bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Protein Z-Dependent Protease Inhibitor (ZPI) In Situ in Different Malignant Tumors.
Authors: Sierko Ewa , Ramlau Rodryg , Zimnoch Lech , Broze George J. , Wojtukiewicz Marek
Published: October 2019

Cancer progression is often associated with thromboembolic complications. Coagulation factors and inhibitors influence various processes involved in malignant tumor growth and metastatic dissemination. Recently a new coagulation inhibitor - protein Z-dependent protease inhibitor (ZPI) has been characterized. ZPI is a plasma proteinase inhibitor in the serpin superfamily. ZPI inhibits factor Xa activity in the presence of calcium and phospholipids. Protein Z augments its activity by more than 1000-fold. ZPI also attenuates the activity of factor XIa in the reaction which does not require the presence of calcium and phospholipids. ZPI inhibits the coagulation response prior to the formation of the prothrombinase complex. Up to now the data on the presence of ZPI in malignant tumors are obscure. The purpose of the study was to elucidate the solid phase interactions between various types of maligant tissues and ZPI that may contribute to tumor progression. The tissues from colon, breast, gastric, renal, pancreatic, endometrial carcinoma, non-small cell lung cancer (NSCLC) as well as from glial neoplasms were obtained at surgical resection during radical treatment. The patients undergoing surgery have not received any previous antineoplastic therapy. Tumor fragments were processed acc. to AMeX method and then embedded in paraffin. Immunohistochemical studies (avidin-biotin complex-ABC - technique) were performed using a monoclonal antibody against ZPI. Expression of ZPI was observed in cancer cell bodies of colon, breast, gastric, renal, endometrial, pancreatic cancer, NSCLC and glial neoplasms. The staining intensity for ZPI was irregular: both strong and weak expression of ZPI was observed. Moreover, various percentages of ZPI-positive cancer cells were revealed in different specimens of examined tissues. Macrophages and mucin also revealed expression of ZPI in the case of colon cancer. The presence of ZPI was demonstrated in the stroma of renal cancer. The obtained results suggest that ZPI being an inhibitor of coagulation might additionally play a role in the regulation of cancer progression.
The Effect of Platelet -, and Leukocyte Count on the Thrombelastograph® (TEG) Parameters.
Authors: Roeloffzen W.W.H. , Kluin-Nelemans J.C. , de Wolf Joost
Published: October 2019

Background. The TEG is used in situations were point of care testing of hemostasis is desired, although its value is still controversially because of insufficient test validation. The main parameters of the TEG are (a) the reaction time (R), the time until the initial fibrin formation and comparable with the coagulation times PT and APTT; (b) clotting time (K), the time until a fixed level of clot firmness is reached; (c) the angle (α) is closely related to K and measures the rapidity of fibrin build up and gives information about the clot strength; R, K and α are prolonged by anticoagulants and factor deficiencies; (d) maximum amplitude (MA) is a measurement of maximum strength or stiffness of the developed clot; it is especially influenced by platelets and fibrin.

Methods. We performed a multivariate analysis using the Cox multiple-regression model to study the effects of Leukocytes, Hb, and platelet count on the TEG parameters.

Results. Ninety native whole blood samples from 19 patients undergoing consolidation chemotherapy were studied; in the post chemotherapy phase in which platelets decreased from normal to < 10 x 109/l samples were taken; in all these cases PT, APTT and Fibrinogen were within normal limits. Platelets significantly influenced all parameters: R (p<0.001, r=−0.5), K (p<0.001, r=−0.7), α (p<0.001, r=+0.7), MA (p<0.001, r=+0.6) whereas Leukocytes influenced MA as well (p<0.001, r=0.3). In normal controls K is 9 ± 3 min (n=110), in patients with platelet count 50–100, 25–50 and <25 x 109/l K was resp. 17 ± 9, 30 ± 13 and 46 ± 10 min. In normal controls MA was 46 ± 7 mm, in patients MA became significant smaller with platelets < 25 x 109/l: 30 ± 5 mm. In patients with leukocytes ranging from 0–0.1, 0.1–1.0, 1.0–3.5 and > 3.5 x 109/l the MA was resp. 44 ± 14, 49 ± 15, 54 ± 9, and 58 ± 8 mm. As the MA is considered the parameter most influenced by platelet count, we calculated the sensitivity, specificity, pos and neg predictive value of MA to detect a platelet count less then 50 x 109/l, they were resp. 35%, 100%, 100% and 73%.

Conclusion. Firstly, platelet count not only influences MA but also the coagulation parameters R, K and α; besides leukocytes influences the clot strength; this is in agreement with the new conceptual cell based model of hemostasis; secondly, the TEG should be considered as additive to platelet count and plasmatic coagulation tests and not as a replacement.
Incidence of Extramedullary Relapse of Acute Myeloid Leukemia Following Transplantation with Busulfan-Based Conditioning Regimens.
Authors: Blum William , Penza Sam , Farag Sherif , Avalos Belinda , Marcucci Guido , Lin Thomas , Byrd John C. , Elder Patrick , Copelan Edward A.
Published: October 2019

Rates of extramedullary relapse are reported to be more than twice as high following transplantation with busulfan-based conditioning regimens (Simpson et al, Bone Marrow Transplantation 1998, Lee et al, Bone Marrow Transplantation 2000) compared to total body irradiation (TBI)-based regimens (Mortimer et al, J. Clin Oncol 1989) in acute myeloid leukemia (AML). Up to 50% of relapses following transplantation with busulfan-based therapies are reported to be extramedullary, with more than 25% as isolated extramedullary relapses. We analyzed 228 consecutive AML patients who underwent allogeneic (n=192) or autologous (n=36) transplantation following busulfan-based conditioning regimens between 2/84 and 12/03. One hundred twenty-three patients were in remission at the time of transplantation, 67 had relapsed disease, and 38 had never achieved remission. One hundred twenty-seven patients received BuCy, 77 received BuCyVP16, and 24 (all autologous) received BuVP16. All patients had a lumbar puncture demonstrating no evidence of leukemia, and all received intrathecal methotrexate prophylaxis prior to receiving busulfan. Seventy-nine patients (35%) relapsed (any site) at a median of 6 months (range, 1 to 44 months) after transplantation. Fifteen patients (7%) relapsed at extramedullary sites, including 7 in the CNS. Ten patients (4%) had isolated extramedullary relapses at a median of 13 months (range, 1 to 44 months) after transplantation. Four had isolated CNS relapses, and 4 had isolated cutaneous relapses. Of patients transplanted in remission, only 2 relapsed at extramedullary sites, including one patient with an isolated CNS relapse.

This retrospective study includes a substantially larger cohort of patients with longer follow-up than prior studies examining extramedullary relapse in patients with AML following transplantation with busulfan-based conditioning. In contrast to other reports, these results demonstrate that the incidence of extramedullary relapse is comparable to that reported with TBI-based conditioning regimens for patients with AML.
A Pilot Trial of Valproic Acid in Patients with Kaposi’s Sarcoma: A Multi-Center Trial of the AIDS Malignancy Consortium.
Authors: Lechowicz Mary Jo , Lee Jeannette , Dittmer Dirk , Krown Susan , Said Jonathan , Ambinder Richard F.
Published: October 2019

PURPOSE: Although the use of highly active antiretroviral therapy (HAART) has led to improvements in the management of AIDS-associated Kaposi’s sarcoma (KS), KS may persist or progress despite HAART. It has been hypothesized that activation of KS-associated herpesvirus (KSHV, HHV8) lytic expression might render tumor cells susceptible to immune surveillance by cytotoxic T cells. Alternatively, it has been suggested that lytic induction could lead to tumor progression. In vitro, valproic acid (VA) and other histone deacetylase inhibitors induce KSHV lytic gene expression in primary effusion lymphoma cell lines. We investigated VA in AIDS/KS patients to assess its safety and its impact on lytic viral gene expression in tumor and viral copy number in blood.

PATIENTS AND METHODS: VA was given orally to patients with AIDS and cutaneous KS on stable antiviral regimens; the dose was titrated to maintain trough concentrations between 50 and 100 mcg/mL. VA was given daily for 28 days followed by a rapid taper and patients were then followed for 6 months. Quantitative real time PCR was used to assess viral DNA in plasma and PBMC, and viral RNA in tumor specimens. Immunohistochemistry was used to assess viral antigen expression in tumor specimens.

RESULTS: 18 patients were treated. 15/18 patients completed therapy; 3 patients discontinued therapy early, one secondary to grade 2 toxicity and 2 to patient preference. One patient showed a partial response and 17 showed stable disease at the completion of therapy. No patients progressed during treatment. There were no differences between KSHV copy number in plasma or PBMC before, during, or after therapy. Similarly, although serial biopsies in some patients showed an increase in lytic gene expression, these changes did not achieve statistical significance. However, in multivariate analyses, viral lytic RNA increased in tumor biopsies on day 8 as a function of VA level. There was no change in HIV viral load with VA treatment.

CONCLUSION : VA was well tolerated in AIDS patients, was not associated with accelerated disease progression, but rarely induced tumor regression after short-term treatment. In patients who achieved the highest serum VA levels there was increased lytic viral RNA expression. These findings support investigation of more potent HDAC inhibitors over longer treatment courses in patients with AIDS-associated KS.
Kikuchi Disease in Korean Children: Clinical Features and Disease Courses.
Authors: Park Jun Eun , Jung Hyun Joo , Kim Chul-Ho , Hong Jeong , Han Jae Ho
Published: October 2019

OBJECTIVES: Kikuchi’s disease (KD) is a benign, self-limiting disorder of unknown origin with lymphadenopathy and fever, and prevalent in young Asian women. The initial presentations mimic malignancies or other serious infections, and the biopsy of lymph nodes affected is the only definite diagnostic tool of KD. We analyzed data from a series of pediatric cases collected at a single medical center and suggest proper guideline of management for patients with KD.

METHODS: The medical records of children under 16 years of age, who underwent lymph node biopsies and received histiopathologic diagnosis of KD at Ajou University Hospital, South Korea from January 2000 to June 2007, were retrospectively reviewed.

RESULTS: Twenty six pediatric patients with KD included 10 boys and 16 girls. In all cases except two, the cervical lymph nodes were involved. Twenty (76.9%) patients had fever as a presenting symptom, and 5 patients (19.2%) had various skin rash. Leukopenia was noted in 8 cases (30.8%), but there was no case of leukocytosis. The levels of ESR and LDH were slightly elevated, but CRP and liver enzymes were within normal range. No KD patients in this study developed any other autoimmune disorders during follow-up period. Eleven (55%) cases with fever improved immediately after excision biopsy of affected lymph nodes. Eight (30.8%) cases treated with steroid showed dramatic responses. Six patients (23.1%) had resolutions of symptoms simply with symptomatic treatments. The recurrence happened in 6 (23.1%) children within a few weeks from the first episode.

CONCLUSIONS: Contrary to previous reports on Asian cases, KD in Korean children is not rare in young boys, and fever is more common presenting symptoms. It’s relationship with autoimmune diseases or infection of EBV is not clear. Symptoms such as fever improved in some cases only by the removal of affected lymph node for excision biopsy. In agreement with previous reports, patients treated with steroid recovered dramatically. Therefore, we suggest an earlier excision biopsy for patients suspicious with KD and an immediate treatment of cases with severe symptoms with steroid. The recurrence rate in this study was higher than that in previous studies, therefore, a spell of follow-up period after disappearance of symptoms is advisable for patients with original diagnosis of KD.
The Brain Transcription Factor OTX1 Is Activated in Specific Non-Hodgkin Lymphoma Subtypes and Normally Expressed in a Germinal Center-Restricted Small Subpopulation of CD138+ Plasmacell-Like Cells.
Authors: Russo Filippo , Omodei Daniela , Acampora Dario , De Filippi Rosaria , Severino Valeria , Di Francia Raffaele , Frigeri Ferdinando , Mancuso Pietro , De Chiara Anna , Franco Renato , Casola Stefano , Pinto Antonio , Simeone Antonio
Published: October 2019

OTX1 and OTX2 are transcription factors containing a bicoid-like homeodomain and play crucial roles in brain development and in few other body districts as in the case of OTX1, which is required in hematopoiesis for development of the erythroid compartment. Previous studies indicated that in human OTX2 is functionally involved in the generation of anaplastic medulloblastomas; OTX1 expression strongly correlates with nodular/desmoplastic medulloblastomas; and none of them is expressed in other brain tumors. Here we have investigated whether OTX1 and/or OTX2 may potentially be involved in tumors of nonneural origin by focusing on human B-cell non-Hodgkin’s lymphomas (NHLs). We analyzed 184 cases (fig 1)encompassing different NHL subtypes according to the World Health Organization classification and discovered that, while OTX2 expression was never detected, OTX1 was selectively activated in virtually all cases of Diffuse Large B Cell Lymphoma, Burkitt Lymphoma and a relevant fraction of Follicular Cell Lymphomas. In DLBCL lymph nodes and tissue infiltrations, OTX1 protein was detected in the nucleus of PAX5+-CD20+ cancer cells. Conversely, in nonmalignant lymph nodes and tonsils, OTX1 was expressed in a numerically very small and non-proliferating cell population restricted to the germinal center. These OTX1+ cells were CD138+-IgG+, about 50% of them also co-expressed both CD10 and CD20 and none of them exhibited PAX5 or BCL6 expression, thus suggesting that OTX1 expression identifies a subset of GC-restricted plasmacell-like cells (fig 2). Interestingly, in contrast with lymphomas, in these cells the OTX1 protein was prevalently localized to the cytoplasm. Collectively, our data suggest that activation of OTX1 expression and its nuclear localization represent together a newly described molecular event occurring in specific NHL subtypes of GC and early post-GC origin (FCL, DLBCL, BL), and lead us to hypothesize that it may be potentially involved (directly or indirectly) in the initiation and/or maintenance of these tumors.

Figure Figure Figure Figure
Discrimination Indices as Screening Tests for Beta-Thalassemic Trait.
Authors: Ntaios George , Chatzinikolaou Anastasia , Saouli Zoi , Girtovitis Fotios , Kaiafa Georgia , Kontoninas Zisis , Agapakis Dimitrios , Savopoulos Christos , Papadopoulos Athanasios
Published: October 2019

Introduction: The two most frequent microcytic anemias are β-thalassemic trait (β-TT) and iron deficiency anemia (IDA). Several discrimination indices have been proposed in order to distinguish between these two conditions. These indices are derived from several simple red blood cell indices, like RBC count, mean cell volume and RBC distribution width (RDW), as these are provided by electronic cell counters. Purpose of the study is to examine the diagnostic accuracy of six discrimination indices in the differentiation between IDA and β-TT.

Methods: The six discrimination indices that were examined were: Mentzer Index, Green & King Index, RDW Index, England & Fraser Index, RDW and RBC count. We calculated these indices on 373 patients (205 males, 168 females) with β-TT and 120 patients (50 males, 70 females) with IDA, as well as their sensitivity, specificity, positive and negative prognostic value, efficiency and Youden’s index.

Results: G&K shows the highest reliability, followed by E&F, RBC count, MI and RDWI. On the contrary, RDW completely failed to differentiate between IDA and β-TT. G & K proved to be the most reliable index, since it had the highest sensitivity (75.06%), efficiency (80.12%) and YI (70.86%) for the detection of β-TT.

Conclusion: These six discrimination indices cannot be relied on for a safe differential diagnosis between β-TT and IDA. They do have high specificity, but their sensitivity for the detection of β-TT is not satisfactory. Consequently, they cannot be used neither as a screening tool for β-thalassemic trait, since they could result in a significant number of false negative results.
Spectrum of Thromboses in Patients with Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms: Continuing Controversy.
Authors: Sullivan Kevin , Kempin Sanford , Li Zujun , Cook William , Marionneaux Steve
Published: October 2019

Thromboembolic disease is common with an annual incidence of 1–3/1000 individuals. Both acquired and genetic risk factors are responsible. A risk factor for thrombosis can now be identified in over 80 percent of patients. Fifty percent of thrombotic events in patients with inherited thrombophilia are associated with an acquired risk factor. Patients with multiple inherited risk factors are at a greater risk. Two common variations of the MTHFR gene (677CàT and 1298AàC) result in amino acid substitutions and enhanced thermolability of the enzyme. The latter, in the presence of folic acid deficiency, results in elevated homocysteine levels. Thrombosis has been observed in these patients, but there is still controversy whether there is a direct association (Bezemer I, et al. Arch Int Med167, 497–501, 2007; den Heijer M et al. J Thromb Haemost3:292–299, 2005). In the present study, we describe the spectrum of thromboembolic disease observed in 39 patients with a variety of MTHFR polymorphisms referred to a specialty center with a personal or family history of thromboses or obstetrical loss, 18 of these patients having no other risk factor other than MTHFR polymorphisms. These polymorphisms include 18 heterozygotes (11 with 677CàT; 7 with 1298AàC), 9 homozygotes (5 with 677CàT; 4 with 1298AàC) and 12 compound heterozygotes (677CàT and 1298AàC). Concomitant risk factors were found in twenty-one patients (factor V Leiden - 6; prothrombin G20210A - 3; protein C deficiency - 1; protein S deficiency - 1; PAI-1 polymorphism - 2; activated protein C resistance - 2; antiphospholipid antibody syndrome - 5; malignancy - 3; coronary artery disease - 1; hemolytic anemia - 1; factor VIII deficiency - 1). Types of thromboembolic manifestations observed in the 39 patients include venous thromboembolism (22), obstetrical loss (7), arterial thrombosis (3), myocardial infarction (3), ischemic CNS lesions (2), and palpable purpura (1), for a total of 38 manifestations. Seven of the patients had a family history of thromboembolic disease. Patients with only MTHFR polymorphisms suffered 16 types of thromboembolic events (16/38=42%). Our observations suggest that MTHFR polymorphisms alone do play a role in clinically relevant thrombotic disease and probably increase the incidence of these diseases in patients harboring other causes of thrombophilia. Therefore, continuing surveillance for this polymorphism as part of a clinical thrombophilia assessment is recommended, particularly if homocysteine levels are elevated.
Correlation of Thrombosis and Prothrombotic Markers with Outcome in Lung Adenocarcinoma Patients: A Prospective Study.
Authors: de Meis Ernesto , Zamboni Mauro M. , Pinheiro Vera R. , Guedes Maria T.S. , Lima Luize G. , Castilho Ilda A.M. , Sousa Amanda F. , Silveira Neura P.R. , Loures Maria A.L. , Levy Roger A. , Monteiro Robson Q.
Published: October 2019

Background: It is largely accepted that cancer patients have a high incidence of thrombosis which is strictly related to prognosis of cancer. Aim: We prospectively evaluated 81 consecutively lung adenocarcinoma patients in order to identify prothrombotic factors and thrombosis incidence.

Methods: Patients were submitted to Doppler examination of arms and limbs every three months or in case of thrombosis suspicion. Additional image evaluations were made in case of inconclusive diagnosis. Levels of C protein, platelets, and fibrinogen, factor VIII and IX, D-dimmer, antitrombin and apoptotic microparticles were evaluated each six months. Samples were also tested for lupus anticoagulant (LA) antibodies and levels of IL-1, IL-6 and Tumor Necrosis Factor (TNF). A group of 30 healthy blood donors was used as control.

Results: From the studied patients 21% had thrombosis, 38% had fibrinogen above 500 mg/dl, 42% had FVIII activity above 200%, 63% had increased D-dimmer, 60% had more than 1x106 microparticles/ml serum, and none of them have decreased levels of C protein or antitrombin. A small fraction of patients had increase of platelet counts. At immunological evaluation 52% of patients had LA detection and 63% showed increased leves of IL-6. Patients with at least one of the variables thrombosis, high levels of IL-6 (p<0.001), FVIII (p=0.05) and LA p=0.02) detection had a worst prognostic. Patients with increased FVIII levels (p=0,05) or LA detection (p=0,028) showed a significant increase in the thrombosis risk, whereas patients with more than 1x106 microparticles/ml showed only a tendency to increased risk of thrombosis (p=0,09).

Conclusion: There is a high incidence of thrombosis in lung adenocarcinoma patients, on which FVIII and LA significantly influenced and microparticles only showed a tendency. However, presence of LA, thrombosis or increased levels of IL-6 or FVIII showed directly interfered with patient’s survival.
Venous Thromboembolic Events in Women Using the NuvaRing®.
Authors: Shum Merrill K. , Rajagopalan Kumar , Lachant Neil A.
Published: October 2019

Venous thromboembolic events (VTEs) are serious adverse effects seen in some women using oral contraceptives. Although the novel vaginal ring, (NuvaRing®) has been reported to be effective and well tolerated, the risks of VTEs are unknown. We report 3 women who developed potentially life-threatening VTEs (2 with pulmonary emboli and 1 inferior vena cava thrombus) while on the NuvaRing®. VTEs occurred 22 days, 3 months and 3 months after NuvaRing® insertion. None had a personal or family history of VTEs. Extensive thrombophilia evaluation showed 1 with heterozygous factor V Leiden and 2 with a transient lupus anticoagulant. 2 had a personal history of multiple miscarriages and all 3 had an elevated body mass index > 26. Searching Pubmed, Ovid and Medline, there have been 10 other reported cases of VTEs, in women using the vaginal ring (see table below). Risk factors reported by other authors include anticardiolipin antibodies, protein S deficiency, heterozygous prothrombin 20210, heterozygous factor V Leiden and prolonged air travel. With limited follow up, the true incidence of VTEs in women using the NuvaRing® is difficult to determine. However, in 2005 the Swedish Medical Products Agency reported a relative incidence of 5.3 cases per 10,000 women years for VTE complications. In summary, we report 3 women who had life-threatening VTEs associated with the use of the NuvaRing®. Longer follow-up of safety studies and post-marketing adverse event reporting are needed to determine VTE risks and incidence while using the NuvaRing®.

Studies reporting VTEs in women using the NuvaRing® Source Number of patients VTE risk factors Family history Site Bjarnadottir et al, 2002 1/121 Unknown Unknown LE DVT Tuppurainen el al, 2004 1/40 Unknown Unknown LE DVT Oddsson et al, 2005 1/512 Unknown Unknown LE DVT Miller et al, 2005 1/429 Anticardiolipin antibodies, protein S deficiency Grandmother with DVT LE DVT Ahrendt et al, 2006 1/499 Double heterozygous factor V Leiden/prothrombin 20210, prolonged air travel Unknown LE DVT Roumen et al, 2006 1/854 Unknown Unknown Cerebral sinus thrombosis Samuelsson et al, 2007 1 Heterozygous prothrombin 20210 None Axillary vein DVT Swedish Medical Products Agency, 2005 4/? Unknown Unknown DVTs Shum et al, 2007 3 Obesity (3), Personal history of miscarriages (2), heterozygous factor V Leiden (1) 1 sister with miscarriage Bilateral pulmonary emboli (2), Iliac vein-IVC thrombus (1)
A NUP98-HOXD13 Fusion Gene Induces a Transplantable Myelodysplastic Syndrome in Mice.
Authors: Chung Yang Jo , Choi Chul Won , Slape Christopher , Fry Terry , Aplan Peter D.
Published: October 2019

The myelodysplastic syndromes (MDSs) are a group of hematologic stem cell disorders characterized by ineffective hematopoiesis and dysplasia. A large number of chromosomal aberrations including deletions, amplifications, inversions, and translocations, some of which involve the NUP98 gene, have been associated with MDS. Recently an MDS mouse model expressing a NUP98-HOXD13 (NHD13) fusion gene was developed, which faithfully recapitulates all of the key features of MDS. Although it is well-established that acute myeloid leukemia (AML) is transplantable, there is no evidence that MDS is a transplantable condition. Therefore, in order to develop evidence for MDS as a hematopoietic stem cell (HSC) disease, we attempted to transfer MDS to normal recipients through bone marrow transplantation (BMT). All the recipients transplanted with bone marrow (BM) cells from NHD13 mice with MDS showed anemia, leukopenia, lymphopenia, and neutropenia when compared to recipients of wild-type (WT) littermates. The homing efficiency of the NHD13 primitive progenitor cells (Lineage negative [Lin−], Sca-1+) was about 2 fold higher than WT, and there was no significant difference in BM cellularity between the recipients of NHD13 and WT BM, indicating that the NHD13 recipients had ineffective hematopoiesis. These phenomena were reproduced in secondary recipients using primary recipients of NHD13 BM as donor mice. In secondary transplantation assays, 3 out of 5 recipients developed acute myeloid leukemia (AML) at 16 weeks post-transplantation. Morphological features of MDS, including nuclear-cytoplasmic asynchrony, binucleate cells, hypersegmented neutrophils, and giant platelets were detected in BM and peripheral blood of NHD13 donor, primary and secondary recipients by cytospin preparations. In competitive repopulation assays, mice transplanted with equal numbers of WT and NHD13 BM cells showed a decreased percentage of NHD13 cells in the peripheral blood, but an increased percentage of NHD13 cells in the BM, again providing evidence of ineffective hematopoiesis of the NHD13 cells. The transplantation of lineage depleted cells from BM has shown that the transplantable cells for MDS reside in the Lin− population of NHD13 BM. These findings demonstrate that MDS can be transferred to healthy recipients by BMT, supporting the concept that MDS originates in a transplantable multilineage hematopoietic stem cell.
Universal Thrombophilia Screening with Risk Stratification and Anticoagulation Management in Pediatric Renal Transplant Recipients.
Authors: Saxena Kapil , Turman Martin , McNall-Knapp Rene Y. , Rianthavorn Pornpimol , Kropp Bradley P. , Sindhwani Puneet , Meyer William H.
Published: October 2019

Objective To identify renal transplant patients at a higher risk for allograft thrombosis and develop a risk stratification protocol for post-operative anticoagulation.

Background According to the North American Pediatric Renal Transplant Cooperative Study data, vascular thrombosis is responsible for 12% of renal transplant failures. Congenital and acquired prothrombotic disorders have recently been found to play a major role in defining outcome of the renal transplants.

Methods In 2007, we initiated universal screening for acquired and congenital thrombophilic risk factors for all renal transplant patients prior to transplant. They were assigned a risk category (Level I-IV) based on the presence of thrombophilic risk factors and their post-operative anticoagulation plan was pre-determined based on their risk level (Table). Low-dose unfractionated heparin drip was initiated a few hours after the transplant and titrated to achieve a 1.5 fold aPTT level. Anticoagulation was changed to low molecular weight heparin (LMWH) on day 2–4, when the risk of major bleed was over, and the serum creatinine had normalized.

Result Nine patients have been screened and 8/9 were found to be at high risk for thrombosis (Level II or III). The prothrombotic risk factors identified were Prothrombin gene mutation (2), MTHFR (8), Lupus anticoagulant (2) and low protein S (2). Five patients have undergone renal transplant, and have been treated with LMWH post-operatively as per plan, with successful outcome. No patient has developed allograft thrombosis. 1/5 patients developed a peri-renal hematoma requiring evacuation, but completed prophylactic LMWH therapy.

Conclusion Renal allograft thrombosis is preventable. Universal screening can stratify risk and allow an effective intervention to reduce that risk. Our Institution has developed a unique risk stratification protocol to identify patients at high risk for allograft thrombosis and prevent this complication by post-operative anticoagulation.

Risk Categorization and Management Plan Risk Level Thrombophilia Management Level I Evidence of Thrombophilia (other than MTHFR) and previous thrombosis Heparin postoperatively and then LMWH bid for 6 months Level II Evidence of Thrombophilia (other than MTHFR), No history of thrombosis Heparin postoperatively, LMWH bid for 3 months, then qd for 3 months Level III MTHFR with normal homocysteine Heparin postoperatively, then LMWH qd for 3 months Level IV No evidence of thrombophilia No anticoagulation
HLA B*1402, Cw*0802 Haplotype and DRB1*1501 Allele Are Significantly Increased in Italian Paroxysmal Nocturnal Hemoglobinuria Affected Patients.
Authors: Alfinito Fiorella , Cosentini Elena , Camerlengo Rosa , Terrazzano Giuseppe , Sica Michela , Risitano Antonio , Costagliola Stefania , Pepa Roberta Della , Palladino Carmela , Luzzatto Lucio , Rotoli Bruno , Notaro Rosario , Ruggiero Giuseppina
Published: October 2019

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder characterized by the emergence and expansion of a GPI-defective clonal haematopoiesis. Its clinical features are haemolytic anaemia, cytopenia and thrombosis. Several data suggests the pathogenetic relevance for auto-immune-mediated mechanisms in the expansion of the mutated clone. Since HLA genes represent a susceptibility factor for autoimmunity, we have investigated HLA genotypes in 42 Italian patients with PNH. This study analyzed the HLA class I and class II genes in a cohort of 42 Italian PNH patients. Data indicate the occurrence of a significant increase of A*33 (9.5% in PNH patients versus 2.3% in controls; p<0.05), A*0201 (52.5% versus 32.2%; p<0.05), B*1402 (23.8% versus 5.3%; p<0.0005) and Cw*0802 alleles (23.8% versus 5.9%; p<0.001) in the PNH patient cohort, as compared with a population of 301 healthy controls of the same ethnical origin. In addition, an increased frequency of DRB1*1501 (21.1% versus 5.3%; p<0.005) and DRB1*01 (31.6% versus 10.9%; p<0.005), without specific association with the DRB1*0101 or DRB1*0102 allele, has been revealed. Notably, the B*1402, Cw*0802 (23.8% versus 5.3%; p<0.0005) HLA -B,-C haplotype was significantly increased. Moreover, the A*33, B*1402, Cw*0802, DRB1*0102 HLA-A, -B, -C, -DR haplotype shows a fifteen times increased frequency in PNH patients, if compared with controls (10.5% versus 0.66%; p<0.005). HLA polymorphism represents a main autoimmune susceptibility factor. In this context, our results suggest that specific peptide/s could be relevant for the selection/dominance of PIG-A mutated clones in our cohort of Italian PNH patients. Alternatively, the linkage of the described allele/s and haplotype with not yet characterized genes and/or with gene/s likely involved in pro-inflammatory cytokine productopn might be hypothesized. The association between certain HLA types and the occurrence of PNH may provide further insight into the mechanism for selective damage to normal (GPI+) hematopoiesis, which is responsible for the clonal expansion of GPI-defective hematopoiesis that ultimately gives rise to clinical PNH.
Identification of Target Genes of an Erythroid Transcription Factor Complex Containing SCL (TAL1).
Authors: Jim Johanna H. , Dhami Pawandeep , King Amanda , Cooper Jonathan , Vetrie Dave
Published: October 2019

Haematopoiesis is the process whereby haematopoietic stem cells give rise to mature blood cell lineages. The SCL (TAL1) gene, originally identified by chromosomal translocations associated with T-cell acute lymphocytic leukaemia, encodes a key transcription factor (TF) which is expressed in various blood lineages and is essential for haematopoietic development. It has been shown that the SCL protein forms a multi-protein complex during erythroid development with other TFs (GATA1, E2A, LDB1, and LMO2) which binds to a sequence-specific motif to regulate the expression of glycophorin A and c-kit. We have used two complementary approaches to identify novel target genes regulated by this TF complex during erythroid development. In the first approach, we have transfected short interfering RNAs (siRNAs) into the K562 cell line to knockdown transiently the TFs of the erythroid complex. For all members of the complex, a knockdown efficiency of at least 70% was confirmed at the mRNA and protein level within 48 hours after transfection. The consequences of the knockdown at the level of gene expression were observed using Affymetrix GeneChips in order to identify downstream events associated with the erythroid complex in transcriptional programmes. In the second approach, chromatin immunoprecipitation (ChIP) was performed for each member of the complex in the K562 cell line and the ChIP material hybridised to a human transcription factor promoter microarray. A number of novel target genes for the SCL erythroid complex have been identified and verified independently using both approaches. Our data shows that members of the erythroid complex are involved in auto-regulation and regulate genes which control chromatin structure and function. These findings demonstrate that the expression of this TF complex is tightly controlled and point to an important role for it in orchestrating fundamental biological processes which have profound effects on gene expression in erythroid development.
Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.
Authors: Qiao Zhenhua , Zhao Xiujuan
Published: October 2019

Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA).

Methods:

MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen.

Results:

There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality.

Conclusions:

morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.
Absence of Oncogenic Transformation in Long-Term Culture Mesenchymal Cells from Bone Marrow of Children.
Authors: Choumerianou Despoina M. , Dimitriou Helen , Perdikogianni Chrysoula , Martimianaki Georgia , Pelagiadis Iordanis , Kalmanti Maria
Published: October 2019

Mesenchymal stromal cells (MSC) have attracted considerable interest in both the scientific and clinical fields because of their potential to regenerate tissues and support hematopoiesis. Although laboratory isolation of MSC is established, the lack of an MSC specific marker and their low frequency in bone marrow necessitates their in vitro expansion in order to obtain a sufficient cell number for applications. During this process their characteristics may be altered and cells may acquire oncogenic properties or abnormal proliferative capacity. We investigated the effect of in vitro expansion on MSC, isolated from BM of children with idiopathic thrombocytopenic purpura (ITP, n=10) and autoimmune neutropenia (AIN, n=6). MSC were maintained in culture for a number of serial passages (P1–P6). We studied the expression profile of tumor suppressor genes (p53, p16, Rb) and the oncogene (H-Ras) along with hTERT, involved in malignant transformation at different passages. The cell line HUVEC was used as normal control for adherent cells and the M5 as positive control for hTERT expression. P2-MSC and P6-MSC (n=9) were grown on soft agar to assess their anchorage dependence. P2-MSC showed normal, relative to GAPDH, expression of p53, p16, Rb and H-Ras (ITP: 0.37, 0.2, 0.53, 0.64 respectively and AIN: 0.41, 0.27, 0.62, 0.75 respectively) compared to HUVEC cells (0.5, 0.23, 0.73, 0.93, respectively) which remained stable upto P6 (ITP P2vsP6: p= 0.63, p= 0.63, p= 0.82, p=0.33, AIN P2vsP6: p= 0.85, p= 0.07, p= 0.14, p=0.07). In all samples, hTERT expression was undetectable both at P2 and P6, and moreover none of the samples showed anchorage independent growth. These findings suggest that expression profile of oncogenes and tumor suppressor genes does not change during in vitro expansion of MSC. Importantly, the similar transcript levels between MSC and HUVEC, the absence of hTERT expression and anchorage independent growth suggest that MSC may not be transformed for a number of passages. In conclusion, although in vivo models are required, such properties encourage MSC use in clinical practice and should be considered as candidates for therapeutic application.
Fog1 and Cebpa Are DNA Targets of GATA-1/PU.1 Antagonism during Leukemia Differentiation.
Authors: Burda Pavel , Curik Nikola , Kokavec Juraj , Pospisil Vit , Skoultchi Arthur I. , Zavadil Jiri , Stopka Tomas
Published: October 2019

PU.1 and GATA1 are hematopoietic lineage-specific transcription factors that play key roles in normal myeloid and erythroid differentiation respectively. Inappropriate expression of PU.1 in proerythroblasts causes its binding to GATA-1 on DNA resulting in a block of erythroid differentiation and development of murine erythroleukemia (MEL) (Rekhtman 1999). Activation of a conditional transgene of GATA-1 (fused with the ligand binding domain of the estrogen receptor, ER) in MEL cells disrupts PU.1-mediated repression in chromatin leading to re-intiation of erythroid differentiation and cell cycle arrest (Choe 2003, Stopka 2005). In this study we show that MEL cells can also be induced to express myeloid differentiation programs upon PU.1-ER activation. Gene expression microarray analysis of GATA-1-ER MEL cells and PU.1-ER MEL cells treated with ER activators allowed us to identify mRNAs that are regulated by both GATA-1 and PU.1 including the set of GATA-1 targets repressed by PU.1, as well as the set of PU.1 targets repressed by GATA-1 in MEL cells. The targets of mutual antagonism of PU.1 and GATA-1 consisted of lineage specific transcription factors, differentiation markers and genes that cause cell cycle arrest and antiapoptotic regulators previously associated with myeloid and erythroid cell differentiation. To determine if PU.1 and GATA-1 directly regulate the lineage specific transcription factor genes, we performed chromatin immunoprecipitation (ChIP) and analyzed the ChIP samples on microarrays and by qPCR. We found that PU.1 and GATA-1 are localized near PU.1 binding sites in the genes for myeloid transcription factors Cebpa and Cbfb and near GATA-1 binding sites in the genes for erythroid transcription factors Fog1 and Nfe2. In addition, further ChIP experiments delineated chromatin architecture near the binding sites for PU.1 and GATA-1 including histone H3 content and acetylation of histone H3K9. We propose that the mutual antagonism of PU.1 and GATA-1 in inhibiting the respective differentiation programs is rendered through specific changes in chromatin structure around lineage specific transcription factors genes. These changes may contribute to the block to differentiation evident in leukemias.
The t(14;18) Is Associated with Germinal Center Phenotype Subset of the Diffuse Large B-Cell Lymphoma Patients in Egypt.
Authors: Abdel-Ghaffar Hasan A. , Abdel-Aziz Sherin M. , Shahin Doaa A. , Board Ezzat S. Sobki , Attwan Nadia I. , Al-Tonbary Youssef A. , Abraham Nader G.
Published: October 2019

Diffuse large B-cell lymphoma (DLBCL) is a generic term for clinically and biologically heterogeneous group of tumors. Identification of high risk patients at presentation will allow effective trials of treatment. Therefore, t(14;18) detection using interphase Florescence in Situ Hybridization (FISH) and Biomed multiplex polymerase chain reaction (PCR) was done on formalin fixed paraffin embedded lymph node archives from pathology department, National Cancer Institute, Cairo, Egypt. Diagnosis were confirmed by pathological review using the diagnostic criteria defined in the revised European-American Classification of Lymphoid Neoplasm / WHO classification. The study was carried out on 26 patients with lymph screen CD 19 +/ CD 5 - / CD 10 ± correlating t(14;18) with the immunophenotypic biological variables, Immunohistochemistry, and the standardized international prognostic index (IPI) with a median follow up for 5 years. Comparison of FISH and PCR techniques showed identical specificity with advantageous sensitivity of FISH over the PCR. Nine patients out of eleven with t(14;18) were associated with Germinal Center (GC) phenotype (CD10+ /Bcl-6 +). However, Only two out of fifteen with non GC phenotype(CD10- /Bcl-6 -) were associated t(14;18). The mean 5 years survival time of patients with t(14;18) was significantly lower (31.18 ± 3.06 month) compared to those without translocation (54.32 ± 2.54 month) (P=0.001). Interestingly, patients with t(14;18) showed Bcl-2 positive (100%) compared to 46.6% in patient without t(14;18) (P=0.004). There is a significant correlation between t(14;18) and the clinicopathological risk criteria of IPI(P=0.01). In our study we demonstrated a detection of t(14;18) by FISH was found to be superior to PCR. The high risk group of GC phenotype together with Bcl-2 expression were associated with t(14;18) and could be used to tailor treatment.
A Study on t(9;22)(q34;q11) in Endothelial Cells in Chronic Myeloid Leukemia.
Authors: Huang Liang , Wu Jingyi , Zheng Miao , Huang Mei , Zhang Yicheng , Zhou Jianfeng
Published: October 2019

Chronic myeloid leukemia (CML) is a disease characterized by t(9;22)(q34;q11), the Philadelphia chromosome (Ph chromosome), and speculated to derive from the hemangioblast, which can give rise to both blood cells and endothelial cells. By using fluorescence in situ hybridization (FISH), we detected Ph chromosome in endothelial cells, which were separated with magnetic beads of CD105+, the marker for endothelial cells, from bone marrow mononuclear cells of 6 CML patients. Moreover, by using Fiction technique, a method combined immunofluorescence with FISH assay, we studied the translocation in vascular endothelium in hepatic and gastroenteric tissue sections and bone marrow trephines of another 7 CML patients. We showed that CD105+ endothelial cells of 6 CML patients with Ph chromosome were identified to harbor the same chromosome aberration as the leukemia cells, 69∼87% of bone marrow endothelial cells carried Ph chromosome. And Ph chromosome was identified in 21∼74% of microvascular endothelial cells in another 7 patients. These results confirmed that CML is derived from the hemangioblast, which can contribute to the maintenance of the blood vascular endothelium. Given specific cytotoxic T lymphocytes can be activated by the immunogenic bcr/abl peptide, we presume that the expression of bcr/abl on endothelial cells in CML patients may be one of the reasons for the incidence of chronic graft versus host disease (cGVHD) after bone marrow transplantation.