bluuebunny/crossref_metadata_2025 huggingface dataset:
High Incidence of Pelvic Deep Vein Thrombosis Detected by Magnetic Resonance Venograpy Post-Caesarean Section.
Authors: Rodger Marc , Avruch Leonard , Olivier Andre , Walker Mark
Published: October 2019

Background Venous thromboembolism (VTE) is the leading cause of maternal mortality in the developed world. The post-partum period is the highest risk period for pregnancy associated VTE and delivery by Caesarean section further increases this risk. The true incidence of deep vein thrombosis (DVT) post- Caesarean section is unknown but felt to be low. The limited number of screening studies conducted to date have not included systematic pelvic vein imaging.

Objectives To determine the incidence of DVT post-Caesarean section using Magnetic Resonance Venograpy (MRV) and bilateral compression ultrasounds.

Methods Prospective cohort study of moderate to high risk women (one or more VTE risk factors) post-Caesarean section. On the day of post-partum discharge we conducted systematic bilateral proximal leg vein compression ultrasound imaging to detect proximal leg DVT and pelvic vein imaging with magnetic resonance venography (MRV). MRVs were independently and blindly interpreted by two radiologists with disagreements resolved by consensus. MRVs were interpreted as demonstrating definite, probable, possible or no thrombosis. Two rater Kappa scores were calculated from initial interpretations (prior to consensus review). Incidence of DVT and 95% confidence interval were calculated.

Results Fifteen patients were recruited. At discharge, there were no proximal DVTs on bilateral leg compression ultrasounds. MRV results are shown in Table I.

Conclusions The incidence of pelvic vein DVT post- Caesarean section is much higher than anticipated (46%). The clinical significance of this finding remains to be determined. However, diagnoses such as septic pelvic vein thrombophlebitis, which depend on demonstrating the presence pelvic vein thrombosis in the setting of post-partum fever may be falsely overdiagnosed. Pelvic vein thrombosis may be a common (and normal) finding post-Caesarean section. MRV may also prove to be a useful surrogate outcome measure in post-partum VTE prophylaxis studies.

Table I: Pelvic Vein Imaging Results Post-C-Section

MRV Consensus Result n/N (%) 95% CI Two Rater Kappa 95% CI= 95% Confidence Interval Definite Thrombosis 7/15 (46%) 21–73% 0.5 Definite or Probable Thrombosis 8/15 (53%) 27–79% 0.6 Definite, Probable or Possible Thrombosis 10/15 (66%) 38–88% 1.0 No Thrombosis 5/15 (33%) 12–62% 1.0
Blood Outgrowth Endothelial Cells (BOEC) Contribute to Tumor Vascularization and Can Be Used for Delivery of Cancer Gene Therapy.
Authors: Dudek Arkadiusz Z. , Bodempudi Vidya , Nelson Curtis M. , Chang Liming , Hebbel Robert P.
Published: October 2019

Continuous and adequate levels of angiogenesis inhibitors at the site of tumor vasculature may be necessary to obtain optimal antitumor effect. Following recent observations that circulating in blood endothelial cells enhance tumor vasculogenesis we have designed grown from peripheral blood endothelial cells to deliver anti-angiogenic antitumor therapy. BOEC, after several-fold expansion, expressed endothelial markers: VE-cadherin, CD31, von Willebrand factor, and P1H12 and responded to functional endothelial assays: uptake of acetylated low-density lipoprotein, and vascular tube formation. BOEC were also found in functional vessels in vivo, when NOD-SCID mice implanted subcutaneously with Lewis lung carcinoma or H226 lung cancer, were injected via tail vein with 5x105 BOEC. Staining for CD31 on tumor sections of BOEC injected mice demonstrated increased blood vessel density (BOEC 1982±1281, sham 328±229 (P<0.001)). In FsaII tumor model, systemic BOEC injection resulted in increased tumor volumes and higher tumor oxygenation when compared with sham injected animals. Injected via tail vein BOEC distributed initially throughout organs with high vascularization, but in 72 hours remained only in tumor, liver, spleen, and bone marrow. Given the ability of BOEC to survive within tumor vasculature, we designed BOEC to deliver endostatin and soluble VEGFR-1 (sFlt1) genes to the tumor’s endothelium. A retroviral MSCV vector was constructed to contain green fluorescent protein (GFP) and human endostatin gene (hES) or sFlt1. The hES or sFlt1 containing BOEC were evaluated for vWF, CD31, and VE-cadherin, and found positive. Transduced with endostatin or sFlt1 gene BOEC had a slower growth rate in culture, produced detectable endostatin (1.84 ng/ml) or sFlt-1 (665 pg/ml), and were able to inhibit MVEC growth in co-culture condition. When endostatin transduced BOEC were injected to mice with tumor tumor vascularization was decreased (EBOEC 290±172 versus BOEC 1982±1281(p<0.001)). This resulted in tumor volume reduction in mice injected with EBOEC (0.364±0.227cm3 versus sham 0.802±0.431cm3 (p=0.102)). This early data suggest that BOEC have the potential for tumor-specific delivery of cancer gene therapy.
Impact of Increased Body Weight on Acute Toxicity and Outcome of Autologous Stem Cell Transplantation for Non-Hodgkin’s Lymphoma: A Southwest Oncology Group Analysis.
Authors: Stiff Patrick , Unger Joseph , Forman Stephen , LeBlanc Michael , Miller Thomas , Press Oliver , Thompson John , Fisher Richard
Published: October 2019

Little is known about the optimal dosing of chemotherapy agents in patients significantly above their ideal body weight (IBW) who undergo high dose therapy with an autologous or allogeneic hematopoietic stem cell transplant. While dose attenuation is often advocated for overweight patients and appears to reduce acute toxicities, its effect on progression-free and overall survival (PFS/OS) is unknown. Due to a high relapse rate after autografts for relapsed/refractory NHL, SWOG has long investigated the use of augmented preparative regimens that utilize high-dose etoposide based on actual body weight (ABW) of 60 mg/kg, along with 12 Gy of TBI and cyclophosphamide (100 mg/kg) which is dosed on unadjusted IBW. We determined acute toxicities and PFS/OS using this approach in a recently completed study (S9438) of 358 patients undergoing autografts for relapsed/refractory NHL, comparing all grade 5 toxicities, grade 3–4 skin toxicities (known to be associated with high dose etoposide) and all other grade 4 toxicities in patients in this study based on % above IBW. The Devine formula for IBW with an adjustment for patients <5 feet was used. Patients at or below their IBW were dosed using their actual body weight. All patients received the preparative regimen as stated followed by autologous peripheral blood stem cells. Overall there were 31 patients with grade 5 toxicities. This group was a mean 42% above their IBW vs 24% for those surviving transplant (p=.001). Increasing % above IBW predicted grade 5 toxicity (p=.002). Even those at or 10% above their IBW had a higher toxic death rate (10.1 vs 3.6%; p=.04). While increasing weight above IBW did not predict for grade 4 lung, liver, cardiac toxicities or infections (p=.12), it did predict for grade 3/4 skin toxicities (p< .001). Skin toxicity, most commonly hand/foot syndrome was seen at rates up to 27.3% vs 7.3% for those ≥ 50 vs < 50% over IBW (p < .001). However, even those just ≥ 10% over IBW had nearly a 4 fold higher risk of skin toxicity (13.4 vs 3.6%; p=.005). While survival by % over IBW is confounded by comorbidities in overweight patients, we found no evidence of a different 2-year PFS or OS for overweight patients, even those ≥ 50% above IBW (PFS=42 vs 45%, p = .28; OS=52 vs 62%, p = .38). These multicenter trial data do establish a correlation of the ABW dosing of etoposide with significant skin toxicity. Without an apparent later PFS/OS benefit to the use of unadjusted etoposide dosing for overweight patients but higher acute skin toxicity and a higher early death rate, etoposide will be dosed using adjusted IBW [ IBW + .4 (ABW − IBW)] in subsequent studies of this regimen.
GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.
Authors: Strouboulis John , Rodriguez Patrick , Bonte Edgar , Krijgsveld Jeroen , Kolodziej Katarzyna , Guyot Boris , Vyas Paresh , de Boer Ernie , Grosveld Frank
Published: October 2019

GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.
Correction of β Thalassemia by Homologous Recombination in Embryonic Stem Cells.
Authors: Ryan Thomas M. , Sun Chiao-Wang , Wu Li-Chen , Ren Jin-Xiang , Townes Tim M.
Published: October 2019

Genetic correction of patient-derived embryonic stem (ES) cells is a powerful strategy for the treatment of hemoglobinopathies such as β thalassemia and sickle cell disease. One genetic strategy for the correction of β thalassemia is to replace mutant or deleted β-globin alleles with a wild-type gene by homologous recombination in ES cells. Thalassemic mice that mimic the disorder have been generated by targeted gene deletion of the adult murine β-globin genes (PNAS 92: 9259–9263). We derived ES cells from our β-globin knockout mice and produced genetically identical mutant mice by injecting the ES cells into tetraploid embryos. These cloned β thalassemic mice have a severe microcytic anemia characterized by a marked reduction of the erythrocyte mean corpuscular volume (MCV), hemoglobin level (Hb), and hematocrit (Hct), and a marked increase in reticulocytes and red cell distribution width (RDW) compared to cloned wild-type control animals. In contrast to the normochromic, normocytic erythrocytes of wild-type clones, erythrocytes in peripheral blood smears of β thalassemic mice were hypochromic and exhibit extreme anisopoikilocytosis. A targeting construct containing 8.7 kb of mouse homology flanking a human γ- and β-globin gene cassette and a hygromycin marker gene was electroporated into the β thalassemic ES cells. After selection, DNA from 48 ES cell colonies was analyzed by PCR to identify homologous recombinants. Nineteen colonies (40%) had correctly integrated the human globin genes into the deleted mouse β-globin locus. Correctly targeted cells were injected into tetraploid blastocysts to produce mice that are derived solely from the corrected ES cells. These cloned mice synthesize high levels of human β-globin polypeptide that corrects the α- to β-globin chain imbalance, thereby eliminating the thalassemic erythrocyte morphology. The MCV, Hb, Hct, RDW, and reticulocyte levels in the blood of these mice are normal. These results demonstrate that a severe hemoglobinopathy can be cured after targeted gene replacement of a mutant gene(s) with a wild-type allele by homologous recombination in ES cells.
Novel Immunomodulatory Drugs (IMiDs®): A Potential, New Therapy for β-Hemoglobinopathies.
Authors: de Parseval Laure Moutouh , Brady Helen , Glezer Emila , Richard Normand , Muller George , Morris Christopher L. , Stirling Dave , Chan Kyle
Published: October 2019

Sickle cell anemia (SCA) and β-thalassemia constitute a public health problem worldwide and new therapies are needed 1. Inhibition of hemoglobin S (HbS) polymerization is a major target for therapeutic approaches in SCA. New experimental therapies including hydroxyurea (HU) have attempted to augment the synthesis of fetal hemoglobin (HbF) and improve upon current treatment 2. We examined whether immunomodulatory analogs of thalidomide (IMiDs®) which have effects on hematopoiesis and are currently under evaluation for the treatment of hematological cancers could stimulate erythropoiesis and hemoglobin synthesis 3. Clinical trial results have demonstrated that Thalidomide and Revlimid™ reduce or even eliminate the need for red blood cell transfusions in some anemic myelodysplastic patients 4,5. We demonstrate here that CC-4047 (Actimid™) and CC-5013 (Revlimid™) are potent inducers of HbF in vitro and synergize with hydroxyurea during erythroid differentiation of CD34 progenitors isolated from human donors. In addition, we show by gene microarray profiling of CD34 differentiated cells that IMiDs® regulate specific erythroid genes that participate in hemoglobin synthesis. These results support that IMiDs®, alone or in combination with current approved therapies, can be a new approach to augment HbF synthesis, and have the potential to provide an innovative therapy for the β-hemoglobinopathies and other anemias.
Induction of Gamma Globin and Erythrocyte Anion Exchane Protein in BFU-E Cultured in the Presence of Hydroxyurea.
Authors: Green Gloria A. , Braden Beau R. , Mba Obianuju , Chivira Stacy A. , Ramezani Laleh , Johnson Cage S.
Published: October 2019

Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell hemoglobinopathy and beta-thalassemias. The clinical efficacy of HU is due primarily to increases in fetal hemoglobin (HbF) levels. HU increases the %HbF and the %F cells. The HU reactive mechanism(s) in erythroid cells, however, have not been clearly defined.

Patients receiving HU therapy develop subpopulations of macrocytic erythrocytes. Our previous studies demonstrate that sickle cell patients treated with HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. We propose that part of the HU reactive mechanism will include the upmodulation of non-gamma globin erythroid proteins that contribute to the macrocytic structures. As part of our investigation of the development of RBCs expressing increased band 3 protein per cell, we have examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors.

To investigate HU induced protein synthesis as a function of HU concentration, erythroid progenitors were cultured in semisolid media containing different concentrations of HU [0–40 micromolar] then assayed for AE1(band 3) and gamma globin. BFU-E were scored and harvested after 15 days in culture, then assayed. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry, these cells were then assessed by laser scanning confocal microscopy. Results show that the frequency of cells positive for band 3 protein was greater in colonies grown in hydroxyurea as compared to controls. The band-3 upmodulation appears to plateau at 12.5 micromolar HU. These cells were assessed for dual and single stains by laser scanning confocal microscopy. Both band-3 protein and spectrin were detected. Laser scanning confocal microscopy revealed spectrin in the majority of cells; both band 3 and spectrin were detected in forty percent of the cells cultured in 12.5 micromolar HU. Band 3 protein detected by Western blots was increased [1–1.5 fold] over untreated control BFU-E harvested during the same time period.

Secondly, the presence of band 3 protein and gamma globin in BFU-E was detected using two-color flow cytometry. BFU-E were cultured in increasing concentrations of HU. Colonies were permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were subsequently labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody. Results show 2–3 fold increase in the % band 3 plus gamma globin positive cells over untreated cells. Collectively, these results suggest that part of the mechanism of HU action in erythroid cells involves the induction of erythroid structural proteins concordant with the induction of gamma globin.
Serum Ferritin a Predictor of Iron Overload in Patients with Thalassemia and Sickle Cell Disease?.
Authors: Pakbaz Zahra , Fischer Roland , Gamino Richard , Fung Ellen B. , Harmatz Paul , Vichinsky Elliott
Published: October 2019

Introduction: Monitoring iron overload by serum ferritin in patients with hemosiderosis is still a routine practice although its limitations are widely studied and well known. Using non-invasive liver iron assessment by quantitative MRI or by biomagnetic liver susceptometry (BLS) with SQUID biomagnetometers would be the better alternative, however, these methods are available at only a few centers worldwide.

Objective: To determine the relationship between serum ferritin (SF) and liver iron concentration (LIC), measured by BLS at CHRCO, in patients with different types of hemosiderosis.

Methods and Patients: A total of 97 patients with thalassemia (TM: 3 to 52 y, 54% females) and 39 patients with sickle cell disease (SCD: 5 to 49 y, 60% female) were prospectively assessed for LIC and SF. Both tests were performed within 2 weeks of each other. Most patients with TM and SCD were chronically transfused, while 10 b-thalassemia intermedia (TI), 5 HbE/β-thalassemia (HbE), and 5 SCD patients were not on transfusion programs. LIC was measured by LTc SQUID biosusceptometer system (Ferritometer®, Model 5700, Tristan Technologies, San Diego, USA) under the standardized Hamburg-Torino-Oakland protocol. A non-parametric test (U-test) was utilized to analyze differences between SF and LIC data.

Results: In chronically transfused TM and SCD patients, the median SF and LIC were very similar (Table I). In TI&HbE patients, ferritin results were disproportionately low with respect to LIC. In order to improve prediction of iron stores by SF, the SF/LIC ratio was calculated. There was a significant difference between the median ratios of the two groups of transfused and non- transfused thalassemia patients, 0.82 vs. 0.32 [μg/l]/[μg/gliver], respectively (p < 0.01). In SCD patients the ratio is significantly (p < 0.01) higher.

Conclusion: Present data confirm ferritin to be a poor predictor of liver iron stores both in sickle cell disease and thalassemia. Relying only on ferritin to monitor iron overload in patients with hemosiderosis can be misleading, especially, in sickle cell disease and non-transfused thalassemia patients. Taking into account disease specific ferritin-LIC relations, could improve the prediction of iron stores. However, assessment of liver iron stores is the ultimate method to initiate and adjust chelation treatment in order to avoid progressive organ injury.

Table I. Median values and ranges ( − ) of serum ferritin (SF) and liver iron concentration (LIC) in transfused (Tx) and non-transfused (non-Tx) hemosiderosis patients.

Patient group n SF μg/l] LIC [mg/gliver ] SF:LIC Thalassemia Tx 82 1721 (209–8867) 3424 (364–7570) 0.82 (0.3–1.8) TI &HbE non-Tx 15 766 (52–2681) 2174 (226–5498) 0.32 (0.1–1.4) SCD Tx 34 2757 (400–9138) 1941 (518–6670) 1.2 (0.6–3.3)
Acute Hyperglycaemia Enhances Platelet Activation: Involvement of Multiple Mechanisms.
Authors: Li Nailin , Sudic Dzana , Forslund Mikael , Razmara Masoud
Published: October 2019

Diabetes mellitus is associated with platelet dysfunction, and hypoglycemic treatments may counteract this diabetic alteration. We thus studied how acute hyperglycemia influences platelet function. Fast blood (n=20) was incubated with different levels of glucose (5, 15, and 30 mM) for 5 min, and without or with in vitro stimuli. Platelet activation was monitored by whole blood flow cytometry, while platelet aggregation was also measured using impedance aggregometry. Blood glucose levels had little influence on unstimulated platelets. Hyperglycemia enhanced ADP- and TRAP-induced platelet P-selectin expression. Hyperglycemia also increased TRAP-induced platelet fibrinogen binding. High glucose levels mildly increased ADP- and TRAP-induced platelet-leukocyte aggregation. The blockade of protein kinase C (PKC) slightly attenuated ADP-induced, and markedly inhibited TRAP-induced platelet activation. PKC blockade had, however, little effect on hyperglycemia effect. Platelet P-selectin expression induced by the direct PKC activator phorbol 12-myristate 13-acetate (PMA) was not augmented by hyperglycemia. Superoxide anion scavenging by superoxide dismutase (SOD) reduced, whistle cyclo-oxygenase (COX) inhibition by naproxen did not reduced the enhancing effect of platelet activation by hyperglycemia. In conclusion, acute hyperglycemia enhances platelet activation in whole blood. Superoxide anions contribute importantly to hyperglycemia-enhanced platelet activation, while other mechanisms not identified presently also contribute to hyperglycemic effects.
Cyclosporin A as Immunosuppressive Treatment Option for Patients with Refractory Auto-Immune Thrombocytopenic Purpura.
Authors: Zver Samo , Zupan Irena P. , Andoljsek Dusan , Cernelc Peter
Published: October 2019

The treatment of auto-immune thrombocytopenic purpura (AITP) remains unsatisfactory in patients refractory to first-line management such as corticosteroids and/or splenectomy. Those patients usually require unacceptably high doses of corticosteroids to maintain a safe platelet count and thus have refractory AITP. Relativelly new immunosupressive treatment modality is cyclosporin A (CsA) and no large studies involving these drug have been conducted. We treated 7 refractory AITP patients with CsA. Their platelet count were without any therapy below 20x109/L and mostly they had signs of subcutaneous and mucosal bleeding. In 6/7 patients splenectomy was performed earlier. In order to maintain »safe« platelet count, they all needed methylprednisolon (MP), at least 32 mg/daily. So at the time treatment with CsA began, all patients were on MP. During next months MP dosage was tapered or withdrew, according to patients platelet count and treatment efficacy. At the endpoint, in 4/7 patients complete remission (CR) was achieved and later CsA was slowly ceased. 3/7 patients are currently in partial remission (PR) of the disease. Also one of them who has more than 30 years history of the disease is in stable PR, on low dose CsA and low dose MP maintanance therapy. Her platelet count is well above 20x109/L. Among CsA treatment related side effects painful lower limb edema was most frequent.

Based on our experience we suggest, that CsA should be recommended in refractory (chronic) AITP patients, because it shows long-term efficacy and good safety profile and is able to sustain remission at low doses or even after CsA discontinuation.
Production of Monoclonal Antibodies Against ADAMTS13 Determining Its Expression in Different Human Tissues.
Authors: Gao Weiqiang , Su Jiang , Bai Xia , Shen Fei , Ruan Changgeng
Published: October 2019

von Willebrand factor-cleaving protease/ADAMTS13 is a plasma metalloproteinase that degrades unusually large von Willebrand factor multimers (UL-vWF) derived from endothelial cells (ECs) and megakaryocytes (MCs) into small peptides circulating in blood. It is documented that transcript mRNAs of the protease are present in many human tissues; however, the protein expression of ADAMTS13 remains to be elucidated. In the present work, the gene of metalloproteinase domain of human ADAMTS13 was cloned into the multiclone site of pET28a(+). After induced by IPTG, the recombinant protein was purified using a Ni-NTA column and the Bal b/c mice were immunized with the protein. Screened with ELISA, three monoclonal antibodies against the metalloproteinase domain of ADAMTS13 were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belonged to IgG1 subclass. The quantity of them in ascites were 4 mg/ml, and their titers were as high as 1×10−5. The data of competitive ELISA showed that SZ-112 and SZ-113 recognized different epitopes of the recombinant protein. Western blot results demonstrated that SZ-112 not only reacted against the recombinant protein, but also recognized the full-length recombinant ADAMTS13 protein that expressed in CHO cell line (the vectors containing the ADAMTS13 cDNA sequences were provided by Prof. Sadler JE). The immunoprecipitation results showed that the two antibodies could react to an approximately 200 KDa protein in platelet lysate. Then, the expression panels of ADAMTS13 in human normal tissues were investigated using immunohistochemistry with the monoclonal antibodies. And the protease was found to be present in many kinds of tissues such as liver, spleen, ovary, prostate, bladder, small intestine, thyroid and thymus with significantly positive staining. The protease was also present in lung, uterus, large intestine and heart but stained weakly. We did not found the protease in brain. In most of these organs, the protease was expressed in epithelium of the tissues. While in liver, spleen and thymus, it was mainly presented in a subgroup of the solid tissue cells. Moreover, the preliminary results showed that the expression of ADAMTS13 slightly decreased in liver tissues of patients suffering form hepatitis type B and cirrhosis. In conclusion, our data indicated that two novel monoclonal antibodies against the metalloproteinase domain of human ADAMTS13 were successfully prepared, and the expression of ADAMTS13 in different tissue and specific locality might be associated with the regulation and function of the protease, which would contribute to the further research of the deficiency mechanism of the proteases in some disorders.
Relevance of Thrombophilic Risk Factors on Clinical Phenotype in Patients with Haemophilia A.
Authors: Krause Manuela , Hahn Anika , Peyvandi Ferrial , Scharrer Inge
Published: October 2019

Introduction: By assuming the hypothesis, that a high coinheritance of genetic haemostatic abnormalities influences the clinical phenotype in patients with coagulation disorders, we analysed the bleeding tendency in adult patients with haemophilia A.

Patients: A total of 49 patients with haemophilia A (severe:20 patients, moderate:12 patients, mild:17 patients; median age:44 years, range:22–78 years) and 80 sex-matched healthy subjects (median age:26 years, range:16–58 years) were studied in our haemophilia treatment center. Two of this patients have an inhibitor against factor VIII. 10 of 49 patients receive the factor VIII replacement therapy on demand. In addition to factor VIII activity, FV G1691A mutation and the FII G20210A variant were investigated.

Results: In our study the prevalence of FV G1691A was significantly higher among patients 6/49 (severe:5, moderate:1) than among control subjects 2/80 [12% vs 2.5%; p=0.03 OR 4.9]. We found no differences in the FII G20210A variant in 2/49 patient (moderate:1, mild:1) as compared to 2/80 among controls [4% vs 2.5%; p=0.62 OR 1.63 ]. In none of our patients both defects together were identified.

Within the last year the onset of bleeding was not different in thrombophilic haemophilia patients (n=8) 64 bleeding episodes/year compared with non-carriers (n=41) 284 bleeding episodes/year [8 vs 7 bleeding episodes/year/patient; p=0.72; OR 1.15].

Conclusion: The FV G1691A was found significantly more frequent in the thrombophilic haemophiliacs than in the general population. In our smal study group we could not demonstrate a relevant influence of the FV G1691A and FII G20210A on bleeding tendency. Further studies are needed to confirm whether FV G1691A and FII G20210A plays a role on the influence of bleeding tendency in haemophilia A patients.
von Willebrand Disease in Children with Glycogen Storage Disease Type Ia.
Authors: Yilmaz Aygen , Artan Reha , Akcam Mustafa , Kupesiz Alphan , Yesilipek M. Akif
Published: October 2019

Glycogen storage disease, Type Ia (GSD-Ia) is an autosomal recessive disease, characterized by hepatomegaly, hypoglycemia, hyperuricemia, and bleeding diathesis. Easy bruising and epistaxis are common and are associated with a prolonged bleeding time as a result of impaired platelet aggregation and adhesion. Two of four of our GSD-Ia patients were evaluated because of bleeding diathesis and both were diagnosed as von Willebrand’s disease (vWd) with normal platelet count, prothrombin time and activated partial thromboplastin time.

Case 1: Six year old boy diagnosed as GSD-Ia when he was 3 month of age and bleeding diathesis was first recognized 5th year of his life. His bleeding time was prolonged (15 minutes). Measurement of serum factor VIII-related antigen (vWf-Ag) revealed a decrease (% 31) and ristocetin cofactor activity was abnormal (% 37).

Case 2: Nine year old girl diagnosed as GSD-Ia when he was 6 month of age and bleeding diathesis was first recognized 4th year of his life. His bleeding time was prolonged (3.5 minutes). Measurement of serum factor VIII-related antigen (vWf-Ag) revealed a decrease (% 17) and ristocetin cofactor activity was abnormal (% 5.7).

Frequent diagnosis of vWd among our GSD-Ia patients might suggest alterations in membrane glycoprotein synthesis. There is no definitive explanation addresses how these alterations actually cause defective platelet aggregation. Although, the presence of association between these two separate diseases might be coincidental, in literature, diagnosis of vWd among GSD-Ia patients had been reported in only one paper. We conclude that GSD-Ia patients may have a metabolically acquired form of vWd as well as an acquired intrinsic platelet defect. Further molecular studies may be necessary to clarify the etiopathogenesis of this association.
Differential Immunodominance of Human and Porcine Factor VIII in Hemophilia A Mice.
Authors: Healey John F. , Parker Ernest T. , Barrow Rachel T. , Lollar Pete
Published: October 2019

Hemophilia A inhibitor patients and patients with acquired hemophilia A recognize immunodominant epitopes in the A2 and C2 domains of human factor VIII (fVIII). Hemophilia A mice also recognize A2 and C2 domain epitopes when immunized with human fVIII using a dosing schedule that mimics clinical use. We compared the immune responses of hemophilia A mice to human and porcine fVIII using a domain specific ELISA. In this assay, monoclonal antibodies are tested against a panel of six single human fVIII domain hybrid human/porcine fVIII molecules that contain the human A1, A2, ap, A3, C1 or C2 domains. With anti-human antibodies, a positive signal with one of the single human domain proteins identifies domain specificity, whereas loss of signal indicates domain specificity of anti-porcine fVIII antibodies. Exon16 (E16) - disrupted hemophilia A mice (n = 3) received six weekly μ10 g/kg intravenous injections of recombinant B-domain deleted human fVIII and a final 25 μg/kg boost. To obtain comparable inhibitor titers, E16 mice (n = 3) received six weekly injections of μ40 g/kg of recombinant B-domain deleted porcine fVIII. Spleens from high titer mice were fused with NS1 mouse myeloma cells and 485 of the resulting hybridomas were analyzed for fVIII domain specificity (Table). Only two hybridomas secreted antibodies specific for the ap domain. Human fVIII elicited a significantly greater number of antibodies to the A2 domain, whereas porcine fVIII elicited a significantly greater number of antibodies to the A1 and A3 domains (p < 0.01, chi square test). The greater number of anti-C2 antibodies to human fVIII was not significant at the 95% confidence level (p = 0.08). The differential immunodominance of human and porcine fVIII epitopes suggests that it may be possible to design a recombinant hybrid human/porcine fVIII molecule that is less immunogenic than human fVIII in the treatment of patients with hemophilia A.

Domain Specificity of Anti-FVIII MAbs

Mouse ID: Immunogen No. of MAbs A1 A2 A3 C1 C2 CR & MD CR: Cross Reactive MD: Multidomain 1- Human fVIII 95 2 16 2 7 21 23 & 24 2- Human fVIII 126 13 23 1 2 27 39 & 21 3- Human fVIII 54 1 15 2 1 10 9 & 15 4- Porcine fVIII 123 39 7 19 8 16 33 & 0 5- Porcine fVIII 27 13 5 0 0 4 2 & 3 6- Porcine fVIII 60 9 6 12 1 9 13 & 10
Portal Vein Thrombosis a Common Complication Following Splenectomy in Adults Patients with CLL or Myeloproliferative Disease.
Authors: Svensson Magnus , Wiren Mikael , Kimby Eva , Hagglund Hans
Published: October 2019

Elective laparoscopic splenectomy (LS) is performed with increasing frequency in relation to open splenectomy (OS). The advantage with LS is reduced morbidity. Moreover, this method is feasible also in patients with splenomegaly, a patient group with more postoperative complications as bleeding, infections and portal vein thrombosis. Portal vein thrombosis (PVT) is a rare but serious complication of splenectomy. We retrospectively reviewed the medical records of 69 consecutive patients splenectomized for haematological diseases during a five-years period (Jan 1999 to Dec 2003) at the Dep. of Surgery Huddinge University Hospital, with the aim of comparing the results and complications after LS and OS, with focus on thromboembolic events. The follow-up for all patients was performed Jan 31. 2004. Thirty-nine patients underwent LS and 30 OS. There were three conversions (7.7%) from laparoscopic to open surgery due to bleeding and splenomegaly. Accessory spleens were found in 16 of 69 (23%) patients, 6 of 39 (15%) in LS and 10 of 30 (33%) in OS. Thromboembolic complications were seen in seven patients; a) deep vein thromboses in the lower leg in two patients with ITP, both with LS and neither had received thromboprophylaxis, b) PVT in five patients, one after LS and four after OS; three with CLL, (two with a concomitant haemolytic anaemia) and two with a myeloproliferative disease. The five patients with PVT had all splenomegaly and had received thromboprophylaxis with low-molecular-weight heparin. PVT was diagnosed from day 6 to day 111 after splenectomy. Three of the five patients had thrombocytosis, 478, 740 and 1459x 10(9) at the time of PVT-diagnosis. In all the splenectomized patients, two patients had overwhelming post splenectomy sepsis (OPSI). One patient with Evans syndrom died of E. coli sepsis four months after splenectomy and one patient with myelofibrosis had severe pneumococksepsis 44 months after splenectomy. Both had recieved preoperative pneumocock vaccination. Further seven patients died during the follow-up period, five due to infections and bleedings, in all related to progressive malignant lymphoma. One patient died of sudden cardiac arrest 15 months postoperative, and one patient performed suicide. Conclusions: Portal vein thrombosis after splenectomy was seen in 13.5 % of patients with haematological malignancies despite thromboprophylaxis. Patients with splenomegaly and myeloproliferative disease or CLL with haemolytic anaemia have high risk of PVT. We recommend careful observation of postoperative thrombocytosis and extended thromboprophylaxis. Ultrasonography or CT should be considered in all patients with abdominal symtoms after splenectomy. Patients should receive pneumocockvaccination and be informed of the lifelong risk of severe infections.
Lymhoma of the Oral Cavity. A Rare Entity with Favorable Prognosis. Ten-Year Experience of a Single Hematology Unit.
Authors: Siakantaris Marina P. , Tzenou Tatiana , Vassilakopoulos Theodoros P. , Kyrtsonis Marie-Christine , Angelopoulou Maria K. , Dimopoulou Maria N. , Kokoris Styliani I. , Dimitriadou Evangelia N. , Korkolopoulou Penelope , Patsouris Efstratios , Tsaftaridis Panayiotis , Variamis Eleni , Laskaris George , Pangalis Gerassimos A.
Published: October 2019

Lymphomas of the oral cavity consist <5% of the malignancies of the region. B-cell lymphoma of the diffuse large cell histology is the predominant type (B-DLCL). The purpose of our study was to describe the clinicopathologic characteristics of a group of patients with oral cavity lymphomas and their treatment outcome. We reviewed the records of 46 patients (21 males and 25 females) with oral lymphomas who were diagnosed and treated in our Service, between 1993 and 2003. The mean age was 57 years and the most prevalent site of involvement was the tonsil (41.3%) followed by the tongue (15.2%), while 8.7% had >1 involved sites. Thirty six patients (pts) (78.3%) had clinical stage I or II and 10 (21.7%) advanced stage disease. A monoclonal lymphocytic population was detected in the blood of 7 pts (15.2%) by PCR (IgH or TCR rearrangement), 4 of them with early stage lymphoma. 56.5% of the lesions were diagnosed as B-DLCL and only 10.8% as peripheral T-cell lymphomas.

Nineteen pts with B-DLCL received CHOP or equivalent chemotherapy regimens and 11 CHOP combined with anti-CD20. Chlorambucil was administered in 4 pts with MALT lymphoma and local radiotherapy as the sole treatment in 1. The pts with T-cell lymphoma received CHOP (3) or interferon alfa-2b (2). Thirty eight pts are in complete remission. There were 3 deaths due to the disease (1 MALT, 1 predominantly small cleaved cell and 1 B-DLCL). Three additional pts relapsed and achieved complete response with a second chemotherapy course and 2 pts developed a lymphoma of different histology. In conclusion, oral lymphoma is a rare disease mainly of a diffuse large B-cell histology and a prolonged survival, when treated with chemotherapy (10-year Event Free Survival 82.5%).
Radiolabeled IL-2-Human Serum Albumin Fusion Protein (Albuleukin™) as a Potential New Reagent for Radioimmunoimaging / Therapy of Hodgkin’s Lymphoma.
Authors: Staak Jan O. , Colcher David , Wang Jianyi , Sung Cynthia , Raubitschek Andrew A.
Published: October 2019

Objectives:

CD25 (IL-2Rα) is overexpressed on the cell surface by a variety of malignant diseases including Hodgkin’s lymphoma (HL) and its surrounding lymphoid infitrate. A soluble form of this receptor (sCD25) may limit a targeting agent’s ability to bind to the tumor cells which may result in decreased therapeutic efficacy. Albuleukin is a fusion protein of recombinant native IL-2 and human serum albumin with a molecular mass of 82kD. Albuleukin possesses the immunomodulatory characteristics of IL-2 with a significantly extended circulatory half-life and better localization in lymphocyte rich tissues than IL-2. We investigated the targeting and pharmacokinetic properties of Albuleukin in a human CD25+ HL xenograft model as a potential novel radioimmunodiagnostic/-therapeutic agent.

Materials & Methods:

Albuleukin was conjugated with a DOTA bifunctional chelating agent. DOTA-Albuleukin was radiolabeled with 111In (specific activity 3μCi/μg; 91–100% labeling efficiencies). Human HL (L540) tumor bearing nude mice were injected i.v. with 111In-Albuleukin (5μCi) with and without unlabeled humanized anti-CD25 (daclizumab; 5μg 4h prior) to investigate its potential impact on pharmacokinetics and tumor targeting. Mice were sacrificed at various time points and tumors, blood and major organs were collected and analyzed for radioactivity. Serum samples were analyzed by size exclusion chromatography (SEC-) HPLC. Serial whole body γ-imaging studies (15μCi) were also performed.

Results:

Biodistribution and imaging studies of 111In-Albuleukin exhibited rapid blood clearance (4%ID/g at 24h), specific tumor localization (18%ID/g at 24h) and favorable tumor:blood ratios (22:1 at 48h). Adding anti-CD25 MAb did not alter the biodistribution profile or targeting of Albuleukin. No breakdown products of Albuleukin or formation of complexes between Albuleukin and sCD25 (with or without anti-CD25) were detected by SEC-HPLC in the mouse serum.

Conclusions:

Unlike small MAb fragments of comparable size, Albuleukin did not accumulate in metabolizing organs such as liver and kidneys. The low affinity of Albuleukin to sCD25 may explain the absence of immunocomplexes, as we have observed with the radiolabeled anti-CD25 alone, and unaltered biodistribution when unlabeled anti-CD25 was added. The good tumor targeting of the Albuleukin may be due to the involvement of the cell surface high affinity receptor including beta- and gamma-chains. The rapid blood clearance in concert with low normal organ uptake and good tumor targeting as well as its presumed low immunogenicity in humans makes 111In-Albuleukin a potential new reagent for radioimmunoimaging, and possibly for treatment of CD25+ lymphoma utilizing 90Y.
High Frequency of Aberrant Phenotype in Multiple Myeloma Patients Detected by Multiparametric Flow Cytometry.
Authors: Calheiros Luiz Arthur , Kimura Eliza Y.S. , Almeida Manuella S.S. , Chauffaille Maria de Lourdes L.F. , Bigonha Jandey G. , Sandes Alex F. , Barreto Wolney G. , Escaramuzi Carolina L. , Colleoni Gisele W.B. , Yamamoto Mihoko
Published: October 2019

Multiple Myeloma (MM) is a B cell lymphoproliferative disease with clonal plasma cell accumulation in bone marrow. Multiparametric flow cytometry (MFC) is an usefull tool to distinguish MM cells from normal plasma cells. Normal plasma cells are characterized by the expression of CD19+, CD45++, CD38++, CD138++, cytoplasmic immunoglobulin light chains (κ and λ) and CD56- while most MM plasma cells lose CD19, CD45 and gain CD56. In addition, many other antigens may be expressed by myeloma cells such as myeloid or lymphoid lineage associated antigens and these abnormal antigen expression is known as aberrant phenotype (AP). We studied 29 MM patients at diagnosis, in attempt to evaluate AP, it’s frequency and relation to prognostic parameters. The following monoclonal antibodies were used: CD45, CD38, CD138, CD56, CD19, CD20, CD22, CD10, CD13, CD14, CD33, CD117, CD28 and CD40, conjugated to FITC, PE, PerCP and APC) and acquisition / analysis were done through flow cytometer (FACS calibur, BD, San Jose) using CELL QUEST software (BD). Plasma cells were identified by the expression of CD38, CD138 and CD45 and the monoclonality confirmed by immunoglobulin light chain restriction. Our results showed presence of at least 2 AP in all cases : 2 AP (7 patients), 3 AP (12 patients), 4 AP( 5 cases), 5 AP (4 cases) and 8 AP in one case. The most frequent APs were CD45−, CD56+, CD117+, CD13+, CD33+, and were observed in 88% of patients. The most frequent AP association was CD56+/CD45− (40%), followed by myeloid antigen associated phenotypes (CD117, CD33, CD13). The lymphoid antigens expression was more observed in patients with large number of AP (>4 AP). CD56- patients presented serum β2-microglobulin and ionic calcium labeling levels higher than CD56+ patients (p=0,02) showing the usefulness of this antigen as prognostic marker. Morphological analysis showed that the majority (55%) of plasmablastic cases expressed >2 myeloid antigens against 18% of mature plasma cell morphology cases. These results allow us to conclude that MM express high frequency of AP, highlighting the importance of CD56 as a prognostic factor. MFC may be useful to the immunological detection of minimal residual disease in a great majority of MM patients and we suggest the panel CD45, CD56, CD117, CD33 and CD13 for this purpose, in addition to CD38 and CD138.
Selective Inhibition of JAK2 Driven Erythroid Differentiation of Polycythemia Vera Progenitors
Authors: Geron Ifat , Abrahamsson Annelie , Barroga Charlene , Kavelerchik Edward , Gotlib Jason R. , Hood John D. , Durocher Jeffrey , Mak Chi Ching , Noronha Glen , Soll Richard , Tefferi Ayalew , Kaushansky Kenneth , Jamieson Catriona H.M.
Published: October 2019

Myeloproliferative disorders (MPDs), diagnosed in 15,000 individuals annually, are characterized by overproduction of lineage-committed blood cells, thrombotic events, occasionally marrow fibrosis or progression to acute myelogenous leukemia and frequently a point mutation (V617F) in the JAK2 kinase. Mutant JAK2 expression results in erythrocyte overproduction in almost all cases of polycythemia Vera (PV). We investigated the molecular mechanisms driving erythroid skewed differentiation and the capacity of a selective JAK2 inhibitor (TG101348) to normalize PV progenitor differentiation. Both JAK2 V617F+ transduced cord blood and PV progenitor erythroid colony formation was potently inhibited by TG101348 (300 nM). Bioluminescent immunocompromised mouse transplantation studies revealed that TG101348 (150 mg/kg) treatment significantly reduced human erythroid engraftment by both PV and JAK2 V617F+ cord blood progenitors. Moreover, the imbalance between GATA-1 and PU.1 transcripts typical of JAK2 V617F+ progenitors normalized after treatment with TG101348. In addition, TG101348 inhibited both JAK2-mediated STAT5 phosphorylation and AKT-regulated GATA-1 phosphorylation in an erythropoietin responsive cell line suggesting a dual mechanism for the erythroid inhibitory effects of TG101348. Because of selective inhibition of JAK2 V617F+ progenitors both in vitro and in vivo, TG101348 may be an effective molecularly targeted inhibitor of JAK2 V617F+ driven MPDs in clinical trials.