bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclosporine A (CSA) Significantly Reduces the Cytotoxic Effects of In Vitro Expanded NK Cells: Implications for Adoptive NK Cell Therapy in the Setting of Allogeneic Hematopoietic Stem Cell Transplantation (HCT).
Authors: Yokoyama Hisayuki , Berg Maria , Lundqvist Andreas , McCoy J. Philip , Srivastava Shivani , Smith Aleah , Childs Richard
Published: October 2019

The ability to expand NK cells in vitro has led to the recent initiation of protocols incorporating adoptive NK cell infusions after HCT. Calcineurin inhibitors such as CSA are commonly used to prevent graft versus host disease (GVHD) in HCT recipients. Recently, Hong et al found the phenotype and function of fresh NK cells cultured in vitro with CSA was altered, with CSA treated NK cell cultures having enhanced cytotoxicity against tumor targets. However, the impact of CSA on in vitro expanded NK cell function and phenotype has not been explored. We analyzed cell proliferation, IFN-gamma production, cell surface immunofluorescent staining and cytotoxicity against K562 and renal cell carcinoma cell lines by in vitro expanded vs freshly isolated NK cells cultured in physiological doses of CSA (40ng/ml, 200ng/ml, 1000ng/ml for 18hrs). Fresh NK cells were obtained from the PBMC of healthy donors using immunomagnetic beads to isolate CD56+/ CD3− cells. NK cells were expanded in vitro using irradiated EBV transformed B cells as feeder cells in media containing IL-2 [500U/ml] for 12–14 days. Comparing CSA containing cultures to controls, there was a significant reduction in IL-2 stimulated fresh NK cell proliferation (stimulation index 0.51± 0.1) and TRAIL expression (MFI 10.4 vs 3.01). Furthermore, an ELISA assay showed fresh NK cells treated with CSA had a significant reduction in IL-2 induced IFN-g production compared to controls (median 231 vs 57 pg/ml, p=0.025). In contrast, in vitro expanded NK cells cultured in CSA showed no significant reduction of proliferation or TRAIL expression. At the highest doses of CSA (1000ng/ml), minimal inhibition of K562 killing of freshly isolated NK cells was observed. In contrast, expanded NK cells cultured in CSA for 18 hours compared to controls had a significant reduction in the killing of K562 cells (E:T=10:1, median 66 vs 43% lysis, p=0.011) and RCC tumor cells (E:T=20:1, 14.8 vs 8.8%, p=0.043).

Figure Figure

These data confirm CSA alters the phenotype and function of CD3−/CD56 + NK cells. Importantly, CSA appears to have a deleterious effect on expanded NK cell tumor cytotoxicity that was not observed with fresh NK cells. These finding suggest the anti-tumor effects of in vitro expanded NK cells could be hindered when adoptively infused in HCT patients receiving CSA.
Cure of Murine Medulloblastoma by Adoptive Transfer of Tumor-Specific T Cells.
Authors: Nicholson Cameron L. , Plautz Gregory E.
Published: October 2019

Medulloblastoma is the most common malignant brain tumor of childhood. Although the current overall survival has increased due to more aggressive therapy, the involved surgery, radiation and in some diseases high-dose chemotherapy have significant acute and long-term toxicities. T cell immunotherapy is a promising adjuvant for medulloblastoma because the immune response is specific for targeted antigens and is highly anatomically focused, occurring at the level of cell-to-cell interactions, thereby sparing adjacent normal brain tissue. Adoptive transfer of tumor-sensitized T cells has demonstrated therapeutic efficacy in animal tumor models, and clinical trials involving transfer of T lymphocytes are ongoing. Previous strategies have employed cultured human medulloblastoma cell lines as therapeutic targets, however priming of T cells using a spontaneously-arising medulloblastoma cell line in an immunocompetent mice model may be more representative of human cancer and more applicable to clinical practice. Spontaneous Murine medulloblastoma cell lines were created from knock-out Ptc+/−p53−/− mice and passaged in vivo. Brain tumors were established by transcranial inoculation of a single-cell suspension of murine medulloblastoma tumor cells in immunocompetent, C57BL/6N mice. Murine Medulloblastoma cell lines were rapidly tumorigenic and consistently fatal following intracranial inoculation in a dose-dependent manner. T-cells were obtained surgically from tumor draining lymph nodes (TDLN) in syngeneic mice and after initial purification, antigen-stimulated CD62Llow T cells were isolated by depletion of CD62Lhigh cells using MACS beads. T-cells were further activated ex vivo by incubation with anti-CD3 monoclonal antibody and exposure to media containing Interleukin-2 and Interleukin-7. Mice carrying 3-day established tumors receive systemic transfer of T cells by injection through the tail vein after 10 days of ex vivo culture. Mice typically receive sub-lethal total body irradiation (5 Gy) from a Cesium irradiator for lympho-depletion several hours before adoptive transfer. Mice were followed for survival and sacrificed when neurological symptoms were apparent as per Institutional Animal Care and Use Committee established guidelines. The activated T cells adoptively transferred to irradiated hosts with 3-day established intracranial MM1 tumors resulting in complete regression of established tumors (see figure). In addition, the above therapy demonstrated a dose-dependent effect, and the mice were cured without any evidence of neurological dysfunction caused by the anti-tumor response. This research suggests anti-medulloblastoma T cell vaccines may be promising for subsequent clinical testing.

Figure 4. Active Immunology of 3-day MMI medulloblastoma Figure 4. Active Immunology of 3-day MMI medulloblastoma
Hematopoietic Stem Cells Mobilization by G-CSF and LTB4 Was Significantly Suppressed by an Oxygen Radical Scavenger or an Inhibitor of NADPH Oxidase.
Authors: Mun Yeung-Chul , Oh Soo Ah , Lee Kyoung-Eun , Yoo Eun-Sun , Chang Hye-Jung , Choi Moon-Young , Ahn Jee-Young , Kim Jae-Hong , Seong Chu-Myong
Published: October 2019

Introduction: We previously had reported that LTB4 was able to mobilize HSC within 4 hours without significant side effects and that LTB4 receptor was involved in both of the pathways of mobilization induced by G-CSF and LTB4 in the murine model. Though the roles of protease (ie. NE, MMPs) seem to be important, the precise mechanisms are still unknown. ROS (reactive oxygen species) have cell signaling roles that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate many biological systems. Because a variety of inflammatory cytokines and chemokines, including G-CSF and LTB4, induce a rapid increase of intracellular ROS, we hypothesized that the role of ROS on HSC mobilization induced by G-CSF and LTB4 might be important.

Methods: MS5, murine stromal cell line cells, or bEnd3, murine microvascular cell line cells, were grown to confluence on the microporous transwell membrane. Murine marrow cells were placed on top of the prepared transwell membrane. The transwells were then seated in wells containing media and G-CSF or LTB4 with or without pretreatment of NAC, an oxygen free radical scavenger, or DPI, an inhibitor of NADPH oxidase-like flavoproteins. Cells that migrated through the stromal or endothelial layer into the wells were assayed for mobilization. NAC and DPI were given to C57BL/6 mice followed by rhG-CSF (5μg, IV) or LTB4 (1μg, IV) after a period of 2 hours. 24 hours after the rhG-CSF injection or 4 hours after the LTB4 injection, peripheral blood samples were obtained via cardiac puncture. The samples were analyzed for TNC using a trypan blue stain and FACS analysis were performed using Sca-1 and lineage markers, including CD45R (B220), CD116, Gr-1 and TER119.

Results: The numbers of migrated cell through the MS5 or bEnd3 were increased by treatment of G-CSF or LTB4. However, increasing effects of G-CSF or LTB4 to the transmigration through the MS5 or bEnd3 were inhibited by pretreatment of NAC or DPI. Comparing the control arm, the numbers of WBC and HSC (Sca-1+Lin−) in blood were decreased in the G-CSF or LTB4 mobilized mice (N=4), in which NAC or DPI were pretreated (Sca-1+Lin− fraction in peripheral blood: 1.91±0.80% in G-CSF alone, 0.55±0.37% in G-CSF with NAC, 0.36±0.09% in G-CSF with DPI, P<0.05; 1.92±0.49% in LTB4 alone, 0.55±0.29% in LTB4 with NAC, 0.67±0.52% in LTB4 with DPI, P<0.05).

Conclusions: Through our data, it is suggested that ROS are involved in the HSC mobilization induced by G-CSF and LTB4 in the murine model. It would be very interesting to test the effect of G-CSF or LTB4 on ROS K/O mice in the future. As for now, we will try to find the critical signal transduction pathway, through which we may find more effective and efficient molecules for HSC mobilization.
AV513 Enhances Tissue Factor Activity by Inhibiting TFPI in Hemophilia A Plasma: Potential TFPI Target Sites and Potentiation of Factor VIII Activity.
Authors: Knappe Sabine , Kripalani Nicholas , Patarroyo-White Susannah , Johnson Kirk W. , Prasad Srinivasa
Published: October 2019

AV513, a sulfated polysaccharide prepared from brown seaweed and enriched for fucoidan accelerates clotting in hemophilia A and B plasma. Prior studies have demonstrated that AV513 functions mainly by inhibiting TFPI activity. In this report, we have further studied AV513 mechanism of action using human plasma thromboelastography (TEG) model and a thrombin generation assay in FVIII deficient plasma. In the TEG assay, AV513 enhanced tissue factor (TF) induced clotting in human hemophilia A pooled plasma in a dose-dependent manner. A dose-dependent potentiation effect by AV513 was observed with reduction of TEG R time to normal levels at 0.5 μg/mL of AV513. An AV513 dose dependent increase in thrombin generation was observed in hemophilia A pooled plasma with a 3-fold increase in peak thrombin generation at 2.5 μg/mL AV513 (basal: 40 nM vs. treated: 140 nM). When TFPI was immunodepleted from hemophilia A plasma (FVIII/TFPI deficient), sub-optimal concentrations of TF enhanced thrombin generation which was not further enhanced by addition of AV513. The increase in thrombin generation in FVIII /TFPI deficient plasma was inhibited by 93% after the addition of exogenous full-length TFPI or by 47% after the addition of truncated rTFPI (amino acids 1–161) that lacks the Kunitz 3 domain and the C-terminal tail. AV513 at low concentrations reversed exogenous full length TFPI activity almost completely in FVIII/TFPI-deficient plasma but had only a small effect on truncated TFPI. In a human plasma TEG combination study of AV513 and low concentrations of rFVIII (ReFacto), AV513 at a concentration of 0.25 μg/mL reduced the TEG R time by 35% in severe hemophilia A plasma compared to 10 mU/mL of rFVIII alone. These results indicate that AV513 functions as a pro-coagulant by inhibiting TFPI at least in part via targeting the C-terminal region. Moreover, AV513, in the presence of low rFVIII (ReFacto) levels, improves severe hemophilia A plasma clotting - as measured by TEG - beyond that observed with either agent alone.
Early Onset of Acute Graft-Versus-Host Disease Indicating a Worse Prognosis in Terms of Chronic Graft-Versus-Host Disease and Survival Compared to Late Onset.
Authors: Sohn Sang Kyun , Moon Joon Ho , Kim Jong Gwang , Chae Yee Soo , Cho Yoon Young , Ahn Byung Min , Kim Shi Nae , Lee Soo Jung , Kim Yun Jeong
Published: October 2019

Background: Acute graft-versus-host disease (GVHD) is an important risk factor for predicting chronic GVHD. The transplant outcome can be influenced by the onset time of acute GVHD in patients who received allogeneic stem cell transplantation (SCT)

Methods: The medical records of one hundred six patients with hematological malignancies who received allogeneic transplantation were retrospectively reviewed.

Results: Fifty four (39.7%) patients developed grade II to IV acute GVHD within D+30 after allogeneic SCT (<D+30 group) and 13 (9.6%) patients manifested acute GVHD after D+30 (≥D+30 group). The cumulative incidence of chronic GVHD was 81.5% and 53.8% in <D+30 group and ≥D+30 group, respectively.(p<0.001) On multivariate analysis, grade II to IV acute GVHD developed before D+30 and primary diagnosis of chronic myeloid leukemia were identified as independent variables predicting chronic GVHD. The overall survival rate was significantly lower in the <D+30 group than grade 0 or I group (p<0.001). But there was no statistical difference between the group with grade 0 or I and ≥D+30 group in terms of the incidence of chronic GVHD (p=0.295). Among the 54 patients with grade II to IV acute GVHD developed at before D+30, 26 (48.1%) patients developed into quiescent chronic GVHD and 20 (37%) patients progressive chronic GVHD. The quiescent chronic GVHD showed a better survival than progressive chronic GVHD (p=0.063).

Conclusion: Acute GVHD of early onset (within D+30) was regarded as a worse prognostic indicator in terms of chronic GVHD and survival.
Study on Early Diagnosis of Invasive Aspergillosis in Patients with Hematological Diseases.
Authors: Sun Ai-ning , Wang Jun , Wu De Pei , Chen Su-ning
Published: October 2019

Objective To evaluate the value of galactomannan(GM) detection and nest PCR for early diagnosis of invasive aspergillosis (IA) in patients with hematological disease.

Methods GM ELISA and nest PCR was performed to serially screen for circulating galactomannan(GM) and DNA of Aspergillus ssp. Respectively, 96 patients were grouped according to case definitions of the EORTC, including 5 proven IA and 28 probable IA cases. Samples of the patients included serum twice weekly(326) and bronchoalveolar lavage fluid(BALF 12). Sensitivity, specificity and predictive values were calculated respectively and compared.

Results

According to the receiver operator characteristic curve(ROC), using a reduced cutoff of 0.5 O.D.I of two-positive GM test result, we can achieved the most optimal results. The sensitivity, specificity, positive predictive value (PPV)and negative predictive value (NPV) of the ELISA test were 85.7%,91.6%,85.7%,91.6%respectively. While for the BALF samples, a increased cutoff 1.0 maybe improve the value of this diagnosis, with sensitivity 50% and specificity 100%.Quantitive results of the serum-based GM antigen can represent the status of IA and were correlated with the prognosis Of IA.Galactomannanemia preceded the development of characteristic findings on CT about 6 days. The nest PCR assay amplifies specifically a region of the 18S rRNA gene that is highly conserved in Aspergillus species and allows detection of down to 5.0 fg/ml of Aspergillus DNA. When two-positive results were used to define an episode as ‘PCR positive’, the sensitivity, specificity, PPV and NPV of the PCR test were 92.9%, 100%,100%,96.0% respectively. For the BALF samples, the sensitivity and specificity decreased to 75%and 60%. The frequency of the consecutively positive results of a patient was correlated with prognosis Of IA. The antifungal therapy usually causes a intermittent positive PCR results. Positve PCR results preceded the development of characteristic findings on CT about 9 days.

Conclusion

The cutoff 0.5 of two-positive serum-based GM ELISA test result is the most optimal cutoff value. GM ELISA test is a rapid,reliable method for early diagnosis and treatment of IA, with good sensitivity(85.7%)and specificity(91.6%). GM ELISA test is a good direction for preemptive antifungal therapy in patients with hematological disease at risk. Using two-positive results to define an episode as ‘PCR positive’, great sensitivity(92.9%)and specificity(100%) could be achieved. The serum-based PCR test is useful for screening for Aspergillus spp. in patients with hematological disease at risk but without antifungal treatment.
HHV-6 Reactivation after Hematopoietic Stem Cell Transplantation in Chinese Patients Is Associated with aGVHD.
Authors: Ye Xiu Jing , Liao Wei Wei , He Jing Song , Huang He
Published: October 2019

Recent reports indicate that human herpesvirus (HHV)-6 reactivation occurs in 40–65% of patients undergoing hematopoietic stem cell transplantation (HSCT).But the complication after HSCT that predispose to HHV-6 viremia are not well characterized. The aim of our research is to study the potential relationship between HHV-6 activation and acute graft-versus-host disease (aGVHD) after hematopoietic cell transplantation (HSCT) in chinese patients. Peripheral blood samples were collected before and weekly after HSCT from 40 consecutive recipients who underwent HSCT between March 2005 and Joungry 2007 (2 autologous and 38 allogeneic transplants) .HHV-6 DNAemia was monitored by real-time PCR. The genotypes of HHV-6 were identified by Hind III restriction assay. Of the 40 patients, HHV-6 DNAemia were detected in only 1 patient (2.5%) before HSCT, and the viral load was 420 copies/ml.After HSCT there were 18(45%) patients detected HHV-6 DNA on a median of day 14.5 (range, 0– 23 days), and the median HHV-6 viral load of 4884.4±374.4 copies/ml (range, 282 – 43400 copies/ml). Respectively, HHV-6B was identified as the predominant variant.Grade I – IV aGHVD occurred in 18 (45%) on a median of day 20 (range, 8–40 days). The median onset time of HHV-6 DNAemia was significantly earlier than that of aGHVD (P <0.05). Compared with that in HHV-6 DNAemia positive [HHV-6(+)] patients, the cumulative incidence of grade I – IV aGHVD was higher (72.2% vs. 27.7%, P <0.05) than in negative [HHV-6(−)] patients. Cumulative incidence of grade I – IV aGVHD was higher in patients with both HHV-6 and CMV positive (CMV+/HHV-6+) than in those with either CMV (CMV+/HHV-6−) or HHV-6 positive (CMV−/HHV-6+) and neither of them positive (CMV−/HHV-6−) [66.7% (10/15),26.7% (4/15) ,66.7% (2/3) and 28.6% (2/7), respectively, P<0.05]. Our data suggest that patients who undergo HSCT are at significant risk for HHV-6 reactivation. HHV-6 viremia occurs early during the post-transplant course, most often within the first 2 weeks. Patients with HHV-6 activation or HHV-6/CMV co-infection maybe involved in the occurrence of aGVHD after HSCT.
A Novel Strategy for the Treatment of Acute Graft-Versus-Host Disease Using the Adsorbent To Remove Excessive Inflammatory Cytokines.
Authors: Takami Akiyoshi , Teshima Takanori , Ushizaki Koshin , Taniguchi Takumi , Endo Tohru , Sakurai Hiroshi , Kondo Yukio , Yamazaki Hirohito , Shimadoi Shigeru , Okumura Hirokazu , Nakao Shinji
Published: October 2019

Acute graft-versus-host disease (aGVHD) remains the major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT). Previous studies show that inflammatory cytokines such as tumor necrosis factor alfa (TNFα), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18) are involved in the pathogenesis of aGVHD, and that the excess of these cytokines is associated with severity and mortality of aGVHD. We hypothesized that removal of these excessive cytokines from patients’ blood at the onset of aGVHD might improve the treatment outcome. A novel absorbent CTR can effectively adsorb small- to middle-sized proteins like cytokines and enterotoxins in vitro. In view of future exploitation of extracorporeal treatment using CTR column, we tested whether CTR could remove these inflammatory cytokines from blood. When the serum containing a mix of recombinant cytokines was incubated with a CTR adsorbent for 2 hrs, 55% of TNFα, 81% of IL-6, 83% of IL-8, and 22% of IL-18 were successfully removed. Next, we measured TNFα, soluble TNFα receptor 1 (TNFR1), IL-6, IL-8, and IL-18 levels in serum samples obtained from 5 patients (median age 38y, range 26–63y) who underwent myeloablative SCT in 4 and non-myeloablative SCT in 1. AGVHD developed in 2 with grade 3 and in 3 with grade 2. When cytokine levels in patients were expressed as a ratio to the mean cytokine level in control serum samples obtained from three healthy individuals, the mean ratios of TNFα, TNFR1, IL-6, IL-8, and IL-18 at the onset of aGVHD were 6.0 (range, 1.2–12.0), 6.5 (2.5–9.0), 274 (3.5–651), 48.3 (11.3–75.2), and 6.7 (3.2–10.8). The CTR adsorption considerably reduced the concentrations of these cytokines except for IL-18 (Figure 1). The adsorption rates of these cytokines were 64% for TNFα, 48% for TNFR1, 59% for IL-6, more than 94% for IL-8, and 0% for IL-18. The efficient removal of inflammatory cytokines suggests that extracorporeal blood purification with CTR column may be effective in the treatment of aGVHD. This treatment strategy may be promising because it essentially has no deleterious effects on immune functions of SCT recipients unlike other GVHD treatments.

Figure Figure
Antifungal Prophylaxis in Allogeneic Stem Cell Transplantation: Results of a Retrospective Cohort Study.
Authors: Wolschke Christine , Lellek Heinrich , Heinzelmann Marion , Kroeger Nicolaus , Zander Axel
Published: October 2019

Background: Patients with haematogical diseases and undergoing stem cell or bone marrow transplantation are at high risk for an invasive fungal infection. The appearance and course of this is a significant threat to the overall success of transplantation. The optimal antifungal prophylaxis for stem cell transplanted patients has not yet been clearly etablished. Whereas oral medication seems to be convenient it is often hampered in the course of administration by mucositis or/and unclear absorption. We analysed the impact of antimycotic prophylaxis in our unit. We started with Voriconazol (tbl.) or Itraconazol (solution) followed by an early switch to liposomal amphotericin B with a dose 1 mg/kgBW, if the patients didn’t tolerate oral medications.

Patients and methods: 48 patients (18f/30m) with a history of possible (n=4), probable (n=1) or proven (n=3) invasive fungal infection according to EORTC-MSG criteria or no (n=40) fungal infection underwent allogeneic stem cell transplantation in our centre between 08/2005 and 05/2006. Mean age was 47 (6–67) and underlying disease was acute leukaemia (18 AML, 5 ALL, 3 NHL, 4 MM, 4 CML, 14 other). Transplants were unrelated in 38 and related in10 cases.

Results: Prophylaxis started on average 6 days prior to transplant (mean 6, range 0–19) with ITZ (n=24), VOR (n=8) or LAMB (n=8). 8 pts. developed early signs of fungal infection precluding analysis within the prophylaxis population. 15/24 pts. on ITZ and 3/8 pts. on VOR needed a switch to i.v.-medication with LAMB. 3/24 pts. starting with ITZ developed a fungal infection (2 possible, 1 probable). Pts. starting with VOR or LAMB required empiric therapy in 1 and 4 cases, respectively. 12 pts. after switching to LAMB required no further antifungal-therapy.

Conclusions: In our experience antifungal prophylaxis with oral medication being switched to LAMB in case of mucositis or side effects is an effective and tolerated option to prevent invasive fungal infection in patients during allogeneic stem cell or bone marrow transplantation.
Should Patients with Myelodysplasia and/or Acute Myeloid Leukemia (MDS/AML) Be Screened for Inherited RUNX1 Mutations Prior to Sibling Donor Allografting?.
Authors: Owen Carolyn J. , Yin John A.L. , Toze Cynthia J. , Forrest Donna L. , Macheta Anthony , Smith Clayton A. , Barnett Michael J. , Andrew T. , Fitzgibbon Jude
Published: October 2019

Background Familial platelet disorder with propensity to myeloid malignancy (FPD/AML) is an autosomal dominant disease characterised by platelet abnormalities and risk of MDS/AML. FPD/AML results from germline mutations in RUNX1. The phenotype is heterogeneous with ∼35% of affected individuals developing MDS/AML. This clinical variability may obscure the diagnosis and prevent appropriate screening. We describe 2 families with novel C-terminal RUNX1 mutations and FPD/AML phenotypes, in which potentially affected siblings were used as haematopoietic stem cell transplant (HSCT) donors prior to identification of the inherited mutation.

Methods DNA was collected from members of two families with histories of thrombocytopenia and MDS/AML. Direct PCR sequencing was performed on exons 1 and 3–8 of the RUNX1 gene. Results Pedigree 1 has 4 affected members from 2 generations. The proband (I-1) had a history of chronic thrombocytopenia and developed MDS at age 63 and died of AML at age 64. Her daughter (II-1) presented with MDS at age 37 and underwent 2 HSCTs from II-2, relapsing 8 months after the first and remaining well 6 years after the second HSCT. Individuals II-2 and II-3 were recently noted to have mild thrombocytopenia with normal bone marrow morphologies. DNA from blood and bone marrow of II-2 and II-3 revealed an out-of-frame 7 base pair (bp) deletion in exon 8 (1007_1013del) of RUNX1 at amino acid 336 (Gly336fsTer564). Pedigree 2 has 2 affected individuals from 2 generations. The proband (I-1) had a history of chronic thrombocytopenia since childhood. She developed MDS at age 52 and received an HSCT from her brother (I-2) in 2007 with normal engraftment and no early transplant complications. Her son (II-2) had presented with AML at age 8 and received an HSCT from his sister (II-1) at that time. He remains well 26 years later. DNA from blood and saliva of I-1 revealed a substitution of C for T in exon 7 (877C>T) of [italic[RUNX1, resulting in a stop codon and an early-truncated protein at position 292 (Arg292Ter).

Conclusions Two novel, C-terminal RUNX1 mutations are described in families with FPD/AML phenotypes. Both families were investigated after affected individuals received HSCT from siblings (who were also at risk of having inherited the mutation). The sibling donors exhibited normal or near-normal platelet counts prior to HSCT, obscuring a FPD/AML diagnosis. Therefore, all patients with MDS/AML should be screened for a family history of thrombocytopenia and/or MDS/AML and considered for investigation of RUNX1 mutations prior to HSCT.

Figure Figure Figure Figure
Phase I/II Gene Therapy Study for Chronic Granulomatous Disease: Results, Lessons and Perspectives.
Authors: Ott Marion G. , Stein Stefan , Schultze-Strasser Stephan , Jauch Anna , Burwinkel Barbara , Schlegelberger Brigitte , Goehring Gudrun , Preiss Carolin , Kramer Rolf , Schwarzwaelder Kerstin , Koehl Ulrike , Kunkel Hanna , Kuehlcke Klaus , Schilz Andrea , Schmidt Manfred , von Kalle Christof , Trasher Adrian , Hoelzer Dieter , Seger Reinhard , Grez Manuel
Published: October 2019

Gene transfer into hematopoietic stem cells has been envisaged as an alternative to stem cell transplantation for the treatment of many genetic diseases of the blood system. In 2004 we initiated a gene therapy trial aimed at the correction of Chronic Granulomatous Disease (CGD), a rare inherited immunodeficiency caused by a functional defect in the microbial killing activity of phagocytes. Gene marking and functional correction of phagocytes were high shortly after transplantation of gene modified cells, leading to the eradication of therapy resistant infections from which patients had suffered for many years. However, one of our patients died 27 months after treatment due to a severe sepsis with multiorgan dysfunction. Although gene marking was still high at this time point, expression of the therapeutic gene, gp91phox, was minimal. This down regulation of transgene expression was due to CpG methylation within the viral LTR. Similar effects were observed in a second patient treated more than 3 years ago. In both patients CpG methylation was restricted to the promoter region of the viral LTR, while CpG dinucleotides within the enhancer region of the viral LTRs were not methylated. As a consequence, gp91phox gene expression was suppressed but the capacity of the viral LTRs to transactivate nearby sequences is still intact. Indeed we were able to detect cellular transcripts at predominant retroviral integration sites leading to an unbalanced clonal distribution of gene marked cells in peripheral blood and bone marrow. A third patient, a 5 years old child was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects.
Allogeneic Hematopoietic Stem Cell Transplantation as Consolidation Therapy for Adult T Cell Leukemia/Lymphoma Patients: A Single-Center Experience in 35 Patients.
Authors: Takemoto Shigeki , Hidaka Michihiro , Nagakura Shoichi , Sakai Tatsunori , Matsui Takahiro , Kawakita Toshiro , Inoue Yoshiko , Tsukamoto Atsuko , Kiyokawa Tetsuyuki , Kawano Fumio
Published: October 2019

Adult T cell leukemia/lymphoma (ATL) has a high incident in Japanese. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) against ATL made the patients’ survival longer than chemotherapy alone. Actually, some of them became long survivors (LS). The efficacy of allo-SCT for ATL was examined and the prognostic factors were discussed. We retrospectively surveyed the data of ATL patients who underwent allo-HSCT at our hospital between June 1997 and June 2007. The results of allo-HSCT in ATL patients (n=35) including acute type (n=18) and lymphoma-type (n=17) were analyzed retrospectively. Donors were HLA-related relation in 24 patients (69%) or unrelated volunteers in 11 patients (31%). Twelve patients (34%) were in complete remission (CR) before conditioning regimen. Twenty-one (60%) received a conventional stem cell transplantation (CST) and 14 (40%) received a reduced intensity stem cell transplantation (RIST). Because transplant related mortality (TRM) and relapse are mainly observed within day 100, we divided the patients into short survivors (SS) who died within day 100 and LS who were alive more than one year after allo-HSCT. The median survival time (MST) of SS and LS were 55.6 (10–99) days and 1241.3 (503–2492) days. Most of LS (8 of 10) showed CR as status of ATL before allo-HSCT and all LS were received CST but not RIST. Response of induction chemotherapy and following CST are important factors for the success of allo-HSCT for ATL as a promising consolidation therapy.

Characteristics of patients ATL subtype Acute type Lymphoma-type 18 17 Gender Male Female 21 14 Age (range) 51.1 yo (41–61) ATL status CR PR PD Relapse 13 16 4 2 Conditioning regimen CST RIST 21 14 Stem cell source PB BM CB  Related 22 2 0  Unrelated 0 9 2 Short survivors TRM PD or Relapse  Related 8 5  Unrelated 5 1 Long survivors Alive Died  Related 6 2  Unrelated 2 0
Successful Hematopoietic Stem Cell Transplantation in a Patient with a Novel DNA Ligase IV Mutation.
Authors: Unal Sule , Cerosaletti Karen , Tekin Mustafa , Cetin Mualla , Uckan-Cetinkaya Duygu , Gumruk Fatma
Published: October 2019

DNA Ligase IV deficiency syndrome (LIG4 syndrome) is a rare autosomal recessive disorder caused by mutations in the DNA ligase IV gene (LIG4), which is essential for the repair of DNA double-strand breaks in mamalian cells by non-homologous end joining (O’Driscoll et al, 2001; Critchlow et al, 1998). LIG4-deficient patients are characterized by microcephaly, growth retardation, developmental delay, low birth weight, dysmorphic facial findings called bird-like face, immunodeficiency, pancytopenia, and pronounced clinical and cellular radiosensitivity (O’Driscoll et al, 2001). Herein, we report two siblings with a novel DNA ligase IV mutation, one of whom underwent hematopoietic stem cell transplantation (HSCT). Case 1, was a 10 year-old girl, whose pancytopenia started at 4 years of age. History revealed a birth weight of 2400 g. There was a first-degree consanguinity between parents. Body weight, height, and head circumference were below the third percentile. Low anterior hairline, prominent nasal bridge and bilateral epicanthus were present. Recurrent upper respiratory infection (URI) history was striking. Serum IgM was low for age. Pancytopenia deepened progressively and HSCT was performed from 6/6 HLA matched father after non-myeloablative conditioning; however autologous reconstruction developed and a 2nd HSCT was performed after busulfan, cyclophosphamide and ATG conditioning. Engraftment was achieved by +12 day. Case 2 was a 6 year-old boy, presented with widespread echymoses at three years of age. Microcephaly, body weight and height below the third percentile, inguinal hernia, prominent nasal bridge and bilateral epicanthi were positive findings. History revealed recurrent URI. Serum IgG and IgM were low for age. DEB and mitomycin-C induced chromosomal analyses were within normal limits, whereas spontaneous breakage was found to be increased. Bone marrow examination revealed hypercellularity in both siblings. Alpha-fetoprotein levels were within normal limits. Mutation analysis for Nijmegen breakage syndrome was negative, however sequencing revealed a novel mutation in the LIG4 gene. A homozygous sequence variant, 1762delAAG, which results in the amino acid deletion 588delK, was identified in case 2. Both parents were heterozygous for the alteration. This novel mutation is located in a highly conserved peptide in the ligase IV protein, adjacent to the ligase domain, with previously unknown function (Wei et al, 1995). Our results demonstrate that non-myeloablative conditioning may not be adequate for LIG4 syndrome patients with hypercellular bone marrow. However, successful treatment of LIG4 syndrome can be achieved by HSCT after more potent conditioning.
Autologous (Auto) and Allogeneic (Allo) Stem Cell Transplantation (SCT) for Pateints (pts) with Advanced Chronic Lymphoctic Leukemia (CLL): A Comparative Study.
Authors: Jimenez Antonio , Farhat Mohamed I. , Gregory Stephanie A. , Kassar Mohamad , Gimelfarb Alla , Batus Marta , Nangia Julie , Shah Neel , Venugopal Parameswaran , Larson Melissa L. , Christopherson Kent W. , Fung Henry C.
Published: October 2019

Background: AlloSCT is a potential curative treatment for pts with advanced CLL but alloSCT is limited by significant acute and chronic treatment related toxicities and the availability of HLA matched donors. AutoSCT is widely used for treatment of acute leukemia and aggressive lymphoma but rarely used for pts with CLL. This retrospective analysis describes the outcomes of 27 patients from our institution who have undergone either autologous or allogenic SCT for treatment of their advanced CLL. We also compare their transplant outcomes.

Patients and methods: Between 08/98 and 07/07, 27 pts (M;22; median age 54.1, range 37–61) with variable RAI-Sawistsky stage CLL at the time of histological diagnosis (RAI I for 14 patients) underwent SCT (10 autoSCT, 17 matched related donors, 3 matched unrelated donors) Fourteen patients (52%) had a normal karyotype, 13q deletion and trisomy 12 occurring in six and four patients respectively. Median disease duration prior to transplant was 41.7 months (range 5–121). The indication for transplant was refractory disease for most patients. Preparative regimens consisted of Cyclophosphamide (Cy) + TBI (6), Fludarabine (Flu)+Cy (6), Rituximab (R) +Cy+TBI (4) R +melphalan + flu (4) and others (7). NCI complete remission (CR) criteria, flow cytometry, cytogenetics and chimerism data, as available, were used to assess response to therapy.

Results: All 27 patients engrafted, with a mean neutrophil engraftment time of 13.5 days (range 9–24). From the group of patients that received allogeneic transplant, eight (47%) developed acute GVHD, with a median time of 18 days after transplant. Seventeen patients (62%) achieved CR on day +100 by marrow morphology, 15 out of 19 by flow cytometry and 13 out of 15 patients had normal chimerism studies. Non relapse mortality (NRM) on day +100 was 14.8% (17.6% for allo, 0% for auto). Estimated actuarial survival at 3 years for all patients was 68% (65% for alloSCT and 72% for the autoSCT group).Disease free survival at 3 years was defined as the percentage of patients who remained in CR 3 years following transplant. DFS was 62% for the whole group (77% for autoSCT and 51% for the patients who received an alloSCT).

Conclusion: In this study, we have demonstrated high remission rate after both autologous and allogeneic SCT, however, the transplant outcomes (day 100 NRM, DFS and OS) favors autologous over allogeneic with toxicity related death accounted for most of the treatment failures after an alloSCT. Our results suggested that AutoSCT warranted further studies, in particular for older patients and those without a suitable donor.
Effects of Graft Compositions on Hematopoietic Reconstitution and Graft-Versus-Host Disease in Related Donor Peripheral Blood Stem Cell Transplantation.
Authors: Feng Ru , Zhan Yu , Yi Zhengshan , Zhang Yongmin , Huang Feng , Fan Zhiping
Published: October 2019

Objective To explore the effects of graft compositions on hematopoietic reconstitution and graft-versus-host disease(GVHD) in human leukocyte antigen(HLA)-identical related donor peripheral blood stem cell transplantation(PBSCT) for malignant hematological diseases.

Methods A retrospective analysis was made on 107 patients with malignant hematological diseases undergoing HLA-identical related donor PBSCT in our department. The main cell doses in grafts were respectively measured by flow cytometry and evaluated their association with hematopoietic reconstitution and GVHD by statistical analysis. Results The measured cell doses were all not in linear correlation with the time of neutrophil reconstitution.There was a significant negative correlation between mononuclear cell(MNC) or CD34+ cell dose and the time of platelet reconstitution (P<0.05)which indicated the more MNC,CD34+ cells may contribute to accelerate platelet reconstitution. However the absolute values of the correlation coefficients were both below 0.4.There was a significant negative correlation between CD34+ or CD34+CD38− cell dose and the development of acute graft-versus-host disease (aGVHD) (P<0.05)which indicated CD34+, CD34+CD38− cells may play negative regulatory roles in the development of aGVHD. Nevertheless the little absolute values of the correlation coefficients also had little usefulness. Each lymphoid subset measured did not correlate with aGVHD.A significant positive correlation was found between CD25+CD4+,CD3+or CD4+CD3+ cell dose or the CD4+ to CD8+ ratio and the development of chronic graft-versus-host disease (cGVHD) (P<0.05) which indicated these lymphoid subsets may play important roles in the development of cGVHD. Furthermore, the correlation coefficient of the CD25+CD4+ cell reached up to 0.76.CD34+, CD34+CD38-cell doses did not correlate with cGVHD.

Conclusion It was concluded that efforts to further accelerate reconstitution in HLA-identical related donor PBSCT by increasing MNC,CD34+, CD34+CD38- cell doses may be counterproductive when the cell doses had attained a threshold. Besides cGVHD may be enhanced by simultaneously increased lymphoid subsets in grafts.
G-CSF Primed Bone Marrow Is Not Superior to Unprimed Bone Marrow as a Source of Stem Cells in HLA Matched Related Allogeneic SCT for β Thalassaemia Major.
Authors: Rajasekar Reena , Mathews Vikram , Lakshmi Kavitha M. , Viswabandya Auro , George Biju , Chandy Mammen , Srivastava Alok
Published: October 2019

G-CSF stimulated BM (G-BM) offers a source of stem cells that has been reported to be associated with faster engraftment and decreased risk of GVHD. It is possible that these differences are related to the immunological composition of these grafts. We compared G-BM and unprimed bone marrow as a source of stem cells in 33 consecutive thalassaemia major patients who underwent a matched related allogeneic SCT treated with a similar conditioning and GVHD prophylaxis regimen. Twenty patients received G-BM from HLA matched donors primed with G-CSF 10ug/Kg/day for 2 days prior to BM harvest and the rest received unprimed BM. Lymphocyte subsets and progenitor cells contained in the grafts was assessed using FACSCalibur and monoclonal antibodies directed against B, NK and T cell subsets. The median age of the cohort was 7 years (range: 2–14) and there were 22 (67%) males. All patients received Bu/Cy/ATG as conditioning regimen. Cyclosporine and short course Methotrexate were used as GVHD prophylaxis. 88% of patients achieved sustained engraftment. The mean follow up was 37 months. Patient and graft characteristics were similar in both HSC sources (Table 1). The target cell dose of 3x108 TNC/Kg could be achieved with smaller volume of the graft in the G-BM group. Time to neutrophil engraftment (median: 16 days, p= 0.260) and platelet engraftment (median: 30 Vs 26 days, p= 0.163) was identical in both the groups. The incidence of acute GVHD (grade II–IV, 10% vs. 15%) and chronic GVHD (12% vs. 9%) were similar in both the groups. 9 patients rejected their graft, six (30%) in G-BM and three (23%) in BM. There were 4 transplant-related deaths within the first 100 days (two in each group). Survival curve analysis showed no difference in overall and event free survival between the two groups. In this cohort of patients, G-BM resulted in rates of engraftment, GVHD, rejection and TRM that were similar to those produced by unprimed BM. G-BM as a HSC source may not add selective advantage to patients with thalassaemia major undergoing a matched related allogeneic SCT.

Comparison of Graft Source Characteristics BM (n=13) G-BM (n=20) P Age (median years: range) 6 (2–14) 8 (2–13) 0.22 Male (%) 9 (69) 13 (65) 1.00 Lucarelli Class III (%) 5 (39) 9 (45) 0.71 Harvest TNC/mm3 23430±9007 39045±13457 0.000 Harvest Volume 417±173 290±98 0.012 TNC / 10e8/kg 4 (2.9–7.6) 4.8 (2.4–8.8) 0.083 CD34 x 10e6/kg 6.89 (3.3–12.9) 8.3 (1.2–16.4) 0.439 Total CD3 cells x 10e6/kg 45.6 (12.7–120.1) 40.4 (26.2 – 139.8) 0.912 NK cells x 10e6/kg 4.71 (0.9–49.8) 6.7 (0.1–15.2) 0.580 Total B cells x 10e6/kg 19 (0.3 – 47.2) 24.3 (0.8 – 62.2) 0.32 Helper T cells x 10e6/kg 20.8 (7.2 – 48.7) 13.7 (15.4 – 77.8) 0.556 Cytotoxic T cells x 10e6/kg 24.8 (7 – 91) 10.6 (8.8 – 51.2) 0.39
Comparative Study of French Unrelated Registered Stem Cell Donors and Cryopreserved Cord Blood Units.
Authors: Balere Marie-Lorraine M.L. , Lapierre Valerie V. , Garnier Federico F. , Marry Evelyne E.
Published: October 2019

The question of the HLA complementarity between unrelated Donors Registries and Cord Blood banks is often raised. On december 31th 2006, three methods were used to qualify and quantify the genetic diversity and complementarity between French marrow unrelated donors (FMUD) and French cryopreserved cord blood units (FCBU). The first one is based on HLA haplotype frequencies performed according to the EM algorithm method, the second on the contribution in new phenotypes (CinNP), the third on the presence of no, one or two frequent haplotypes within MUD/CBU phenotypes. Preliminary operations on HLA typings were necessary to homogenize data: split level, exclusion of only typed at class I level donors. HLA haplotype frequencies comparison does not lead to a global significant difference between FMUD and FCBU. The 5 most frequent haplotypes are identical in both populations: 01-08-17, 29-44-07, 03-07-15, 02-44-04, 02-07-15. Then some haplotypes, characteristic of either Nord or South of Europe, were more frequent among the FMUD than among the FCBU: 02-44-07, 02-51-13, 11-35-01, 02-60-13. On the contrary, some haplotypes of Middle East and Eastern Europe were more frequent among the FCBU than among the FMUD: 30-18-17, 24-35-11, 02-13-07, 33-65-01. In 2006 CinNP does not differ significantly between FMUD and FCBU. 49% of new MUD/CBU bring new HLA-ABDR low resolution split level phenotypes to the FMUD and FCBU populations present the year before, with differences between regions: Martinique and Reunion islands have the largest CinNP, Britanny the smallest. Effectiveness in term of CinNP have been studied for France during the past 6 years, by 2 year sections: 2001–2002, 2003–2004, 2005–2006. As expected, in order to reach an equivalent rate of CinNP, a growing number of donors must be registered every year. The 25 most frequent haplotypes among the FMUD and FCBU were calculated, and called “frequent haplotypes” (FH) among the French population. We could then categorize individuals according to the number of FH they had: none, one or two. 85% of harvested FMUD for a national patient between 2000 and 2006 had one or two FH versus 54% of infused FCBU. 50% of harvested FMUD/ infused FCBU for an international patient had no FH, versus 26% for a national patient. FMUD registry and FCBU banks are complementary: they answer to various HLA haplotypes needs for national and international patients. Incontestably, international patients benefit from French MUD/CBU, which present HLA characteristics not or under-represented in their Registry of origin.
Regulation of Progenitor Cell Proliferation and Granulocyte Function by microRNA-223.
Authors: Camargo Fernando D. , Johnnidis Jonathan J. , Harris Marian H. , Stehling-Sun Sandra , Wheeler Robert T. , Lam Michael , Kirak Oktay , Fleming Mark D.
Published: October 2019

MicroRNAs are abundant in animal genomes and have been predicted to have important roles in a broad range of gene expression programs. Despite this prominence, there is a dearth of functional knowledge regarding individual mammalian microRNAs. Using a loss-of-function allele in mice, we report here that the myeloid-specific microRNA-223 (miR-223) negatively regulates progenitor proliferation and granulocyte differentiation and activation. miR-223 mutant mice have an expanded granulocytic compartment resulting from a specific and cell-autonomous increase in the number of granulocyte-monocyte progenitors (GMPs). We show that Mef2c, a transcription factor that promotes GMP renewal, is a target of miR-223 and that genetic ablation of Mef2c suppresses progenitor expansion and corrects the neutrophilic phenotype in miR-223 null mice. In addition, granulocytes lacking miR-223 are hypermature, hypersensitive to activating stimuli and display increased fungicidal activity. Consequent to this neutrophil hyperactivity, miR-223 mutant mice spontaneously develop inflammatory lung pathology and exhibit exaggerated tissue destruction following endotoxin challenge. Our results present a new paradigm in miRNA-mediated gene regulation, in which a microRNA can negatively modulate the proliferation and differentiation program of the lineage to which it is most strongly associated.
Effects of Recipients’ Body Weight on Engraftment after Unrelated Bone Marrow Transplantation: Analysis of Clinical Data of 4065 Patients from the Database of the Japan Marrow Donor Program.
Authors: Yamashita Takuya , Ohashi Kazuteru , Akiyama Hideki , Sakamaki Hisashi
Published: October 2019

In allogeneic bone marrow transplantation from unrelated donor of Japan Marrow Donor Program (JMDP), the standard nucleated cell dose for transplantation is regulated as 3.0x10E8 bone marrow nucleated cells/recipient’s body weight. But if a recipient is heavyweight, it it often diffecult to get enough bone marrow from the donor for transplantation. Infused cell number and recipients’ body weight may infuence the engraftment and outcome of transplantation. We retrospectively analysed the clinical data of 4065 adult transplant recipients (median age 35, range 16–70) from extracted the database of JMDP. Based on the previous studies, the possible factors which could influence on engraftment were collected. The correlations of these factors with engraftment and neutrophil recovery after transplantation were analysed by using multiple logistic regression. Variables correlated with an increased possibility of engraftment were: incrased recipient’s body weight (p<0.001), increased transplanted cells/recipient’s body weight (p=0.0016), younger recipient’s age (p=0.0019) and using TBI for conditioning (p=0.0358). Variables correlated with early attaining of neutrophil>500 were: use of G-CSF after transplantation (p<0.0001), increased transplanted cells/recipient’s body weight (p<0.0001), and no use of MTX for GVHD prophylaxis (p<0.0001). But in the subset analysis of G-CSF use group, first 25 percentile group reached neutrophil>500 significantly earlier than other group (p<0.0001). These results suggested that recipients’ heavy body weight positively influenced on engraftment, but obesity of recipients might delay neutrophil recovery. This study may contribute to consider the adequate cell dose for unrelated bone marrow transplantation and assess the value of recipients’ body weight as a risk factor of hematopoietic cell transplantation.