoagulant which may explain the lower levels seen, although a lupus screen was negative. No variation within the coding sequence of the factor XI gene was detected. Two of the patients were heterozygous for the −403 G>T promoter polymorphism, whilst the remaining patient was homozygous for the −403 T allele and heterozygous for the −273 C>G polymorphism. Analysis of >50 factor XI alleles in patients of Afro-Caribbean origin failed to detect this base change in individuals with normal factor XI levels.

Purine-rich sequences, such as that in exon 1 affected by the −53 G>A substitution, are known to form extremely stable minihairpin loops. These sequences /structures have been shown to be important as splicing enhancers and in mRNA stability, particularly in making them more resistant to nucleases. Within the 5′ untranslated region (5′-UTR) of genes they have been demonstrated to be important in modulating translation efficiency. The -53 G>A substitution is located just 10 bases prior to the start of the factor XI mRNA and any of these mechanism could potentially explain the causative nature of this change. The -53 G>A substitution is predicted to cause ‘slippage’ within the postulated minihairpin loop, potentially making it unstable. Further work is on-going to try and prove and explain the causality of this mutation.

We speculate that the -53 G>A base change affects the normal processing of factor XI mRNA and, possibly in combination with the promoter polymorphisms, results in a mildly reduced plasma factor XI level.