bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombotic Microangiopathy after Allogeneic Stem Cell Transplantation: A Single Centre Experience.
Authors: Ngirabacu Marie Christine , Kwan John , Leroy Rita , Huynh Phuong , Bron Dominique
Published: April 2020

Introduction Transplant associated microangiopathy (TMA) is a severe complication occurring after allogeneic stem cell transplantation(alloSCT). It is recognized to have a poor prognosis and no effective treatment has been defined.

Methods and Population In this study, we analysed the outcome of patients who developed TMA after alloSCT in our institution from 1996 to 2007. A total 199 patients underwent allogeneic transplantation. To diagnose TMA, we used the criteria proposed by the International Working group of TMA: > 4% schistocytes in blood, de novo or prolonged thrombocytopenia, sudden and persistent increase in lactate dehydrogenase concentration, decrease in serum haptoglobin and decreased haemoglobin.

Results From 1996 to 2006, the overall incidence of TMA in our institution was 19%(37/199). According to the type of transplant, the incidence of TMA was: 18%(14/79) for sibling myeloablative SCT(MSCT); 27%(10/37) for unrelated MSCT; 22.5%(9/40) in haploidentical SCT; 20%(2/10) for sibling non myeloablative SCT(NMSCT) and 20%(2/10) for unrelated NMSCT. Median age of patients was 37 years (range: 16–63). Male to female sex ratio was 1:1.8. As conditioning for transplantation, 60% of patients had received total body irradiation (49% for the alloSCT and 11% for previous treatments). At time of diagnosis of TMA, 35% of patients presented with neurological symptoms (unexplained headache, epilepsy, impaired concentration, drowsiness and/or confusion). Fourty-nine percent (49%) of patients also had cytomegalovirus reactivation; 54% were treated with steroids for acute Graft-versus-host disease and 100% of patients were treated with ciclosporine. The overall mortality rate in the TMA group was 86%, but TMA related mortality was 46%. In patients who died from TMA, median survival post-TMA diagnosis was 16 days(range: 4–60). 88% of those patients had been treated with plasma exchange, 12% by defibrotide. Four of the six patients(66%) treated with defibrotide died from haemorrhagic complications. Only 1 patient treated with defibrotide achieved a complete remission (CR). Of those patients who did not achieve complete remission, 65% had been conditioned with TBI versus 41% in patients who achieved CR.

Conclusion: In our series, the incidence of TMA is significant (19%). The most important risk factors of developing TMA are use of cyclosporine, steroid treatment, CMV reactivation, the use of TBI as conditioning and unrelated donor transplantation. From this study we are unable to define the most effective treatment for TMA. The use of defibrotide was associated with a high risk of haemorrhage in our series and should be used with caution. Because of its incidence and poor prognosis, randomized trials should be utilised to define effective treatment.
Elucidating the Molecular Architecture of the FA-Core Complex Utilizing Biochemical Purification Approach.
Authors: Ali Abdullah M. , Du Changhu , Singh Thiyam R. , Meetei Ruhikanta A.
Published: April 2020

FA is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. FA is genetically heterogeneous, represented by at least thirteen different complementation groups. The genes corresponding to groups A, B, C, D1 (BRCA2), D2, E, F, G, I, J, L, M and N have been cloned. The Fanconi proteins FANC -A, -B, -C, -E, -F, -G, -L, -M along with another newly discovered protein FAAP100 forms a core complex (CC) in the nucleus that is required for the monoubiquitination of two other Fanconi proteins FANCD2 and FANCI. In addition to their presence in nucleus, most of the Fanconi proteins are also found in the cytosol however their role in cytosol is not clear. Also, whether the FA-core complex proteins exist as a single complex or sub-complexes in the cytosol is not clear. The goals of this study are to

develop a method of purification of FA core complex from nuclear and cytosol identify individual components of cytosolic and nuclear complex(s) of the FA proteins.

We have established a two-step purification method using 6XHis and FLAG tags for the biochemical and functional characterization of the FA core complex proteins. In an attempt to isolate interacting partners of FANCC, FANCG, FANCL and FAAP100 proteins; we have established four different HeLa cell lines; HeLa-HF-FANCC, HeLa-HF-FANCG, HeLa-HF-FANCL and HeLa-HF-FAAP100, stably expressing HF-FANCC, HF-FANCG, HF-FANCL and HF-FAAP100 recombinant proteins respectively. Affinity purification was carried out to isolate the complexes from the cytosol and nuclear extracts prepared from stable cell lines. The polypeptides purified from the complexes were identified by mass spectrometry. The results suggest that, in addition to the existence of the FA-core complex containing FANC -A, -B, -C, -E, -F, -G, -L and 100, the FA proteins also exists as sub-complexes in the cytosol. FANCL was found to form a sub-complex with FANCB and FAAP100 in cytosol. Also FANCC, a predominantly cytosolic protein was found to form a sub-complex with FANCE in cytosol. We found several novel proteins that interact with FA proteins.
Responses and Outcome Following Immunosuppressive Therapy in Large Granular Lymphocyte Leukemia-Associated Pure Red Cell Aplasia: A Nationwide Cohort Study in Japan for the PRCA Collaborative Study Group.
Authors: Fujishima Naohito , Sawada Ken-ichi , Hirokawa Makoto , Oshimi Kazuo , Matsuda Akira , Teramura Masanao , Karasawa Masamitsu , Nakao Shinji , Urabe Akio , Omine Mitsuhiro , Ozawa Keiya
Published: April 2020

Background: Large granular lymphocyte (LGL) leukemia accounts for a significant part of secondary PRCA. Cyclophosphamide (CPA) therapy has been reported as being useful in the literature. However, because of its rarity, the long-term response and relapse rate after immunosuppressive therapy including CPA in LGL leukemia-associated PRCA are largely unknown, and optimal management of this disorder remains unclear.

Objectives: We conducted a nationwide survey of immunosuppressive therapy for PRCA in Japan to elucidate the long-term response and overall survival (OS) of this disorder. This report is a summary focusing on CPA therapy for LGL leukemia-associated PRCA.

Methods: The questionnaires were sent to 109 medical institutions in Japan to estimate the number of patients with newly diagnosed acquired PRCA. From a total of 185 patients consisting of 73 idiopathic and 112 secondary PRCA cases, we evaluated 14 patients with LGL leukemia for this report. Endpoints of this study were the response rate and the duration of the response to immunosuppressive therapy and overall survival.

Results. Efficacy of the first remission induction therapy was seen in 6 out of 8 patients treated with CPA (75%), 1 out of 4 patients with cyclosporine (CsA) (25%), and 0 of 2 with corticosteroid (0%), respectively. The median initial dose of CPA for the responding patients (n=6) was 100 mg. The initial dose of CsA for the responding patient was 3.7 mg/kg. Two patients, who failed to respond to the initial CPA therapy, received CsA therapy resulting in the response in one patient. Three patients, who failed to respond to the initial CsA therapy, received CPA therapy resulting in the response in all of three patients. Two patients, who failed to respond to the initial prednisolone therapy, received CsA therapy resulting in the response in one patient. We classified the patients with LGL-associated PRCA into the CPA-group (n=9) and the CsA-group (n=3) according to the response to initial or salvage therapy. Relapse was seen in two CPA-responders following discontinuation of maintenance therapy (23, 45 months), whereas no patients relapsed on maintenance CPA therapy. The estimated median duration of the response to CPA was 125 months. The median OS has yet been reached with the median observation period of 89 months.

Conclusion: CPA showed an excellent response in LGL leukemia-associated PRCA and CPA-containing regimens were effective to prevent relapse of anemia. However, most patients are still receiving CPA for maintenance therapy, and it remains uncertain whether CPA can induce maintenance-free hematological response.
Predicting Response and Survival for Severe Aplastic Anemia Patients Treated with Immunosuppression.
Authors: Scheinberg Phillip , Wu Colin O. , Nunez Olga , Young Neal S.
Published: April 2020

Prognosis in acquired aplastic anemia is predicted by blood counts. The popular “Camitta criteria” were developed about 40 years ago, prior to the introduction of immunosuppressive therapies (IST) and the wide application of hematopoietic stem cell transplantation (HSCT), both of which have markedly improved survival in bone marrow failure. The hematologic response rate to anti-thymocyte-globulin (ATG) has been reported by many centers to be 60–70%; however, a practical and reliable predictor of response to IST is not available. Therefore, the decision to pursue HSCT or IST in SAA often relies on the patient’s age, availability of an HLA-matched sibling donor and in the presence of comorbidities. We conducted a retrospective analysis of 316 patients who received initial IST with a horse ATG-based regimen at the NIH Clinical Center from 1989 to 2005 for criteria that were predictive of hematologic response at 6 months and survival long-term. In multivariate analysis, younger age and higher baseline absolute reticulocyte count (ARC) and absolute lymphocyte count (ALC) were predictive of response at 6 months. Patients with a baseline ARC ≥ 25,000/uL (96 patients) had a much greater probability of response at 6 months following IST compared to the 91 patients with an ARC < 25,000/uL and an ALC < 1,000/uL (80% vs. 41%, respectively; p < 0.001). Those with an ARC < 25,000/uL and an ALC ≥ 1,000/uL (129 patients) formed an intermediate risk group with a probability of response to IST of 62%. This higher likelihood of response translated to better survival in patients in the high ARC and ALC group (92% at 5 years) compared to those with a low ARC and ALC (53%). The probability of response in patients younger than 18 years of age was 74% regardless of the pre-treatment blood counts. When the predictive criteria of the ARC and ALC was used in pediatrics patients only, the ALC was not predictive, however a high baseline ARC remained a significant predictor of response in the pediatric cohort with a response rate of 90% (in those with an ARC ≥ 25,000/uL) vs. 65% (in those with an ARC < 25,000/uL; p=0.02). In the post-ATG era, baseline ARC and ALC together serve as simple predictor of response following IST, which should guide in stratifying risk among patients with SAA. These criteria should be useful for comparison between studies and in clinical decision making, particularly regarding timing of transplantation, as the indication for matched sibling HSCT (now offered to older patients) and alternative donor HSCT (in patients who lack an HLA-matched sibling) broadens.
Long Term Outcome of Pediatric Patients with Severe Aplastic Anemia Treated with Anti-Thymocyte Globulin and Cyclosporine.
Authors: Scheinberg Phillip , Wu Colin O. , Nunez Olga , Young Neal
Published: April 2020

Acquired severe aplastic anemia (SAA) is a fatal bone marrow failure disorder which commonly affects the young. An increased incidence of SAA is observed in children and young adults between the ages of 15 and 25. In those who have a histocompatible sibling donor, hematopoietic stem cell transplantation (HSCT) is often performed; for the remainder of patients immunosuppressive therapy (IST) with an anti-thymocyte globulin (ATG) based regimen is the treatment of choice. The overall response rate (children + adults) to ATG is 60–70%, with prolonged survival achieved in 70–80% of cases. Most of the reports on the efficacy of IST with ATG in children are of small cohorts. Here we report the outcome of children under the age of 18 treated with horse ATG + cyclosporine (CsA) at our institution from 1989 to 2006. Overall 77 received horse ATG as the initial IST. The overall response rate at 6 months was 74% (57/77); the cumulative incidence of relapse at 10 years was 33% and the median time to relapse was 558 days. The cumulative incidence of evolution following IST was 8.5%; all 3 cases occurred among partial responders. Cytogenetic abnormalities that were detected after IST were: monosomy 7 in two patients and deletion 13q in one patient. Overall there were 13 deaths (17%): four occurred within the 3 months following IST in patients who had a pre-treatment ANC of less than 100/uL and nine deaths occurred further than 6 months of IST. The median time to death was 570 days. The overall 10-year survival for the entire cohort was 80%; long term survival in patients who responded to IST was 89%. We conducted a separate analysis in the age group of 18–21. In total, 35 patients in this age group were treated with h-ATG-based IST from 1989 to 2006. The overall hematologic response rate was 60% (21/35) of which 7 relapsed (33%). The overall response rate among children (< 18) was about 75% which is higher than what is observed in older patients at our institution (response rate of about 60%). The response rate in young adults approximates the overall adult response rate. The long term survival in pediatrics patients who respond to IST is very favorable at about 90%. IST remains a very good alternative in pediatric patients who lack an HLA-matched sibling donor and should be offered as initial therapy prior to an alternative HSCT.
TCR Vbeta Repertoire Analyses in Childhood Aquired Servere Aplastic Anemia: A New Predictor for IST Response?.
Authors: Hubner Bernd , Fuhrer Monika , Gombert Michael , Schuster Friedhelm R. , Harbott Jochen , Borkhardt Arndt
Published: April 2020

Acquired Severe Aplastic Anemia (aSAA) is a rare disease characterized by pancytopenia and bone marrow hypocellularity. There is good clinical and laboratory evidence that a T-cell mediated autoimmune mechanism directed against stem- and progenitor cells located in bone marrow plays a major role in the pathogenesis of aSAA. Immunosuppressive therapy (IST) and bone marrow transplantation (BMT) are the two treatment options. One of the key issues selecting the appropriate treatment is the long latency period of 4–6 months before a response to IST. In a large multicenter prospective study in children we could show that the severity of the disease (i.e. degree of granulocytopenia < 200/μl) is predictive for response to IST. The spectratyping (immunoscope)-technique reflects the T-cell diversity by determining the T-cell receptor (TCR) CDR3 length polymorphisms. Since the grade of polymorphism is proportional to the number of peaks detected, it is possible to determine a complexity score which reflects the diversity of the T-cell population. Herein, we prospectively analyzed the clonality of Vbeta TCR-repertoires in bone marrow of children with aSAA (n=6). After treatment with IST we correlated the initial diversity of the T-cell repertoire with their response 4–6 months after the start of IST. Moreover, we addressed the question weather the bone marrow T-cell repertoire is skewed by the expansion of specific cells, which indicates an antigen driven immune response located in BM. For that we compared the complexities of bone marrow (BM) and respective peripheral blood (PB) derived lymphocytes. The Vbeta profiles in bone marrow compared to PB were skewed in aSAA patients (n=4) most apparent in the CD8 population (figure). The complexities in the healthy control group (n=3) were similar in both compartments. Furthermore, in patients with a haematological response (n=3) a decrease in complexity of T-lymphocytes was present in BM compared to IST non-responders (n=3). The decreased complexity reflects a high number of monoclonal expansions in BM as determined by sequencing analysis of 37 (CD4) and 55 (CD8) CDR3 regions. However, we could not detect preferential usage of specific Vbeta families, J-beta segments or uncommon features in CDR3 size compared to V(D)J-rearranged sequences in the NCBI Prot database. Our findings presented here provide evidence for BM specific proliferation of lymphocytes possibly due to an antigen driven immune response. Moreover, the TCR complexity at diagnosis is associated with a later IST response and could thus be used as an additional predictor for the appropriate treatment.

Figure Figure Figure Figure
Oxidative Metabolism in Red Cells Deficient in Antioxidant Enzymes.
Authors: Johnson Robert M. , Ho Ye-Shih , Yu Dae-Yeul , Kuypers Frans A. , Ravindranath Yaddanapudi , Goyette Gerard W.
Published: April 2020

Since 1–2% of the red cell’s oxyhemoglobin undergoes spontaneous heterolytic dissociation into metHb and superoxide every day, and superoxide is readily converted to H2O2 by superoxide dismutase, the red cell is unavoidably exposed to high levels of reactive oxygen species (ROS). We have used a series of gene-disrupted mice to examine the oxidative defenses of the red cell. Work with glutathione peroxidase deficient [GSHPx(−/−)] red cells has been reported earlier. We here add results with red cells from mice that were catalase deficient (−/−), catalase heterozygotes (+/−), double knockouts [GSHPx(−/−) and catalase (−/−)], or peroxiredoxin II deficient. Catalase(−/−) cells were readily oxidized by exogenous H2O2, as monitored by methemoglobin formation, but no methemoglobin was formed when these cells were exposed to organic peroxides. Catalase heterozygotes were not distinguishable from wild type cells in these assays. This is consistent with the clinical finding that partial catalase deficiency is a benign condition. Red cells lacking both GSHPx and catalase exhibited more metHb formation in response to an H2O2 generating system than did either single knockout, indicating that both enzymes contribute to the defense against exogenous H2O2. Adding catalase deficiency to GSHPx deficiency did not increase Hb oxidation by organic peroxides. With peroxiredoxin-deficient cells, PrxII(−/−), preliminary results found an increased flux of the endogenously generated H2O2 through catalase, which could be detected using 3-amino-1,2,4-triazole (3-AT) inhibition. Earlier work (Lee et al, Blood101:5033, 2003) has demonstrated increased ROS with cell and membrane damage in PrxII(−/−) red cells, but exposure to a flux of H2O2 generated by glucose oxidase produced no increase in metHb formation over that seen in wild type cells. Thus, Hb may be less sensitive to oxidation than is the cell membrane. Prx(−/−) red cells were not more sensitive than wild type red cells to Hb oxidation by organic peroxides. These findings show that all three oxidant defense enzymes play multiple roles in the erythrocyte. All participate in detoxifying endogenously generated H2O2. Calculations with a model of erythrocyte oxidative reactions (Johnson et al, Free Rad. Biol. Med.39:1407, 2005) are consistent with the experimental findings, predicting H2O2 disposal by all three enzymes. Thus, it is not accurate to identify any one of these enzymes as the sole oxidant defense mechanism in the erythrocyte, as all participate in H2O2 removal. GSHPx in addition has a unique role in detoxifying organic peroxides. The data indicate that peroxiredoxin plays a minor role in the red cell’s defense against Hb oxidation. However, PrxII(−/−) mice are anemic, in contrast with the hematologically normal GSHPx(−/−) and catalase(−/−) mice. This suggests that the primary role of PrxII in the red cell may lie in protecting the cell membrane against oxidative damage.
Microvascular Endothelial Cells Express Phosphatidylserine Receptor: A Functionally Active Receptor for Phosphatidylserine-Positive Erythrocytes.
Authors: Setty Yamaja B.N. , Betal Suhita Gayen
Published: April 2020

Phosphatidylserine (PS)-positive erythrocytes adhere to both endothelial cells and the sub-endothelial matrix components. While thrombospondin mediates these interactions, it is not known whether PS-associated erythrocyte-endothelial adhesion occurs in the absence of plasma ligands. Using ionophore-treated PS-expressing control erythrocytes, we demonstrate that PS-positive erythrocytes adhered directly to human lung micro-endothelial cells in the absence of plasma ligands, that this adhesion was further enhanced following endothelial activation with IL-1α, TNF-α, LPS, and hypoxia (2.5- to 8-fold increase), and that this adhesive interaction was selective to erythrocyte-PS. We next explored whether micro-endothelial cells express an adhesion receptor that recognizes cell surface expressed PS (PSR) similar to that expressed on activated macrophages. Using RT-PCR and Western blotting, we demonstrate constitutive expression of both PSR mRNA and protein which were up-regulated in a time-dependent manner following endothelial activation (with maximal increases of 3-fold in mRNA and 2-fold in protein noted at 4 and 6 hour, respectively). While minimal PSR expression was noted on un-stimulated cell surface (8% positivity), endothelial activation up-regulated surface expression of this receptor (35–40% positivity in IL-1α and TNF-α activated cultures). In antibody blocking studies, using both artificially generated PS-positive erythrocytes and also using PS-positive erythrocytes from patient with sickle cell disease, we demonstrate that PSR was functionally active supporting PS-mediated erythrocyte adhesion to activated endothelial cells. Our results demonstrate the existence of a novel functional adhesion receptor for PS on the micro-endothelium which is up-regulated by such pathologically relevant agonists as hypoxia and cytokines.
Cell Membrane Sulfatide Promotes Sickle Cell Adhesion to Endothelium.
Authors: Guchhait Prasenjit , Thiagarajan Perumal , Lopez Jose A.
Published: April 2020

Sickle cell disease (SCD) affects millions of people worldwide, and is associated with significant morbidity and mortality. Although the clinical manifestations of the disease are very complex, much of the cause can be ascribed to occlusion of small vessels by the sickle red blood cells (RBCs). More than 30% of all deaths in SCD are due to the vasoocclusion, which results in ischemia, multiorgan failure and strokes. The proximate cause for vasoocclusion appears to be an increased adhesiveness of sickle cells to the vessel wall, and we postulate that the exposure of sulfatide on sickle cells accounts for their adhesive phenotype. Sulfatide binds with high affinity to many of the adhesion proteins known to be involved in cell adhesion to subendothelium and endothelium, including von Willebrand factor (VWF), thrombospondin (TSP), laminin and P-selectin. We therefore compared the expression and distribution of sulfatide in sickle cells to that in normal RBCs. When examined by flow cytometry using a previously described Alexa fluor-conjugated single-chain variable fragment (scFv) antibody, PA38, we found that sickle cells displayed more sulfatide on surface than normal RBCs (mean fluorescence 1.6±0.5 Vs. 0.9±0.3, p<0.05, n=6). When we examined sulfatide distribution by confocal microscopy using the labeled PA38, we found it to label more intensely in sickle cells than the normal RBCs and to be distributed heterogeneously, with areas of intense staining. The heterogeneous distribution suggested that the sulfatide might exist within membrane-microdomains/lipid rafts. We tested this possibility by sucrose density centrifugation of detergent lysates (1% Triton X-100) of erythrocyte ghosts from sickle and normal cells and found that sulfatide was distributed in raft fractions, as defined by being in the fractions containing the raft marker flotillin-1. Consistent with an important role for sulfatide in sickle vaso-occlusion, we found that both normal and sickle RBCs attached under flow to the surface of histamine-activated human umbilical vein endothelial cells (HUVEC). The sickle RBCs adhered more avidly, as they were able to rest higher shear stresses (1.86 and 2.5 dyne/cm2) than the normal RBCs before detaching Greater than 50% of the initial adhesion was inhibited by treatment with the anti-sulfatide scFv, PA38. We obtained similar results in terms of the greater shear resistance of sickle cells and the ability of PA38 to inhibit adhesion when we compared the adhesion of sickle and normal RBCs to surfaces coated with the adhesive ligands such as VWF (the ultra-large form) and laminin. Thus, our study elucidates an important role of red cell membrane sulfatide in sickle cell adhesion to the endothelium and to adhesive ligands, and suggests that this mechanism is important pathophysiologically in the development of sickle vaso-occlusion. Sulfatide distribution into lipid rafts may allow the formation of adhesive patches that facilitate adhesion.
The Ligand Occupancy of Endothelial Protein C Receptor Switches the PAR-1 Dependent Signaling Specificity of Thrombin from a Disruptive to a Protective Response in Endothelial Cells.
Authors: Rezaie Alireza R. , Bae Jong-Sup , Yang Likui , Manithody Chandrashekhara
Published: April 2020

It has been hypothesized that activated protein C (APC) exerts its cytoprotective and antiinflammatory activities through the endothelial protein C receptor (EPCR)-dependent cleavage of protease activated receptor 1 (PAR-1) on vascular endothelial cells. Noting that

the activation of protein C on endothelial cells requires thrombin, relative to APC, thrombin cleaves PAR-1 with ∼3–4-orders of magnitude higher catalytic efficiency, and PAR-1 is a target for the proinflammatory activity of thrombin, it is not understood how APC can elicit a protective signaling response through the cleavage of PAR-1 when thrombin is present.

In this study, we demonstrate that EPCR is associated with caveolin-1 in lipid rafts of endothelial cells and that its occupancy by the Gla-domain of protein C/APC leads to its dissociation from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway through coupling of PAR-1 to the pertussis toxin sensitive Gi-protein. Thus, when EPCR is bound by protein C/APC, the PAR-1 cleavage-dependent protective signaling responses in endothelial cells can be mediated by either thrombin or APC. These results provide a new paradigm for understanding how PAR-1 and EPCR participate in protective signaling events in endothelial cells.
Identification of a New Genetic Determinant Controlling Human Hematopoietic Stem Cell Engraftment.
Authors: Takenaka Katsuto , Prasolava Tatiana K. , Wang Jean C.Y. , Mortin-Toth Steven M. , Khalouei Sam , Gan Olga I. , Dick John E. , Danska Jayne S.
Published: April 2020

Transplantation of human hematopoietic stem cells (HSC) has been the most important clinical application of stem cell biology. A key discovery enabling successful HSC transplantation was the recognition that HSC engraftment is controlled by human leukocyte antigen (HLA) polymorphism. Although HLA disparity plays a key role in graft rejection, graft failure can occur even in patients receiving an HLA-identical transplant, suggesting that additional, as yet uncharacterized, factors modulate engraftment. We have identified a new genetic determinant that controls the outcome of human HSC transplantation. Xenotransplantation in the non-obese diabetic/severe combine immune-deficient (NOD.SCID) mouse is the best functional assay for human HSC, based on their ability to repopulate recipient animals. We show that NOD.SCID mice provide significantly better support for human hematopoietic grafts than mice of different strain backgrounds carrying equivalent immunodeficiency mutations. To identify the molecular basis for this strain-specificity, we used positional genetics combined with in vitro and in vivo assays of human hematopoiesis. We generated reciprocal congenic strains between NOD and the related non-obese diabetes resistant (NOR) strain which is 88% identical to NOD, and found that support of human hematopoiesis is conferred by variation in a single gene, signal regulatory protein α (Sirpα) on chromosome 2. Human HSC were unable to engraft NOD.SCID mice carrying the NOR allele of Sirpα. NOD SIRPα displays 24 amino acid variations compared to NOR and C57BL/6, concentrated in the immunoglobulin IgV-like domain of this Ig-superfamily member. SIRPα is expressed on myeloid cells and mediates signals that modulate diverse macrophage functions. Indeed, lentiviral gene transfer of the NOD Sirpα variant into NOR macrophages restored their ability to support human hematopoiesis in long-term chimeric stroma-based assays. CD47, the only known cellular ligand of SIRPα, is ubiquitously expressed and modulates multiple cellular actions on hematopoietic cells including platelet activation and adhesion, and leukocyte adhesion and cytokine production. NOD SIRPα demonstrates enhanced binding to human CD47 compared to the NOR protein, suggesting that differential interaction with CD47 underlies the strong effect of Sirpα variation on support of human hematopoiesis in vitro and in vivo. Our findings reveal a novel SIRPα-dependent, macrophage-mediated mechanism critical in HSC transplantation. Future analysis of Sirpα variants within human populations could identify alleles associated with hematopoietic support in BM failure syndromes or correlated with outcomes in clinical transplantation.
Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells.
Authors: Bae Jong-Sup , Rezaie Alireza R.
Published: April 2020

It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.
Endogenous Elevations of Short Chain Fatty Acids (SCFAs) Can Up-Regulate Embryonic Globin Gene Expression.
Authors: Bhatia Himanshu , Hallock Jennifer , Sterner Lauren , Miyazaki Toru , Dean Ann , Little Jane
Published: April 2020

Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders. Since SCFAs can affect embryonic and fetal globin gene expression, we examined their role during development. Murine globin gene expression, β-type (embryonic βH1, and epsilon-y, εY, and adult βmajor), and alpha (α)-type (embryonic zeta, ζ, >α, adult α), were compared between wildtype (wt) and transgenic mice, in which a key enzyme for SCFA metabolism, PCCA, had been knocked out (PCCA−/−, (Miyazaki et al, 2001). E10.5 PCCA−/− yolk sac (n= 9), showed increased α, βH1 and ζ gene expression, at respectively 2-, 2.6- and 1.6-fold relative to wt (n=13, p<.05), and εY gene expression, at 1.7-fold (p=0.07). The embryonic-to-adult globin gene switch was modestly delayed in yolk sacs from E12.5 PCCA−/− (n=9) vs. wt (n=4) and E 14.5 PCCA−/− (n=6) vs. wt (n=6). % embryonic β-type globin gene expression (% βH1 and εY of total β globin) was 77±6 PCCA−/− and 74±3 wt at E12.5, p=n.s., and 42±13 PCCA−/− and 21±3 wt at E14.5, p<.05; % emvbryonic α-type expression (% ζ of total α) was 32±3 PCCA−/−, 25±1wt at E12.5, p<.05 and 7±2 PCCA−/− and 4±1 wt at E14.5, p<.05). Embryonic globin gene expression in E 12.5 and 14.5 fetal livers was not different between PCCA−/− and wt embryos. Cultures of pooled E14.5 wt fetal liver cells (FLCs, n=4 separate experiments), however, suggested that embryonic globin genes can be activated in FLCs. The percent of total β-type globin gene expression that was embryonic after culture with butyrate (1mM) was 11.6±2.6%, with propionate (2.5 mM) was 3.6±0.2%, and insulin/erythropoietin or basal media was 0.03±0.03% and 0.42±0.26% respectively (p<.05 relative to SCFAs). Dose-response with propionate (n=2 seaparate experiments) suggest inadequate endogenous propionate levels for activation in PCCA −/− fetal liver, as % embryonic β-type globin gene expression rose above basal levels only at concentrations of 1 to 5 mM (2.5 mM maximal) but not at <0.6 mM. We conclude that endogenous SCFAs, at levels achievable in vivo can activate embryonic globin gene expression during development in the murine yolk-sac. However, higher levels than achievable endogenously currently are necessary to produce this effect in murine fetal livers.
Regulation of TLR Expression and Activation in Human Mesenchymal Stromal Cells by IFN-a, IFN-g and TNF-a.
Authors: François Moïra , Romieu-Mourez Raphaëlle , Boivin Marie-Noëlle , Galipeau Jacques
Published: April 2020

Members of the toll-like receptor (TLR) family bind to pathogen-associated molecular patterns and subsequently induce the activation of the innate and adaptive immune responses. We investigated here the expression and role of TLRs in mesenchymal stromal cells (MSC), which are bone marrow-derived mesenchymal progenitors with recently described antigen presenting cell (APC) properties [Stagg et al, Blood (March 2006), Romieu et al., JI (in press)]. We observed that human MSCs express basal levels of TLR3 and TLR4, and these responded to their respective ligands, e.g. polycytidylic acid (poly I:C) or lipopolysaccharide (LPS), by the production of IL-6, IL-8 and IL-1b but not of active IL-12 p70 dimer. Exposure of MSCs to TNF-a increased the expression of TLR2 and TLR4 while IFN-a strongly upregulated TLR3 expression. By contrast, IFN-g had only a marginal effect on the expression of any TLR. IFN-a and poly I:C, as well as IFN-g and poly I:C or LPS were observed to work in synergy for the up-regulation of expression of several cytokines or inflammatory mediators, such as IL-12 p35 (IL-12A), TNF-a, RANTES, TRAIL, IFN-b and NOS2A in MSC. In contrast, none of these treatments induced the upregulation of IL-12 p40 (IL-12B), which may explain the lower levels of IL-12 p70 produced in MSCs compared to primary macrophages. Since c-Rel/p50 NF-kB dimers as well as IFN-induced IRF-1 were shown to control IL-12B and/or IL12A promoters, respectively, we next investigated expression of these transcription factors in MSCs. Results showed that MSC expressed the ubiquitous RelA NF-kB subunit but displayed significantly lower expression of c-Rel compared to macrophages. IRF-1 was upregulated following treatments with IFN-g or, to a lesser extent, with IFN-a. Therefore, the lower levels of IL-12 p70 seen in MSCs compared to macrophages may be explained by the absence of c-Rel in MSCs, which hinders the upregulation of IL-12A and IL-12B after exposure to single TLR ligands. However, the induction of IRF-1 by IFN-g or IFN-a may explain the synergistic effect observed in the upregulation of IL-12A in MSC treated with IFN-g or IFN-a and TLR ligands. In conclusion, TLR expression in human MSCs plays a role in their immune activation, although the outcome of this activation is different from the result of macrophage activation by TLR.
IREM-1 Is a Cell Surface Receptor Expressed in AML Blasts: Potential for Targeted Immunotherapy in AML.
Authors: Zhao Xiaoxian , Korver Wouter , Singh Shweta , Hsi Eric D. , Abo Arie
Published: April 2020

Background: Acute myeloid leukemia (AML) in adults is associated with a poor prognosis, with many patients suffering from treatment-resistant relapse. Novel targeted therapies are needed. Immune Receptor Expressed by Myeloid Cells (IREM-1), a member of the CD300L gene family, is an inhibitory receptor expressed by myeloid cells. We have generated monoclonal antibodies (mAbs) specific for IREM-1, characterized IREM-1 expression in AML blasts and explored the therapeutic potential of these antibodies in AML.

Methods: mAbs were generated against the extracellular domain of IREM-1. Fresh peripheral blood or bone marrow cells from AML patients, healthy donors, or cell lines OCI-AML-2 (IREM-1+) and K562 (IREM-1−) were used for expression studies by flow cytometry (FC) with FITC-conjugated mAbs. Immunohistochemistry (IHC) was carried out on Tissue Microarrays (TMAs) of normal human tissues. mAbs were evaluated for their ability to induce lysis in Complement-dependent cytotoxicity (CDC) assays against cell lines in vitro and AML patient cells ex vivo.

Results: By IHC, normal tissues lacked expression of IREM-1, with the exception of spleen and tonsil. However, using FC, expression of IREM-1 was demonstrated on monocytes, granulocytes and dendritic cells, but not on lymphocytes and platelets. FC of AML blasts showed that 18/24 (75%) cases expressed IREM-1 (range: 23.8–93.2%). The 6 negative cases of IREM-1 include 2 AML cases of inv (16) (p13q22). IREM-1 was not detectable in acute lymphocytic leukemia (ALL) blasts (n=5). CD34+ hematopoietic stem cells of control bone marrows were largely negative (n=7), only one case shown low level expression. Functionally, anti-IREM-1 antibodies mediated CDC in OCI-AML-2, while no effect was seen in IREM-1 negative K562 cells. Human embryonic kidney 293 cells only became susceptible to anti-IREM-1 antibody mediated killing after transfection with IREM-1. Furthermore, an anti-IREM-1 mAb showed dose-dependent CDC activity in all IREM-1+ AML cases tested (n=4), while no CDC activity was observed against IREM-1− AML cases (n=3) and ALL blasts (n=3).

Conclusion: Our study identifies IREM-1 as a novel cell surface antigen expressed in majority of AML blasts. We have generated mAbs that are capable of mediating CDC activity against AML blasts. Currently we are testing the killing activity of anti-IREM-1 chimeric Ab and undertaking mouse xenograft model to evaluate the therapeutic impact of this target in vivo, which would offer opportunities for therapeutic intervention.
A Single Nucleotide Polymorphism in CD19 Defines a Novel Target for Immunotherapy of B Cell Malignancies with CD4+ Cytotoxic T Cells.
Authors: Spaapen Robbert M. , Lokhorst Henk M. , van den Oudenalder Kelly , Otterud Brith , Dolstra Harry , Leppert Mark , Minnema Monique C. , Bloem Andries C. , Mutis Tuna
Published: April 2020

The Graft-versus-Tumor (GvT) effect of HLA-matched allogeneic stem cell transplantation (allo-SCT) is largely mediated by donor-derived alloreactive CD4+ and CD8+ T cells. Major targets of this curative effect are the minor Histocompatibility antigens (mHags) expressed on the malignant cells. Here we report the first mHag encoded by a hematopoietic gene and recognized by HLA class II (HLA-DQA1*05/B1*02)-restricted CD4+ T cells. This antigen is encoded by a single nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, a highly important target antigen for immunotherapy of almost all B cell malignancies. We identified this antigen using a novel and powerful genetic strategy, in which a phenotype-genotype correlation scanning was the key step for fine-mapping the genetic locus defined by pair-wise linkage analysis. In functional assays, CD4+ T cells specific for the CD19L-encoded mHag mediated effective peptide-dependent maturation of DCs and polarized them to produce significant levels of interleukin-12. In another assay, the CD19L-mHag-specific T cells facilitated the proliferation of a CD8+ mHag-specific T cell clone in an antigen-dependent manner. Even more important, they also lysed CD19L-positive malignant cells, illustrating the therapeutic advantages of targeting this CD19 L -derived, HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.
Leukemia-Reactive Cytotoxic CD8+ and CD4+ T-Cells Specific for Novel WT-1 Epitopes Are Generated In Vitro by Sensitization with Overlapping Pentadecapeptides (15-mers) Spanning the Wilms Tumor Protein.
Authors: Doubrovina Ekaterina , Dupont Jacob , Kanaeva Elena , O’Reilly Richard J.
Published: April 2020

The Wilms Tumor protein, WT-1 is differentially expressed in 60–80% of acute leukemias and CML. Evidence also suggests WT-1 expression in clonogenic leukemia stem cells, making WT-1 an attractive target for T-cell therapy. However, to date, only a limited number of immunogenic epitopes have been identified. To address this limitation, we employed a pool of 15-mers with 10 amino acid overlaps spanning the complete sequence of WT-1 loaded on autologous monocyte derived dendritic cells or EBV transformed BLCL to sensitize T-cells from normal donors of varied HLA genotypes. Specific 15-mers eliciting WT-1 specific responses were identified by quantitating IFNγ+ CD8+ or CD4+ T-cell responses to secondary stimulation with 15-mer subpools organized in a mapping grid with single overlaps. Phenotypes of T-cells producing IFNγ+ in response to identified specific 15-mers were determined by FACS analysis. We identified the HLA class I or II allele presenting each epitope by assessing the cytolytic activity and/or cytokine production of the pooled-peptide sensitized T-cells in response to 2° stimulation with a panel of PHA blasts loaded with the identified 15-mer, each sharing a single HLA allele with the T-cell donor. T-cells from each of 15 donors tested generated WT-1 peptide reactive T-cells in response to the WT-1 peptide pool. Of 23 epitopes identified that elicited T-cell responses, 19 were heretofore unrecognized epitopes presented by HLA A0201(N=4), A0301(N=2), A2402 (N=2), B3501(N=3), B0701(N=1), B4402(N=2), B3808(N=1), DRB, 0401(N=2), DRB10402(N=1) and DRB1 0701(N=1). Each of the 15 new epitopes presented by class I alleles elicited IFNγ+ CD8+ T-cells that were also cytotoxic against leukemic blasts sharing the restricting allele. All four epitopes presented by class II HLA alleles elicited IFNγ+ CD4+ T-cell responses, of which 2 were also cytotoxic against HLADR+ leukemia blasts expressing the restricting allele. Responses to these newly identified epitopes were comparable to or exceeded those elicited by the known immunogenic peptide epitope 126–134RMFPNAPYL presented by HLAA0201. Like RMF-specific T-cells, T-cells specific for 4 of the new epitopes tested to date do not exhibit activity against normal hematopoietic progenitor cells. Thus, by sensitizing T-cells from unselected normal donors, with autologous DCs loaded with a pool of overlapping 15-mers spanning WT-1, we have generated T-cells specific for a series of new epitopes of WT-1, each, presented by a distinct class I or II HLA allele, which also exhibit HLA-restricted cytotoxic against WT-1+ leukemic cells. Clinical trials of adoptive therapy with WT-1 specific T-cells generated by this approach are being initiated for adoptive therapy.
A Novel Measurement of the Rate of Spontaneous Mutations in Human Myeloid Cells.
Authors: Araten David J. , Mulloy James C. , Krejci Ondrej , DiTata Kimberly
Published: April 2020

The mutation rate (μ) is likely to be a critical parameter in leukemogenesis, but has not previously been measured in human myeloid cells. Using the PIG-A gene as a sentinel, we have recently measured the mutation rate in B-lymphoblastoid cell lines (BLCLs) and shown that it is elevated in cell lines derived from lymphoid malignancies. Here we have measured μ in normal human CD34+ cells derived from cord blood samples that have been transduced with an AML-ETO fusion gene, growing under the influence of cytokines. PIG-A is an X-linked gene essential for the synthesis of glycosylphosphatidylinositol (GPI). Due to X-inactivation in females and hemizygosity in males, a single mutation can produce the GPI (−) phenotype. From patients with PNH, it is known that a broad spectrum of mutations can inactivate PIG-A. Mutants do not express GPI-linked surface proteins such as CD55 or CD59 and do not bind to the FLAER reagent (which binds GPI directly), but do express transmembrane proteins (e.g. CD45). To measure the rate of new mutations in vitro, we first flow-sorted each culture to collect CD59(+) cells, in order to exclude pre-existing mutants. Then we expanded the collected cells over 3 weeks; using cell counts, we calculated population doublings in culture (d), which ranged from 5.1 to 7.4. Cells were then stained sequentially with biotinylated FLAER, murine anti-CD55 and anti-CD59 antibodies, rabbit anti-mouse PE and streptavidin-PE secondary conjugates, and CD45-FITC. Live cells were identified by forward and side scatter, propidium iodide exclusion, and expression of CD45. The mutant frequency (f) was calculated as the number of GPI (−) cells [which do not bind anti-CD55, CD59 or FLAER] divided by the total number of cells analyzed. An average of 1.2 million cells were analyzed to detect rare mutants. The mutation rate was calculated by the formula μ = f/d. Among 5 cultures derived from 2 different cord blood samples, the mean mutation rate was 12.4 × 10−7 (range 5.1 to 21.4 × 10−7) per cell division. This value is consistent with mathematical models of the mutation rates in human populations as well as our previous experimental determination of μ in BLCLs. We also analyzed 6 cell lines in which p53 had been disrupted by various methods, including shRNA, a dominant-negative p53 mutant, and introduction of HPV E6/E7. Overall, disruption of p53 resulted in a 30% increase in the mutation rate (p=.02). We conclude that spontaneous mutations in human myeloid cultures are rare but measurable and that disruption of p53 results in a modest increase in μ. We predict that the ability to measure this critical parameter in human hematopoietic cells will facilitate the investigation of the role of specific oncogene and tumor suppressor mutations in inducing hypermutability.