bluuebunny/crossref_metadata_2025 huggingface dataset:
The Spectrum of Hypercoagulable States in Minority Patients with Unexplained Thrombosis.
Authors: Ghuman Damanjit K. , Cohen Alice J.
Published: April 2020

The association of genetic risk factors with hypercoagulable states in minority populations has not been well defined. With an estimated prevalence of anywhere between 2-15% in healthy individuals, activated protein C resistance (APCR/Factor V Leiden) is considered to be the most common risk factor for venous thromboembolism( VTE) in the white population. It has also been postulated that this mutation is extremely rare in non-white populations. The prevalence of the prothrombin gene mutation G20210A in the white population is estimated at 0.7–4%, protein C and S deficiencies at 2% each and antithrombin III deficiency at 0.1–0.5% but unknown in Blacks with VTE though case control studies have identified protein C and protein S deficiencies in this population. This study is a retrospective review of all patients with thrombophilia registered at the Hemophilia Treatment Center between 1999–2005. 45/164(27%) of patients with thrombophilia were identified to be from minority groups. Of these minority patients 23/45(51%) had an identifiable primary hypercoagulable state. This group included 7/23(30%) males and 16/23(70%) females. The mean age of the patients was 35 years (range 12–80 years ). 4/23( 17%) were smokers and only 4/23(17%) had a family history of thrombosis with no documented hypercoagulable states in any family members. The majority of the patients were of African American descent 16/23(69%), 5/23(22%) were Hispanic and 2/23(9%) were Asians. 16/23(69%) of the patients had documented deep venous thrombosis/pulmonary embolus, 1/23(4%) had arterial thrombosis, 3/23(13%) had fetal loss, and 2/23(9%) were asymptomatic. APCR was the most common diagnosis in 8/23(35%) of the patients, followed by antiphospholipid antibody syndrome in 7/23(30%) of the patients. Protein S deficiency was diagnosed in 5/23(22%), hyperhomocysteinemia in 4/23(17%), Protein C deficiency in 1/23(4%), antithrombin III in 1/23(4%), and prothrombin gene mutation in 1/23(4%) of the patients. 4/23(17%) of the patients were found to have two coexisting hypercoagulable diagnoses. Recurrent VTE occurred in 7/23(30%) of the patients.

Conclusion: Primary hypercoagulable states are not rare in minorities. In this study, APCR was found to be the most common identified abnormality, followed by antiphospholipid antibody and protein S deficiency. Similar to the white population, thrombophilia in minorities occurred more commonly in young female patients. Work up for primary hypercoagulable states should be considered in minority patients with unexplained thrombosis. Further studies are warranted to determine the true prevalence of hypercoagulable states in minority populations.
Economy Class Syndrome: A “Pilot” Study.
Authors: Kristinsson Sigurdur Y. , Vidarsson Brynjar , Love Thorvardur J. , Rafnsson Vilhjalmur , Hrafnkelsson Jon , Onundarson Pall T.
Published: April 2020

Introduction: Air travel associated venous thromboembolism (VTE) has been termed the economy class syndrome (ECS). Risk factors for ECS are divided into i) patient related; age, previous VTE, thrombophilia, malignancy and ii) cabin related; hypoxia, low humidity, dehydration and prolonged sitting. No study to date has looked at the incidence of VTE in healthy frequent flyers. The aim of this study was to see if professional pilots are at increased risk of VTE.

Materials and methods: A retrospective cohort of 206 pilots working for a single airline operating on medium duration (3–7 hour) international routes from 1991–2000 was studied. Landspitali University Hospital is the only hospital serving Reykjavik, Iceland and its neighboring communities, and it is the sole primary hospital for approximately 130.000 people age 18 and over. Databases of clinical and radiographic diagnoses made at Landspitali University Hospital were searched for codes consistent with deep vein thrombosis (DVT) and pulmonary embolism (PE) occurring from 1991 to 2000. A computerized database of pilot personal identification numbers was then record linked to this VTE cohort from Landspitali University Hospital to see if any of the pilots had been diagnosed with VTE. For comparison purposes, a subgroup of the hospital database cohort and the underlying Reykjavik population was defined to match the gender and age characteristics of the pilot group. Expected number of cases with VTE among pilots was then calculated by extrapolating the observed prevalence of VTE among this subgroup to the pilot cohort. Finally, the relative risk of VTE for the pilots was calculated with p-value and 95% confidence interval.

Results: Two hundred and six male pilots (median age 37 years, range 26–69) were included in the study. A total of 2263 patients were diagnosed with VTE at the Landspitali during the 10 year period, 931 with DVT and 1509 with PE, of whom 177 had both DVT and PE. There were 46,016 males aged 26 to 69 years living in the service area. Of these, 447 developed VTE. Extrapolating from the observed proportion of males age 26–69 diagnosed with VTE in the greater Reykjavik area, the expected number of VTE in the pilot cohort was found to be two. Of the 206 pilots, none was diagnosed with DVT or PE at Landspitali University Hospital during the study period. The relative risk of VTE in the pilot group was found to be 0, with no applicable lower limit to the 95% confidence interval, an upper limit of 1.83, and a p-value of 0.14.

Conclusion: Professional airline pilots flying medium range distances do not have an increased risk of VTE and therefore they seem not to develop the ECS. This study does not point to an increased risk of VTE in healthy travelers in association with medium range flights.
Factor VIII Levels Measured at the Time of Diagnosis of Acute Idiopathic Venous Thromboembolism Are Higher Then When Measured Six-Months Post Diagnosis.
Authors: Kovacs Michael J. , Keeney Michael , MacKinnon Karen
Published: April 2020

Background: Thrombophilia screens are performed frequently in persons with a history of acute venous thromboembolism especially in those for whom the etiology is unprovoked or idiopathic. The optimal timing of the thrombophilia screen is controversial. Elevation of Factor VIII levels are a more recently described thrombophilia that is felt to be hereditary, however, the exact mode of inheritance is not certain. Factor VIII is also known to be elevated as an acute phase reactant. The accuracy of assessing Factor VIII levels at the time of diagnosis of acute venous thromboembolism is not known. The purpose of this study was to determine if there is a difference in Factor VIII levels measured at the time of diagnosis of acute venous thromboembolism as compared to six months later while patients are on oral anticoagulation.

Methods: Consecutive patients with a first episode of idiopathic acute venous thromboembolism were eligible. Patients were excluded if they were <18 years of age or had already been started on oral anticoagulants. Plasma was collected within 48 hours of diagnosis in.105 mmol sodium citrate, double spun at 1,500 G and frozen at −70 Celsius for batch testing. Factor VIII levels were assessed with a three point assay on an ACL 9000 (Beckman Coulter, Mississauga). A seven-point reference curve was used for all factor assays. Linear regression showed r2 values were always > 0.99 on calibration lines. Controls at two levels, 1.00 U/mL normal pooled plasma and 0.32 U/mL were run with all assays. All patients were treated with dalteparin at 200u/kg sc daily for 5–7 days and simultaneously initiated on warfarin for six months. At the six-month point repeat Factor VIII assessments were performed while the patients were still receiving oral anticoagulation with warfarin.

Results: There were 61 patients (37 male) and the mean age was 50.4 years (18–85 years). Thirty patients had deep vein thrombosis, 23 pulmonary embolism and 8 patients had both diagnoses. The patients’ Factor VIII levels at baseline and six months were compared. At baseline the mean Factor VIII level was 1.77 units/ml and at six months it was 1.59 units/ml. The 95% confidence interval for difference in means was 0.04 – 0.32. These results were statistically significant, (paired t-test p=.01).

Conclusion: This study confirms that caution should be used in interpreting Factor VIII levels drawn as part of a thrombophilia screen at the time of diagnosis of acute idiopathic venous thromboembolism. Factor VIII levels will be lower six-months later when patients are stable on oral anticoagulation.
Antiplatelet Treatment with Aggressive Lipid Lowering Therapy and Reduction of Clinical Event after Ischemic Stroke.
Authors: Corinaldesi Giorgio , Corinaldesi Christian
Published: April 2020

The objective of the present study is to investigate the effects of anticoagulation and inhibition of platelet aggregation, which is strongly related with ischemic stroke, with aspirin 100 mg/once daily and clopidogrel 75 mg/once daily together with atorvastatin 80 mg/once daily then evaluating the event free survival in patients who had stroke with LDL-C > 130 mg/dl. An hypolipidaemic diet was given to every patient. As known statins other than reducing LDL-C (silent killer), has many other effects characterized by a wider and wider checklist: reduction of plasmatic levels of C-reactive protein, of MCP-1, and the monocyte traffic, of IL-6, TNF-alfa, of S-VCAM-1, of COX-2 and MMP-9, and upregulate the TPA, and downregulate the PAI-1, statins also reduce the expression of CD40 on the endothelial surface, thus augmenting the EPC (endothelial progenitor cells) involved in neo-vascularization of ischemic tissue, with a final protective effects over the vascular endothelium, including the plaque stabilization.

We have studied 67 patients with a previous ischemic stroke aged between 58 and 72 years (25% smoker, 32% with hypertension, 14% with diabetes, 6% with obesity), with LDL-C higher than 130 mg/dl, evaluating in particular the lipidic profile (total cholesterol, HDL, LDL, TG, Lpa), fibrinogen and d-dimer for a total period of 36 months. We observed a clear reduction of lipidic parameters: Total-C <42%, LDL-C <46%, TG <30%, ApoB <35%, d-dimer <10%, and we did not observe any increase in transaminases, CPK, or fibrinogen.

We observed 12 patients with relapsing clinical events requiring emergency hospitalization: 1 exitus for second stroke two months after the first event, 11 cases of RIND, with less than 4 transitory ischemic episodes, with the total length of events significantly reduced after the first 18 weeks of treatment. We have observed 22 adverse effects events: 4 cases of epistaxis, 2 case of tooth bleeding, 3 of abdominal pain, 9 of muscular pain, 2 of chest pain, 2 case of headache, however, these events did not influence the course of the pharmacological treatment; 55 patients did not refer any clinical problem, every patient has been followed on the basis of a three months period, and on demand.

The present study has demonstrated a secure efficacy of the aggressive combined treatment (antiplatelet + statin) in the management of patients with a previous stroke, showing a symptomatologic benefit (reduction of the total ischemic events and of the emergencies) with an overall improvement of the quality of life.

PARAMETER BEFORE AFTER TOTAL-C 320.0 mg/dl 180.0 mg/dl LDL-C 148.0 mg/dl 82.4 mg/dl HDL-C 38.6 mg/dl 44.2 mg/dl TG 180.0 mg/dl 108.2 mg/dl FIBRINOGEN 460.0 mg/dl 370.0 mg/dl FASTING GLUCOSE 110.0 mg/dl 96.0 mg/dl
Combined Use of Warfarin and Aspirin Decreases Mortality in Patients with Peripheral Arterial Disease.
Authors: Pasquale Donald , Jain Abhishek , Chikkappa G.
Published: April 2020

Anti-platelet therapy with aspirin (ASA) is standard therapy for patients with peripheral arterial disease (PAD). While ASA is not felt to impact the natural history of PAD, it decreases mortality from stroke, myocardial infarction, and other co-morbidities in the patients with PAD. Anticoagulation with warfarin (WAR) is indicated in arterial and venous thrombo-embolic conditions such as deep vein thrombosis and atrial fibrillation, but is generally considered not beneficial in patients with PAD. We retrospectively identified patients via ICD9 code who were diagnosed with PAD between January and December 2000, and assessed their course through July 2005. Of 530 individuals, 45 had a history of taking ASA and WAR concomitantly, 116 of taking WAR alone, 219 of taking ASA alone, and 150 received neither drug. Age, mortality, prevalence of hemorrhage and of co-morbid conditions (smoking, diabetes mellitus, atrial fibrillation, stroke, myocardial infarction, hypertension, coronary artery disease, hyperlipidemia, congestive heart failure, renal failure, and chronic obstructive pulmonary disease) that could affect mortality or hemorrhage were determined. Results are illustrated in the table.

Group WAR+ASA WAR ASA Neither n=45 n=116 n=219 n=150 ICH= Intracranial ST= Soft Tissue * Mean ± SD Mortality n (%) 12 (26.7) 51 (44.0) 91 (41.6) 71 (47.3) Hemorrhage N (%) 6 (13.3) 10 (8.6) 6 (2.7) 17 (11.3) Type of Hemorrhage 3-GI 8-GI 6-GI 13-GI 2-ICH 1-Nose 3-ICH 1-ST 1-ST 1-Hematuria Age* 73 ± 9 73 ± 9 73 ± 9 72 ± 11 Co-Morbid Conditions/Patient* 5.4 ± 1.7 4.1 ± 1.6 3.5 ± 1.6 2.9 ± 1.7 INR* 2.2 ± 0.7 2.2 ± 0.5

The higher number (not statistically significant) of co-morbid conditions in patients receiving WAR was because most of these individuals had atrial fibrillation. We observed a significant decrease in mortality in patients receiving WAR+ASA compared to patients on WAR alone (Chi-square, p=0.044) and neither drugs (p=0.014), and a trend for decreased mortality compared with patients on ASA alone (p=0.062). However, we observed an increased prevalence of hemorrhage in patients receiving WAR with or without ASA compared with individuals receiving ASA alone (WAR vs ASA p=0.016; WAR+ASA vs ASA p=0.002). We suspect that the frequent hemorrhagic events observed in individuals on neither drug may in part explain why these individuals were not on ASA and/or WAR. This retrospective data suggests combination therapy with WAR+ASA may be better than either agent alone or no therapy for reducing mortality in patients with PAD. Further, this data suggests that the pathogenesis of PAD may involve both platelet and non-platelet mediated hypercoagulable states.
Discordance between the 1st and 2nd Low Molecular Weight Heparin Standards. Implications for Dosing.
Authors: Jeske Walter P. , Cunanan Josephine , Hoppensteadt Debra , Walenga Jeanine M. , Iqbal Omer , Florian-Kujawski Michelle , Fareed Jawed
Published: April 2020

Soon after the introduction of low molecular weight heparins (LMWHs) as antithrombotic agents, a LMWH standard to cross-reference the potency of commercially available LMWHs was developed. For the past 15 years, this standard has been used by manufacturers to cross-reference their branded LMWHs. The 1st LMWH standard (85/600) was produced by nitrous acid digestion of porcine mucosal heparin. This standard had characteristics that were comparable to dalteparin. Because of the widespread use of the first standard, the supplies are now depleted necessitating the introduction of new standard material. The WHO has introduced a 2nd LMWH standard which is also produced by nitrous acid digestion and which mimics the commercially available LMWH reviparin. The molecular weight profile and the biologic activities, including SERPIN affinity, of these two standards are very distinct. When used to cross-reference commercially available LMWHs such as enoxaparin, the new standard consistently overestimates the anti-Xa potency by 5–10% in comparison to the first standard. Such an overestimation may lead to a reduction in dosage and have a significant impact on the clinical dosing of LMWH in various indications. As many generic versions of LMWHs are currently being introduced, a systematic study was carried out to cross-reference three commercially available generic products from India and South America against the first and second LMWH standards using amidolytic anti-Xa and anti-IIa assays. Differences were observed in the slopes of the concentration-response curves for these standards in both the anti-Xa and anti-IIa assays. The potencies of the generic LMWHs ranged from 141 to 152 U/mg in relation to the first standard and from 132 to 143 U/mg in relation to the second standard. On average, the anti-Xa potency was 6.5% lower when calculated with the 2nd standard. The differences in anti-IIa potency were further amplified and exhibited wider variation. On average, the anti-IIa potency was 18.9% lower when calculated with the 2nd standard. These studies clearly demonstrate that the limitations in the use of the first and second LMWH standards for cross-referencing commercially available LMWHs and their potential generic versions. If the 2nd standard is used, it should first be cross-referenced against the first standard in order to properly assign the anti-Xa and anti-IIa activities. This study also underscores the need for developing individual standards for each of the branded products. Such standards should be defined in terms of not only anti-Xa and anti-IIa activity, but also in terms of anticoagulant activity in global clotting assays such as the USP assay, activated clotting time (ACT), aPTT and Heptest. Such designations will be particularly useful in evaluating the potential equivalence of generic versions of branded LMWHs.
Use of Venipuncture Blood Sample for INR Testing on a Point of Care Machine.
Authors: Dhami Mandeep S. , Bulgaru Anca , Jagathambal K. , Kapur Dinesh , Norris Francine , Slater Dennis E. , Cavanaugh Jeri
Published: April 2020

Point of care (POC) testing of International Normalized Ratio (INR) for monitoring warfarin therapy is rapidly becoming procedure of choice for patients requiring long term oral anticoagulation. This method allows for fingerstick blood sample to be used for INR testing at the point of service with immediate dose modification as needed. Most patients prefer a fingerstick method for blood draw to venipuncture. It is not known if venipuncture blood sample can be used for testing on POC machines. A significant number of patients in an oncology practice need additional laboratory testing on the same day as INR testing. These patients therefore get a fingerstick for POC INR testing and a venipuncture for other tests. We compared results of INR from venipuncture sample run on a POC machine (CoaguChek monitor) using CoaguChek test strips (ISI-2.0) with the INR performed on MDA analyzer (ISI-2.0). Blood samples from 24 patients on warfarin therapy were drawn from antecubital vein by a clean stick in a 10 cc plastic syringe. A drop of blood was immediately placed on the CoaguCheck test strip. Next the same blood sample was used to fill a 3.2% sodium citrate tube for testing on MDA analyzer. Results were analyzed using regression analysis; the correlation coefficient (r), slope and intercept were determined and following graph of the regression analysis was generated (see figure 1).

Conclusions: These results show a good correlation (r-value> 0.9) between the results of INR obtained on CoaguChek POC machine and the MDA analyzer using venipuncture blood sample. The preferred method to obtain blood sample for POC INR monitoring must remain fingerstick sample as per manufacturer’s recommendations. However, for those patients who need more than one blood sample done on the same day, a venipuncture sample as described above gives acceptable results when tested on CoaguCheck machine.

Figure Figure
Binding Properties of a Human Anti-Ib ScFv Antibody (HIb-1) to Native, Recombinant, and Transgenicially Expressed Human Platelet GP Ibα Antigen.
Authors: Kim Antony Y. , Bell George , Lovelock Joshua , Favre Elizabeth G. , Biswas Tapan K. , Yousef Mohammed , Miller Jonathan L.
Published: April 2020

HIb-1 is a single chain antibody (ScFv) originally obtained from the Griffin.1 library. ScFv from this library are derived from the in vitro recombination of germline human immunoglobulin VH and VL chain segments whose diversity has been greatly increased by mutagenesis directed at the CDR3 regions. HIb-1 was selected from the library by sequential biopanning first against CHO cells surface-expressing human GPIbα, and then against human platelets. Previous studies using Western blot analysis have shown that HIb-1 binds to an epitope within the 45 kDa amino-terminal portion of GPIbα (Lapan, Lyle, and Miller, 1999, Thromb. Haemost. 82S:393). HIb-1 displays inhibitory activity against both ristocetin-induced and shear-induced platelet aggregation. Biacore surface plasmon resonance analysis was used to establish dissociation constants between HIb-1and either the extracellular domain of GPIbα (i.e., “glycocalicin” or GC) derived from normal human platelets or the CHO-cell secreted GPIbα1–483 recombinant extracellular protein. With native GC immobilized on dextran sulfate, the KD for binding by analyte HIb-1 was ~600nM. With the wild-type recombinant GPIbα1–483 bound to dextran sulfate, the KD was only slightly higher at ~900 nM. We additionally studied a double mutant increase-of-function GPIbα1-483 containing the Gly233→Val233 and Met239→Val239 substitituions. The KD of HIb-1 binding to the double mutant was virtually identical to that of the wild-type, at ~900 nM. Binding of HIb-1 to native GC immobilized in an ELISA assay format was also performed. HIb-1 showed saturable binding, with a half-maximal binding concentration of approximately 170 nM. Flow cytometric analysis was also performed on platelets from murine GPIbα-null (i.e., “Bernard-Soulier”) mice that had been rescued with the human GPIbα transgene (Ware, Russell, and Ruggeri, 2000, PNAS, 97:2803), as well as upon normal mice. Murine platelets were gated using a rat anti-murine GPIIb/IIIa antibody. HIb-1 did not bind to normal murine platelets, but showed strong binding to platelets from the transgenic animals, comparable in intensity to that seen with the GPIbα mab SZ-2 (for which quantitative bead analysis indicated approximately 8,000 GPIbα receptors per platelet). It should be noted that the binding capabilities of HIb-1 to human GPIbα are those of a monovalent ScFv molecule of approximately 30 kDa, and may be sufficient for studies of in vivo GPIbα inhibition either in the case of acute injury model or of longer-term models involving repeated dosing. Model systems of more chronic inhibition, however, may require conversion of the ScFv into IgG molecules, in order to achieve greater avidity.
Transfusion Requirements in Nonmyeloablative Allogeneic Stem Cell Transplantation for Hematologic Malignancies.
Authors: Brown Jason W. , Triulzi Darrell J.
Published: April 2020

Nonmyeloablative allogeneic stem cell transplants (NM-ASCT) are increasingly being utilized to treat hematologic malignancies in settings where high dose, fully ablative regimens would not be tolerated. We identified 10 male and 13 female patients (n=23) ranging in age from 19 to 63 years old (mean=48) representing all patients whom had undergone NM-ASCT in the last five years. NM-ASCT were performed for acute myelogenous leukemia (n=7), non-Hodgkin’s lymphoma (n=6), Hodgkin’s disease (n=4), multiple myeloma (n=3), myelodysplastic syndrome (n=2), and acute lymphoblastic leukemia (n=1). The majority of patients had undergone at least one previous stem cell transplant (n=16). The mean number of stem cells infused per kilogram at time of transplant was 4.72 X 106 ±1.97 X 106 stem cells and mean time to engraftment was 15.8 ±5.15 days (3 consecutive days with ANC >500). All of the subjects required both platelet and red cell transfusion during the period of analysis. Mean transfusion requirements in the 7 days prior to transplantation were 3.1 ±5.9 units of platelets and 1.2 ±1.5 units of packed red blood cells. Mean platelet transfusion requirements 90 days post transplant were 73.7 ±79.7 units and mean red cell requirements 11.6 ±8.3 units. Total mean transfusion requirements during the time period analyzed were 76.9 ±82.2 units of platelets and 12.8 ±8.9 units of packed red blood cells. There was no significant correlation between the number of stem cells infused per kilogram on day of transplant, the number of previous transplants, or the time to engraftment with red blood cell or platelet transfusion requirements. Our data illustrate a higher proportion of patients requiring platelet transfusions (100% vs. 23%) and red blood cell transfusions (100% vs. 63%) as well as a higher platelet transfusion requirement (median 48 vs. 0 units) and packed red blood cell transfusion requirement (median 11 vs. 2 units) than other authors have reported in a 60 day period in HLA-matched sibling NM-ASCT [Weissinger et al. Blood 98(13):3584-8, 2001]. As is illustrated by the percentage of patients undergoing previous fully ablative stem cell transplants, our patients represent a heavily pretreated population and this may account for the higher transfusion requirements seen in this analysis. Although patients undergoing NM-ASCT receive reduced intensity therapy, in our analysis their transfusion requirements remain substantial.
PTPN2, Distinctively Expressed in GCB-Like and ABC-Like DLBCL, Is the Nuclear Phosphatase of STAT6.
Authors: Lossos Izidore S. , Lu XiaoQing , Tiganis Tony
Published: April 2020

Gene expression profiling studies sub-classified diffuse large B-cell lymphomas (DLBCL) into two clinically distinct types: Germinal Center B cell (GCB)-like and Activated B-cell (ABC)-like tumors, characterized by long and short survival, respectively. We have recently reported distinct responsiveness of GCB-like and ABC-like DLBCL cell lines to IL-4 stimulation. In GCB-like lymphoma cells, IL-4 leads to nuclear accumulation of phosphorylated signal transducer and activator of transcription 6 (STAT6) and induction of IL-4 target genes, while in ABC-like tumor cells there is an increased nuclear dephosphorylation of pSTAT6 and no induction of IL-4 target genes (Lu et al: Blood2005;105:2924). Furthermore, GCB-like and ABC-like DLBCL tumors exhibit a different expression profile of protein tyrosine phosphatases (PTPs). Among the distinctively expressed PTPs, only 45kDa isoform of protein tyrosine phosphatase non-receptor type 2 (PTPN2, also known as T-cell PTP-TCPTP) localizes to the nucleus. Despite PTPN2–45 having an apparent exclusively nuclear localization in resting cells, specific stimuli can induce PTPN2–45 shuttling to the cytoplasm, thus gaining access to the cytoplasmic substrates, such as the epidermal growth factor (EGF) receptor, the insulin receptor, Src family kinases, adaptor protein p52 Shc and Janus protein tyrosine kinases (JAK 1 and 3), thereby regulating multiple intracellular signaling pathways. In contrast, STAT1, 5A and 5B are the only to date known PTPN2-45 nuclear substrates. Substrate identification is a crucial step in delineating the functional spectrum of PTPN2 in vivo. Herein, we demonstrate that STAT6 may serve as a physiological nuclear substrate of PTPN2-45. Over-expression of PTPN2 leads to nuclear STAT6 dephosphorylation. By using a nuclear restricted version of PTPN2 (pEGFP-PTPN2), we show that the dephosphorylation of STAT6 is independent of PTPN2 induced JAK dephosphorylation. We demonstrate interactions between endogenous PTPN2 and STAT6 and delineate the STAT6 and PTPN2 protein domains responsible for the interaction: the catalytic domain of PTPN2 and the C terminal transactivation domain of STAT6. Furthermore, we demonstrate that over-expression of PTPN2 ameliorates IL-4 induced expression of its target genes in GCB-like DLBCL tumor cells. We observe tight control of PTPN2 at different differentiation levels of B cell lymphocytes thus suggesting that distinct expression of PTPN2 in GCB-like and ABC-like DLBCL tumors stems from different cellular origin of these lymphomas. Our findings identify STAT6 as a new nuclear target of PTPN2 and suggest that distinct PTPs expression profiles may contribute to different biological characteristics of GCB-like and ABC-like DLBCL.
Survey of Red Blood Cell Transfusion Practices in Medically Ill Patients.
Authors: Yuen Maggie M. , Anderson David R. , Patrick Ward , Rose Caren
Published: April 2020

Although red cell transfusion is a relatively common treatment used in medically ill patients, much is not known about clinical determinants used to guide its use. A cross-sectional self-administered survey was used to assess the red blood cell transfusion practices of academic medical internists, subspecialty physicians and internal medicine residents who practice on the Medical Teaching Unit (MTU) at the Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia. We evaluated transfusion thresholds before transfusion and the number of red cell units ordered for four clinical scenarios: chronic obstructive pulmonary disease (COPD), coronary artery disease (CAD), gastrointestinal bleeding and delirium. Clinical characteristics were varied to further clarify clinical determinants of transfusion. The response rate among the forty-nine medical internists and subspecialty physicians was 59%. Among internal medicine residents the response rate was 100% (n=14). The primary area of practice for the majority of respondents was general internal medicine. Most staff physicians attended on the MTU for greater than four weeks. Baseline hemoglobin transfusion thresholds averaged from 75.6 +/− 9.8 g/L in a patient with acute delirium to 91.4 +/− 11.4 g/L in the gastrointestinal bleeding scenario. Range of baseline hemoglobin transfusion thresholds within scenarios was as wide as 60 to 120 g/L. Between the four scenarios, baseline hemoglobin transfusion thresholds differed significantly (p<0.03) except between the CAD and gastrointestinal bleeding scenarios. Clinical factors age and gender did not significantly (p>0.05) alter hemoglobin transfusion thresholds in the CAD and delirium scenarios, but were significant (p<0.02) modifiers of hemoglobin transfusion thresholds along with oxygen saturation, lactic acidosis, hemodynamic stability, requirement for urgent surgery, and chronic surgery in all other scenarios. Comparing staff physicians and internal medicine residents, there was no significant difference between the two groups for baseline hemoglobin transfusion thresholds. In conclusion, among physicians caring for medically ill patients, there is significant variation in red blood cell transfusion practices. Consistent with research in the critical care setting, pre-transfusion hemoglobin, along with other clinical factors, continues to be an important determinant of red cell transfusion.
The Common Practice of Transfusing 1–2 Units of Fresh Frozen Plasma (FFP) to Patients with Mildly Prolonged Prothrombin Times (PT) Does Not Lead to Clinically Significant Changes in Their PT Values.
Authors: Darabi Kamran , Uhl Lynne
Published: April 2020

INTRODUCTION: Various guidelines define appropriateness of FFP administration based on prothrombin time (PT) greater than 1.5 times normal. However, FFP is often transfused to patients with PTs that are in the upper range of normal or mildly prolonged. There is a paucity of data on whether transfusion of FFP to such patients will change their PT.

METHODS: We retrospectively reviewed all FFP transfusions that were administered to patients with a PT below 17 seconds (normal reference range 11.3–13.6 seconds) at a tertiary care hospital in 2004. Patients were included in our analysis if their PT was measured prior to and within six hours of transfusion and if they received no more than two units of FFP. We identified 143 consecutive cases that met these criteria.

RESULTS: 66 patients received one unit and 77 patients received 2 units of FFP. The median decrease in PT after the infusion of one or two units of FFP was 0.4 seconds and 0.6 seconds respectively. The pretransfusion PTs in 6 patients ranged between 12.2–13 seconds (subgroup 1), in 17 patients 13–14 seconds (subgroup 2), in 46 patients 14–15 seconds (subgroup 3), in 37 patients 15–16 seconds (subgroup 4), and in 37 patients 16–17 seconds (subgroup 5). Patients in subgroups 1 and 2 were noted to have higher PT values after the administration of FFP (median prolongation of 0.6 and 0.1 seconds respectively). The PT shortened in subgroup 3 by 0.4 seconds (median) and in subgroups 4 and 5 by 0.9 seconds (median) when compared to the pretransfusion PT, which represent clinically insignificant corrections.

CONCLUSION: Transfusion of blood products is not without risks; given that PT values of less than 17 seconds do not significantly change following transfusion of one to two units of FFP, this clinical practice should be avoided.
Rational Development of Serologic Assays for Anti-HTLV Antibodies.
Authors: Poiesz Michael J. , Choi David , Hengst James , Ehrlich Garth , Dube Syamalima , Poiesz Bernard J.
Published: April 2020

Both HIV and HTLV virions are comprised mainly of gag proteins (p24 and p19 for HTLV) (Table 1); while both virions lack their primary RNA transcriptional regulatory proteins (Tax and Tat for HTLV and HIV, respectively), HIV virions contain considerably more surface and transmembrane envelope proteins than do HTLV virions. When subjected to high speed centrifugation almost all detectable viral RNA in HTLV or HIV conditioned, media is contained within a dense, precipatable particle (Table 2). HIV particle associated RNA can be gently immunoprecipitated with beads bound with either anti-gp120 env or anti-gp41 env antibodies indicating the presence of both proteins on the viral envelope. Only minimal immunoprecipitation of HTLV-I RNA was observed with beads bound with either anti-gp46 env or gp21 env antibodies indicating a relative paucity of these two proteins on the viral envelope. 1237 (0.08%) of the VBD samples were positive for HTLV antibodies when analyzed by EIA.

Of these, 105 (0.007%) were confirmed as true positives by WB and PCR (50 HTLV; 55 HTLV-II), while the remainder were deemed false positive. When examined by WB and RIPA, 170 (15%) of the false positives gave completely negative signals. 712 (63%) scored positive against p19gag only; 203 (7.9%) against p24 only; 18 (1.6%) both p19 gag and p24 gag; 22 (1.9%) against gp21 env only; 3 (0.3%) both p19 gag and gp21 env. Of the people at risk for HTLV infection, 350 (14%) were positive for HTLV antibodies when analyzed by EIA. Of these, 348 were confirmed as true positives (150 HTLV-I; 198 HTLV-II). Of these, 345 (99.1%) were DNA PCR positive and all were WB positive. However, an additional 11 HTLV-I and 73 HTLV-II EIA negative, PCR positive persons were identified. In the EIA negative PCR positive individuals, approximately one half made antibodies to either HTLV Tax, gp46env and/or gp21 env proteins. Epitope mapping of seroreactivities to HTLV linear and recombinant peptides using 20 each of select EIA true seronegative, true seropositive, falso seronegative, and false seropositive samples indicated that an optimal HTLV antigen prep would omit HTLV p19 gag, and include whole p24 gag and Tax, and recombinant gp46 and gp21 env peptides encoded by nucleic acids 5290–5829 and 6169–6390, respectively.
BMP4/Madh5 Dependent Signaling Is Required for the Expansion of a Population of Stress Erythroid Progenitors in the Fetal Liver.
Authors: Paulson Robert F. , Porayette Prashanth
Published: April 2020

Fetal liver hematopoiesis is primarily erythropoiesis, which robustly produces erythrocytes to meet the growing need of the developing embryo. In many ways fetal liver erythropoiesis resembles stress erythropoiesis in the adult, where in response to acute anemia, a unique population of stress erythroid progenitors is rapidly expanded in the spleen. The development of these stress progenitors requires BMP4/Madh5 dependent signals. Spleen stress progenitors exhibit properties that are distinct from bone marrow steady state progenitors in that they are able to rapidly form large BFU-E colonies, which require only Epo stimulation for their generation. Mice mutant at the flexed-tail locus exhibit a defective stress erythroid response because of a mutation in Madh5. In addition to this defect, flexed-tail mice also exhibit a severe fetal-neonatal anemia. We have analyzed fetal liver erythropoiesis in flexed-tail and control embryos. We show that BMP4 is expressed in the fetal liver and its expression correlates with the time of maximum erythropoiesis. In flexed-tail mutant embryos the expression is delayed and this correlates with both a delay and a defect in the expansion of erythroid progenitors. Our analysis also shows that the fetal liver contains two types of erythroid progenitors. One type exhibits the properties of stress BFU-E found in the adult spleen, which are compromised in flexed-tail embryos and a second type that is similar to bone marrow steady state BFU-E. These data demonstrate that BMP4 dependent signaling drives the expansion of erythroid progenitors in the fetal liver in a manner similar to stress erythropoiesis in the adult spleen.
Periodicity of Differentiation and Self-Renewal Suggest Homeostatic Feedback Control of Commitment in a Hematopoietic Progenitor Cell Line.
Authors: Cross Michael , McIvor Zoe , Labani Marion , Alt Ruediger , Niederwieser Dietger
Published: April 2020

FDCPmix cells are karyotypically normal, non-leukemic, murine multipotent progenitors which undergo extensive self-renewal in the presence of a high concentration of interleukin-3 (IL-3), and progressive commitment and differentiation along all myelo-erythroid lineages upon substitution with the appropriate cytokines. Although clonal, the FDCPmix cells are characterised by a variable degree of heterogeneity in the population. This heterogeneity is apparent firstly from the variable background of morphologically differentiated cells which accumulate in self-renewal (high IL-3) conditions, and secondly from the low and variable incidence of colony-forming cells (CFU) which are capable of extensive self-renewal in semi-solid media and which are generally assumed to be the founder cells of the population. In order to define the variability in more detail, early passage FDCPmix cells were thawed into standard culture conditions and passaged back to 6x104 cells per ml every 2 days over a period of nine months. The degree of background differentiation and the incidence of colony forming cells were determined at weekly intervals. Based on our unpublished observations that glucose concentration can influence progenitor cell fate, parallel cultures were maintained in IMDM (4g/L glucose) and DMEM (1g/L glucose) and all CFU assays were performed using both IMDM and DMEM based semi-solid media. In both IMDM and DMEM cultures, the level of background differentiation increased from 0 to an initial peak of 15% (IMDM) to 30%(DMEM) over the first 5 weeks, followed thereafter by further peaks at 3–4 week intervals. There was no corresponding pattern in the CFUIMDM frequency. However, a similar 3–4 week periodicity (displaced from that of the background differentiation) was evident in both the frequency of CFUDMEM and particularly in the ratio of CFUDMEM to CFUIMDM. The high frequency of CFUDMEM and the low frequency of differentiated cells over the first 5 weeks of culture corresponded to the period in which the FDCPmix cells underwent a homogenous and rapid differentiation in response to erythroid or myeloid growth factors. These patterns of periodicity in both IMDM and DMEM cultures are suggestive of a delayed feedback phenomenon in which the accumulation of differentiating cells causes a decrease in the ratio of commitment-to-self-renewal of the founder cells, and subsequent decrease in differentiating cell numbers, with the delay between commitment and differentiation being responsible for overshoot, and the relatively long periods of phasing observed. Since the cultures were diluted regularly into fresh media, the basis of such a feedback is likely to involve cell-cell contact rather than diffusible factors. In vivo, a local cell census taken in this way may represent a means of restricting the local concentration of stem cells in a given niche.
Stem Cell Aging Studies Reveal Changes in the Proteasome.
Authors: Miller Alison R. , Liang Ying , Van Zant Gary
Published: April 2020

To investigate aging mechanisms in murine hematopoietic stem cells (HSCs), a microarray analysis was performed on sorted Lin-cKit+ Sca-1+ cells of young and old mice of two strains: long-lived C57B6L/6 (B6) and short-lived DBA/2 (D2). Following analysis by two-way ANOVA with a FDR of 5%, the data was organized using gene ontology software. The following age-related transcriptional changes were found to be of significance: increases in 20S (α and β) and 26S proteasome subunits, ribosomal proteins, mitochondrial enzymatic proteins, and carbohydrate metabolism, and a decrease in ubiquitination proteins. In general, B6 changes were more dramatic than those of D2. These data are surprisingly consistent with well-established theories of aging in post-mitotic cells, most commonly neurons, where the function of the proteasome decreases while the number of proteasome subunits increases in a compensatory manner to maintain protein turnover. Eventually, the proteasome can become over-loaded and the lysosomal pathway must assume the load of additional protein degradation. It is common for protein aggregates, lysosomal dysfunction, and damaged mitochondria to be observed in neurons with inhibited proteasome function. Our data are consistent with the initial steps in this theory, but the later, dysfunctional consequences of proteasome inhibition have not yet been observed in HSCs. It is interesting that this pattern would be present in stem cells, since they are responsible for self-renewal and differentiation throughout the lifetime of the organism. It is possible that the lack of protein aggregates or dysfunctional mitochondria is actually due to dilution by cell division, especially considering that the proteasome is responsible for degradation of many cell cycle proteins and that genes involved in energy production were up regulated. Therefore, it is possible that HSCs show similar aging trends with regard to the proteasome but do not have the same cellular consequences as post-mitotic cells. It is also interesting that B6 and D2 showed the same aging trends but B6 appears better able to compensate for proteasome alterations. For example, B6 increases the beta catalytic subunits of the 20S proteasome core more so than its short-lived counterpart. Additionally, B6 are known to have more HSCs in old age than D2, which is consistent with more cells being able to either compensate for decreased proteasome function or to increase cell cycle to dilute out any deleterious effects of proteasome inhibition. These data suggest that HSCs share common aging trends with other cell types; however, they are able to maintain “healthy aging” more effectively than post-mitotic cells, especially in the case of B6. It is tempting to speculate that the longevity of D2 mice could be limited by its alterations in proteasome function and its decreased ability to compensate other cellular responses in order to maintain homeostasis.
Different Gene Expression Profiles of CD133+ Cells from Cord Blood and Mobilized Peripheral Blood.
Authors: Giorgetti Alessandra , Agnelli Luca , Mattioli Michela , Lombardi Luisa , Neri Antonino , Bicciato Silvio , Giordano Rosaria , Rebulla Paolo , Lazzari Lorenza
Published: April 2020

Cord blood (CB) is a valid alternative to mobilized peripheral blood (mPB) as a source of hematopoietic stem cells for clinical transplantation. The qualitative differences among CD133+ cells from these sources have been demonstrated by in vitro and in vivo studies but the molecular phenotype of these cells is only partially understood. In view of better exploiting the peculiarities and advantages of both stem cell sources, we performed a comparative analysis of gene expression of CD133+ cells from CB and mPB using Affymetrix HG-U133A arrays. The CD133+ cells were selected using the MiniMacs immunomagnetic separation system (Miltenyi Biotec) from 6 CB units and 6 mPB samples. Since microarray results are strictly dependent on the purity of the cell population and on the RNA quality, rigorous quality controls were applied. The purity of CD133+ cells was more than 93% in all samples. In addition, after extraction, the RNA was purified by the RNeasy system (Qiagen) and the RNA quality was evaluated using the RNA 6000 Nano LabChip kit (Agilent Technologies). Given the low amount of RNA due to the limited numbers of CB CD133+ cells, we used a double-cycle amplification protocol for both sources to obtain biotin-labeled target for hybridization starting from as low as 100 ng of total RNA. Probe level data have been converted to expression values using Affymetrix MAS 5.0 algorithm. Differentially expressed genes have been identified using Significance Analysis of Microarrays (SAM). The cut-off for significance has been determined tuning the Delta parameter on the false positive rate and controlling the q-value for the gene list. We found that mPB CD133+ cells expressed cell cycle driving, transcription factor and DNA synthesis genes at distinctly higher levels than CB CD133+ cells, indicating a higher cycling activity in mPB CD133+ cells. Furthermore apoptosis driving genes were up-regulated in mPB CD133+ cells, probably due to G-CSF mobilization. Conversely in CB CD133+ cells we found an overexpression of genes involved in immunoresponse and immunomodulation. This finding could justify the peculiar immunological properties of CB. Finally, many genes related to different metabolic pathways such as alcohol, sterol, nucleotide, lipid and sugar biosynthesis, were found up-regulated in CB CD133+ cells, maybe due to the neonatal origin of these cells. In conclusion, mPB CD133+ cells include a more committed and cycling population while CB CD133+ represent a cell subset with higher potentialities probably due to their immaturity. This suggest that even if CB has a lower number of stem cells, CB CD133+ cells are more susceptible to different stimuli and potential fates.