bluuebunny/crossref_metadata_2025 huggingface dataset:
The Ubiquitin-Ligase APC-Cdh1 Is Required for Maintaining Genomic Stability and Is Transcriptional Repressed by AML1/ETO.
Authors: Waesch Ralph , Engelbert Dirk , Schnerch Dominik
Published: April 2020

Accurate DNA replication and chromosome segregation is essential during cell division in order to provide genomic stability and avoid malignant growth. We found that ubiquitin-dependent proteolytic cell cycle control by the E3-ubiquitin-ligase anaphase-promoting complex (APC) activated by Cdh1 (APC-Cdh1) is required for maintaining genomic integrity and viability in proliferating human cells. Lentiviral-delivered stable expression of short hairpins targeted against Cdh1 causes an apoptotic phenotype associated with p53-stabilization and p53-dependent transcriptional up-regulation of the cyclin-dependent kinase inhibitor p21. Cdh1-depleted cells enter mitosis delayed compared to controls suggesting the activation of a DNA-damage checkpoint in S- and G2-phase. Depletion of Cdh1 leads to premature accumulation of cyclin A2 and cyclin B1 in G1- and S-phase. This may interfere with loading of pre-replication-complexes onto origins of replication in G1 and cause chromosomal instability when cells progress into mitosis. In addition, stabilization of the Cdh1 target Aurora A at physiological levels is sufficient to cause centrosome overduplication and subsequent polyploidization in Cdh1 and p53 deficient cells. Genetic instability is a hallmark of cancer cells and deregulation of APC-Cdh1 may be involved, since we observe downregulation of Cdh1 in t(8;21) acute myeloid leukemia. Moreover, expression of AML1/ETO in an inducible system leads to downregulation of Cdh1 indicating transcriptional repression of Cdh1 by AML1/ETO. Consistent with these data AML1/ETO positive cell lines show numerical chromosomal aberrations. Thus, disruption of the central mitotic control machinery leads to genetic instability by several mechanisms and the APC may be an important suppressor of tumor progression in AML1/ETO positive leukemia.
Arsenic Trioxide Targets the MDS1 and EVI1- Containing Oncoproteins for Degradation by Proteasome Dependent and Independent Mechanisms.
Authors: Shackelford David , Kenific Candia , Waxman Samuel , Ren Ruibao
Published: April 2020

The EVI1 gene was first identified as a common site of retroviral integration in murine myeloid tumors and expresses a zinc finger-containing transcriptional regulator. It also encodes for MDS1/EVI1, a longer, alternatively spliced form of EVI1. In blood development EVI1 is normally expressed in hematopoietic stem cells (HSCs) but not in committed cells. However, aberrant expression of EVI1 or MDS1/EVI1 has been frequently found in myelodysplastic syndrome (MDS), and in cases of chronic myeloid leukemia during blast phase (CML-BP) and acute myelogenous leukemia (AML). In addition, the MDS1/EVI1 gene fuses to the AML1 (Runx1) gene as a consequence of the t(3;21)(q26;q22) translocation found in patients with CML-BP and therapy related MDS and AML, producing the AML1/MDS1/EVI1 (AME) or AML1/MDS1 fusion genes. We have previously shown that expression of the AME fusion protein in mouse bone marrow cells by retroviral transduction impaired hematopoiesis and eventually induced an AML. EVI1 was also reported to induce MDS in mice. These results indicate that the EVI1 and AME oncoproteins play causal roles in t(3;21)- and EVI1-positive hematological malignancies. Therapies targeting oncoproteins that play causal roles in tumorigenesis have been shown to be effective in treating cancers. Arsenic trioxide (ATO) has been found as an effective treatment for patients suffering from acute promyelocytic leukemia (APL) that harbor the t(15;17) translocation. The success of ATO in treating APL patients has prompted many to consider expanding the drug’s use to target other translocations and/or mutations found in hematological malignancies. In this study we found that ATO could target the EVI1, MDS1/EVI1, AML1/MDS1 and AME oncoproteins for degradation. We further found that ATO targeted EVI1 for degradation through the ubiquitin-proteasome pathway. Interestingly, ATO induced degradation of MDS1-containing oncoproteins via a proteasome-independent mechanism. Consistent with the downregulation of AME, the ATO treatment induced differentiation and apoptosis in AME leukemic cells in vitro and reduced splenomegally in mice transplanted with AME leukemic cells. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1- and EVI1-positive hematological malignancies. We are currently studying the mechanism of increased ubiquitination of EVI1 by ATO, as well as the proteasome-independent mechanism by which ATO downregulates MDS1-containing proteins. Such research will help further the identification of new therapeutic targets and the development of new therapeutic strategies for human cancer.
HOXB9 Is Aberrantly Expressed in Blast Cells in a Subset of Acute Myeloid Leukemia Patients and Supports Proliferation of AML Cell Lines
Authors: Heinrichs Stefan , Schoch Claudia , Neuberg Donna S. , Look A. Thomas
Published: April 2020

Studies in mice and humans suggests that HOX genes integrate the myelodysplastic and leukemogenic activities of different oncogenes. HOXA9 has been identified as a major downstream target in patients with MLL translocations and amplifications. Since upregulation of HOXA9 and other specific HOX genes is more frequent than MLL deregulation, additional upstream pathways have to be postulated. Given the fact that HOXA9 is targeted by several yet to be identified mechanisms, we asked whether other members of the HOX9 paralog group are also highly expressed in myeloid malignancies. Microarray gene expression analysis of 449 AML patients revealed that besides HOXA9 (61% of all cases), HOXB9 is upregulated in 15% of the cases, whereas the overexpression of HOXC9 and HOXD9 expression is a very rare event (>1% of all cases). High levels of HOXB9 expression did not correlate with low levels of HOXA9 expression ruling out the possibility that HOXB9 compensates for a lack of HOXA9 activity in the leukemogenesis of a HOXA9 negative AML cases. Moreover, within the cytogenetically defined subgroups inv(16), t(15;17), t(8;21) that have no HOXA9 expression at all, HOXB9 was also low or absent. The analysis of 27 AML cell lines revealed a gene expression pattern of HOXA9 and HOXB9 that reflected the frequency of overexpression found in patient samples (14/27 showed HOXA9 expression; 6/27 showed HOXB9 expression). Interestingly, the absolute expression level of HOXB9 mRNA in cell lines is about 10fold higher than HOXA9 as measured by real-time RT-PCR. Western-blotting with an antibody recognizing both HOXA9 and HOXB9 confirmed this result on the protein level. However, the analysis of the absolute levels in normal CD34+ bone marrow cells revealed an inverse ratio, with very low levels of HOXB9. Taken together these results indicate that HOXB9 expression is aberrant in the leukemic clone.

To identify downstream pathways driven by HOXB9 overexpression we designed two highly efficient siRNAs targeting HOXB9 and knocked down its expression by retroviral transduction. In comparison to cells expressing a control siRNA, the resulting cell lines showed <5% of the control HOXB9 protein levels and a reduction in the cellular growth rate. Consequently, HOXB9 expression is required for the rapid growth of these AML cells. To gain insight into the molecular mechanisms underlying this phenotype we are performing global gene expression analysis. Gene set enrichment analysis (GSEA) and related approaches will be used to extract relevant candidate pathways for further analysis.

Our results indicate that aberrant expression of HOXB9 inhibits the differentiation of leukemic myeloid progenitor cells and maintains the cells of the leukemic clone in an undifferentiated and rapidly proliferative state.
Methylation-Independent Silencing of the Tumor Suppressor p15INK4B by CBFb-SMMHC in Acute Myeloid Leukemias with inv(16).
Authors: Markus Jan , Garin Matthew T. , Galili Naomi , Raza Azra , Thirman Michael J. , LeBeau Michelle M. , Rowley Janet D. , Wolff Linda
Published: April 2020

The tumor suppressor INK4B(p15) gene is silenced by CpG island hypermethylation in a majority of acute myeloid leukemias (AML). This silencing can be reversed by the treatment with hypomethylating agents, and these agents are currently being tested for therapeutic intervention. So far, it was not investigated whether or not the INK4B is hypermethylated in all cytogenetic subtypes of AML. Our experiments, which compare levels of INK4B methylation in AML with inv(16), t(8:21) and t(15;17) reveal a strikingly low level of methylation in all leukemias with inv(16). This contrasts with significant levels of DNA methylation in a high proportion of the AML from the other two groups. Surprisingly, even though there is a lack of INK4B methylation in samples from patients with inv(16), expression of the gene is very low when compared to that of PBL from healthy individuals or HL60 cells. Subsequent experiments uncovered a novel mechanism to explain the low level of INK4B expression in the inv(16) AMLs. Overexpression of the aberrant chromosome 16-associated gene CBFb-MYH11 in U937 cells results in failure to induce INK4B in response to vitamin D3. Furthermore, CBFb-SMMHC, encoded by CBFb-MYH11 directly represses transcription from an INK4B promoter in a reporter assay. Electromobility shift assays in the AML-derived cell line ME-1 and U937 cells expressing the fusion gene demonstrate that the repression is due to a change in the composition of the complexes recognizing the binding sites for the transcription regulator CBF. In conclusion, we have found that methylation is not the only way to induce silencing of the tumor suppressor p15INK4B in AML. In inv(16)-containing AML, loss of gene expression is accomplished by the direct transcriptional repressor activity of CBFβ-SMMHC.
Long-Term Outcomes of Post-Surgical Deep-Vein Thrombosis in Asia: The AIDA-Extension Study.
Authors: Piovella Franco , Wang Ching-Jen , Lee Lai Heng , Lee Won Chul , Turpie Alexander G. , Gallus Alexander , Planes Andre , Passera Raffaella
Published: April 2020

Background: We have recently demonstrated in a large multinational, multiethnic study that the incidence of venographic deep-vein thrombosis (DVT) in Asian patients undergoing major orthopedic surgery of the lower limbs and not receiving thromboprophylaxis was similar to that observed in orthopedic patients in Western countries. Overall, 407 patients (20–99 years) undergoing THR (n=175), TKR (n=136) or HFS (n= 96) were recruited in 19 centres across Asia (Indonesia, South Korea, Malaysia, Philippines, Taiwan and Thailand). 72.5% of the enrolled patients had adequate venograms. Total DVT was diagnosed in 121 of 295 evaluable patients (41.0%, [95% confidence interval: 35.4–46.7]). Proximal DVT was found in 30 patients (10.2% [7.0–14.2]). However the long-term outcomes following deep vein thrombosis such as recurrence of DVT, incidence of pulmonary embolism (PE), and post-thrombotic syndrome (PTS) remain unknown. PTS has received little attention in the literature, and particularly in Asia.

Objectives: To assess the long-term complication rates (at 1 and 2 years) of patients with objectively assessed DVT compared to those observed in patients without post-operative DVT.

Methods: Annual visits were performed to record the occurrence of DVT/PE, incidence of PTS, cumulative incidence rates and related predictive factors. PTS scoring were assessed using the Villalta scale (signs and symptoms). In addition in a subset of 18 out of 27 (66%) patients from South Korea with centrally adjudicated venographic DVT, a second venogram was performed at 1 year in order to verify possible spontaneous modifications of the thrombus (none of these patients had undergone any anticoagulant treatment).

Results: From the 332 patients having completed the AIDA study in the 19 centres involved in the extension study, 236 patients (71.1%) agreed to participate in the extension phase and were assessed at year 1. Year 2 follow up visits were completed in May 2005. Complete data analysis of the final study results are ongoing and should be completed by September 2005. Of the 18 patients who underwent a second follow-up bilateral or unilateral ascending venography at 1 year ± 3 months post-operatively, 11 (61%) patients demonstrated resolution of the thrombus, with 3 (16%) patients showing incomplete resolution or persistence of the venous thrombosis. The venograms of the remaining four (23%) patients presented with development of collateral circulation confirming the progressive state of PTS. The majority of patients with centrally adjudicated venographic DVT demonstrated positive resolution following venographic assessment at year 1. The high incidence of thrombus resolution may have been due to the fact that most patients were diagnosed with distal DVT, which would suggest that a high percentage of DVT of the distal veins may resolve spontaneously compared to other types of DVT.
Body Size Measures and the Risk of Venous Thrombosis: The Longitudinal Investigation of Thromboembolism Etiology (LITE).
Authors: Cushman Mary , O’Meara Ellen , Folsom Aaron R. , Heckbert Susan R. , Zakai Neil , Rosamond Wayne D.
Published: April 2020

Background: Obesity is associated with an increased risk of venous thrombosis (VT), however little is known about which components of obesity are important. Proposed hypotheses include an association of adipocyte products with hemostatic imbalance, increased body or leg size impairing venous return, increased inflammation, and differences in venous vessel walls in obese persons. We studied the associations of measures of body size and composition with risk of VT in the LITE study.

Methods: The LITE is a prospective study evaluating VT risk factors in 21,680 participants aged 45–100, in the Atherosclerosis Risk in Communities (ARIC) study and the Cardiovascular Health Study (CHS). Baseline body size measures were obtained using standardized methods and bioelectric impedance was used to calculate fat and fat-free mass in CHS participants. 729 participants reporting prebaseline VT or baseline warfarin use were excluded. VT events during 12.6 years of follow up were validated by medical record review and classified as idiopathic or secondary. Body size measures were evaluated as risk factors using Cox proportional hazards models, adjusting for age, sex, race and diabetes status.

Results: There were 451 VT events during follow-up (1.8 per 1000 person-years); 182 were idiopathic, 315 were deep vein thrombosis (DVT) and 136 pulmonary embolus (PE) +/− DVT. All body size measures were associated with increased risk of VT (table), with height having the weakest association. Those with severe obesity (body-mass index above 40 kg/m2) were at the highest risk, with a nearly 3-fold increased risk compared to those of normal weight. Both fat and fat-free mass were risk factors.

Conclusion: In this prospective study, multiple measures of body size, and measures of body composition including fat and non-fat components, were risk factors for VT. Findings suggest a multicausal pathogenesis for obesity-related thrombosis. Further study is required to determine reasons for the association of body size and composition with VT.

Relative Risk of VT by Body Size Categories (Quartiles (Q) Except when Noted; lowest quartile is the reference group) Q1 Q2 Q3 Q4 * P <0.05; ** categories are <25, 25–20, 30–40, >40 kg/m2 Body-mass index** 1.0 (reference) 1.3* 2.1* 2.9* Weight 1.0 (ref) 1.2 1.5* 2.5* Height 1.0 (ref) 1.1 1.2 1.4* Waist-hip ratio 1.0 (ref) 1.3 1.5* 1.6* Waist Circumference 1.0 (ref) 1.2 1.3 2.1* Hip Circumference 1.0 (ref) 1.3* 1.5* 2.2* Calf Circumference 1.0 (ref) 1.3 2.0* 2.3* Fat-Free Mass 1.0 (ref) 1.2 1.4 1.9* Fat Mass 1.0 (ref) 1.1 0.8 1.7*
European Collaborative Paediatric Database on Cerebral Sinus Venous Thrombosis (CVT): Risk of Recurrent Venous Thrombosis (Update 2005).
Authors: Kenet Gili , Kirkham Fenella , Bidligmaier Christoph , Nowak-Gottl Ulrike
Published: April 2020

The present European database study was designed to evaluate the risk of recurrent venous thrombosis (rVT) in 407 consecutively recruited CVT children from the Israel, United Kingdom, and Germany. Children were recruited between January 1996 and January 2003. 330 of them were prospectively followed at least 12 months. Median(range) age at first thrombosis onset was 5.3 years (newborn to < 18; male: 55%). The median(range) follow-up time was 33months (12–216).

Acute antithrombotic treatment (AT) with UFH or LMWH was applied in 252 of 330 patients (76.4%), followed by secondary AT in 212 subjects (64%). The latter was carried out in the majority of cases with LMWH (77.8%: enoxaparin or dalteparin: 2–4 hours anti-Xa activity 0.3–0.5 U/ml; duration: 6 months [3–12]), vitamin K-antagonists (17.5%; duration: 12 months [(4-lifelong]), or aspirin (1.5%).

18 of 330 surviving children (5.5%) suffered a second venous thrombosis 6 months (0.5–114) after first CVT. The rate of recurrences was no different in the three European countries (p=0.94). A significant positive correlation between higher age at first CVT and rVT was calculated (r=0.34; p=0.0002), and recurrence was significantly associated with the duration or secondary AT (p=0.0025). In contrast, no significant associations between rVT and mode of treatment (p=54), 3–6 months patency rates (p=0.08), basic diseases (p=0.2), or the presence of prothrombotic risk factors (p=0.8) were found. At the time of recurrence six of 18 patients were on AT, whereas in the remaining 12 patients rVT occurred 8 to 114 months after first thrombosis and after withdrawal of AT. In 87.5% of cases rVTs were associated with HIT, infections diseases, malignancies, diabetes type I, nephrotic syndrome, trauma, and the use of oral contraception. Spontaneous rVT occurred in two further children. Prothrombotic risk factors were found in 7 of 15 patients (46.6%) tested. In conclusion, in this European database study the rate of rVT in children with CVT is 5.5%. Since rVT was associated with age, AT therapy and co-morbid risk factors, the need for appropriate guidelines for prophylactic administration of AT in risk situations in children suffering a first CVT deserves consideration.
Continuing Reduction in Level of Residual Disease after 4 Years in Patients with CML in Chronic Phase Responding to First-Line Imatinib (IM) in the IRIS Study.
Authors: Goldman John M. , Hughes Timothy , Radich Jerry , Branford Susan , Hochhaus Andreas , So Charlene , Gathmann Insa , Wehrle Elisabeth , Kaeda Jaspal
Published: April 2020

Background and methods: The IRIS study initiated in June 2000 compared treating newly diagnosed CML-CP pts with IM or interferon+ara-C. Pts who achieved a complete cytogenetic response (CCyR) had BCR-ABL transcript numbers in the blood measured serially by real-time quantitative PCR. Results were expressed as a log reduction from a standardized baseline value for untreated pts. We showed previously that by 1 year an estimated 40% of pts who started IM at 400 mg/day had achieved a major molecular response (MMR) defined as ≥3 log reduction in BCR-ABL levels. Of the 136 pts who achieved MMR at one year, all survived at 42 months without evidence of progression to advanced phase whereas 5% of the 94 pts who failed to achieve MMR at one year had progressed. Although routine PCR sampling was interrupted January 2003, (24 months after end of study recruitment), 6-monthly monitoring was resumed in August 2004 for all pts on study regardless of CCyR status. PCR data analyzed up to February 2005 are included here.

Objectives: To evaluate level of BCR-ABL transcripts after approximately 4 years on IM in pts who had transcript numbers measured in CCyR at 1 year. Of the 553 pts who started treatment with IM, this analysis is based on the 101 pts who had achieved CCyR within 1 year, who received first-line treatment at least for 24 months and who had blood collected for measuring transcript numbers at 1 year and 4 years after starting treatment.

Results: At 1 year BCR-ABL transcript levels had fallen by at least 3 log in 47 (46%) of the 101 pts. At the most recent measurement at or after 44 months from start of study (median 49 months, range 44 – 55), 76 (75%) pts showed a reduction of at least 3 logs. Of these 76 pts, 39 (51%) had already had a ≥3 log reduction at year one, whereas 37 (49%) had not. Conversely 8 of the 47 pts with ≥3 log reduction at 1 year had log reductions <3 at 4 years (see table).

Table 1. BCR-ABL log reduction

For all pts the mean ± SD log reduction was 3.0 ± 1.0 at 1 year and 3.4 ± 1.1 at 4 years (p=0.0008, paired t-test). At the 1 and 4 year points 20 (20%) and 36 (36%) respectively of these pts had achieved ≥4 log reductions. In this subset responding well to IM, the majority of pts have BCR-ABL transcript levels that continue to decline with 35% falling more than 1 log and 18% falling 0.5–1 log between 1 and 4 years; however in 13% levels increased more than 1 log between the two time-points.

Summary:. Pts with CCyR and a moderate decline of their BCR-ABL transcript levels at 1 year still have a good chance of achieving an MMR at 4 years. The full analysis (data cut-off of 31-July-2005) will be presented together with the relationship of transcript levels to clinical outcomes.
Incidence, Timing of Onset and Severity of Post-Thrombotic Syndrome after Acute Symptomatic Deep Vein Thrombosis: Two-Year Results from a Canadian Multicenter Prospective Cohort Study (The VETO Study).
Authors: Kahn Susan R. , Ducruet Thierry , Arsenault Louise , Miron Marie Jose , Roussin Andre , Desmarais Sylvie , Joyal France , Kassis Jeannine , Solymoss Susan , Desjardins Louis , Johri Mira , Shrier Ian , Lamping Donna L. , Ginsberg Jeffrey S.
Published: April 2020

Background and Objectives: The post thrombotic syndrome (PTS) is a chronic complication of deep venous thrombosis (DVT). It is difficult to provide DVT patients with long-term prognostic information as there is little longitudinal data on the occurrence of PTS after DVT. We performed a prospective multicenter study of long-term outcomes after DVT (the Venous Thrombosis Outcomes [VETO] Study) to determine the incidence, timing of onset and severity of PTS.

Methods: Consecutive patients with objectively diagnosed acute DVT were recruited at 7 participating hospital centres from April 2001-July 2002. Patients attended study visits at Baseline, 4, 8, 12 and 24 months. Clinical data were recorded and standardized assessments for PTS (using Villalta scale) were performed by trained study personnel at each follow-up visit. Patients were classified as having developed PTS if the ipsilateral Villalta score was ≥ 5 on at least 2 visits or at the final follow-up visit, and having severe PTS if a score of >14 or a venous ulcer was documented on any one occasion. The Kaplan-Meier survival method was used to estimate the cumulative probability of PTS and of severe PTS over time.

Results: The study cohort consisted of 359 patients. The mean age was 56 years, 50% were male, 2/3 were outpatients, 55% had proximal DVT and 20% had previous DVT. The Kaplan-Meier estimates of cumulative risk of PTS were 29% at 4 months, 33% at 8 months, 35% at 12 months and 37% at 24 months. Corresponding risks of severe PTS were 6.6%, 6.9%, 6.9% and 6.9%, respectively. When the analysis was stratified according to history of DVT, the cumulative incidence of PTS in patients without previous DVT was 25%, 28%, 30% and 31% at 4, 8, 12 and 24 months. Corresponding rates of PTS in patients with previous DVT were 45%, 51%, 53% and 57%.

Conclusions: More than one third of patients in our cohort developed PTS within 2 years of DVT, and of these, 1 in 6 was classified as severe. Most cases of PTS and of severe PTS were apparent by 4 months after DVT. Patients with previous DVT had a higher frequency of PTS and of late onset PTS. Our study provides longitudinal data on prognosis that is relevant both to patients with DVT and their treating physicians.
Factor XIII Val34Leu Polymorphism and the Risk of Myocardial Infarction: A Meta-Analysis.
Authors: Shafey Mona , Anderson Josdalyne L. , Scarvelis Dimitrios , Doucette Steve P. , Gagnon France , Wells Philip S.
Published: April 2020

Genetic factors involving blood coagulation are thought to contribute to the pathogenesis of myocardial infarction. A common polymorphism of Factor XIII, Factor XIII Val34Leu, may be protective against developing an acute myocardial infarction, but various studies show conflicting results. We performed a meta-analysis to determine whether the Factor XIII Val34Leu polymorphism is associated with a decreased risk of myocardial infarction. 93 articles were reviewed after a MEDLINE search of the literature (1966 through April Week 1 2005) and 12 case-control studies were selected. We included studies involving patients with objectively diagnosed myocardial infarctions (according to the WHO criteria) provided Factor XIII genotyping data were available. Inclusion decisions, quality assessment and data extraction were conducted by two reviewers. Hardy-Weinberg equilibrium was verified in all studies. Hypothesizing that the Leu allele was protective we performed 3 analyses with the Val/Val genotype as the reference group. Pooled odds ratios and their 95% confidence intervals were determined using the method of DerSimonian-Laird. Prior to pooling, heterogenity testing was performed using the I2 statistic. Funnel plots ruled out the possibility of publication bias. These studies included a total of 8743 patients, of which 3663 were MI patients and 5080 were healthy controls. Using the random effects methods, protective effects were seen with the Leu/Val genotype alone (OR for MI 0.79, 95% CI 0.68–0.93) and with Leu/Val and Leu/Leu genotypes (OR 0.79, 95% CI 0.68–0.93). There was also a protective effect with the Leu/Leu genotype alone, but this was not statistically significant (0.83, 95% CI 0.61–1.12) likley due to the low frequency of this genotype. Removing the first published study (Kohler 1998) due to potential publication bias assocaited with the first study evaluating a genotype did not significantly change the results. Our analysis found that the Factor XIII Leu allele confers a small but significant protective effect against myocardial infarction. The polymorphism may be useful in profiling an individual’s susceptibility for arterial thrombosis. It remains to be elucidated whether or not routine testing for the FXIII Val34Leu polymorphism will be clinically relevant.
The Thrombospondin-1 N700S Polymorphism Does Not Alter von Willebrand Factor Multimer Size in Patients Suffering an Acute Myocardial Infarction.
Authors: Zwicker Jeffrey , Peyvandi Flora , Palla Roberta , Lombardi Rosanna , Canciani Maria Teresa , Bauer Kenneth , Lawler Jack , Mannucci P.M.
Published: April 2020

An uncommon missense variant of thrombospondin-1 representing an asparagine (Asn-700) to serine substitution (Ser-700) has been identified as a potential genetic risk factor for myocardial infarction (MI). Thrombospondin-1 (TSP-1) is a glycoprotein stored in human platelet alpha granules and has been shown to regulate von Willebrand factor (VWF) multimer size. The TSP-1 domain responsible for VWF-reducing activity was localized to the calcium-binding C-terminal sequence at position Cys 974. Considering the proximity of the N700S TSP-1 polymorphism and the demonstration that the Ser-700 polymorphism alters the calcium binding affinity of this domain, we investigated whether altered VWF-reducing activity of Ser-700 polymorphism underlies the observed prothrombotic phenotype. Plasma samples were analyzed from a cohort of patients (N=31) who survived an acute myocardial infarction at age 45 or younger compared with age-matched controls (N=19). The relative percentage of high molecular weight VWF multimers did not differ significantly between individuals homozygous for either Ser-700 or Asn-700 (p=.20). Similarly, the relative percentage of high molecular weight multimers was 31.2% for the Ser-700 homozygous MI patients (N=21) versus 31.1% (p=.95) in the Asn-700 healthy controls (N=10). These findings were confirmed using the collagen binding assay to assess VWF activity relative to antigen concentrations. The mean ratio of CBA/Ag was .98 in the Ser-700 patients versus 1.0 in the Asn-700 control group (p=.22). Mean plasma thrombospondin-1 concentrations did not differ between the two groups. The VWF reducing capacity of TSP-1 was also assessed in vitro using recombinant TSP-1 N700S constructs containing the last EGF-like repeat, all type-3 repeats (calcium-binding domains), and C-terminal sequence. Incubation of Ser-700 peptide, Asn-700 peptide, or platelet purified full-length TSP-1 with ultra-large VWF (using either TTP plasma or recombinant ultra-large VWF) did not result in any significant reduction of VWF multimer size under static conditions. In conclusion, the association between coronary artery thrombosis and the TSP-1 Ser-700 polymorphism is not mediated through altered VWF reducing activity.
Mechanical Ventricular Assistance Rescues Endocardial Thrombomodulin Expression and May Restore Thromboresistance.
Authors: Kim Antony Y. , Favre Elizabeth , Tian Xiaojing , Paul Jonathan , Raman Jaishankar , Miller Jonathan L.
Published: April 2020

Heart failure is associated with a significant risk for thrombosis and an increased risk for thromboembolic events. Disturbance in blood flow, endothelial dysfunction or injury, and a hypercoagulable state are each present in heart failure and may contribute to the prothrombotic milieu. More recent evidence has linked heart failure with an active thrombogenic state, as demonstrated by elevated levels of thrombin-antithrombin complexes, prothrombin fragment F1+2, and fibrinopeptide A, all indices of thrombin activation. Thrombomodulin (TM), an integral membrane protein found on the endocardial endothelium (EE), is a potent thrombin inhibitor via activation of the protein C pathway. We hypothesized that the loss of thromboresistance on the ventricular endocardium, which has previously been described in cardiomyopathy, would be restored by placement of a temporary ventricular assistance device (VAD). Using left ventricular apical tissue obtained from heart failure subjects (CHF) undergoing a VAD as a bridge to heart transplant (n=4), and then from apical tissue of the same subjects at the time of explantation (post-VAD), we quantified TM protein expression using an anti-human TM antibody on EE by digital immunohistochemical analysis. TM expression levels were expressed as arbitrary units (AU) reaching signal intensity threshold per millimeter (mm) of left ventricular (LV) perimeter (AU/mm). Control TM staining of LV endocardial segments (n=2) obtained from the apex of normal hearts not used for transplantation after being excised from potential donors was 1482 and 1587 AU/mm (avg=1534 AU/mm). In CHF cardiac tissue obtained at the time of VAD placement (pre-VAD), TM protein expression on the ventricular EE of the LV apex was markedly decreased compared to control (275±67 AU/mm, p<0.01 vs. control LV, using unpaired t-test). Endocardial TM protein expression returned to near baseline levels (1182±32 AU/mm) in post-VAD cardiac samples explanted at the time of transplant from the same subjects (following VAD placement of 1–4 months in all subjects). The post-VAD TM level represented a highly significant rise over pre-VAD TM level (p<0.01 using paired t-test). Persistent staining of adjacent tissue sections in all cardiac samples for von Willebrand factor, an endothelial marker, provided clear evidence that endocardial integrity was maintained and endocardial cell loss was not responsible for the reduction of TM expression. The present study demonstrates that TM protein expression on the endocardial surface is reduced by greater than 80% in heart failure patients. Mechanical ventricular assistance restores endocardial TM levels up to 77% of control LV. The observable restoration of endocardial TM by mechanical ventricular assistance may rescue the loss of thromboresistance that is characteristic in heart failure.
Inducible Nitric Oxide Synthase Plays a Stimulatory Role in Platelet Activation.
Authors: Marjanovic Jasna A. , Stojanovic Aleksandra , Brovkovych Viktor , Skidgel Randal A. , Du Xiaoping
Published: April 2020

Platelets generate nitric oxide (NO) in response to agonist stimulation. Previous reports have shown that the endothelial nitric oxide synthase (eNOS) plays a role in agonist-stimulated platelet NO production and in platelet activation. Here we show that platelets from eNOS knockout mice (eNOS−/−) showed only partial reduction in thrombin-induced NO production compared to wild type platelets (50% reduction), indicating the presence of another NOS isoform in platelets. More importantly, we show that resting platelets express functional inducible nitric oxide synthase (iNOS), which participates in platelet activation. Compared to wild type platelets, thrombin-induced NO production was reduced by 54% in platelets isolated from iNOS knockout mice (iNOS−/−), indicating an iNOS-dependent NO production in platelets induced by thrombin. Since thrombin-induced NO production occurs during the first 3 min of thrombin stimulation, our findings provide the first evidence for a short-term regulation of iNOS activity independent of transcription regulation. In contrast, previous description of iNOS activation was primarily at the transcriptional level and required much longer time of induction. To determine the role of iNOS in platelet activation, platelets from wild type and iNOS−/− mice were stimulated with low concentrations of agonists. iNOS−/− platelets exhibited lower aggregation and secretion response compared to wild type control, indicating that iNOS plays a stimulatory role in platelet activation. We also examined the effect of iNOS inhibitors on platelet activation. Human and mouse platelets preincubated with iNOS specific inhibitors, 1400W and aminoguanidine, exhibited a dose-dependent inhibition of platelet secretion and aggregation induced by either low-dose thrombin or collagen. Furthermore, the inhibitory effect of iNOS-specific inhibitors was only shown in wild type mouse platelets, but was lacking in iNOS−/− platelets. Thus, activation of both iNOS and eNOS is important in agonist-induced NO production which stimulates platelet secretion and aggregation.
AKT Mediates Platelet Activation by Stimulating Nitric Oxide Synthesis and cGMP Elevation.
Authors: Stojanovic Aleksandra , Marjanovic Jasna A. , Brokovych Viktor , Skidgel Randal A. , Hay Nissim , Du Xiaoping
Published: April 2020

Phosphoinositide 3-kinase (PI3-K) and Akt play important roles in platelet activation. However, the downstream mechanisms for their roles are unclear. We have recently shown that nitric oxide (NO) synthase 3 (NOS3) and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3-K-mediated Akt activation plays a critical role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knockout platelets. Akt-1 knockout or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of sodium nitroprusside (SNP) or cGMP analogs. Similarly, PI3-K inhibitors diminished elevation of cGMP and also inhibited platelet secretion and the second-wave platelet aggregation, which was also partially reversed by cGMP analogs and by SNP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3-K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3-K-Akt pathway is a major upstream mechanism responsible for activating the NO-cGMP pathway. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3-K, Akt, NOS3, sGC, and cGMP-dependent protein kinase.
Inhibition of the Binding of von Willebrand Factor to the Platelet Glycoprotein Ib-IX with an Inhibitor of 14-3-3 and Glycoprotein Ib-IX Interaction.
Authors: Dai Kesheng , Bodnar Richard , Berndt Michael , Du Xiaoping
Published: April 2020

Platelet adhesion and activation are essential for thrombosis and hemostasis. In arteries and capillaries where blood flow shear rate is high, initial platelet adhesion is dependent on the binding of von Willebrand factor (VWF) to its platelet receptor, the glycoprotein (GP) Ib-IX-V complex (GPIb-IX), which consists of four subunits, GPIbα, GPIbβ, GPIX and GPV. We have shown previously that a phosphoserine-dependent intracellular signaling molecule, 14-3-3, interacts with the C-terminal SIRYSGHSL610 sequence in the cytoplasmic domain of GPIbα, and this interaction is dependent upon phosphorylation at Serine 609 of GPIbα. Here we show that a short cell-permeable myristoylated phospho-peptide corresponding to the 14-3-3 binding sequence of GPIbα inhibits VWF binding to platelets and VWF-mediated platelet adhesion. This peptide also specifically inhibits VWF-dependent platelet agglutination induced by ristocetin but has no significant effect on platelet aggregation induced by platelet agonists such as ADP and collagen. Furthermore, intravenous injection of this phospho-peptide caused significantly prolonged bleeding time in mice. Thus, 14-3-3 interaction with GPIbα plays critical roles in VWF binding function of GPIb-IX and hemostatic function of platelets. These results also suggest a new type of anti-platelet drugs that may potentially be useful in treating thrombosis.
Human Mesenchymal Stem Cells (MSC) Home at Injured Sites after Local Irradiation and Contribute To Reduce Radiation-Induced Intestinal Lesion.
Authors: Chapel Alain , Semont Alexandra , Francois Sabine , Mouiseddine Moubarack , Thierry Dominique
Published: April 2020

Some patients who undergo radiotherapy may develop side effects that can be life threatening. Tissue complications can result in functional alterations of organs caused by radiation-induced stem cell depletion. Stem cell therapy is a promising approach to improve radiotherapy-enhanced tissue complications. The multipotential of Mesenchymal Stem Cells (MSC), their easy isolation and high ex vivo expansive capacity make these cells good candidates for replenishment of the depleted stem cell compartment during radiotherapy. In this study, we make the assumption that radiation-induced normal tissue injuries might play a role in the recruitment of MSC for tissue repair. We isolated MSC from the human bone marrow (hMSC) and transplanted them via the systemic route into immunotolerent NOD/SCID mice. Using two models of radiation-induced lesion (total body irradiation: TBI, total abdominal irradiation: TAI), we have studied the link between tissue damage and hMSC implantation. Tissue alterations were studied by histological analysis. hMSC in tissues of transplant was quantified by real-time PCR assay 14 days after their injection. In unirradiated NOD/SCID mice, hMSC homed significantly in lung (0.06% of total lung cells), muscle (0.07%) and bone marrow (0.14%). Following TBI of NOD/SCID mice at a sublethal dose (3.2 Gy), we observed an increase of engraftment in brain (0.07%), heart (0.05%), liver (0.11%), muscle (0.12%) and bone marrow (0.37%) compared to unirradiated mice. TAI (8 Gy) induced a significant increase of hMSC engraftment levels in kidney (0.10%), stomach (0.12%), liver (0.44%), spleen (0.94%), and small intestine (0.17%) compared to TBI mice. Histological study show that MSC reduce radiation-induced intestinal lesion: following AI, MSC accelerate the renewal of small intestine in participating to their regenerative process. From 3 days after radiation exposure, when hMSC are injected, the integrity of small intestine is already regained. The villus length is significantly increased (1.7 fold compared with control). We also observe many proliferating zone). 15 days after irradiation, the villus length remains elevated and is significantly increased in comparison with uninjected or unirradiated group. We then demonstrated that hMSC could accelerate and favour small intestinal regeneration after radiation-induced their damages. We conclude that mainly MSC implantation takes place in radiation-injured organs of exposed areas. We demonstrate the capacity of hMSC to improve the spontaneous renewal of NOD/SCID mice small intestine. The characterization of the capacity of MSC in intestinal functional recover is under progress in our laboratory.
Intravascular Injection of Murine Mesenchymal Stem Cells in Non-Injured Neonatal Mice Results in Bone Forming Nodules within Pulmonary Vessels.
Authors: Badillo Andrea T. , Tsai Anthony Y. , Flake Alan W.
Published: April 2020

Murine mesenchymal stem cells (mMSC) are capable of differentiation into multiple cell types in vitro, and as a result have been proposed as ideal candidates for the development of cellular based therapy for tissue repair. The distribution, engraftment, and differentiation properties of these cells in vivo have yet to be conclusively defined. Reports of mMSC engraftment and differentiation are confounded by issues of cell population purity, questions of fusion, a reliance on pathologic injury models to demonstrate significant engraftment levels, and immunofluorescent based cell tracking modalities which are prone to artifact and rely on subjective interpretation. In this study, we utilize a non-injured neonatal mouse model to demonstrate the fate of culture expanded, purified mMSC derived from the bone marrow of adult GFP transgenic mice. Following intravascular injection into Swiss Webster neonatal mice on the first day of life, mMSC were tracked with fluorescent stereomicroscopy and corresponding tissue specimens were collected and processed for histology and immunoperoxidase staining for GFP. mMSC were found to concentrate within the lungs with few, isolated cells visualized outside the lungs. Histological examination confirmed the presence of GFP expressing cells within the pulmonary vasculature but occurring as aggregates of bone. These nodules were found as early as 9 days after injection and persisted to 42 days after injection. These nodules were located within the confines of the vessel wall and were surrounded by an inflammatory infiltrate. There was no evidence of tissue invasion nor did these cells demonstrate malignant features. These bony aggregrates were also demonstrated after injection into C57Bl/6 congenic controls. Our findings indicate that mMSC are filtered by the pulmonary vasculature following intravenous injection thereby limiting their capacity to reach other tissues. The large adherent phenotype of these cells makes them prone to vessel lodgment, and this lodgment results is aberrant osteogenesis. These findings have major implications for intravascular stem cell based therapies for tissue repair.
Bortezomib Plus Rituximab in Patients with Indolent Non-Hodgkin’s Lymphoma (NHL): A Phase 2 Study.
Authors: de Vos Sven , Dakhil Shaker , McLaughlin Peter , Saleh Mansoor , Belt Robert , Flowers Christopher , Knapp Mark , Hart Lowell , Patel-Donnelly Dipti , Glenn Martha , Gregory Stephanie A. , Holladay Charles , Boral Anthony , Zhang Tracy , Goy André
Published: April 2020

Background: Bortezomib (VELCADE®, Vc) a novel, first-in-class proteasome inhibitor, is being investigated in the treatment (tx) of non-Hodgkin’s lymphoma (NHL). Preclinical data with combined rituximab (R) and Vc suggest additive cytotoxic activity. This phase 2 study investigated the response rate to R plus Vc, weekly or twice-weekly, in patients (pts) with relapsed follicular (FL) or marginal zone (MZL) NHL.

Methods: Eligibility criteria included CD20+ FL or MZL with measurable disease, and Karnofsky Performance Status (KPS) ≥50% (ECOG 0–2). Pts on any prior regimen including R had to respond and show TTP of ≥4 months. Pts were randomized (1:1) to Vc 1.3mg/m2 twice-weekly on days 1, 4, 8, and 11 of a 21-day cycle (Arm A) or Vc 1.6mg/m2 weekly on days 1, 8, 15, and 22 of a 35-day cycle (Arm B) for up to 15 weeks (5 and 3 cycles in arms A and B, respectively). Starting from day 1, R 375mg/m2 was administered weekly for 4 weeks. Response was evaluated by International Workshop Criteria.

Results: 81 pts were enrolled between May 2004 and August 2005. At data reporting for this abstract, 46 (23 each arm) had received study drug, and 42 (20 Arm A, 22 Arm B) were evaluable for response. Of these 42 pts, 83% had FL and 17% had MZL; 62% were male; 55% were aged >60 years (range 33–80); 93% had KPS ≥90%; 26% had lactic dehydrogenase level >ULN. 71% of pts received prior R (alone or in combination), and 79% of pts received prior CHOP, RCHOP, CVP or RCVP. Of 46 treated pts, 9 (39%) in Arm A and 16 (70%) in Arm B completed all planned cycles. Median Vc dose received was 15.9mg/m2 (61% of max. expected) in Arm A, and 19.0mg/m2 (99% of max. expected) in Arm B. Overall response rate was 50% (1 CR + 9 PR) in Arm A, and 45% (2 CR + 8 PR) in Arm B. 12 pts had PD (6 each arm) and 1 pt died (Arm A). Time to event analyses, including TTP and duration of response, are pending further follow-up. Tx was well tolerated in both arms; grade ≥3 AEs were seen in 10 pts in Arm A and 5 pts in Arm B. The most common grade ≥3 AEs were gastrointestinal toxicities, neutropenia, thrombocytopenia and peripheral neuropathy, but no grade ≥3 thrombocytopenia or neutropenia were observed in Arm B. SAEs were seen in 7 pts in Arm A versus 2 pts in Arm B.

Conclusions: Weekly and twice weekly Vc plus R are active and well tolerated treatments for relapsed/refractory CD20+ indolent NHL. The more convenient weekly regimen appears to offer a similar response rate with less toxicity. Final efficacy and safety data for both regimens will be presented.
Outcome of Bone Marrow Transplantation for Myelofibrosis.
Authors: Ballen Karen , Sobocinski Kathleen A. , Zhang Mei-Jie , Arora Mukta , Horowitz Mary M. , Giralt Sergio ,   the Chronic Leukemia Working Committee of the Center for International Blood Marrow Transplant Research
Published: April 2020

Myelofibrosis is a myeloproliferative disorder characterized by splenomegaly, bone marrow fibrosis and immature white and red blood cells. Allogeneic transplantation is the only curative therapy. In this study, we analyzed the outcomes of 320 patients receiving allogeneic hematopoietic stem cell transplants for myelofibrosis between 1989 and 2002, using the databases of the Center for International Bone Marrow Transplant Research (CIBMTR), a research affiliation of the International Bone Marrow Transplant Registry (IBMTR) and the National Marrow Donor Program (NMDP). This is the largest report of transplantation for myelofibrosis. Patients received a variety of conditioning and graft versus host disease prophylaxis regimens. Most patients received ablative conditioning with either TBI (n=117) or Busulfan (n=150) and cyclophosphamide. Bone marrow was the graft source in 208 patients. 170 transplants were from an HLA-identical sibling donor, 117 from an unrelated donor (MUD), and 33 from an alternative related donor. Median ages at transplant were 45 (<1–73), 47 (1–69) and 40 (<1–65) years, respectively. Median follow up times for survivors were 41 (3–136), 48 (4–124) and 32 (7–118) months, respectively. Both early and long-term survival rates were higher after HLA-identical sibling transplantation. 100-day mortality was 22% after sibling transplants, 42% after MUD transplants, and 27% after alternative family donor transplants. Corresponding 5 year overall survival rates were 39%, 31% and 31%. In multivariate analysis of 215 adult recipients of myeloablative transplants, having an HLA-identical sibling donor, Karnofsky performance score greater than or equal to 90%, younger age, more recent date of transplantation, and absence of blasts in peripheral blood prior to transplantation correlated with better survival. Among 18 patients with all of these favorable factors, the five-year probability of survival was 81%. In conclusion, 1) allogeneic transplantation cures approximately 1/3 of patients with myelofibrosis; 2) young patients with HLA-matched sibling donors have superior survival; 3) results have improved over the last decade. Future research directions will focus on the use of nonmyeloablative conditioning regimens for myelofibrosis.