bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-Cell Mutational Profiling Describes the Molecular Heterogeneity of Clonal Evolution in MDS during Therapy and Relapse
Authors: Aleshin Alexey , Durruthy-Durruthy Robert , Medeiros Bruno C. , Eastburn Dennis J. , Greenberg Peter L
Published: October 2019

Background: Myelodysplastic syndromes (MDS) are a collection of clonal diseases of dysfunctional hematopoietic stem cells, characterized by ineffective hematopoiesis, cytopenias, and dysplasia. Increased understanding of the mutational landscape of MDS has led to initial improvements in prognostic models based on clinical and cytogenetic variables. However, bulk sequencing techniques are limited in their ability to delineate clonal complexity and identify rare drug resistant subclones. To better understand clonal heterogeneity and clonal evolution of MDS we applied a high-throughput single cell sequencing technique to both diagnostic and longitudinal MDS samples.

Methods: Samples were examined for 5 patients with MDS at diagnosis and, when available, progression. Mutational bulk sequencing was performed by NGS panel sequencing and exon sequencing was available in select cases. Single cell processing was performed using the Tapestri (Mission Bio) platform. Briefly, individual cells were isolated using a microfluidic approach, followed by barcoding and genomic DNA amplification for individual cancer cells confined to droplets. Barcodes are then used to reassemble the genetic profiles of cells from next generation sequencing data. We applied this approach to individual MDS samples, genotyping the most clinically relevant loci across upwards of 10,000 individual cells.

Results: Single-cell sequencing was able to be performed successfully on all samples tested and recapitulated bulk sequencing data. We observed high concordance between bulk variant allele frequencies (VAFs) and sample level VAFs derived from single cell sequencing data (r2 = 0.98). Additionally, single cell analysis allowed for resolution of subclonal architecture and tumor phylogenetic evolution beyond what was predicted from bulk sequencing alone. Single-cell SNVs were able to resolve host and donor cell populations after bone marrow transplant and accurately predict chimerism and disease relapse. Furthermore, we were able to resolve the co-occurance of molecular alterations within subclones and establish zygosity of individual mutations at a single cell level. Rare subclones associated with disease relapse, were able to be identified in initial diagnostic samples that were frequently under the limit of detection of bulk NGS.

Conclusions: Our results suggest more molecular complexity in MDS tumor samples than implied from bulk sequencing methods alone and indicates utility of single-cell sequencing for identification of resistant clones and longitudinal therapy monitoring.

Disclosures

Aleshin: Mission Bio, Inc.: Consultancy; Natera, Inc.: Employment. Durruthy-Durruthy:Mission Bio, Inc.: Employment, Equity Ownership. Medeiros:Genentech: Employment; Celgene: Consultancy, Research Funding. Eastburn:Mission Bio, Inc.: Employment, Equity Ownership.
Correlation of Circulating Microparticles with Coagulation and Fibrinolysis Is Healthy Individuals
Authors: Albert Venencia , Subramanian Arulselvi , Pati Hara Prasad
Published: October 2019

Introduction

Microparticles (MPs) are small membrane blebs that are released in response to cellular activation or apoptosis from the cell surface by proteolytic cleavage of the cytoskeleton. Previous studies have reported low levels of naturally produced cell-derived microparticle in healthy individuals. This study was conducted to assess whether circulating microparticles induce thrombin generation leading to low grade activation of the coagulation system in healthy individuals.

Materials & Methods

Flow cytometry analysis of three phenotypes of Platelet derived (PMP), Endothelial derived (EMP) and TF bearing (TFMP) microparticles was done in in the 20 healthy individuals. Triple gating strategy (i) particle size(<0.5µm) (ii) expression of cell surface markers (PMP, CD42a+; EMP, CD62E+; TF bearing MP(TFMP), CD142+) and (iii) phosphatidylserine(Annexin V+) was used. Plasma concentrations of thrombomodulin (TM), Tissue Factor (TF), Tissue factor pathway inhibitor (TFPI), Protein C (PC), Free Protein S (PS), Thrombin antithrombin (TAT) complexes, soluble fibrin monomer (sFM) was done assess the status of coagulation system. Spearman's rank correlation was done examine the role of cell-derived microparticles as critical effectors of thrombosis.

Results: the study group comprised of 75% males of the age group (mean±SD27.3±4.32. Total Microparticles (Annexin V+) enumerated in plasma was 1125±355 (count/µl). 52.8% were TFMP [median (IQR)] [380.3(301.8-710.1) (count/µl)]. Highest number of MP originated from activated platelets [249.1(198.9-404.5) (count/µl)], followed by endothelial cells [140.9(124.9-286.0) (count/µl)]. A statistically significant and inverse correlation of EMP's with TAT complex was observed [12.7(6.8-20.1) ng/ml] (r = -0.46, p = 0.01), PS (93.4±20.6 %) (r = -0.52, p = 0.01) and TM (11.4±4.47 ng/ml) (r = -0.56, p = 0.008). Also, TFMP significantly correlated with TF [3.9(3.0-5.0)] (r = 0.46, p = 0.03) and PC (90.4±17.7) (r=-0.42, p=0.04). No correlation was observed between PMP numbers and coagulation pathway markers.

Conclusion: Sub optimal degree of coagulation and natural anticoagulation pathway are occurring in healthy individuals. Microparticles moderately correlated with TF levels. Low EMP's levels correlated with the anticoagulation markers and thrombin generation, suggesting that EMPS may have a role in inhibition of thrombosis. The impact of endothelial vesiculation on basal coagulation should be studied further.

Disclosures

No relevant conflicts of interest to declare.
Ectopic expression of Delta4 impairs hematopoietic development and leads to lymphoproliferative disease
Authors: Dorsch Marion , Zheng Gang , Yowe David , Rao Prakash , Wang Yanjun , Shen Qiong , Murphy Curran , Xiong Ximing , Shi Qiuju , Gutierrez-Ramos Jose-Carlos , Fraser Chris , Villeval Jean-Luc
Published: October 2019

Notch signaling plays a critical role in cell fate determination in many developmental systems, including the hematopoietic system. We and others have recently cloned a novel Notch ligand called Delta4. In this study, we show the effect of retrovirus-mediated ectopic expression of Delta4 in hematopoietic cells. Lethally irradiated mice transplanted with bone marrow cells expressing Delta4 initially suffered from leukopenia and thrombocytopenia. Although all lineages were affected, the deficit in B cells and platelets was the most durable and profound. A rapid expansion of CD4+CD8+ cells occurred shortly after transplantation. CD4+CD8+ cells progressively invaded all tissues analyzed except the thymus, which surprisingly was atrophic. CD4+CD8+cells were mainly non–Delta4-transduced cells, strongly suggesting that the disease was not cell autonomous. Around 15 weeks after transplantation, mice died from this severe lymphoproliferative disorder, which was not transplantable in late-stage disease into secondary recipients. Mice transduced with a soluble form of Delta4 behaved like control mice. Characterization of early hematopoietic development revealed that Delta4 expression impaired formation of day-12 spleen colony-forming units (CFU-Ss) and, to a greater extent, pre–CFU-Ss. No effect was observed on myeloid colony-forming cells (CFU-Cs), indicating that Delta4 specifically acted on the earliest hematopoietic stem cell compartment. These results show that constitutive expression of Delta4 in hematopoietic cells impairs the development of B cells, platelets, and early stem cells and induces a lethal lymphoproliferative disease.
Acute thrombocytopenia after treatment with tirofiban or eptifibatide is associated with antibodies specific for ligand-occupied GPIIb/IIIa
Authors: Bougie Daniel W. , Wilker Peter R. , Wuitschick Elizabeth D. , Curtis Brian R. , Malik Mohammad , Levine Stewart , Lind Richard N. , Pereira Jaime , Aster Richard H.
Published: October 2019

Acute thrombocytopenia is a recognized complication of treatment with GPIIb/IIIa inhibitors whose cause is not yet known. We studied 9 patients who developed severe thrombocytopenia (platelets less than 25 × 109/L) within several hours of treatment with the GPIIb/IIIa inhibitors tirofiban (4 patients) and eptifibatide (5 patients). In each patient, acute-phase serum contained a high titer (range, 1:80-1:20 000) IgG antibody that reacted with the glycoprotein IIb/IIIa complex only in the presence of the drug used in treatment. Four patients had been previously treated with the same drug, but 5 had no known prior exposure. Pretreatment serum samples from 2 of the latter patients contained drug-dependent antibodies similar to those identified after treatment. No tirofiban- or eptifibatide-dependent antibodies were found in any of 100 randomly selected healthy blood donors, and only 2 of 23 patients receiving tirofiban or eptifibatide who did not experience significant thrombocytopenia had extremely weak (titer, 1:2) tirofiban-dependent antibodies. In preliminary studies, evidence was obtained that the 9 antibodies recognize multiple target epitopes on GPIIb/IIIa complexed with the inhibitor to which the patient was sensitive, indicating that they cannot all be specific for the drug-binding site. The findings indicate that acute thrombocytopenia after the administration of tirofiban or eptifibatide can be caused by drug-dependent antibodies that are “naturally occurring” or are induced by prior exposure to drug. These antibodies may be human analogs of mouse monoclonal antibodies that recognize ligand-induced binding sites (LIBS) induced in the GPIIb/IIIa heterodimer when it reacts with a ligand-mimetic drug.
Normal Arp2/3 complex activation in platelets lacking WASp
Authors: Falet Hervé , Hoffmeister Karin M. , Neujahr Ralph , Hartwig John H.
Published: October 2019

Arp2/3 complex is believed to induce de novo nucleation of actin filaments at the edge of motile cells downstream of WASp family proteins. In this study, the signaling pathways leading to Arp2/3 complex activation, actin assembly, and shape change were investigated in platelets isolated from patients with Wiskott-Aldrich Syndrome (WAS), that is, who lack WASp, and in WASp-deficient mouse platelets. WASp-deficient human and mouse platelets elaborate filopodia, spread lamellae, and assemble actin, identical to control WASp-expressing platelets. Human platelets contain 2 μM Arp2/3 complex, or 8600 molecules/cell. Arp2/3 complex redistributes to the edge of the lamellae and to the Triton X-100–insoluble actin cytoskeleton of activated WASp-deficient platelets. Furthermore, the C-terminal CA domain of N-WASp, which sequesters Arp2/3 complex, inhibits by half the actin nucleation capacity of octylglucoside-permeabilized and activated WAS platelets, similar to its effect in WASp-expressing cells. Along with WASp, platelets express WAVE-2 as a physiologic activator of Arp2/3 complex and a small amount of N-WASp. Taken together, our findings show that platelets activate Arp2/3 complex, assemble actin, and change shape in the absence of WASp, indicating a more specialized role for WASp in these cells.
Differential Expression of CD180 and sIgM by B Chronic Lymphocytic Leukaemia Cells (B-CLL) Using Mutated and Unmutated IgVh Genes.
Authors: Porakishvili Nino , Kulikova Nino , Manoussaka Maria , Jewell Andrew P. , Youinou Pierre Y. , Nedelec S. , Yong Kwee , Nathwani Amit , Heelan B. , Hamblin Terry , Wallace P. , Ely P. , Clarke Ed , Lydyard Peter M.
Published: October 2019

Introduction. B cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease as shown by differential expression of a variety of surface and cytoplasmic markers. In a search for markers that could define biological activity of different B-CLL subsets, we have studied the surface expression of the Toll-like receptor (TLR) family member CD180 in relation to other surface markers and mutation status of IgVh genes.

Methods. Seventy eight B-CLL patients (68 untreated and 10 treated) and 15 age-matched controls were studied in three different clinics. CD19+ B cells were stained using indirect immuno fluorescence for CD180, surface IgM (sIgM), CD79b and CD38, analysed by flow cytometry and data expressed as the relative antibody binding sites (RBS)/cell for each marker. Monoclonal anti-CD5 antibodies were also used with anti CD180 to determine levels of expression of CD180 in control CD5+ cells. IgVh mutation was determined for 47 patients.

Results B-CLL cells had variable levels of CD180 expression, but this was always less (1036 ± 935 RBS/cell) than that expressed by normal blood B cells (5548 ± 2271 RBS/cell) and was stable for up to 18 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated (M) compared with those using unmutated (UM) IgVh genes. This was in contrast to the higher levels of expression of sIgM by B-CLL cells using UM than M IgVh genes (Figure).

Conclusions. CD180 is expressed at higher levels on B-CLL cells using M than those using UM IgVh genes and is in contrast to the level of expression of sIgM which is higher on B-CLL cells using UM versus M genes.

This differential expression of CD180 supports the notion that B-CLL cells using UM IgVh genes represent a population of cells actively responding to signals (perhaps to self antigens) via their surface IgM.

Figure Figure
Outcome with the Hyper-CVAD and Rituximab Regimen in Burkitt (BL) and Burkitt-Like (BLL) Leukemia/Lymphoma.
Authors: Thomas Deborah A. , Cortes Jorge , Faderl Stefan , O’Brien Susan , Beran Miloslav , Koller Charles , Garcia-Manero Guillermo , Younes Anas , Fayad Luis , Wierda William , Pierce Sherry , Giles Francis J. , Kantarjian Hagop
Published: October 2019

The prognosis has improved for BL or BLL, particularly with use of short, intensive, multi-agent chemotherapy programs. Outcome of 26 non-HIV Burkitt leukemia patients (pts) treated with the intensive chemotherapy hyper-CVAD showed an overall CR rate of 81% with an induction mortality of 19% owing to systemic fungal infections in patients aged 60 years and older [Thomas et al, JCO 17:2461, 1999]. In addition, older age was an adverse feature predicting relapse with shorter 3-year overall survival compared with younger pts (17% versus 77%). Thus, the program was modified to give the induction course in laminar air flow rooms for pts aged 60 years or older. Given the high expression of CD20 in BL and BLL, rituximab was added to the regimen. From November 1999 to April 2004, 40 pts with newly diagnosed BL (n=28) or BLL (n=12) were enrolled. The median age was 47 years (range, 17–77 years) with 23% older than 60 years. Nine pts were HIV-positive. Advanced Ann Arbor stage III/IV was present in 80% (10% had CNS involvement). The median white blood cell count (WBC) was 8.3 x 109/L; 38% had WBC > 10 x 109/L. Rituximab was given at a standard dose of 375 mg/m2 days 1 and 11 of courses 1 and 3 with hyper-CVAD (fractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone) and days 1 and 8 of courses 2 and 4 with high dose methotrexate and ara-C. A total of 8 courses was planned along with 2 intrathecal prophylactic treatments of methotrexate and ara-C per course (total 16 intrathecals). The overall complete response rate in 22 evaluable non-HIV pts (2 too early, 7 with CR at start either due to one course of prior therapy or resected disease) was 95%, with no detectable difference between BL and BLL subtypes. No induction deaths were observed in the non-HIV group. After a median follow up of 18 months (range, 3 – 50+ months) in the non-HIV group, 2 relapses were observed. Six of 9 HIV pts achieved CR (2 resistant, 1 early death) but either died in CR of HIV-related infections (n=3) or relapsed (n=1), with 2 in continuous CR at 5 and 42+ months. Seventeen of the 31 non-HIV pts have been followed over one year from start of therapy without recurrence; one pt died in CR related to co-morbid conditions. Toxicity profile was similar to hyper-CVAD alone. Rituximab added to the hyper-CVAD regimen appears to improve outcome in BL and BLL.
Telomerase Inhibition with a Novel G-Quadruplex-Interactive Agent, Telomestatin: In Vitro and In Vivo Studies in Acute Leukemia.
Authors: Tauchi Tetsuzo , Shin-ya Kazuo , Sashida Goro , Sumi Masahiko , Nakajima Akihiro , Ohyashiki Junko H. , Ohyashiki Kazuma
Published: October 2019

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. Genetic experiments using a dominant-negative form of human telomerase (DN-hTERT) have demonstrated that telomerase inhibition can result in telomere shortening followed by proliferation arrest and cell death by apoptosis in leukemia cells (Tauchi et al. Clin Cancer Res, 8; 3341, 2002). Recently, we have demonstrated that treatment with a G-quadruplex-interactive agent, telomestatin reproducibly inhibited telomerase activity in the BCR-ABL positive leukemic cell lines (Tauchi et al. Oncogene, 22; 5338, 2003). In the present study, we investigated the mechanisms of apotosis induced by telomerase inhibition in acute leukemia. We first examined the levels of activated p38MAP kinase, MKK3/6, ASK1 and JNK in DN-hTERT-expressing U937 cells (PD, population doubling, 25) and telomestatin-treated U937 cells (PD25). Activation of p38MAP kinase and MKK3/6 was found in DN-hTERT-expressing U937 cells (PD25) and telomestatin-treated U937 cells (PD25), however, activation of JNK and ASK1 was not detected in these cells. We have also found the activation of caspase-3 cascades and Bax translocation in DN-hTERT-expressing U937 cells (PD25) and telomestatin-treated U937 cells (PD25). To examine the effect of p38MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580, which specifically inhibits p38MAP kinase but not the Erk or JNK pathway. DN-hTERT-expressing U937 cells stopped proliferation on PD25, however, in the presence of SB203580, a significant increase in growth rate was observed. Treatment of SB203580 also reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25). These results suggest that p38MAP kinase has a critical role for the induction of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the effect of telomestatin on the growth of U937 cells in xenograft mouse model. Systemic intraperiotoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited marked apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity. Taken together, these results lay the foundations for a program of drug development to achieve the dual aims of efficacy and selectivity in vivo.
Overexpression of BHLHB1 (OLIG 2) Causes Precursor T-Cell Lymphoblastic Lymphoma/Leukemia (pre-T LBL) in the Presence of LMO1 Overexpression.
Authors: Lin Yingwei , Deveney Ramona , Aplan Peter D.
Published: October 2019

The human BHLHB1 (basic domain, helix-loop-helix protein, class B, 1; also known as OLIG 2) was initially cloned by virtue of its activation in pre-T lymphoblastic lymphoma/leukemia (pre-T LBL) with a t(14; 21)(q11.2; q22). BHLHB1, whose expression is normally restricted to neural tissues, is activated in this translocation by its juxtaposition with the TCRA locus. A survey of patients with pre-T LBL (Ferrando et al., Lancet 363:535) indicated that rare patients without a known t(14;21) overexpressed BHLHB1. We generated transgenic mice that expressed a full-length BHLHB1 cDNA driven by the lck promoter. As anticipated, Northern blot analysis showed ectopic BHLHB1 expression in the thymus and bone marrow. We followed a total of 69 transgenic offspring from 3 independent founder lines, all of which showed expression of BHLHB1 in the thymus by Northern blot, for one year. None of these mice developed evidence of T-cell malignancy. Since SCL, like BHLHB1 a class B bHLH protein cooperates with LMO1 to induce pre-T LBL in mice, we considered the possibility that BHLHB1 might also cooperate with LMO1 to induce T-cell tumors. Therefore, we crossed the lck-BHLHB1 mice with lck-LMO1 mice. Approximately 47% (8/47) of BHLHB1-LMO1 double positive mice have developed pre-T LBL by 14 months, while 19% (3/16) of BHLHB1 single positive mice and 7% (1/15) of LMO1 single positive have developed pre-T LBL or died unexpectedly within this time period. The leukemia is highly aggressive, and displays massive thymic enlargement, diffuse lymphadenopathy, and organ infiltration (spleen, liver, kidney, and lung). The immunophenotype of the leukemic cells is CD3-positive and CD4/8-double positive, and Southern blot analysis of these tumors demonstrated clonal TCRB gene rearrangements. We have been able to culture cells from these tumors for over 6 months in the absence of supplemental cytokines, suggesting that these cells are immortal.

We compared the gene expression pattern of the T-cell tumors from BHLHB1-LMO1 double transgenic mice with that of normal thymus tissue using a long oligonucleotide set containing >20, 000 features. Several genes were consistently upregulated at least 4-fold in the tumor tissue, including several translation initiation/elongation factors, transferrin receptor, CCL8 (a chemokine ligand), BST1 (an antigen that lymphocyte growth), granzyme A (a product of cytolytic T-cells), and SAA3 (an acute phase reactant).

Several studies have shown that the LMO1 and LMO2 each collaborate with SCL to induce pre-T LBL in almost 100% of mice within 6 months. Similar to the situation seen with SCL, we have now demonstrated that LMO1 and BHLHB1 collaborate to induce an aggressive pre-T LBL in mice. Since BHLHB1 overexpression can inhibit E2A function, it seems reasonable to suggest that BHLHB1 is exerting its leukemogenic effect through an inhibition of E-proteins important for T-cell differentiation such as HEB or E2A.
An Alternatively Spliced Survivin Variant Is Positively Regulated by p53 and Sensitizes Leukemia Cells to Chemotherapy.
Authors: Zhu Ningxi , Gu Lubing , Findley Harry W. , Li Fengzhi , Zhou Muxiang
Published: October 2019

Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53–143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53–143. When EU-4/p53–143 cell culture was shifted from 37.5°C to the wt-p53-permissive temperature (32.5°C), the expression of survivin and survivin-DEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a pro-apoptotic factor and its expression is upregulated by p53.
Factor XIII Val34Leu Polymorphism Is Protective Against Venous Thromboembolism; a Meta-Analysis.
Authors: Anderson Josdalyne L. , Scarvelis Dimitrios , Doucette Steven , Wells Philip S.
Published: October 2019

Controversy over whether the factor XIII Val34Leu polymorphism confers protection against venous thromboembolism (VTE) is a persistent debate in the current thrombosis literature. If an accurate estimate of the protective effect were established, routine screening for this mutation in patients with VTE could provide more precise individual risk-profiling, but presently, it remains undetermined whether it is worthwhile to screen for this polymorphism. We performed a meta-analysis to determine whether the 34Leu allele of the factor XIII gene is associated with a decreased risk of VTE. Studies were identified by searching MEDLINE (1966 through to June Week 2 2004) followed by a manual search of reference lists from previously retrieved articles, symposia proceedings, and abstracts from major thrombosis conferences. Studies were selected for review if they involved unselected objectively diagnosed VTE patients with factor XIII Val34Leu genotypes reported and were a case-controlled study. Inclusion decisions, quality assessment and data extraction were conducted by two reviewers and consensus was achieved by discussion or by a third independent reviewer. Hardy-Weinberg equilibrium of the study control populations were verified. Four of the studies did not report whether they had verified Hardy-Weinberg equilibrium (Balogh, Catto, Margaglione, and Zidane). Authors were contacted for missing data. Forty-five studies were reviewed for eligibility and ten studies met the inclusion criteria. Pooled Odds Ratios and their 95% confidence intervals (CI) were determined using the method of DerSimonian-Laird. Prior to pooling, heterogeneity testing was performed using the Breslow-Day statistic and was insignificant (p = 0.06) which validated testing our FXIII 34Leu recessive model using the random effects model. Funnel plots ruled out the possibility of publication bias. The random effects model gave a combined odds ratio of 0.75 (95% CI= 0.55 to 1.01). Testing our dominant model using a random effects model gave a combined odds ratio of 0.86 (95% CI=0.76 to 0.98). Heterogeneity testing was insignificant (p=0.16). Our meta-analysis found significantly lower odds of VTE with the presence of FXIII 34Leu allele using a dominant model (i.e. the presence of at least one Leu allele). This polymorphism should be given consideration as part of routine risk profiling in patients at risk for VTE.
Transcriptional Profiling Reveals a Critical Role for Gfi1 in STAT3-Dependent Dendritic Cell Development and -Function.
Authors: Rathinam Chozhavendan , Geffers Robert , Yuecel Raif , Buer Jan , Welte Karl , Moeroey Tarik , Klein Christoph
Published: October 2019

Dendritic cells (DCs) comprise heterogenous and functionally diverse populations of antigen presenting cells. Their developmental pathways remain largely unknown. Using a transcriptional profiling approach, we identified Gfi1 as a novel critical transcription factor in GM-CSF-dependent DC differentiation. Gfi1 is expressed in precursor and mature DCs, as seen in Gfi+/GFP mice, in which one Gfi1 allele is replaced by the GFP cDNA.

Gfi1−/ − mice showed a global reduction of myeloid and lymphoid DCs in all lymphoid organs whereas epidermal Langerhans cells were enhanced in number. Gfi1−/ − DCs showed marked phenotypic and functional alterations, as exemplified by decreased MHC class II expression, absent upregulation of costimulatory molecules upon stimulation and reduced ability to stimulate specific T-cell responses. In contrast, Gfi1−/ − DCs exhibited an increased activation profile as assessed by enhanced secretion of IL12. In vitro, Gfi1−/ − hematopoietic progenitor cells were unable to develop into DCs in the presence of GM-CSF or Flt3L. Instead, they differentiated into macrophages, as evidenced by morphology, expression of cell surface markers, and functional properties. These findings suggest that Gfi1 is a critical modulator of DC versus macrophage development. Analysis of hematopoietic chimeras upon transplantation into congenic recipient mice established a cell-autonomous and non-redundant role for Gfi1 in DC development. Furthermore, upon retroviral gene transfer into Gfi1−/ − progenitor cells, the developmental defect could be reconstituted in vitro and in vivo. The inability of Gfi1−/ − hematopoietic progenitor cells to develop into DCs was associated with decreased STAT3 activation, as shown by Western blot and EMSA assays. In conclusion, we have identified Gfi1 as a critical transcription factor that controls DC versus macrophage development and dissociates DC maturation and -activation.
The Use of a pH Meter for Bacterial Screening of Whole Blood Platelets.
Authors: Yazer Mark H. , Triulzi Darrell J.
Published: October 2019

Since March 2004 the American Association of Blood Banks has required blood collection and transfusion services to have in place measures that limit and detect bacterial contamination of platelet products. At our institution we employ a pH meter to screen all whole blood platelets (WBP) at time of issue. A pH of less than 7.0 is considered a failure and the unit is quarantined and cultured. We undertook a large scale study to establish the rate of pH screening failure and rate of culture positivity of WBP. We studied a representative sample of such units for various laboratory parameters to establish the cause of the pH failure. The number of WBP subjected to pH testing between May and July 2004 was 21,751 and there were 305 failures (1.40%). The average age of the WBPs that failed pH screening was 4.6 days and the average pH was 6.57. The relationship between platelet age and rate of pH failure is shown in the Table. All WBP units that failed the pH screen were cultured; during the study period four culture-positive WBP units were identified (1.3%); in one case S. aureus was isolated from the WBP unit after 4.0 hours of culture and was also identified in the associated RBC unit after 45.6 hours. Diptheroids, coagulase negative Staphylococcus and B. subtilis were isolated from the other three culture positive WBP units however, as the accompanying RBCs remained sterile they were suspected to be contaminants. A series of 38 WBPs that failed pH testing during the study period were evaluated for platelet and WBC concentration and found to have an average WBC and PLT concentration of 2.6x103/mm3 and 1371.0x103/mm3 respectively. The WBC concentration among the WBPs that failed pH screening was more than double the concentration of the WBPs that had acceptable pH (1.2x103/mm3, p=0.005). Additionally, the units that failed pH testing contained approximately 15% more platelets than those WBP units with acceptable pH (1189.6x103/mm3, p<0.05). Our experience with pH screening of WBPs at time of issue revealed a 1.3% bacterial contamination rate of WBPs that failed pH screening. The remaining units that failed pH screening are likely attributable to significantly higher WBC and PLT concentrations. The pH screening method lacks specificity in evaluating bacterial contamination of WBPs.

Relationship of WBP age and pH failure

Age of WBP (days) % of total WBP issued % of pH failures Rate of pH failure by age (%) 2 8.9 1.6 0.3 3 16.3 5.6 0.5 4 30.3 23 1.1 5 44.3 70 2.2
The Molecular Cytogenetic Analysis of MGUS Provides Additional Evidence for a Novel, 14q-Translocation Independent Pathogenetic Pathway in Clonal Plasma Cell Disorders.
Authors: Liebisch Peter , Kirschenlohr Meike , Wendl Christiane , Dohner Hartmut
Published: October 2019

Introduction: There is growing evidence for two almost mutually exclusive pathways in the pathogenesis of multiple myeloma (MM): the first one is characterized by the activation of oncogenes through one of five "primary" IgH translocations, while the second one seems to be designated by the presence of various chromosomal extra copies ("hyperdiploidy") in the absence of a primary 14q-translocation. However, the pivotal chromosomal gains in hyperdiploid tumors and the crucial genetic events in the transition of MGUS to overt MM are unknown.

Aims: i) to evaluate the incidences of chromosomal aberrations using a myeloma-specific DNA probe in pts. with MGUS ii) to compare the FISH data from the present MGUS series with our MM FISH data previously determined on a large series of tumors with the goal to distinguish primary aberrations (already present in MGUS and therefore potentially involved in the process of immortalization) and secondary genetic changes (not or infrequently detectable in MGUS but common in MM, therefore potentially participating in the process of MGUS-MM transition).

Material and Methods: Bone marrow aspirates from 50 pts. diagnosed with MGUS were obtained during routine diagnostic procedures. In the majority of cases, a positive selection of plasma cells using immunomagnetic beads (CD 138) was performed. FISH was combined with immunocytology using light-chain fluorescence antibodies for precise detection of plasma cells carrying the respective clonal light chain (kappa or lambda). At least 50 to 100 plasma cells were evaluated for each probe. The DNA probe set comprised probes mapping to chromosome bands 1p22, 1q21, 6q21, 8p11, 9q34, 11q25, 13q14, 14q32, 17p13, and 22q11.

Results: The most frequent chromosomal aberrations in our MGUS series were (in order of decreasing prevalence; + denotes gains, mostly trisomies, - denotes losses, mostly deletions): t(14q32)/46%, +9q/35%, +11q/27%, +1q/25%, and 13q-/19%. The incidences of all other aberrations were lower than 8%. Comparing the incidences of aberrations in MM and MGUS, the greatest difference was found for 13q- (43% vs. 19%), while it was <15% for all other abnormalities. Of note, 22q- was present in 13% of cases with MM, while no pt. with MGUS exhibited this abnormality.

Conclusions: i) The high incidence of +9q and +11q in MGUS suggests these chromosomal extra copies as early genetic events and provides further evidence for a novel, 14q-translocation independent pathogenetic pathway in clonal plasma cell disorders. ii) Our data indicate that 13q- and 22q- could be relevant for the transition of MGUS to MM. iii) +1q, previously proposed as a marker of advanced MM, is already detectable in 25% of pts. with MGUS.
Non-Heme Induction of HO-1 in Macrophages Does Not Apparently Alter Cellular Iron Levels.
Authors: Sheftel Alex D. , Kim Sangwon F. , Ponka Premysl
Published: October 2019

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The predominant isozyme in the digestion of erythroid heme is the inducible form, HO-1. The products of heme catabolism by HO-1 are ferrous iron, biliveridin (subsequently converted to bilirubin), and carbon monoxide (CO). In addition to its function in the recycling of erythrocyte iron, this microsomal enzyme has been implicated in a number of cytoprotective mechanisms mainly in the context of oxidant insult. HO-1 can be induced not only by heme, but also by cytokines, heat shock, metals, and other cellular stressors, especially those that generate reactive oxygen species (ROS). Implicit in all the reports of HO-1 cytoprotection to date are effects on the cellular handling of heme and , therefore, iron. While bilirubin is an antioxidant, ferrous iron is known to catalyze the production of hydroxyl radicals through Fenton chemistry. Thus, the release of iron from heme could be counterproductive in protecting the cell from oxidative stress. Furthermore, there are a number of non-heme stimulators of HO-1 induction, bringing to question the source of substrate for this enzyme in these paradigms. In the present study, HO-1 was induced by either sodium arsenite or hemin in the murine macrophage-like cell line, RAW264.7. Both of these inducers elicited a transient increase in both the mRNA and protein levels of HO-1, however, only hemin exposure induced an increase in the synthesis rate of the iron storage protein, ferritin. This increase in ferritin production rate, as measured by 35S-methionine incorporation, was attenuated by the HO inhibitor, tin-protoporphyrin IX (SnPP) as well as the cell permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone (SIH). Additionally, treatment of the cells with hemin was able to elicit a decrease in the activity of iron regulatory proteins (IRPs) that could be blocked by preincubation with SnPP. Sodium arsenite had no effect on IRPs. These results suggest that iron released from hemin by HO-1 stimulates ferritin synthesis via the IRE/IRP system, while an increase in the enzyme via a non-heme inducer does not lead to iron release from endogenous heme sources.
Myocardial Infarction Induced by Intravenous Iron Dextran Infusion in Absence of Coronary Artery Disease.
Authors: Ahluwalia Manmeet S. , Patel Sandeep , Daw Hamed
Published: October 2019

A 37-year-old-female presented with history of fatigue for 2–3 months and excessive vaginal bleeding. Further evaluation of the patient revealed iron deficiency anemia secondary to blood loss because of dysfunctional uterine bleeding. She was scheduled for intravenous (IV) iron dextran infusion because of her inability to tolerate oral iron. The possible risks were explained to the patient who decided to proceed with the infusion. Immediately after the infusion the patient developed anaphylactic type reaction and complained of severe chest pain and shortness of breath. Electrocardiogram (EKG) done did not show any evidence of ST changes suggestive of ischemia. However her troponin was elevated at 2.7 (0–0.25). Patient underwent a cardiac catheterization that did not show any evidence of coronary artery disease (CAD). There was however mild impairment of left ventricular systolic function with severe hypokinesis of the apex and distal anterior and anterolateral wall with ejection fraction of around 45 %. Patient most likely developed a non-ST elevation myocardial infarction (MI) secondary to IV iron-dextran induced anaphylactic reaction and hypotension. She was treated with steroids and anti histaminics. She was discharged home second day after catheterization.

Discussion: IV iron infusion is often given to iron deficient patient intolerant to oral iron. Patients report number of adverse events from IV iron dextran infusion that can range from injection site reactions to nausea, diarrhea, allergic type reactions including rash, uticaria, dyspnea, hypotension and in most severe cases, shock and death. Severe anaphylactic reaction may be seen in approximately 0.6% of patients. This risk of anaphylactic reactions appears to be probably secondary to dextran that is known to be antigenic. The exact mechanism is not well understood although cross reactivity to antibodies formed in response to polysaccharides generated by gastrointestinal organisms has been offered as an explanation. Although cases of anaphylactic reactions secondary to IV iron have been reported in literature this is the first case to the best of our knowledge in which patient was documented to have MI in absence of CAD. MI was most likely induced by coronary vasospasm and hypotension secondary to anaphylactic reaction. Hence caution must be exercised when adminstering IV iron-dextran infusion. This should only be given when appropriate resuscitative equipment is immediately available. Alternative IV iron preparations should be considered when possible.
New Longer Acting Erythropoiesis Stimulating Proteins (ESPs) Correct Anemia with a Less Frequent Dosing Schedule in a Mouse Model.
Authors: Hartley Cynthia A. , McElroy Patricia , Khaja Raheem , Molineux Graham
Published: October 2019

Introduction: Recombinant human erythropoietin (rHuEPO) has become a standard treatment for the alleviation of anemia resulting from chronic renal failure and chemotherapy induced anemia. Treatment with rHuEPO corrects anemia, resulting in a broad range of clinical benefits including improved quality of life. Darbepoetin alfa (DA), a super-sialylated erythropoietic stimulating protein (ESP) with a 3-fold longer serum half-life than rHuEPO, is approved for use in the treatment of anemia. Another ESP investigated was a hyperglycosylated analog of DA having a greater number of carbohydrate chains and sialic acids (AMG205).

Objective: Our intention was to develop and characterize new ESPs and examine their relative activity and ability to maintain normal hemoglobin levels in a mouse model with stable anemia (hemoglobin 2g/dL below initial levels, > 98 days).

Methods and Results: Clearance of ESPs was reduced and in vivo potency was increased in proportion to number of carbohydrate chains; AMG205 was cleared the slowest and was the most potent of the 3 test molecules. A regimen of repeated low dose exposure to cisplatin (CDDP) was established that resulted in a stable reduction of hemoglobin levels by 2–3 g/dL (12.23±0.75g/dL). The hemoglobin range in control animals not treated with CDDP in this experiment was (14.53±0.57g/dL). Animals made anemic by repeated administration of CDDP were treated with rHuEPO (20μg/kg), DA (10μg/kg) or AMG205 (10μg/kg). Each group consisted of 15 mice. Following the initial injection, hemoglobin was measured every 2–3 days, and subsequent injections were administered when hemoglobin values were < 13 g/dL. Animals were so treated for 98 days. In this manner the number of injections of each ESP required to maintain normal hemoglobin was determined. Mice treated for 98 days required more injections of rHuEPO to maintain normal Hb levels than DA (13 vs.7 injections), while mice treated with AMG205 required 5 injections.

Conclusion: Normal hemoglobin levels were maintained over a 3 month period with repeated administration of the ESPs investigated. The ESPs with increased carbohydrate content had reduced clearance, increased in vivo potency and could maintain hemoglobin within a normal range with fewer injections than rHuEPO.
Folic Acid Supplementation and Twin Pregnancy in Patients with Sickle Cell Disease.
Authors: Ballas Samir K. , Baxter Jason , Riddick Gaye
Published: October 2019

Patients with sickle cell disease (SCD) usually take 1mg of folic acid daily. The rationale for this approach is to maintain effective erythropoiesis with a stable hemoglobin level. Other potential advantages of folate therapy in patients with SCD include the prevention of hyperhomocysteinemia that may predispose to thrombotic events, which, in turn, may lead to painful episodes. Moreover, folate supplementation during pregnancy is known to prevent neural tube defects in infants. The major disadvantage of folate supplementation in patients with SCD is that it may mask vitamin B12 deficiency. Another controversial effect of folic acid supplementation pertains to its potential effect on the number of twins coming to term. We reviewed our database on patients with SCD to determine the effect, if any, of folic acid supplementation on twin pregnancies. The data were collected prospectively since 1981. All patients routinely took 1.0 mg of folic acid orally on a daily basis. Random testing of the level of folic acid in the steady state in women with SCD including those who became pregnant showed increased levels to > 20ng/ml (Normal range: 3.0–18.0 ng/ml) in most patients. Pregnant patients also took additional perinatal vitamins that also contained folic acid. We selected those pregnant patients in whom the outcome of pregnancy was either a liveborn or stillborn at or after 20 weeks’ gestation. We found that 46 patients with SCD became pregnant 60 times between 1981 and 2002 and who met the defined criteria mentioned above. The average maternal age at delivery was 26 years. Fifty-six pregnancies (93%) ended in liveborn and the remaining four (7%) in intrauterine fetal death. Five pregnancies (8.3%) resulted in the delivery of twins. This is a significantly higher rate of multiple births compared to other pregnant women. The reported rate of multiple births is between 0.34 and 1.1% both in Black and Caucasian women respectively. All twin births were dizygotic in nature. Patients with SCD take higher amounts of folic acid on a regular basis for a longer period of time before and after pregnancy than other pregnant women. This may explain why twin pregnancies are higher in these patients. The reason why folate therapy is associated with twinning is unknown at the present. Further studies may clarify the pathogenetic pathway of this phenomenon.
The Role of Reactive Oxygen Species in γ-Globin Gene Activation by Fetal Hemoglobin Inducers.
Authors: Hsiao Cheng-Hui , Li Wei , Lou Tzu-Fang , Lee Edward , Pace Betty S.
Published: October 2019

Enhancing γ-globin expression is an effective therapeutic strategy for sickle cell disease. Fetal hemoglobin (HbF) inducers such as sodium butyrate (NaB), trichostatin A (TSA) and hydroxyurea (HU) have been studied extensively to ascertain molecular and cell signaling mechanisms for γ-gene activation. Reactive oxygen species (ROS) including nitric oxide, superoxide, hydroxyl radicals and hydrogen peroxide stimulate signaling through the cyclic guanosine monophosphate (cGMP) and p38 MAPK pathways. Indirect evidence supports HU as a nitric oxide donor thus providing a mechanism for HbF induction via cGMP activation. Recently, we and others demonstrated a role for p38 signaling in γ-gene induction by HU as well as NaB, TSA and apicidin. However, the ability of HbF inducers to stimulate ROS formation as a mechanism for γ-globin activation via cGMP or p38 MAPK signaling has not been determined. To clarify this mechanism we measured ROS levels using 2′,7′-dichlorofluorescin diacetate which emits fluorescence at a 529 nm wavelength when activated by hydrogen peroxide. We correlated ROS levels with p38 phosphorylation and γ-globin mRNA levels measured by western blot and real-time PCR respectively. Studies were also completed in the absence and presence of myxothiazol, a known inhibitor of ROS formation to confirm our findings. The positive controls menadione and hemin increased ROS levels 4-fold and 3-fold with a concomitant 6.5-fold and 4-fold increase in γ-globin mRNA respectively. Pretreatment with myxothiazol inhibited maximal HbF induction by both agents. By contrast, NaB and TSA did not increase ROS levels in K562 cells therefore they activate γ-globin by a ROS-independent, p38 MAPK-dependent mechanism. We concluded that ROS play a major role in the mechanism for HbF induction by hemin and the vitamin K derivative, menadione. Investigations were completed which confirmed the ability of HU to stimulate ROS formation as well. Studies are underway to determine the role of hydrogen peroxide and/or nitric oxide in HbF induction by HU via cGMP/p38 MAPK cell signaling.