bluuebunny/crossref_metadata_2025 huggingface dataset:
Blocking Complement-Mediated Hemolysis Using RNA Aptamers That Bind Complement Component C8.
Authors: Nishimura Jun-ichi , Nimjee Shahid M. , Jiang Haixiang , Howard Thad A. , Pitoc George A. , Rusconi Christopher P. , Omine Mitsuhiro , Chao Nelson J. , Frank Michael M. , Sullenger Bruce A. , Ware Russell E.
Published: October 2019

A recent pilot study in patients with paroxysmal nocturnal hemoglobinuria (PNH), using a humanized monoclonal antibody that binds human complement C5 and inhibits terminal complement protein activation, suggests that blocking complement is a potentially effective therapeutic option for PNH. However, since C5 is critical for proper regulation of inflammatory responses as well as complement activation, terminal complement proteins with more restricted function may represent better targets. The pore-forming C5-9 complex includes several protein targets, but persons with C9 deficiency have measurable evidence of in vitro complement activation, indicating that the C5b-8 complex can cause lysis in the absence of C9. Blocking complement at C9 may not, therefore, completely prevent complement-mediated hemolysis and adequately protect PNH erythrocytes. Accordingly, we used in vitro selection methodology to identify high affinity nuclease-resistant RNA aptamers that bind specifically to human complement C8. Aptamers bound C8 with a Kd of 1.4nM and 54.1% after round 7. C8 aptamers identified after rounds 4 to 7 of selection were cloned and sequenced, and aptamer clones were screened for in vitro binding and complement-inhibitory activity. Aptamers were first incubated with purified C8 at a concentration sufficient to induce 50–70% hemolysis, then antibody-sensitized erythrocytes and C8-depleted serum were added. One C8 aptamer clone (4–101) had strong inhibitory hemolytic activity with 91.9% inhibition at 250nM, compared to only 12.3% inhibition observed with random aptamers at 250nM. Aptamer clone 4–101 bound C8 with a Kd of 15nM and a Bmax of 74.2%. Inhibition of hemolysis by C8 aptamer clone 4–101 was enhanced by adding a previously published C5 aptamer clone f8/c11, (TCTCATGCGCCGAGTGTGAGTTTACCTTCGT, Immunopharmacology42:219, 1999) in a modified human serum hemolytic assay using total human serum: 54.4% inhibition was observed using C8 aptamer 4–101 at 500nM, versus 83.0% inhibition using C8 aptamer 4–101 with C5 aptamer f8/c11 in combination at 500nM. Thus, C8 aptamer clones can efficiently inhibit complement-mediated lysis, with enhanced activity in combination with a known C5 aptamer. These data indicate that combinatorial blocking aptamers that bind terminal human complement proteins can efficiently inhibit the complement pathway, and therefore represent a novel potential therapeutic option for patients with PNH. Based on these in vitro data, therapeutic C8 aptamers should be considered for the in vivo treatment of PNH patients with massive hemolysis. C5 inhibitors could still be useful, however, as an adjunct therapy if C8 aptamers insufficiently inhibit complement-mediated lysis.
Subcutaneous Dosing of CRA-027483, a Small Molecule FVIIa Inhibitor, Prevents Arterial Thrombosis in a Baboon Model.
Authors: Marzec Ulla M. , Sukbuntherng Juthamas , Dalrymple Stacie A. , Young Wendy B. , Vijaykumar Dange , Young Peter R. , Hanson Stephen R.
Published: October 2019

Vessel injury may expose tissue factor leading to the activation of FVII, initiation of coagulation, and thrombosis. A small reversible inhibitor of FVIIa (CRA-027483), dosed by subcutaneous (SC) injection, was investigated in an arterio-venous shunt thrombosis model in non-anticoagulated awake baboons. Drug pharmacokinetics (PK) and pharmacodynamics (PD) were studied in non-shunted baboons; PK and PD results were closely correlated with ~ 100% bioavailability following SC drug administration. Thrombosis was initiated by interposing within the shunt a segment of porous expanded (poly)tetrafluoroethylene vascular graft (ePTFE, 4mm ID), filled with relipidated tissue factor. Upon initiation of blood flow at 100mL/min (wall shear rate =256/sec) thrombus growth was monitored by gamma camera imaging of autologous Indium-111 labeled platelets for 1 hour. Fibrin accumulation was quantified using trace amounts of homologous fibrinogen labeled with I-125. Both bleeding time and prothrombin time (PT), the PD marker, were monitored throughout. Animals were dosed SC 90min prior to the initiation of thrombus formation, a regimen that was predicted from PK/PD to result in a fixed concentration of CRA-027483 during the thrombosis phase. Dosing of baboons with CRA 027483 at 1mg/kg, 2mg/kg and 4mg/kg SC (4–5 animals in each study group) inhibited total platelet deposition on the tissue factor surface by 13±4%, 43±30% and 67±26% respectively, vs. control results. Fibrin accumulation was reduced by 22±9%, 50±34%, and 76±19% respectively for these doses. PT values remained stable throughout the thrombosis phase and increased 1.3-, 1.6- and 2.1-fold over baseline with the escalating doses. Bleeding time measurements were slightly prolonged at the higher doses (7.3±1.6min and 7.1±2.5min for the 2mg/kg and 4mg/kg doses, respectively) as compared to the pre-drug measurement of 3.8±0.7min. We conclude that SC administration of CRA-027483, a reversible small molecule inhibitor of FVIIa, effectively reduces tissue factor-initiated thrombus formation in baboons with minimal hemostatic impairment.
The Oral, Direct Factor Xa Inhibitor BAY 59-7939 Does Not Cross-React with Anti-Heparin/PF4 (Heparin-Induced Thrombocytopenia) Antibodies.
Authors: Walenga Jeanine M. , Hoppensteadt Debra , Iqbal Omer , Neville Brian , Jeske Walter P. , Prechel Margaret , Messmore Harry L. , Misselwitz Frank , Bakhos Mamdouh
Published: October 2019

BAY 59-7939 is an orally bioavailable, small-molecule, direct Factor Xa (FXa) inhibitor in advanced clinical trials for the prevention and treatment of thromboembolic disorders. Unfractionated heparin and the low molecular weight heparins (LMWHs) are the current standards of care for patients requiring anticoagulation. However, their use can be restricted by heparin-induced thrombocytopenia (HIT), which may be associated with severe thrombotic complications. It has been reported previously that fondaparinux, a heparin-derived pentasaccharide that indirectly inhibits FXa, does not cross-react with anti-heparin/PF4 (HIT) antibodies. However, we have shown that increased sulfation of fondaparinux does result in strong cross-reactivity with HIT antibodies, leading to platelet activation/aggregation. Previous studies have shown that direct thrombin inhibitors (DTIs), such as argatroban and lepirudin, do not cross-react with HIT antibodies. Current guidelines for patients who have HIT recommend use of a DTI to prevent or treat associated thrombosis. This study was performed to evaluate whether BAY 59-7939 cross-reacts with HIT antibodies, in order to examine its potential as an alternative anticoagulant for the management of patients with HIT. The effect of BAY 59-7939 on platelet activation mediated by HIT antibodies was examined in sera collected from 63 patients diagnosed with HIT (HIT sera), using platelet aggregation assays, the [14C]serotonin release assay, and flow cytometry for the detection of platelet P-selectin expression and platelet microparticle formation. Heparin, the LMWH enoxaparin, fondaparinux, and the DTI melagatran were included for comparison. BAY 59-7939 did not activate platelets or cause aggregation with any of the HIT sera tested, establishing that there is no interaction between BAY 59-7939 and HIT antibodies. As expected, heparin strongly activated platelets and caused their aggregation, and gave a positive response with 100% of the HIT sera tested. Enoxaparin showed positive responses with 73% of the sera. Of all the HIT sera tested, one exhibited a weak positive response with fondaparinux. As has been observed with other DTIs, melagatran did not cause any platelet activation or aggregation responses with the HIT sera. This study clearly demonstrates that BAY 59-7939, a novel, orally active, direct FXa inhibitor, does not interact with preformed HIT antibodies. Therefore, BAY 59-7939 has potential as a new option for the prevention and treatment of thrombosis in patients with HIT.
TTP889, a Novel Orally Active Partial Inhibitor of FIXa Inhibits Clotting in Two A/V Shunt Models without Prolonging Bleeding Times.
Authors: Rothlein Robert , Shen Jane M. , Naser Najih , Gohimukkula Devi R. , Caligan Thomas B. , Andrews Robert C. , Schmidt Ann Marie , Rose Eric A. , Mjalli Adnan M. M.
Published: October 2019

Based on previous studies showing the efficacy of FactorIXa (FIXa) blockade using an active site-blocked form of this coagulation enzyme, we speculated that partial inhibition of the intrinsic coagulation pathway would offer a novel approach to attenuate intravascular clot formation without promoting untoward bleeding. Here we describe the anticoagulant activity of TTP889, a small molecule partial inhibitor of FIXa activity. TTP889 is orally absorbed with a PK profile that is conducive to once daily dosing. It is selective for FIXa in that it shows little to no activity against several other proteases in the clotting cascade including FXa, FXIa, FXIIa or FVIIa in a unique clotting assay. In vivo, TTP889 inhibited fibrin deposition in a rat arteriovenous (A/V) shunt model. In this model, vehicle treated rats had 104mg ± 43 of fibrin deposited on a silk thread after a 15 minute shunt while TTP889 treated rats had significantly less fibrin deposited (39mg ± 18 p=<0.001). Furthermore, TTP889 inhibited clotting in a porcine A/V shunt model where pressure across a hemodialysis filter that was shunting the carotid artery to the jugular vein was used as an indirect marker of clot formation. In this model, TTP889 at 0.3mg/kg performed as well as 150U/kg of heparin over the 90-minute shunt. Of additional importance, TTP889 has no effect on bleeding times as measured by APTT or ACT assays ex-vivo or by bleeding time and volume from incisions in the skin or spleen in vivo. Together, these data support that TTP889 is a selective partial inhibitor of FIXa activity that offers a novel approach to attenuate clot formation associated with intravascular clotting without promoting untoward bleeding. TTP 889 is currently in Phase II and is being evaluated for the prevention of DVT in hip fracture patients with treatment starting one week post surgery and continued for 3 weeks.
Host Inflammation Increases Alloimmunization to Transfused Red Blood Cells.
Authors: Hendrickson Jeanne E. , Chadwick Traci E. , Roback John D. , Hillyer Christopher D. , Zimring James C.
Published: October 2019

Background: Alloimmunization to RBC is a serious sequelae of transfusion, which can result in hemolytic transfusion reactions and difficulty identifying compatible units for future transfusion. Although any given unit of RBC contains numerous alloantigens, only 6% of transfusion recipients mount a measurable alloantibody response. The factors that determine whether a given transfusion recipient will generate antibodies remains undetermined. Activation of the innate immune system through inflammation is known to have a significant effect on immune responses in other settings. Accordingly, we hypothesized that recipient inflammation would modulate humoral responses to transfused RBC.

Methods: Hen egg lysozyme (HEL) is a well studied antigen. In order to convert HEL into a model blood group antigen, we utilized transgenic mHEL mice, which express HEL on the surface of RBC as a transmembrane protein. Leukoreduced mHEL RBC were transfused into B10. BR recipient mice (H-2k MHC haplotype). To induce host inflammation, recipient mice received an intraperitoneal injection of poly(I:C), which is a double-stranded RNA that mimics inflammation commonly seen with viral infection. The inflammatory effects of poly (I:C) were confirmed by observing activation of macrophages using flow cytometry. Plasma was obtained from recipient mice 7 and 14 days post-transfusion, and anti-HEL antibody titers and isotypes were determined by HEL specific ELISA.

Results: Inflammation of mice with poly (I:C) resulted in a significant increase in the magnitude of anti-HEL IgG synthesis. Whereas mice receiving mHEL RBC alone had only a weak anti-HEL IgG response, treatment with poly (I:C) prior to transfusion resulted in a 15 fold increase in anti-HEL IgG (p value =.0003). In addition, whereas IgG1 and IgG3 were the predominant anti-HEL IgG subtypes in mice receiving mHEL RBC alone, IgG3 and IgG2b predominated in mice treated with poly (I:C) prior to mHEL transfusion. Thus, treatment of transfusion recipients with an agent that mimics inflammation commonly seen in viral infections significantly increased humoral immunity and altered IgG subtype composition.

Discussion: The findings reported here demonstrate that host inflammation with a viral-like agent increases alloimmunization to transfused RBC and suggests that co-existing infections in humans may increase humoral immunization to transfused RBC. In addition, since different IgG subtypes have different hemolytic potentials, the observed deviation may influence the clinical significance of alloantibody formation. These findings provide the rational basis for possible intervention using immunomodulatory agents to prevent alloimmunization or to divert the response to a non-hemolytic IgG subtype.
Mantle Cell Lymphoma: Prognostic Capacity of the Follicular Lymphoma International Prognostic Index.
Authors: Moller Michael B. , Pedersen Niels T. , Christensen Bjarne E.
Published: October 2019

Background: The International Prognostic Index (IPI) is the most commonly used prognostic model in mantle cell lymphoma. However, the prognostic value of IPI is limited. The recently published Follicular Lymphoma International Prognostic Index (FLIPI) is built on variables (age, stage, lactic dehydrogenase, anemia, and nodal disease) which also are pertinent to mantle cell lymphoma. This study was conducted to evaluate the prognostic value of FLIPI in patients with mantle cell lymphoma.

Patients and Methods: A population-based series of 93 patients with mantle cell lymphoma diagnosed in a 7-year period were studied. End points of the study were response to therapy, overall survival, and failure-free survival according to IPI and FLIPI.

Results: Applied to the whole series, FLIPI identified 3 risk groups with markedly different outcome with 5-year overall survival rates of 65%, 42%, and 8%, respectively (P < .0001; log-rank 28.13; figure below). Notably, the high-risk group comprised 53% of patients. In contrast, IPI only allocated 16% of cases to the high-risk group and had a lower overall predictive capacity (log-rank 24.8). When both FLIPI and IPI were included in a multivariate analysis, only FLIPI was related to survival. In patients treated with CHOP-based regimens (n = 45) FLIPI also had superior predictive capacity compared to IPI (log-rank, 18.51 versus 11.37), and again only FLIPI retained significance in multivariate analysis. Multivariate analysis of failure-free survival also identified FLIPI, and not IPI, as independently significant.

Conclusion: FLIPI is the superior prognostic model as compared to IPI and should be the preferred clinical prognostic index in mantle cell lymphoma.

Overall survival according to FLIPI risk groups Overall survival according to FLIPI risk groups
FDG-PET May Be Helpful in the Determination of Transformation to an Aggressive Subtype in Low-Grade Lymphoma.
Authors: Kostakoglu Lale , Goldsmith Stanley J. , Chadburn Amy , Vidan Erez , Schuster Michael , Christos Paul , Coleman Morton
Published: October 2019

The therapeutic management of the patient with transformed low-grade lymphoma is quite different from those with pure indolent disease. In cases of transformation FDG uptake characteristics may help identify an area of transformation in indolent disease and thereby impact management. Our objective was to evaluate the potential of FDG-PET imaging in the determination of transformation

Methods: A total of 60 patients with low-grade lymphoma (48 patients) and those with known transformation to DLCL (12 patients) underwent FDG-PET imaging prior to therapy. In the indolent group, 27 patients were diagnosed with a follicular histology and 11 with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and 10 were with marginal zone lymphoma. A total of 370 lesions were evaluated and SUV’s were obtained for all visualized lesions. Mean values were calculated on a patient and total site basis. Additionally, mean SUV values were calculated for lesions with the highest SUV’s, in groups with indolent and transformation histology.

Results: Results are summarized in the Table. There was a significant difference in the mean SUV’s between the group with transformation and that with indolent lymphoma (7.6 ± 4.6 vs. 4.5 ±2.5). More importantly, the mean values for SUV in the lesions with the highest FDG uptake were significantly different between the two groups (13.8 ±5.3 vs 5.8±2.9). Among the patients who had a biopsy taken from the lesions with the highest SUV’s the range was 8 to 27 and the mean value was 14.9±8.0.

Conclusions: In patients with indolent lymphoma, areas of significant increase in FDG (particularly SUV’s >12) should be considered targets for biopsy.

Differences in SUV’s Between Transformed and Indolent Lesions Patient Number Mean SUV Mean SUV in Hottest Lesions SUV Range in Hottest Lesions SUV: standardized uptake values Patients with Transformation 12 7.6+/−4.6 13.8+/−5.3 8–27 Patients with Indolent Lymphoma 48 4.5+/−2.5 5.8+/−2.9 2–12
Insertional Mutagenesis Identifies Genes That Promote the Immortalization/Self-Renewal of Primary Bone Marrow Progenitor Cells.
Authors: Du Yang , Jenkins Nancy A. , Copeland Neal G.
Published: October 2019

Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of primary mouse bone marrow progenitor cells, which normally have only limited self-renewal. Transduction of bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only a neo marker gene, followed by serial passage in liquid culture containing SCF and IL-3, produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologues and are validated human myeloid leukemia genes.

Integrations are located in intron 1 or 2 where they promote expression of truncated proteins lacking the PR domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Similarly, immortalized cells that are blocked at a very early hematopoietic progenitor stage can be produced by serially passaging the infected cells in SCF and FLT3L. In this case, viral integration deregulates the expression of Setbp1, a gene that is thought to regulate the activity of SET, which is mutated in acute human undifferentiated leukemia.

Interestingly, recurrent retroviral vector integrations at the Evi1 locus have also been recently identified in blood cells of rhesus monkeys transplanted up to 7 years earlier with retrovirally infected CD34+ bone marrow cells (Calmels et al, Blood. in press), and similar results have also been reported for mice (Kustikova et al, Science 2005 308:1171-4). Our results suggest that these cells were selected in transplant recipients due to their increased self-renewal or engraftment potential, which resulted from insertional mutations at Evi1. These studies highlight a new potential problem for human gene therapy, the selection of immortalized cells in transplant recipients that carry activating mutations in human leukemia oncogenes and a possible concomitant increased propensity for developing hematopoietic cancer as the patients age. Based on these studies, it will be important to assay the safety of other retroviral vectors, such as SIN vectors, that may be less apt to activate the expression of an oncogene following viral integration. The immortalization assay we describe here provides a means for assessing this safety.
Analysis of YY1 and XIAP Expression, Proteins That Regulate Resistance, in AIDS-NHL Tissue Arrays.
Authors: Huerta-Yepez Sara , Vega Mario , Gui Dorina , Said Jonathan , Bonavida Benjamin
Published: October 2019

HIV infection is associated with an increased risk of Non-Hodgkin’s B cell lymphoma (AIDS-NHL). AIDS-NHL may arise, in part, because the patients may be immunocompromised and tumor escape takes place. The standard treatment for NHL is chemotherapy, however, many patients become refractory to such treatments. Alternative treatment modalities include immunotherapy, though, even in the presence of an effective anti-tumor response, the tumor may develop mechanisms of resistance to immune-mediated cytotoxicity (e.g., Fas-ligand, TRAIL) and resistance to apoptosis. We have shown that overexpression of the transcription factor Yin-Yang 1 (YY1) is involved in the regulation of tumor cell resistance to FasL-induced apoptosis. The direct role of YY1 was demonstrated in cells transfected with siRNA YY1 which were sensitized to Fas-induced apoptosis (Vega, et al., 2005, Journal of Immunology (In Press)). In addition, we have also shown that overexpression of YY1 and X-linked inhibitor of apoptosis (XIAP) regulate the resistance of tumor cells to TRAIL-induced apoptosis (Ng and Bonavida, 2002, Molecular Cancer Therapeutics1:1051–1058, Huerta-Yepez, et al., 2004, Oncogene23:4993–5003). Hence, we hypothesized that one mechanism of AIDS-NHL immune escape may be due to overexpression of YY1 and XIAP. Tissue arrays containing formalin fixed, paraffin embedded sections from AIDS lymphoma were obtained from the AIDS and Cancer Specimen Resource (ACSR) of the National Cancer Institute (NCI). These arrays consisted of 21 Burkitt, 29 Large Cell Lymphoma, and 6 Small Cell Lymphoma. Immunohistochemistry was performed for the expression of YY1, and XIAP. The arrays were scored and both the percent of positive cells and the intensity were recorded and the data were analyzed statistically. The findings reveal that YY1 and XIAP were overexpressed in the majority of the AIDS-NHL patient specimens. In addition, there was a significant correlation between YY1 and XIAP expression in all 3 types of lymphoma. These studies and studies based on in vitro findings with AIDS-NHL cell lines suggest that overexpression of YY1 and XIAP may be involved in the pathogenesis of AIDS-NHL and are potential markers for tumor unresponsiveness to immune-mediated cytotoxic therapies. Furthermore, inhibitors of YY1 and XIAP expression/activity may be targets for therapeutic intervention when combined with immunotherapy.
Characterization of Platelet TFPI.
Authors: Maroney Susan A. , Friese Paul , Alliman Sarah , Dale George L. , Mast Alan
Published: October 2019

The accumulation of blood borne tissue factor within a blood clot is thought to be important for normal fibrin generation and may cause propagation of the blood clot into an occlusive thrombus. Tissue factor pathway inhibitor (TFPI), the primary inhibitor of tissue factor activity, is present in platelets where it may have an important role in down regulating the activity of circulating tissue factor. Western blot analysis of gel filtered platelet lysates demonstrates the presence of full-length, 43 kDa TFPI. The lower molecular weight, C-terminally truncated forms of TFPI present in plasma are not present in platelet lysates suggesting that full length TFPI is selectively adsorbed from plasma or that TFPI is synthesized within megakaryocytes. Real time PCR analysis of cDNA produced from highly purified platelet mRNA demonstrates transcripts for both TFPI and TFPI-beta, a truncated form of TFPI produced by alternative splicing. TFPI mRNA is about 3000-fold more abundant than TFPI-beta mRNA. TFPI is not released from platelets following stimulation with the thrombin receptor activating peptide SFLLRN (TRAP) indicating that it is not stored as a soluble alpha-granule protein. Instead, it appears to be associated with platelet membranes. It remains largely insoluble following lysis of platelets by repeated freeze thaw or exposure to hypotonic buffer. There is little to no expression of TFPI on the surface of quiescent platelets as assessed by flow cytometry. TRAP stimulated platelets have surface TFPI that is detected by flow cytometry, but the amount varies between experiments. TFPI is consistently detectable by flow cytometry on the surface of coated-platelets that are produced by stimulation with thrombin/convulxin or thrombin/calcium ionophore. Platelet TFPI activity was measured in eight individuals by determining the rate of factor X activation by factor VIIa/tissue factor. Comparison of quiescent and coated-platelets demonstrates a significant increase in the amount of TFPI activity on the surface the coated-platelets (11.0+/−0.3 vs. 38.7+/−17.2 pmole/10 million platelets; p<0.005), but no significant increase in platelets activated with 20 or 200 micromolar TRAP. Since TFPI associates with the endothelial surface through a GPI-anchor, coated-platelets were treated with phospahtidyl inositol specific phospholipase C (PIPLC) to determine if it would remove TFPI. In experiments where PIPLC removed 56% of the CD59 from the platelet surface, only 22% of the TFPI was removed indicating that if platelet TFPI is GPI-anchored, it is expressed in a manner that is relatively protected from PIPLC. Confocal and electron microscopy studies demonstrate that TFPI is distributed throughout the cytoplasm of quiescent platelets without localization to granules suggesting that it may associate with the platelet cytoskeleton.
Measurement of Blood-Borne Tissue Factor Procoagulant Activity in Healthy Individuals and Cancer Patients Using a Novel Assay.
Authors: Tilley Rachel E. , Holscher Todd , Belani Rajesh , Nieva Jorge , Mackman Nigel
Published: October 2019

Tissue factor (TF) is the cellular initiator of the extrinsic pathway of coagulation. Recent studies have reported circulating blood-borne TF (BBTF) antigen by methods including ELISA and flow cytometry. Whether this BBTF is active has been rarely addressed due to the lack of a simple functional assay. We have developed a new assay that measures BBTF procoagulant activity. Whole blood was collected into tubes containing citrate and corn trypsin inhibitor (FXIIa inhibitor). First, we compared the procoagulant activity obtained in detergent lyzed platelets and microparticles with peripheral blood mononuclear cells (PBMCs) following a 5 hour ex vivo stimulation of whole blood with LPS. Platelets and microparticles had 3% (23 ± 10 mU/mL) and 0.1% (1.0 ± 0.4 mU/mL), respectively, of the procoagulant activity of PBMCs (1048 ± 200 mU/mL). The procoagulant activity of PBMCs and platelets was inhibited by >90% in the presence of an anti-TF polyclonal antibody. Next, we determined the time course of TF-dependent procoagulant activity of microparticles and platelets after ex vivo LPS stimulation of whole blood. In both cases, a dramatic increase in microparticle and platelet procoagulant activity was observed after 12 hours of LPS stimulation, with a maximum activity observed at 48 hours (48 mU/mL for microparticles and 1028 mU/mL for platelets at 48 hours). The majority (>90%) of this procoagulant activity was TF-dependent. Therefore, we subsequently analyzed combined platelet and microparticle (P+MP) fractions for BBTF activity. We determined functional BBTF activity in P+MP fractions from the blood of healthy individuals with and without LPS ex vivo stimulation. In healthy individuals, very low levels of BBTF procoagulant activity was detected in the absence of LPS stimulation (0.22 ± 0.09 mU/mL) and this activity was decreased on average by 28% with an anti-TF antibody (0.16 ± 0.1 mU/mL; p = 0.01). In contrast, P+MP from LPS stimulated blood demonstrated on average 30 fold higher procoagulant activity, of which >90% was TF-dependent. Cancer is associated with an increased susceptibility to develop pathological thrombosis. Using our new assay, we demonstrate a significant elevation of the procoagulant activity of P+MP fractions from advanced (stage IV) solid tumor patients of varying histology’s (n=14; 0.88 ± 0.55 mU/mL) compared with normal subjects (p <0.001). Furthermore, we showed that on average 50% of the procoagulant activity was TF-dependent (procoagulant activity was reduced to 0.57 ± 0.35 mU/mL in the presence of an anti-TF antibody; p < 0.01), suggesting that circulating BBTF in cancer patients has the potential to contribute to thrombosis in vivo. In summary, we have developed a novel assay that measures BBTF activity. This assay may be useful in the detection of a pre-thrombotic state in cancer patients.
Long-Term Follow-Up of Patients Treated by Gene Therapy for X-Linked Chronic Granulomatous Disease.
Authors: Ott Marion G. , Grez Manuel , Stein Stefan , Siler Ulrich , Koehl Ulrike , Kuehlcke Klaus , Schmidt Manfred , Schilz Andrea , Kunkel Hana , Naundorf Sonja , Brinkmann Andrea , Hassan Moustapha , von Kalle Christof , Trasher Adrian , Seger Reinhard , Hoelzer Dieter
Published: October 2019

Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector.

Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.
Exosite-Specific Inhibition of Thrombin Using Photo-Crosslinked Fluorescent Reporter Peptides.
Authors: Yegneswaran Subramanian , Tiefenbrunn Theresa , Fernandez Jose’ A. , Dawson Philip E.
Published: October 2019

Multi-functional enzymes such as thrombin acquire specificity from exosite-dependent interactions. Peptide inhibition studies of protein-protein interactions are useful to identify exosite functions. We developed a novel general approach for exosite analysis using photocrosslinking of exosite inhibitory peptides that also carry a fluorescent tag and reporter. Here we demonstrate the method’s feasibility in studying the thrombin-hirugen (enzyme-peptide) complex. Thrombin contains two positively-charged exosites, named exosites I and II. Hirugen, the C-terminal residues 54 to 65 of hirudin, binds exosite I of thrombin. A series of desulfo-hirugen analogs were synthesized with either an additional fluorescein-(benzoyl) phenylalanine-(abbreviated Fl-bF-), or Fl-bF-mercaptopropionic acid or Fl-bF-lactic acid moiety conjugated to the N-terminal end of the peptide; i.e. a fluorescent label followed by a photocrosslinker and a cleavable linker in succession were conjugated to the N-terminus of hirugen analogs. Each hirugen analog was bound and photocrosslinked to thrombin, and the resulting covalent complex was purified from the reactants to homogeneity. These thrombin-hirugen adducts hydrolyzed small oligopeptide substrates (CBS 34-47 or S2366) with an efficiency similar to that of native thrombin, showing that the active site was unaffected by labeling. However, these adducts were significantly inhibited in assays of fibrinogen clotting and of thrombomodulin-mediated protein C activation, implying that exosite I was blocked, causing reduced accessibility for the substrate, fibrinogen, or for the cofactor, thrombomodulin. Exosite II mediates thrombin binding to heparin. The thrombin-hirugen adduct bound to and eluted from heparin-Sepharose like native thrombin, suggesting that exosite II was unaffected by the photocrosslinked hirugen analog. Treatment of the thrombin-(Fl-bF-mercaptopropionic acid) hirugen adduct that contains a thioester bond with the mild nucleophile hydroxylamine released the amino acids C-terminal to the thioester bond from the adduct, effectively releasing hirugen residues 54–65 from the complex while retaining the fluorescein reporter near exosite I (Fl-thrombin). This treatment did not affect the amidolytic activity of Fl-thrombin. Additionally, Fl-thrombin could be inhibited by unlabeled hirugen suggesting that exosite I was uncovered by release of the peptide by hydroxylamine treatment. In summary, our results show that exosites of clotting factors, e.g., thrombin, can be specifically targeted and labeled with fluorescent reporters. This novel technology may have broad applicability for studies of protein-protein interactions that regulate coagulation.
The CXCR4-Antagonist AMD3100 Augments the Number of Mobilized Peripheral Blood Progenitor Cells (PBPC) When Added to a G-CSF Standard Mobilization Regime and AMD3100-Mobilized PBPC Result in Rapid Hematopoietic Reconstitution after Autologous Transplantation.
Authors: Fruehauf Stefan , Seeger Timon , Topaly Julian , Herrmann Doris , Dillmann Falk , Humpert Per , Calandra Gary , Laufs Stephanie , Goldschmidt Hartmut , Ho Anthony D.
Published: October 2019

Sufficient mobilization of peripheral blood progenitor cells (PBPC) is pivotal for successful autologous transplantation. G-CSF has gained a confirmed and dominant role in standard mobilization regimens. Recent reports provided evidence for the importance of the SDF1/CXCR4 axis in hematopoietic stem cell trafficking. AMD3100 is a CXCR4 antagonist that induces rapid mobilization of CD34+ cells in healthy volunteers. We initiated a phase II study assessing the safety and potential of AMD3100 in patients with multiple myeloma (MM) and non-Hodgkin’s lymphoma (NHL). At the time of the report 6 patients with MM and 4 patients with NHL were enrolled (5 female, 5 male; age median 44, range 44–71 yrs; prior chemotherapy regimens median 3, range 1–8). All patients with MM were in stage IIA or IIIA. Patients with NHL were in stage IIB, IIIA, IIIE or IV. Mobilization treatment consisted of 5 days G-CSF (10 μg/kg, s.c. AM) and a single dose of AMD3100 (240 μg/kg, s.c.) in the evening of day 4, 10–11 hours prior to leukapheresis. As expected, following 4 days of G-CSF treatment the CD34+ cell count in the peripheral blood increased 22-fold (range 7,8–33) and there was a correlation between baseline and day 4 PB CD34+ counts (r=0,88). Addition of AMD3100 led almost to a tripling of circulating CD34+ cells within 10 h after administration (2,8-fold increase, range 1,85–4,74). On the other hand, there was no mobilization of B-cells (CD19) -thus giving no indication for the co-mobilization of tumor cells- and no mobilization of NK/T-cell subsets (CD2, CD3, CD4, CD8). Patients with low starting PB CD34+ counts profited most. There was no association between the SDF1 1-3A polymorphism and the mobilization efficiency following AMD3100+G-CSF vs. G-CSF mobilization: 21% of patients showed the heterozygous G/A phenotype and the remainder the G/G phenotype. Interestingly, SDF1-serum levels in patients increased significantly after addition of AMD3100. Per leukapheresis procedure 4,3 (range 2,6–12,1) * 10e6 CD34+ cells/kg body weight (bw) were collected. Adverse effects were mild, one patient reported of nausea and emesis, WHO grade I. To date, four patients have been transplanted after high-dose chemotherapy (Melphalan 200mg/m2 or BEAM) with 4,7 (range 2,4–6,05) * 10e6 CD34+ cells/kg bw. Hematopoietic reconstitution (leukocytes >1/nl and thrombocytes >20/nl) was observed within a median of 14 (range 12–17) and 13 (range 10–15) days, respectively and is sustained in all patients. Thus CD34+ cells mobilized with AMD3100 appear to be fully functional. In conclusion AMD3100 is a seminal new drug development in the field of stem cell transplantation with the highest potential in poorly mobilizing patients.
Mobilization of Human Hemopoietic Progenitor Stem Cell Via Modulating CXCR4/SDF-1 Using Galactofucan Sulfate.
Authors: Irhimeh Mohammad R. , Lowenthal Ray M. , Fitton J. Helen
Published: October 2019

Mobilization of peripheral blood progenitor cells for hematopoietic rescue following autologous transplantation is usually achieved with the chemokine G-CSF. Engraftment potential is increased when higher levels of the receptor CXCR4 are noted on the hematopoietic progenitor stem cells (HPCs). It was previously reported that intravenous treatment with fucoidan or dextran sulfate, which are sulfated high molecular weight compounds, increased numbers of circulating mature white blood cells and HPCs in mice and nonhuman primates. This treatment also led to an increase in the pro-inflammatory cytokines IFN-γ and IL-12 levels in mice. In vitro treatment of bone marrow mononuclear cells with IFN-γ can up-regulate the expression of CXCR4 on granulocyte precursors and monocytes. We obtained ethics approval and informed consent to study the mobilization effect of orally ingested GFSTM (Galactofucan Sulfate), a seaweed-derived fucoidan, in healthy human volunteers in a single blinded placebo controlled phase I/II clinical study. Flow cytometry was used to monitor CXCR4 receptor on CD34+ stem cells. When moderate quantities (3 g/day) of seaweed containing 10% GFSTM were ingested, a slight increase in the total number of HPCs (CD34+) in the peripheral blood (PB) was observed, from 1.38 to 1.69 cells/μL (p=0.22, n=6). In addition, there was a small increase in the percentage of HPCs that expressed CXCR4 surface receptor, from 0.59 to 1.47 cells/μL, which is equivalent to 43% to 63% (p=0.19, n=6). Moreover, when 3 g/day of 75% GFSTM was ingested, a greater increase in the total number of HPCs (CD34+) in PB was observed, from 1.65 to 1.84 cells/μL (p=0.04, n=23). Furthermore, the percentage of HPCs that expressed CXCR4 increased from 0.746–1.652 cells/μL, which is equivalent to 45% to 90% (p=0.0002, n=23). Cytokine analysis, which was performed using ELISA to test for SDF-1 and IFN-γ, showed a significant increase in the plasma level of these cytokines. SDF-1 level was elevated from 1979 to 2068 pg/mL (p=0.051, n=10) and the level of IFN-γ from 9.04 to 9.90 pg/mL (p=0.007, n=10). These results suggest that GFSTM may modulate CXCR4 or disturb the SDF-1 gradient between bone marrow and PB. IFN-γ might play a role in the up-regulation of the expression of CXCR4 on CD34+ cells. To the authors’ knowledge, this is the first report of mobilization of HPCs by disruption of CXCR4/SDF-1 interaction using oral fucoidan.
Molecular Profiling of CD34+ Cells from Diagnosis in CML Patients Identifies Low Expression of CD7 with High Expression of Proteinase 3 or Elastase as Predictors of Longer Survival.
Authors: Yong Agnes S.M. , Szydlo Richard M. , Goldman John M. , Apperley Jane F. , Melo Junia V.
Published: October 2019

Although most patients with chronic myeloid leukemia (CML) have the same initial molecular abnormality, the BCR-ABL fusion gene, the duration of chronic phase (CP) varies widely. To identify the possible molecular basis of this heterogeneity, we studied CD34+ cells collected at diagnosis from 68 CML-CP patients. In all cases the Philadelphia chromosome was the only cytogenetic abnormality identified. Cryopreserved cells had all been collected by leukapheresis within 3 months of diagnosis, when the patients still had high leukocyte counts prior to definitive treatment. Although the majority of patients were diagnosed in the pre-imatinib era and treated with hydroxyurea and/or interferon-α, 32 (47%) received imatinib during the course of their disease. The median age at diagnosis was 45.2 years (range 17.6–68.3), and the male: female ratio was 1.8: 1 (44 males, 24 females). Total RNA was extracted from CD34+ purified cells. Gene expression profiling using Affymetrix HG-U133A microarrays was performed on two subsets of patients, one comprising patients with an ‘aggressive disease’ who developed blastic transformation (BT) within 3 years of diagnosis (n=10) and, at the other extreme, patients with an ‘indolent disease’ whose BT occurred 7 or more years from diagnosis (n=9). There was no difference in the proportion of the CD34+ subsets of CD38+ Lin+, CD38+ Lin− or CD38− Lin− cells between patients in the indolent as compared to the aggressive CML groups. Microarray data were analysed using a combination of MAS 5.0, dChip, RMA and GeneSpring 5.0 software, and revealed 20 genes differentially expressed in patients with aggressive and indolent disease, which were validated by quantitative real-time polymerase chain reaction (qPCR). The correlation between microarray and qPCR expression measures was highly significant for 18/20 genes (p<0.005, Spearman’s Rho). In the complete cohort of 68 patients, a multivariate Cox regression model identified the combination of low CD7 expression with high expression of proteinase 3 (PR-3) or elastase as associated with longer survival (p=0.0004 and p=0.0006, respectively). The levels of cytoplasmic PR-3 and surface CD7 protein expression by flowcytometry were also of prognostic value (p=0.029 and p=0.031, respectively). Patients who had more than 11% CD34+ cells expressing surface CD7 protein at diagnosis had a poor survival. Conversely, patients with more than 2% CD34+ cells expressing cytoplasmic PR-3 had a superior survival. This differential pattern of gene expression probably reflects the intrinsic heterogeneity of the disease; if so, assessing expression levels of selected genes at diagnosis may be valuable in predicting duration of survival in patients treated upfront with imatinib and the newer tyrosine kinase inhibitors.
Fetal Hematopoietic Development in the Mouse Aorta-Gonad-Mesonephros Region Is Inhibited by Homeodomain-Interacting Protein Kinase 2.
Authors: Ohtsu Naoki , Nobuhisa Ikuo , Mochita Miyuki , Taga Tetsuya
Published: October 2019

Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of mouse embryo. Our previous studies showed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine 727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in the hematopoietic development. HIPK2 transcripts were found in the fetal hematopoietic tissues such as the AGM region at embryonic day (E) 10.5 and E11.5 and the E14.5 fetal liver as detected by RT-PCR and whole mount in situ hybridization. The functional analysis of HIPK2 was done by introducing wild-type and its mutant constructs into the primary culture of the mouse AGM region. Retrovirus mediated forced expression of HIPK2 with green fluorescent protein (GFP) in the AGM culture remarkably inhibited the emergence of GFP+ nonadherent cells. In coculture of virus-infected GFP+ nonadherent cells with OP9 stromal cells, no cobblestone-like area-forming colonies were detected in the HIPK2-infected cells, while control GFP virus had no effect on the colony formation. Similarly, when GFP+ nonadherent cells were sorted and cultured in semisolid medium, colonies were not formed from the HIPK2-GFP virus-infected AGM culture. These data suggest that HIPK2 suppresses differentiation of adherent endothelial-like cells to colony-forming hematopoietic cells or, alternatively HIPK2 inhibits proliferation of newly developed hematopoietic progenitors. Development of cells positive for CD45 (an immature hematopoietic cell maker) cells was suppressed by forced expression of HIPK2 in the AGM culture. To determine the domain important for the inhibitory activity of HIPK2, we constructed a series of point and deletion mutants. Overexpression of HIPK2 mutants in the AGM culture showed that a certain structure of the kinase domain as well as its C-terminally located structure but not the kinase activity is essential for the HIPK2-mediated inhibition of hematopoiesis. When nonadherent cells in the AGM culture were fractionated for their expression levels of CD45 and c-Kit (a receptor for stem cell factor), CD45lowc-Kit+ cells have a potential to form hematopoietic cell colonies. In the HIPK2-overexpressed AGM culture, emergence of CD45lowc-Kit+ cells was significantly inhibited. These data indicated that HIPK2 plays a negative regulatory role in the development of AGM hematopoiesis in mouse embryo.