bluuebunny/crossref_metadata_2025_split huggingface dataset:
Presence of a T383G Mutation in the Vitamin K Epoxide Reductase Gene (VKORC1) in a Patient Resistant to Four Different Vitamin K Antagonists.
Authors: Samama Michel-Meyer M.M. , Bodin Laurent L. , Horellou Marie Helene M.H. , Parent Florence F. , Kereveur Anne A. , Beaune Philippe P. , Loriot Marie Anne M.A. , Conard Jacqueline J. 
Published: October 2019

Abstract
Resistance to vitamin K antagonists is a rare disorder which until recently has not been associated to a genetic factor. The existence of inherited warfarin resistant rat strains has been related to mutations of the genes involved in the vitamin K cycle. Vitamin K dependent carboxylase was cloned in 19911, while human vitamin K epoxide reductase complex 1 (VKORC1), another enzyme implicated in vitamin K cycle, has been cloned more recently2,3. Mutations in this gene are responsible for both human and rat warfarin resistance 3. In 1997, we reported 4 a 63-year old patient with recurrent pulmonary embolism and deep vein thrombosis without known hereditary or acquired thrombophilia who was found resistant to warfarin (up to 45mg/day), fluindione (up to 80mg/day), acenocoumarol (up to 12 mg/day) and phenprocoumon (up to 30 mg/day). With phenprocoumon, long-acting vitamin K antagonist, drug concentration reached 85 mg/L (usual range 1–5 mg/L) but the INR remained around 1. Daily low molecular weight heparin (LMWH) was continued 3 months. Treatment was discontinued on 2 occasions and a recurrent thrombotic episode was observed. The patient is now receiving a single daily dose of low molecular weight heparin (80 mg Enoxaparin, body weight 120 kg, 67 IU/kg once a day) for the past 10 years without thrombotic or bleeding episodes. Osteodensitometry remains normal after 9 years of treatment. Careful biochemichal investigation had demonstrated a deficiency in vitamin K dependent carboxylation and absence of blockade of vitamin K epoxide reductase by phenprocoumon. This year, we sequenced the three exons of VKORC1 in this patient and detected a heterozygous T383G transversion resulting in a leucine to arginine substitution (L128R). This mutation has been recently identified by Rost3 et al. in an individual with warfarin resistance. It was not found in 259 control subjects that we had tested. Interestingly, another gene mutation of the VKORC1 can be responsible for a combined deficiency of vitamin K dependent clotting factors 3. In conclusion, a resistance to all vitamin K antagonists, including warfarin up to 45 mg/day is extremely rare and the mutation T383G of the VKORC gene has been reported in only one patient before the case reported here. Testing for mutation in VKORC1 will help in explaining some cases of anti vitamin K resistance.
Development of Non-Anticoagulant Derivatives of Biotechnology Derived Sulfaminoheparosan Derivatives to Investigate the Non-Anticoagulant Effects of these Agents.
Authors: Cornelli Umberto , Maddineni Jyothi , Vana Laurel , Hoppensteadt Debra , Florian-Kujawski Michelle , Fareed Jawed 
Published: October 2019

Abstract
It is now well known that heparin and related glycosaminoglycans produce some of their non-anticoagulant effects such as the anti-inflammatory, antiproliferative and regulatory actions independent of endogenous serpine (AT and HC-II). Sulfaminoheparosans (SAH) represent a class of biotechnology derived bacterial capsular polysaccharide (K5) derivatives which are epimerized and sulfated to mimic heparin’s biologic effects and exhibit affinity to both AT and HC-II. These SAH can be produced to exhibit molecular profile mimicking the low molecular weight heparins (LMWHs). The disaccharide unit structure of epimerized SAH is GlcA-GlcNSO36SO3(3,6)H) in comparison to heparin which is IdoA2SO3-GlcNSO36SO3. The anticoagulant and antiprotease profile of a 6 kDa SAH has been found to be similar to Tinzaparin (Maddineni et al. Clin Appl Thromb Hemost 10(1):27–37,2004). As this agent also exhibits various other biologic effects such as the release of TFPI, modulation of adhesion molecules and antiproliferative effects, it was hypothesized that the non-anticoagulant forms of this agent may exhibit some of these effects. In order to produce the non-anticoagulant derivatives in 6 kDa SAH, desulfated derivatives were prepared by removing sulfate groups on the position 2 alone (2 desulfated) and positions 2 and 6 (2,6 desulfated derivatives). Additional modifications in the 2,6 desulfated derivatives included the presence of either free amino group or N-acetyl in position 2. These modifications did not result in the molecular weight profile of the desulfated derivatives. However, in comparison to the parent 6 kDa SAH both the 2-desulfated and 2,6 desulfated derivatives were weaker in the anticoagulant (PT, PTT, Heptest) and antiprotease (anti-Xa, anti-IIa) and protease generation assays. 2,6 desulfation produced a strong decrease in the anticoagulant effects. Furthermore, the desulfation was proportional to a decrease in the affinity to AT and HC-II. None of these agents produced any activation of ADP induced platelet aggregation, however in whole blood flowcytometric studies ADP induced aggregation was augmented by 6 kDa SAH whereas the mono and disulfated derivatives produced variable effects. Heparinase I and II did not produce any digestion of any of these derivatives. Incubation of the 6 kDa SAH and the 2 desulfated and 2,6 disulfated derivatives produced varying degrees of inhibition of the Lewis-lung carcinoma cell cultures which was not dependent on degree of sulfation. These studies suggest that the non-serpine mediated effect of these agents may be independent of the sulfation pattern and the relative anticoagulant effects of these sulfaminoheparosan.
Intracellular Co-Localization of Factor VIII and Von Willebrand Factor Is Necessary for In Vivo FVIII Secretion.
Authors: Lamont Patricia A. , Ragni Margaret V. 
Published: October 2019

Abstract
Although the extracellular association of Factor VIII (FVIII) and Von Willebrand Factor (VWF) is well established, the intracellular interaction of FVIII and VWF is not well understood. Recently, the importance of intracellular co-localization of FVIII and VWF for in vitro FVIII secretion was demonstrated in endothelial cell lines. Whether intracellular co-localization of FVIII and VWF is required for in vivo FVIII secretion, however, is not known. We previously showed that liver transplantation leads to phenotypic cure of hemophilia A, by virtue of FVIII production in the allograft liver. Because FVIII is synthesized only in the allograft liver but not in endothelial cells of transplant recipients, and VWF is synthesized in extrahepatic tissue, this is an ideal model to study whether co-localization of FVIII and VWF is required for in vivo FVIII secretion. We, therefore, studied FVIII and VWF response after desmopression (DDAVP) infusion, administered at 0.3 mcg/kg by intravenous infusion over 30 minutes, in each of two men with severe hemophilia A (FVIII:C <0.01 U/ml) who had undergone orthotopic liver transplantation for endstage liver disease six months earlier. Both men had HIV and hepatitis C co-infection and were clinically well, with mildly elevated liver function tests, and FVIII:C levels >30% following transplantation. Coagulation studies, drawn before and after DDAVP, revealed that VWF:RCoF and VWF:Ag, but not FVIII:C, increased after DDAVP administration (see Table). The prolonged aPTT and correction in a 1:1 aPTT mix confirmed the absence of an inhibitor in these subjects. The lack of FVIII response to DDAVP supports previous in vitro work, and demonstrates for the first time that intracellular co-localization of FVIII and VWF is essential for in vivo FVIII secretion. These data also suggest that extrahepatic FVIII synthesis is necessary for in vivo response of the DDAVP releasable pool of FVIII. By contrast, co-localization does not appear to be necessary for VWF secretion. Although it is not possible to exclude that a chronic, exhaustive post-transplant increase in VWF may have limited VWF response to DDAVP, it is clear that FVIII did not increase following DDAVP. These findings have important implications for the design of gene therapies for hemophilia A and Von Willebrand Disease.
Subject Demographic Sample aPTT aPTT mix FVIII:C VWF:RCoF VWF:Ag 01-BW 32yoM Hem A Pre-DDAVP 44.4 sec 37.7 sec 0.50 U/ml 2.17 U/ml 2.42 U/ml HIV+/HCV+ Post-DDAVP 44.8 sec 37.4 sec 0.48 U/ml 2.91 U/ml 2.91 U/ml 02-PB 36yoM Hem A Pre-DDAVP 49.5 sec 38.0 sec 0.32 U/ml 1.61 U/ml 2.16 U/ml HIV+/HCV+ Post-DDAVP 50.8 sec 38.5 sec 0.30 U/ml 2.20 U/ml 2.50 U/ml
The Effect of Telomerase Antisense on the Differentiation of CD34+ Cells from Bone Marrow to Blood Cells.
Authors: Park Kyogn Hwa , Kim Byung Soo , Choi In Keun , Kim Seok Jin , Oh Sang Cheul , Seo Jae Hong , Choi Chul Won , Shin Sang Won , Kim Yeul Hong , Kim Jun Suk 
Published: October 2019

Abstract
Background: This study was designed to evaluate the effect of adenovirus-mediated telomerase antisense (TA) expression on cellular proliferation and apoptosis of CD34+ cells in the ex-vivo culture condition collected from normal bone marrow donors.
Methods: We used adenovirus vector containing recombinant antisense telomerase template RNA. Mononuclear cells were obtained from 21 bone marrow donors with normal marrow profile. Each samples were divided into two groups, consisted of the study group treated with telomerase antisense oligonucleotides(TA) and control group without treatment of TA. And then, CD34+ cells were purified from each sample with MACS method and were cultured with LTC-IC method. We evaluated the total and viable cell number, apoptosis degree and blood cell morphology at 2 weeks after LTC-IC culture, and compared between control and experimental groups.
Results: Total cell number of study group (2.3–3.3(median:2.6)X104) was not significantly different from that of control group(2.1–3.5(median:2.7)X104). Viable cell percentage of control and study groups were 71–83(median:76)% and 69–84(median:77)%, respectively, without significant difference. Apoptosis degree of study group (25–38%, median:32%) was significantly higher than that of control (13–27%, median 18%), Blood cell morphology of study group was more differentiated without blast cells (0%) than that of control group showing blast cells (4–9%, median 7%) at 2 weeks of culture.
Conclusion: We found that adenovirus-mediated antisense expression of telomerase RNA might accelerate the differentiation of hematopoietic stem cells with significant effect on the apoptosis degree. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.
Cell Cycle Related Stem Cell Gene Expression.
Authors: Dooner Gerri J. , Colvin Gerald A. , Dooner Mark S. , Quesenberry Peter J. 
Published: October 2019

Abstract
We have previously reported that marrow stem cells show changes in engraftment (Habibian, et al J Ex. Hem, 188:393–398, 1998), homing (Cerny et al., J Hematother Stem Cell Res11:913–922, 2002) and differentiation (Colvin et al., J Cell Phys199:20–31, 2004) phenotype as they transit a cytokine-driven cell cycle. mRNA and surface expression of adhesion proteins also change (Becker et al., Exp Hematol27:533–541, 1999). We have evaluated gene expression by Real-time PCR of murine lineage negative, Sca+ (Lin-Sca+) stem cells stimulated by Il-3, Il-6, Il-11 and Steel factor (at 0, 24 and 48h) and lineage negative Rhodamine low, Hoescht low (LRH) stem cells stimulated by TPO, Flt-3 and Steel at various points in cell cycle transit (0,32,40,48h). In Lin-Sca+ cells (4experiments, time 0) expression of the following genes in descending order was as follows: IKAROS, L-selectin, Pu-1, Gata-2, Pecam, Cd84, Rock-1, c-fms, FOG, Cxcr4, c-kit, Cd4. The following were either not expressed or expressed at very low levels: Il-11, Ccr4, Sdf-1, Gata-1, P-selectin and Vecam. A pattern of depressed gene expression in S-phase (24h) with subsequent recovery (48h) was seen with c-fms and c-kit. With LRH cells (2 experiments, time 0) approximate descending rank order of gene expression was Cd45r, Cd34, G-CSFR, Mac-1, GM-CSFR and Flt-3. Il7r was not detected. With cycle progression Cd34 and Sca-1 were markedly elevated while Mac-1 and c-mpl were decreased. The expression of GM-CSFR, G-CSFR, Cd45r and Cd4 showed variable fluctuation. Il-7r was negative throughout. These data show that primitive marrow stem cells express a wide variety of “hematopoietic genes”, that expression modulates with cell cycle transit and perhaps most importantly that observed changes in gene expression are reversible. This is consistent with the continuum theory of stem cell regulation.
Genome-Scale Analysis of Hematopoietic Regulatory Sequences by Digital Analysis of Chromatin Structure.
Authors: Stamatoyannopoulos John A. , Humbert Richard , Hawrylycz Michael , Wallace James , Dorschner Michael O. , Kohli Ajay , Li Qiliang , McArthur Michael , Sabo Peter J. 
Published: October 2019

Abstract
In vivo, cis-regulatory elements coincide with focal disruptions in chromatin structure that manifest as DNaseI hypersensitive sites (HSs). We developed a novel stochastic methodology for comprehensive, genome-wide mapping of DNaseI HSs in vivo. We show that 19–20bp genomic DNA tags can be used to effect genome-wide localization of individual DNaseI cutting events in nuclear chromatin, and that such tags can be generated and sequenced in large numbers from limited numbers of starting cells. We analyzed 257,000 tags from a hematopoietic cell line (K562) and applied a quantitative algorithm to discriminate statistically significant tag clustering events. Such tag clusters identified both known and novel functional elements of hematopoietic genes across the genome. A unique feature of this approach is that it permits unbiased evaluation of the chromatin context of regulatory sequences from disperse genomic loci. We observed surprisingly large differences in the chromatin structural configuration of a variety of active erythroid and hematopoietic regulatory sequences, suggesting a discrete hierarchy of nuclear organization that is not apparent with conventional assays. This approach can be applied readily to generate a comprehensive catalogue of cis-regulatory sequences active in hematopoietic stem cells.
Autologous Peripheral Blood Stem Cell Implantation as Therapeutic Angiogenesis for Severe Limb Ischemia Autologous Peripheral Blood Stem Cell Implantation as Therapeutic Angiogenesis for Severe Limb Ischemia.
Authors: Han Zhong Chao , Huang Pingping , Li Shangzhu 
Published: October 2019

Abstract
It is estimated that 100 million individuals suffer from severe limb ischemia worldwide. Here, we report a novel therapy for severe limb ischemia by transplanting autologous peripheral blood stem cells (PBSCs). Thirty patients with different limb ischemia were enrolled and randomized to either stem cell injection or standard therapy in the study. The patients in the transplant group received intramuscular injections of autologous PBSCs collected after G-CSF-induced stem cell mobilization. Lower limb pain, ulcer repair and wound healing and blood perfusion were improved in the cell treated group. All cell treated patients achieved successful limb salvage. Analysis by digital subtraction angiography demonstrated the formation of new collateral vessels in the ischemic limbs of transplanted patients after cell injection. In contrast, no significant improvement was observed in the control patients. These results provide pilot evidence indicating that transplantation of autologous PBSCs is a simple and effective therapeutic angiogenesis for limb ischemia. To determine the mechanism of action, several animal experiments were performed by transplanting human PBSCs via tail vein into nude mice. It was found that the transplanted cells survived and incorporated into capillary networks in murine ischemic limb. Laser Doppler Perfusion Image (LDPI) and histological analysis revealed that the transplantation of human PBSCs augmented blood flow and neovascularization in the ischemic hindlimb, ameliorated necrosis and improved limb salvage. Our results demonstrate that PBSCs are excellent cell population capable of augmenting neovascularization.
The Effect of Telomerase Antisense for the Differentiation of Embryonic Stem Cells to Hemopoietic Stem Cells.
Authors: Park Kyong Hwa , Kim Byung Soo , Choi In Keun , Kim Seok Jin , Oh Sang Cheul , Seo Jae Hong , Choi Chul Won , Shin Sang Won , Kim Yeul Hong , Kim Jun Suk 
Published: October 2019

Abstract
Background: This study was designed to evaluate the effect of adenovirus-mediated telomerase antisense(TA) expression on cellular proliferation and apoptosis of the ex-vivo culture from human embryonic stem cells to hematopoietic stem cells.
Methods: Cultured embryonic stem cell lines (SNU-3 cell line) to hemopoietic stem cells were classified to control and study groups. Control group was that of which embryonic body were cultured to LTC-IC assay not treated with telomerase antisense oligonucleotides(TA). Study group was that of which embryonic body were cultured to LTC-IC assay treated with TA. We evaluated CD34+ cell number and viable cell percentage and the degree of apoptosis of each samples at 5 days after LTC-IC assay and the results were compared between each groups. These studies were tried for 17 times.
Results: The number of CD34+ cells in study group 0.8–2.4(median: 1.5X103) showed no significant difference with that in control group 0.6–2.9(median: 1.6X103). Viable cell percentage of control and study groups were 76–89(median: 83) % and 78–90(median: 85) %, respectively, without significant difference. And there was no difference in the degree of apoptosis (absorbance, A405nm – A490nm) between control and study groups; 0.092–0.134(median: 0.129) vs 0.087–0.152(median 0.138), with reference to negative control: 0.020, positive control: 0.186. The colony count of CFU-GM in control and study group was 0.1–0.5(medium: 0.3) X103 and 0.2–0.5(medium: 0.3) X103, respectively without no significant difference.
Conclusion: We found that adenovirus-mediated antisense expression of telomerase RNA didn’t interrupt the differentiation of embryonic stem cells to hematopoietic stem cells without significant effect on the degree of apoptosis. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.
Enhanced HDAC (Histone Deacetylase) Enzyme Activities in Blood CD 34 + Cells in Patients with Agnogenic Myeloid Metaplasia.
Authors: Wang Jen-Chin , Hemavathy Kirugaval , Suppiah Kathir , Chang Tsong S. , Novetsky Allan D. 
Published: October 2019

Abstract
We previously reported that Blood TIMP-1, TPO levels are elevated and blood MMP-3, bone marrow SDF-1 levels are decreased in patients with Agnogenic Myeloid Metaplasia (AMM). We postulated that these finding are likely related to the defects in TGF-β 1 pathway especially the TGF-β Type II (TGF β RII) receptor defects, resulting in enhanced TGF-β 1 activity. Decreased TGF β RII mRNA has been reported in blood CD 34 + cells previously. HDAC have been found lately to play an important role in the transcriptional repression and silencing of genes in cancer and HDAC inhibitors have proven to be useful tools in the treatment of cancer by reversre the silenced genes. We therefore measured HDAC activity in Blood CD 34 + cells to further explore the mechanism of the TGF β R II defects in this disease. HDAC activity was measured using the HDAC Colorimetric Activity Assay/Drug discovery Kit (BIOMOL Research Lab. Inc. PA). Nine patients with AMM and other patients with myeloproliferative diseases and normal volunteers used as controls were studied. The HDAC activities were expressed as the amount of the released Deacetylated Products (uM) as shown in Table 1.
This preliminary study suggests that HDAC activity is enhanced in some patients with AMM, and could be responsible for the decreased TGF β RII expression. HDAC inhibitors could therefore serve as valuable tools in the treatment of this disease.
Table 1
AMM MPD Controls Mean ± SE 19 ± 9.2 1.05 ± 0.54 2.10± 0.71
Xeroderma Pigmentosum as a Model for Treatment of Bone Marrow Failure in Fanconi Anemia and Aplastic Anemia.
Authors: Lambert W. Clark , Centurion Santiago A. 
Published: October 2019

Abstract
We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.