bluuebunny/crossref_metadata_2025 huggingface dataset:
Routine Automated Processing of Cord Blood Units Using the Sepax Cell Processing System.
Authors: Karandish Safa , Berrios Nery , Kiran Sufira , Ayuste Charisse , Hamblin Toby , Ceriani Alessio , Shpall Elizabeth J. , McMannis John D.
Published: October 2019

We recently incorporated the use of the automated Sepax Cell Processing System (Biosafe SA, Switzerland) for red cell and volume reduction of cord blood units (CBU) before cryopreservation. Now that we have been routinely using this new technique in the laboratory for about six months, we decided to compare the results of this method with our standard manual processing method (Rubinstein, et al, PNAS1995,; 92: 10119–22).

For both methods, hespan is added to the cells at final concentration of 20% (v/v). With the Sepax system, after addition of hespan, the cell bag is connected to the Sepax tubing set with the final freeze bag pre-attached to the set. After completion of the automated procedure, buffy coat is collected in the final freeze bag. Cryoprotectant solution is then added directly to the freeze bag. In the manual method, buffy coat and white cell rich plasma layer is collected after first centrifugation step and the white cells are separated from the plasma after the second centrifugation step. Cryoprotectant solution is then added to the cells before transfer to the final freeze bag for cryopreservation.

The following are summary of results for each method:

Table 1 Manual (n=1160) Sepax (n=311) Pre-Processing volume (ml) 107±30 114±28 Pre-Processing TNC (×10e6) 1196±577 1315±519 TNC Recovery (%) 80±8 83±8 TNC Viability (%) (7-AAD) 97±3 98±3 Total CD34 (×10e6) 4.3±4 4.9±3.6 Total DFU (×10e6) 70±0.9 61.5±20 Post Processing RBC Volume (ml) 9±2 7.3±2 Total Processing Time (including Setup) 60 minutes 30 minutes

It is important to note that there was not a significant difference in TNC Recovery over a wide range of Pre-Processing Volume (66–206ml) or Pre-Processing TNC (440 – 3559×106).

Since the Sepax device is an automated procedure, issues could arise (i.e. short term loss of electrical power) that would require us to reprocess the CBU before cryopreservation. The Sepax system allows for recovery and reprocessing of the cell using the ‘Purge mode’. We used 5 CBU units to evaluate TNC recovery and viability after purging the cells once and reprocessing.

Table 2 TNC Recovery (%) TNC Viability (%) First Buffy Coat 85±5 98±0.9 Post Purge and reprocessing 76±5 98±0.9

Although the TNC recovery was lower after the second procedure, it was still within acceptable limits and the viability of the cells had not changed. These data demonstrates that both methods are equivalent with respect to cell recovery. However, the Sepax System substantially reduces processing time and hands-on operator intervention. Additionally system provides, closed-system processing, bar code reading capability and run data print-out suitable for GMP manufacturing settings.
Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes.
Authors: Jansen Valerie M. , Ramachandran Shaji , Desgardin Aurelie , He Jin , Parekh Vishwas , Jane Stephen M. , Cunningham John M.
Published: October 2019

Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.
Prior Rituximab Reduces Relapse and Improves Survival Following High Dose Chemotherapy and Stem Cell Transplantation for Relapsed Composite Low and Intermediate Grade (Including Transformed) Lymphoma.
Authors: Ramadan Khaled M. , Connors Joseph M. , Al-Tourah Abdulwahab J. , Gascoyne Randy D. , Song Kevin , Barnett Michael J. , Nantel Stephen H. , Nevill Thomas J. , Shepherd John D. , Sutherland Heather J. , Lavoie Julye , Forrest Donna L. , Hogge Donna E. , Voss Nicholas J. , Brinkman Ryan , Smith Clayton A. , Toze Cynthia L.
Published: October 2019

The effect of prior exposure to rituximab (ritux) on relapse (REL) and survival following stem cell transplantation (SCT) in patients (pts) with REL composite low and intermediate grade non-Hodgkin lymphoma (L/I-NHL) is unknown. Fifty-four pts with REL L/I-NHL underwent high-dose chemotherapy (CT) and SCT Jan ’89 to June ’05. Follow-up is complete to April ’6 with a median of 32 mo. Ritux was added to CT regimens since 2001 and given at 375 mg/m2. Eighteen pts received ritux at initial diagnosis of NHL or after REL with salvage CT prior to SCT. Pts proceeded to high dose CT with allogeneic (allo) (n=12) or autologous (auto) SCT (n=6). This group was compared with a group of pts not receiving ritux pre-SCT (n=36)(Table 1).

Eleven pts (61%) are alive in the ritux group compared with 11 pts (31%) in the non-ritux group. The 2 and 4-y OS for pts who received ritux were 52% and 52%, vs 36% and 24% (p=.12, RR=.52) for pts who did not receive ritux. The EFS were significantly different with 2 and 4-y EFS for the ritux group 56% and 56% vs 24% and 18% (P=.038, RR=.42) for the non-ritux group (figure 1). No effect was seen on TRM. The risk of REL post SCT was significantly lower in the ritux group (p=.017)(figure 2). Two of 18 pts (11%) had NHL REL in the ritux vs 18 of 36 (50%) in the non-ritux group. The hazard rate of REL in pts who received ritux was 20% that of pts in the non-ritux group. Significance maintained in multivariate analysis (p=.016).

No impact was seen on graft-versus-host disease (GVHD). Fifty percent and 61% of pts developed acute GVHD grades 2–4 and 50% and 64% of pts developed chronic GVHD in the ritux and non-ritux groups respectively.

In conclusion, prior treatment with ritux in pts with relapsed composite L/I-NHL undergoing SCT was associated with reduced risk of REL and improved survival without associated increase in toxicity. Further analysis is underway to clarify the nature of this important finding.

Table 1: Clinicopathological characteristics of ritux and non-ritux groups Parameter$ Rituximab group, n=18 (%) Non-rituximab group, n=36 (%) $ all p values are >0.1. *at diagnosis Median age at SCT 48 y 44 y M:F 2.6:1 1.6:1 Allo-SCT 12(67) 28(78) BM involvement* 10(56) 22(61) B symptoms* 7(39) 10(28) Prior purine analogue therapy 8(44) 11(31) Residual disease prior to SCT 9(50) 14(39) TBI in conditioning 13(72) 30(83)

Figure Figure

Figure Figure
Important Role for Absolute Lymphocyte Count in Predicting Risk of Relapse in Pediatric Acute Lymphoblastic Leukemia Post Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Ishaqi Mohammed Kashif , Afzal Samina , Dupuis Annie , Doyle John , Gassas Adam
Published: October 2019

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently an established treatment for children with relapsed or high risk acute lymphoblastic leukemia (ALL). Relapse is a major risk post HSCT and an important cause of treatment failure with abysmal outcome. An important therapeutic mechanism of allogeneic HSCT is the immune-mediated destruction of recipient leukemic cells by donor lymphocytes, the graft-versus-leukemia-effect (GVL). Our hypothesis was to determine if delayed lymphocyte recovery measured by the absolute lymphocyte count (ALC) at two time points post HSCT at day 21 and day 30 correlates with leukemia relapse. We reviewed 136 consecutive paediatric patients with ALL who received allogeneic HSCT between 1994 and 2005 in the Hospital for Sick Children, Toronto, Canada. All patients were in complete morphological remission prior to HSCT and remission status at time of HSCT were as follows; Complete remission 1 (CR1, n=36); Complete remission 2 (CR2, n=79); Complete remission 3 (CR3, n=21). Conditioning regimens included single dose of VP16 (60mg/kg infused over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP16/TBI) in 49 patients (1994–1998) and cyclophosphamide 50mg/kg infused over 1 hour daily for 4 days followed by the same dose of fractionated TBI (CY/TBI) in 83 patients (1999–2005) and 4 patients received other conditioning regimens. Fifty-six patients received matched sibling donor (MSD), 12 patients received one antigen mismatched related donor (MMRD), 64 received unrelated donors and 4 patients received cord progenitor stem cells. Two groups were identified based on absolute lymphocyte count at day 21 and day 30 post HSCT. Patients with absolute lymphocyte count <0.3×109/L at day 21 (n=104) had more than 5 times risk of relapse compared to those with ALC count >0.3 × 109/L (n=32) (Hazard ratio 0.19; P=0.0004). Patients with ALC <0.3×109/L (n=48) at day 30 were more than twice likely to relapse compared to those with ALC >0.3×109/L (n=88) (Hazard ratio 0.46; P=0.01).

Conclusion: ALC, at day 21 and day 30 post HSCT identifies a patient population of pediatric ALL with delayed lymphocyte recovery and a significant risk of relapse post HSCT. Such patients could be considered for early intervention targeted to prevent relapse such as reduction of immunosuppressive therapy or other means of immunotherapy to enhance the graft versus leukemia effect.
Optimizing Immunotherapy in Multiple Myeloma: Restoring the Function of Patient’s Monocyte-Derived Dendritic Cells by Inhibiting p38 or Activating MEK/ERK MAPK and Neutralizing Interleukin-6 in the Progenitor Cells.
Authors: Yi Qing , Wang Siqing , Hong Sungyoul , Yang Jing , Qian Jianfei , Zhang Xiang , Shpall Elizabeth , Kwak Larry W.
Published: October 2019

Previous studies have demonstrated that circulating dendritic cells (DCs) in myeloma patients are functionally abnormal. However, the phenotypic and functional properties of monocyte-derived DCs (MoDCs) from myeloma patients, which are commonly used for immunotherapy in patients, are still poorly defined. This study was undertaken to examine the quality of MoDCs from myeloma patients compared with cells from healthy donors. We analyzed and compared cells from 12 newly diagnosed, stage II or III myeloma patients and 10 healthy donors. MoDCs were generated by culturing adherent cells or purified CD14+ monocytes in medium containing GM-CSF and IL-4. A cocktail of IL-1β, IL-6, TNF-α, and PGE2 was used to induce DC maturation. We found that patient-derived MoDCs are phenotypically and functionally defective. Compared with their normal counterpart, patient-derived, immature and mature MoDCs expressed significantly lower levels of CD1a, CD40, CD80, and HLA-DR. The mature cells were also poor at activating alloreactive T cells, presenting recall antigen and activating autologous antigen-specific T cells, and inducing myeloma-specific cytotoxic T-lymphocyte response. These abnormalities may be attributed to elevated production of autocrine cytokines such as IL-6, constitutively activated p38 and STAT3, and inhibited MEK/ERK signaling pathways in the progenitor cells. Treatment with neutralizing IL-6-specific antibody and more importantly, p38 inhibitor, or both, could correct these abnormalities. Treating patient-derived cells with these agents not only significantly increased cell yield, but also produced MoDCs that were as functional as their normal counterpart. We further showed that the p38 inhibitor inhibited the activity of p38 and drastically increased MEK1/2 and ERK1/2 phosphorylation and activation, and a combination of IL-6 neutralizing antibody with the p38 inhibitor further upregulated phosphorylation of MEK and ERK. By examining the role of STAT3 in MoDC differentiation, we found that STAT3 activation did not impair differentiation of patient-derived cells. These results indicate that the p38 inhibitor improved the generation and function of the MoDCs via inhibiting p38 and activating Raf/MEK/ERK signaling pathways, independent of the STAT3 pathway. Thus, this study has delineated the mechanistic defects of MoDCs from myeloma patients, and identified ways for restoring the function of the cells to improve the efficacy of DC-based immunotherapy against multiple myeloma.
Widening the Applicability of Human Neutrophil Elastase and Proteinase 3 Peptide Vaccines by Elucidating Immunogenic Non-HLA-A2 MHC Class I Restricted Epitopes.
Authors: Hewitt Rachel E. , Melenhorst J. Joseph , Price David A. , Gostick Emma , Hensel Nancy F. , McCoy J.P. , Barrett A. John
Published: October 2019

Human Neutrophil Elastase (HNE) and proteinase 3 (Pr3) belong to a group of myeloid tissue restricted serine proteases, aberrantly expressed by myeloid leukemia cells of interest as a source of myeloid-restricted antigens applicable to immunization strategies. HNE shares with Pr3 the antigenic PR1 peptide sequence inducing HLA-A*0201 restricted cytotoxic T cells (CTL) against leukemic cells. Our previous studies with full length HNE protein have demonstrated its potential for inducing both CD4+ and CD8+ T cell responses in healthy individuals including HLA-A2 negative individuals, suggesting an immunogenic potential of HNE beyond the PR1 peptide (Fujiwara H et al Blood 2004). To find epitopes other than PR1 in HNE and Pr3, peripheral blood mononuclear cells (PBMC) from 15 AML patients were screened at diagnosis or relapse with a peptide matrix consisting of 15mers overlapping by 11 amino acids spanning HNE and Pr3.The corresponding Pr3 and HNE 15mers were also used to generate and test short term T cell lines derived from patient samples. Responses to the peptide matrix pools were measured by a CFSE flow cytometry-based proliferation assay where the proliferating fraction in response to peptide pools was compared to the background co-stimulatory antibody only control for each assay. A positive response was defined as a proliferating fraction minimum 0.1% of the total CD8/CD3 population after subtraction of background proliferation and at least one and a half times background for each assay. These steps helped to control for variability between samples. Using this strategy we identified PR1 and 6 new candidate epitopes contained within HNE and Pr3. Corresponding immunogenic Pr3 sequences in 2 epitopes differed by only 3 amino acids from the immunogenic HNE sequence. CD8 short term T cell lines generated to these epitopes were restricted by HLA-A2, B35 and A68. Optimal peptide length was predicted using RANKPEP (http://www.mifoundation.org/Tools/rankpep.html) and confirmed with flow cytometric assays measuring responses to candidate epitopes, by interferon-gamma, tumor necrosis factor-alpha, IL-2 production and degranulation in response to HLA-matched or mismatched EBV-transformed B cell antigen-presenting cells pulsed with relevant and irrelevant peptides. This study confirms the presence of multiple immunogenic epitopes in HNE and Pr3 and represents the first step in developing a multi-peptide leukemia vaccine broadly applicable to individuals of diverse HLA type.
Rapid Regression of Bone Marrow Fibrosis after Dose-Reduced Allogeneic Stem Cell Transplantation in Patients with Myelofibrosis.
Authors: Kroeger Nicolaus , Thiele Juergen , Zander Axel , Kvasnicka Michael
Published: October 2019

Myelofibrosis with myeloid metaplasia [MMM] is a myeloproliferative disease of which bone marrow histology is characterized by substantial collagen fibrosis and osteosclerosis. Bone marrow histology changes in MMM are reactive and cytokine mediated. Studies suggested a pathogenic role of transforming growth factor β [TGF-β], platelet derived growth factor, and basic fibroblast growth factor. In the current study we investigated the dynamic and kinetic of fibrosis regression in 18 patients with myelofibrosis who underwent allogeneic stem cell transplantation after a dose-reduced conditioning. The conditioning regimen consisted of Busulfan 10 mg/kg, Fludarabin 180 mg/m2, and Antithymocyte globulin 30 – 60 mg/kg followed by peripheral stem cell transplantation (SCT). 18 patients with a median age of 45 [range 35 to 64] were included and bone marrow histology was investigated prior SCT, between day+30 and +40 after engraftment, on day+100, and 1 year after stem cell transplantation. The grading of myelofibrosis [MF] was performed according to the recently published European Consensus on Grading Bone Marrow Fibrosis [Thiele et al., Haematologica2005;90:1125–1132]. This consensus distinguishes between:

MF - 0 Scattered linear reticulin with no intersections [cross-overs] corresponding to normal bone marrow MF - 1 Loose network of reticulin with many intersections, especially in perivascular areas MF - 2 Diffuse and dense increase in reticulin with extensive intersections, occasionally with only focal bundles of collagen and/or focal osteosclerosis MF - 3 Diffuse and dense increase in reticulin with extensive intersections with coarse bundles of collagen, often associated with significant osteosclerosis

At time of transplantation all patients suffered from advanced myelofibrosis 2° [39 %] or 3° [61 %], resulting in a median MF grade of 3 [range 2–3]. After engraftment on day+30 a regression of MF grade to a median of 2 [range 0–3] was observed. Grade 2 or less was observed in 80 % and grade 1 or 0 in 40 % of the patients. On day+100 the median grade of MF was 1 [range 0–3] and 93 % of the patients had an MF-grade of 2 or less, while 73 % of the patients had MF grade-1 or 0. One year after transplantation the median MF-grade was 0 [range 0–2] and 100 % of the patients had a MF-grade of 2 or less and 92 % of the patients had MF grade 1 or 0. We conclude that allogeneic stem cell transplantation after a dose-reduced conditioning induces a rapid and nearly complete regression of bone marrow fibrosis within the first year after transplantation.
Efficacy and Safety of Once Weekly Epoetin Beta 30 000 IU in 691 Patients with Solid or Lymphoid Malignancies Receiving Chemotherapy: Results from the NAUTICA Study.
Authors: Spaeth Dominique , Salles Gilles , Facon Thierry , Garidi Reda , Pichon Francois , Damaj Ghandi , Bergougnoux Loic
Published: October 2019

Anemia is the most frequent hematologic complication of cancer, affecting 68% of patients at some time (Ludwig et al, Eur J Cancer 2004). Anemia causes debilitating fatigue and has a major impact on quality of life. Epoetin beta* (NeoRecormon®) is an effective treatment for anemia that has traditionally been administered three times weekly; however, a once weekly (QW) schedule would be more convenient and may encourage patient compliance. This open-label, single-arm study evaluated the efficacy and safety of epoetin beta 30 000 IU QW in patients with solid or lymphoid malignancies. Adult patients with anemia (hemoglobin [Hb] levels 8–12 g/dL), a WHO performance status 0–2 and who were scheduled to receive chemotherapy were enrolled from 87 centers in France between 31 Dec 2003 and 30 Aug 2005. Patients received SC epoetin beta 30 000 IU QW for up to 16 weeks. Primary efficacy parameter was change in Hb level. Hb response was defined according to patients’ baseline Hb level: in patients with Hb levels of 11–12 g/dL, response was defined as achievement of Hb level of ≥ 13 g/dL; in patients with Hb levels of <11 g/dL, response was defined as Hb increase of ≥ 2 g/dL or Hb level of ≥ 12 g/dL. Of the 691 patients included in the intent-to-treat population, 540 had baseline Hb levels <11 g/dL and 151 had baseline Hb levels of 11–12 g/dL. Fifty-three percent had solid tumors and 47% had lymphoid malignancies. The median duration of treatment was 14 weeks. Mean (±SD) baseline Hb level was 10.1±1.1 g/dL. At endpoint, mean Hb level had increased to 12.0±2.2 g/dL. Hb levels increased rapidly in the subgroup of patients with baseline Hb <11 g/dL, which corresponds to the intervention level recommended by the EORTC (figure). Hb response was seen equally in patients with lymphoid malignancies (60%) or solid tumors (61%). Median time to response was 49 (range 10–130) days. Hb response was seen with all types of chemotherapy. Epoetin beta was well tolerated. Seven percent of patients presented with thromboembolic events, a rate consistent with information provided in the current label for epoetin beta. Epoetin beta 30 000 IU QW is effective in anemic patients with solid tumors or lymphoid malignancies. This regimen provides a convenient and well tolerated treatment which may encourage patient compliance.

Figure. Increase in Hb levels during epoetin beta therapy: patients with baseline Hb levels <11g/dl (n=540) Figure. Increase in Hb levels during epoetin beta therapy: patients with baseline Hb levels <11g/dl (n=540)
Alemtuzumab-Cyclosporin A for Bone Marrow Failure Syndrome.
Authors: Kim Hawk , Shin Soojin , Park Jeo-Hoo , Min Young Joo
Published: October 2019

Patients with bone marrow failure syndrome are suffered from cytopenia and resulting requirement of transfusion. Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the standard therapy for severe aplastic anemia (AA) with suitable sibling donor. However, it is true in many cases that alloHSCT can not be performed even in very severe AA due to no suitable donor. In that regards, immunosuppressive therapy (IST) is another option for those patients. Alemtuzumab (ALM) targets not only B-cell but also T-cell, which is known to play an important role in pathogenesis of bone marrow failure syndrome (BMFS). Although there were several reports of ALM for immune cytopenia and pure red cell aplasia, no report were published regarding ALM for BMFS. We studied the feasibility of ALM and cyclosporine A (CsA) for patients with BMFS. Inclusion criteria were transfusion-dependent BMFS including AA, MDS, PNH. Prior IST or other drugs for BMFS except for ALM were not excluded. ALM was infused for 3 days (10mg on day 1, 20mg on day 2, and 30mg on day 3) along with CsA for at least 6 months. Prophylactic ciprofloxacin, acyclovir, and bactrim were given for first 2 months. Without partial response (PR) or more within 6 months, subsequent alloHSCT or antithymoglobulin therapy was allowed. Total 12 patients were enrolled. Female were 9 (75%). Median age was 43 (16–74) years. Disease were 9 severe AA, 2 moderate AA and 1 hypoplastic MDS. All patients were transfusion-dependent. Median amount of transfused red cell, platelet concentrate and platelet apheresis prior to ALM-CsA were 2, 2 and 7 units, respectively. Prior therapy were none in 9 (75%), anti-thymoglobulin in 2 (16.7%), and danazol in 1 (8.3%). Only 1 patient could not receive full scheduled CsA because the patient underwent alloHSCT. Median follow-up was 326 (140–466) days. Response to ALM-CsA were no response in 7 (58.3%), PR in 4 (33.3%), and CR in 1 (8.3%). Therefore, overall response rate was 5/12 (41.7%). All responsive patients showed their responses within 3 months. Median time to response was 47 (24–71) days [Figure 1]. PR was converted to CR in 1 patient at 12 months after treatment. Neither fever nor skin rash during treatment did not affect the possibility of response to ALM-CsA (p=0.523, p=0.523, respectively). Toxicity during ALM was fever in 3 (25%), skin rash in 3 (25%), G1 ALT elevation in 7, G1 AST elevation in 4, and hyperbilirubinemia in 3 (G1 in 2, G3 in 1). There were no anaphylaxis, serum sickness or CMV reactivation.

In spite of small number, AML-CsA showed comparable response rate within 3 months after treatment. Therefore in conclusion, ALM-CsA is feasible and tolerable therapy for BMFS.

Figure Figure
Cyclosporine A Effect the CD28 Expression on CD8+T Cells of Patients with Aplastic Anemia through down Regulate FLIP.
Authors: Huang Haiwen , Wu De Pei , Chen Guanghua , Fu Jinxiang
Published: October 2019

Objective The exact reason of aplastic anemia is still unknown. Recently more and more researchers realized that immune factors play an important role in aplastic anemia. In this study, we detected the expression of CD28 on different subgroups of T cells in patients with aplastic anemia and furthermore studied the effect of cyclosporine A to CD28 and FLIP on these cells. Method 21 patients and 15 healthy controls were investigated. The Expression of CD4CD28 and CD8CD28 on patients’ T cells was detected with FCM; patients’ CD8+T cells were purified with Mini MACR system, then the expression of FLIP, caspase-8 in CD8+T cells were analyzed by RT-PCR; we also detected the expression of CD28, FLIP and caspase-8 in patients’ CD8+T cells after incubation with cyclosporine A.

Result: The percentage of CD8+CD28−T cells in the patients is significantly higher than that of normal controls (30.45±5.26% versus 19.32±4.72%, p<0.05) and it can be induced to return to normal after effective treatment with Cyclosporine A; the expression of FLIP in patients’ purified CD8+ T cells is higher than that in healthy controls’ CD8+ T cells, but there is no difference in the expression of caspase-8; after being cultured with Cyclosporine A, the expression of FLIP in patient’s CD8+T cells was down regulated and the quantity of CD28 on the surface of patient’s CD8+T cells increased significantly (28.3±5.1% versus 40.6±7.71%40.6±7.71%).

Conclusion: The increased expression of FLIP in patients’ CD8+T cells is related to the increased percentage of CD8+CD28−T cells in patients’ peripheral blood. Cyclosporine A can down regulate the FLIP expression and correct the abnormal percentage of CD8+CD28−T cells in patients with aplastic anemia.
Role of JAK 2 Mutation Detection in Budd-Chiari Syndrome (BCS) and Portal Vein Thrombosis (PVT) Associated to MPD.
Authors: Kiladjian Jean-Jacques , Cervantes Francisco , Leebeek Frank W.G. , Chevret Sylvie , Cazals-Hatem Dominique , Plessier Aurélie , Marzac Christophe , Cassinat Bruno , Raffa Sebastian , Garcia-Pagan Juan Carlos , Murad Sarwa Darwish , Janssen Harry L.A. , Valla Dominique , Casadevall Nicole , Fenaux Pierre
Published: October 2019

Background: MPD is found in up to 50% of BCS and 30% of PVT, but its diagnosis is often difficult, bone marrow biopsy (BMB) and endogenous erythroid colony (EEC) formation being the most reliable tests in our experience (Br J Haematol, 2005;129:553) and that of others. We assessed the diagnostic and prognostic value of V617F JAK2 detection in this situation.

Methods: Underlying prothrombotic factors were studied in 241 pts (104 BCS, 137 PVT) from 3 centers (Paris, Barcelona, Rotterdam). Based on BMB results (blindly reviewed), 31% of patients had MPD, 67% no-MPD, and 2% were uncertain. Based on combined BMB+EEC results, 29% had MPD, 41% no-MPD and 31% had discordant features. V617F JAK2 mutation was studied in all patients by quantitative RT-PCR.

Results: V617FJAK2 was detected in 45% of BCS and 34% of PVT (p=.08). Mean % circulating V617F allele was 37% and 28% in BCS and PVT pts, respectively (p<.04). In pts diagnosed with MPD using conventional criteria, V617F mutation was found in 84% pts with positive BMB, and in 97% pts with concordant BMB+EEC results (table). In pts previously diagnosed as no-MPD, V617F was present in 19% pts with negative BMB and in 7% pts with both negative BMB and EEC. This discrepancy was greater in BCS patients, where V617F was found in 25% pts previously not diagnosed with MPD. V617F was also found in 58% of pts with discordant BMB and EEC results, allowing to increase the rate of definite MPD diagnosis. On the other hand, 16% pts with wild type JAK2 had MPD features on BMB and 20% had positive EEC. Taking into account V617F JAK2 in addition to BMB and EEC, definite MPD incidence increased from 30 to 49% overall; from 32% to 60% in BCS patients; and from 29% to 49% in PVT patients. V617F JAK2 was significantly associated with higher Hb, WBC, and platelet counts, larger spleen size, lower serum EPO, and higher red cell mass. Other prothrombotic states were found in 19/92 V617F patients and 29/144 wtJAK2 patients (NS). Baseline BCS prognostic scores (Child Pugh, Clichy and Rotterdam scores) did not significantly differ between V617F and wtJAK2 patients. BCS and PVT survival was not affected by the presence of V617F JAK2.

Conclusion: Detection of V617FJAK2 (1) is almost as common in PVT as in BCS but the % of mutant allele is higher in BCS; (2) is not fully redundant with EEC or BMB findings and increases the diagnosis of underlying MPD by 28% in BCS and by 20% in PVT; (3) has no discernible impact on medium term outcome of BCS or PVT. (Supported by EU: QLG1-CT-2002-01686).

% of V617F JAK2 positive All patients BCS PVT BMB / EEC + / + 97 100 94 − / − 7 25 0 Discordant 58 53 62 BMB + 84 75 92 − 19 27 12 EEC + 77 84 72 − 25 38 18 inconclusive 33 36 30
Enalapril Therapy Prevents Cardiac Remodelling of Sickle Cell Disease Patients.
Authors: Saad Sara T. , Lima Carmen S. Passos , Ueti Osvaldo M. , Ueti Adriana A. , Franchini Kleber G. , Costa Fernando F.
Published: October 2019

Angiotensin-converting enzyme (ACE) inhibitors are capable of decreasing cardiac remodelling in patients with cardiac dysfunction, however their effects on sickle cell disease (SCD) are unknown. Thus, this study aimed to investigate the cardiac effects of enalapril on SCD patients. Thirteen adult patients with sickle cell disease (SCD) and microalbuminuria (3M/10F); 11 sickle cell anemia patients (SCA), 1 Sβ thalassemia patient (Sβ) and 1 patient with hemoglobin SC (SC) were treated with enalapril. Thirteen other SCD patients (4M/9F) without microalbuminuria, matched according to age, diagnosis (11 SCA, 1 Sβ and 1 SC), and levels of hemoglobin, hematocrit and fetal hemoglobin, did not receive enalapril and were followed up as the control group in the same period of study. In the treated group, enalapril (5mg) was administered to 9 SCD patients during the entire study period. One patient did not complete follow-up, and higher doses (7.5mg to 20mg) were administered to 3 patients who developed systolic BP over 120 mmHg during the study period. Median age (28.5 vs 29.0; P= 0.580), baseline values of hemoglobin (8.5 vs 8.4g/dL, P= 0.600), hematocrit (25.0 vs 23.5%, P= 0.500), fetal hemoglobin (4.4 vs 4.1%, P=0.720) and mean blood pressure (MBP; 80 vs 93mmHg, P= 0.13) were similar in treated and untreated patients Echocardiograms were performed before the study entry and once a year in patients and controls. Comparisons of groups were performed at the beginning of the study and after 36 months of follow-up using the Wilcoxon-signed rank test and Spearman coefficient. At 36 months of follow-up, MBP was lower than the baseline value (93 mmHg vs 87 mmHg, P= 0.018) in the treated group. Significant increases in left ventricular mass (192 vs 231g, P= 0.005), posterior left ventricular wall thickness in end-diastole (8.5 vs 10.0mm, P= 0.013), left ventricular mass index (114.4 vs 131g/m2, P= 0.043), interventricular septal wall thickness in end-diastole (9.0 vs 9.5mm, P= 0.036) and aortic root dimension (28 vs 32mm, P= 0.009) values were seen in untreated, but not in enalapril treated patients. No major changes were seen in left ventricular ejection fraction, left ventricular systolic diameter, left ventricular diastolic dimension and left atrial diameter, in both groups, along the observational period. No significant correlation was detected between the data here presented. At the end of the study, no symptoms or signals related to cardiac failure were found in any of the enrolled patients. These results indicate a trend toward cardiac and aortic root remodeling in untreated SCD patients and suggest that long-term treatment with ACE inhibitors has beneficial effects on the cardiac remodeling of SCD patients and could be indicated for adult patients, if an increase in baseline blood pressure occurs.
A New Sequence Change in the Hs2-Lcr (G→A − 10.677) and Gg-Globin Gene Promoter Region (T→C − 157 and a 4 Bp Deletion − 222 to − 225) in Sickle Cell Anemia Patients with Fetal Hemoglobin Level and bS-Globin Gene Haplotypes Diversity.
Authors: Adorno Elisangela V. , Neto Jose P. Moura , Lyra Isa , Zanette Angela D. , Santos Leandro F. , Seixas Magda O. , Reis Mitermayer G. , Costa Fernando F. , Goncalves Marilda S.
Published: October 2019

The sickle cell anemia has a very high prevalence in Bahia, a state of Brazil. The HbF levels and bS-globin gene haplotypes of 125 sickle cell anemia patients were investigated and the Gγ and Aγ gene promoter and HS2-LCR regions of ten patients were selected for DNA sequence. The study was approved by the Oswaldo Cruz Research Foundation’s human research ethics committee. The bS- globin gene haplotypes were investigated using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques; the HbF levels were estimated by high performance liquid chromatography (HPLC). The Gg and Ag-globin gene promoter and HS2-LCR regions were sequenced in an automatic sequencer. The distribution of bS- globin gene haplotypes showed 64 (51.2%) patients with genotype CAR/Ben; 36 (28.8%) Ben/Ben; 18 (14.4%) CAR/CAR; two (1.6%) CAR/Aty; two (1.6%) Ben/Cam; one (0.8%) Car/Cam; one (0.8%) CAR/Arab-India and one (0.8%) Sen/Aty. Among these patients, four CAR/CAR had HbF ≥10.0%; three Ben/Ben had HbF ≤ 5.0%; 11 CAR/Ben had HbF ≥15.0% and 18 had HbF ≤ 5.0%; two Cam/Ben patients had HbF levels ≥15.0%. Ten individuals presenting HbF and bS-globin gene haplotypes diversity were selected to sequence the Gg and Ag-globin gene promoter and HS2-LCR regions. The HS2-LCR sequence analyses demonstrated a G→A change (−10.677) (GeneBank DQ873522) in five sickle cell anemia patients with Ben haplotype and high HbF levels (Table 1); the CAR/Ben and Ben/Ben patients with low HbF levels did not have this sequence variation; among the small patient group investigated, there were two CAR/CAR patients with high HbF levels without this substitution. The Gg gene promoter sequence analyses showed a T→C substitution (−157) (GeneBank DQ873521) among all patients and a 4 bp deletion (−222 to −225) (GeneBank DQ873519) related to Cam haplotype (Figure 1). The HS2-LCR sequence change reported here is located in the proximity to a binding site of the GATA-1 and a ubiquitous trans-acting factor and can constitute a motif associated to the Ben chromosome and may play an important role in g-globin gene transcription regulation. The polymorphic site on Gg-globin gene promoter region can be a common sequence characteristic among the Brazilian sickle cell anemia patients. The results described here suggest new polymorphisms in the HS2-LCR and Gg gene promoter and confirms the genotypic heterogeneity among Brazilian sickle cell anemia patients, justifying further studies to investigate its correlation with HbF synthesis and clinical profile in sickle cell anemia patients.

Figure Figure
Correlation between Dose-Dependent Reductions in Serum Transaminase (ALT) and Serum Ferritin Levels during Long-Term Chelation Therapy with Deferasirox (Exjade®, ICL670).
Authors: Brissot P. , Deugnier Y. , Cianciulli P. , Cario H. , Jeng M. , Rabault B.
Published: October 2019

Introduction: Hepatic siderosis, a result of chronic transfusion therapy, leads to hepatocellular injury and progression to chronic liver disease. Increases in serum transaminase levels are indicative of progressive liver damage. The effect of deferasirox (Exjade®, ICL670), a novel, once-daily oral chelator, on liver iron content and pathology in chronically transfused patients has been assessed in Phase II and III clinical studies. A sub-analysis of pooled data from the planned 4-year extension of two pivotal studies sought to correlate the long-term changes in serum ALT levels with those of serum ferritin in iron-overloaded patients during treatment with deferasirox.

Methods: In these two clinical studies, 480 patients with β-thalassemia (β-thal), myelodysplastic syndromes (MDS), Diamond-Blackfan anemia (DBA), and other transfusion-dependent anemias received deferasirox at 5, 10, 20 or 30 mg/kg/day according to baseline liver iron concentrations for 12 months. In the extension phases, doses were titrated according to patient response, with dose escalation for patients who did not receive sufficient drug in the core study to reduce iron burden. Serum ferritin and ALT levels were assessed at baseline and monthly during the core and extension phases. For this analysis, serum ferritin and ALT levels were analyzed in the combined patient groups who received deferasirox 5 and 10 mg/kg/day, and in those who received 20 and 30 mg/kg/day.

Results: Throughout the entire period, deferasirox at 20 and 30 mg/kg/day resulted in a negative iron balance in transfused patients with secondary iron overload. Changes from baseline to week 128 (2.5 years) for serum ferritin and ALT levels are presented. The figure shows that deferasirox at 20 and 30 mg/kg/day decreased median serum ferritin. These decreases were mirrored by decreases in ALT, with a Spearman’s rank-correlation 0.67. Patient numbers shown are baseline/week 128. A number of patients had not reached week 128 of treatment at the time of data cut-off.

Figure Figure

Decreases in ALT were also observed after dose escalation in the extension phase in patients originally assigned deferasirox 5 and 10 mg/kg/day. There were no consistent relevant changes in other biochemical liver parameters, such as serum aspartate transaminase, in this patient population.

Conclusions: With long-term follow-up of this large patient population, correlation between decreases in serum ferritin and ALT levels was high, indicating an association between iron burden and liver dysfunction, and further emphasizing the potential beneficial effect of deferasirox on hepatic function.
Thrombogenicity of Microparticles Derived from Vascular Cells.
Authors: Isobe Hirotaka , Perkmann Thomas , Oskolkova Olga , Bochkov Valeri , Binder Bernd R.
Published: October 2019

Microparticles (MPs) are released from cells during processes such as apoptosis or during cell activation. These MPs contain phospholipids, proteins and even nucleic acids derived from their parent cells. They are found circulating in plasma but also in tissues such as atherosclerotic plaques. It is thought that MPs contain and transfer tissue factor and can thereby induce blood clotting.

In this study we analyzed clot promoting properties of MPs generated from vascular cells in vitro. MPs were generated from endothelial cells (EC), smooth muscles cells (SMC), monocytes (U937), erythrocytes (RBC) or platelets (Pl) by inducing apoptosis or by calcium ionophore activation; they were subsequently isolated by differential centrifugation. Thrombogenicity of the MPs was evaluated using a thrombin generation assay (Technothrombin® TGA) and MP free plasma as substrate. MPs displayed a different thrombin generating potential depending on the parent cells. MPs derived from RBCs (~400nM peak thrombin/105 MPs/ml plasma), ECs (~300nM), SMCs (~300nM) and Pls (~300nM) were more thrombogenic than MPs derived from U937 (~200 nM). In addition EC, SMC and U937 MPs all expressed tissue factor but EC MPs induced thrombin generation in a tissue factor and FVII independent manner. EC MPs even expressed active tissue factor pathway inhibitor and functionally inhibited tissue factor dependent thrombin generation. Since the higher thrombin generation induced by MPs derived from EC as compared U937 derived MPs could not be explained by a different activity of tissue factor, we were interested whether lipids contained in the microparticles could account for the differences in thrombin generation. We therefore analyzed thrombin generation induced by lipids isolated from MPs and parent cells and could show that lipids from EC MPs and SMC MPs exhibited higher thrombin generation than those from U937 MPs. Upon analysis of lipids by thin layer chromatography and mass spectrometry we found that in general microparticles are enriched in cholesterol, sphingomyeline and phosphatidylserine over the parent cells and that EC and SMC MPs were enriched in negatively charged phospholipids (different species of phosphatidylserine and phosphatiylglycerol) as compared to MPs derived from U937 cells. When thrombogenicity was, however, evaluated in vivo by injecting MPs into mice it was found that the highest capability to induce thrombin-antithrombin (TAT) complexes had MPs derived from SMCs; also U937 MPs induced an increase in TAT levels, while EC MPs – although more thrombogenic than U937 MPs in vitro – did not induce TAT complex formation by themselves but were only synergistic in vivo.

From these data we conclude that thrombin formation in vivo depends on the initiation of the tissue factor FVII pathway, while the extent of thrombin formation is dependent on negatively charged phospholipids contained to a higher extent in MPs derived e.g. from ECs.
S-MRI Score as a Simple Method To Assess Bone Marrow Involvement in Gaucher Disease.
Authors: Roca Mercedes , Mota Juan , Alfonso Pilar , Giralt Manuel , Pocovi Miguel , Giraldo Pilar
Published: October 2019

Background: Semiquantitative MRI is a very useful procedure to evaluate the bone marrow burden in Gaucher disease (GD). Score systems have been applied to obtain a parameter to evaluate the severity of bone disease. Our purpose was applied a simple, reproducible and accurate score to evaluate completely bone marrow involvement in GD patients

Patients and Methods: MRI was performed in spine, pelvis and femora at diagnosis in 54 adult consecutively diagnosed as GD1, 61.1% females. Three MRI patterns and punctuation in each location were defined: normal: 0; non-homogeneous infiltration subtypes reticular:

mottled: diffuse: and homogeneous infiltration: This score was called as Spanish-MRI (S-MRI):

Two independent observers applied the S-MRI and Bone Marrow Burden (BMB) score and to compare the differences between both systems by using non parametric Mann-Whitney test. Correlation rank test was calculated.

Results: In 46 patients (85.2%) bone involvement was observed, 39 (72.3%) has spine affected, 35 (64.8%) pelvis and 33 (61.2%) femorae, 14 patients had bone infarcts, 14 avascular necrosis, 2 vertebral fracture and 2 bone crisis. Correlation analysis between S-MRI and BMB was significant (r2:.675; p= .0001). No evidence of correlation was observed between CT activity and S-MRI and neither between CT activity and BMB. We have analysed the relationship between the patient genotype and bone infiltration according S-MRI, the location and complications. Patients with genotype N370S/rare allele (n=22), have more bone complications (90.1%) Infarcts: 9; AVN: 8, Bone crisis: 2; and vertebral fracture: 1. The mean S-MRI in this group was 7.9. Patients N370S/L444P (n=16) were the higher group with spine involvement (87.5%) mean S-MRI: 8.7. By contrast all patients homozygous for N370S (n=8), have femorae involvement, but a low S-MRI 7.5. The remaining 5 patients have complex genotypes (rare/rare), nevertheless they are not significant data(p=.335, p=.795)

Conclusions: S-MRI is a simple method that provides useful information to evaluate bone infiltration and detect silent complications. Our results were correlated with the BMB score with higher sensitivity and specificity and provide accuracy to classify the degrees of the bone disease

This work has been performed with the collaboration of the Spanish Group on Gaucher Disease and partially sponsored by a grant from FEETEG.
Toll-Like Receptor 2 Signalling Regulates Oxidative Burst of Human Neutrophils, but Not Cytokine Response of Dendritic Cells after Contact with Aspergillus fumigatus.
Authors: Loeffler Juergen , Mezger Markus , Wozniok Iwona , Kurzai Oliver , Einsele Hermann
Published: October 2019

Toll-like receptors (TLRs) play a crucial role for detecting conserved pathogen-associated molecular patterns comprising fungal structures. Activation of polymorphonuclear granulocytes (PMNs) as well as mobilization of antigen presenting cells is important for successful clearance of fungal infections. Here, the role of TLR2 and TLR4 for activation of PMNs and immature dendritic cells (iDCs) through Aspergillus fumigatus was analyzed.

Generation of iDCs was achieved by cultivating monocytes in the presence of GM-CSF and IL-4. PMNs were obtained from fresh blood. In blocking studies, cells were preincubated with anti-TLR2 and / or anti-TLR4 antibody before stimulation with Aspergillus fumigatus germinating conidia. For RNA interference (RNAi) experiments, iDCs were transfected with short-interfering RNA (siRNA) via electroporation and gene expression was controlled by quantitative real-time PCR.

Oxidative burst of PMNs was induced after contact with Aspergillus or TLR2 ligand zymosan. Release of oxygen intermediates could be significantly reduced if PMNs were preincubated with anti-TLR2 antibody.

In contrast to monocytes, cytokine secretion (IL-12 and TNF-α) of iDCs was not altered if anti-TLR antibodies were used. To study the role of TLR2 and TLR4 more detailed, an RNAi system for iDCs was established to downregulate gene expression. Determination of siRNA transfection rate revealed an efficiency of about 85% and allowed significant downregulation of TLR2 and TLR4 (> 90%). In accordance to blocking studies, expression of IL-10, IL-12 and TNF-a was not reduced after TLR2 or TLR4 siRNA transfection and stimulation with Aspergillus.

We conclude that TLR2 plays an important role in the induction of the oxidative burst of PMNs whereas cytokine release of iDCs seems to be independent of TLR2 / TLR4 signalling. The established siRNA system allows the detection of receptors responsible for activation of iDCs.