bluuebunny/crossref_metadata_2025 huggingface dataset:
Analytical Characterization of Infusion Volume Reduced Humate-P®/Haemate® P.
Authors: Metzner Hubert J. , Kanzy Ernst-Jürgen , Kalbass Antje , Lind Holger , Mueller Heinz-Georg , Kumpe Gerhard , Schulte Stefan
Published: October 2019

Commercially available von Willebrand factor (VWF)/factor VIII concentrates are therapy of choice in severe von Willebrand disease (VWD), clinically characterized by serious bleedings, as well as in some cases of hemophilia A. Humate-P®/Haemate® P is one of the few products approved for therapy and prophylaxis in both indications, VWD and hemophilia A. As it was desirable to further improve the convenience of application for the patients by reducing the application volume, an infusion volume reduced (i.v.r.) Humate-P®/Haemate® P was developed increasing the product concentration while keeping the well-known positive product characteristics. Eight i.v.r. Humate-P®/Haemate® P batches were produced and vials of each batch were reconstituted using the reduced volume of water for injection, i.e. 5 mL (250 IU F VIII), 10 mL (500 IU F VIII), for 15 mL (1.000 IU F VIII). These lots were compared analytically with 18 lots of current Humate-P®/Haemate® P. The analytical comparison demonstrated that the i.v.r. product contains twofold increased concentrations of the active ingredients VWF and F VIII (see table). As the purification and virus inactivation processes have not been changed, the quality of the active ingredients is maintained. This e.g. could be shown by a high VWF:RCo/F VIII:C ratio of about 2.4 for the volume reduced as well as the current product. Additionally, the VWF:RCo/VWF:Ag ratio and the high molecular weight (HMW) multimer structure of i.v.r. Humate-P®/Haemate® P are equivalent to current Humate-P®/Haemate® P (see table) and close to that of NHP indicating a comparably good efficacy of i.v.r. and current products. Based on the analytical investigations described here, it can be concluded that the development of i.v.r. Humate-P®/Haemate® P resulted in a product of unchanged quality of the active ingredients but very high VWF concentration. This allows the application of VWF in small volumes and, consequently, will furthermore improve the convenience of VWD and hemophilia A treatment.

Table Humate-P®/Haemate® P i.v.r. Humate-P®/Haemate® P Parameter 250/500 IU (n=12) 1000 IU (n=6) 250/500 IU (n=5) 1000 IU (n=3) F VIII:C (1-stage), IU/mL 24.5±1.3 31.2±2.0 51.2±0.8 66.3±4.2 VWF:RCo, IU/mL 58.3±4.1 78.3±4.0 119.8±10.9 159.3±14.2 VWF:RCo/VWF:Ag 0.78 ± 0.08 0.81 ± 0.06 VWF:RCo/F VIII:C (1-stage) 2.43 ± 0.12 2.36 ± 0.15 VWF HMW Multimers (>10) 75 ± 5.5% of NHP 77 ± 3.5% of NHP
Validation of TSE Agent Removal by Immunoaffinity and Ion Exchange Chromatography Steps of the ReFacto Manufacturing Process.
Authors: Booth James , Tannatt Molly , Vicik Steve , Kelley Brian
Published: October 2019

ReFacto® (moroctocog alfa), a recombinant Factor VIII approved for treatment of hemophilia A is produced by a mammalian cell culture process that includes therapeutic-grade human serum albumin (HSA) in the cell culture medium. The plasma-derived HSA used in the cell culture process is sourced from countries whose blood supply does not pose a significant transmissible spongiform encephalopathy (TSE) risk, and is manufactured utilizing a fractionation process that has demonstrated the capability to significantly reduce the level of experimentally added TSE infectivity. While to date, no case of TSE has been identified with a history of exposure to fractionated HSA, Wyeth has conducted a study to demonstrate that the ReFacto manufacturing process has significant capacity to remove a TSE agent if it were present in the HSA. The ReFacto purification process consists of five chromatographic steps, two of which were validated for removal of a TSE agent: the immunoaffinity step (8A4 Sepharose chromatography) and anion exchange step (Q Sepharose chromatography). The GLP study was performed using appropriately qualified lab-scale systems, which model the performance of the production equipment. Brain homogenates from scrapie-infected hamsters (263K strain) were added to loads of the respective chromatography steps and levels of the scrapie agent in the product pools were determined using a validated Western blot quantitation method. Duplicate runs were performed to assess the consistency of the TSE agent reduction. The results show that both chromatography steps provide significant clearance of the TSE agent, with greater than a billion-fold (9 log) reduction in infectivity provided by the two process steps in combination. The consistency of the duplicate run results was very good (less than 0.7 log difference between runs). While the risk of TSE contamination of HSA is only theoretical, these data provide a high degree of assurance that in the event of an adventitious TSE contamination of the HSA used in the ReFacto cell culture process, the purification steps would remove the infectious agent to safe levels.
Late Increase of Peripheral CD34+ Cells Number during Acute Liver Failure Recovery.
Authors: Drénou Bernard , Corlu Anne , Desille Yoann , Sicot Christèle , Fardel Olivier , Amiot Laurence , Lorho Richard , Camus Christophe , Guillygomarc’h Anne , Boudjema Karim , Guyader Dominique
Published: October 2019

Human Hematopoietic Stem Cells (HSC) of bone marrow origin are reported to transdifferentiate into various cell types, including cells of hepatic lineage, and to be involved in hepatic repopulation. As it has been previously reported in the course of myocardial infarction, we hypothesized that CD34+ cells would appear early in severe acute liver failure and could predict a favourable outcome.

Methods: We quantified circulating CD34+ cells in peripheral blood of 146 patients without and with severe hepatic failure (HF) defined by factor V <50%: 20 acute hepatitis with HF, 26 acute alcoholic hepatitis with HF, 22 major right hepatectomy (6/23 developped HF) and 78 controls (31 healthy subjects, 23 cirrhosis, 24 cirrhosis with chronic HF). CD34+ cells were quantified by flow cytometry, according to ISHAGE guidelines in both one and dual platform approaches. Phenotypic and functional analyses were performed in selected cases. In case of acute HF, quantification was done every day until correction of liver function. One patient underwent liver transplantation. The number of circulating CD34+ cells was correlated with factor V level. Results were presented using median and range.

Results : At inclusion, the numbers of CD34+ cells /mm3 were similar in different groups (healthy controls = 1.0 [0.1– 4.0]; cirrhosis = 0.9 [0.1–5.1]; cirrhosis with chronic HF = 1.0 [0.1–3.7]; alcoholic hepatitis with HF = 1.2 [0.1–6.0]; liver resection 24H after surgery = 0.8 [0.2–3.2]; acute hepatitis with HF = 0.7 [0.2–3.1]. For patients with acute hepatitis with HF, the number of CD34+ increased over time, from 0.7 [0.2–3.1] to 1.8 [0.5–9.6] at the 4th day after admission and was significantly (p <10–3) correlated with correction of factor V. There was no correlation between the number of CD34+ and the time between admission and onset of the disease. There was no significant increase over time in the hepatectomy group nor in the alcoholic hepatitis group. CD34+ cells detected during acute hepatitis regeneration presented an immature phenotype and coexpressed CD33, CD90, CD133, CD117, CD135 (FLT3-R), CD38, HLA-DR and not V-Cadherin. Clonogenic progenitor cell assays did not reveal specific features and correlation between number of CD34 and BFU-E colonies were in favour of their hematopoietic potentiality.

Conclusion: By contrast with data reported in acute myocardial infarction in which early mobilisation of CD34+ cells was observed, we found that the increase of circulating CD34+ HSC is a late event occurring at the time of the recovery of liver function in acute severe hepatic failure. The hematopoietic potentiality of mobilized CD34+ cells suggests that they do not play a major role in liver repopulation in fulminant acute hepatitis.
Mechanisms by Which NK T Cells Become the Predominant T Cell Subset in Mice after Irradiation.
Authors: Yao Zhenyu , Liu Yinping , McIntire Jennifer , Strober Samuel
Published: October 2019

Previously, we found that the percentage of NK T cells among all T cells in the spleen of mice treated with fractionated irradiation to the lymphoid tissues (Total lymphoid irradiation; TLI) with a total dose of 4,080 cGy increased markedly due to greater reduction in the absolute number of non-NK T cells as compared to NK T cells. The underlying mechanisms of the change in the T cell subsets after irradiation remained to be established. In the current study, C57BL/6 mice were given escalating single doses of 240, 1,000, 2,000 and 3,000 cGy total body irradiation (TBI). Splenocytes were harvested at 4 or 24 hours after irradiation, and the percentage and absolute number of NK T and non-NK T cells was determined. At the same time, the intracellular level of the anti-apoptotic protein, Bcl-2 was assayed by flow cytometry. In some studies, the turnover rate of NK T cells and non-NK T cells was examined by injection of BrdU and intracellular staining. At 4 hours after all doses of irradiation, neither the NK T nor non-NK T cell subset had a significant change in percentage or absolute number as compared to untreated controls. However, at 24 hours the percentage of NK T cells among all T cells had progressively increased with increased doses of TBI from 3% in the untreated controls to 65% in mice given 3,000 cGy. Whereas the absolute number of non-NK T cells decreased at least 1000 fold, the absolute number of NK T cells decreased approximately 50 fold after 3,000 cGy. The BrdU incorporation of NK T cells from irradiated mice was markedly reduced as compared to untreated mice, and was similar to that of non NK T cells in these irradiated mice. 8–12% of NK T cells and non NK T cells in untreated mice expressed a high level Bcl-2. As the dose of TBI increased progressively, the percentage of Bcl-2hi cells increased progressively to 89% amongst NK T cells and 70% amongst non-NK T cells. At each irradiation dose, the percentage of Bcl-2hi cells amongst NK T cells was higher than amongst non-NK T cells. There were 40×103 Bcl-2hi NK T cell and 10×103 Bcl-2hi non-NK T cells surviving per spleen at 24 hours after 3000 cGy TBI. The absolute number of Bcl-2hi NK T cells decreased by about two fold while the absolute number of Bcl-2hi non-NK T cells decreased by about 100 fold. These results indicate that the increased percentage of NK T cells amongst all T cells after irradiation is due to greater radioresistance rather than to more rapid replenishment of NK T cells as compared to non-NK T cells. We are investigating whether Bcl-2 plays a critical role in the extraordinary radioresistance of the NK T cells.
Peripheral Blood Progenitor Cell Apheresis Increases Circulating Stem Cells.
Authors: Chang Naibai , Pei Lei , Xu Xiaodong , Wei Jianping , Gu Xichun , Liu Hui
Published: October 2019

Peripheral blood hematopoietic stem cell transplantation has been replacing the bone marrow source stem cell transplantation for the treatment of hematological malignancies. Many efforts were made to enhance the stem cell number in peripheral blood and optimize the peripheral stem cell collection. Hematopoietic stem cell circles between bone marrow and peripheral blood during maturation. Thus the recruitment of hematopoietic stem cell in peripheral blood is a factor influencing the efficacy of peripheral blood stem cell apheresis. The aim of this study was to evaluate the effects of apheresis on the number of circulating stem cells. There were 41 consecutive cases involved. The mobilization regimen was chemotherapy + G-CSF. All patients underwent at least two apheresis. The volume of blood processed was calculated automatically by COBE Spectra cell separator. Blood samples were drawn just before each apheresis. CD34+ cells both in blood samples and in apheresis products were analyzed by multicolour flowcytometry. CFU-GM was cultured at same time. Data from the 1st and 2nd collection was compared. CD34+ /CD38− cells in peripheral blood increased from 48±12/ul to 133±40/ul (p<0.05) and CD34+/HLA-DR− cells from 45±12/ul to 97±29/ul (p<0.05) after apheresis. But CFU-GM was unchanged (154±35/105 cells to101±20/105 cells). CD34+ /CD38− cells, CD34+/HLA-DR− cells and CFU-GM in apheresis products were slightly increased but not reached the statistical significance(2.10±2.31x108 to 2.89±2.08x108, 2.09±2.35x108 to 2.32±1.61x108, 1.28±1.53x107 to1.93±3.11x107 respectively).

Conclusions: Peripheral blood stem cell apheresis promotes stem cell recruitment in peripheral blood and increases circulating hematopoietic stem cells.
Atypical Presentations of EBV Reactivation and EBV-Associated PTLD Post-HSCT: Implication for Routine EBV Surveillance after Allogeneic HSCT?.
Authors: Dobbelstein Christiane E. , Kamal Haytham M. , Dammann Elke , Hertenstein Bernd , Weissinger Eva M. , Ganser Arnold
Published: October 2019

Allogeneic hematopoietic stem cell transplantation (HSCT), an effective treatment for leukemia and other disorders, produces profound immune deficiency in the early period after transplantation. Viral reactivations such as Epstein-Barr virus (EBV) are a major cause of morbidity and mortality after HSCT. EBV-associated posttransplant lymphoproliferative disease (PTLD) is a life-threating and often fatal complication occurring after allogeneic HSCT with a cumulative incidence of 1% at 10 years. Most cases occur during the first year after transplantation with the incidence highest at 2–3 months after HSCT. Typically PTLD presents as a disseminated disease with fever, lymph node enlargement, respiratory disease and feature of hepatitis. The common lymphadenopathy usually leads to diagnosis, with confirmation on viral tests. Here we retrospectively evaluate the patients at our institution since January 2002 to date (07/2005) who were allogeneic transplanted. Of the total 236 patients who were transplanted during this time period eight patients (3.4 %) developed a significant EBV reactivation with treatment need. Of the eight patients six had a fulminant course. Of these five patients died (EBV reactivation/PTLD is at least probable cause of death). Since at least two of the patients with the fulminant course presented atypically with fever as initial symptom and without other manifestations of PTLD like lymph node enlargement, these cases underline the need of an immediate pre-emptive therapy without any time delay for further diagnostic procedures e. g. diagnostic lymphadenectomy. While at present there is no recommendation of the EBMT for routine surveillance after HSCT, our data indicate the need for a standardized surveillance strategy.

UPN diagnosis conditioning GvHD prophylaxis HSCT max. viral load (EBV-DNA copies/ml) treatment outcome abbreviations: RIT: radioimmuno therapy; FLAMSA: fludarabine, amsacrine, cytosine arabinoside, cyclophosphamide, TBI 4Gy; TCD: T-cell depletion; (*): diagnosis post mortem. 888 AML Flu, Mel ATG, CsA, MTX, MMF MUD-PBSCT 11.0 x 10e6 (d +51) rituximab CR 908 AML TBI, Cy ATG, TCD MRD-PBSCT 1.75 x 10e6 (d +724) rituximab, foscavir, cyclophosphamide death 932 multiple myeloma Flu, Mel ATG, CsA, MTX MUD-PBSCT 2.3 x 10e6 (d +101) rituximab CR 973 AML RIT, TBI, Cy ATG, TCD MRD-PBSCT 10.0 x 10e6 (d +110) none (*) death 1140 multiple myeloma Flu, Mel ATG, CsA, MTX MUD-PBSCT 0.07 x 10e6 (d +278) rituximab death 1264 ALL TBI, Cy ATG, CsA, MTX MUD-PBSCT 1.75 x 10e6 (d +111) rituximab death 1271 AML FLAMSA ATG, CsA, MMF MMUD-BMT 7.0 x 10e6 (d +91) rituximab death 1300 aplastic anemia Flu, Cy, TBI ATG, CsA, MTX MRD-BMT 0.75x 10e6 (d +26) rituximab CR
Identifying Patients at Risk of Early Relapse after Submyeloablative Allogeneic HSCT.
Authors: Mehta Jayesh , Gordon L. , Tallman M. , Winter J. , Williams S. , Evens A. , Frankfurt O. , Grinblatt D. , Kaminer L. , Meagher R. , Singhal S.
Published: October 2019

Since graft-vs-tumor effects are the mainstay of sustained disease control after submyeloablative (mini) allogeneic transplantation, rapidly-progressive malignant disease may not be curable with SMAT. Figure 1 shows the cumulative incidence of chronic GVHD and relapse (n=35) in 63 patients undergoing mini-allografts for hematologic malignancies (acute leukemia, lymphoma/CLL, myeloma).

Figure Figure

The pattern suggests that a substantial number of relapses occur before chronic GVHD and associated GVT get established. The purpose of this analysis was to see if patients at risk of early relapse (relapse within 100 days; n=15 in this series) could be identified based on pre-transplant characteristics (diagnosis, chemosensitivity, Hb <9, platelets <100, abnormal LDH, albumin <3, performance status). In Cox analysis, the only factor predictive of early relapse was Hb <9 at the time of transplant (7 of 15 vs 8 of 48 with Hb ≥9; RR=3.1; P=0.029). The patients were also retrospectively classified by their physicians to have “slow” (n=33) or “rapid” (n=30) disease pre-transplant based on clinical impression. 11 early relapses occurred in patients with “rapid” disease and 4 in those with “slow” disease. When disease tempo was introduced in the Cox analysis as an additional variable, it was of borderline significance (P=0.051); with Hb <9 remaining as the sole predictive factor.

Figure Figure

The curves showing the temporal relationship between chronic GVHD and early relapse based upon Hb or disease tempo suggest that patients with Hb ≥9 or those with “slow” disease stand to benefit more than the others from graft-vs-tumor effects. Accordingly, restriciting mini-allografts to patients with Hb ≥9 or those with “slow” disease would be expected to improve outcome in terms of disease-free survival. However, neither of these two factors is particularly satisfactory: Hb <9 identifies only half the patients relapsing early, and the subjective assessment of disease tempo by the treating physician, while identifying a greater proportion of patients at risk of early relapse, excludes almost half the patients from the allograft. These data suggest that studies are needed in larger groups of patients to develop more robust (sensitive as well as specific) criteria to identify patients at risk of early relapse after mini-allografts. Since such patients are unlikely to benefit from a standard mini-allograft procedure, they may be better suited for alternatives such as conventional-intensity allografts, tandem autograft and mini-allograft procedures, or novel immunotherapeutic approaches applied very early after a mini-allograft.
Impact of High-Resolution HLA Matching on Outcomes of Unrelated Donor Hematopoietic Stem Cell Transplantation.
Authors: Kang Seok Yun , Kim Hyeoung Il , Lee Hyun Woo , Jang Jun Ho , Park Joon Seong , Choi Jin Hyuk , Lim Ho Yeong , Kim Hugh C.
Published: October 2019

Objective: We assessed the impact of high-resolution genotypic results of human leukocyte antigen (HLA) for all major class I and II loci between donors and recipients in the outcome of unrelated hematopoietic stem cell transplantation (HSCT).

Method: Between 1999 and 2005, high-resolution genotyping for HLA-A, -B, -C and -DRB1 was performed for 23 unrelated HSCT. All the patients were typed as HLA identical by serologic technique and then they were also typed HLA identical by high resolution technique. Unrelated bone marrow transplantation using DNA-based high resolution HLA compatibilities were considered in the analyses of clinical outcomes such as hematopoietic engraftment, acute GVHD, and survival. And then, we compared with patients who received related HSCT (same institute, same duration) and also unrelated HSCT data from IBMTR.

Results: Median follow up duration was 9 months (1–51). Fifteen patients were male and 8 were female. Median age was 22 years (range 6–52). Median time from diagnosis to transplantation was 7 months (range 4–63). Eight patients of acute myeloid leukemia (AML), 6 of chronic myeloid leukemia (CML, 2 of 6 were in blast crisis), 4 of acute lymphoid leukemia (ALL), 3 of severe aplastic anemia and each case of juvenile myelomonocytic leukemia and myelodysplastic syndrome were enrolled. Median value of total nucleated cell and CD34 positive cell count was 3.51 (1.06–20.7) x 108/kg and 4.88 (1.33–46.9) x 106/kg, respectively. The conditioning regimen and prophylaxis for graft versus host disease (GVHD) were not different from conventional HSCT except one case of non-myeloablative transplantation. Median value of granulocytic (absolute granulocyte count > 500/mm3) and platelet (> 20,000/mm3) engraftment were D + 16, D + 17, respectively. Grade II acute GVHD developed in 4 patients (2 patients subsequently proceded to chronic GVHD). Treatment related mortality was 2 out of 23 patients (8.7%). Median value of overall survival duration was 30 months. For AML patients, 3-year survival rate was 72.9 %.

Conclusions: Our survival data for unrelated HSCT based on high resolution genotyped HLA matching was superior to unrelated HSCT. Although the sample size is small, the survival data of AML patients (CR1 at transplant) was superior to the survivals of related HSCT, as well as that of unrelated HSCT. The findings were that transplantation using unrelated donors selected by high-resolution genotype identity improves the transplantation outcomes similar degree to the result of the related HSCT.
Simultaneous Transplantation of Three Cord Blood Units in Adults with High Risk Acute Leukemia.
Authors: Wiktor-Jedrzejczak Wieslaw , Rokicka Malgorzata , Urbanowska Elzbieta , Torosjan Tigran , Gronkowska Anna , Graczyk-Pol Elzbieta , Tomaszewska Agnieszka , Paluszewska Monika , Tormanowska Magdalena , Krol Malgorzata , Krol Maria , Jolkowska Justyna , Witt Michal
Published: October 2019

Adult patients with high risk acute leukemia have allogeneic hematopoietic transplantation as the only therapeutic option with some curative potential. Those who lack related or unrelated marrow donor are deprived of this possibility. Patients of smaller size may benefit from the transplantation of single cord blood unit, but for heavier patients two units may be required as reported by Barker et al. (Blood2005;105:1343). For very heavy patients even more units would be necessary to approximate the preferred threshold of 37 millions of cells per kilo. Moreover, cord blood units have variable size and it is always a risk that the only HLA-matched units for given patients would be small and two units would not provide adequate number of cells even for patients of moderate size. This is yet another argument to attempt transplantation of more than two units. Three patients have so far been included. Patient No 1 was 24 yrs old male (body weight 87 kg) with resistant AML transplanted with 3 units (two matched at 4/6 HLA antigens, one matched at 3/6 HLA antigens) on March 29, 2004 following conditioning with standard BuCy4 + ATG regimen. This patient died d. +42 of Gram negative sepsis without hematopoietic reconstitution. Patient No 2 is 22 yrs old male (body weight 100 kg) with Ph+ ALL in complete remission, who was transplanted on October 11, 2004 with 3 units (two matched at 4/6 HLA antigens, one matched at 3/6 antigens) following conditioning with TBI, etoposide and ATG. This patient had neutrophil recovery d. +27, and platelet recovery d. +49. He is alive and well 9 months after the transplant fully chimeric with progeny of only one of transplanted units. Patient No 3 is 31 yrs old male (body weight of 100 kg) with ALL relapsed after autologous BMT with multiple cytogenetic abnormalities. He was transplanted May 24, 2005 with 3 units (one matched at 5/6 HLA antigens, two matched at 4/6 HLA antigens) following conditioning with treosulfan, cyclophosphamide, melphalan and ATG (he earlier received TBI prior auto-BMT). Patient obtained neutrophil recovery d. +36 and as of d.+60 still has low platelets. No data on chimerism are yet available. These data confirm that transplantation of more than two cord blood units is feasible and may provide therapeutic option for patients with high risk leukemia who lack marrow donor and for whom two cord blood units do not provide sufficient number of cells. As in the case of the transplantation of two units patients seem to be reconstituted with progeny of only one “winning” unit.
HIV-1 Vector Mediated Expression of Human B-Domain Deleted FVIII in Primary Canine Marrow Mononuclear Cells.
Authors: Schuening Friedrich G. , Srinivasakumar Narasimhachar , Zaboikin Michail M. , Zaboikina Tatiana N.
Published: October 2019

We wished to determine if primary canine marrow mononuclear cells (MNC) would support the expression of human FVIII and thereby serve as a potential target for gene therapy of hemophilia A. To this end, we created HIV-1 vectors that express B-domain deleted human FVIII (BDFVIII) using the viral LTR promoter or constitutively active cellular promoters: the mouse phosphoglycerate kinase (PGK) or elongation factor 1α (EF1α) promoters. The vector that expressed BDFVIII from the LTR also encoded a functional single-exon Tat protein [Srinivasakumar and Schuening (2000) J. Virol.74:6659–6668]. These promoters have been previously shown, by us and others, to express transgenes at high levels in hematopoietic cells [Srinivasakumar et. al., (2002). J. Virol.76:7334–7342]. Separate vector stocks were prepared for each of the above vectors by transient transfection of 293T cells [Srinivasakumar (2002) Methods Mol. Med. 69:275–302] and their titers were determined by real-time quantitative PCR of genomic DNA isolated from vector transduced 293T cells (Table 1). Each vector stock was then used for infection of primary canine marrow MNC by three spin-infections over 2 days on CH296-coated 6-well plates. The medium was replaced at the end of the transduction procedure. Conditioned medium was harvested 3-days after transduction and assayed for functional FVIII activity using the Coamatic Factor VIII kit (Chromogenix, Monza, Italy). The results, shown in Table 1, indicate that vector-derived FVIII could be detected in the supernatants of canine marrow MNCs transduced with all HIV-1 vectors. Highest levels of functional FVIII were obtained with the Tat-encoding bicistronic HIV-1 vector. These results demonstrate that canine marrow MNC support expression and secretion of functional human FVIII. Further experiments are warranted to determine if the levels of FVIII observed in vitro will translate into therapeutic benefit in vivo in canine models of hemophilia A.

Table 1. Expression of human BDFVIII in canine marrow mononuclear cells transduced by HIV-1 vectors.
High-Throughput DNA Analysis for Red Blood Cell Antigens Facilitates the Transfusion Support with Antigen-Matched Blood in Sickle Cell Disease Patients.
Authors: Castilho Lilian , Hashmi Ghazala , Reid Marion E. , Shariff Tasmia , Seul Michael , Pellegrino Jordao , Costa Fernando F.
Published: October 2019

Background: Transfusion dependent patients as those with Sickle Cell Disease (SCD) patients become alloimmunized and have the potential to form additional antibodies with such frequency that antigen-negative blood is preferred to prevent further alloimmunization. Blood group genotyping is playing a supporting role in the routine blood banks, especially for provision of antigen-matched blood for these patients. However, current techniques for genomic typing are all labor-intensive and require manual set up and analysis by gel electrophoresis. As a result, DNA microarrays are being developed for the single nucleotide polymorphisms (SNPs) detection in the blood group genes to provide a fast procedure and an automated analysis of numerous blood group polymorphisms. We evaluated the usefulness of DNA microarray to provide a means to precisely match donor blood to the antigen-negative type of SCD patients. Method: A total of 12 DNA samples from patients with SCD (homozygous for HbS) and 84 DNA samples from blood donors, were analyzed by the HEA Beadchip (Hashmi et al, 2005) containing a total of 18 SNPs (FYA/B, FY-GATA, FY265, DOA/B (nt 378, 624, 793), COA/B, LWA/B, DIA/B, SC1/SC2, M/N, S/s, LUA/B, KEL1/2, JKA/B, DO323, DO350, HgbS) in a single reaction. Results: A genotype result was obtained for all SNPs tested on 96 samples within 4 hours of the start of testing. Results obtained by Beadchip analysis in donors were used to provide antigen-matched blood for FYA/B, FY-GATA, FY265, DOA/B, M/N, S/s KEL1/2, JKA/B, for all 12 SCD patients. This technology provided a fast procedure and facilitated the transfusion support with antigen-matched blood in SCD patients allowing the reduction of alloimunization to blood group antigens. Conclusion: This high-throughput DNA analysis has the potential not only to increase the inventory of antigen-negative blood but also to facilitate the matching of RBC component to the recipient’s blood type. It also contributes to the management of transfusions in SCD patients by allowing a more accurate selection of donor units. The application of microarray technology in transfusion medicine may have a tremendous impact on further improvement of the safety of blood transfusion.
A New Methodology To Screen Hemoglobinopathies Using Capillary Blood Samples.
Authors: Bento Celeste , Relvas Luis , Rebelo Umbelina , Vazão Helena , Campos Joana , Ribeiro Leticia
Published: October 2019

The prevalence of β-thalassemia carriers among the Portuguese was around 2–3%, but nowadays, due to the immigrant populations coming from Africa, Brazil, East Europe and Asia, with different forms of thalassemias and Hb variants, hemoglobinopathies are much more common and with different genetic characteristics. Concerned with the risk of having an increasing number of patients with the severe forms of hemoglobinopathies, we decided to screen carriers, after personal informed consent, among pregnant women until 18-weeks of gestation and young adults attending primary care services. The goal of the project is to screen approximately 50000 blood samples in order to:

know the hemoglobinopathies frequency and heterogeneity in the population living in Central Portugal (~2500000 inhabitants), and, based on these data, to develop a future cost/effective strategy for carrier identification; identify carriers to provide genetic information and counseling.

In primary care medical centers there are no facilities to collect venous blood samples and we needed to establish a methodology to identify Hb variants and to correctly quantify HbA2 and Hb F in capillary blood samples, which has to be send by ordinary mail to our Lab Center. Working with the “HbA1c Capillary Collection System” from BioRad, we settled an accurate procedure to perform HPLC analysis on capillary blood stored at room temperature until 7 days after collection. To validate the technique we tested, in diverse conditions, more than 200 random and known controls samples: for the same individuals, HPLC results in capillary blood, collected with this system, and in EDTA peripheral blood samples, were identical. This methodology will detect β- and δβ-thalassemias and Hb variants; molecular characterization can be done through blood spots in filter paper (Guthrie spots) collected at the same time. Within 24–48 hours after sample reception in the Lab, results are reported to the respective physician, who is going to establish the correlation with the hematological parameters.

Carriers identified through the screening will be urged to have their partner tested. If they are both carriers, they will be sent to our out patient clinic in order to evaluate the risk of having a child with a clinically significant hemoglobinopathy. If indicated, underling mutation will be identified, genetic counseling provided and prenatal diagnosis offered.

The project, supported by the national program Saúde XXI/FEDER/FSE, started last April, was preceded by training sessions for doctors and nurses working in primary care centers and maternity hospitals. Brochures and posters are distributed for public information. As reported in some European surveys, we are willing to be effective in preventing the birth of affected infants with sickle cell disease and severe forms of thalassemia in Portugal.
8 and 11Gauge Bone Marrow Biopsy Needle Performance Characteristics.
Authors: Goldenberg Alec , Kelley Priscilla , Ibrahim Sherif , Sen Filiz , Liu Cynthia
Published: October 2019

Introduction. An ideal biopsy needle consistently recovers representative specimens of adequate length with minimal dependence on patient characteristics. We have shown that bone marrow biopsy needle gauge influences the length and quantity of recovered specimens. A needle that performs independently of patient characteristics should yield specimens demonstrating a normal distribution of specimen lengths. We evaluated the influence of age and needle gauge on the specimen length distribution curves of patients undergoing bone marrow biopsies using SNARECOIL needles.

Methods. 88 bone marrow core specimens were recovered from 72 patients using 11G SNARECOIL bone marrow biopsy needles. The mean age of the patients was 61.4 and the m/f ratio was 45/27. 53 specimens were recovered from 39 patients (mean age = 47.1, m/f = 27/12) ≤ 64 and 35 specimens were recovered from 33 patients (mean age = 78.6, m/f = 18/15) ≥ 65. 106 patients underwent 127 bone marrow biopsy procedures using 8G SNARECOIL needles. The m/f ratio was 56/50 and the mean age of the patients was 63.1 years. 72 specimens were recovered from 56 patients (mean age = 51.4, m/f = 30/26) ≤ 64 years old and 55 specimens were recovered from 49 patients (mean age = 76.1, m/f = 26/23) ≥ 65. 40 additional specimens were prospectively recovered from 39 patients ≥ 65 (mean age = 74.3, m/f = 19/20) with 8G SNARECOIL needles.

Results. The mean specimen lengths of the 11G and 8G specimens were statistically the same (mean±SEM, 1.97±0.07 cm vs. 1.99±0.05 cm, respectively, p = 0.8). However the 11G specimen length frequency distribution curve deviated markedly from a normal distribution (skewness(skw) = 0.52) while the 8G specimen distribution was nearly normal (skw = 0.04). While the frequency distribution curves of older patients (≥ 65) biopsied with 8G needles showed minimal deviation from normality (skw = 0.12,) the distribution curve of older patients (≥65) biopsied with 11G needles deviated markedly from a normal curve (skw = 0.64). 40 specimens prospectively obtained from patients ≥ 65 demonstrated a mean specimen length of 1.77±0.09 cm with a near normal specimen length frequency distribution curve (skw = 0.07).

Conclusions. 1. Although commonly reported, mean specimen lengths do not completely characterize the performance of biopsy needles. 2.0 Specimen length frequency distribution curves provide added characterization of needle performance in defined patient cohorts. 3.0 In the older patient population, near-normal 8G specimen frequency distributions and skewed 11G distributions suggest that 8G specimens may provide more representative sampling.
A Randomized Phase III Trial of Low Dose Liposomal Amphotericin B (L-AmB) as Prophylaxis of Invasive Fungal Infections (IFI) in Neutropenic Patients (pts).
Authors: Penack Olaf , Schwartz Stefan , Martus Peter , Reinwald Mark , Schmidt-Hieber Martin , Thiel Eckhard , Blau Igor W.
Published: October 2019

Background The prophylactic use of systemic antifungal agents reduces morbidity and mortality after allogeneic hemopoietic stem cell transplantation (HSCT). However, the efficacy of this approach in pts receiving intensive chemotherapy or autologous HSCT is yet unproven. This trial was designed to evaluate the efficacy of L-AmB prophylaxis in high-risk neutropenic pts.

Methods: 231 pts with hematological malignancies and expected neutropenia (N) of more than 10 days (d) following intensive chemotherapy or autologous HSCT were enrolled, 219 pts became neutropenic and were randomized to receive either 50 mg L-AmB i.v. every second d (arm A) or no systemic antifungal prophylaxis (arm B). Treatment with L-AmB started 1–3 d prior onset of N and was continued until neutrophil recovery, breakthrough IFI, intolerable toxicity or death. The level of significance was 0.05 (two-sided) for all statistical analyses. Calculations were performed using commercially (SPSSWIN 12.0) software, the calculations for GEE were performed using the software MAREG.

Results Pt. characteristics: Eligible pts 219; arm A: 110; arm B: 109. Reasons for exclusion were: Absence of N (8), infection prior N (3) and pts decision (1). Baseline characteristics were balanced for age (mean 53.8 years), underlying disease (119 AML, 27 ALL, 64 NHL, 9 other), duration of N (mean 14.8 D) and treatment modality (primary 149, secondary 42, transplant 28). Primary endpoint: The incidence of proven and probable IFI was 5 of 110 pts (4.6%) in arm A and 22 of 109 pts (20.2%) in arm B (p = 0.001, RR = 2.9, CI 1.3 – 6.5). Key secondary endpoints: Pneumonia of unknown origin occurred in 6 pts (5.5%) vs. 28 pts (25.7%) (p < 0.001), the incidence of possible, probable and proven IFI was 11 pts (10.2%) vs. 42 pts (39.6%) (p < 0.001), systemic antifungals were used in 24 pts (22%) vs. 64 pts (59%) (p < 0.001), and death occurred in 4 pts (3.7%) vs. 9 pts (8.2%) (p = 0.16) in arm A vs. arm B. Toxicity: No grade 3 or 4 toxicity was noted. Laboratory abnormalities, including creatinine and liver function tests, were not different between the treatment groups.

Conclusion: Intermittent application of low dose L-AmB is save. The significant lower incidence of IFI in pts treated with L-AmB prophylaxis supports its use in prolonged N.
Prognostic Significance of Minimal Residual Disease (MRD) in Children with High Risk Acute Lymphoblastic Leukemia(ALL) A Children’s Oncology Group Study.
Authors: Borowitz Michael J. , Devidas Meenakshi , Bowman W. Paul , Larsen Eric , Pullen Jeanette , Camitta Bruce
Published: October 2019

MRD is known to be an important prognostic factor in childhood ALL. In POG 9906 we studied a select group of 271 patients with NCI high risk precursor B-ALL further defined by an age/sex/WBC algorithm (Shuster et al. Cancer Res Therapy Control9:101,1999) to select those at highest risk; historical data suggested that patients selected by this algorithm would account for about 12% of patients and have a 44% 4y EFS. Patients with Ph+ ALL or favorable genetics (TEL-AML1 translocation or trisomies 4 and 10) were excluded. All patients received 4-drug induction with vincristine, prednisone, L-asparaginase and daunomycin, plus intrathecal methotrexate, and consolidation with cyclophosphamide, ara-C and 6-MP followed by 2 cycles of interim maintenance and delayed intensification similar but not identical to so-called “augmented BFM”. MRD was measured in peripheral blood on day 8 (PB) and in bone marrow at day 29 (BM) by 4-color flow cytometry as previously described (Leukemia17:1566,2003). BM MRD data were available on 240 patients, and PB data on 243. At cutoffs of 1%, 0.1% and .01%, MRD in BM was associated with increased relapse rate (p=.0002). At a cutoff of .01%, 83/240 (35%) patients were MRD positive; these had 3 y EFS of 58±6% compared to 79±5% for MRD negative patients (p=.0008). The EFS of the 28 patients with MRD >1% was 44±14%. There were numerous (25/52) extramedullary events, mostly in CNS. MRD predicted 17/27 marrow relapses but only 8/25 other relapses (p=.03). Overall, 21% of MRD positive patients (>.01%) had a marrow relapse compared to 6% of MRD negative ones. Day 8 PB MRD was also prognostic, with the 49 MRD negative patients having a 3 y EFS of 89±6% compared to 70±5% for the 194 patients with MRD >.01% (p=.008). End induction BM MRD is a strong prognostic factor even among patients with especially high risk ALL and is a better predictor of marrow than of extramedullary relapse. Absence of MRD in day 8 PB identifies a subgroup of approximately 20% of these high risk patients who have an exceptionally good EFS, particularly given their very poor expected outcome.

EFS by Day 28 Marrow MRD EFS by Day 28 Marrow MRD
Toxicity of the Oncoprotein LMP1 of Epstein Barr Virus as a New Mechanism of LMP1 Regulation Explaining the Heterogeneity of LMP1 Protein Levels in EBV-Transformed B-Cells.
Authors: Christophe Le Clorennec , Tan-Sothea Ouk , Chantal Jayat-Vignoles , Ibtissam Youlyouz-Marfak , Eric Adriaenssens , Jean Coll , Dominique Bordessoule , Jean Feuillard
Published: October 2019

Epstein Barr Virus (EBV) oncoprotein LMP1 has been described as the major transforming protein in various cell types, by rerouting the TNF-receptor family signalling pathway. Despite this undoubtedly role in EBV-associated transformation of cells, toxicity of LMP1 is a well known experimental feature, poorly studied, may be because it contradicts the dogma of its transformation power. This phenomenon may be important to understand in the view of natural immune control of the EBV burden in the immunocompetent host (Le Clorennec, 2006). Moreover it is noteworthy that, at the single cell level, expression of LMP1 is very heterogeneous both in EBV-associated tumours and in cell lines.

To understand the toxic effect of LMP1 in LCLs, we cloned wild type LMP1 into a novel double inducible episomal vector pRT-1, from which the gene of interest is expressed under the control of a bi-directional doxycyclin regulatable promoter with a truncated version of NGFR (NGFRt) as a surrogate marker of inductibility.

We first found that increasing levels of doxycyclin concentration induce over-expression of CD95 that parallels induction of LMP1. Induction of LMP1 resulted in induction of CD95 aggregation, FADD phosphorylation, activation of caspase 8, 9 and 3, followed by late stages of apoptosis as demonstrated by PARP cleavage and sub-G1 peak. Immunoprecipitation of CD95 and caspase 8 suggested that LMP1 induction was associated with formation of CD95-dependant DISC.

Induction oxygen species (OS) has been shown to induce aggregation and autoactivation of CD95. We found that inhibition of the formation of oxygen species (OS) by catalase resulted in a decrease of LMP1-dependant apoptosis, whereas treatment of cells with H2O2 had the opposite effect. We also show that induction of latency III is associated with induction of OS. Levels of OS in latency III EBV-infected B-cells were significantly increased by induction of LMP1.

Therefore, our results are in agreement with a model according which (i) EBV latency III increases the pool of OS of the infected B-cell, rendering the cell permissive to CD95 auto-activation, (ii) increasing levels of LMP1 induces both expression of the pro-apoptotic CD95 and survival of the cell through NF-kappa B activation, and (iii) increasing CD95-autoactivation due to inappropriate over-expression of LMP1 finally overwhelms the antiapoptotic effect of NF-kappa B.

From this model, it is expected that at the single cell level, LMP1 expression results from an equilibrium between NF-kappa B dependant survival of cells and CD95-dependant apoptosis, explaining the heterogeneity of LMP1 expression from cell to cell and pointing a new mechanism of regulation of LMP1.
Ubiquitin-Conjugating Enzyme Ubc13 Is a Critical Component of TRAF-Mediated Inflammatory Responses.
Authors: Fukushima Toru , Matsuzawa Shu-ichi , Kress Christinia L. , Bruey Jean Marie , Krajewska Maryla , Lefebvre Sophie , Zapata Juan M. , Ronai Ze’ev , Reed John C.
Published: October 2019

Ubc13 is an ubiquitin-conjugating enzyme responsible for non-canonical ubiquitination of TRAF-family adapter proteins involved in Toll-like Receptor (TLR) and TNF-family cytokine receptor signaling. Gene ablation was used to study the function of Ubc13 in mice. While homozygous ubc13 gene disruption resulted in embryonic lethality, heterozygous ubc13+/− mice appeared normal without alterations in immune cell populations. Haploinsufficient ubc13+/− mice were resistant to lipopolysaccharide (LPS)-induced lethality, and demonstrated reduced in vivo ubiquitination of TRAF6. Macrophages and splenocytes isolated from ubc13+/− mice exhibited reduced LPS-inducible cytokine secretion and impaired activation of TRAF-dependent signal transduction pathways (NF-kB, JNK, p38 MAPK). These findings document a critical role for Ubc13 in inflammatory responses, and suggest that agents reducing Ubc13 activity could have therapeutic utility.
MUNC13–4 Mutations in Patients with Hemophagocytic Lymphohistiocytosis Are Scattered over the Functional Domains of the Protein.
Authors: Santoro Alessandra , Cannella Sonia , Bossi Giovanna , Gallo Federico , Trizzino Antonino , Pende Daniela , Dieli Francesco , Bruno Giuseppa , Stinchcombe Jane C. , Putti Caterina , De Fusco Carmela , Danesino Cesare , Moretta Lorenzo , Notarangelo Luigi D. , Griffiths Gillian , Aricò Maurizio
Published: October 2019

Mutations of PRF1 and MUNC13–4 genes, involved with cellular cytotoxicity, are associated with the familial form of Hemophagocytic Lymphohistiocytosis (HLH) type 2 (FHL2) and 3 (FHL3). In all patients with HLH, in which PRF1 mutations had been excluded, we screened MUNC13–4 mutations.

The 32 exons and their proximal flanking regions of MUNC13–4 gene were sequenced by “cycle sequencing” approach (BigDye Terminator, Applied Biosystems).

Of 60 patients, 21 had MUNC13–4 mutations. 5 were reported mutations: 753+1G>T, 817C>T (R273X); 1389+1G>A; 1822del 12 bp (del V608-A611); 2346–9delGGAG (R782FsX12). Of 16 novel mutations 2 (2212C>T and 2650C>T) introduce a stop codon (at Q738 and Q884); 4 cause frameshift: 441delA (P147fsX14), 532delC (Q178fsX70), 3082delC (L1028fsX), and 3226insG (H1076fsX51). Of 8 missense mutations [175G>A (A59T), 419T>C (I140T), 610A>G (M204B), 1241G>T (R414L), 1847A>G (E616G), 2039G>C (R680P), 2570T>G (F857C), 2782C>T (R928C)], 6 fall within the protein functional domains; each of the 2 falling outside was associated with 2 additional pathogenic mutations.

The remaining novel 1992+5 G>A and 2448insC −12 fall outside the coding region but are close enough to induce alternative splicing. We identified polymorphisms 279C>T and 3198A>G. The 2599A>G (K867E) transition was found in several unrelated families, including one homozygous parent. To assess its frequency we studied 50 consecutive newborns, of which 64% were heterozygous.

To understand the effects of the mutations we analysed Munc13–4 protein expression from CTL and/or NK cells by western blot using an antibody raised against amino acids 1–262. Trace amounts of protein could be detected only in 5 patients. Analysis of granule polarisation in 2 patients with trace amounts of Munc13–4 protein showed many more docked granules visible than in controls, consistent with a block in granule secretion in these patients.

Median age at diagnosis of FHL3 was 6.5 months, but 8/21 (38%) patients were diagnosed when older than 5 years, with one young adult of 18 years. CNS disease was present in 10 patients; NK was markedly reduced or absent in all patients tested. MUNC13–4 mutations were found in 35% of our HLH patients non type-2. Mutations were almost entirely different from those reported so far and scattered over different exons, but in far most cases they fall within the protein functional domain. Since these patients may develop the disease during adolescence or even later on, not only pediatric but also as adults hematologists should include FHL-2 and 3 in the differential diagnosis of children and young adults with fever, cytopenia, splenomegaly and hypercytokinemia.