bluuebunny/crossref_metadata_2025 huggingface dataset:
Alternative Donor Stem Cell Transplantation In Aplastic Anemias and Refractory Cytopenias Is Save and Feasible With T- and B-Cell Depleted Haploidentical Grafts
Authors: Feuchtinger Tobias , Teltschik Heiko-Manuel , Pfeiffer Matthias , Mueller Ingo , Schrauder Andre , Handgretinger Rupert , Lang Peter
Published: October 2019

Allogeneic stem cell transplantation is still the curative treatment option for the majority of patients with severe aplastic anemia and refractory cytopenias. However, a HLA-matched donor is not available for all patients. Alternative donor transplantation has been an experimental treatment option, limited by high rejection rates and transplant related mortality. We performed a prospective clinical trial to evaluate the safety and feasibility of haploidentical stem cell transplantation, since haploidentical family members are always available donors. We investigated a cohort of 10 pediatric patients with severe aplastic anemia or myelodysplatic syndrome transplanted (refractory cytopenia) with T-cell depleted grafts between 2004 and 2011. 7 patients had myelodyslastic syndrome with refractory cytopenia (MDS-RC), 3 had severe aplastic anemias (SAA) refractory to immunosuppresive treatment. 3 patients received a 2nd SCT after rejecting the graft from matched donors. Median age was 11.4 years. Standard conditioning regimen consisted of Fludarabin 3-4x40mg/m2, Thiotepa 1-3x5mg/kg, Melphalan 2x70mg/m2 (n=8) and serotherapy using OKT3 (n=5) and ATG (n=5). 8 patients received additional total lymphoid irradiation (TLI 7 Gy) to prevent graft rejection. In vitro graft manipulation was carried out by direct depletion using antiCD3/19 magnetic microbeads. A median number of 10.1x106 CD34+ progenitor cells and 27x103 T-cells/kg body weight (BW) were transfused. Pharmacological GvHD prophylaxis (graft vs. host disease) was carried out with Mycofenolate until day 60, if residual T-cells in the graft exceeded 25000/kg BW. Primary engraftment occurred in all patients Median time to reach 500/µl neutrophiles was 9 days (9-11). Independence from platelet substitution was reached after 13 days (8-16). Three patients rejected the graft later on. 6/10 patients had no signs of acute GvHD or GvHD grade I, 2 patients had GvHD grade II. TRM at day +100 and after 1 year was 0% and 20%, respectively. Event free survival (EFS) at 3 years was 80%.

Conclusions

Haploidentical SCT with T-cell depleted grafts is a therapeutic option for refractory cytopenias and severe aplastic anemia after nonresponse to immunosuppressive treatment if no HLA-matched donor is available. Recovery of neutrophiles and platelets were fast and TRM was low, even if retransplantation was necessary. Since spontaneous outcome of these conditions are poor, alternative donor SCT is a realistic option for these patients.

Disclosures:

No relevant conflicts of interest to declare.
Differential Gene Expression Analysis Predicts For Response To Bortezomib In The Treatment Of Steroid Refractory Chronic Graft-Versus-Host Disease (cGVHD)
Authors: Miller Alan M , Schneider Reva , Vance Estil A. , Agura Edward , Powell Sheila , Pascual Virginia , Munagala Indira , Qin Huanying
Published: October 2019

We conducted a trial of bortezomib in patients with steroid-refractory cGVHD. Patients received 1.6 mg/m2 q wk x 4 followed by one week of rest, for up to six cycles, with some going on to a maintenance phase of treatment every other week for an additional 6 months. Twenty-one patients were registered on trial two patients withdrew before beginning treatment. Nineteen patients received between two and twelve months of treatment. The treatment was well tolerated with no grade 3 or higher adverse events (AE). Six patients had either CR or near CR. Average Rodnan scores on the 8 patients with sclerosis completing 2 or more cycles of therapy decreased from 22.6 ± 12.8 to 5.9 ± 6.2, p<0.005. One patient with non-healing suppurating lesions of his lower extremities had marked healing, another had significant decrease in chronic diarrhea. Weekly bortezomib for cGVHD was well tolerated and resulted in early improvement in a subset of patients with long standing refractory disease.

Differential gene expression analysis was performed on the blood of patients prior to starting therapy, after two cycles and after their final cycle of therapy. All samples were run simultaneously at the end of the trial. Patients were classified as High (CR or near CR), intermediate (PR or stable disease), or poor responders (progression or not evaluable) based on clinical response. At baseline cGVHD patients express an overall decrease in T cell markers such as lineage (CD4), chemokine receptors (CCR6, CXCR3 and CCR7), and co-stimulatory molecules (CD40L, ICOS, CD28, and CD27), as well as decreases in IL23 and Granzyme K message compared to healthy controls. Increases were seen in myeloid/macrophage markers (CD163 and OSCAR). Baseline profiles of good responders were closer to those of healthy controls as compared to poor responders (figure below). Inflammation, cell death and apoptosis modules are up regulated and immune response modules are down-regulated in low and medium responders

HC – Healthy Control; DC – Disease Control (allogeneic BMT recipients without cGVHD)

Weekly bortezomib for cGVHD appears to be well tolerated and results in early improvement in long standing refractory disease in a subset of patients. Gene expression profiles at the initiation of treatment correlated with response and may be useful in predicting which patients will respond to bortezomib.

Disclosures:

Miller: Millennium Pharmaceuticals: Research Funding. Off Label Use: Bortezomib for chronic graft vs. host disease.
Palifermin For Prevention Of Oral Mucositis Has No Negative Effect On Long-Term Outcome In Patients With Hematological Malignancies Undergoing HSCT - Long-Term Follow-Up To 15 Years
Authors: Stiff Patrick J , Spielberger Ricardo
Published: October 2019

Background

Palifermin has been shown to reduce the incidence and duration of severe oral mucositis in hematological stem cell transplantation (HSCT) recipients. A follow-up study was performed to rule out a potential long-term safety risk of palifermin use.

Objectives

to compare long-term disease outcome (overall survival, disease progression and incidence of secondary malignancies) between palifermin and placebo.

Methods

This is a long-term follow-up study of patients with hematological malignancies undergoing HSCT and treated with palifermin or placebo to prevent oral mucositis. The aim of the study was to detail any potential late complications due to palifermin exposure. Patients were enrolled between 1997-2003 into four randomized, placebo-controlled phase II/III studies conducted at 31 sites in Australia, Europe and the US. The survival outcomes were compared using hazard ratios (HRs) estimated with Cox model including the treatment group, baseline age, disease type (Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, leukemia), ECOG performance status, country and presence of prior radiotherapy as covariates. The incidence of secondary malignancies was compared with chi-squared test.

Results

A total of 672 patients were randomized to the parent studies (429 palifermin, 243 placebo) and 543 (345, 198) were enrolled to the long-term follow-up. The median follow-up time for subjects alive was 7.9 years (range 0.1-14.9) for palifermin and 8.8 (0.1-14.8) for placebo. No significant differences were seen for either overall survival (HR = 1.01; 95% confidence interval (CI) 0.78-1.31; p=0.921) or progression-free survival (HR=1.04; 95% CI 0.83-1.31; p=0.733). Secondary malignancies were reported by 13% (palifermin) vs. 11% (placebo) of the patients (p=0.477). The most common secondary malignancies were acute myeloid leukemia/myelodysplastic syndrome (5% vs. 5%) and skin cancers (2% vs. 2%).

Conclusion

The overall survival, progression-free survival, and the incidence of secondary malignancies were comparable between palifermin and placebo in patients undergoing HSCT. After a follow-up of up to 15 years, no negative effect of palifermin on long-term outcomes was observed.

Disclosures:

No relevant conflicts of interest to declare.
Estimation Of Serum Erythropoietin Levels in Anaemic HIV Infected Patients in Lagos, Nigeria
Authors: Olu-Taiwo Adebola , Akanmu Suleimon , Amusu Olatilewa , Ogunro Samuel
Published: October 2019

Background

Erythropoietin(EPO) response to anaemia has been reported to be suboptimal in HIV infected anaemic patients.

Objectives

To assess the relationship between EPO levels and degree of anaemia, CD4+ cell count and viral load of HIV infected treatment naive subjects and if there is any association with inflammatory cytokines.

Subjects and Methods

A total of 120 subjects aged 18-66 years, comprises (1) HIV infected treatment naive subjects who had anaemia with Hb level <10g/dL. Control subjects were: (2) HIV infected non-anaemic, (3) non-HIV infected anaemic and (4) non-HIV infected non-anaemic individuals. Full blood count using automated counter; CD4+ cell count by semi-automated flow cytometer; and viral load were estimated. Serum erythropoietin levels, serum ferritin levels, CRP levels, TNF-α levels and IL-6 levels were estimated. Appropriateness of EPO response was determined by calculating EPO response ratio.

Results

Majority (72%) of the HIV infected anaemic patients had a normocytic hypochromic type of anaemia. A weak positive correlation between EPO levels and CD4+ cell count (r=0.075, p=0.572) was obtained. A significant negative correlation between EPO levels and haemoglobin levels (r=-0.268, p=0.003). Twenty-seven (71.05%) of the 38 HIV infected anaemic subjects had EPO response ratio less than 0.8. Serum ferritin level was significantly higher in HIV infected anaemic subjects compared to the controls (p=0.000). Serum interleukin-6 level was significantly higher in HIV infected anaemic subjects compared to HIV infected non-anaemic subjects (p=0.003). Serum C-reactive protein level was significantly higher in HIV infected anaemic subjects compared to non-HIV infected non-anaemic (p=0.004) and non-HIV infected anaemic subjects and(0.001). Serum tumour necrosis factor-α level was significantly higher in HIV infected anaemic subjects compared to other arms of the study population (p=0.000).

Conclusion

The present study revealed that HIV infected anaemic subjects had reduced serum EPO levels. However, serum inflammatory cytokines levels were raised in the same subjects.

Disclosures:

No relevant conflicts of interest to declare.
Pulmonary Hypertension in Sickle Cell Disease: Correlation With Echocardiographic Assessment and Serum Brain Natriuretic Peptide Concentration
Authors: Alkindi Salam , Jabeen Zeba , Pathare Anil , Al-Huneini Mohammed , Al Khabori Murtadha , Al-Farsi Khalil , Al Riyami Adil , Al-Raesi Majdah , Dennison David
Published: October 2019

Background & Purpose

Pulmonary hypertension (PH) in sickle cell disease (SCD) is an important risk factor for complications including sudden death. In this study, we aimed to determine the prevalence of PH and correlate the echocardiographic parameters and general laboratory data with markers of hemolysis and serum NT pro-brain natriuretic peptide [BNP] concentrations in Omani SCD patients.

Methods

A cohort of 163 SCD patients [Mean age 25.4±8.4 years], in steady state was prospectively screened for PH with Doppler echocardiography (defined as a tricuspid regurgitation jet flow velocity of ≥ 2.5 m/sec and/or mean pulmonary artery pressure [mPAP] ≥25 mmHg). After a written informed consent, all patients were investigated with a complete blood count, renal chemistry, hemolytic parameters including LDH, haptoglobin, liver function tests, coagulation studies, HPLC studies including HbS and HbF level estimations, X ray Chest, ECG, ABG, Pulmonary function tests, Pulse oximetry, and Serum NT pro-BNP levels.

Results

In the evaluable patient cohort of 116 subjects [63 females, 53-males], the prevalence of PH was 5.2%. No statistically significant differences were detected in Hb levels, ECG, chest radiography, pulmonary function tests between patients with and without PH. However, plasma NT pro-BNP levels were significantly correlated with PAH [r=0.934, p<0.000], TRV jet [r=.671, p<0.000], Abnormal ECHO [r=0.672, p<0.000], and direct Bilirubin levels [r=0.278,p<0.009] in SCD patients. Furthermore, in SCD patients with PH, there was a statistically significant increase in plasma NT pro-BNP levels [ p<0.001], ALT [p<0.02], S. Creatinine [p=0.045] and Total bilirubin [p<0.0001] and direct Bilirubin levels [p<0.0001][Table].

Conclusions

Serum NT pro-BNP is a strong indicator of PH in SCD patients. Doppler ultrasound echocardiography is a useful initial screen for PH in SCD patients. The correlation between PH and hemolytic markers suggests an implication in the pathogenesis of PH.

Disclosures:

No relevant conflicts of interest to declare.
Nitrite Regulation Of Platelet Reactivity Under Physiological Conditions
Authors: Park Ji Won , Piknova Barbora , Nghiem Khanh , Lozier Jay N. , Schechter Alan N.
Published: October 2019

Nitric oxide (NO) is generated by serial reduction pathway of nitrate and nitrite or by activity of endogenous nitric oxide synthase (NOS), and modulates platelet function and hemostasis. We have shown by aggregometry and flow cytometric analysis of P-selectin that nitrite in the presence of erythrocytes inhibits platelet aggregation and activation after its reduction to NO at low oxygen tension [PLoS One. 2012; 7: e30380. 10.1371/journal.pone.0030380]. We then investigated how nitrite may affect overall clotting processes by regulating platelet function using thrombelastography (TEG), a method used to assess platelet function, fibrin clot formation, and fibrinolysis in blood and/or plasma. We measured the effect of NO donors and nitrite on three TEG parameters, reaction time (R, time to initial fibrin formation), α angle (velocity of clot growth) and maximum amplitude (MA, clot strength), in healthy volunteers. The NO donor (DEANONOate) inhibited all three TEG parameters in response to two independent platelet activators (ADP and AA), in platelet rich plasma (PRP) or whole blood diluted with plasma to yield 20% hematocrit (Hct) to model blood in the microcirculation in vivo. At DEANONOate concentrations ranging from 0-1 μM, R values were progressively prolonged from 1.9 to 3.6 (p=0.008), α angle was decreased from 17.8 to 9.5 (p=0.01) and maximum clot amplitude was reduced from 9.0 to 4.4 (p=0.001) in 20% Hct with ADP stimulation. In contrast, nitrite did not affect clotting parameters in PRP, but exhibited moderate inhibitory effects in 20% Hct at concentrations from 0-10 μM; R values were slightly prolonged from 1.6 to 2.7 (p=0.12), α angle was decreased from 21.8 to 12.8 (p=0.07) and maximum clot amplitude was reduced from 11.0 to 5.0 (p=0.02). This inhibitory effect of nitrite on clotting was greatly enhanced under hypoxic conditions (blood pO2 46.5±11.6 mmHg); R values from 1.4 to 4.0 (p=0.003), α angle from 21.7 to 7.8 (p=0.002), and maximum clot amplitude from 10.0 to 3.4 (p=0.0003). These results suggest that the nitrite effect may be greatest in the microcirculation and be important in differences between arterial and venous clotting. In conclusion, our results show TEG parameters indicate NO inhibition of platelet-mediated blood clotting and that the physiological effect of factors which determine NO bioavailability, such as reduction of blood and tissue nitrite, could be used to predict hemostasis.

Disclosures:

Schechter: National Institutes of Health: Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases., Dr. Alan Schechter is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. Patents & Royalties.
A Case Of Vitamin C Deficiency Manifesting With Easy Bruising: Suggestion For a Supplementation Protocol
Authors: Zaidi Aisha , Moffett Padmini
Published: October 2019

Contrary to most animals which are capable of obtaining ascorbic acid (vitamin C) by metabolizing glucose, humans require an exogenous source of vitamin C. It is a water-soluble vitamin found in various foods, including citrus fruits (oranges, lemons, grapefruit), green vegetables (peppers, broccoli, cabbage), tomatoes, and potatoes. It is essential for growth and repair of tissues, particularly blood vessels. Deficiency of vitamin C is relatively rare; the third National Health and Nutrition Examination Survey (NHANES III, 1988-1994) found approximately 13% of the US population to be vitamin C deficient. Smoking cigarettes lowers the amount of vitamin C in the body, so smokers are at a higher risk of deficiency. Additionally, alcoholics, the elderly, patients with psychiatric disorders and individuals who experience social isolation are also at risk. Symptoms can manifest after 3 months of deficient intake of vitamin C -containing foods. Since ascorbic acid is required for collagen synthesis, deficiency usually manifests with bleeding gums, ecchymoses, petechiae, coiled hairs and hyperkeratosis. Diagnosis can be established when plasma levels of vitamin C fall below 0.2mg/dl. There are currently no evidence based guidelines for supplementation once low levels are detected. We describe a case of a 33 year old Caucasian female seen in our clinic for easy bruising of 10 years duration. She would wake up in the morning with bruised arms and legs without any inciting trauma. She denied prolonged bleeding after dental extractions, frequent nose bleeds, menorrhagia any genitounrinary or gastrointestinal blood loss. Her exam revealed ecchymoses on her arms and legs with no evidence of perifollicular hemorrhage or gingival hyperplasia. Workup including CBC, Coagulation profile and Von Willibrand factor levels were all within normal limits. She had a vitamin C level of 0.1mg/dl. She was subsequently advised to increase vitamin C intake through fruits and vegetable and was started on vitamin C supplementation with 500mg twice a day for four weeks. Her repeat vitamin C level on her one month follow up was noted to be 1.9mg/dl. She noticed complete resolution of her symptoms. This case illustrates the fact that vitamin C deficiency should be excluded in adults presenting with bruising. It also offers a supplementation regimen that resulted in improvement of clinical symptoms as well as laboratory value normalization.

Disclosures:

No relevant conflicts of interest to declare.
Acquired Hemophilia A. Experience Of a Single Center
Authors: Alzate Mauricio A , Meschengieser Susana S , Blanco Alicia , Grosso Silvia , Lazzari María A , Sánchez-Luceros Analía
Published: October 2019

Introduction

Acquired hemophilia A is a rare and serious autoimmune disease. Morbidity and mortality are associated with advanced age, comorbidities, toxicity of treatment and bleeding severity. Treatment goals are control of the bleeding and eradication of the inhibitor, while treating the underlying condition if it is present.

Objective

To describe the baseline characteristics of acquired hemophilia A patients and to assess the response to treatment.

Patients and Methods

Between November 1991 and April 2013, 27 patients were diagnosed with acquired hemophilia A (mean age 59, range 21-86; 18 women - 66%) in the Departamento de Hemostasia y Trombosis. Five patients were lost from follow-up. APTT mixing studies with normal plasma (1:1) and time-temperature dependent effect on the APTT were performed for a-FVIII diagnosis. Whenever possible, inhibitor activity was titrated by Bethesda method at diagnosis (BU/mL). Medical records were reviewed to evaluate the initial symptoms, underlying diseases, treatments and outcome.

Results

The mean follow-up was 86 weeks (range 1-640). Underlying etiologies included: idiopathic 70.4%, postpartum 14.8%, malignancy 11.1%, autoimmune disease 3.7%. All patients had bleeding at diagnosis. The most frequent sites of bleeding were: muscular 32%, soft tissue 18%, urinary tract 9%, gastrointestinal tract 6%; being from multiple sites in 9%. At diagnosis, the mean value for FVIII was 6% (range 1-40), and inhibitor titer 220 BU/mL (range 2.2-1173). Initial therapeutic scheme included glucocorticoids in 97% of the patients, 13 in monotherapy (mean age 53 years ± 19), 13 with cyclophosphamide (63 years ± 18) (p= ns), and human immunoglobulin in 1 patient. This last patient died after 1 week of diagnosis due to uncontrolled gastrointestinal bleeding (previous to the era of rVIIa). As a second-line therapy, rituximab was used in 3 patients.

Sixty-three (63%) patients achieved complete response (CR) (inhibitor titer < 0.6 BU/mL without bleeding episodes), and 23% achieved partial response (PR) (reduction in inhibitor titer > 50% without bleeding episodes), without differences between monotherapy or combined. Overall, women responded more frequently than men (93.3% vs. 71.4%, p= ns). All patients that received rituximab achieved CR.

Conclusions

In this study, the overall response rate was higher than 80%. In most cases, the disease has a prolonged course like other autoimmune diseases, with remissions and relapses.

Disclosures:

No relevant conflicts of interest to declare.
Dysregulation Of Thrombotic and Hemostatic Factors In End Stage Renal Disease
Authors: Bansal Vinod , Hoppensteadt Debra , Ahmed Syed , Fareed Jawed
Published: October 2019

Introduction

Increased cardiovascular events occur in ESRD patients on maintenance hemodialysis. The objective of this study was to utilize newly introduced cardiac biomarker chips and immunoenzymatic methods, profiling various biomarkers of inflammation and thrombogenesis in a defined ESRD patient population.

Material and Methods

Blood samples from seventy two ESRD patients were drawn prior to maintenance hemodialysis. Commercially available ELISA kits for Tissue Plasminogen Activator – Plasminogen Activator Inhibitor Type-1 complex (tPA-PAI-1 complex), Plasminogen Activator Inhibitor Type-1 (PAI-1), Myeloperoxidase, (MPO), Thrombomodulin, (TM), Interleukin-1 beta (IL-1β), Anti-Annexin V, human sL-selectin, and Inter-Cellular Adhesion Molecule 1 (ICAM-1). Functional methods were used for measuring Antithrombin and Von Willebrand factor (vWF) activity were used. The biochip arrays (Randox, Evidence System, United Kingdom)for cardiac markers, included creatine kinase-MB (CK-MB), Myoglobin (Myo), Heart-type fatty acid binding protein (hFABP) and cardiac troponin I (cTn1). The cerebral array included C-reactive protein (CRP), D-Dimer (DDMER), Neuron Specific Enolase (NSE), Neutrophil Gelatinase-Associated Lipocalin (NGAL), soluble Tumor Necrosis Factor Receptor 1 (TNFRI) and Thrombodulin (TM).

Results

ESRD patients showed assay dependent decrease in markers such as tPA-PAI-1, Anti-Annexin V, L-selectin and Antithrombin activity (ranging from 18 to 46%). The levels for MPO, TM, CK-MB, MYO, FABP, CRP, DDMER, NSE, NGAL, TNFR1, and vWF activity all showed a significant increase in the ESRD patients (ranging from 25%-5587%). No changes were observed in the IL-1B, ICAM1, or cTn1 in ESRD patients compared to normal groups.

Conclusions

These studies underscore the role of inflammatory and thrombotic mediators in ESRD. The cardiac biochip array revealed a remarkable elevation of the FABP and as well as a notable increase in MYO. The cerebral array revealed a remarkable elevation of the TNFR1.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Analysis Of Patients With Deep Venous Thrombosis
Authors: Hosseeini Soudabeh , Kalantari Ebrahim , Dorgalaleh Akbar , Rozei Arash , Jafari Marzieh , Naderi Majid , Tabibian Shadi , Bamedi Taregh
Published: October 2019

Background

Deep venous thrombosis (DVT) refers to the formation of a thrombus within a deep vein that frequently occurs after surgical procedures, trauma, in the presence of cancer and immobilization conditions. DVT is a major health problem that causes high rate of morbidity and mortality in the general population. Hyper coagulation states such as antithrombin-III, protein-C and protein-S deficiencies, contribute to formation of DVT.

Congenital and acquired gene mutations are other risk factors that stimulate formation of thrombus. Our aims in this study was to molecularly analyze the patients with DVT and assess the impact of common mutations of MTHFR (C677T) (A1298C), PAI-1, Prothrombin 20210 and FV Leiden mutation on occurrence of deep venous thrombosis.

Methods

This long-term study was conducted from June 2009 to July 2013 on 221 patients with deep venues thrombosis. Two hundred and twenty-one age and sex matched individuals were also chosen as control group. The diagnosis of venous thromboembolic disease was based on patient’s history, clinical findings and D-dimer test. Finally deep venous thrombosis was confirmed with Doppler ultrasonography. In addition, all participants were asked to complete a standardized questionnaire on acquired risk factors for venous thrombosis.

After confirmation of DVT, both groups were assessed molecularly for five mutations including, MTHFR C677T, MTHFR A1298C, PAI-1 4G/5G, Prothrombin 20210 and FV Leiden. The relationship between these mutations and the risk of DVT was calculated using logistic regression and expressed as an OR with a 95% confidence interval (CI).

Results

The mean age of patients and control group were 38±0.8 and 3.7± 0.7 years. Our results revealed that the MTHFR C677T (OR 2.9, 95% 95% CI 1.1 to 7.5) and MTHFR A1298C in heterozygote manner (OR 4.3, 95% CI 1.4 to 13.7) were each associated with an increased risk of DVT. The OR associated with being a carrier of the PAI-1 4G/5G genotype was 2.9 (95% CI 1.14 to 7.5). There was a 4-fold increased risk of DVT associated with Prothrombin 20210 mutation in heterozygote manner (OR 4.3, 95% CI 1.4 to 13.7).

For patients who were heterozygous for FV Leiden mutation OR DVT was 2.6 (95% CI 1.3 to 5.0).

Conclusion

Our findings suggest that genetic risk factors have a contributory role on occurrence of DVT.

Disclosures:

No relevant conflicts of interest to declare.
Thrombolytic properties of staphylokinase
Authors: Matsuo O , Okada K , Fukao H , Tomioka Y , Ueshima S , Watanuki M , Sakai M
Published: October 2019

We evaluated the properties of recombinant staphylokinase in comparison with those of tissue-type plasminogen activator (t-PA) and streptokinase (SK). The presence of fibrin(ogen) fragment FCB-2 in the reaction mixture increased plasminogen activation by staphylokinase more than 20-fold. Such characteristics are similar to those of t-PA. On the other hand, SK was not affected by the presence of FCB-2. The thrombolytic properties of staphylokinase were studied in a system consisting of a radioactive human plasma clot (125I-fibrinogen-labeled) suspended in the circulating citrated plasma. Significant thrombolysis (50% in 3 hours) was obtained with 2 micrograms/mL of staphylokinase and 4.45 micrograms/mL t-PA, as compared with 12 micrograms/mL for SK. The relative molar potency of staphylokinase, calculated from the molecular weight, was about two times more effective than that of SK, but about half of that of t-PA. Systemic fibrinolytic activation and fibrinogen breakdown was not observed with staphylokinase or t-PA, but was observed with SK. The thrombolytic efficiency of staphylokinase, which was calculated as the ratio of the degree of thrombolysis/the degree of fibrinogenolysis, was about five times greater than that of SK, and about half of that of t-PA. These findings suggest that staphylokinase has higher specific thrombolytic properties and lesser fibrinogenolytic properties than those of SK.
Dihydrotestosterone exerts a depressive influence on the production of interleukin-4 (IL-4), IL-5, and gamma-interferon, but not IL-2 by activated murine T cells
Authors: Araneo BA , Dowell T , Diegel M , Daynes RA
Published: October 2019

The present study examined the effects of the androgen steroid, dihydrotestosterone (DHT), on murine T-cell production of a number of lymphokines. Direct exposure of murine T cells to DHT in vitro was found to reduce the amount of interleukin-4 (IL-4), IL-5, and gamma- interferon (gamma IFN) produced after activation with anti-CD3 without affecting the production of IL-2. Exposure of T cells to either androstenedione or testosterone (the metabolic precursors of DHT) affected no change in the biosynthesis of either of these lymphokines. We have determined that macrophages possess 5 alpha-reductase, and are thus competent to metabolize testosterone to DHT. This physicochemical information is complemented by a functional analysis of macrophage metabolism of testosterone. By incubating bone marrow macrophages with testosterone, before their use as accessory cells, the IL-4 and IL-5 producing potential of the activated T cells cocultured with them was depressed. That the observed effect was mediated by the conversion of testosterone to DHT was further corroborated by illustrating that the inhibition of IL-4 production was abrogated if 4MA, a specific 5 alpha- reductase inhibitor, was added to macrophage cultures containing testosterone. The biologic role of DHT in lymphokine and immune response regulation in vivo was addressed using several lines of investigation. First, transdermal delivery of DHT to groups of mice altered the capacity of T cells residing in the draining lymph nodes, only, to produce lymphokines. Second, treatment of either aged mice or the T cells isolated from them with a combination of dehydroepiandrosterone and DHT restored the capacity of their T cells to produce IL-2, IL-4, and gamma IFN to levels equivalent to that of younger mice. Finally, we observed a difference between males and females of a given age to produce IL-2, IL-4, and gamma IFN, with both IL-4 and gamma IFN production being elevated in females. Collectively, our findings indicate that DHT, similar to other steroid hormones, may play an important role in lymphokine regulation in vivo.
The t(1;22) (p13;q13) is nonrandom and restricted to infants with acute megakaryoblastic leukemia: a Pediatric Oncology Group Study
Authors: Carroll A , Civin C , Schneider N , Dahl G , Pappo A , Bowman P , Emami A , Gross S , Alvarado C , Phillips C
Published: October 2019

We report the nonrandom occurrence and frequency of the t(1;22)(p13;q13) in acute myeloid leukemia (AML) and its close association with the French-American-British M7 subtype of AML in infants (less than 1 year). This chromosomal abnormality occurred in 6 of 252 (2.4%) children and adolescents with AML (6 of 28 infants, 22%; 6 of 18 M7 AML cases overall, 33%; and 6 of 6 M7 cases in infants). Infants with AML of M7 subtype and the t(1;22) often presented with prominent abdominal masses. Two of these infants were not treated and died early. Three of four treated infants entered complete remission with therapy for AML; the remaining infant died of hemorrhage on day 8. Of the three infants who entered remission, only one remains alive and disease free at 5+ months. The other two infants relapsed in the bone marrow at 5 and 2 months from the start of therapy, respectively. We conclude that M7 AML with the t(1;22) usually presents in infants with extensive infiltration of abdominal organs by leukemic cells and may confer a poor prognosis despite intensive AML-directed treatment. Identification of this nonrandom translocation exclusively in infants with acute megakaryoblastic leukemia (AMkL) implies that it may serve as an additional diagnostic marker for this disease and links it to the pathogenesis of AMkL in infants.
Identification and characterization of a functional receptor for interferon-gamma on a megakaryocytic cell line
Authors: Monte D , Wietzerbin J , Pancre V , Merlin G , Greenberg SM , Kusnierz JP , Capron A , Auriault C
Published: October 2019

We have previously shown that human interferon-gamma (Hu-IFN-gamma) induces platelets to become efficient effector cells, capable of killing young larvae of the parasite Schistosoma mansoni. Recently, binding sites for IFN-gamma on platelets have been characterized. We show here the presence of high-affinity receptors for IFN-gamma on the surface of the human megakaryocytic Dami cell line. Scatchard analysis indicated the presence of about 11,000 binding sites per cell, with a kd of 3 +/- 0.5 x 10(-10) mol/L; the apparent molecular weight of the receptor was 90 Kd. Receptor-bound 125I Hu-recombinant IFN-gamma was rapidly internalized and degraded when the temperature was increased from 4 degrees C to 37 degrees C. The half-life of this receptor was about 7 hours, and pretreatment of cells with IFN-gamma or phorbol myristate acetate had very little effect on the surface receptor number and no detectable effect on IFN-gamma receptor messenger RNA (mRNA) expression. The receptor was functional, because 24 hours of treatment with IFN-gamma led to the increase of HLA class I mRNA expression and to the initiation of HLA class II mRNA expression. These effects were selective because platelet glycoprotein Ib, IIb, or IIIa mRNA expression and cell proliferation were unaffected.
Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells
Authors: Moore KA , Deisseroth AB , Reading CL , Williams DE , Belmont JW
Published: October 2019

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.
Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein
Authors: Meszaros K , Aberle S , Dedrick R , Machovich R , Horwitz A , Birr C , Theofan G , Parent JB
Published: October 2019

Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (> or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.
Regulated Secretion in Platelets: Identification of Elements of the Platelet Exocytosis Machinery
Authors: Lemons Paula P. , Chen Dong , Bernstein Audrey M. , Bennett Mark K. , Whiteheart S.W.
Published: October 2019

To further characterize the molecular mechanisms of platelet function, we have sought to identify some of the proteins that mediate the secretory events of the platelet release reaction. We report that platelets contain the general elements of the membrane transport apparatus: N-ethylmaleimide sensitive fusion protein (NSF ), p115/transcytosis-associated protein (p115/TAP), and the soluble NSF attachment proteins (α- and, γ-SNAP). The cDNAs encoding two of these proteins, α- and γ-SNAP, have been cloned from a human platelet-derived cDNA library. Platelet membrane extracts possess SNAPreceptor (SNARE) activity, suggesting that the class of proteins (SNAREs) proposed to provide the specificity for vesicle docking and membrane fusion are present in platelets. To identify these proteins, we have used specific antibodies against known SNAREs to probe platelet extracts. Syntaxin 2 and 4 can be readily detected in platelet membrane preparations and are shown to participate in 20 S complex formation. Syntaxin 1, 3, and 5 could not be detected. Other known SNARE and SNARE-associated proteins such as vesicle-associated membrane protein (VAMP)/synaptobrevin 2, SNAP-25, synaptophysin, or synaptotagmin I could not be immunochemically detected in platelet membrane preparations. The presence of both the general transport proteins (NSF and SNAPs) and specific transport proteins (syntaxin 2 and 4) indicates that platelet exocytosis uses a molecular mechanism similar to other secretory cells such as neurons. However, the subcellular concentrations of these proteins suggest that, unlike neuronal secretion, granule-to plasma membrane docking may be the limiting step in platelet exocytosis.
Role of A2a Extracellular Adenosine Receptor-Mediated Signaling in Adenosine-Mediated Inhibition of T-Cell Activation and Expansion
Authors: Huang Steve , Apasov Sergey , Koshiba Masahiro , Sitkovsky Michail
Published: October 2019

Accumulation of adenosine and of deoxyadenosine in the absence of adenosine deaminase activity (ADA) activity results in lymphocyte depletion and in severe combined immunodeficiency (ADA SCID), which is currently explained by direct cell death-causing effects of intracellular products of adenosine metabolism. We explored the alternative mechanisms of peripheral T-cell depletion as due to inhibition of T-cell expansion by extracellular adenosine-mediated signaling through purinergic receptors. The strong inhibition of the T-cell receptor (TCR)-triggered proliferation and of upregulation of interleukin-2 receptor α chain (CD25) molecules, but not the direct lymphotoxicity, were observed at low concentrations of extracellular adenosine. These effects of extracellular adenosine (Ado) are likely to be mediated by A2a receptor-mediated signaling rather than by intracellular toxicity of adenosine catabolites, because (1) poorly metabolized adenosine analogs cause the accumulation of cAMP and strong inhibition of TCR-triggered CD25 upregulation; (2) the A2a, but not the A1 or A3, receptors are the major expressed and functionally coupled adenosine receptors in mouse peripheral T and B lymphocytes, and the adenosine-induced cAMP accumulation in lymphocytes correlates with the expression of A2a receptors; (3) the specific agonist of A2a receptor, CGS21680, induces increases in [cAMP]i in lymphocytes, whereas the specific antagonist of A2a receptor, CSC, inhibits the effects of Ado and CGS21680; and (4) the increases in [cAMP]i mimic the adenosine-induced inhibition of TCR-triggered CD25 upregulation and splenocyte proliferation. These studies suggest the possible role of adenosine receptors in the regulation of lymphocyte expansion and point to the downregulation of A2a purinergic receptors on T cells as a potentially attractive pharmacologic target.
A BCR-ABLp190 Fusion Gene Made by Homologous Recombination Causes B-Cell Acute Lymphoblastic Leukemias in Chimeric Mice With Independence of the Endogenous bcr Product
Authors: Castellanos A. , Pintado B. , Weruaga E. , Arévalo R. , López A. , Orfao A. , Sánchez-Garcı́a I.
Published: October 2019

BCR-ABLp190 oncogene is the result of a reciprocal translocation between chromosomes 9 and 22 and is associated with B-cell acute lymphoblastic leukemia (B-ALL) in humans. Current models expressing the BCR-ABLp190 chimeric gene fail to consistently reproduce the phenotype with which the fusion gene is associated in human pathology, mainly due to the difficulty of being expressed in the appropriate cell type in vivo. We have used here homologous recombination in ES cells to create an in-frame fusion of BCR-ABLp190 that mimics the consequences of the human chromosomal translocation by fusion of BCR-ABL coding sequences into the bcr endogenous gene. The chimeric mice generated with the mutant embryonic stem cells systematically develop B-ALL. Using these chimeric mice, we further show that BCR-ABL oncogene does not require the endogenous bcr product in leukemogenesis. Our results show that BCR-ABLp190 chimeric mice are a new model to study the biology of the BCR-ABL oncogene and indicate the efficacy of this strategy for studying the role of specific chromosome abnormalities in tumor development.
Hematopoietic Cell Phosphatase Negatively Regulates Erythropoietin-Induced Hemoglobinization in Erythroleukemic SKT6 Cells
Authors: Sharlow Elizabeth R. , Pacifici Robert , Crouse Jill , Batac Jennifer , Todokoro Kazuo , Wojchowski D.M.
Published: October 2019

In an increasing number of hematopoietic cytokine receptor systems (T-cell receptor, B-cell receptor, and macrophage colony-stimulating factor, stem cell factor, interleukin-3, and erythropoietin [EPO] receptors), inhibitory roles for the protein tyrosine phosphatase hematopoietic cell phosphatase (HCP; SHPTP1, PTP1C, and SHP1) have been defined in proliferative signaling. However, evidence exists to suggest that HCP also may exert important effects on blood cell differentiation. To investigate possible roles for HCP during late erythroid differentiation, effects of manipulating HCP expression or recruitment on EPO-induced hemoglobinization in erythroleukemic SKT6 cells have been investigated. No effects of EPO on levels of HCP, Syp, Stat5, the EPO receptor, or GATA-1 expression were observed during induced differentiation. However, the tyrosine phosphorylation of JAK2, the EPO receptor, and Stat5 was efficiently activated, and HCP was observed to associate constitutively with the EPO receptor in this differentiation-specific system. In studies of HCP function, inhibition of HCP expression by antisense oligonucleotides enhanced hemoglobinization, whereas the enforced ectopic expression of wild-type (wt) HCP markedly inhibited EPO-induced globin expression and Stat5 activation. Based on these findings, epidermal growth factor (EGF) receptor/EPO receptor chimeras containing either the wt EPO receptor cytoplasmic domain (EECA) or a derived HCP binding site mutant (EECA-Y429,431F ) were expressed in SKT6 cells, and their abilities to mediate differentiation were assayed. Each chimera supported EGF-induced hemoglobinization, but efficiencies for EECA-Y429,431F were enhanced 400% to 500%. Thus, these studies show a novel role for HCP as a negative regulator of EPO-induced erythroid differentiation. In normal erythroid progenitor cells, HCP may act to prevent premature commitment to terminal differentiation. In erythroleukemic SKT6 cells, this action also may enforce mitogenesis.
Safety of Autologous, Ex Vivo-Expanded Human Immunodeficiency Virus (HIV)-Specific Cytotoxic T-Lymphocyte Infusion in HIV-Infected Patients
Authors: Lieberman Judy , Skolnik Paul R. , Parkerson G. Robert , Fabry Jessica A. , Landry Bernard , Bethel James , Kagan Jonathan ,   the DATRI 006 Study Team
Published: October 2019

We infused six human immunodeficiency virus (HIV)-seropositive subjects with autologous CD8+ cytotoxic T cells (CTLs) enriched for HIV-specific cytotoxicity targeted against a diversity of HIV epitopes in gp120, gag p17 and p24, and nef. There was no toxicity and no subject deteriorated clinically. In the first 2 weeks, CD4 counts increased for all subjects and plasma viremia decreased in five of six subjects. Twenty-four weeks later, the mean values of all measures of viral burden and surrogate markers of HIV infection were either unchanged or improved, but none of the changes was statistically significant. Two subjects continued to have decreased cell-associated viral burden and another subject had more than doubled CD4 cell count. HIV-specific CTL activity increased in most subjects. The increase in CD4 T-cell counts in the first weeks after the infusion suggests that antiviral CTLs of diverse specificities do not play a significant role in CD4 T-cell decline. The lack of any acute toxicity or adverse effect on viral burden suggests that therapy with antiviral CTLs deserves further study.
Ability of Early Acting Cytokines to Directly Promote Survival and Suppress Apoptosis of Human Primitive CD34+CD38− Bone Marrow Cells With Multilineage Potential at the Single-Cell Level: Key Role of Thrombopoietin
Authors: Borge Ole J. , Ramsfjell Veslemøy , Cui Li , Jacobsen Sten E.W.
Published: October 2019

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38− human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38− but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38− progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38− long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38− cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.