bluuebunny/crossref_metadata_2025 huggingface dataset:
Lenalidomide Up-Regulates SPARC and Inhibits In Vitro Growth of the Malignant Clone in Myelodysplastic Syndrome Patients with 5q Deletion.
Authors: Pellagatti Andrea , Jädersten Martin , Forsblom Ann-Mari , Cattan Helen , Christensson Birger , Emanuelsson Emma K. , Merup Mats , Nilsson Lars , Samuelsson Jan , Sander Birgitta , Wainscoat James S. , Boultwood Jacqueline , Hellström-Lindberg Eva
Published: April 2020

The immunomodulatory drug lenalidomide induces cytogenetic remissions in 75% of patients with myelodysplastic syndrome (MDS) and del(5)(q31) through unknown mechanisms. We investigated the in vitro effects of lenalidomide on growth and maturation in differentiating erythroblasts from MDS patients with del(5)(q31) (n=13) and from healthy controls (n=10). Lenalidomide selectively inhibited growth of del(5q) erythroblasts, while not affecting normal cells, including cytogenetically normal cells from MDS del(5q) patients. The inhibitory effect was more pronounced in erythroid than in myeloid cells. In order to gain insight into the mode of action of lenalidomide and to identify the molecular targets of this drug, we have investigated the gene expression profiles of the lenalidomide-treated and untreated intermediate erythroblasts from MDS del(5q) patients (n=9) and from healthy controls (n=8). GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), covering over 47,000 transcripts representing 39,000 human genes, were used. Treatment with lenalidomide significantly influenced the pattern of gene expression in del(5q) intermediate erythroblasts, with up-regulation of VSIG4, PPIC, TPBG, and SPARC in all samples, and down-regulation of many genes involved in erythropoiesis, including HBA2, GYPA, and KLF1, in most samples. Up-regulation of SPARC (median 4.4-fold, range 2.4–9.5) is of particular interest since SPARC, a gene with known tumor suppressor functions, is both anti-proliferative and anti-angiogenic, and is located at 5q31–q32, within the commonly deleted region in MDS 5q- syndrome. Activin A was one of the most significant differentially expressed genes between lenalidomide-treated cells of MDS del(5q) patients and healthy controls. Activin A is a member of the transforming growth factor-beta superfamily, with pleiotropic functions including apoptosis of hemopoietic cells. We conclude that lenalidomide specifically inhibits growth of del(5q) erythroid progenitors, while not affecting cytogenetically normal cells. These novel findings suggest that up-regulation of SPARC and Activin A may underlie the potent effects of lenalidomide, in particular growth inhibition and anti-angiogenesis, in MDS with del(5)(q31). The localization of the SPARC gene to the CDR of the 5q- syndrome is intriguing and, in relation to the findings of the present study, we suggest that SPARC may well play a role in the molecular pathogenesis of the 5q- syndrome.
Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.
Authors: Adams Gregor B. , Alley Ian R. , Chabner Karissa T. , Chung Ung-il , Marsters Emily S. , Weinstein Lee S. , Kronenberg Henry M. , Scadden David T.
Published: April 2020

During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.
Suspension of Vitamin K Antagonists Is Not Required for Cataract Surgery: A Prospective Study in a Cohort of 135 Consecutive Patients on Long-Term Anticoagulant Therapy.
Authors: Pollio Berardino , Demarie Giuseppe A. , Ocello Patrizia , Delios Grazia , Tucciarone Marco , Aitoro Gabriele , Perna Rossella , D’Ardia Stefano , Ladetto Marco , Bauchiero Luigi , Girotto Mauro
Published: April 2020

The perioperative management of oral anticoagulant therapy (OAT) often arouses controversy between surgeons and internists.

In geriatric patients, cataract surgery for those who are taking vitamin K antagonists is a common clinical procedure. Phacoemulsification requires a 3 mm incision involving a tissue devoid of blood vessels. This study reports the experience of an Italian Anticoagulation Management Service (AMS) with 135 anticoagulated patients on long-term anticoagulant therapy who underwent phacoemulsification performed by the same ophthalmologist team from January 2001 to December 2005. The patients received either topical (30%) or peribulbar (70%) anaesthesia. Data were collected by physicians with specialized software, but the dosage of oral anticoagulant was manual. Two oral vitamin K antagonisists are available in Italy: acenocumarol and warfarin. We prepared all patients in accordance with the following standardized protocol :

the scheduled dose was always omitted the day before surgery an INR measurement was provided 3–5 days before the invasive procedure; if the patient’s INR was below 3, we simply omitted the scheduled dose of the day before cataract surgery if the patient’s INR was above 3, we withheld two or more scheduled doses to allow the INR to fall to 2.5 or less 1 hour before cataract surgery, INR was measured if the patient’s INR was below 2.5, phacoemulsification was performed

Results: This standardized procedural protocol allowed the surgeon to carry out phacoemulsification with INR always below 2.5. We observed only one peribulbar bleeding (0.7%) during peribulbar anaesthesia before the corneal incision was made. No thromboembolic complications were registered during three months of follow up. We compared our results with the data of an earlier cohort of 7014 conventional patients who underwent this eye surgery in the same ophthalmologic institute. We did not observe statistical differences between the two groups with regard to hemorragic complications.

Conclusions: The risk of thromboembolism when antithrombotic therapy is interrupted is a well-grounded concern, particularly for patients with mechanical heart valves. Low molecular weight heparin bridging is a valid but more complicated alternative. Our study demonstrates the feasibility and safety of this simple standardized protocol which avoids OAT interruption. Therefore, we conclude that in patients receiving OAT, it is not necessary for the anticoagulant effect to wear off before cataract surgery is performed.
rNAPc2, a Novel Inhibitor of Tissue Factor/Factor VIIa Complex, Suppresses Lung Metastasis and Synergizes with 5-FU or Bevacizumab in Mouse Models of Colorectal Cancer.
Authors: Zhao Jingsong , Aguilar Gerard , Deitcher Steven R. , Funk Walter , Abo Arie
Published: April 2020

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastasis, anti-angiogenesis, and anti-thrombosis activities. rNAPc2 has been previously shown to inhibit both the primary growth and metastasis of murine B16 melanoma and Lewis lung carcinoma in mice. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with the progression of malignancy. To explore the therapeutic potential of rNAPc2 during tumor growth and progression, we tested rNAPc2 efficacy in experimental colorectal cancer in mice. Both primary and metastatic colorectal tumor models were used in the current study and rNAPc2 was given to mice via daily intraperitoneal injections. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). The doses and dosing regimens of rNAPc2 used in these murine models of colorectal cancer were well tolerated by recipient mice without major complications of hemorrhage or any other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.
Safe Interruption of Ongoing Thrombosis by the Protein C Activator Thrombin Analog, W215A/E217A; a Comparison to Enoxaparin in Primates.
Authors: Marzec Ulla M. , Bush Leslie , Tucker Erik , Gruber Andras , Hanson Stephen R. , Di Cera Enrico
Published: April 2020

Antithrombotic agents are effective but pose significant bleeding risk, thus safer new agents are needed. Since the infused double mutant human thrombin (W215A/E217A; WE) binds endothelial thrombomodulin, activates protein C locally in the intraluminal boundary layer, and prevents thrombus formation without hemostasis impairment, we investigated whether WE could also interrupt the growth of existing thrombi. WE was compared to standard interventional doses of the low molecular weight heparin, enoxaparin (Sanofi) in awake baboons. Thrombosis was initiated by interposing a two part device consisting of a 2 cm, 4 mm ID polyethylene terephthalate vascular graft (DVG) followed by a 2 cm silicone rubber expansion chamber, 9 mmID. Upon initiation of blood flow at 100 mL/min, wall shear rate of 265 sec-1 in the 4 mm ID segment (arterial type flow) and 29 sec-1 in the expansion chamber (venous type flow), platelet thrombus was monitored by gamma camera imaging of autologous 111In-labelled platelets for 60 min or 90 min. Fibrin accumulation was quantified by homologous 125I-labelled fibrinogen. Hemostasis was assessed as template bleeding time prolongation. APTT was also monitored. Treatments started 25 min after the initiation of thrombosis by enoxaparin, 0.5 mg/kg (LD) and 1 mg/kg (HD) IV bolus or WE, 7.5 μg/kg/hr (LD) and 15 μg/kg/hr (HD) infusion (1/3 dose given as a bolus followed by IV infusion over 60min) and compared to saline infused controls. All interventions were given systemically, downstream from the device and 5–6 studies comprised each study group. Treatment with enoxaparin at 25 min after initiation of thrombus growth resulted in a reduction of platelet deposition of 19 % with the LD and 76 % with the HD after 35 min of therapy in the expanded venous type segment. WE reduced venous thrombosis by 45 % and 65 % at the two doses studied and was further reduced to 65 % and 80 % following 65 min of WE infusion as compared to controls. Arterial type thrombosis was reduced by WE by 34 % and 39 % respectively for the two doses studied. HD enoxaparin reduced arterial type thrombosis by 37 %. Fibrin accumulation paralleled the platelet deposition. APTT was prolonged 1.57- and 1.87-fold respectively for the WE doses without any bleeding time prolongation. Enoxaparin prolonged the APTT 1.4- and 1.75-fold, similar to WE but with a 1.13- and 1.75-fold prolongation of the bleeding time. In conclusion, the double mutant human thrombin, WE, effectively interrupted both venous and arterial type growing thrombi at very low doses without detectable compromise to hemostasis. Likewise, enoxaparin at the doses tested had similar efficacy but with a significantly prolonged bleeding time at the 1 mg/kg bolus dose. WE might provide a safer alternative to heparins in the treatment of acute progressive thrombosis.
Pediatric Experience with Fondaparinux in Deep Venous Thrombosis.
Authors: Grabowski Eric F. , Bussel James B.
Published: April 2020

Over the past two years we have employed daily SC fondaparinux as longterm anticoaguation in five pediatric patients who presented with deep venous thrombosis (DVT; 3 cases) or DVT combined with heparin-induced thrombocytopenia (HIT) confirmed by ELISA for HIT antibody ( 2 cases). The Table summarizes characteristics of the patients. In all cases, anti-factor Xa levels were obtained 3–4 hrs after the third dose and were in the range of 0.5 to 1.2 IU/ml, calibrated to fondaparinux. Indications for fondaparinux were outpatient treatment of HIT or suspected HIT (2 cases), a desire for true once-daily outpatient dosing of a SC agent (3 cases), or a desire to minimize volume of agent given SC (2 cases). Dosing for Pt 1 was based upon a calculated enhanced renal excretion of fondaparinux in very young children, assuming drug metabolism to vary as (body mass}0.75 rather than as body mass. Pt 5 had previously required 10,000 IU of a low MW heparin SC bid, in view of her heterozygous ATIII deficiency, and suffered recurrent hematomas at the injection sites. These almost entirely resolved with use of fondaparinux and a consequent 60% reduction in the daily injected volume of anticoagulant. There have been no episodes of recurrent thrombosis and no bleeding complications after a mean pt follow-up of 8.5 months. Thus, fondaparinux can be employed safely and effectively in both young children and adolescents, with and without HIT.

Patient Characteristics Pt Number Age HIT Risk factors Mo on fondaparinux 1 13 mo present anticardiolipin antibody 8 2 14 yrs suspected anticardiolipin antibody 4.5 3 17 yrs absent FV Leiden, May-Thurner Syndrome, elevated Lp(a), BCPs 18 4 17 yrs absent cervicle rib (subclavian vein thrombosis) 10 5 18 yrs absent ATIII deficiency, atretic IVC, elevated Lp(a), BCPs 2
Effects of the Factor Xa Inhibitor Apixaban on Venous Thrombosis and Hemostasis in Rabbits.
Authors: Wong Pancras C. , Watson Carol A. , Crain Earl J. , Luettgen Joseph M. , Ogletree Martin L. , Wexler Ruth R. , Lam Patrick Y.S. , Pinto Donald J. , Knabb Robert M.
Published: April 2020

Apixaban is a small-molecule, potent, reversible and direct inhibitor of human coagulation factor Xa (FXa) (Ki = 0.08 nM) with greater than 30000-fold selectivity over other coagulation proteases. It is orally bioavailable in rats, dogs, chimpanzees and humans, and is currently in clinical development for prevention and treatment of thromboembolic diseases. The objective of this study was to compare antithrombotic and bleeding time effects of apixaban and warfarin in rabbits. We selected rabbits as the animal model because apixaban has similar potency in inhibiting human and rabbit FXa, but is less potent in inhibiting rat and dog FXa (Ki (nM): 0.16, rabbit; 1.3, rat; 1.8, dog). We induced the formation of venous thrombus by placing threads in the vena cava, and bleeding by the incision of cuticles in anesthetized rabbits. Apixaban was infused IV 60 min before the initiation of thrombosis. Warfarin was administered orally for 4 days before the study. Thrombus weight averaged 73±5 mg and bleeding time averaged 183±7 s in the vehicle group (n=6 per group). Apixaban and warfarin inhibited the formation of venous thrombus in a dose-dependent manner. The estimated IC50 for apixaban was 0.16±0.04 μM. At antithrombotic doses studied, apixaban did not alter blood pressure and heart rate, suggesting hemodynamic effects are not likely to be involved in its antithrombotic activity. At the antithrombotic ID80, apixaban and warfarin increased bleeding time by 9±4% and 516±24%, respectively (n=6 per group). At this dose, apixaban increased ex vivo activated partial thromboplastin time and prothrombin time to 1.4±0.1 and 1.7±0.1 times control, respectively, and warfarin increased prothrombin time to 4.5±0.1 times control. Apixaban at antithrombotic doses selectively inhibited ex vivo FXa but not thrombin activity. In summary, these findings demonstrate potent activity of apixaban as a selective FXa inhibitor exhibiting potential strong efficacy in prevention of venous thrombosis at doses that preserve hemostasis and produce less increases in systemic anticoagulation than warfarin.
Induction of the NKG2D Ligands Expression by Histone Deacetylase Inhibitor and Its Impact on the Susceptibility of Leukemic Cells to the Cytolytic Activity of NKG2D-Expressing Cells.
Authors: Kato Naoko , Tanaka Junji , Sugita Junichi , Toubai Tomomi , Miura Yoko , Ibata Makoto , Shono Yusuke , Ota Shuichi , Kondo Takeshi , Asaka Masahiro , Imamura Masahiro
Published: April 2020

Innate immune cells such as natural killer (NK) cells play a crucial rule in antitumor immune responses. NK cells have functionally opposite receptors for activating and inhibiting signals to exert cytotoxicity. NKG2D is a C-type lectin-like activating receptor and is expressed in various immune cells, including NK cells, NKT cells, CD8+α/β+T cells, and γ/δ+T cells. NKG2D recognizes its ligands, MHC class I-related chain A and B (MICA, MICB). NKG2D ligands are not present on normal cells but are induced by various stresses such as viral infection. Moreover NKG2D ligands are expressed in many malignant cells including hematological malignancies. It has been suggested that involvement of NKG2D in NK or CD8+Tcell-mediated cytotoxicity correlates with the expression levels of ligands on target cells. Therefore induction of NKG2D ligands may lead to enhance the sensitivity to NKG2D-mediated cytotoxicity. In this study, we could enhance expression levels of MICA and MICB by treatment with the histone deacetylase inhibitor trichostatin A (TsA) in lymphoid leukemic cell line BALL1 and some primary patients’ lymphoid leukemic cells. Treatment of BALL1 and patients’ leukemic cells with TsA for 12 hr increased MICA and MICB mRNA expression at least by more than 2 fold by Real-time PCR. Treatment of BALL1 with TsA for 12 hr increased MICA and MICB protein expression on the cell surface by more than 2 fold by flow cytometry analysis. These results suggested that expression of MICA and MICB is partly regulated by histone acetylation. Chromatin immunoprecipitation assay revealed that treatment with TsA resulted in increased acetylation of histone H3 and decreased association with the counteracting enzymes of histone acetyltransferases HDAC1 at the MICA and MICB promoter in BALL1 cell and patients’ leukemic cells. To examine the impact of the cytolytic activity of NKG2D-expressing cells on leukemic cells in which expression of NKG2D ligands was induced by TsA treatment, we performed standard 4 hr 51Cr release assays using BALL1 cells and patients’ leukemic cells. Up-regulation of NKG2D ligands by TsA treatment led to enhance the susceptibility of BALL1 and patients’ leukemic cells by 2 or 3 times to the cytolytic activity of NKG2D-expressing cells. Blocking experiment using specific antibodies for MICA and MICB inhibited the NKG2D-expressing cell-mediated cytolytic activity against BALL1 cells. Our results suggest that regulation of NKG2D ligands expression by treatment with chromatin-remodeling drugs may be an effective strategy to enhance the susceptibility of leukemic cells to the cytolytic activity of NKG2D-expressing cells for immunotherapy.
Platelet Factor 4 (PF4) Antigenic Complexes on the Macrophage Surface: Implications for the Pathogenesis of Heparin Induced Thrombocytopenia (HIT).
Authors: Rauova Lubica , Cines Douglas B. , Poncz Mortimer
Published: April 2020

HIT is the most frequent antibody-mediated drug-induced thrombocytopenia and is unique in the associated prevalence of thrombosis. HIT is caused by antibodies to a normal host protein, PF4, complexed to heparin or cellular glycosaminoglycans (GAGs). We have shown that binding of HIT antibodies and a monoclonal HIT-like antibody KKO to platelet surface chondroitin sulfate (CS) follows a bell-shaped curve with respect to PF4 concentration. At external PF4 concentrations below the peak value of <50 μg/mL, heparin dissociates PF4 and thereby decreases surface antigenicity, whereas at PF4 concentrations >50 μg/mL, heparin initially increases antigenicity. Surface GAGs on macrophages differ from those on platelets in several ways, including having a higher affinity for PF4. In addition, the binding of HIT-IgG elicits the elaboration of tissue factor. Therefore, we characterized the binding of HIT IgG antibody to macrophages exposed to different concentrations of PF4. Binding of PF4 to the surface of human/murine macrophages also followed a bell-shaped curve with peak antigenicity at the same PF4 concentration as seen on platelets. However, HIT antigenticity persisted at lower PF4 concentrations. For example at a PF4 concentration of 6 μg/mL, six times as much KKO bound to macrophages as to platelets. PF4-coated monocytes were also more resistant to the abrogation of KKO binding by heparin. At therapeutic concentrations of heparin, HIT antigenicity actually increased on macrophages exposed to optimal concentrations of PF4, while platelet surface antigenicity was nearly eliminated. Enzymatic removal of surface GAGs showed that unlike platelet surfaces, where CS is the major GAG involved in HIT antigenicity, a more complex pattern was seen on macrophages that included heparan sulfates, CS and likely dermatan sulfates. Thus, these studies show that expression of surface HIT antigenic complexes on macrophages develop at much lower concentrations of PF4 than platelets and that these complexes are more resistant to removal by heparin. The enhanced avidity of macrophages for PF4/GAG complexes may also contribute to the persistent risk of thrombosis for at least several days after heparin exposure has been stopped. Whether measurement of antigenic complexes on macrophages would identity patients at higher risk of thrombosis and whether reduction in the risk of thrombosis requires measures directed at the integrity of these complexes in addition to use of direct thrombin inhibitors are under investigation.
Assessment of the Clinical Benefit of Prophylactic Platelet Transfusion in Thrombocytopenia Using Novel Methodology To Measure Thrombus Formation and Thrombin Generation.
Authors: Cauwenberghs Sandra , Feijge Marion A.H. , Heemskerk Johan W.M. , van Pampus Elisabeth , Curvers Joyce
Published: April 2020

Patients developing severe thrombocytopenia during chemotherapy treatment are prophylactically transfused with platelets. Two novel methods were developed for measuring improved hemostasis in thrombocytopenic patients and to identify patients with aberrant responsiveness. In whole blood, platelet adhesion and thrombus formation on collagen was measured under flow. In platelet-rich plasma, platelet-dependent coagulation was assayed by automated measurement of thrombin generation. Thirty-eight thrombocytopenic patients were transfused with platelets stored in plasma or synthetic medium (PASII), and the transfusion effect on hemostatic activity was evaluated. Experimental conditions were established, where the outcome of flow and thrombin generation tests linearly increased with the platelet concentration (R2=0.98, p=0.001 and R2=0.98, p=0.008 respectively), and informed on the activation properties of platelets. In 35 out of 38 patients, transfusion with platelets in plasma or synthetic medium resulted in increased adhesion and thrombus formation under flow, and in increased platelet-dependent coagulation. The increase in platelet count after transfusion predicted 57% (P=0.001) of the improvement in function. Transfusion with platelets in plasma or synthetic medium increased the hemostatic activity with 0.63±0.30% and 0.68±0.48%, as determined with either platelet function test, with subtle differences between the two storage media. In acute graft-versus-host-disease, platelet-dependent coagulation was higher than in other patients, while thrombus formation was normal. In conclusion, altered thrombus formation and/or aberrant coagulation at low platelet counts can be detected with these novel developed and validated techniques.
Lack of Alloantibody Response to Red Blood Cell Antigens in Juvenile Mice Following Transfusion: Non-Immunogenic or Tolerogenic Response?.
Authors: Hendrickson Jeanne E. , Josephson Cassandra D. , Chadwick Traci E. , Zimring James C.
Published: April 2020

Introduction: Alloimmunization to red blood cell (RBC) antigens occurs in approximately 6% of chronically transfused adults, with some patient populations having significantly higher rates of alloimmunization. Despite these numbers, multiple studies have failed to detect alloantibodies to RBC antigens in neonates following transfusion, even after repeat exposures from the same donor. This is consistent with two possible scenarios:

RBC antigens are non-immunogenic in neonates, or RBC antigens are tolerogenic.

If early exposure to RBC antigens is a tolerogenic stimulus, then the potential clinical implications are considerable. To allow a controlled testing of these hypotheses, we have constructed a murine model of alloimmunization to RBC antigens in pediatric populations.

Methods: Weight-adjusted volumes of leukoreduced blood from mHEL donors, expressing membrane bound hen-egg lysozyme as a unique RBC antigen, were transfused into B10.BR recipients at 3 weeks (juveniles) or 3 months (adults) of age. To assess the effects of inflammation, which we have recently reported augments RBC alloimmunization in adult mice, half of the recipients were treated with Poly (I:C), a double-stranded RNA that mimics viral inflammation. Two weeks post-transfusion, RBC alloimmunization was determined by measuring serum anti-HEL IgG levels by ELISA.

Results: In 2 out of 2 experiments, as part of an ongoing study, whereas all 10 adult mice mounted a low level alloantibody response by ELISA, only one out of 10 juvenile mice made a detectable alloantibody response. The non-responsiveness of juvenile mice was not significantly altered by inflammation, as only 2 out of 10 juvenile mice treated with Poly (I:C) mounted an alloantibody response.

Conclusion: Our data demonstrate that transfused RBCs are weakly immunogenic in adult mice, but only rarely result in alloimmunization in juvenile mice. This is consistent with the pattern of alloimmunization seen in adult versus neonatal humans. Since our model system detects alloantibodies by ELISA, which is 2000 fold more sensitive than agglutination assays using mHEL RBCs, these data support the notion that the majority of juvenile mice do not make alloantibodies to the mHEL RBC antigen. Additionally, inflammation enhances alloimmunization in juvenile mice to a significantly lesser extent than in adult mice. Ongoing studies will utilize the ability of this model to assess if juvenile transfusion recipients are tolerized, by challenging with an immunogenic stimulus (transfusion in the presence of inflammation or subcutaneous injection of HEL/CFA ). If these studies demonstrate that tolerance to RBC antigens occurs after exposure in the neonatal or juvenile period, then the potential clinical implications are significant.
Determinants of PF4/Heparin Immunogenicity in a Murine Model of HIT.
Authors: Arepally Gowthami M. , Qi Rui , Hollingsworth John , Suvarna Shayela
Published: April 2020

Heparin-Induced Thrombocytopenia (HIT) is a life-threatening immune-mediated reaction caused by platelet factor 4 (PF4)/heparin complexes. In recent in vitro studies, we have shown that human and monoclonal HIT antibodies recognize and optimally bind to ultra-large complexes (ULC; MW>670kDa) formed at equimolar ratios of PF4 and heparin. We undertook the following studies to determine if the in vivo immunogenicity of PF4/heparin complexes showed similar requirements for stoichiometric molar ratios of PF4:heparin (PHR). Using a previously described murine immunization model, mice were immunized with antigen by retro-orbital injection daily for 5 days without adjuvant. Blood was collected at baseline and at weekly intervals for 4–6 weeks. Plasma was tested for antibody development by a murine PF4/heparin (mP+H) ELISA. To determine the effects of PHR on mP+H antibody production in vivo, PF4 was mixed with unfractionated heparin (UFH) at molar ratios of 2:1, 1:1 or 1:2 and injected into mice (n=5/cohort). Antibody levels (mean A450nm ± SD) of mice injected with PHR 2:1 (0.79 ± 0.77) were higher than mice injected with PHR 1:1 (0.23± 0. 2), or PHR 1:2 (0.09 ± 0.02, p<0.05 for PHR 2:1 v. PHR 1:2). Because in vitro studies showed that UFH was far more capable of generating ULCs when mixed with PF4 than low-molecular weight heparin (LMWH) or fondaparinux, mice (n=5/cohort) were injected with buffer, or mPF4 mixed with UFH, or enoxaparin (a LMWH) or the pentasaccharide, fondaparinux at doses that are therapeutically equivalent to doses given to humans. Antibody levels (mean A450nm ± SD) were significantly higher in mice injected with UFH (3.340 + 0.15) than in mice injected with buffer (0.0451 ± 0.013, p<0.001 for buffer v UFH), LMWH (0.8526 ± 0.44, p<0.001 for LMWH v UFH) or the pentasaccharide (0.80 ± 0.0.7, p<0.001 for pentasaccharide v UFH). Because PF4/heparin ULCs are composed of repeating structural antigenic units, we next investigated the role of toll-like receptors (TLRs), a family of pattern recognition receptors (PRRs) that recognize repeating structural determinants on microbes and foreign antigens. To determine if mP+H complexes engage TLRs, we injected mP+H into wild-type (WT, n=5) or mice deficient in MyD88 (n=7), an adaptor molecule critical for intracellular signaling by most TLRs. Antibody levels (mean A450nm ± SD) did not significantly differ between WT (1.5 ± 0.6) and MyD88 null mice (0.9 ± 0.5). In summary, these studies indicate that in vivo antibody responses to mP+H complexes are critically dependent on stoichiometric ratios favoring ULC formation and that immune activation by PF4/heparin is independent of MyD88 signaling pathways.
Notch 4 Plymorphism Associated with Risk of Aplastic Anemia.
Authors: Kim Hee Nam , Choi Jeong-Hwa , Yun Ju-Hyun , Kim Yeo-Kyeoung , Lee Il-Kwon , Yang Deok-Hwan , Shin Myung-Geun , Kook Hoon , Hwang Tai Ju , Park Moo-Rim , Jo Deog-Yeon , Won Jong Ho , Kim HyeoungJoon
Published: April 2020

Aplastic anemia (AA) is a heterogeneous disease characterized by bone marrow aplasia and peripheral blood pancytopenia, a profound deficit of hematopoietic stem and progenitor cells. Although the causes for aplastic anemia are multiple, the pathogenic mechanism leading to the disease might be limited to two main pathways: environmental factors and genetic susceptibility. The mammalian Notch family has four homologs, Notch1 to 4. Notch4 signaling plays a role in lineage commitment in hematopoiesis. The human Notch4 gene is located at the MHC on chromosome 6p21.3. Many single nucleotide polymorphisms (SNP) have been mapped to the Notch4 locus. Therefore, we hypothesized that some of these variants could potentially influence the expression level of Notch4 protein. To investigate the role of the two Notch4 rs2071282 (+2763 C>T, Pro204Leu) and 422951(+3323 A>G, Thr320Ala) polymorphisms in susceptibility to AA, a case-control study was conducted in Chonnam National University Hwasun Hospital between March 2002 and June 2006. We genotyped in 151 AA patients and 552 healthy control subjects using PCR-RFLP. Here, we report that rs2071282 CT genotype is significantly associated with increased risk for AA. Using subjects with the rs2071282 CC as a reference group, the odds ratio (OR) of CT is 1.77 (95% CI 1.05–2.98, p=0.032). The haplotype CG is significantly associated with the risk of AA. Using subjects with the haplotype CA, the OR of haplotype CG is 0.70 (95% CI 0.50–0.98, p= 0.037).

In conclusion, we were able to identify two polymorphisms of Notch4, therefore, theses results suggest that Notch polymorphism may play an important role in the susceptibility to AA.

Distribution of Notch polymophisms in aplastic anemia patients and controls Control (%) AA(%) OR (95% CI) p Rs 422951 AA 302(54.8) 94(62.3) 1 AG 216(39.2) 49(32.5) 0.72(0.478–1.098) 0.128 GG 33(6.0) 8(5.3) 0.54(0.23–1.279) 0.163 Total 551 151 Rs 2071282 CC 481(87.1) 120(81.1) 1 CT 68(12.3) 28(18.9) 1.769(1.049–2.982) 0.032 TT 3(0.5) 0 - - Total Haplotype frequency for AA and controls Haplotype Control (%) AA(%) P (Chi-square) OR (95% CI) p *Haplotype frequencies were estimated from genotype data using Gibbs- algorithm.*OR and 95%Cis were estimated using based logistc regression and adjusted for sex and age C-A 743(67.7) 206(70.0) 0.062 1 C-G 281(25.6) 60(20.4) 0.70(0.50–0.98) 0.037 T-A 72(6.5) 28(9.5) 1.49(0.90–2.47) 0.126
ULBP and MICA/B as Novel Indicators for Both Diagnosis of Immune-Mediated Marrow Injury and Favorable Response to Immunosuppressive Therapy in Bone Marrow Failure Syndromes.
Authors: Hanaoka Nobuyoshi , Kawaguchi Tatsuya , Horikawa Kentaro , Nagakura Shoichi , Tsuzuki Yasuchika , Yonemura Yuji , Nakakuma Hideki
Published: April 2020

Bone marrow failure syndromes (BFS) including aplastic anemia (AA), myelodysplastic syndromes (MDS), and paroxysmal nocturnal hemoglobinuria (PNH) are considered to harbor immune-mediated marrow injury. Indeed, immunosuppressive therapy (IST) with antithymocyte globulin and cyclosporine ameliorates the hematopoiesis in BFS patients, despite its limit: infrequent relapse of marrow failure after IST, resistance of some patients to IST, and untoward effects like infection. To overcome the difficulties, molecular targeted therapy is alternative. However, neither incitement of marrow injury nor target molecules on marrow cells recognized by cytotoxic lymphocytes has been identified. We have currently suggested that NKG2D ligands such as ULBP and MICA/B serve as triggers for immune-mediated marrow injury in PNH (Hanaoka, Blood2006;107:1184). ULBP and MICA/B are stress-inducible membrane proteins that appear in infection and transformation. The ligands share NKG2D receptor on lymphocytes such as NK, CD8+ T, and γδ T cells and promote activation of the lymphocytes. Cells expressing the ligands are then deadly injured by NKG2D+ cells (Groh, PNAS USA 1996; Cosman, Immunity 2001). In this background, we attempted to confirm the clinical significance of the expression of NKG2D ligands in BFS. The ligands were detected by flow cytometry on the granulocytes and marrow cells in 47 (53%) of 89 patients with BFS: 28 (56%) of 50 patients with AA; 11 (65%) of 17 patients with PNH; 8 (36%) of 22 patients with MDS; and none of 17 healthy individuals. The membrane expression of the ligands was supported by their increase in plasma. It is then conceivable that blood cells were exposed to a certain stress to induce NKG2D ligands in the patients, leading to NKG2D-mediated marrow injury. There was a close association of the expression of NKG2D ligands with both progression of marrow failure and favorable response to IST. Thus, we propose that the NKG2D ligands not only are feasible predictors for both immune-mediated marrow injury and IST effects, but also serve for potential targeted therapy in BFS.
Predictive Markers of Myelodysplastic Syndrome in the Bone Marrow of Patients with Fanconi Anemia.
Authors: Al-Rahawan Mohamad M. , Alter Blanche P. , Bryant Barbara J. , Elghetany M. Tarek
Published: April 2020

Fanconi Anemia (FA) patients are at increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Impaired cell cycle regulation may have a role in this predisposition. Over-expression of p53, Ki-67 and survivin proteins has been reported in association with MDS. We analyzed bone marrow samples from 138 individuals: 31 FA, 17 acquired aplastic anemia (AA), 40 sporadic low-grade MDS (MDS-1: 35 refractory cytopenia with multilineage dysplasia [RCMD], 5 RCMD with ring sideroblasts [RCMD-RS]), 11 high-grade MDS (MDS-2: refractory anemia with excess blasts [RAEB]), 12 AML, and 27 normal. To identify cell cycle regulation abnormalities that predict risk of MDS or AML in FA patients, we analyzed bone marrow morphology, cellularity, and immunohistochemical expression of cell cycle regulation proteins: p53 (cycle arrest and apoptosis), Ki-67 (cell proliferation), caspase-3 (apoptosis), and survivin (anti-apoptosis). Protein expression was classified as over-expressed if it was detected in a percentage of cells exceeding the highest values observed in normal marrow. Between group comparisons were made using the Fisher exact test. More FA marrows were hypocellular versus sporadic MDS-1, MDS-2 and AML (p <0.0001 in all). p53 was over-expressed at similar rates in FA, MDS-1, MDS-2 and AML patients (12/25 vs. 22/35, 4/11 and 8/12; p = 0.3, 0.7 and 0.3, respectively); p53 was not over-expressed in AA (0/13, p = 0.003). Ki-67 was also over-expressed at similar rates in FA, MDS-2 and AML patients (9/18 vs. 3/11 and 7/12, p = 0.3 and 0.7), in whom over-expression was higher than in AA or MDS-1 (0/12 and 4/35). Survivin expression was more common in FA than in MDS-1, MDS-2, AML or AA (12/16 vs. 2/32, 0/10, 2/12 or 1/13; p <0.01 for all comparisons). In contrast, the caspase-3 over-expression was higher in MDS-2 than in FA (4/11 vs. 0/18, p = 0.014). 7/31 FA patients had RCMD marrow changes; however, there was no correlation between morphologic features of myelodysplasia and the over-expression of cell cycle markers in these patients. p53 over-expression was frequent in FA, MDS-1, MDS-2, and AML; this distinguishes these disorders from AA. Previous studies found no p53 gene mutations in FA; thus, p53 over-expression observed here may represent upregulation of p53, or diminished p53 protein catabolism. Ki-67 over-expression may be a marker for higher grade MDS. The prevalence of over-expression of survivin was unique to FA, distinguishing it clearly from any of the acquired disorders (AA, MDS or AML), and may be a marker for any inherited bone marrow failure syndrome. Our cell cycle marker results suggest that patients in general in whom there is over-expression of Ki-67 and/or p53 may be at higher risk of evolution to MDS/AML; a longitudinal study is needed to address this prediction.
Long-Term Follow up of Children with Apalstic Anemia Treated with Immunosuppressive Therapy.
Authors: Pongtanakul Bunchoo , Das Prabodh K. , Dror Yigal
Published: April 2020

Background: Immunosuppressive therapy (IST) is the alternative first line treatment in children with aplastic anemia (AA) who have no HLA match siblings available. The long-term outcome of patients with AA who survive after IST is unknown. We evaluated outcomes of children with AA treated with IST at long-term follow up.

Methods: we retrospectively reviewed the hospital records of children with AA from 1984 to 2004, treated at our institution with horse-derived antithymocyte globulin (hATG) 160 mg/Kg over 4 days, short course of prednisone and cyclosporine (CS).

Results: Forty two patients were treated with IST (25 boys, 18 girls). The median age at diagnosis was 8.5 years. Twenty nine (69%), eight (19%) and five (12%) patients were diagnosed with severe, very severe, and moderate AA, respectively. Nine patients (21%) had hepatitis associated AA. Twenty seven patients (64%) received one course of ATG and fifteen (36%) received 2 courses (8 received 2 courses of hATG and 7 received 1 course hATG and 1 course rabbit derived ATG). Eleven patients (26%) required G-CSF. Median follow up time was 53.3 months (range 3–244 months). Twenty six patients (62%) had a complete response (CR), eight (19%) had a partial response (PR) and eight (19%) had no response (NR). Two patients relapsed after one course of IST and needed a second course of IST and both of them had a partial response. Median time to discontinuation of CS was 13 months. Nine patients (21%) died (7 with NR and 2 with PR) and 33 patients (79%) are alive. Two patients developed myelodysplastic syndrome (MDS) 21 and 19 months post IST; both received long-term G-CSF (18 and 14 months) and had PR after 2 courses of IST. Five of 33 patients (15%) who survived had significant hypertension after CS was discontinued and one required continuous antihypertensive medication.

Conclusion: The results of this study shows promising response in children with AA treated with IST. Hypertension and MDS are late complication. Longer follow up in these patients is warranted to definite the accurate rates of the late complications and risk factors.
Monitoring Immune Function in the Allogeneic Transplant Recipient: Predicting Graft vs Host Disease (GvHD) and Infection.
Authors: Anaissie Elias J. , Diaz Jose A. , Grazziutti Monica L. , Miceli Marisa H. , Fox-Cottler Michele , Barlogie Bart , van Rhee Frits
Published: April 2020

Background: GvHD, intensity of immunosuppression (ISP) and infection are major causes of toxicity post allogeneic stem cell transplantation (allo-SCT). Yet, no method reliably predicts these complications. We explored the use of a novel FDA-approved immune function assay to determine whether an immunologic response to changes in immunosuppressive therapy for GvHD could predict risk of GvHD and infection and allows tailoring ISP and infection prophylaxis. The ImmuKnow is an assay of global T-cell function that measures increased ATP production after stimulating CD4+ T-helper cell and has been shown useful for managing ISP therapies in solid-organ transplantation.1 We hypothesized that ImmuKnow could assist in predicting risk of GvHD and infection.

Patients and Methods: Between March 2004 and March 2006, 24 adult allo-SCT patients (pts) (mean age 48 years; range 23–70; 50% females) underwent blood sampling for determining ImmuKnow at various times post-HSCT (267 assays, mean of 11/pt). Results were correlated with clinical course (infection, GvHD or event-free). Underlying diseases included acute myelogenous leukemia (n=9), chronic myelogenous leukemia (n=4), myeloma (n=3) and others (n=8).

Results: during the testing period, GvHD was documented in 10 pts [(acute, n=3; chronic, n=7] and 20 infections occurred [bacteremia (n=9); CMV viremia (n=4); others (n=7)]. The average immune function (ng/mL ATP) of GvHD pts was significantly higher than that of event-free pts (385 +/− 102 vs. 189 ng/mL +/−78 respectively), p<0.001. Pts with infections (13 pts, 71 events) had a mean 86 ng/mL ATP (+/−87), also statistically different (p<0.001) from event-free. ROC analysis showed that the positive predictive value (PPV) for GvHD with ImmuKnow > 279 ng/mL was 74 with a sensitivity of 90% (95% CI, 76.9–97.2) and specificity of 83% (95% CI 73 - 91) and an odds ratio (OR) for GVHD of 42 (95% CI, 13 - 136, p<0.001). For infection, ImmuKnow values < 94 ng/mL were 70% sensitive (95% CI, 59 - 80) and 87% specific (95% CI, 77 - 94) with a PPV of 85 and OR of 17 (95% CI, 8–39, p<0.001).

Conclusions: these preliminary data suggest that the ImmuKnow assay is an objective marker of risk for infection and GvHD post-HSCT. Tailoring intensity and duration of ISP to reduce risks of GvHD and infection in an individual patient may be possible and may improve outcomes. Validation of these preliminary data is ongoing.
Influence of Chromosomal Aberrations on Response to First Line Therapy in B-Cell Chronic Lymphocytic Leukemia: Interim Analysis of PALG-CLL3 Randomized Study Comparing Cladribine Plus Cyclophosphamide vs Fludarabine Plus Cyclophosphamide.
Authors: Robak Tadeusz , Blonski Jerzy Z. , Wawrzyniak Ewa , Palacz Aleksandra , Gora-Tybor Joanna , Jamroziak Krzysztof , Piszcz Jaroslaw , Kloczko Janusz S. , Koczkodaj Dorota , Kowal Malgorzata , Dmoszynska Anna , Mital Andrzej , Hellmann Andrzej
Published: April 2020

Impact of cytogenetic abnormalities on treatment with different purine nucleoside analogs in patients (pts) with B-cell chronic lymphocytic leukemia (B-CLL) is largely unknown. One of objectives of PALG-CLL3 trial, comparing cladribine plus cyclophosphamide (CC) with fludarabine plus cyclophosphamide (FC) in previously untreated progressive B-CLL, was to verify the response to treatment in subsets of pts characterized by common cytogenetic abberrations. Chromosomal abnormalities were assessed using fluorescence in situ hybridization (FISH) on interphase nuclei of lymphocytes on whole blood smears prior to the start of the study treatment. Pts were screened for trisomy 12, deletions (del) 11q, del 13q and del 17p using DNA probes: CEP12, LSI: ATM, D13S319 and p53 (Vysis), respectively. For the purpose of the present interim analysis complete cytogenetic results were available in 133 pts out of 423 pts included to the study. In this group the chromosomal aberrations were detected in 102 pts (77%) including single abnormalities observed in 69 pts (52%) and two or more aberrations in 33 pts (25%). Thirty-one pts (23%) exhibited a normal interphase FISH pattern. The most frequent single abnormality was del 13q found in 38 pts (29%), while del 17p, trisomy 12 and del 11q were identified in 14 pts (11%), 11 pts (8%), and 6 pts, (5%), respectively. The most frequently observed associations of chromosomal aberrations were: del 13q with del 11q (11 pts, 8%) and del 13q with del 17p (10 pts, 8%). Four pts (3%) revealed three chromosomal abnormalities including association of trisomy 12/del 11q/del 13q in two pts, trisomy 12/del 11q/del 17p in one pt and del 11q/del 13q/del 17p in one pt. Overall, treatment was completed and response assessed in 113 out of 133 pts with known FISH pattern. In this group of pts del 17p was the only chromosomal abnormality that correlated significantly with treatment outcome. Pts with del 17p (21, 19%) had lower probability to achieve a complete response (CR) (0.044). Interestingly, in independent analyses of both treatment arms, the negative impact of 17p was seen in pts treated with FC (p=0.002), but not in pts treated with CC (p=0.6). Moreover, comparing response rates between treatment arms we found that CC was superior to FC in terms of complete response in pts with del 17p (57% CR in CC v 14% CR in FC arm, p=0.04). In conclusion, chromosomal abnormalities can be detected in majority B-CLL pts requiring treatment. Our preliminary results suggest that CC combination may have some advantage in terms of CR achievement in B-CLL pts harboring del 17p.