bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Classification in Human Peripheral Blood Using the Amnis ImageStream® System.
Authors: Morrissey Philip , George Thaddeus , Basiji David , Frost Keith , Ortyn William , Williams Douglas
Published: October 2019

Amnis Corporation’s ImageStream® system combines the sample handling and quantitative power of flow cytometry with high-resolution brightfield, darkfield, and fluorescence cellular imagery. The system simultaneously generates up to six images of each cell in flow and can acquire data sets consisting of tens of thousands of cells in just a few minutes, while offering fluorescence sensitivity equal to or better than flow cytometry. The image data are analyzed using Amnis’ IDEAS® software, which automatically calculates over 200 morphometric and photometric features and associated statistics for each cell, identifying unique cell groups based not only on their fluorescence intensity signature but also on their morphological characteristics. The software offers the ability to view the imagery associated with any cell in a scatter plot, perform “virtual cell sorts” of user-specified sub-populations, and generate custom features of biological significance (e.g. N/C ratio). The ImageStream platform’s ability to quantitate morphologic and immunofluorescent differences between very large numbers of cells in suspension make it particularly well suited for hematology.

In the present study, human peripheral blood mononuclear cells were stained with a fluorescent DNA binding dye to reveal nuclear morphology, as well as fluorescently labeled mAb to various CD markers. Five images of each cell were acquired: brightfield (transmitted light), darkfield (laser side scatter), and three fluorescent colors for nuclear imagery and quantitation of the CD marker abundance. The object of the study was to identify morphometric parameters in the brightfield, darkfield, and nuclear imagery that would prove useful in hematologic cell type classification. The mAb to CD antigens provided a positive control for use in the evaluation of the of the various morphometric parameters. Parameters with discriminating power included cellular size and texture, darkfield intensity and granularity, and nuclear fluorescence intensity, texture, and shape. Cell types that could be automatically discriminated using these parameters in lieu of immunofluorescent markers included neutrophils, eosinophils, monocytes, and lymphocytes (including putative activated lymphocytes).

In addition to forming the basis for an advanced ImageStream hematology platform, it is envisioned that the automated morphometric classification of blood cells will act as the foundation for a wide range of image-based cellular assays performed in peripheral blood (e.g. NF-kB translocation, apoptosis, mAb compartmentalization), allowing the differential quantitation of assay results in various cell types for the purposes of basic research, drug discovery, and clinical diagnostics.
Deletion of Bcl11a Gene Reveals Roles in Lymphoid, Myeloid, and Leukemic Cell Development.
Authors: Ortiz Mariaestela , Liu P. , Tessarollo L. , Rachel R.A. , Nakamura T. , Stull Steven W. , Jenkins N.A. , Copeland N.G. , Keller J. R.
Published: October 2019

B-cell lymphoma/leukemia 11a (Bcl11a) gene, encodes a zinc finger protein that is expressed in normal B- and T-lymphocytes, monocytes, megakaryocytes, and physically interacts with the human B-cell proto-oncogene, BCL6. Bcl11a is activated by fusion of IG the locus in human B-cell lymphomas carrying the (2;14)(p13:q32.3) translocation. To determine the function of Bcl11a in normal hematopoiesis and proliferation of leukemic cells, we generated a Bcl11a knockout mouse model. This showed that Bcl11a is essential for postnatal development and normal lymphopoiesis. While Bcl11a+/− mice are fertile, Bcl11a−/− die a few hours after birth from unknown causes. To evaluate the role of Bcl11a in hematopoiesis, fetal liver (FL) cells were analyzed by FACS for the expression of lineage specific cell surface markers. B-cell maturation was blocked prior to the development of pro-B cells (B220+/CD43+), while myeloid and erythroid lineages were normal. The Bcl11a−/− FL also showed defects in T-cell maturation (reduced CD4+ CD8− and increased CD4− CD8+ fetal thymocytes). Lethally irradiated mice transplanted with Bcl11a−/− FL cells showed normal myeloid and erythroid development, while B- and T-cell development was blocked. Thus, defects in lymphoid development were intrinsic to the hematopoietic stem cells (HSC). We established that this defect in lymphopoiesis was not due to homing to the thymus, as shown by failure of T-cell repopulation when Bcl11a−/− FL cells were injected into the thymus. Greater than 80% of the mice transplanted with Bcl11a−/− FL cells died between 15 and 27 weeks, while 100% mice transplanted with Bcl11a+/− survived. Histopathology of moribund mice showed enlarged thymus with lymphoblasts infiltrating the bone marrow, liver, lung, heart, and kidney. Furthermore, analysis by real time-PCR of thymocytes from the sick mice showed increased expression of Notch1, a negative regulator of lymphoid development. Leukemic cells were tumorogenic when injected into SCID-NOD mice, and were immortalized by co-culturing on OP9 stromal cells. Immortalized leukemic cell lines showed high expression of T-cell markers CD3e, CD4, CD8, and B-cell marker CD43. Finally, myeloid progenitor cell growth (CFU-c, 3H-Thymidine incorporation, and viable cell growth) was inhibited by over-expression of Bcl11a in bone marrow cells from 5-FU treated mice. Thus, these data show that Bcl11a is required for B and T cell differentiation and may also function as a T-cell tumor suppressor gene. Furthermore, these results are the first indication of a possible suppressive role for Bcl11a during normal myeloid progenitor development and differentiation. This project has been funded in part from NCI, NIH Contract # NO1-CO-56000, with SAIC-Fredrick, Inc.
Retrovirus-Transduced T Cell Blasts Have Not Only Antigen-Presenting Capabilities but Also Suppressor Regulatory T Cell-Inducing Capability.
Authors: Okamura Takayuki , Leen Ann M. , Silli Uluhan , Gottschalk Stephen M. , Vigouroux Stephane , Heslop Helen E. , Brenner Malcolm K. , Rooney Cliona M.
Published: October 2019

EBV-associated T/NK-lymphoproliferative diseases (LPD) are relatively rare but their prognosis is very poor. In order to understand the pathogenesis and rationalize immunotherapy, it is important to know whether EBV-infected T/NK cells have antigen-presenting capacities and how they escape the EBV-specific immunity that controls virus-infected B cells and epithelial cells. As a model for T cell antigen presenting cells (T-APC), we used activated T cells expressing pp65, the immunodominant antigen of CMV, after retroviral transduction. Two to three days after activation with CD3/28, T cells were transduced with retrovirus supernatants for 24h to 48 hr on retronectin-coated plates, and then expanded for one to two weeks with IL-2 before use as APCs or target cells. As control APCs, we used LCLs transduced with a retrovirus vector expressing pp65 (pp65-LCL). Pp65-expressing T cells functioned as target cells, since they were readily killed by pp65-specific cytotoxic T cells (CTL), reactivated using Adenovirus-pp65-pulsed dendritic cells. Furthermore, pp65T-APCs could reactivate pp65-specific CD8+ CTL from autologous PBMC. However, the pp65T-APC could not sustain prolonged pp65-specific CTL expansion. The ratio of CD4+: CD8+ T cells increased in responders after the 2nd stimulation with pp65-T-APC, and after the 3rd stimulation, the CD8+ CTL had virtually disappeared so that most of live cells were CD4+ T cells. The majority of the CD4+ T cells co-expressed CD25, the glucocoticoid-induced TNF receptor (GITR) and intracellular antigen CD152, all markers of T regulatory cells. To determine if they had regulatory function, the CD4+CD25+ population was isolated and shown to inhibit the proliferation of autologous PBMCs stimulated by pp65-LCL. In contrast, the CD4+CD25− fraction did not inhibit proliferation. Transwell experiments revealed that the inhibition required cell-to-cell contact. CFSE analysis showed that the CD4+CD25+ cells expanded while inhibiting the proliferation of stimulated PBMC. Taken together, these findings indicated that T-APC could reactivate antigen specific CD8+ CTL, but at the same time they activated regulatory CD4+ T cells, which prevented the long-term expansion of the CD8+ CTL. Similar results were found when the LMP2 antigen of EBV, which is expressed in T/NK-LPD, was used as antigen. Thus the immune escape of EBV-associated T/NK-LPD might result in part from their induction of regulatory T cells which inhibit T cell-specific CTL generation and function. T cells in general may evade immune-mediated recognition of their “novel” T cell receptors in the same way.
Sensitization of Human T Cells with Overlapping Pentadecapeptides Spanning the Wt1 Protein Induces Expansion of Leukemocidal T Cells Specific for Both Previously Identified and Novel WT1 Epitopes.
Authors: Doubrovina Ekaterina , Dupont Jacob , Trivedi Deepa , Kanaeva Elena , O’Reilly Richard J.
Published: October 2019

The oncofetal protein, WT1, is differentially expressed in 60–80% of acute leukemias and CML. Previously, four peptide epitopes presented by HLA-A0201 or HLA-A2402 have been identified. We used a pool of 15-mers with 10 amino acid overlaps spanning the full sequence of WT1, loaded on autologous monocyte-derived dendritic cells or EBV-transformed BLCL to sensitize and raise T cell lines from a series of normal donors expressing or not expressing HLA-A0201 or HLA-A2402. Specific 15-mers eliciting responses were identified by quantitating IFNg+ CD8+ or CD4+ T cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Responses against targets loaded with the identified stimulating 15-mer and sharing single HLA class I or II alleles permitted identification of restricting HLA alleles. Patients inheriting HLA-A0201 have generated CD8+ T cells in response to the complete pool which are selectively reactive against single 15-mers containing the known immunogen 252–260RMFPNAPYL. However, in certain donors, responses to 15-mer 28, containing a heretofore unreported epitope 136–144ALLPAVPSL, (which has a high predicted affinity for HLA-A0201) have been dominant. By mapping responses of T cells sensitized with the complete pool, defining the presenting HLA alleles and delineating immunogenic sequences within specific 15-mers, additional novel epitopes presented by HLA- B0801 and DRB10301 have also been defined. T cells generated by this approach also lyse both peptide-loaded targets as well as primary WT1+ leukemic cells expressing the restricting HLA allele. Thus, sensitization with pools of overlapping synthetic 15-mers spanning WT1 may selectively stimulate WT1-specific CD4+ and CD8+ T cell lines restricted by HLA alleles other than HLA- A0201 or A2402 that are leukemocidal and therefore of potential use for adoptive immunotherapy.
Effectiveness of Splenectomy and Platelet Autoantibodies.
Authors: Sikorska Anna , Maslanka Krystyna , Misiak Andrzej , Konopka Lech , Zupanska Barbara
Published: October 2019

The role of the spleen in pathophysiology of Idiopatic Thrombocytopenic Purpura (ITP) remains incompletely defined. Splenectomy has a fairly favourable initial response rate of 60% to 80%, and the longer the follow-up, the lower the success rate. As yet, there is no consistently effective method of predicting an individual response to splenectomy. So far not many observations have been published and if so, they were based on very small groups of ITP.

The aim of this work was to evaluate the response to splenectomy in 151 patients with ITP in relation to the detection of platelet autoantibodies. 151 ITP patients before and after splenectomy were observed for a period of 16 years.

Autoantibodies were examined by the platelet immunofluoresce test (PIFT) and/or by the monoclonal antibody immunobilization of platelet antigen (MAIPA) assay, sometimes in addition the HPA genotyping was performed using the PCR.

The response to splenectomy was regarded to be very good if platelet count was >150 G/l during at least 6 months follow-up. However, some patients were followed-up even for 15 years. Overall, platelet antibodies were detected in 34.3% (52/151) of patients. In 76% out of 52 patients the antibodies were detected by PIFT, in 63% by MAIPA; both tests were positive in 27% of patients. Most antibodies reacted with GPIIbIIIa.

Splenectomy was effective in 57% (30/52) of patients with antibodies and in 75.8% (75/99) of patients without detectable antibodies (p<0.01). In the effective splenectomy and antibodies group, in 23 patients the antibodies were examined during a follow-up and in 10 of them (43.5%) were not detected. In the uneffective splenectomy group only in 4 out of 18 (22.2%) examined patients the antibodies were not found. The differences between these groups were not significant.

Our data show that autoantibody platelet examination is not very helpful for predicting the effectiveness of splenectomy.
Erythroid Inhibition by AML1-ETO: Repression of GATA-1 Function by a Novel Non-Transcriptional Mechanism Involving Targeted Protein Degradation.
Authors: Choi Youngjin , Elagib Kamaleldin E. , Goldfarb Adam N.
Published: October 2019

AML1-ETO (A/E) is the fusion product of a chromosomal translocation, t(8;21) frequently associated with FAB M2 acute myeloid leukemia (AML). The fusion combines the runt domain of the hematopoietic transcription factor RUNX1 with almost the entire transcriptional repressor ETO. Clinical cases of AML with t(8;21) are distinguished by blockade in erythroid differentiation. In addition, enforced expression of A/E in primary human erythroid progenitors impairs differentiation. Existing paradigms postulate that A/E exerts its leukemogenic effects through recruitment to RUNX binding sites of cofactors such as corepressors, histone deacetylases (HDACs), and DNA methyltransferases (DNMTs), causing repression of RUNX target genes. However, this paradigm fails to explain effects of A/E on erythropoiesis as erythroid genes generally lack functional RUNX sites. We have published a physical and functional interplay between RUNX1 and the erythroid master regulator GATA-1 (Blood 101:4333). Furthermore, A/E physically interacted and functionally interfered with GATA-1. In the current studies we have examined domain and cofactor requirements for A/E inhibition both of GATA-1 function and of erythroid differentiation. Deletional mutagenesis of A/E demonstrated that the zinc finger (NH4) and runt domains were absolutely required for GATA-1 inhibition. Treatment with HDAC and DNMT inhibitors failed to affect A/E repression of GATA-1. RNAi knockdown of all known NH4 interactors, HDACs 1-3, N-CoR, SMRT-A, and SMRT-B also failed to affect A/E inhibition of GATA-1. Inducible expression of A/E in MEL cells caused downregulation of endogenous GATA-1 protein and mRNA, an effect dependent on induction of erythroid differentiation. A coexpressed GATA-1-GFP fusion showed downregulation with identical kinetics to endogenous GATA-1. Interestingly, proteasome-specific inhibitors effectively prevented the downregulation of endogenous GATA-1 and GATA-1-GFP caused by induction of A/E coupled with erythroid differentiation. Fluorescence microscopy showed a striking relocation of GATA-1-GFP from the nucleus to discrete, paranuclear bodies upon joint induction of A/E expression and erythroid differentiation. Our findings indicate that A/E inhibition of GATA-1 occurs through a previously undescribed mechanism that involves GATA-1 redistribution to novel cellular structures followed by proteasome-mediated degradation. These findings expand the paradigm of A/E leukemogenicity to include a non-transcriptional mechanism in which a growth inhibitor/tumor suppressor, GATA-1, is targeted by A/E for proteolytic degradation in a manner reminiscent of human papilloma virus E6 targeting of p53 for degradation in cervical carcinogenesis.
Murine Platelets Retain High Amounts of Alpha Granule Proteins on Their Surface upon Stimulation with Multiple Agonists.
Authors: Jobe Shawn M. , Leo Lorie , Dicknite Gerhard , Lentz Steven R. , Di Paola Jorge
Published: October 2019

Human platelets exposed to two strong agonists have recently been noted to form a unique subpopulation characterized by increased amounts of bound alpha granule constituents, including fibrinogen and factor V. We have identified and characterized a similar population of platelets in the mouse utilizing thrombin and the GPVI agonist, convulxin, as dual stimulants. We demonstrate that these platelets are analogous to those previously characterized in humans and examine their formation in both Fc Receptor γ-chain (FcRγ) and Factor XIIIa null mice. C57/Bl6 mouse platelets were exposed to convulxin, thrombin, and ionomycin singly or in combination at concentrations previously demonstrated to give maximal aggregation. Platelets were then examined by flow cytometry. Upon stimulation by any of the agonists, all platelets exhibited moderate amounts of fibrinogen binding and equivalent P-selectin exposure. Approximately 70% of convulxin and thrombin stimulated platelets (CoaT platelets) and 10% of thrombin stimulated platelets retained high amounts of alpha granule proteins, including fibrinogen and vWF. Interestingly, despite their high amount of bound fibrinogen, CoaT platelets demonstrated decreased binding of the GPIIb/IIIa activated state-specific antibody, JON/A, relative to single agonist stimulated platelets. Further characterization identified this platelet population as having high phosphatidylserine exposure (increased annexin V binding) and increased ability to bind the actin specific marker, phalloidin. All of these characteristics were present within two minutes of agonist stimulation. CoaT platelets could not be generated in the FcRγ-chain knockout mouse demonstrating the importance of this signaling pathway in their formation. Since human CoaT platelet formation is inhibited both by transglutaminase inhibitors and a monoclonal antibody to Factor XIIIa, CoaT platelet formation was examined in the FXIIIa null mouse. The absence of FXIIIa did not affect CoaT platelet formation, aggregation, or phosphatidylserine exposure. These findings demonstrate the presence of a unique subpopulation of platelets in the mouse formed on exposure to dual agonists analogous to the previously described CoaT platelets. This population is characterized by high amounts of bound alpha granule proteins, decreased binding of a GPIIb/IIIa activation specific mAb, and increased phosphatidylserine exposure; and in the mouse is not dependent on FXIIIa.
Microvesicles Derived from Activated Platelets Enhance the Invasive Potential of Breast Cancer Cells.
Authors: Marquez-Curtis Leah A. , Wysoczynski Marcin , Ratajczak Mariusz Z. , Janowska-Wieczorek Anna
Published: October 2019

There is increasing evidence that platelets contribute to cancer metastasis, and yet platelet concentrates are frequently transfused to cancer patients to treat thrombocytopenia after chemotherapy. Recently we reported that microvesicles derived from activated platelets (PMV) transfer various surface receptors/adhesion molecules to normal and malignant target cells and modulate their biological responses (Blood2001; 98:3143; Exp Hematol2002; 30:450). In this work, we hypothesized that the interaction of PMV with cancer cells increases their invasive and metastatic potential. PMV were isolated from outdated platelet concentrates and pre-incubated with human breast cancer cell lines (MDA-MB-231, BT-549 and T47D), and the effect of PMV on the invasive/metastatic potential of these cancer cells was evaluated. We determined (i) the transfer of the platelet-derived antigen CD41 to cancer cells and the adhesion of these cells to human umbilical vein endothelial cells (HUVEC), (ii) the expression of matrix metalloproteinases (MMPs) by breast cancer cells and their ability to cross the reconstituted basement membrane Matrigel, (iii) the expression of CXCR4, the cognate receptor of the a-chemokine SDF-1, produced in bone marrow, in these cell lines after incubation with PMV, and (iv) the effects of PMV on the interactions of the tumor cells with stroma. We found that PMV transfer platelet-derived CD41 integrin to the surface of breast cancer cells and promote their adhesion to HUVEC. Preincubation with PMV upregulates the mRNA for MMP-9 and protein secretion in invasive breast cancer cells (MDA-MB-231 and BT-549) and enhances their trans-Matrigel chemoinvasion. PMV also transfer CXCR4 to the surface of the breast cancer cells and stimulate the trans-Matrigel migration of MDA-MB-231 cells towards SDF-1, which was abrogated by AMD3100, a CXCR4 antagonist. Finally we found that PMV increase activation of the latent form of MMP-2 constitutively secreted by fibroblastic cells in co-cultures of tumor cells with bone marrow stroma. Thus, we conclude that PMV may enhance the invasive and metastatic potential of breast cancer cells. Because concentrations of PMV are known to be higher in old platelet concentrates than in fresh ones, we recommend that cancer patients should preferably be transfused with fresh platelet concentrates only.
Unique Expression of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) on Embryonic Stem Cells.
Authors: Han Zhong Chao , Li Zong Jin , Wang Zack Z. , Zheng Yi Zhou , Xu Bin , Yang Ren Chi , Scadden David T.
Published: October 2019

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as endothelial marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. Here, we demonstrate that undifferentiated ES cells expressed high level PECAM-1, but low levels of other endothelium markers, including VE-cadherin, Flk-1 and CD34. Kinetic analysis revealed that the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. PECAM-1 was expressed in the vascular-like area along with other endothelium makers during differentiation of ES cells. In the absence of angiogenic growth factors, embryonic bodies (EBs) were lack of vascular-like structures, and PECAM-1 was detected in the cells without organized structure. Furthermore, undifferentiated ES cells were found to express all eight known alternatively spliced isoforms of PECAM-1, among them, the expression of PECAM-1 isoforms lacking exon 15, (D15), or 14&15 (D14&15) was predominant. Only the expression of PECAM-1 isoform lacking exon 12&14&15 (D12&14&15) was significantly increased as vascular development of ES cells. These results indicate that PECAM-1 is a constitutive marker of undifferentiated murine ES cells and suggest that the diversity in the expression of cytoplasmic PECAM-1 isoforms may affect its function during vasculogenesis and angiogenesis.
Effect of Native and Cleaved Forms of Antithrombin on Nuclear Factor κB Activation in Endothelial Cells.
Authors: Ni Hongyu , Chauhan Balwant , Olson Steven
Published: October 2019

Antithrombin (AT) is a single chain plasma glycoprotein that belongs to a superfamily of serine protease inhibitors (Serpin). AT is the major inhibitor of the serine proteases of the coagulation cascade, notably thrombin and factor Xa. In addition, AT has also been demonstrated to have anti-inflammatory properties. Recently, cleaved and latent forms of AT have been shown to function as antiangiogenic agents. However, the molecular mechanisms by which native AT and cleaved AT exert their anti-inflammatory and antiangiogenic effects remains unknown. In this study, we have investigated the effects of both native and cleaved forms of AT on endothelial cell proliferation and nuclear factor κB (NF-κB) activation in the endothelial cells. Bovine pulmonary artery endothelial cells (BPAE) was used in the study. Human α-antithrombin was purified from plasma by affinity chromatography on heparin Sepharose, followed by anion-exchange chromatography. Cleaved form of AT was prepared by digestion of purified human AT with human neutrophil elastase, followed by chromatography on a heparin Sepharose column. The endothelial cells were stimulated with basic fibroblast growth factor (bFGF) or tumor necrosis factor-α (TNF-α) in culture and incubated with either native AT or cleaved AT. Endothelial cell proliferation was measured by the MTT (methylthiazolyldiphenyl-tetrazolium bromide) cell proliferation assay as well as by counting cell numbers before and after the treatment. The results demonstrate that both native AT and cleaved AT could inhibit bovine pulmonary artery endothelial cell proliferation. These results contrast with those reported with other types of endothelial cells such as human umbilical vein endothelial cells (HUVEC) in which only cleaved form but not native form of AT inhibits cell proliferation. NF-κB activation was detected by the ELISA-based assay using antibodies specific for the activated form of p50 and p65 subunit of the NF-κB. In native AT, the inhibitory activity of endothelial cell proliferation was associated with down-regulation of NF-κB as measured by decreased nuclear p65. However, the cleaved AT showed minimal effect on the NF-κB activation. Our results suggest that although both native and cleaved AT inhibit endothelial cell proliferation, they might use different signal transduction pathways. Moreover, our findings suggest that native and cleaved AT may have differential effects on different types of endothelial cells.
Rituximab in the Treatment of Adults with Chronic Idiopathic Thrombocytopenic Purpura (ITP) and Autoimmune Hemolytic Anemia (AIHA).
Authors: Zalzaleh Ghassan , Jajeh Ahmad , Tamoseviciene Diemante
Published: October 2019

Corticosteroids have been the first line of treatment of ITP since 1950, however some patients do not respond to this treatment (refractory) and some will relapse after its discontinuation. For such patients second line treatments were introduced. Some patients will continue to be refractory to this treatment and need other therapy modality.

Rituximab is a chimeric monoclonal antibody directed against the CD20 antigen exposing B Lymphocytes, causing its depletion. This could alter the production of auto-antibodies in some Auto-Immune diseases and thus could be used in their treatment. Few medical centers had reported using Rituximab in the treatment of refractory (ITP) and (AIHA), yet its definite role could not be determined, and here we share our experience.

Patients with documented diagnosis of ITP or AIHA who were refractory to at least two lines of therapy including steroids were offered to receive Rituximab (375mg/m2 weekly for 4 weeks). 15 patients were enrolled, 10 with ITP, 4 with AIHA, 1 with Coombs negative Hemolytic anemia, and 1 with pure red cell aplasia. One had both ITP and AIHA. 10 were females and 5 males. 5 were >60 years old and 10 were < 60 years old. 2 out of the 10 patients with ITP had also Chronic Lymphocytic Leukemia (CLL). Duration of follow up ranged from 2 months to 17 months (average 7 mos). Of the 10 patients with refractory ITP treated with Rituximab overall response was 60%. 4 were NR (no response), 2 were MR (minimal response: Platelets increased to <50000), 2 were PR (Partial response: Platelets increased to <100000) and 2 were complete response (Platelets became normal). 3 patients of 6 with Hemolytic anemia or PRC aplasia had NR, 1 had MR (Hct <30), and 2 had partial response (Hct 30–35). No complete response was observed in this group. In 3 patients with hemolytic anemia and CLL 1 had MR, 1 had PR and 1 had NR. 2 patients with hemolytic anemia who had NR died as a complication of their disease (one with septic shock and one with severe autoimmune flare up). Only one patient with refractory ITP had mild allergic side effects and did not complete 4 doses. No Rituximab related mortality was observed.

CONCLUSION:

Rituximab therapy had a variable but valuable effect in the treatment of patients with chronic refractory ITP and refractory/ relapsed AIHA. Overall response in our group reached 60%. No clinical or laboratory parameters were found to predict response, although there was a suggestion that males, younger age, and no history of splenectomy have a better chance of response. As we lack an effective alternative treatment in chronic refractory ITP and AIHA, Rituximab use could be a valid option in view of its mild toxicity. Further follow up of our patients and input from other institutions in this regard are needed.
A Ten Year Survey of Chronic Immune Thrombocytopenic Purpura in Canadian Children, 1984–2003: Report of the Canadian Chronic ITP Working Group.
Authors: Ali Syed Kaiser , Belletruti Mark , Barnard Dorothy , Chan Anthony , David Michele , Luke Brian , Price Victoria , Ritchie Bruce , Wu John
Published: October 2019

Chronic Immune Thrombocytopenic Purpura (C-ITP) is a heterogeneous group of disorders manifesting as thrombocytopenia of greater than six months duration. To better understand its natural history, and detect trends of response to therapy and of outcomes, a ten-year retrospective survey of C-ITP diagnosed in Canadian children from age 6 months to under 20 years was conducted in 2004. A standardised form for capture of anonymous data on each patient was completed and returned from 11 institutions, including the three largest Canadian Pediatric Hematology/Oncology centres. Of the total of 243 forms received, 213 cases met the inclusion criteria. Male:female::1:1.3. Mean age at diagnosis of C-ITP was 7.9 years (range 8 months - 17.7 years). Mean platelet count at diagnosis and mean duration of C-ITP were 46 x 109/L (range 1 - 145 x 109/L) and 2.9 years (range 0 - 9.7 years) respectively. Primary ITP was the underlying diagnosis in 85 % of cases. Internal hemorrhage was documented in 1 % of patients. Treatments (either singly or in combination) with drugs, IVIG, anti-D, or monoclonal antibody, were administered in 91 % of cases. Five percent of patients received blood transfusions for treatment of bleeding episodes; 18.1 % underwent splenectomy. Information gained from this data base will be used in developing management guidelines for C-ITP, and will provide the foundation for establishing a Web-based Canadian National C-ITP Registry.
Impedance-Based Whole Blood and Optical-Based Platelet Rich Plasma Aggregation Methods for the Assessment of Celexocib Effects on Platelet Function: A Preliminary Report.
Authors: Dyszkiewicz-Korpanty Anna M. , Frenkel Eugene P. , Sarode Ravindra
Published: October 2019

Cyclooxygenase-2 (COX-2) is the inducible cyclooxygenase isoform expressed by cells involved in inflammation. Platelets normally lack COX-2; however, the enzyme is expressed in megakaryocytes and newly formed platelets and may play a role in clinical syndromes associated with high platelet turnover (1, 2). Selective COX-2 inhibitors (coxibs), have become the mainstay for treatment of patients with arthritis and also have been widely used in cancer chemoprevention. Coxibs were found to have a safer GI toxicity profile when compared with classic NSAIDS; however, in some patients they may increase the risk of GI bleeding (3). The underlying mechanism has not yet been elucidated. Moreover, the role of coxibs in hemostatic alterations has been a subject of controversy. The aim of the study was to investigate the possible anti-platelet effects of celecoxib (200 mg/d for 7 days) in 7 healthy volunteers (2 females, 5 males; mean age = 35.7 ± 8; mean platelet count = 230 ± 23; mean Ht = 42.3 ± 6). A very sensitive whole blood impedance platelet aggregation assay, previously unused for this purpose, was employed for the assessment of celecoxib influence on platelet function. The test was performed along with a traditional, optical-based platelet rich plasma (PRP) aggregation method. Arachidonic acid (0.5; 0.25 mM), Collagen (2; 1mg/mL) and ADP (20, 10 mM) were used as platelet agonists in both tests.

Celexocib did not affect platelet function in neither whole blood nor in PRP. The preliminary results of this study indicate that the increased bleeding tendency in patients treated with celecoxib is probably not related to platelet dysfunction.

Assay Arachidonic Acid Collagen ADP 0.5[mM] 0.25[mM] 2[μg/mL] 1[μg/mL] 20[μM] 10[μ M] WB Impedance 17.5±4.7 20.9±3.8 25.4±3.9 25.9±6.1 14.3±2.6 14.1±5 Aggregation: Pre-drug Post-drug 18±4 21±5 27.1±4.7 24±4 18.7±6.2 16.3±7 PRP Optical 118.1±10.3 118.1±6.7 101.4±13.7 110±2.2 104±15 108±8.9 Aggregation: Pre-drug Post-drug 119.6±6.7 112.9±10.2 105.4±11.5 110.4±7.5 102.1±18.3 107.7±9.6
Determination of TAFI in Plasma Using a New Chromogenic Method.
Authors: Wilmer Marianne , Buehler Beatrice , Peter Wikstroem , Hugo Ziegler
Published: October 2019

TAFI (thrombin-activatable fibrinolysis inhibitor) is a proenzyme similar to carboxypeptidase B and is activated to TAFIa by thrombin and probably plasmin also. Thrombomodulin accelerates TAFI activation by thrombin about 1250-fold. TAFIa is an important inhibitor of fibrinolysis by cleaving C-terminal lysine and arginin residues in the fibrin clot. By this the carboxy-terminal plasminogen binding sites in the fibrin clot are degraded, plasminogen activation by t-PA and in consequence fibrinolysis is most effectively inhibited. Whether elevated TAFI levels correlate with the risk of thrombosis is currently under study. To clarify the role of TAFI in various diseases a simple and rapid assay for determination of TAFI concentration in plasma is desirable. With Pefakit® TAFI we developed a new chromogenic method for determination of TAFI in plasma samples.

Method: TAFI is activated by thrombin/thrombomodulin complex in the presence of calcium ions. The activated TAFI cleaves a 3-thia-arginine peptide substrate whose cleavage products subsequently react with Ellman’s reagent. Color development is followed up by measuring absorption at 405 nm. The method is the result of synthetic work and substrate screening. The assay was optimized to determine TAFI in human plasma. The concentration of TAFI in plasma sample corresponds to the initial reaction rate of substrate cleavage. TAFI level in each sample can be calculated from a linear standard curve.

Results TAFI was measured in plasma of about 50 patients. The plasma levels of TAFI determined with Pefakit® TAFI correlate well with a commercially available chromogenic assay (Actichrome TAFI).

Conclusion: Pefakit® TAFI is a fast and precise method to determine TAFI concentration in plasma. There is a linear correlation between TAFI concentration and measuring signal. Because of its simple practicability the assay can be adapted to most of the common automated analyzers for routine application.
Successful Treatment of Intractable Gastrointestinal Hemorrhage with Low Dose of Recombinant FVIIa (Novoseven®) in Pediatric Patient without Coagulation Deficiencies.
Authors: Kim Ji Yoon , Lee Kun Soo , Kwon Soon Hak , Choe Byung Ho , Sohn Sang Kyun
Published: October 2019

Recombinant FVIIa (Novoseven®) was initially developed for the treatment of bleeding in patient with congenital hemophilia with antibodies against factors VIII and IX and FVII deficiency. The mechanism of action is to induce hemostasis at the site of injury both by forming complexes with exposed tissue factor and by directly activating factor X upon the surface of an activated platelet. More recently it has begun to use gradually in the management of bleeding patient without congenital coagulation defect. We report the successful use of recombinant factor VIIa in a pediatric patient who has intractable gastrointestinal bleeding despite of conventional medical therapy.

Case: The patient is a 13-year-old female and her body weight is only 10 kg, with a prior history of cerebral palsy and epilepsy. Recently she presented a recurrent massive hematemesis and melena probably due to stress ulcer for 8 days. She had no personal or family history of a suggestive bleeding disorder. The patient had a normal platelet count and coagulation screening test. Her hospital course was complicated by recurrent gastrointestinal hemorrhage but an endoscopic intervention was not permitted due to her poor condition. Despite of conventional medication with omeprazole and sucralfate over 20 days, she suffered from recurrent hematemesis and melena. In the gastrointestinal bleeding scan with 99mTc-labeled RBCs, small intestinal hemorrhage was found. The following day, the patient was given three doses of Novoseven® at 100 μg/kg, as bolus doses with 2 hour interval, which resulted in resolution of hematemesis and melena after last dose administration. Although she had mild whole body rash after third dose administration, it was subsided soon after medication of antihistamine and steroid. No evidence of hemorrhage was documented at a follow-up gastrointestinal bleeding scan that was taken after 4 days later of treatment. The patient made a full recovery and no further treatment was required.

Conclusion: We suggest that low dose of recombinant FVIIa (Novoseven®) were effective in treatment of pediatric intractable gastrointestinal hemorrhage.
Germline Polymorphisms Provide Strong Prognostic Information in Intermediate Risk Acute Myeloblastic Leukemia (AML).
Authors: Urbano-Ispizua Alvaro , Brunet Salut , Monzo Mariano , Perea Granada , Navarro Alfons , Esteve Jordi , Berlanga Juan , Ribera Josep-Maria , Bueno Javier , Llorente Andreu , Besalduch Juan , Guardia Ramon , Tormo Mar , Pedro Carmen , Sanchez Juan-Maria , Torres Pio , de Llano Maria-Paz Queipo , Font Llorens , Moraleda Jose-Maria , Marti Josep-Maria , Vivancos Pilar , Peñarubia Maria-Jesus , Nomdedeu Josep , Montserrat Emili , Sierra Jorge
Published: October 2019

In this study we tested whether distinct patterns of functional genomic polymorphisms in genes involved in drug metabolic pathways (GSTT1, SULT1C2, CD-EX, TOP2A, SXR-1, SXR-2), DNA repair (XPD, XPA, XPG, ERCC1), angiogenesis (VEGF-1, VEGF-2), and multidrug resistance (MDR1-1, MDR1-2) predict clinical outcome in patients with AML. Two hundred adult patients ≤60 year-old with primary AML (M3 excluded), enrolled in the AML-99 protocol of the CETLAM group between November-98 and July-03, have been studied. Induction therapy consisted of idarubicin, intermediate dose ara-C (IDAC) and VP16. Intensification included mitoxantrone and IDAC. Patients were following treated with high-dose ara-C or transplantation depending on the risk category. According to the MRC cytogenetic classification, 32 patients had a favourable karyotype, 23 had abnormalities indicating poor prognosis, and 110 patients were included in the intermediate prognosis (IP) group. We focused on IP group patients with the aim of improving its prognostic stratification. The characteristics considered were: age (<50 years vs ≥50), WBC (≤20x109/l vs >20x109/l), FAB classification, MLL rearrangement, internal tandem duplication of FLT3 (ITD-FLT3), induction courses to achieve complete remission (CR) (1 vs 2), and germline polymorphisms of the above-mentioned genes. Prognostic variables in the univariate analysis (Kaplan-Meier method) with a p value ≤0.2 (log-rank test) were included for the multivariate analysis (proportional hazard method). Of the 110 patients included in the IP group, 86 (78%) achieved a CR, 11 (10%) were chemoresistant and 13 (12%) died during induction. After a median follow-up of alive patients of 24 months (range 5–64), overall survival was of 31% at 5 years. In the multivariate analysis, adverse prognostic variables for survival were polymorphism of XPA (RR=3.4; p=0.02) and of MDR1-1 (RR=2.1; p=0.02), and WBC >20x109/l (RR=2.1; p=0.02). Increased risk of relapse was associated with polymorphism of SULT1C2 (RR 4.1; p=0.004), ITD-FLT3 (RR 3.3; p=0.003), VEGF2 (RR 2.8; p=0.04) and MDR1-1 (RR 2.4; p=0.02). Finally, in the multivariate analysis for refractoriness to chemotherapy, XPA polymorphism was the only independent factor increasing the risk (RR=14; p=0.02). In conclusion, germline polymorphisms, which can easily be analyzed from DNA of peripheral blood, have independent prognostic value in intermediate risk AML.
Generation of Prothrombinase According to Hicks and Pitney Test. Comparison of the Levels of F1+2 Simultaneously in Hemophiliacs with and without Thrombophilic Mutations and Normal Subjects.
Authors: Makris Pantelis P.E.M. , Girtovitis Fotios F.I.G. , Tsaousidou Maria M.K.T. , Pithara Eleftheria E.P. , Foka Zoi Z.I.F. , Samama Michel Meyer M.M.S.
Published: October 2019

It has been seen that a significant number of hemophiliacs show a co-existence of mutations related with an increased risk of thrombosis (Foka et al 2003). It has also been suggested that the existence of these genetic defects that manifestate with either hemorrhage or thrombosis, may actually protect these patients from recurrent bleeding episodes. In order to study the coagulation mechanism in these patients the Hicks & Pitney test was selected, that examines the generation of prothrombinase. Materials and Methods. 64 subjects were studied (56 hemophiliacs 42±15 year old and 8 normal subjects 45±10 year old). 37 without mutations, and 19 others with mutations (12 with FVLeiden and FII G20210A and 7 homozygotes in MTHFR). The test was applied to 2 groups (one with hemophiliacs and one with normal subjects). The levels of F1+2 and TAT were also measured in every incubation phase, as markers of the generation of prothrombinase and thrombin.

It can be seen in the charts that follow that hemophiliacs with no mutations show a gradual prothrombinase generation. On the contrary, hemophiliacs with mutations show a rapid generation that is also expressed by the concomitant increase in the levels of F1+2 and TAT. A similar response was observed in the group of normal subjects.

This can be explained as an adjustion of the coagulation mechanism of hemophiliacs with coexistence of thrombophilic mutations, possibly capable of preventing the high frequency and severity of bleeding episodes.

Figure Figure
Human Factor IX Expression and Hemophilia Phenotypic Correction in Mice: A Comparison of AAV Serotypes.
Authors: Chao Hengjun , Chen Wei , Tunstead James R. , Walsh Christopher E.
Published: October 2019

Recombinant adeno-associated virus (rAAV) has been extensively studied as a vector for hemophilia gene transfer. AAV serotype 2 targeting the liver and skeletal muscle has been used in clinical trials for hemophilia B patients. Previously we reported that the use of recombinant non-AAV2 serotype vectors generated persistent expression of supra-normal levels of canine factor IX (cFIX) in immunodeficient mice (Mol Ther ‘2000), and resulted in sustained and complete hemophilia B phenotype correction in immune competent hemophilia B mice (Mol Ther ‘2001). In those studies AAV serotypes 1–5 were tested by intramuscular injection. In this study we tested rAAV serotypes 1–8 for hepatic transduction and FIX production in both C57BL/6 and FIX knockout mice. Animals received intrapotal vein injection of 1x1011 virion particles of rAAV 1 thru 8 carrying a human FIX cDNA linked to a chicken beta actin enhancer-CMV promoter and bovine growth hormone polyA. Animals were followed for 8 months and assessed for plasma levels of human FIX by ELISA. A rapid, sustained and maximal rate of hFIX expression was observed within 1–2 weeks with all vectors except AAV2 where the peak expression occurred at 8–10 weeks. hFIX expression was sustained for all serotyped vectors (n=5 animals tested at each AAV serotype). Animals that received serotypes 7 and 8 maintained hFIX at physiological levels (100% hFIX ~ 5000 ng/ml). A differential hFIX expression pattern emerged with rAAV7 (6213 ng/ml) > 8 (5111 ng/ml) > 5 (2367 ng/ml) > 1 (1090 ng/ml) > 4 (377 ng/ml) > 2 (314 ng/ml) > 3 (232 ng/ml). rAAV7 and rAAV8 generated 20 times more hFIX per virion particle than the rAAV2 vector. We did not detect anti-human FIX antibody in any of the experimental mice. To assess why hFIX production differed between each serotype we performed immunohistochemical staining of the mouse liver using a fluorescent-tagged anti-human FIX antibody. Based on this assay, rAAV7 and AAV8 transduced 30% of hepatocytes while rAAV2 transduced less than 2% of the hepatocytes. We subsequently tested FIX production and effect on hemophilia phenotype of rAAV 7 and 8 (1x1011 virion particles) in hemophilia B mice; the data was similar to that observed using the C57Bl/6 mice. Human FIX levels (>5000 ng/ml) were again sustained during the 6–8 month observation period. No anti-human FIX antibody was detected in these hemophilia B mice. Results of clotting function by aPTT testing demonstrated a normalization of clotting time in all the rAA8/hFIX treated hemophilia mice. Survival of all treated mice by tail clip (lethal for all non-treated knockout animals) confirmed that phenotypic correction had been achieved. Our results again demonstrate that AAV serotypes have differential transduction rates and that new serotypes with greater mouse hepatocyte transduction rates are more efficient for hemophilia gene transfer.
Ten-Year Follow-Up of Inhibitor Induction in Previously Untreated Patients (PUPs) on Recombinate.
Authors: Aledort Louis M. , White Gilbert C. , Rivard Georges E.
Published: October 2019

After contamination of factor concentrates with blood-borne viruses, U.S. FDA requirements for new product licensure was based on the safety and efficacy of new biologicals as determined in previously untreated patients (PUPs). While PUPs provided an excellent model for assessing contamination of new products HIV, hepatitis, and other blood-borne viruses, they were not a good model for risk of inhibitor development, since inhibitor development in one form or another was unpredictable and occurred in up to 30-35% of PUPs. More recently, the Factor VIII & IX Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis has recommended that inhibitor induction potential of new factor replacement products is best studied in previously treated patients (PTPs). At the conclusion of the five-year study of 73 PUPs on Recombinate (Baxter) [Bray GL, Gomperts ED, Courter SG et al. A multicenter study of recombinant factor VIII (recombinate): safety, efficacy, and inhibitor risk in previously untreated patients. Blood1994; 83: 2428–35.], the Data Safety and Monitoring Board (DSMB) independently initiated a five-year follow-up to determine how many new inhibitors occurred, and how many transient inhibitors recurred during this time. Data collection was a simple form to determine presence or absence of inhibitor yearly; if an inhibitor was reported to develop, did the subject remain on Recombinate or use other recombinant factor concentrate, and the inhibitor titer. Sixty-five of the original PUPs had data reported. For all 5 years, there were 21, for 4 years, 20, for 3 years, 5, for 2 years, 9, for 1 year, 10. For the 65 who have provided adequate data: Two subjects had a recurrence of their transient inhibitor with titers ranging from 0.78 to 1.3. They both stayed on Recombinate. They lost these inhibitors over the ensuing two years. Three subjects developed a new inhibitor. Their titers ranged from 0.9 to 4 Bethesda units. All stayed on Recombinate. None of these inhibitors were of high titer (>5.0 BU). These patients are PTPs, and on continued recombinant factor replacement, few new inhibitors occur, and those that did were low-titered, and some that disappear recur. Thus, five-year follow-up does not tell the whole story and continues to raise the issue of how many inhibitors are acceptable to regulatory agencies for licensure of a new product.