bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral Quinolone Switch after 8 Days of Aminoside Is an Efficient and Safe Antibiotherapeutic Strategy for Febrile Neutropenia after Hematopoetic Stem Cell Transplantation.
Authors: Prebet Thomas , Bilger Karin , Gomez Fréderic , Sebban Catherine , Soumelis Valérie , Furhman Catherine , Michallet Mauricette , Biron Pierre
Published: October 2019

Management of febrile neutropenia is one of the important points during the management of autologous or allogeneic hematopoietic stem cell transplantation (HSCT). In this study, we prospectively evaluated the safety and effectiveness of an empiric antibiotherapeutic scheme that associated ticarcillin-clavulanic acid (TIM) and isepamycin (ISE) for 7 days followed by a switch at day 8 to TIM and ofloxacin (OFX), additional therapy (glycopeptide or antifungal therapy) was allowed according to clinical features or documented infection. All the steps of the strategy were evaluated. Ninety six patients (73 autologous HSCT and 23 allogeneic HSCT) were enrolled. Fifty three percents of the patients presented a Fever of Unknown Origin. Bacteriologically proven infections were documented in 32 patients mainly on blood cultures (66%) with an high frequency of gram positive bacterias (71%: 38% of coagulase negative staphylococci, 16% of streptococci, 6% of S. aureus). Median time to neutrophil recovery was 14 days and 24 days respectively for autologous and allogeneic HSCT. We observed an overall effectiveness of the strategy in 78% of the cases. First step strategy was successful in 64 patients (65%) but only 26 patients kept the TIM/ISE bi therapy alone (Glycopeptide and antifungal therapy added in 29 and 7 patients). Quinolone switch was performed in 57 patients (59%) and was effective in 35 patients (61%). The success rate of TIM/OFX after success of TIM/ISE was 95%. Forty five percents of the patient could keep an oral form of OFX. Glycopeptide and antifungal therapy were necessary in 58% and 16% of cases with an effectiveness of 79% and 57% respectively. Failures were mainly observed after front-line treatment (TIM/ISE: 34 Failures) with addition of other kind of antibiotics in 95% of the cases. In 11 patients (32%) the additional antibiotic was netromidazole for clinical symptoms conclusive with anaerobic bacteria. A significantly better effectiveness was found in short duration aplasia (<15days, p=0.008) and in autologous HSCT (p=0.0041). Tolerance was good with only 6 grade III toxicities, no severe nephrotoxicity (8 grade I events, 7 in allogeneic setting) and no infectious death was observed. These results showed

(i) that the choice of the first-line beta lactamin molecule should be discussed

(ii) that oral quinolone switch is safe and effective and

(iii) furthermore pointed out the importance of different antibiotic strategies in autologous (and allogeneic HSCT using Reduced Intensity conditioning) in comparison to standard myeloablative allogeneic HSCT.
Long-Term Outcome after LACE (Lomustine, Ara-C, Cyclophosphamide, Etoposide) Conditioned Autologous Stem Cell Transplantation for Relapsed or Refractory Hodgkin’s Lymphoma: A Single Centre Experience.
Authors: Perz Jolanta B. , Giles Chrissy M. , MacDonald Donald , Apperley Jane F. , Kanfer Edward J.
Published: October 2019

Introduction: The majority of patients with Hodgkin’s lymphoma are cured with initial therapy. However, in patients with primary refractory or relapsed disease, high-dose therapy followed by autologous stem cell transplantation has been shown to be the best option. We analysed patients (pts) who underwent autologous stem cell transplantation (ASCT) following LACE (Lomustine 200 mg/m2, Ara-C 4 g/m2, Cyclophosphamide 4.8 g/m2, Etoposide 1 g/m2) conditioning for relapsed or refractory Hodgkin’s lymphoma at the Hammersmith Hospital, London, between 1991 and 2004.

Patients and methods: 67 pts (46 m, 21 f) initially diagnosed with Hodgkin’s lymphoma (stage I; n=2, stage II; n=29, stage III; n=22 and stage IV; n=14) received first-line chemotherapy with ABVD or COP/ABVD (n=20), BEMOP-CA (n=29), COPP or similar (n=14) or mantle radiotherapy alone (n=4). High dose chemotherapy (HDC) with LACE and ASCT was undertaken in 45 of these pts in 1st relapse, 15 pts in 2nd or subsequent relapse and 7 pts with refractory disease. Median age at the time of HDC was 32 y (17 – 70 y). Prior to ASCT further chemotherapy achieved a complete or partial remission in 41 pts (chemosensitive), but 26 pts had no significant response (chemoresistant). Stem cells were mobilised with Etoposide (1.8 g/m2) and G-CSF in 56 pts, and bone marrow harvest was performed in the other 11 pts.

Results: Two pts suffered a treatment-related mortality (TRM) within the first 100 days (3%). Two pts (3%) developed secondary malignancy (acute myeloid leukaemia). With a median follow-up of 43.3 months (range 0.5 – 145.5 months) the cumulative probabilities of overall survival (OS) and progression free survival (PFS) at both 5 and 10 years was 70% and 62% respectively. Pts who had chemosensitive disease at the time of ASCT had a better OS (p=0.008) and PFS (p=0.08) when compared with pts who had chemoresistant disease. Median PFS has not yet been reached for chemosensitive pts but was 23.4 months for chemoresistant pts. Median OS has not yet been reached for either group.

Conclusions: The outcome for patients with relapsed or refractory Hodgkin’s lymphoma following high dose chemotherapy and ASCT has been sufficiently encouraging to suggest that ASCT should be considered early in chemosensitive patients. However, new therapeutic strategies are needed to improve the clinical outcome of patients with chemoresistant disease.
Evaluation of Costs of Healthcare in HIV Patients with Anemia.
Authors: Dickson Michael , Mody Samir H. , Zilberbeg Marya
Published: October 2019

Background: Although the prevalence of mild to moderate anemia in HIV+ patients (pts) is approximately 22% in the HAART era, the incremental economic burden of anemia is not well understood. This study provides an estimate of the incremental cost of care for anemia in HIV+ pts.

Methods: A longitudinal, retrospective, cohort design was employed to identify subjects in a commercial database of medical and pharmacy claims who were ≥18 years, HIV+, and enrolled in 1 healthcare plan continuously for 18 months from 1996 to 2004. Pts with anemia (A+) were identified using ICD-9 diagnosis codes or receipt of transfusion(s) in the absence of major GI bleeding, trauma or surgery. A control period of 6 months immediately prior to the 12-month study period served to evaluate comorbidity burden and disease severity. To isolate health care costs, including in- and out-patient visits and pharmacy costs, specific to anemia, multivariate regression analyses were conducted to control for demographics, comorbidities, and disease burden.

Results: Of the 2,195 HIV+ subjects identified, 364 (17%) were A+. While there was no significant gender effect, each 1-year increase in age was associated with a 2.1% rise in average monthly cost. A+ cohort was associated with a 118.5% higher average cost compared to A−. The unadjusted average monthly cost for A− subjects in the post-index period was $466 compared with $1,200 for the A+ cohort. In a regression model, anemia maintained an independent and significant association with increased costs (p < 0.0001).

Conclusion: The presence of anemia is associated with a substantial increase in costs among HIV+ patients, independent of demographic differences, disease severity and comorbidity burden. Prospective studies are needed to evaluate the effect of anemia correction on healthcare costs in this population.
Diving Suit: Sterile, Ambulatory, Personnal Device for Protective and Preventive Isolation of Nosocomial Invasive Fungal Infections (IFI) in Neutropenic Patients Hospitalised in Laminar Air Flow Room Who Need a Disruption of Isolation.
Authors: Thiebaut Anne , Perraud Michel , Lyonnet Denis , Ozil Samuel
Published: October 2019

BACKGROUND : Patients with long neutropenia present high risk to develop invasive pulmonary aspergillosis (API) of poor prognosis. Pulmonary computed tomographic Scan (CT scan) allows earlier diagnosis of API and improve management in these patients (D. Caillot, JCO, 1997). However, CT scan need an isolation breaking which is potentially dangerous. We propose a diving suit to maintain the preventive isolation.

MATERIAL AND METHODS : The diving suit is sterile, personnal, ambulatory and transparent to permit monitoring, visual and conversational contact. It is supplied with air with a self-contained station of ventilation (12 hours). The Air contamination is controlled with 2 HEPA filters. Fithteen patients treated for hematologic malignancies like acute leukaemia have tested it after giving inform consent.

RESULTS : The diving suit has been validated for air contamination, physiological (CO2), CT scan feasibility and patients acceptation. All patients (5 male, 10 females) presented a severe neutropenia (PMN < 0.5 G/l during more than 10 days) at time of CT scan. Three patients have had already pulmonary CT scan before using diving suit and 2 claimed to be claustrophobic. No patient describe dyspnea, either pain nor discomfort. All patients feelt reassured and agreed for a new CT scan with this clothing. Two patients have had another CT scan with diving suit for suspicion of pulmonary aspergillosis. Diving suit was very easy to manipulate and did not disrupt monitoring or treatments administration. Diving suit was also compatible with Doppler and Radio Magnetic Nuclear.

CONCLUSION : Pulmonary CT scan needs very often to be performed in neutropenic patients to assess API diagnosis. An ambulatory, personnal protective clothing allows no disruption of isolation for immunocompromized patients during a CT scan. It can also be proposed for medical staff protection when treating patients with SARS or other highly contagious agents.
A Novel Splicing Factor RBM-9 Regulates Protein 4.1R Exon 16 Splicing.
Authors: Yang Guang , Huang Shu-Ching , Benz Edward J.
Published: October 2019

Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to embedded membrane proteins. A regulated splicing event, the inclusion of exon 16 that encodes for peptides critical for spectrin/actin binding, occurs during late erythroid differentiation. We showed earlier that an intricate combination of enhancer and silencer elements direct exon 16 splicing. Regulated expression of splicing factors, SF2/ASF and hnRNP A/B, has also been implicated in mediating exon 16 splicing. In this study, we attempted to characterize the mechanism involved in exon 16 splicing through UGCAUG, an intronic splicing enhancer present in three copies in the intron downstream of exon 16. We first used a wild-type minigene construct consisting of exons 13, 16, 17 and their respective flanking introns that mimics endogenous exon 16 splicing during the induced differentiation of mouse erythroleukemia cells (MELC). Mutational analysis showed a dose-dependent effect of UGCAUG on exon 16 splicing: the presence of all three copies had the most effect. These results were recapitulated with an internal chimeric exon in a test neutral reporter system “DUP4-1”, suggesting that the enhancing effect could be attributed directly to UGCAUG. Furthermore, we identified a novel splicing factor, RBM-9, from MELC that enhanced the internal exon splicing in an UGCAUG-dependent manner in both the exon 16 minigene and DUP4-1 reporter systems. Our characterization of RBM-9 revealed that diverse isoforms of RBM-9 are generated by the utilization of alternative translation initiation sites and tissue-specific alternative splicing; different isoforms from various tissues exhibited differential exon 16 splicing enhancing activities. MELC-RBM-9 enhanced exon 16 splicing the most among all RBM-9 isoforms tested. Inhibition of RBM-9 expression by RBM-9-shRNA reversed its enhancing activity on exon 16 inclusion in MELC. RBM-9-shRNA also reduced exon 16 splicing in a dose-dependent manner in HeLa cells. Furthermore, purified RBM-9 specifically binds to the UGCAUG sequence in a gel-mobility shift assay. Finally, expression of RBM-9 is upregulated and correlates with exon 16 inclusion during MELC differentiation. These results suggest that a novel splicing factor, RBM-9, enhances erythroid differentiation stage-specific exon 16 splicing by interacting with the splicing enhancer UGCAUG.
Variables Affecting Kinetics of Minimal Residual Disease Clearance in Children with Lymphoblastic Leukaemia; Results of the United Kingdom Medical Research Council (UK MRC) Protocols ALL97, ALL97/99 and ALL2003.
Authors: Vora9 Ajay J. , Richards S. , Hancock J. , Mitchell C. , Eden O. B. , Kinsey S. , Hann I. , Lilleyman J. S. , Goulden N.
Published: October 2019

We studied the influence of patient, leukaemia and treatment characteristics on the kinetics of Minimal Residual Disease (MRD) clearance in children with lymphoblastic leukaemia treated using an intensive risk stratified approach. UK MRC protocol ALL97 (1997–1999), and its amended version ALL 97/99 (1999–2002), compared the efficacy and toxicity of dexamethasone (DEX) with prednisolone (PRED), and 6-thioguanine (TG) with 6-Mercaptopurine (MP) in a randomised fashion. The trial produced a 5 year event-free survival (EFS) of 80%, with better systemic and Central Nervous System outcomes in DEX compared with PRED recipients but no difference between TG and MP recipients. Several changes to the risk stratification and treatment regimens during the period of the trial provided an opportunity to determine their impact on MRD clearance. We compared this with clearance in those treated on the successor trial ALL 2003 (more intensive induction containing DEX and Pegylated Asparaginase). The variables investigated for their potential influence on MRD status at the end of induction (EOI) were: NCI Risk; Asparaginase intensity (Erwinia Asparaginase [ERW] in ALL 97 and early part of ALL97/99 vs native or Pegylated E. Coli Asparaginase [E. Coli] in later part of ALL 97/99 and ALL2003); DEX vs PRED; and marrow response at day 8/15 of induction (Slow Early Response [SER] >25% blasts vs Rapid Early Response [RER] ⩽ 25% blasts). MRD was assessed using either a semi-quantitative sequence-specific PCR (ALL97) or Real-Time Quantitative PCR (ALL99 and ALL 2003) of antigen receptor gene re-arrangements at EOI. MRD status was defined as NEG if no MRD was detected by two markers sensitive to 10−4; POS if > 10−4, and Positive Outside Quantitative Range (POQR) if positive < 10−4. Results were available from retrospective testing in 66 ALL97 and 76 ALL97/99 patients, and 204 ALL2003 patients monitored prospectively. There was no significant difference in the proportions of patients MRD NEG, POS or POQR in steroid or NCI sub-groups. Significantly more ERW Asparaginase recipients were MRD POS compared with E.Coli (p< 0.0005). Although SER was predictive of slow MRD clearance, 80/235 (34%) RERs were MRD POS and 9/27(33%) SERs were MRD NEG. Relapse risk at 4 years is 8% in NEG v 28% in POS group in ALL97, 0% in NEG/POQR, versus 15% in POS group in ALL97/99 (p=.001) 2 of the ALL2003 patients have relapsed; one was POS and the other POQR. In the context of intensive risk stratified treatment of childhood ALL, type of Asparaginase influenced rapidity of MRD clearance, whilst NCI risk and type of steroid had no significant influence. Patients with detectable MRD < 10−4 at EOI have the same low risk of relapse as those who have undetectable MRD.
A Novel, Efficacious Therapeutic Approach to Patients with Acute Promyelocytic Leukemia (APL) Using Differentiating Agent and Metronomic Chemotherapy (CT).
Authors: Banavali Shripad D. , Goyal Lovenish , Nair Reena , Biswas Ghanashyam , Amare-Kadam Pratibha , Baisane Chanda , Mahadik Shashikant , Gujral Sumit , Karanth Narayan , Arora Brijesh , Bhagwat Roshni , Kurkure Purna , Parikh Purvish
Published: October 2019

Background: The prognosis of APL has improved markedly with the use of All-Trans-Retinoic Acid (ATRA), but many patients in developing countries are in the high-risk group at presentation and because of inadequate infrastructure die during aggressive induction therapy due to either sepsis or bleeding and/or ATRA syndrome. There is growing appreciation of pivotal role of angiogenesis in APL (Blood2001; 97: 3919). The objective of our study was to evaluate the efficacy of low-dose oral metronomic CT during induction and maintenance therapy along with ATRA in APL.

Materials and Methods: From 2003, 23 patients (ages 1 yr to 43 yrs; median 24 yrs; 13 females, 10 males; median WBC count 5.09 X 109/L, range 0.5 to 113 X 109 /L; 7 low risk, 7 intermediate risk & 9 high risk) with APL who could not go for standard CT were included. ATRA 45 mg/m2/d was given for 90 days. Oral CT was given with Prednisolone 40 mg/m2/d, Etoposide 50 mg/m2/d and 6-TG 40 mg/m2/d (PET) for 21 days as induction therapy with ATRA. Consolidation was with 3 cycles of single agent Daunorubicin at 45 mg/m2/d on d 1, 2, 3 every 21 days. This was followed by 6 cycles of oral ET/PET. ATRA was continued for 15 days every 3 months for total of 18 months.

Results: The protocol was well tolerated on out-patient basis and after the initial admission at presentation, only 6 patients required readmission. Complete morphological, cytogenetic and molecular remission was achieved in 21 patients (91.3%) at a median of 40 days. There were 2 septic deaths during induction; only 1 patient developed ATRA-syndrome once started on PET. There was 1 remission death because of disseminated chickenpox and 1 patient had CNS relapse. Projected Event Free Survival (EFS) and Overall Survival (OAS) at 24 months is 73% and 84% respectively which is significantly better than our previous results (Am J Hematol1999; 60:87). Interestingly the CR rate and OAS for the 9 high risk patients is 100% so far.

Conclusions: There is a valuable role of single hospital trials in exploring innovative therapies in APL. This pilot study contributes to discussion of 4 more debated issues (1) Is there a role for Ara-C in the treatment of APL. (2) How can one improve the therapy in high risk patients without increasing toxicity, (3) What is the ideal duration of maintenance therapy, and (4) What is the ideal therapy for elderly patients and for those who are known to have appreciable mortality from aggressive chemotherapy.
Meta-Analyses of Randomised Controlled Trials (RCT) of Warfarin and Low Molecular Weight Heparin (LMWH) for the Prevention of Venous Thromboembolism (VTE) and Death in Cancer Patients.
Authors: Wheatley Keith , Gross Laura E. , Hills Robert K. , Young Annie M.
Published: October 2019

VTE is a common clinical problem in cancer patients. Uncertainty remains as to whether prophylactic treatment of oral anticoagulants (mainly warfarin) or LMWHs, given by subcutaneous injection, is beneficial and practice varies widely. It has been proposed that anticoagulant therapy may also improve survival in cancer patients. The largest trial of VTE prophylaxis in cancer patients with central venous catheters (CVCs), WARP, has just been completed and showed no apparent benefit for low dose warfarin - among 811 patients randomised to low dose warfarin versus not, the VTE rate was 5% in both arms (odds ratio [OR]=1.04, 95% confidence interval [CI]=0.56–1.92, p=1.0) and mortality was not reduced (OR=1.02, CI=0.74–1.4, p=0.8) (Young et al., ASCO, 2005, LBA8004). This result needs to be put in the context of other RCTs of prophylaxis with anticoagulants, so we performed a meta-analysis to evaluate the effect of warfarin and LMWH on VTE rates and cancer mortality. Computerized searches for trials were performed, including MedLine, Embase, NCI trials register, ASCO and ASH meeting abstracts. Standard meta-analysis methods were used and the results presented as ORs and CIs, with heterogeneity of treatment effect between trials examined using tests for interaction. In 5 trials (n=1355 patients) of warfarin v. not, there was no clear evidence of a decrease in VTEs (OR=0.68, CI=0.46–1.01, p=0.06). An additional 3 trials (n=2034 for all 8 trials) had survival data and there was no clear evidence of a decrease in mortality (OR=0.88, CI=0.77–1.01, p=0.06). In 5 trials (n=793) of LMWH v. not, VTE rate was reduced (OR=0.53, CI=0.32–0.87, p=0.01). 6 trials (n=1295) contained survival data and mortality was reduced with LMWH (OR=0.79, CI=0.67–0.92, p=0.003). In 3 trials (n=985) of LMWH v. oral anti-coagulation (mainly with warfarin), VTE rates were lower with LMWH (OR=0.46, CI=0.32–0.67, p<0.0001) but no mortality benefit was observed (OR=0.89, CI=0.72–1.1, p=0.3). The results of these preliminary meta-analyses suggest that LMWH is the preferred form of prophylaxis for VTE in cancer patients and that overall, there may be an anti-tumour effect, leading to a survival benefit.
The Bcl-2 Family Protein Inhibitor, ABT-263, Promotes Apoptotic-Like Response in Isolated Platelets.
Authors: Tse Chris , Zhang Haichao , Chen Jun , Fryer Ryan M. , Nelson Richard , Nimmer Paul , Reinhart Glenn A. , Smith Morey L. , Tahir Stephen K. , Rosenberg Saul H. , Elmore Steven W.
Published: October 2019

Platelets are relatively short-lived, anucleated cells derived from megakaryocytes that are essential for proper hemostasis. Although they lack a nucleus, platelets possess a highly organized structure with multiple cellular organelles including mitochondria and a number of specific granules. Upon recruitment to sites of endothelial injury, platelet activation stimulates their adhesion, aggregation and release of granule components leading to coagulation. Many of the cellular changes observed during platelet activation and platelet senescence resemble those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, cell shrinkage and microparticle formation (analogous to membrane blebbing). ABT-263 is an orally bioavailable Bcl-2 family protein inhibitor currently in clinical development. This compound is a potent antagonist of the anti-apoptotic proteins Bcl-xL, Bcl-2 and Bcl-w and induces apoptosis in cancer cells dependent on these proteins for survival. ABT-263 induces a unique thrombocytopenia in multiple species that is characterized by a rapid clearance of circulating platelets with recovery to normal platelet counts within several days after treatment cessation. To elucidate the mechanisms underlying this thrombocytopenia, a series of in vitro experiments were performed using freshly isolated platelets comparing ABT-263, its enantiomer (which has significantly lower activity against Bcl-2 family proteins) and known platelet activators. ABT-263, but not the enantiomer control, reduced the viability of dog and human platelets and activated key apoptotic processes, including cytochrome c release, caspase 3 activation, and PS externalization. ABT-263 did not induce aggregation of isolated platelets and did not induce surface expression of GPIIb/IIIa that is essential for platelet adhesion to fibrinogen during clot formation. While ABT-263 had no effect on markers of lysosomal- or alpha-granule release, increases in a marker of dense granule release were apparent at high concentrations. Taken together these data suggest that ABT-263 induces an apoptotic-like response in platelets that is distinct from platelet activation. Furthermore, the lack of effect of the enantiomer control compound suggests this effect is mediated through inhibition of antiapoptotic Bcl-2 family members. Loss of membrane asymmetry and PS externalization has been suggested to be the primary signal for the recognition and clearance of senescent platelets by phagocytes of the reticuloendothelial system. Induction of apoptosis in platelets by Bcl-2 family protein inhibitors such as ABT-263 may coopt this process inducing a premature platelet senescence resulting in a novel type of thrombocytopenia, in vivo.
Focal Adhesion Kinase (FAK) Is Required for CXCL12-Induced Dhemotaxis and Adhesion to VCAM-1 in B Lymphocytes and Hematopoietic Stem/Progenitor Cells.
Authors: Silberstein Leslie E. , Dykxhoorn Derek , Le Yi , Honczarenko Marek , Lieberman Judy , Glodek Aleksandra
Published: October 2019

The chemokine CXCL12 (SDF-1) and its receptor CXCR4 play a critical role in hematopoietic progenitor cell migration and positioning within the bone marrow (BM). However, CXCL12/CXCR4-induced signaling pathways in blood cell migration are poorly characterized. Based on previous studies of BM progenitor B cells indicating a strong correlation between FAK signaling and CXCL12 induced adhesion to VCAM-1, we speculated that FAK might be an important signaling component in CXCL12-induced chemotaxis and integrin-mediated adhesion. Here, we used two approaches to reduce FAK expression in (human and mouse) progenitor B cells and mouse Sca-1+, Kit+, Lin− stem/progenitor cells. FAK specific siRNAs reduced FAK expression by 80% and abolished both CXCL12-induced chemotaxis and adhesion to VCAM-1 in the pro-B cell line, REH. FAK knock-down did not change expression levels of CXCR4 and VLA-4 integrin. FAK expression was rescued by transfection with wild type, chicken FAK, which also restored both CXCL12-induced chemotaxis and adhesion. Furthermore, we found that in FAK deficient cells CXCL12-induced activation of the GTP-ase Rap1 was reduced, suggesting the importance of FAK in CXCL12-mediated inside-out integrin activation. CXCL12-mediated chemotaxis was also impaired in primary progenitor B cells and hematopoietic stem/progenitor cells (HSC/P) isolated from FAK floxed mice, in which FAK was deleted by Cre-mediated excision of FAK floxed alleles. Cre-mediated FAK deletion did not affect cell viability or induce apoptosis. These studies suggest that FAK may function as a key intermediary in signaling pathways controlling hematopoietic cell migration and lineage development.
A Screen for Mll-AF9 Cooperating Mutations in Leukemogenesis Using MLV-Based Mutagenesis.
Authors: Bergerson Rachel J.S. , Collier Lara S. , Cox Tony , Hudson Wendy A. , Allaei Raha , Wolff Linda , Kersey John H. , Adams David J. , Largaespada David A.
Published: October 2019

Human translocations involving the MLL gene are associated with a variety of myeloid and lymphoid leukemia. The MLL-AF9 translocation is common in acute myeloid leukemia (AML). C57BL/6 mice with a Mll-AF9 knock-in mimic the phenotype observed in human patients with this translocation: 75% develop AML but only after a preleukemic phase and prolonged latency. A minority of these mice develop acute lymphoblastic leukemia (ALL) (Dobson, et al. EMBO J.1999, 18(13): 3564). This model provides a system for understanding the evolution of AML initiated by a MLL fusion oncoprotein, including the identification of cooperating genetic events required for AML induction. The recombinant retrovirus MOL4070LTR (M4070) induces AML in some strains of mice (Wolff, et al. J Virol.2003, 77(8): 4965). We hypothesized that this virus could cooperate with the MLL-AF9 oncoprotein to accelerate AML by acting as an insertional mutagen, providing second “hits” in leukemia progression. We bred Mll-AF9 heterozygous C57BL/6 males to 129/SvJ wild type (WT) females, and injected the 280 offspring at 3 days of age with either M4070 virus (212) or a control supernatant (68). All mice were genotyped and observed for disease progression. Infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virus treated WT mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. The gross pathology and preliminary analysis of the surface immunophentoype by flow cytometry and Southern blot indicate infected Mll-AF9/+ animals present with primarily myeloid leukemia while infected WT animals present with lymphoid leukemia. To determine what genes, when mutated by M4070 insertion, can cooperate with Mll-AF9, retroviral insertion sites have been cloned from over 140 diseased tissues from our accelerated leukemia animals using a shotgun-based, linker-mediated, cloning protocol. More than 1,000 independent insertions have been isolated and are being analyzed to identify common insertion sites (CIS). So far, we have found several CIS enriched in Mll-AF9/+ AML accelerated by M4070 insertion. We plan to verify nearby genes for cooperation with MLL-AF9 in AML development using human microarray data and mouse modeling.
Analysis of MLL Amplification and 5q Deletions in AML/MDS Cell Lines Identifies Novel Targets.
Authors: Schneider Bjoern , Nagel Stefan , Kaufmann Maren , Quentmeier Hilmar , Matsuo Yoshinobu , Drexler Hans G. , MacLeod Roderick A.F.
Published: October 2019

Genomic amplifications of the 11q23 region occur in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) where MLL and a few neighboring genes, notably DDX6, are deemed salient targets. However, the extent to which amp(lified)-MLL and translocated MLL share effector targets remains to be established. Even less is known about the target(s) of deletions affecting the long arm of chromosome 5 (5q-) which reportedly partner amp-MLL. We analyzed three AML/MDS cell lines by cytogenetics (conventional and FISH) in parallel with real time q(uantitative)-PCR at both 11q23 and 5q2 to measure copy number and expression of salient target genes together with putative downstream targets. The cell lines comprised: MOLM-17 (transforming-MDS), SAML-2 (therapy-related AML), and UOC-M1 (AML-M1). All three cell lines exhibited approximately four-fold genomic amplification of 11q23 including MLL and DDX6, while the amplicon extended telomerically to include FLI1 (11q24) and HNT (11q25) in MOLM-17 and UOC-M1 only. Expression, quantified relative to AML/MDS cell lines without MLL rearrangement, revealed that of the genomically amplified genes only MLL was generally overexpressed, namely by 9.5x (MOLM-17), 5.1x (UOC-M1), and 4.6x (SAML-2). In addition to the highest MLL expression, in MOLM-17 FLI1 (3.8x) and DDX6 (2.8x) were significantly upregulated. Expression was also quantified among reputed MLL target genes, and showed that in the three cell lines MEIS1 was upregulated in MOLM-17 only (by 6x), and CDKN2C in all cell lines (by about 2x), while HOXA9 and CDKN1B showed near-normal levels of expression. All three cell lines carried 5q- with a common deleted region at 5q31 extending from 134.2–137.5 Mbp. Of a panel of genes recently identified as 5q- deletion targets (centromere-TIGA1, CAMLG, C5orf15, C5orf14, BRD8, HARS, KIAA0141, CSNK1A1, RBM22-telomere), only C5orf15 (function unknown) and BRD8 (a component of the nua4 histone acetyltransferase complex involved in transcriptional activation) were generally downregulated - to about 0.25x, and about 0.4x normalized expression levels, respectively. Both genes lie within the common deleted region. In summary, we have characterized amp-MLL and 5q- in MOLM-17, the first MDS cell line to be described with these rearrangements, together with two AML cell lines with similar cytogenetic profiles. Our data suggest that MLL is the only clear object of 11q23 amplification hitherto identified and CDKN2C its sole unequivocal target in AML/MDS cell lines. It is possible that MEIS1 is also targeted for activation in specific cell types or disease phases in MDS. These findings also highlight C5orf15 and/or BRD8 as possible leukemogenic accomplices targeted for downregulation in accompanying 5q-. These findings may point to differences in signalling pathways targeted by amp-MLL in AML and MDS.
A CK2-Dependent Mechanism for PML Degradation upon Cellular and Oncogenic Stress.
Authors: Scaglioni Pier P. , Yung Thomas M. , Cai Lu F. , Erdjument-Bromage Hediye , Kaufman Andrew J. , Singh Bhuvanesh , Teruya-Feldstein Julie , Tempst Paul , Pandolfi Pier Paolo
Published: October 2019

The PML tumor suppressor controls key pathways for growth suppression, induction of apoptosis and cellular senescence. PML loss occurs frequently in hematopoietic and solid tumors through unknown post-translational mechanisms. PML loss often correlates with tumor progression. Casein kinase 2 (CK2) is a stress activated serine/threonine protein kinase that is oncogenic and frequently over-expressed in human tumor of multiple histological origins including non Hodgkin’s lymphomas, acute leukemias and multiple myeloma. In addition, CK2 over-expression due to gene amplification has been reported to be a powerful adverse prognostic factor in non-small cell lung cancer. We recently reported that PML undergoes ubiquitin/proteasome mediated degradation in immortalized and tumor derived cell lines (Cell. 2006, 126:269). PML degradation depends on direct CK2 phosphorylation of Ser517 in the PML C-terminal degron. PML mutants that are resistant to CK2 phosphorylation display increased tumor suppressive functions in assays measuring apoptosis, replicative senescence and in xenograft models. Now, we report the identification and characterization of novel cellular and oncogenic stress signaling pathways that control the CK2/PML degradation pathway. This analysis allowed us to determine the signaling cascades upstream of CK2. In addition, we found an inverse correlation between CK2 kinase activity and PML protein levels in cancer-derived cell lines and primary specimens. Significantly, CK2 pharmacological inhibition enhances PML tumor suppressive property in vivo and in vitro. These data identify a key post-translational mechanism that controls PML protein levels and provide therapeutic means toward PML restoration through CK2 inhibition. The implications of these new findings during the physiologic response to cellular stress and in the pathogenesis of hemopoietic malignancies will be discussed at the meeting.
Protein Phosphatase 2A Inactivates Bcl2’s Antiapoptotic Function by Dephosphorylation and Up-Regulation of Bcl2-p53 Binding.
Authors: Deng Xingming , Gao Fengqin , Flagg Tammy , May W. Stratford
Published: October 2019

DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membranes results in a Bcl2 conformational change and loss of its antiapoptotic function. Our data now indicate that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (10 nM), or specific disruption of PP2A activity by the expression of SV40 small tumor antigen enhances Bcl2 phosphorylation and suppresses the cisplatin-stimulated Bcl2-p53 interaction in association with prolonged cell survival. By contrast, C2-ceramide, a potent PP2A activator, reduces Bcl2 phosphorylation and increases Bcl2-p53 binding and promotes apoptotic cell death, suggesting that PP2A may function as a physiological regulator of Bcl2 by, at least in part, affecting its association with p53. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses Bcl2 phosphorylation in association with increased p53-Bcl2 binding and apoptotic cell death. By contrast, specific depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation, suppresses p53-Bcl2 interaction and prolongs cell survival. Purified PP2A can directly enhance the formation of the p53-Bcl2 complex in vitro in an okadaic acid-sensitive manner, supporting a direct mechanism. Importantly, PP2A directly interacts with Bcl2 at its BH4 domain which may function as the PP2A ‘docking site’ to potentially ‘bridge’ PP2A to the flexible loop domain which contains the physiological serine 70 phosphorylation site. Thus, PP2A may provide a double whammy to Bcl2’s survival function by both dephosphorylating and enhancing p53-Bcl2 binding. Therapeutically stimulating Bcl2 dephosphorylation and/or increasing Bcl2/p53 binding by activating PP2A may represent an efficient and novel antineoplastic approach.
JAZ Is Required for Interlukin-3 Withdrawal-Induced Apoptosis by Directly Activating p53.
Authors: Yang Mingli , Qiu Jingxin , Li Ying , Jia Jinghua , May W. Stratford
Published: October 2019

By screening a murine interleukin-3 (IL-3)-dependent myeloid cell cDNA library, we previously identified JAZ (Just Another Zinc Finger Protein), a novel zinc finger (ZF) protein that localizes in the nucleus and preferentially binds dsRNA rather than DNA. Forced overexpression of JAZ induces apoptosis but the mechanism was not known. JAZ is differentially expressed in CD34+ primary human and mouse bone marrow cells including mononuclear myeloid and lymphoid cells but not in multinuclear megakaryocytes, indicating that JAZ may function in hematopoietic progenitor cells. Since IL-3 withdrawal induces apoptosis in factor-dependent myeloid and lymphoid cells, we tested whether endogenous JAZ is involved. Hematopoietic cell lines including murine NFS/N1.H7, 32D myeloid and BaF3 lymphoid cells were deprived of IL-3 for 0, 0.5, 1, 2, 4, 6, 8, 12, 24 and/or 48 hr. Results reveal that JAZ expression is upregulated prior to induction of cell death. While a role for p53 in hematopoietic progenitor cell response to apoptosis-inducing stress has been postulated, the mechanism is not clear. Therefore, we assessed whether p53 expression or activation can be affected by JAZ. Results reveal that in association with JAZ upregulation, IL-3 withdrawal also induces p53 expression and importantly, up-regulates its transcriptional activity as assessed by increased BAX expression. To verify p53 dependency, p53-deficient murine M1 and human K562 leukemic cells were also tested. These p53-deficient cells are highly insensitive to serum withdrawal-induced cell death. Importantly, siRNA-mediated ‘knock-down’ of endogenous JAZ (by 70–80%) attenuates stress-induced cell death in NFS/N1.H7 but not M1 cells. These data point to a necessary role for JAZ in IL-3 or growth factor withdrawal-induced hematopoietic cell death in a p53-dependent manner. Further analysis using co-immunoprecipitation studies indicates that endogenous JAZ and p53 associate upon IL-3 withdrawal. Furthermore, using p53+/+ and p53−/− isogenic murine embryonic fibroblasts (MEFs), we clearly show that JAZ not only directly interacts with p53 but also stimulates its transcriptional activity, resulting in mediation/acceleration of stress-activated, p53-dependent apoptosis. Therefore, we propose that the nuclear factor JAZ may be a novel regulator of p53 in the hematopoietic cell response to stress leading to apoptosis. Since only about 15% of hematologic-malignancies express mutant (transcriptionally inactive) p53, it may be possible to therapeutically target wild-type p53 through a mechanism involving JAZ.
Endothelial Cell-Derived Microparticles in the Pathogenesis of Chemotherapy-Associated Hypercoagulability.
Authors: Lechner Daniel , Kollars Marietta , Eichinger Sabine , Kyrle Paul Alexander , Weltermann Ansgar
Published: October 2019

Background: Cisplatin-based chemotherapy is a risk factor of venous thromboembolism in cancer patients. The underlying pathogenesis remains unclear. We hypothesized an apoptotic effect of cisplatin on endothelial cells (EC) inducing a release of small membrane vesicles, so-called microparticles (MP) which are known to cause hemostasis activation.

Objectives:

To quantify the release of MP from EC following administration of cisplatin and to investigate MP-associated procoagulant mechanisms.

Methods: Two EC lines (HUVEC, HMVEC-L) were exposed to cisplatin (1, 2.5, 5, 10, and 20 μM) for up to 120 h. Cell viability was assessed by quantification of mitochondrial dehydrogenase activity, counts and procoagulant activity of MP were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen levels were determined by ELISA.

Results: EC viability decreased in a dose- and time-dependent manner and was accompanied by an increasing release of MP into culture media (maximum: HUVEC + 544%; HMVEC-L + 1738%). In parallel, procoagulant activity of media increased by up to 150% (HUVEC) and 493% (HMVEC-L), respectively. The procoagulant activity was almost abolished by annexin V but was not suppressed by a monoclonal TF-antibody. TF antigen levels on MP were persistently low even at high cisplatin concentrations.

Conclusion: At pharmacologically relevant concentrations, cisplatin induced a marked release of procoagulant MP from EC. Negatively charged phospholipids but not TF on MP were decisive for total thrombin generation. Further studies are warranted to investigate the cisplatin-induced release of EC-derived MP in vivo.
Regulation of Tissue Factor by NF-kB Transcription Factor p50 Is Essential for the Pathogeneses of Deep Vein Thrombosis and Arterial Restenosis.
Authors: Geng Jian-Guo , Wang Jian-Guo , Mackman Nigel , Slungaard Arne , Huo Yuqing , Key Nigel S.
Published: October 2019

NF-kB transcription factors regulate the expression of tissue factor (TF), a principal initiator for the coagulation cascade. Dominant among the five cellular members of NF-kB transcription factors is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a 350-dalton antagonist that targets reduced cysteine62 of p50 for inhibition of NF-kB activation) and genetic deletion of p50 potently attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. The direct binding of p50/p65 heterodimer to the TF-kB site in human TF promoter was demonstrated by p50 and p65 antibody ‘supershift’ using electrophoretic mobility shift assay and immunoprecipitation of the promoter of the human TF gene from chromatins of TNF-a-stimulated human umbilical vein endothelial cells. Andro-treated and p50 null mice both exhibited suppressed TF expression, blunted fibrin deposition, reduced venous thrombosis, and decreased neointimal hyperplasia. Blockade of TF activity by an anti-murine TF antibody also attenuated venous thrombosis and neointimal proliferation in vivo. Our findings thus indicate that NF-kB transcription factor p50 critically regulates TF activity in the pathogeneses of deep vein thrombosis and arterial restenosis, and suggest that specific inhibitors of p50, such as Andro, have the potential to be therapeutically valuable for preventing and perhaps treating arterial and venous thrombosis.
Heparin-PF4 Antibodies Are Frequent after Vascular Surgery, but Not a Frequent Cause of Graft Thrombosis or Thrombocytopenia.
Authors: Alexy Tamas , Tucker Sony , Boyle Soames , Rochanda Leanne , Hood Douglas , Rowe Vincent L. , Weaver Fred A. , Liebman Howard A.
Published: October 2019

Surgical intervention for atherosclerotic disease of the aortoiliac and femoral-popliteal arteries has significantly reduced the incidence and complications of peripheral ischemia. However, surgical correction of obstructive arterial lesions can be complicated by either early graft failure due to acute thrombosis or late stenosis of the vascular bypass graft. Despite improvements in surgical technique, early graft failure is still observed in up to 10% of patients and up to 30 % can subsequently develop a graft stenosis. The etiology of early graft closure is less clearly understood. Anatomic or structural defects in the new graft may result in early thrombosis. Underlying prothrombotic conditions have also been suggested as contributing factors to early graft thrombosis. Recently, we have observed recurrent thrombotic events occurring in vascular grafts in two patients who were subsequently diagnosed with the syndrome of Heparin-Induced Thrombocytopenia. Because of these events we undertook an IRB approved, prospective evaluation of HIT antibodies in patients undergoing vascular surgery. Patients undergoing infra-inguinal bypass surgery for atherosclerotic vascular disease were enrolled after informed consent. Blood samples were obtained the day before surgery, day of hospital discharge, at days 14 and 28 after surgery. Platelet counts were obtained on the day of discharge and on post-surgery day 14. Plasma samples were assayed for PFA-heparin antibodies by ELISA (Stago Diagnostica) and platelet activating antibodies by platelet aggregometry. All assays were performed in duplicate.

Seventy-nine patients were enrolled. Six (7.7%) patients provided only the pre-surgical blood specimen and only 68 (86%) patients provided all four blood specimens. All but 5 patients reported previous heparin exposure. Of the 73 patients in which post-surgical blood samples were taken, 28 (38%) became ELISA positive for PF4-Heparin antibodies. When tested for platelet activating antibodies, 7 (10%) were positive. All patients who developed a positive ELISA had antibody detected by day 14. Four patients tested positive by ELISA prior to surgery, with 3/4 having positive aggregation assays. One ELISA negative patient developed early graft closure. No patient developed thrombocytopenia or a 50% or greater decrease in platelet count. Patients undergoing vascular surgery frequently develop PF4-Heparin antibodies of which up to 10% patients have platelet activating antibodies. However, both thrombocytopenia and vascular graft thrombosis due to HIT is a rare event.
Hepatocytes Ingest Longterm Refrigerated Platelets: A Novel Platelet Clearance Mechanism.
Authors: Rumjantseva Viktoria , Josefsson Emma C. , Wandall Hans , Hartwig John H. , Stossel Thomas P. , Hoffmeister Karin M.
Published: October 2019

We previously reported that the lectin domain of αMβ2 receptors on hepatic macrophages mediates rapid clearance of washed murine platelets transfused after refrigeration for 2 hours, recognizing exposed βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered platelet GPIbα molecules and that the same receptors elicit phagocytosis of refrigerated human platelets human macrophages in vitro. A platelet-associated galactosyltransferase catalyzes the covering of βGlcNAc residues with galactose in the presence of UDP-galactose, thereby blocking clearance of cold mouse platelets in vivo and phagocytosis of human platelets in vitro. These intriguing findings contradicted earlier evidence that refrigeration of human platelets procured for transfusion only promotes their rapid clearance after prolonged (>8h) incubation and also are inconsistent with the well-known recognition system for exposed galactose residues through asialoglycoprotein (ASGP) receptors. Reconciling these contradictions, we report that the absence of plasma during storage accounts for the differences in time of exposure to cold to promote clearance and that mouse platelets cold-stored in plasma also only clear rapidly after long-term (48h) storage. We also found that hepatic clearance of long-term cold-stored (LTCS) mouse platelets occurs in hepatocytes. Streptavidin-POD staining revealed abundant LTCS biotinylated platelets in hepatocyte phagosomes. Furthermore, cells of the hepatocyte HepG2 line avidly ingest fluorescently-labeled LTCS human platelets (7-fold above the baseline of room-temperature-stored platelets), as evidenced by flow cytometry, fluorescent microscopy and by time-laps video microscopy. Long-term cold storage increases by ~1.7-fold platelet binding of the galactose-specific lectin RCA I, implying that with long-term cold storage, exposed galactose residues cluster sufficiently to induce recognition by hepatocyte ASGPR receptors. The results define a new clearance mechanism, representing the first example of blood cell removal by a non-myeloid cell. Since we find that human platelets also express a cell surface sialotransferase that adds sialic acid to galactose residues, we suggest that a combination of sialylation and glactosylation, achievable by addition of sugar substrates alone, might accommodate long-term cold storage of platelets for transfusion.