bluuebunny/crossref_metadata_2025 huggingface dataset:
The Src-Abl Kinase Inhibitor Dasatinib (BMS-354825) Shows Anti-Proliferative and Anti-Apoptotic Effects in Chronic Lymphocytic Leukemia (CLL) Cells In Vitro.
Authors: Veldurthy Aditya , Patz Michaela , Pallasch Christian P. , Wendtner Clemens M. , Hallek Michael , Krause Gunter F.
Published: October 2019

Introduction: Dasatinib is an ATP competitive, dual-specific Src-Abl kinase inhibitor used for the treatment of imatinib-resistant Bcr-Abl-positive leukemias. Originally it was developed from the aminothiazole scaffold as an inhibitor of Src family kinases (SFKs). Since the aberrant expression and activity of the SFK Lyn seems to enhance the survival of CLL cells, dasatinib is currently tested in clinical studies for CLL. In order to explore the anti-leukemic potential of dasatinib we tested its effects on freshly isolated CLL cells and on CLL cell lines.

Methods and Results: In freshly isolated CLL cells, dasatinib showed a dose and time dependent reduction of global tyrosine phosphorylation which was paralleled by a decreased phosphorylation of the activating tyrosine residue of SFKs. The comparison of the inhibitory effect of several protein tyrosine kinase inhibitors on overall tyrosine phosphorylation in cellular lysates of freshly isolated CLL cells showed that 0.1 μM dasatinib had a more pronounced effect than 5 μM of nilotinib, or 10 μM of PP1 or imatinib. In the CLL cell lines, Mec1 and JVM-3, 0.1 μM of dasatinib appeared to interfere with survival signaling, since decreased levels of the activated phosphorylated forms of Akt, Erk1/2 and p38 were observed after exposure to dasatinib for 2 hours. In these cell lines, dasatinib treatment increased p53 protein levels and induced PARP cleavage and caspase activity. Pro-apoptotic effects of dasatinib were observed by an increase of annexin V-positive cells and a decrease of metabolic activity measured as XTT reduction, alone and in combination with fludarabine. Among six patient samples, two clones were particularly sensitive to dasatinib and fludarabine-resistant. While in these two samples the apoptosis induction by 5 μM dasatinib surpassed that by 5 μM fludarabine, the same dose of the two drugs led to similar apoptosis induction in the fludarabine-sensitive samples. Dasatinib treatment sensitized CLL cells for fludarabine-induced apoptosis. This enhancement of apoptosis induction was more pronounced in fludarabine-resistant patient samples and JVM-3 cells than in Mec1 and fludarabine-sensitive CLL cells.

Conclusion: The Src-Abl kinase inhibitor dasatinib shows potent inhibitory effects on the survival of CLL cells in vitro.
Marked Sensitivity of Chronic Lymphocytic Leukemia (CLL) to Apoptosis Induced by BCL2 Antagonist ABT-737.
Authors: Vogler Meike , Majid Aneela , Walewska Renata , Dyer Martin J.S. , Cohen Gerald M.
Published: October 2019

Most cases of CLL express BCL2 constitutively at levels comparable to those seen in follicular lymphoma with t(14;18)(q32;q21) and BCL2 is considered to play a pivotal role in the suppression of apoptosis in this disease. The BCL2 antagonist ABT-263 has recently entered phase I/IIa clinical trials for potential treatment of CLL. ABT-263 and ABT-737 are rationally designed BH3 mimetics that are thought to act by displacing pro-apoptotic BIM from BCL2 and thus activating the apoptotic cascade. While being resistant towards some commonly used apoptotic stimuli like etoposide or TRAIL, we found that all primary CLL cells are highly and rapidly sensitive to ABT-737, with an average EC50 in 60 patients of only 6.7 nM and after only 4 h of treatment. ABT-737 induced BAX/BAK oligomerization followed by rapid and extensive cytochrome c release, caspase activation and chromatin condensation. Incubating unfractionated samples in a whole blood assay did not significantly alter sensitivity. Notably, sensitivity of CLL cells to ABT-737 was independent of the patient’s stage of disease, p53 mutation or function, cytogenetics, or IGVH mutational status. Furthermore, ABT-737-induced apoptosis was observed irrespective of previous treatment or in vivo resistance to fludarabine, suggesting that ABT-737 might induce apoptosis even in patients with highly progressive and otherwise resistant disease. We also analyzed expression levels of BCL2 and MCL1 in CLL patients by western blot, since MCL1 is considered to be the most important factor for resistance towards ABT-737. Interestingly, sensitivity to ABT-737 was independent of BCL2 and MCL1 levels in CLL, indicating that expression of MCL1 was not sufficient to render CLL resistant to ABT-737. In addition, we further induced expression of MCL1 by either IL4 or interferon γ treatment, which resulted only in a minor delay of apoptosis that was easily overcome by treatment with slightly higher concentrations of ABT-737. Hence, our data suggest that in contrast to previous reports obtained in a variety of cell lines, MCL1 upregulation is not sufficient to induce resistance to ABT-737 in primary CLL cells. Next, we investigated whether exposure to ABT-737 resulted in a displacement of BH3-only proteins from BCL2. Surprisingly, we could not detect a significant displacement of BIM from BCL2. In contrast, we found that the multidomain proapoptotic protein BAK is directly bound to BCL2 in CLL and this interaction is abrogated by ABT-737, indicating that ABT-737 can directly displace BAK but not BIM from BCL2. Taken together, our results indicate that targeting BCL2 with ABT-263 or ABT-737 may be a highly promising strategy for treatment of CLL.
Phase I Clinical Trial of a Novel, Long-Acting Antithrombotic Drug: A Human Monoclonal Antibody Partially Inhibiting FVIII Activity.
Authors: Verhamme Peter , Pakola Steve , Jacquemin Marc , Saint-Remy Jean Marie , Stassen Jean Marie , Collen Desire , Riisbro Rikke , Jensen Thomas , Glazer Steven
Published: October 2019

Current treatments for preventing thrombotic disease are still inconvenient and associated with a high risk of bleeding. Improved anticoagulant agents are therefore required. TB-402 is a monoclonal antibody (fully human IgG4-antibody) that targets factor VIII (FVIII) and represents a novel type of anticoagulant agent. In vitro, TB-402 only partially inhibits human FVIII even when TB-402 is in excess over FVIII. Preclinical studies confirmed this plateau inhibition and also established the antithrombotic efficacy of TB-402 (Jacquemin M, et al. J Thromb Haemost.2006; 4:1047). Plateau inhibition even in setting of excess TB-402 may allow for improved safety with decreased risk of overdose and decreased need for monitoring. The long half-life of the antibody creates the possibility for a once a month administration. TB-402 may therefore represent a safer and more convenient agent than other available anticoagulants. A Phase I study has completed enrolment of 56 healthy male volunteers. This randomised, double-blind, placebo-controlled, single dose, dose escalation study evaluated the safety and the pharmacokinetic/pharmacodynamic profile of TB-402. Volunteers were treated with a single intravenous administration of placebo or TB-402 at doses of 0.015, 0.1, 0.5, 2.5, 12.5, 37.5, 188, 620 or 1,860 μg/kg. All doses were evaluated in 18–45 year old volunteers, with the highest dose also evaluated in an older age, 55–75 years, cohort. Study drug has been well tolerated in both the young and the older age cohorts, with no safety issues observed. A plateau effect in terms of FVIII level inhibition has been observed. At plateau, FVIII levels were generally decreased by 1/3 to 2/3 from baseline. aPTT prolongation (generally 1.1–1.2 times baseline) was also observed whereas the Prothrombin time (PT) was not modified. aPTT prolongations at doses of ≥ 620 μg/kg were generally maintained for at least 4 weeks. Long half-life of drug and associated anticoagulant effect are supported by the finding of consistent and prolonged effect on aPTT. In conclusion, this study demonstrates that in healthy young and older volunteers a single administration of TB-402 results in a prolonged anticoagulation effect without the risk of overdosing or spontaneous hemorrhage. TB-402 will next be evaluated in patients at risk for venous thromboembolism.
High Dose Methylprednisolone and Rituximab Is an Effective Therapy in Advanced Refractory Chronic Lymphocytic Leukaemia Resistant to Fludarabine Therapy.
Authors: Dungarwalla Moez , Kanagasabapathy Pamala , Kulkarni Samar , Evans Steve O. , Riley Unell , Morgan Gareth J. , Catovsky Daniel , Dearden Claire E. , Matutes Estella
Published: October 2019

The management of refractory advanced chronic lymphocytic leukaemia (CLL) continues to pose a considerable challenge to the clinician. CLL patients who are refractory or become resistant to fludarabine containing regimens currently have a poor prognosis. High dose methylprednisolone (HDMP) has been shown to be an effective therapeutic option in patients who are resistant to fludarabine particularly those with bulky lymphadenopathy or refractory cytopenias who are unsuitable for alemtuzumab therapy. We have used HDMP in combination with the anti-CD20 monoclonal antibody, Rituximab, to treat 14 patients with advanced refractory CLL. Eight of fourteen patients were male. The median age was 62.5 years (range 30–71). Twelve patients had bulky lymphadenopathy with nodal masses greater than 5 centimetres in diameter. Nine patients had Binet stage C and the remainder were stage B. Autoimmune manifestations were present in five of the patients, and in one of them fludarabine therapy was contraindicated due to active autoimmune haemolysis. FISH analysis was performed to detect 17p, 11q and 13q deletions and showed 11q23 deletions in five patients. The overall response rate was 93% with a median progression free survival (PFS) of seven months. Two patients achieved a CR, another a nodular PR and 10 patients had a PR. Responses were seen in all 4 patients who had not responded to alemtuzumab as a single agent. Only one patient failed to respond. The median survival was 20 months. All five patients with 11q23 deletions responded, and the mean progression free survival in this subgroup was 10.5 months. These results compare extremely favourably with our own previous study (Thornton et al, 1998) that used HDMP alone in the management of advanced/refractory CLL where only 43% of patients responded and no patients achieved a CR. We have demonstrated that the combination of HDMP with rituximab is more effective than HDMP alone (p<0.01) in advanced refractory CLL. Although HDMP in combination with rituximab causes little or no myelosuppression, 6/14 patients (43%) developed opportunistic fungal or viral infections and this included fungal mould infections, fungal yeast infections and opportunistic viral infections. Three patients died of pulmonary fungal infections, although 2 of these patients had been retreated with alternative regimens. While the immunosuppressive effects of high dose steroids are well recognised, it appears that the addition of rituximab has made these patients more prone to opportunistic infections. In summary, HDMP in combination with rituximab is an effective rescue regime in purine analogue refractory CLL particularly in patients with bulky lymphadenopathy who are unsuitable for alemtuzumab, and in patients with 11q23 deletions.
The Influence of CYP2C9 and VKORC1 1173C/T Genotype on Reversal of Anticoagulation (INR) Following Phytonadione Administration.
Authors: Limdi Nita A. , Acton Ronald T. , Beasely Timothy M. , Adler Brian
Published: October 2019

Context: Excessive anticoagulation increases the risk of hemorrhagic complications associated with warfarin therapy. Oral, subcutaneous or parenteral phytonadione is used to reverse both symptomatic and asymptomatic excessive anticoagulation. Multiple reports have addressed the influence of phytonadione dose and route of administration on time course of anticoagulation reversal.

Objective: To determine the influence of CYP2C9 and VKORC1 1173C/T on anticoagulation reversal following phytonadione administration.

Design and Setting: Prospective cohort study of 578 patients newly initiated on warfarin (target INR: 2–3).

Main Outcome measures: INR difference (pre-treatment INR- post-treatment INR) produced by phytonadione after accounting for age, gender, race, CYP2C9 and VKORC1-1173C/T genotype, warfarin dose, comorbidity, concomitant therapy with CYP2C9 inhibitors, phytonadione dose and time (hours) between pre-treatment and post-treatment INR measurement.

Results: During follow-up (August 2003-April 2007) various treatment modalities were used to reverse anticoagulation. Phytonadione monotherapy was administered 28 times to reverse anticoagulation in 24 patients (15 women, 14 African American), mean age 66.4±16.9 yrs. The mean phytonadione dose was 4.1mg (±2.7) and the mean duration between pre-treatment and post-treatment INR measurements was 53.4 (±26.7) hours. Administration of phytonadione produced a median INR decline of 5.5 (range 0.2–10.9). Because 3 of the 24 patients were measured more than one time, a linear mixed model using SAS PROC MIXED was performed in order to account for the dependency in the observations. The mixed model statistically adjusted for age, gender, race, CYP2C9 genotype, warfarin dose, comorbidity, concomitant therapy with CYP2C9 inhibitors, phytonadione dose and time (hours) between pre-treatment and post-treatment INR measurement. The extent of anticoagulation reversal (INR decline in response to phytonadione administration) was significantly smaller among patients with variant VKORC1-1173C/T genotype compared to those with wild-type genotype (p=0.0027). The INR decline was also smaller among patients with variant CYP2C9 genotype, although this finding was of marginal statistical significance (p=0.069).

Conclusion: The efficacy of phytonadione in reversing anticoagulant effect of warfarin is significantly influenced by the individuals VKORC1-1173C/T genotype.
Immunogenicity of Human Factor VIII in Rhesus and Cynomolgus Monkeys.
Authors: Skupsky Jonathan , Lozier Jay N. , Donahue Robert E. , Metzger Mark N. , Azimzadeh Agnes M. , Pierson Richard N. , Scott David W.
Published: October 2019

Up to 30% of hemophilia A patients form inhibitory antibodies to therapeutic doses of human factor VIII (fVIII), thus rendering therapy ineffective. Advances in identifying the nature of the fVIII mutations (inversion, stop codons, etc), HLA linkage and other factors have suggested these as important predictors of inhibitor responsiveness in patients. The ability to predict which patients may respond with inhibitors would be important in order to identify suitable patients for tolerance therapies prior to exposure to fVIII and the development of high inhibitor titers. Animal models to evaluate the immunogenicity of fVIII would also be useful in this regard. We and others have previously shown that mice with normal hemostasis produce antibodies to fVIII in response to therapeutic doses of human fVIII given intravenously (i.v.), a normally tolerogenic route (Lin, Soukhareva, and Scott, ASH 2004) or in an adenovirus vector (Brown et al. JTH 2004; Lozier and Zhang, ASH 2005). To establish whether hemostatically normal non-human primates would respond to human fVIII and provide a model for future tolerogenic therapy, we immunized rhesus and cynomolgus monkeys with fVIII using a variety of routes. In the case of rhesus monkeys, fVIII was administered via three routes: i.v. in PBS, intramuscularly (i.m.) in alum, or i.m. in a fVIII-expressing adenoviral vector. All three routes led to significant titers as measured by ELISA, and all six animals responded to i.v. boosting with fVIII in PBS. Cynomolgus macaques (3) that received multiple doses of fVIII i.v. also produced antibody to fVIII, albeit of lower titer. Importantly, T cell proliferation to human fVIII in vitro could be elicited from cynomolgus peripheral blood cells. These results set the stage for future tolerogenic therapies in both naïve and fVIII primed non-human primates.
Dendritic Cells as a Novel Target for Erythropoietin: Studies in a Human System.
Authors: Sagiv Sari Prutchi , Lifshitz Lilach , Orkin Ruth , Newmann Drorit , Mittelman Moshe
Published: October 2019

The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system have been described for some time; however, the mechanism of action by which EPO affects lymphocyte number and function has yet to be elucidated. Our search for possible mechanisms by which EPO affects these parameters led us to the novel discovery that EPO receptors (EPO-R) are expressed in dendritic cells (DCs), the most potent antigen presenting cells and most efficient T cell primers. Furthermore, we show that EPO has direct effects on the phenotype and function of human DCs. When added in vitro, EPO increased the percentage of peripheral blood originated DCs that express the co-stimulatory molecules CD80, CD86 and CD40. We also show that EPO up-regulates the level of expression of these molecules, as well as that of HLA-DR in monocyte-derived DCs. When added to immature DCs, EPO alone can induce their maturation. Furthermore, we demonstrate that EPO enhances DC function, as revealed by increased antigen uptake, secretion of interleukin (IL)-12 and stimulatory function in allogeneic mononuclear cell proliferation. We propose that DCs may represent a missing link which might explain previously observed immunomodulatory actions of EPO. Hence, our findings are of crucial importance and may open new clinical avenues for EPO in the optimization of ex-vivo DC-based vaccines, or when administered to patients in order to enhance immune system responses.
PHF23: A Novel Erythropoietin-Induced Gene Associated with AML and MDS.
Authors: Hodges Vivien M. , Rainey Susan , Smyth Victoria , Mills Ken , Lappin Terence R.J. , Maxwell A. Peter
Published: October 2019

Chromosomal translocations in a number of hematopoietic malignancies, including AML and MDS, result in the formation of NUP98 gene chimeras. Recently, fusion of NUP98 with a novel gene, PHF23 or plant homeodomain (PHD) finger 23, was reported in a cryptic translocation in a patient with AML (Reader et al., 2007). PHF23 is one of three known NUP98 fusion partners, associated with AML, which contain PHD finger motifs. In this study, we report a more detailed characterization of PHF23, which we originally identified by differential display PCR as an erythropoietin-induced gene in a murine model of terminal erythroid differentiation. In silico sequence analysis revealed a five exon gene containing a nuclear localization signal (NLS) as well as the C-terminal PHD motif. PHF23 mapped to chromosome 17p13.1, <450 kb from the TP53 gene, in a region frequently disrupted in cancer. Quantitative PCR analysis indicated Phf23 expression is not restricted to hematopoietic cells but demonstrates widespread tissue distribution, with increased expression levels in more proliferative tissues (∼6 fold increase in gene expression in the testis over mRNA levels in the liver). A ubiquitously expressed Phf23 splice variant (with mRNA concentrations ∼30–45% lower than full-length Phf23 gene expression levels) was generated using an alternative 3′ splice site within exon four, which exactly corresponded to the chr17 translocation breakpoint in the AML patient described. PHF23 is evolutionarily conserved, particularly within the PHD finger domain (>76% sequence identity between human and zebrafish PHD motifs), and is significantly related to another potentially oncogenic protein, PHF13/SPOC-1, associated with ovarian cancer. PHD motifs act as zinc fingers and the expanding family of PHD-containing proteins are implicated in chromatin regulation. Cellular localization studies using fluorescently tagged PHF23 indicated that both the full-length and spliced isoforms are located exclusively within the nucleus. We predicted that PHF23 has a similar structure to PHD motifs of known histone effector proteins, which influence transcription through recognition of specific epigenetic marks. This was demonstrated by immunoprecipitation and histone peptide binding assays showing that PHF23 associated with histone H3, primarily di- and tri-methylated H3K4, which are characteristic marks of active transcription. Microarray analysis of PHF23 mRNA expression levels in AML and MDS patient samples indicated no defined association with AML, however a positive correlation was demonstrated with a subset of MDS patients exhibiting erythroid dysplasia. This study suggests that PHF23, a novel histone effector protein, may play a role in aberrant transcription during defective hematopoiesis.
siRNA Mediated Knockdown of the Chimeric Transcription Factor E2A-PBX1 Identifies Potential Target Genes.
Authors: Chen Chun-Liang , Connell Harkness , Racoma Ira , Regula Brandi , Foley Katherine , Townsend Jessica , Ross Mary Elizabeth
Published: October 2019

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. Several chromosomal translocations have prognostic significance and are used to classify patients for risk-directed therapy. The translocation t(1;19) which results in the fusion of TCF3 (E2A) and PBX1 genes occurs in 5% of pediatric ALL. E2A-PBX1 has been shown previously to have transcriptional activity. However, very little is known about what genes have altered expression in the presence of E2A-PBX1. To explore this question, we assessed genome wide gene expression after siRNA mediated knock down of E2A-PBX1. An E2A-PBX1 containing cell line (697) was transfected with E2A-PBX1 specific siRNA utilizing an Amaxa nucleofector2. Biologic replicates were performed by transfection of independent cultures. 70–80% reduction of E2A-PBX1 at mRNA and protein levels in 697 cells was reproducibly achieved using nucleofection and a combination of E2A-PBX1 specific siRNAs. Genome wide gene expression was assessed by Affymetrix U133 2.0 Plus arrays. Hybridizations were prepared and performed according to current Affymetrix protocols in the Functional Genomics Core at the Research Institute. Microarray data was normalized with RMA. Differentially expressed genes were selected using Significance Analysis of Microarrays (SAM). 78 probe sets demonstrated change in expression in E2A-PBX1 siRNA mediated knockdown relative to both mock transfection and nontargeting siRNA controls at a FDR of ≤ 5%. The 78 probe sets represent 49 known genes and 8 ESTs. Genes of specific interest upregulated by E2A-PBX1 include WNT-16, ANKS1B(EB-1), FAT, and RORB. Other investigators have previously cloned these genes by representational differential display from E2A-PBX1 expressing cell lines. We confirmed knockdown of these messages by E2A-PBX1 siRNA using qRT-PCR. Gene expression was calculated using the ΔΔCt method and normalization to ABL. Additionally, these genes are part of the E2A-PBX1 gene expression profile derived from primary pediatric leukemia samples previously published. [Ross, Blood 2003] An additional 34 candidate genes were further verified using qRT-PCR. The direction of change in gene expression correlated with microarray results in 30/34 genes evaluated. Other classes of genes upregulated by E2A-PBX1: kinases (FGFR2, MAP3K1), phosphtases (PPPIR14C), transcription factors (FOSL2, IRX2, EBF3, BMI1, BCL6), cell cycle-related genes (FBXW7, ETV5), Ras and Rho family genes (RAB8B). While B cell surface antigen expression (HLA-DRA, CD22) was decreased by E2A-PBX1. We have utilized siRNA to E2A-PBX1 to identify potential target genes of E2A-PBX1. This data suggests E2A-PBX1 can alter expression of a variety of classes of genes. Some of these genes may be potential targets for molecularly targeted therapy.
Elevated Fibrinogen Predicts Atherosclerosis.
Authors: Green David , Foiles Nancy , Chan Cheeling , Schreiner Pamela J. , Jacobs David , Liu Kiang
Published: October 2019

Elevated levels of hemostatic factors are observed in patients with atherosclerosis, but whether they promote plaque formation or are a consequence of the disease is uncertain. To examine this issue, we used data from a large biracial cohort of young adults (Coronary Artery Risk Development in Young Adults [CARDIA]) followed up for 13 years, to examine the relationships of hemostatic factors - fibrinogen, factors VII and VIII, and von Willebrand factor (vWF) - with coronary artery calcium (CAC) and carotid intimal-medial thickness (IMT). Complete data were available on 1382 participants, whose mean age was 32 years at enrollment. The age, race, and gender-adjusted prevalence of CAC for increasing quartiles of fibrinogen levels was: 14.0%, 15.0%, 19.6%, and 28.4% (p <0.001 for trend). After further adjustment for BMI, smoking, systolic BP, and total cholesterol, the prevalence of CAC for increasing quartiles of fibrinogen was 15.5%, 16.0%, 19.0%, and 26.4% (p <0.001 for trend). Similar trends were observed for IMT (age, race, and gender-adjusted, p<0.001; multivariable adjusted, p=0.022). When race and gender subgroups were further analyzed, the prevalence of CAC was associated with fibrinogen levels in women and white men after age adjustment, and in women on multivariable analysis. IMT scores adjusted for age were associated with elevated fibrinogen levels in all except black men, and in black women after multivariable adjustment (p=0.003). While the prevalence of CAC was not associated with increasing quartiles of FVII, FVIII, or vWF, IMT scores were associated with elevated FVII on multivariable analysis in white women (p=0.006) and with vWF antigen in white men on age-adjusted (p=0.004) and multivariable analysis (p=0.013). There were no significant associations of hemostatic factors with either the prevalence of CAC or IMT in black men. Participants were categorized as to whether they had 0, 1, or more than 1 hemostatic factors in the highest quartile. After adjustment for age, race, and gender, hemostatic group classification was associated linearly with the prevalence of CAC (p<0.001 for trend) and IMT score (p=0.01 for trend). In conclusion, the main finding from this study is that elevated levels of fibrinogen in persons aged 25 to 37 are associated with the later appearance of subclinical markers of cardiovascular disease. These associations were observed in whites and black women, but not black men. We suggest that atherosclerosis became established during the 13 year observation period, and that increased fibrinogen may have been a contributing factor or a marker for disease development.
Impact of Inherited Thrombophilia on Symptomatic Venous Thrombo-Embolism (VTE) in Children: A Systematic Review & Meta-Analysis of 37 Studies Including 2470 Pediatric Patients.
Authors: Young Guy , Friedrichs Frauke , Chan Anthony , Kenet Gili , Simioni Paolo , van Ommen Helen , Krumpel Anne , Bonduel Marianne , Goldenberg Neil , Male Christophe , Albisetti Manuela , Manco-Johnson Marilyn , Brandao Leo , Nowak-Gottl Ulrike
Published: October 2019

Background: Inherited thrombophilia (IT) has been described as a risk factor for venous thromboembolism (VTE) in children. So far the majority of studies performed in the field were either retrospective or prospective on small numbers of patients. Thus, the results are contradictory or inconclusive mainly due to lack of statistical power. The aim of this study was to better estimate the impact of IT on early VTE onset and recurrence in children as a prerequisite to develop primary and secondary treatment options.

Methods: A systematic search of publications listed in the electronic databases (Pubmed, Medline, EMBASE, Web of Science, The Cochrane Library) up to August 2007 using key words in combination both as MeSH terms and text words, was conducted. Citations were screened by two independent group members and those meeting the inclusion criteria were retained. Articles were included if published after 1990, when pediatric VTE was started to be systematically investigated.

Findings: Twenty case-control and 17 cohort studies from 13 countries met the inclusion criteria. In these studies > 70% of patients had at least one clinical risk factor. The summary odds ratios (OR) and 95% confidence intervals (CI) of included studies under a fixed-effects and random-effects model showed statistically significant associations between the IT traits investigated and VTE onset (table). For the rare event of VTE recurrence, 1227 patients (eight studies) were evaluated: at the present state due to high heterogeneity, a trend towards association with recurrent VTE was found for ≥2 IT traits in the fixed-effects model (0R/CI: 2.8/1.6–4.8).

Interpretation: The present meta-analysis gives evidence that the detection of inherited thrombophilia is clincially meaningful in children with VTE and underlines the importance of a pediatric thrombophilia screening program.

Summary of Data Risk Factors OR/CI:fixed model OR/CI:random model patients/controls 2470/4119 N/A FV G1691A 3.5/2.9–4.2 3.2/2.3–4.4 FII G20210A 2.2/1.5–3.3 2.2/1.5–3.4 Protein C defiiciency 9.8/5.9–16 9.9/6.1–16.1 Protein S deficiency 7.1/3.9–13.2 6.8/3.7–12.7 Antithrombin deficiency 7.9/3.8–16.6 7.3/3.4–15.3 Lipoprotein(a) 4.4/3,2–5.9 4/2.4–6.6 ≥ 2 risk factors 12.6/7.3–21.8 11.6/6.2–20.2
An Ex Vivo Perfusion Model To Study the Treatment of Thrombotic Microangiopathy during Pig-to-Human Xenogenic Kidney Transplantation.
Authors: Ramackers Wolf , Friedrich Lars , Schüttler Wolfgang , Bergmann Sabine , Ganser Arnold , Winkler Michael , Tiede Andreas
Published: October 2019

Early rejection of xenogenic organs is associated with thrombotic microangiopathy and changes of coagulation resembling disseminated intravascular coagulation (DIC). Here, we used an ex vivo perfusion circuit as a model of pig-to-human kidney transplantation to study the nature and treatment of this pathology. Porcine kidneys were obtained following in situ cold perfusion with HTK organ preservation solution and immediately connected to a perfusion circuit containing porcine (“autologous”) or human (“xenogenic”) AB blood supplemented with complement component C1 inhibitor (1 U/ml) and heparin (1 U/ml). Perfusion of porcine kidneys with autologous blood was feasible for >240 min in all experiments. In contrast, perfusion of porcine kidneys with xenogenic human blood was limited by a dramatic increase of flow resistance after 30 to 240 min. Increased concentrations of C3a as a marker for complement activation were associated with early perfusion failure. In addition, a dramatic increase of thrombin-antithrombin complex (TAT) and D Dimer (DD) was observed together with consumption of platelets, fibrinogen and antithrombin (AT). Histological examination demonstrated extensive thrombotic microangiopathy. Supplementation recombinant human activated protein C (rhAPC, 300 ug/l*h, n=3) or recombinant human antithrombin (rhAT, 3 U/ml, n=3) abolished the increase of flow resistance and allowed for a xenogenic kidney survival of >240 min in all experiments. Increase of DD was abolished and the consumption of fibrinogen was abrogated by both treatments as compared to control, whereas the increase of TAT was abolished only by rhAPC. Histological examination revealed no evidence of thrombotic microangiopathy for both treatments as compared to control. In conclusion, the perfusion model introduced here is a suitable tool for studying coagulopathy during early rejection in xenotransplantation. Thrombotic microangiopathy and DIC-like activation of coagulation is associated with an increased flow resistance and failure of perfusion in this model. Pharmacological intervention such as the supplementation of rhAPC or rhAT can be studied using this model and has been shown to prevent coagulopathy and thrombotic microangiopathy.
Correlation between FOXP3 Gene Polymorphisms in Donors, and the Severity of Acute Graft-Versus-Host Disease in Patients after Related Allogeneic Stem Cell Transplantation.
Authors: Perfecto-Avalos Yocanxochitl , Escoboza Jose R. Borbolla , Villela-Martiez Luis M. , Scott Sean P. , Vela-Ojeda Jorge , Gonzalez-Ramella Oscar , Baltazar-Arellano Severiano , Lopez-Hernandez Manuel A.
Published: October 2019

INTRODUCTION: Acute Graft-versus-host disease (aGVHD) is a major complication of allogeneic stem cell transplantation (alloSCT). Risk factors include patient age, sex matching, CMV status and degree of match. Regulatory T cells are critical for immune tolerance processes such as aGVHD, and express the transcription factor FOXP3, a member of the forkhead/winged-helix family, identified as a key regulatory gene required for the development and activity of these cells. It has been suggested that genetic expression of FOXP3 is inversely correlated with the severity of the GVHD. We studied donor’s DNA looking for 5 polymorphisms on the promoter region of the FOXP3 gene, and we tried to correlate them with presence and degree of aGVHD.

PATIENTS AND METHODS: We studied donors of stem cells for allogeneic stem cell transplants. We looked for the presence of the following polymorphisms by PCR: POL01 −5906 T/A rs2869211; POL03 −3279 A/C rs3761548 ; POL04 −2383 C/T rs3761549 ; POL05 −1383 C/T rs2232364 ; POL06 −924 A/G rs2232365.

RESULTS: Our sample consisted of 31 donors, all siblings. In them we found only 2 of the 5 FOXP3 polymorphisms, either as homozygous or heterozygous. These polymorphisms were found in 15/31 donors, with 12 being homozygous (38.7%), and 3 heterozygous (9.7%). These genes polymorphisms were POL03 y POL06. The most observed polymorphism was POL-06 with 9 cases, while POL-03 was found in 6 donors. Only sex difference and CMV status had an elevated hazard ratio for developing GVHD (HR=1.18, CI95%: 0.18 to 7.64; p=0.85) and (HR=3.0, CI95%: 0.07 to 126; p=0.46) respectively. We found no statistically significant difference in the incidence of GVHD between patients who had received cells from donor with or without a FOXP3 polymorphism (p=0.87). When we analyzed the risk of presenting GVHD, results suggest that having one of the 2 positive polymorphisms of the FOXP3 gene could have a protective effect for the patient. For POL03 HR=0.87, CI95%:0.18 to 4.14; p=0.86, and for POL06, HR=1, CI95%: 0.37 to 2.64, p=0.67.

CONCLUSIONS: Even though our sample is still small to make conclusive remarks, we believe that our results point toward some level of “protection” from acute GVHD in patients receiving cells from donors expressing POL03 and POL06. It is also worthwhile mentioning the relatively high frequency of these polymorphisms, as well as the absence of the other 3.
Neutralization of Macrophage Migration Inhibitory Factor (MIF) Reduces GVHD but Preserves GVL.
Authors: Miklos Sandra , Mueller Gunnar , Lindner Petra , Chang Ya-Yi , Holler Ernst , Bucala Richard , Hildebrandt Gerhard C.
Published: October 2019

Acute graft versus host disease (aGVHD) is the major complication after allogeneic (allo) stem cell transplantation (SCT). The pathophysiology of aGVHD involves injury to host tissues by both inflammatory cytokines and donor-derived cellular effectors. Macrophage migration inhibitory factor (MIF) is produced by various cell types and has a broad range of proinflammatory properties including the induction of T cell activation and inflammatory cytokine secretion. In this study, we tested the hypothesis, that MIF contributes to systemic inflammation and mortality after allo-SCT by using a well established murine model (B6→B6D2F1). Lethally (1300cGy) irradiated B6D2F1 (H-2bxd) mice received SCT either from syngeneic (syn) (B6D2F1) or allo (B6; H2-b) donors. One half of animals after allo SCT was treated with polyclonal antibodies against mouse MIF from day 0 until day 14, whereas syn animals and the other half of allo recipients were treated with control IgG. The severity of GVHD was assessed after SCT by survival and a clinical scoring system that incorporates changes in weight loss, fur texture, skin integrity, mobility and posture. As expected, syn-SCT recipients all survived and were indistinguishable from naïve, untransplanted controls, whereas animals receiving allo-SCT plus control IgG developed significant aGVHD and none of the animals survived by day +28. By contrast, in allo recipients treated with anti-MIF antibodies clinical GVHD (day 21: 4.4±0.4 vs. 5.7±0.5) and weight loss (24.6% vs. 45.4%) were less severe, and this reduction in clinical GVHD translated into significantly improved survival (p<0.05). In addition, serum IFNγ (4397±995 vs. 6070±525 pg/ml) and TNFα (113.9±12.7 vs. 159.2±22.5 pg/ml) levels of anti-MIF treated recipients were decreased by day 7. Alloantigen-specific T cell activation in vitro was significantly suppressed in the presence of anti-MIF, as determined by a reduction in T cell proliferation (38517±2411 vs. 65076±3959 cpm), IFNγ (832±117 vs. 4604±179 pg/ml) and TNFα (189.0±7.5 vs. 522.9±6.1 pg/ml) secretion. We next challenged syn and allo recipients with P815 tumor cells (H-2d) at the time of transplantation. No tumor cell elimination was seen after syn SCT. In contrast, FACS analysis revealed, that clearance of P815 cells was comparably high in allo recipients treated with either control or anti-MIF IgG. In summary, our data strongly suggest, that neutralizing MIF may be a novel approach to reduce GVHD but maintain GVL.
Down Regulation of CXC Chemokine Receptor 3 Impedes the Ability of Anti-CXCR3 Antibody To Block Graft-Versus-Host Disease.
Authors: He Shan , Cao Qi , Jin Min , Emerson Stephen G. , Zhang Yi , Zhang Yanyun
Published: October 2019

Graft versus host disease (GVHD) is caused by infused donor T cells that primarily target specific organs, such as skin, liver and intestine. While it has been demonstrated that chemokines are critical to directing in vivo trafficking of host-reactive donor T cells into GVHD target organs, blocking GVHD using antagonist antibody (Ab) against chemokine receptors remains an elusive goal. We found that in vivo administration of anti-CXCR3 Ab for 1 week (named short-term treatment) only partially prevented acute GVHD in B6/SJL mice receiving donor C3H.SW naïve CD8+ T cells, with 45% of them dying from the disease. Flow cytometry analysis showed that CXCR3+ CD8+ T cells occurred in the spleens and livers of these GVHD B6/SJL mice by day 3 and peaked by day 7 after transplantation, and persisted throughout the disease course. When donor CXCR3+ CD8+ T cells were recovered from these GVHD B6/SJL recipients at day 14 after transplantation (named day-14 CXCR3+ CD8+ T cells) and adoptively transferred into secondary B6/SJL mice, they caused severe GVHD in these secondary recipients with 90% mortality. Interestingly, short-term treatment using anti-CXCR3 Ab protected 78% of B6/SJL mice receiving these donor day-14 CXCR3+ CD8+ T cells. Thus, besides CXCR3+ T cells, some other pathogenic T cells have developed during GVH reactions. We found that a substantial population of CXCR3− CD8+ T cells occurred by day 14 (termed day-14 CXCR3− CD8+ T cells) after transplantation in the spleens (71.1%) and livers (81.6%) of B6/SJL recipients with ongoing GVHD. As compared to donor day-14 CXCR3+ CD8+ T cells, donor day-14 CXCR3− CD8+ T cells expressed lower levels of CCR2, CCR3, CCR5, CD127, granzyme B and FasL, and produced less IFN-g and TNF-a. Furthermore, adoptive transfer of donor day-14 CXCR3− CD8+ T cells caused delayed onset of GVHD as compared to those recipients of day-14 CXCR3+ CD8+ T cells, with 55% of them dying from the diseases. Prolonged treatment of B6 mice receiving day-14 CXCR3− CD8+ T cells with anti-CXCR3 Ab up to 3 weeks (named long-term treatment) significantly inhibited the development of GVHD. More than 70% of these recipients survived without clinical signs of GVHD. In vivo experiments showed that 23% of day-14 CXCR3− CD8+ T cells were converted into CXCR3+ by 12 hours after adoptive transfer into B6/SJL recipients, and up to 60% by 24 hours. Interestingly, about 25% of CXCR3+ CD8+ T cells were also converted into CXCR3− by 24 hours after their transfer. We found in ex vivo cultures that both dendritic cells and interleukin-2 accounted for this conversion between CXCR3+ and CXCR3− subsets, which was inhibited by addition of Ab specific to MHC-I or CTLA-4 Ig. Taken together, these results suggest that alloantigen-induced repression of chemokine receptor(s) may present a major barrier to the success of GVHD prevention and treatment when using antagonists against chemokines or chemokine receptors.
Discovery and Validation of STAT-3 and ERK1/2 Phosphorylation as Critical for the Function of Alloactivated T Cells in Acute Graft-Versus-Host-Disease Via a Novel Technique for Drug Discovery.
Authors: Lu Sydney X. , Alpdogan Onder , Campos Roberto , Wang Xiao , Gao Guo-Jian , Chow Melanie , Lin Janine , Suh David , King Christopher , Hubbard Vanessa M. , Bautista Johanne L. , Kochman Adam A. , Zakrzewski Johannes L. , Smith O. Marsinay , Grubin Jeremy , Chow Andrew , Patel Neel , van den Brink Marcel R.M.
Published: October 2019

Graft-versus-host-disease (GVHD) is a serious complication of allogeneic bone marrow transplantation (allo-BMT). The physiology of GVHD is dominated by alloactivated donor T cells, yet current treatments are often nonspecific and offer limited efficacy with relatively high toxicities. We screened for novel drug targets in alloactivated T cells in a murine allo-BMT model using a flow cytometric technique for the in vivo analysis of intracellular signaling. We defined the signaling profile of normal T cells and alloreactive T cells during GVHD, focusing on pathways involved in T cell receptor (TCR), costimulatory, and cytokine signaling. This analysis revealed that although proteins in multiple pathways (MAP kinases, PI3K, Jak/STAT signaling) were all heavily phosphorylated in alloactivated T cells, phosphorylation of STAT-3 and ERK1/2 were particularly prominently increased in donor alloactivated CD4 T cells. We further analyzed the importance of STAT-3 and ERK1/2 signaling in alloactivated T cells via the use of small-molecule inhibitors of STAT-3 (curcurbitacin E/I) and ERK1/2 phosphorylation (SL327). Treatment with these inhibitors attenuated T cell proliferation in response to anti-CD3+CD28 stimulation and in mixed leukocyte reactions in vitro in a dose-dependent fashion (figure 1).

Figure 1 Figure 1.

Pre-incubation of donor splenocytes with cucurbitacin E significanly reduced T cell activation (CD25, CD69) at 24 hours in adoptive transfer experiments in vivo (p<0.05). To rule out any direct toxicity, we analyzed recovered cells for apoptosis by Annexin-V staininga dn detected no significant toxicity (figure 2).

Figure 2 Figure 2.

We conclude that flow cytometric analysis of signaling pathways in single cells represents a novel methodology to assess the in vivo signaling profiles of specific cell populations in order to select drug targets for further study. STAT-3 and ERK1/2 phosphorylation may also represent potential targets to selectively inhibit donor T cell alloactivation and proliferation in GVHD.
Flow Cytometry Analysis of Peripheral Blood CD4+/CD25+/FOXP3+ T Regulator Cells from 11 Patients Treated with Ipilimumab Following Relapse of Malignancy after Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Zhou Jiehua , Zhong Ruikun , Corringham Sue , Sapp Teresa , Soiffer Robert , Mitrovich Rachel C. , Lowy Izzy , Bashey Asad , Ball Edward D.
Published: October 2019

We conducted a multi-center dose-escalation trial to assess whether CTLA-4 blockade is safe and efficacious as immunotherapy in patients with relapse of malignancy following allogeneic hematopoietic stem cell transplantation (allo-HCT). We hypothesized that since CTLA-4 is a negative regulator of effector T-cell activation, inhibition of CTLA-4 ligation with a blocking human monoclonal antibody (ipilimumab) will augment graft-versus-malignancy. Here we report on the effects of a single dose of ipilimumab on in vivo T cell subsets from 11 patients treated at the highest dose level (3 mg/kg) and compare these findings with those in normal volunteer donor lymphocytes. We analyzed the expression of intracellular CTLA-4 and FOXP3 on CD4+/CD25+ Treg cells, intracellular cytokines and surface markers for T-cell activation on peripheral T cells from 11 patients and 9 normal donors. Peripheral blood mononuclear cells (PBMC) from patients before (day 0) and after the treatment at day 1, 3, 7, 14 and 30 were stained with a panel of antibodies and analyzed by flow cytometry. Lymphocytes from normal donors at time zero and 3 days after culture in IL-2 (200u/ml) were used as controls. The pre-treatment expression of intracellular CTLA-4 was significantly higher in CD4+ T cells from patients than normal controls and was increased further after antibody treatment from 7.1±3.8% at day 0 to 18.2±7.1% at day 30 (p=0.02). Although the expression of FOXP3 in CD4+/CD25+ T cells was higher in patients than in normal donors (6.3±4.8% compared with 2.0±1.6%, p=0.02), there was no significant change in the levels following ipilimumab infusion. The expression of CD4+/CD25high in the patients was 7.7±2.8%, higher than the normal donors (2.3±1.1%). However, 6/11 cases had increased expression while others had decrease or no change, overall there was no statistically significant change. CD4+/CD25low activated T cells were elevated in 10/11 patients before ipilimumab (42.1±8.5%). Their levels were not affected by CTLA-4 blockade. CD8+/CD69+ activated T-cells were significantly increased in 8/11 patients within the 30 days after ipilimumab treatment but typically returned to baseline values on longer follow-up. CD4+/CD69+ and CD4+/HLA-DR+ T-cells were unchanged following ipilimumab treatment. These data show that a single dose of ipilimumab enhances levels of some subsets of activated T cells without a significant effect on cells with a T-regulatory phenotype.
Vβ Spectratype Analysis of Tissue-Infiltrating T Cells Responding to Minor Histocompatibility Antigens Involved in Graft-Versus-Host Disease.
Authors: Zilberberg Jenny , Loise Gichuru N. , Friedman Thea M.
Published: October 2019

Lethal graft-versus-host disease (GVHD) can be induced between MHC-matched murine strains expressing multiple minor histocompatibility antigen (miHA) differences. In the C57BL6 (B6)->BALB.B strain combination, both CD4+ and CD8+ donor T cells can mediate severe lethal GVHD, whereas in the B6->CXB-2 model, in which the CXB-2 strain expresses a subset of the BALB.B miHA, only the CD8+ T cells directly potentiate lethality. We have previously used TCR Vβ CDR3-size spectratype analysis to examine the alloreactive B6 CD4+ and CD8+ T cells, isolated from the lymphohematopoietic compartment after transplantation into both BALB.B and CXB-2 recipients. However, since tissue-specific expression of miHA can potentially elicit differential T cell responses, we have extended our T cell repertoire analysis to examine the responses involved in target tissue damage. Infiltrating host-presensitized B6 CD4+ and CD8+ T cells were isolated post-transplant from the intestines, livers and spleens of lethally irradiated (9 Gy; split-dose) BALB.B and CXB-2 recipients. The results indicated some overlapping Vβ CDR3-size skewing in both the CD4+ and CD8+ T cell repertoires between the BALB.B and CXB-2 recipients within the tissues of each recipient strain. Most notably, spectratype analysis demonstrated tissue specific responses unique to each of the BALB.B and CXB-2 infiltrates. In situ observations of the tissue infiltrating alloreactive T cells were performed by fluorescent microscopy of transplanted B6 T cells constitutively expressing eGFP into BALB.B and CXB-2 recipients, in conjunction with immunohistochemical staining of skewed Vβ families. TUNEL staining was also performed to confirm apoptosis of tissue epithelium. These analyses confirmed the increased infiltration of skewed CD4+ and CD8+ Vβ families within the target tissues.