bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression Profiles with Signatures of Tumor Biology and Chemotherapy Sensitivity May Provide a Novel Approach to Maximize Response to Induction Therapy in Patients with Acute Myeloid Leukemia.
Authors: Rao Arati V , Tuchman Sascha A , Potti Anil , Rizzieri David A. , Mano Hiroyuki , Delwel Ruud , Valk Peter , Lowenberg B.
Published: April 2020

Gene expression profiling in acute myeloid leukemia (AML) patients has previously identified prognostically important clusters driven by the presence of chromosomal lesions, genetic mutations (CEBPA), or abnormal oncogene expression (EVI1). Several of these patients despite response to induction chemotherapy have poor disease-free survival (DFS), and relapse rates, thus emphasizing the need for novel therapeutic strategies. Gene expression analysis was conducted by applying signature profiles reflecting deregulation of oncogenic signaling pathways ( Myc, Ras, Src, PI3K), profiles representing an altered tumor environment (angiogenesis, TNF-α), and signatures of chemotherapy sensitivity (adriamycin and etoposide) to a cohort of 345 newly diagnosed AML patients previously treated on protocols of the Dutch–Belgian Hematology–Oncology Cooperative group and the Japan Adult Leukemia Study Group. Four distinct clusters were identified based on oncogenic signaling and tumor environment pathways. There was a statistically significant difference in overall survival (OS) (p < 0.0195) and DFS (p<0.0304) between the four groups. Patients in cluster 1 had the worst OS at 13.5 months and DFS of just 8.4 months. Patients in this cluster had increased activation of Myc and PI3K pathways and low levels of Src activation. Patients in clusters 2 and 4 had the best OS and DFS. Fifty-one percent of patients in cluster 4 had a nucleophosmin mutation (NPM1 +) and demonstrated higher activation of the Ras and TNF pathways compared to cluster 2, while patients in cluster 2 had increased activation of Myc when compared to Cluster 4. Interestingly, patients in Cluster 3 were most sensitive to the anthracycline, adriamycin, yet had a fairly poor DFS of just 14.4 months. While many biologic factors may play a role, one reason for poor DFS might be increased Src activation in this cluster and also much lower rates of NPM1 (12%) mutations. We could hypothesize that patients in this cluster might benefit from induction with chemotherapy in combination with a Src-inhibitor. This analysis has demonstrated distinct patterns of altered tumor biology and chemotherapy sensitivity amongst patients with clinically relevant subtypes of AML. Additional results evaluating chemosensitivity to cytarabine, and other nucleoside analogues are pending.

Figure Figure
The Effects of Continued Azacitidine (AZA) Treatment Cycles on Response in Higher-Risk Patients (Pts) with Myelodysplastic Syndromes (MDS)
Authors: Silverman Lewis R. , Fenaux Pierre , Mufti Ghulam J , Santini Valeria , Hellström-Lindberg Eva , Gattermann Norbert , Sanz Guillermo , List Alan F. , Gore Steven D. , Seymour John F , Backstrom Jay , McKenzie David , Beach C. L.
Published: April 2020

Background. The international, phase III, multicenter AZA-001 trial demonstrated AZA is the first treatment to significantly extend overall survival (OS) in higher-risk MDS pts (Fenaux, Blood2007;110:817). The current treatment paradigm, which is based on a relationship between complete remission (CR) and survival, is increasingly being questioned (Cheson, Blood2006;108:419). Results of AZA-001 show CR is sufficient but not necessary to prolong OS (List, J Clin Oncol26:2008;abstr 7006). Indeed, the AZA CR rate in AZA-001 was modest (17%), while partial remission (PR, 12%) and hematologic improvement (HI, 49%) were also predictive of prolonged survival. This analysis was conducted to assess the median number of AZA treatment cycles associated with achievement of first response, as measured by IWG 2000-defined CR, PR, or HI (major + minor). The number of treatment cycles from first response to best response was also measured.

Methods. Pts with higher-risk MDS (FAB: RAEB, RAEB-T, or CMML, and IPSS: Int-2 or High) were included. Pts were randomized to AZA (75 mg/m2/d SC × 7d q 28d) or to a conventional care regimen (CCR). AZA treatment was continued until disease progression (or unacceptable toxicity), regardless of hematologic response. Erythropoiesis stimulating agents were not allowed.

Results. In all, 358 pts were randomized (179 to AZA and 179 to CCR). Of the 179 AZA pts, 91 (51%) achieved a CR, PR, or HI. For the 91 pts who achieved an IWG response, the median number of cycles to first response was 3 (range: 1 – 22), 81% of pts achieved a first response by 6 cycles, and 90% achieved a first response by 9 cycles. For 57% of responders (n=52), their first response was their best response; the remaining 43% (n=39) had an improvement in their response status at a median of approximately 4 additional treatment cycles (range 1–11 treatment cycles) after their first response.

Conclusions. While many pts achieving a hematologic response with AZA do so in early treatment cycles, continued AZA dosing can further improve pt responses. In the AZA-001 study, a significant OS benefit was observed compared with CCR. In this study, AZA pts received a median of 9 treatment cycles (range 1–39). For those achieving a response of HI or better, 90% did so by 9 cycles; more than 40% of responders later achieved an improved response. In the absence of unacceptable toxicity or disease progression, continued AZA treatment is appropriate and may maximize patient benefit.
Identification of Genes Associated with Inhibitor Development in Hemophilia a by Microarray Analysis.
Authors: Hwang Sung Ho , Kim Hugh C , Lee Hyun Woo , Yoo Ki young , Kim Hyon J , Kim Hye Sun
Published: April 2020

The risk of factor VIII (FVIII) inhibitor development increases by the disease severity, presence of family history and patient-related environment. Recently, FVIII genotypes and genes related to immune response were found to be decisive risk factors for inhibitor development. To identify the differentially expressed genes (DEGs) for developing inhibitory antibodies in hemophilia A, we analyzed the gene expression profiles between inhibitor and non-inhibitor by microarray technique. The results show that the 384 genes were up-regulated and 161 genes were down-regulated in inhibitor patients as compared with non-inhibitor patients. The 545 of DEGs were classified by the functional gene grouping using Panther classification method. Of interest was the finding that the expression levels of immunity and signal transduction related genes were significantly modified in inhibitor patients. For 7 genes, validation of these bead-array data was carried out by semi-quantitative RT-PCR. Finally, we performed Real Time PCR analysis for the significantly changed DEG450. As a result, expression of DEG450 was significantly down-regulated in inhibitor patients. We demonstrate that inhibitor development is closely related to the expression levels of immune-related genes and that these genes can be used as biomarker genes for prediction of inhibitor development
Mapping of Conformational Epitopes, and Analyze of the Distribution of Ig Isotypes and Subclasses Using a Semi-Quantitative Multiplex Assay in 6 Cases of Postpartum Acquired Hemophilia.
Authors: Lavigne-Lissalde Geraldine , Lapalud Priscilla , Pouplard Claire , Granier Claude , Borg Jeanne-Yvonne , Rotschild Chantal , Gruel Yves , Schved Jean-François
Published: April 2020

Background and objectives: Acquired hemophilia (AH) is a severe life-threatening autoimmune disease due to the development of polyclonal autoantibodies (autoAbs) which neutralize the procoagulant activity of coagulation factor VIII (FVIII). The most common epitopes for autoAbs to FVIII are located in the A2, A3 and C2 domains of FVIII. We have recently developed a multiplex assay based on the Luminex technology that allows simultaneously the detection and epitope mapping of anti-FVIII antibodies (Lavigne-Lissalde et al, Thromb; Haemost. 2008). This technology is fast and requires only 100 μl of plasma and we showed that most autoAbs to FVIII are monospecific and preferentially bind to the light chain (LC) of FVIII. The aim of the present study was to evaluate the epitope specificity and IgG isotype/subclasses of autoAbs to FVIII detected in the post partum period.

Methods: Acquired hemophilia was diagnosed in six women (median age: 28 years) included in a French cohort (SACHA) and who exhibited prolonged activated partial thromboplastin time, low FVIII levels (range = 0.5–21 IU/dL) and inhibitor titers between 1.5 and 40 Bethesda units (BU). The epitope mapping was studied in each patient by testing 5 plasma samples collected (at inclusion and after 1, 3, 6 and 12 months of follow up) using a multiplex assay as previously described (Lavigne-Lissalde et al, Thromb; Haemost. 2008). A panel of monoclonal antibodies that bind to LC, heavy chain (HC) and the C2 domain of FVIII was employed and the use of specific immunoconjugates also allowed to define the isotype (IgG, A, M) and subclass (IgG1 to IgG4) distributions of autoAbs to FVIII. Results were expressed in mean fluorescence intensity (MFI).

Results: The autoAbs detected in the post partum in the six women studied recognized both and simultaneously HC and LC with predominant epitopes located in the LC and the C2 domain in 5 cases. At the inclusion, the autoAbs were mixtures of IgG1 and IgG4 in all patients, but the latter subclass was predominant in most cases. In addition, IgM Abs were also present in 3 women. No correlation between the titer of inhibitor (in BU) and the level of autoAbs binding (in MFI) could be evidenced. The evolution was good in all patients but one case for whom an increase in IgG4 binding to the FVIII C2 domain was demonstrated whereas inhibitor titer decreased (from 40 to 2.6 BU). Importantly, no modification in the epitope specificity between inclusion and 12 months of follow-up was found.

Conclusion: The study shows that autoAbs to FVIII developed in the context of pregnancy are mainly IgG4 specific to the C2 domain. In addition, the epitope mapping of anti-FVIII antibodies by multiplex approach could contribute to a better understanding of the pathophysiology of acquired hemophilia.
Efficacy and Safety of Rituximab in Adult Patients with Idiopathic Relapsed or Refractory Thrombotic Thrombocytopenic Purpura (TTP). Results of a Spanish Multicenter Study.
Authors: de la Rubia Javi er , Moscardo Federico , Gómez María J , Guardia Ramon , Rodriguez Pilar , Sebrango Ana , Zamora Concha , Deben Guillermo , Goterri Rosa , Lopez Rafaela , Peña Francisco , Pujol Micola
Published: April 2020

Background: Therapeutic plasma exchange (PE) is the mainstay of treatment of thrombotic thrombocytopenic purpura (TTP). In many patients, prolonged PE is required to achieve remission and between 30% and 60% of patients relapse after a variable period of months to years, with a further episode of TTP or a chronic picture of recurrent relapses. Various immunosuppressive agents have been used in acute refractory and chronic relapsing cases. Despite advances in treatment, mortality remains at 15–20%. Limited clinical data from small groups of patients suggest that the administration of anti-CD20 antibody (Rituximab) may also be useful in acute refractory and chronic relapsing TTP. However, whether rituximab could be and effective strategy for adults with relapsed or refractory TTP remains unresolved.

Patients and Methods: We have studied the clinical response to rituximab in 22 adult patients (16 F/6 M; median age, 43 years, range 24–72) from 11 Spanish centers who presented with acute refractory (13 cases) or an acute relapse (9 cases) episode of idiopathic TTP. Second-line therapies previously administered included vincristine (11 cases) and splenectomy (2 cases). At the time of the study, five (28%) patients presented neurological symptoms, three (17%) renal failure, and two (17%) fever. On admission, every patient received daily PE and corticosteroids during the acute episode.

Results: Rituximab was administered a median of 16 days (range, 7–53) after starting PE. It was administered immediately after PE at a dose of 375 mg/m2 each week for a median (range) of 4 (1–8) doses. No severe acute or delayed toxicity associated with rituximab was observed. Two (9%) patients died due to TTP-related causes 24 and 48 hours after relapse and were not evaluable for response. The remaining 20 (91%) patients achieved complete remission in a median of 21 days after initiating rituximab (range, 2–35). After a median follow-up of 13 months (range, 1.5–33), 17 patients are in remission and 3 patients have relapsed at 7, 29, and 29 months after rituximab administration.

Conclusion: Based on these results, rituximab appears a safe, effective, targeted therapy associated with a high response rate in adult patients with acute refractory/relapsing idiopathic TTP.
Proof of Concept for the Anti Von Willebrand Factor Aptamer in Patients with Relapsing Thrombotic Thrombocytopenic Purpura (TTP).
Authors: lma Bernd Ji , Jilma Petra , Paulinska Petra , Gilbert James C. , Hutabarat Renta , Wagner Trish , Knoebl Paul
Published: April 2020

Background: Thrombotic thrombocytopenic purpura (TTP) has a high morbidity and mortality rate despite current standard therapy comprising plasma exchange (PEX). Aim of this prospective clinical trial was to test the safety and efficacy of the anti von Willebrand Factor aptamer ARC1779 in patients with relapsing TTP, and to find possible proof of concept.

Methods: Three patients with relapsing TTP (with detectable anti-ADAMTS13 autoantibodies) received bolus primed continuous intravenous infusions of ARC1779 (0.002 mg/kg/min) on top of standard PEX therapy until remission of TTP was induced, or for 14 days whatever came first. ARC1779 concentrations were quantified by a highperformance liquid chromatography/ultraviolet assay, the inhibitory effect of ARC1779 on vWF activity was evaluated with an ELISA kit (READDS vWF Activity ELISA Test Kit, Corgenix, Inc, Westminster, Colo), and platelet function was assessed with the platelet function analyzer (PFA-100).

Results: ARC1779 was well tolerated without any evidence for bleeding in these patients. Median ARC1779 concentrations of approximately 10μg/mL in plasma were reached under steady state, and inhibited the collagen binding site of the vWF A1 domain by >95%. While plasma exchange (PEX) removed some 50% of the drug from the blood, ARC1779 concentrations could be restored by subsequent mini boluses.

Platelet counts in all patients normalized with concurrent ARC1779 and PEX: 2 days after initiation of ARC1779 in two patients, and after 6 days in the third. In the third, the time course of response (see Figure) provides support for the concept that ARC1779 can restore platelet counts, and thus interferes with the disease process. While PEX was stopped when platelet counts reached 125/nL, 4 more days of therapy with ARC1779 increased platelet counts to 260/nL. Discontinuation of the ARC1779 infusion was associated with an immediate drop in platelet counts, so that PEX had to be restarted after 2 days (Figure).

Conclusion: These data indicate that ARC1779 effectively inhibits VWF and thereby increases platelet counts in TTP patients. Addition of ARC1779 to PEX may have the potential to accelerate recovery from organ dysfunction in TTP and thereby decrease morbidity/mortality.

Figure Figure
Graft-Versus-Host Tolerance in Dogs Following T-Cell Depleted Bone Marrow Transplantation with Absorbed ATG or CD6-Antibody.
Authors: Zorn Julia , Kolb Hans Jochem
Published: April 2020

Graft-versus-host disease (GvHD) is the major obstacle of allogeneic stem cell transplantation. Depletion of T-cells from the graft reduces the risk of GvHD, but results in a higher risk of leukemia relapse. Adoptive immunotherapy with donor lymphocyte transfusion (DLT) has been shown to control leukemia in patients after T-cell depleted allogeneic stem cell transplantation. However, GvHD may occur, if DLT is given too early after transplantation. In canine models of DLA-identical and DLA-haploidentical bone marrow transplantation, we compared different methods of T-cell depletion (TCD) and investigated the potential of DLT at different times after transplantation to induce GvHD. T-cell depletion was performed either with absorbed anti-thymocyte globuline (aATG) or with a combination of CD6-antibody and baby rabbit complement. ATG was absorbed with erythrocytes, liver, kidney and spleen for eliminating antibodies against stem cells. CD6-antibody (M-T606) and rabbit complement depleted T-cells effectively without affecting hematopoietic progenitor cells. Unlike aATG, monoclonal CD6-antibody spares natural killer (NK) cells and some CD8-positive cells. Treatment of bone marrow with aATG prevented GvHD in 9 dogs following DLA-identical transplantation. DLT on days 1 and 2 or 21 and 22 induced fatal GvHD in two dogs each. However, it did not induce GvHD when given on days 61 and 62 and later. In DLA-haploidentical bone marrow recipients, non-manipulated marrow produced fatal GvHD in all dogs (n=7), whereas marrow treated with aATG (vol:vol 1:100 and 1:200) produced fatal GvHD in 5 out of 16 dogs only. CD6-depletion prevented GvHD in 3 of 3 DLA-haploidentically transplanted dogs. DLT produced fatal GvHD in one dog each, when given on day 3, 7 or 14 after CD6-depleted haploidentical bone marrow transplantation. However, it produced fatal GvHD in only 2 of 4 dogs transfused on day 20 post grafting. Thus, DLT could be given earlier in DLA-haploidentical animals transplanted with CD6-depleted marrow than in DLA-identical animals transplanted with aATG treated marrow without producing GvHD. These findings support the hypothesis that graft-versus-host tolerance can be induced earlier with grafts not depleted of NK cells. NK cells in the graft may inactivate host dendritic cells necessary for the induction of GvHD. In grafts depleted with aATG, NK cells are depleted as well, because aATG still retains broad specificity despite extensive absorptions. This leaves host DCs unaffected. Transfused donor T-cells encountering this environment will thus be activated which results in severe GvHD. In contrast, monoclonal CD6-antibody spares NK cells, so that donor lymphocytes cannot be activated by host DCs at the time of DLT and thus won’t trigger GvHD. CD6-depletion is the preferred method if adoptive immunotherapy with DLT is planned.
Donor T Cell Production of IL-21 Accelerates Graft-Versus-Host Disease Lethality.
Authors: Bucher Christoph , Vogtenhuber Christine , Jasperson Lisa , Panoskaltsis-Mortari Angela , Goren Emily , Sivakumar Pallavur , Blazar Bruce R.
Published: April 2020

IL-21, produced by T-cells, binds to the common gamma chain family member, IL21R, expressed on immune and colonic epithelial cells. IL-21 signaling results in the maturation, activation and proliferation of T, B, NK-cells and DCs. IL-21 has been implicated in Th17 generation/amplification and also modulating Treg differentiation. However, the relevance of this in disease is unclear. Therefore, we wanted to study the effects of IL-21 depletion and the role of Th17 and Treg cells in the context of GVHD. A lethally irradiated, complete MHC disparate model (B6 to B10.BR) using donor bone marrow cells alone or with the addition of wild-type (wt) CD4+CD25- Effector T-cells and irrelevant or anti-IL-21 Ab from days 0 to 25 twice per week. The administration of anti-IL-21Ab lead to a significant delay of weight loss and mortality (P<0.0001). To determine whether IL-21 deficiency accelerates or inhibits GVHD, studies were performed comparing GVHD-inducing ability of wt or IL-21 knockout (IL21−/−) donor CD4+CD25- T cells. A striking survival advantage of recipients of IL-21−/− vs wt CD4+CD25- T cells was observed (100% vs 0% surviving, P < 0.0001). Subsequent studies were performed using whole T cells, a more clinically relevant donor T cell graft source. Whereas recipients of wt T-cells all died of GvHD within 50 days of GvHD, recipients of IL-21 −/− T-cells showed significantly less weight loss and superior long-term survival (P < 0.0001). Histopathological examination of recipients of CD25-depleted T-cells on d14 revealed significantly reduced GvHD scores of the colonic mucosa and a trend towards lower GvHD scores in the small intestine, liver and the spleen in recipients of IL-21−/− cells. Although flow cytometry analysis of mononuclear cells showed no changes in the frequency of IL-17 producing cells in spleen, liver and colon, the frequency of interferon gamma producing CD4+ T- (Th1) cells was significantly lower in the spleen and the colon of recipients of IL-21−/− vs. wt T-cells. Moreover, increased Treg frequencies were seen in the colon of IL-21−/− vs wt CD25-depleted T cells (mean values: 7.5% vs. 2.1%; P < 0.003). We conclude that IL-21 production by either CD25- depleted T-cells or CD4+CD25- T cells is sufficient to increase GVHD mortality and that Treg inhibition, rather than Th17 generation or amplification, are likely contributing to GVHD lethality acceleration. We also conclude that IL-21 neutralization represents a novel approach for GVHD inhibition that warrants further investigation.
Large Scale Selective Ex Vivo Expansion of CD4+CD25+FOXP3+ Regulatory T Cells from Peripheral Blood.
Authors: Nagamura-Inoue Tokiko , Ogami Kazuo , Yokoyama Kazuaki , Izawa Kiyoko , Kobayashi Seiichiro , Nakayama Shin , Ishige Ikuo , Inoue Yusuke , Ooi Jun , Uchimaru Kaoru , Takahashi Satoshi , Tojo Arinobu
Published: April 2020

CD4+CD25+Foxp3+ regulatory T cells (Treg) play an important role in allograft- and self-tolerance and thus have the potential for therapeutic application in immunological and allergic disorders. However, the frequencies of Treg in peripheral blood are very low. Here we attempted the ex vivo expansion of Treg to enable the adaptive immunoregulatory therapy in humans. CD4+ T cells from peripheral blood of healthy donors or patients with chronic graft-versus-host disease (GVHD) were isolated by anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured using a plastic plate coated with anti-CD28 and anti-CD3-MAbs in the medium containing recombinant human (rh) IL-2 and rhTGF-b. After one week of culture, expanding cells were once detached from the plate and subjected to the fresh medium including rhIL-2 and rhTGF-b but not MAbs. After 2-weeks of culture, phenotypic and functional analyses were performed. Mixed lymphocyte reaction was done using CFSE-labeled responder T cells and autologous or allogeneic dendritic cells (DC) with or without expanded Treg-rich populations. Xenogeneic -GVHD in NOD-Scid mice was induced by the injection of human T cells expressing luciferace transgene, followed by in vivo bioluminescence imaging (BLI) analysis using a CCD camera.

Our expansion procedure with TGF-b yielded 45-83% purity of Foxp3+CD25+CD4+Treg co-expressing CTLA-4, CD54 and GITR, while 8-42% purity without TGF-b(p<0.001). These cell populations also displayed CD45RO+CD45RA−CD26high+ memory phenotype. An average expansion rate of Treg was 62,200 fold (25,500–97,900) in healthy donors during the culture periods (n=5). Thus, we obtained an average of 4.7x108 Treg from the initial number of 5x105 CD4+ T cells in peripheral blood. Additionally, from peripheral CD4+ T cells in patients with chronic GVHD, Treg could be expanded equivalently to healthy donors. The resulting Treg-rich populations inhibited the proliferative response of CFSE-labeled T cells to autologous and allogeneic DC (Figure 1). The ex vivo expanded Treg-rich populations had the inhibitory effect on xeno-reactive T cells expressing luciferase transgene in a xenogeneic GVHD model (Figure 2). Our procedure has allowed efficient ex vivo expansion of Treg-rich populations from a small volume of peripheral blood, and will be applicable to clinical use.

Figure 1. MLR inhibited by expanded Treg Figure 1. MLR inhibited by expanded Treg Figure 2. Xenogeneic GVHD diminished by expanded Treg. Figure 2. Xenogeneic GVHD diminished by expanded Treg.
Investigating the Mechanisms Underlying FVIII Antibody Production in Hemophilic Mice Following Recombinant and Plasma-Derived FVIII Infusion
Authors: Qadura Mohammad , Waters Braden , Burnett Erin , Chegeni Rouzbeh , Othman Maha , Lillicrap David
Published: April 2020

The formation of Factor VIII (FVIII) antibodies after FVIII treatment in hemophilic patients is currently the major treatment-related complication faced by hemophilic patients. It has been reported that the use of plasma-derived FVIII (pdFVIII) in patients and animal models of hemophilia A will result in reduced anti-FVIII antibody formation compared to recombinant FVIII (rFVIII). In an attempt to explain this phenomenon, an initial hypothesis relating to the uptake of FVIII by antigen presenting cells has been formulated. In contrast, here we examine the hypothesis that the cytokine microenvironment induced by pdFVIII has a critical influence on the reduction of anti-FVIII antibody titers in hemophilia A mice. Micro-array and confirmatory quantitative RT-PCR experiments performed on CD11c+ dendritic cells, isolated 24 hours after rFVIII or pdFVIII infusion, show that pdFVIII causes a different immune gene expression profile than rFVIII administration. Both treatments caused the up regulation of pro-inflammatory gene expression. However, the rFVIII and pdFVIII treatments caused the up regulation of genes that induce Th1 and Th2 responses, respectively. Moreover, after administering rFVIII or pdFVIII concentrates to mice, we observed that each treatment induced a distinct T cell splenic cytokine micro-environment. Recombinant FVIII induced the release of Th1 cytokines and IL10, while pdFVIII induced the release of Th2 cytokines and TGF-α. In this mouse model, we observed the formation of high titers of anti-human VWF antibodies in the pdFVIII-treated mice and we propose that in these animals, VWF competes with FVIII for antigenic presentation. We further investigated the potential of antigenic competition by treating mice with FVIII and increasing concentrations of another unrelated protein, recombinant human factor IX (FIX). Our studies have shown an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies allude to new and additional mechanisms that may contribute to the reduction of anti-FVIII antibody development in hemophilia A mice treated with pdFVIII.
Quality of Life Scores Pre and Post Autologous Stem Cell Transplant for Patients with Lymphoid Malignancies
Authors: Dabney Jane , Rybicki Lisa , McLellan Linda , Foster Larry , Visnosky Michelle , Curtis Julie , Bolwell Brian
Published: April 2020

We performed prospective quality of life assessments on 304 patients with non-Hodgkin’s lymphoma (NHL), Hodgkin’s disease (HD), or Multiple Myeloma (MM), undergoing an autologous stem cell transplant (ASCT) from June 2003 through December 2007. Patients underwent QOL assessment one to two weeks prior to stem cell mobilization for ASCT, and 40–44 days (Day 42) post ASCT. We used the Functional Assessment of Cancer Therapy–Bone Marrow Transplant (FACT-BMT) assessment tool which includes five component scores: physical well being (PWB), social well being (SWB), emotional well being (EWB), functional well being (FWB), and additional concerns (AC). The total scores the sum of the score of the five components. We also administered a Profile of Mood State (POMS) scale which measures depression, vigor, anger, tension, confusion, and fatigue. Of 304 total patients, 65% had NHL, 21% MM, and 14% HD; median age was 53; 60% were male; 25% received prior radiation therapy; preparative regimens were busulfan based or melphalan; all patients received peripheral stem cells alone, median inpatient length of stay was 21 days. Using the FACT scale, there was a significant change from baseline data to day 42 scores. 218/304 patients completed baseline and Day 42 evaluations. Compared to baseline, patients had a significantly increased day 42 EWB score (p < 0.001), which occurred despite the fact that the functional well being scores decreased in a statistically significant fashion (p = 0.002). Using the POMS tool, statistically significant changes from baseline to day 42 were a reduction in depression (p < 0.001), a reduction in anger (p < 0.001), a reduction in tension (p < 0.001), and a reduction in confusion (p < 0.001), which occurred despite the fact that patients also had a reduction in vigor (p < 0.001). We conclude that using these two QOL tools despite a reduction in functional well being and vigor one month after ASCT, other parameters of coping, including emotional well being, depression, anger, and tension, improve as patients are recovering from the transplant experience. We hypothesize that this reflects resolution of pre transplant fears and anxiety. Whether these scores can be further improved with additional intervention requires further study.
A Comparison of IEF and MSMS for Clinical Hemoglobinopathy Screening in 40,000 Newborns
Authors: Daniel Yvonne , Turner Charles , Farrar Lisa , Dalton R. Neil
Published: April 2020

Currently, newborn haemoglobinopathy screening is carried out using HPLC or isoelectric focusing (IEF). We have previously described a rapid and specific electrospray mass spectrometry–mass spectrometry (MSMS) technique, using multiple reaction monitoring (MRM) based peptide analysis, for simultaneous detection of the clinically significant haemoglobinpathies; haemoglobin (Hb)S, HbC, HbE, HbDPunjab and HbOArab. Here we report the results of a comparison of 40,000 newborn blood spots screened by both IEF and MSMS.

For both IEF and MSMS analysis, blood spots (3.2mm) were punched into separate 96 well plates. IEF was performed using the Resolve haemoglobin test kit (PerkinElmer Life Sciences, Waltham, USA) and Isoscan imaging system. For MSMS analysis, the blood spots were digested for 30min at 37°C with a trypsin reagent, and diluted in mobile phase (acetonitrile: water, 50:50, with 0.025% formic acid. Sample, 2μl, was injected directly into the mobile phase (flow rate 80μl/min) and analysed, in positive ion mode, using a Sciex API4000 (Applied Biosystems, Warrington, UK). Specific MRM transitions for HbS, HbC, HbE, HbDPunjab, HbOArab, normal beta, alpha, gamma and delta chains were acquired; total acquisition time per sample was 60 sec. This enabled identification of sickling disorders and thalassaemia major, as well as assessment of transfusion state and potential identification of HbLepore and HbBarts.

40,000 blood spot samples for routine newborn haemoglobinopathy screening were analysed in parallel. HbS was detected in 199 samples; 8 were HbS/HbF only and 3 HbSC. HbC was detected in 39 samples, HbDPunjab in 52, HbE in 48. No HbOArab or HbLepore mutations were detected by either method. There have been no discrepancies between the analytical techniques. Using MSMS, mutation positive samples can be re-run in product ion scan mode to provide peptide sequence and hence unequivocal confirmation of the haemoglobin variant. In addition, 5,000 samples were analysed on a Sciex API4000 Q trap; using the information dependent acquisition facility provided “real time” peptide sequencing thus removing the requirement for re-injection.

Sample preparation is very quick and simple for both methods, but the consumable costs associated with the MSMS technique are <10% of those for IEF. The capital cost of MSMS can be offset by high throughput and/or integration with current inherited metabolic disease screening by MSMS. The specificity of the MSMS analysis implies that haemoglobinopathy detection can be limited to specified conditions, based on agreed screening policy, and can eliminate the need for costly and time consuming second line testing.

This study demonstrates that newborn haemoglobinopathy screening can be carried out rapidly, easily, and cost effectively using MSMS technology. It enables rationalisation of technology platforms in newborn screening by consolidating screening for haemoglobinopathies and inherited metabolic diseases onto MSMS.
Engineering Kunitz Domain of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis: An Antifibrinolytic Agent with Potential to Replace Aprotinin
Authors: Bajaj Madhu S , Ogueli Godwin I , Schmidt Amy E , Shanker Sreejesh , Bajaj S. Paul
Published: April 2020

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin and therefore it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this report, structure based investigations and analysis of the serine proteases profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ (nomenclature of Schechter and Berger, BBRC, 27:157–162, 1967) position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu17 (bovine pancreatic trypsin inhibitor/aprotinin numbering) in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with the wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in E. Coli, folded and purified to homogeneity. Amino-terminal sequences and mass spectra revealed proper folding of the KD1-WT and KD1-L17R. As compared to KD1-WT, the KD1-L17R neither prolonged the activated partial thromboplastin time of normal plasma nor it inhibited factor XIa, plasma kallikrein or factor VIIa/tissue factor. Further, KD1-L17R inhibited plasmin with ~4-fold increased affinity. In a mouse liver laceration model of bleeding from small vessels, KD1-L17R reduced total blood loss by 84% compared with KD1-WT, which reduced total blood loss by 10%. Moreover, in this bleeding model, KD1-L17R was more effective than aprotinin (70% reduction), which has been used as an antifibrinolytic agent to decrease blood loss during major surgery. In this model, KD1-L17R was also more effective than the lysine analogue tranexamic acid (52% reduction). In additional studies, in a tail transection model of bleeding from a large vessel, KD1-L17R reduced total blood loss by 70% and was more effective than KD1-WT (46% reduction), aprotinin (43% reduction) and tranexamic acid (67% reduction). Notably, as compared to aprotinin, renal toxicity manifesting as multifocal tubular necrosis by histopathology was not observed with KD1-L17R or KD1-WT. In conclusion, KD1-L17R is a specific inhibitor of plasmin without anticoagulant properties and is more effective in reducing blood loss compared with known antifibrinolytic agents in clinical use.
The Small GTPase Rap1A Is Central for Migration and Adhesion of Mesenchymal Stromal Cells (MSCs)
Authors: Deak Erika , Ruester Brigitte , Göttig Stephan , Puccetti Elena , Ruthardt Martin , Seifried Erhard , Scheele Jürgen , Henschler Reinhard
Published: April 2020

MSCs are currently intensively investigated for their tissue regenerative and immunomodulatory properties, and for many applications are applied via the intravenous route. We have previously shown that MSCs roll and adhere on endothelial cells both in vitro and in vivo, using part of the array of the adhesion receptors which are known to mediate adhesion of hematopoietic stem and progenitor cells during mobilization from bone marrow and homing after transplantation (Blood108:3938, 2006). To identify further gene products which are potentially relevant for coordinated circulation behaviour of MSCs, we kept human MSCs under shear stress by continuous stirring for 0, 3, 6 and 12 h, isolated RNA and performed cDNA microarrays. We found approx. 500 out of 21.000 sequences regulated by shear stress. Interestingly, differentiation-associated genes (osteogenic, lipogenic) were generally downregulated. Several adhesion receptors, including integrins α4 and α7 were time-dependently upregulated. Among G proteins and -associated factors, the Guanosin Nucleotide Exchange Factors (GEFs) for the small GTPase Rap1, RAP-GEF2 and PDZ-GEF1 were both strongly upregulated by shear stress, whereas Rho GTPase regulator RGS-3 was downregulated. We next isolated MSCs from murine bone marrow from Rap1A deficient or wild type mice. Wild type MSCs were found to express Rap1A RNA and protein. However, it was not possible in 5/5 cases to isolate MSCs by the culture expansion method from Rap1A−/− mouse bone marrow. Yet we were able to derive and analyze MSCs from Rap1A+/− mice, and found that their binding to immobilized HUVEC endothelial cells in laminar flow chambers was approximately threefold reduced compared to wild type controls. Similarly, shear stress resistant adhesion to both, Vascular Cell Adhesion Molecule (VCAM)-1 and Intercellular Adhesion Molecule (ICAM)-1 was about 50% decreased compared to wild type MSCs isolated from littermate mice. Moreover, SDF-1-induced adhesion strengthening on VCAM-1 and ICAM-1 was also decreased in the Rap1A+/− MSCs. Transfection of MSCs with an expression vector encoding dominant negative Rap1 (Rap1N17) decreased, whereas pretreatment of MSCs with the Rap1 specific EPAC activator, 8-Br-(4PCT)-cAMP increased adhesion of MSCs to VCAM-1. These data demonstrate an essential role of the GTPase Rap1A for integrin-mediated adhesion of MSCs. Interference with Rap1 signalling thus provides a means to modulate adhesion, and potentially, homing behaviour of MSCs.
Id1 Provides a Proper Hematopoietic Progenitor Niche Function
Authors: Suh Hyung Chan , Ji Ming , Gooya John , Lee Michael , Klarmann Kimberly , Keller Jonathan R.
Published: April 2020

Development of hematopoietic stem cells (HSC) and their progeny is maintained by the interaction with cells in the microenvironment. In addition to hematopoietic cells, Id1 is expressed in stromal cells known to support hematopoiesis, and is involved in cell proliferation, differentiation and senescence. Therefore, to investigate the role of Id1 in hematopoiesis, we examined hematologic phenotypes of Id1−/− mice. In this study, we found increased neutrophils and macrophages, and decreased B cells and platelets in peripheral blood, and decreased BM cellularity. While the percentages of hematopoietic stem cells (HSC) in Id1−/− mice were increased relative to the Id1+/+ mice, their total numbers and function appeared normal. For example, Id1 was not required for self-renewal or repopulation of HSC. In contrast, we found that there were increased numbers of hematopoietic progenitor cells (HPC) in S phase of cell cycle in Id1−/− mice BM, suggesting that the loss of Id1 within HPC promotes proliferation. However, purified Id1−/− HPC had the same proliferation potential as Id1+/+ HPC when cultured in vitro. In transplantation experiments, we proved that BM microenvironment in Id1−/− mice is defective by showing that the Id1+/+ HSC showed impaired hematopoietic development in Id1−/− mice, while the Id1−/− HSC had normal repopulation potential in an Id1+/+ microenvironment. In agreement with these findings, Id1−/− BM stromal cell cultures supported enhanced proliferation of hematopoietic progenitors. Furthermore, quantitative PCR showed that SCF, M-CSF, OPN, SDF-1 and TGF-α mRNA expression was decreased in Id1−/− stromal cells relative to Id1+/+ stromal cells, while G-CSF, GM-CSF, and VEGF mRNA expression was significantly increased. Id1−/− BM showed decreased number of mesenchymal stem/progenitor cells. Thus, Id1 does not play a role in maintaining HSC, but is involved in regulating hematopoietic progenitor niche. Funded by NCI contract No. N01-CO-12400.
Asthma in Pediatric Sickle Cell Acute Chest Syndrome: In An Inner City London Hospital
Authors: Bartram Jack L , Fine-Goulden Miriam R , Green Dido , Ahmad Rahail , Inusa Baba PD
Published: April 2020

Acute Chest Syndrome (ACS) is the second most common cause of hospitalisation in patients with Sickle Cell Disease (SCD) and up to 25% of those admitted will require intensive care management. ACS is a leading cause of death in SCD. It may also play a role in the development of chronic lung disease in SCD patients and the prevalence of Asthma in SCD patients is high. The pathogenesis of ACS is complex. Previous work has suggested a relationship between asthma and higher risk of ACS in children with SCD. Data in the UK is limited. Our aim therefore was to describe the presentation, course and outcome of ACS in our local SCD pediatric population, compared with those children who had ACS with SCD and physician diagnosed Asthma (Asthma).

Methods: The data collection took place at The Evelina Children’s Hospital, which is part of St Thomas’ Hospital, a large teaching hospital in Central London, England. There are over 400 children with SCD registered, and around 30 new SCD births per year. A retrospective analysis of patient hospital electronic and paper records was performed of 63 ACS presentations over a three year period from 2003 to 2006. Inclusion in the study required a new infiltrate on chest radiograph plus acute respiratory symptoms in a patient with SCD under the age of 16 years. The group included 16 (25%) presentations in children with SCD and Asthma.

Results: No Known Asthma 47 Presentations; Mean age 6.2 yrs (range 1–15yrs); HbSS 87%, HbSC13%; Previous ACS 26% (n=12); Mean length of stay 5.4 days (range 1–27); Mortality 0; Mean C-Reactive protein (CRP) on admission 70 (normal <5); Mean oxygen saturations on presentation 92% in air (40% of patients presented with saturations <92% in air)

Physician Diagnosed Asthma 16 Presentations; Mean age 4.6 (range 1–15yrs); HbSS 94%, HbSC 6%; Previous ACS 63% (n=10); Mean length of stay 5.4 (range 2–14); Mortality 0; Mean CRP on admission 41; Mean oxygen saturations on presentation 92% in air (50% of patients presented with saturations <92% in air)

DISCUSSION: Demographics: Comparable in terms of age and haemoglobin genotype. Presentation: Patients with asthma were more likely to have had previous ACS. Children with asthma presented with a lower CRP. Treatment: The treatment in both groups including the use of blood transfusion, and need for transfer to intensive care were comparable. However there was an observed difference in the use of inhaled bronchodilators (non asthma 21% v asthma 50%). Steroids were rarely used (4%) to treat the patients who did not have a pre-existing diagnosis of asthma, however were used to treat most (94%) of those patients with asthma. Outcome: Length of stay was comparable, no deaths in either group.

CONCLUSION: Although patients in our study group with asthma had a higher frequency of previous ACS episodes, we did not demonstrate that patients with asthma suffer a more severe course of illness.
Exercise-Induced Pulmonary Hypertension in Sickle Cell Anemia: a Study with Exercise Stress Echocardiography
Authors: Nunes Newton , Campos Orlando , Figueiredo Maria Stella , Cancado Rodolfo Delfini , Vicari Perla , Carvalho Antonio C C , Bordin Jose Orlando
Published: April 2020

Background: Pulmonary hypertension (PH) at rest is a risk factor for death in patients with sickle cell anemia. Exercise treadmill stress echocardiography (ETSE) is able to detect latent PH.

Objective: To investigate the occurrence of exercise-induced PH in patients with sickle cell anemia and normal pulmonary pressure (PP) at rest, and its relationship with clinical and echocardiographic variables.

Design: observational transversal study.

Patients and Methods: We studied 44 patients (22 men, mean age = 25.3 y.o.) with sickle cell anemia and normal PP at rest (tricuspid regurgitant jet velocity: TRV < 2.5 m/s). They were divided into 2 groups: (G1) normal PP after ETSE (TRV ≤ 2.7 m/s); G2: exercise-induced PH (TRV > 2.7 m/s). The cut off points of TRV at rest and exercise were derived from healthy control subjects, matched for age, sex, and body surface area. Data obtained from ETSE were correlated with clinical, echocardiographic and ergometric variables, including left atrial volume indexed for body surface area and E/E’ ratio derived from pulsed wave and tissue Doppler. Student’s t test e chi-square test were used for statistical analysis. Multivariate logistic analysis was used to determine the independent predictors of abnormal PP response to exercise. Statistical significance was defined as a p < 0.05.

Results: The exercise-induced PH occurred in 57% of patients (G2), with levels of TRV ranging from 2.8 to 4.5 m/s (3.4 ± 0.4m/s), significantly higher than those observed in G1 (2.5 ± 0.3m/s, p < 0.001). Exercise-induced PH was significantly related to higher creatinine levels, larger right ventricular diastolic area, TRV ≥ 2.3m/s at rest, left atrial volume index ≥ 41ml/m2, and E/E’ ratio ≥ 6.3. When the multivariate analysis was performed, TRV at rest [OR 6.5 (1.3–33.6), p = 0.03], left atrial volume index [OR 6.4 (1.2–33.5), p = 0.03]; and E/E’ ratio [OR 5.6 (1.2–26.5], p = 0.03) remained independently related to exercise-induced PH.

Conclusion: Patients with sickle cell anemia and normal PP at rest may exhibit exercise-induced PH which was independently related to increased baseline PP levels, increased left atrial volumes end indirect parameters of left ventricular and diastolic pressure. The clinical relevance of these findings deserves future studies.
Suppressed CEBPA Function Is Associated with Favorable Prognosis in Normal Karyotype AML Patients
Authors: Fos Jose , Bonadies Nicola , Pabst Thomas , Mueller Beatrice U
Published: April 2020

The myeloid transcription factor CEBPA is crucial for normal differentiation of granulocytes. In addition, genomic mutations and deregulated expression of CEBPA have been reported in specific subsets of patients with acute myeloid leukemia (AML). We here investigated the prognostic significance of CEBPA mRNA, CEBPA protein, and CEBPA function in 105 consecutive de novo AML patients with a particular focus on AML patients with a normal karyotype. We found that the DNA binding activity of CEBPA in normal karyotype AML patients as determined by an ELISA-based assay conferred significant prognostic information: normal karyotype AML patients with suppressed CEBPA function showed a better OS (p=0.0068) and DFS (p=0.0138) compared to patients with conserved CEBPA function. In addition, suppressed levels of the 42kDa CEBPA protein in these patients tended to predict favorable outcome, whereas differences in p30 CEBPA protein levels and in mRNA expression did not affect the outcome. Finally, AML patients with suppressed CEBPA function had more frequently FLT3-ITD and an unmutated nucleophosmin status identifying CEBPA function as an independent prognostic marker for normal karyotype AML. This is the first study comprehensively assessing CEBPA transcription factor function in leukemic cells from AML patients. Our data suggest that suppressed CEBPA function is associated with favorable prognosis in normal karyotype AML patients independently of other molecular markers, and we propose assessment of CEBPA binding activity to be integrated into clinical practice. Moreover, our results highlight the importance of posttranscriptional mechanisms of CEBPA modulation in AML.