bluuebunny/crossref_metadata_2025 huggingface dataset:
Abdominal Aortic Aneurysm Repair in Renal and Liver Transplant Recipients
Authors: Premaratne Sobath , Hopkins Jonathan , Duddy Martin , Tai Ket Sang , Kay Mark , Rogoveanu Radu , Nicholl Phil , Tiwari Alok
Published: October 2019

Background:

Abdominal aortic aneurysm (AAA) repair in patients with organ transplant remains a challenge. We looked at AAA repair in patients with organ transplants at our tertiary liver and kidney transplant unit.

Methods:

A retrospective analysis of a prospectively maintained database was undertaken from January 2008 to July 2018. We looked at patient demographics, type of repair, and technical success including reinterventions, perioperative transplant organ function, and 30-day and 1-year survival rate. Eight of 662 patients who underwent AAA repair had a solid organ transplant. Of these, 5 were kidney transplants, 2 liver transplants, and 1 had kidney and liver transplant; 75% were male; and average age was 63.4 (range: 49-83). All patients had asymptomatic AAAs, and 6 were treated with standard endovascular repair, 1 standard repair with iliac branch device, and 1 open repair. Adjunctive techniques such as CO2 angiograms, deployment of main body through contralateral iliac, low-profile sheaths, custom-made stent grafts, and temporary axillo-femoral shunting were used to protect transplant organs. Thirty-day survival was 100% with 1 death at 5 months from liver failure, and 1 patient has a persistent type-2 endoleak 3 years after the procedure.

Conclusion:

Abdominal aortic aneurysm repair in patients with organ transplants can be undertaken using adjunctive endovascular and open surgical techniques.
Quality-of-Life Outcomes of Older Adults with Severe Disabilities
Authors: Friedman Carli
Published: October 2019

People with disabilities who age may have unique experiences and face different challenges than people without disabilities who acquire disabilities as they age. These unique challenges may be especially pertinent for people with severe disabilities who already face disparities in quality of life. Yet, there is little research specifically about the quality of life of older adults with severe disabilities, a population that has continued to grow due to advances in medicine and social supports. This study’s aim was to explore quality-of-life outcomes of older adults with severe disabilities. We analyzed Personal Outcome Measures® data from 800 people with severe disabilities aged 55 years old and older. Findings revealed most older adults with severe disabilities had less than half of the quality-of-life outcomes present. Specifically, we found that while most older adults with severe disabilities were healthy, safe, and free from abuse and neglect, the overwhelming majority were lacking in community integration, relationships, choice, and opportunities. Our findings suggest the lack of quality-of-life outcomes among older adults with severe disabilities were largely attributed to an absence of supports. When older adults with severe disabilities received individualized organizational supports, they had better quality of life.
Lifestyle Medicine Around the World: Lifestyle Medicine in Israel
Authors: Malatskey Lilach , Hekselman Igal , Afek Shani
Published: October 2019

In 2015, the Centers for Disease Control in the United States declared that 7 of 10 deaths per year are caused by chronic diseases; in Israel, the situation is consistent with this assessment. Healthy Israel 2020, an Israeli Ministry of Health initiative, places physicians at the forefront of health promotion and preventive medicine. In 2012, the Israeli Society of Lifestyle Medicine (ISLM) was established under the auspices of the Israel Association for Family Physicians. This decision was taken because we, the authors, wanted to promote change in the primary care services through the recruitment of key leaders for implementation of lifestyle medicine (LM), including improved attitudes, knowledge, motivation, and skills of primary care physicians and health providers. Today, the ISLM is an active member of the Israeli Medical Association, promoting educational activities, physician’s health, and well-being initiatives; developing tools for health-promoting clinics; and more. Our future plans are to incorporate LM as an integral part of daily practice in all sectors of the medical profession in Israel. This is challenging, but we see this as the only way to effectively combat the noncommunicable disease epidemic.
State-dependent age replacement policy for a system subject to cascading failures
Authors: Lee Hyunju
Published: October 2019

A cascading failure is a failure in a system of interconnected parts in which the failure of a part triggers the failure of successive parts. Although some initial and empirical studies for probabilistic modeling and analysis of the cascading failures have been performed in the literature, general modeling frameworks and relevant maintenance policies have been barely reported yet. However, in this article, based on a general framework for modeling cascading failures, we study the optimal preventive maintenance problem for the system. A new state-dependent age replacement policy is developed. The comparison with the ordinary age replacement policy is also discussed.
Transnationalistic memories: Frédéric Boissonnas’ photographic construction of a Greek-Christian European Thessaloniki in Salonique et ses basiliques (1919)
Authors: Wilson Colette
Published: October 2019

Frédéric Boissonnas’ album, Salonique et ses basiliques, captures the city at a very precise moment in its history. The album’s photographs were taken between 1912 and 1913, that is, just at the end of the Ottoman and the beginning of Greek rule, but before the great fire of 1917 that destroyed the city centre, removing virtually all traces of the Turkish and Jewish quarters. The album also predates the forced exchange of Muslim and Christian populations in 1923 that finally brought the transculturalism of the city to an abrupt end. Through a close reading of Boissonnas’ photographs, within the theoretical framework of past transnationalistic and present-day transcultural memories, this article argues that political and ideological allegiances directed the creation, dissemination and consumption of an artistic product. The article concludes with a reflection on the city’s return to its transcultural roots.
Effects of Lighting, Liquid Color, and Drink Container Type on Volume Perception
Authors: Chen Yi-Lang , Lee Yi-Chien
Published: October 2019

This study explored the effects of geometric features (i.e., proportionate size and elongation) of tumblers and goblets on volume perception under different lighting environments and with different colors of liquid. Sixty individuals (30 men and 30 women) participated in an experiment that demonstrated volume perception of a specified amount (100 ml or 200 ml, depending on the container) by pouring water or red wine into different pairs of glasses. The results revealed the goblet and tumbler pairs in both the proportionate-size and elongation groups produced significant effects on overall volume perception; by contrast, the effects of lighting and liquid color were only observed in specific groups. The geometric features of the glasses yielded inconsistent results for different pairs of glasses; these dissimilar results may have been caused by differences in visual cues (glass rim or height) affecting volume perception under different experimental settings. In addition, this study revealed that men underestimated the volume more than women did and thus poured more liquid into the glasses. In practical application, these study results should be considered in conjunction with the context and purpose of drink and container selection to understand the commensurate illusory effects.
Traits and characteristics of highly successful medical leaders
Authors: Sharma Shyam S
Published: October 2019

Background

Medicine attracts a broad range of personality traits but the inner thoughts of its leaders have rarely been studied. The BMJ has been asking perceived leaders in the field a set of structured questions on a weekly basis. Those responses have proved insightful into the characteristic traits of high profile doctors.

Methods

We analysed the responses of each medically qualified doctor interviewed weekly by BMJ Confidential following the use of a set of structured questions about their likes and dislikes. These structured questions allowed us to cross analyse responses.

Results

From 2013 to 2017, 134 medically qualified doctors were identified by the BMJ to be suitable for inclusion in their weekly BMJ Confidential series. These individuals were selected because they were deemed by the BMJ to be leaders in their clinical, medico-political or academic fields. Of the cohort, 91% were white and 69% male. Clinical mistakes by these individuals were not uncommon (28%) over the course of their careers. Conceit and arrogance were despised most (16%) whereas politics was of interest but not political correctness. The founder of the NHS Aneurin Bevan was identified as the best Secretary of State for Health while the worst was Andrew Lansley (26%) followed by the former health secretary Jeremy Hunt.

Conclusion

Medicine attracts a broad range of personalities, but the characteristics of its perceived leaders seem less diverse.
Does Size Matter? An Analysis of the Effect of Lumbar Disc Herniation Size on the Success of Nonoperative Treatment
Authors: Gupta Anmol , Upadhyaya Shivam , Yeung Caleb M. , Ostergaard Peter J. , Fogel Harold A. , Cha Thomas , Schwab Joseph , Bono Chris , Hershman Stuart
Published: October 2019

Study Design:

Retrospective study.

Objective:

In this study, we examined whether the size of a lumbar disc herniation (LDH) is predictive of the need for surgical intervention within 2 years after obtaining an initial magnetic resonance imaging (MRI) scan. We hypothesized that a fragment that occupied a larger percentage of the spinal canal would not predict which patients failed conservative management.

Methods:

Using the ICD-10 code M51.26, we identified patients at a single academic institution, across the 2-year period from 2015 to 2016, who received a diagnosis of primary lumbar radicular pain, had MRI showing a disc herniation, and underwent at least 6 weeks of nonoperative management. Patients experiencing symptoms suggesting cauda equina syndrome and those with progressive motor neurological deficits were excluded from analysis, as were patients exhibiting “hard” disc herniations. Within the axial view of an MRI, the following measurements were made on AGFA-IMPACS for a given disc herniation: the length of both the canal and the herniated disc along the anterior-posterior axis, the average width of the disc within the canal; the total canal area, and the area of the disc herniation. Data analysis was conducted in SPSS and a 2-tailed reliability analysis using Cronbach’s alpha as a measure of reliability was obtained.

Results:

A total of 368 patients met the inclusion and exclusion criteria for this study. Of these, 14 (3.8%) had L3-L4 herniations, 185 had L4-L5 herniations (50.3%), and 169 had L5-S1 herniations (45.9%). Overall, 336 (91.3%) patients did not undergo surgery within 1 year of the LDH diagnosis. Patients who did not receive surgery had an average herniation size that occupied 31.2% of the canal, whereas patients who received surgery had disc herniations that occupied 31.5% of the canal on average. A Cronbach’s alpha of .992 was observed overall across interobserver measurements. After controlling for age, race, gender, and location of herniation through a logistic regression, it was found that the size of the herniation and the percentage of the canal that was occupied had no predictive value with regard to failure of conservative management, generating an odds ratio for surgery of 1.00.

Conclusions:

The percentage of the spinal canal occupied by a herniated disc does not predict which patients will fail nonoperative treatment and require surgery within 2 years after undergoing a lumbar spine MRI scan.
Multiple environmentalities and post-politicization in a Canadian Mountain Park
Authors: Youdelis Megan
Published: October 2019

This research explores the centrality of multiple environmentalities at multiple scales in the post-politicization of conservation governance in Jasper National Park, Canada. Austerity politics in Canada contributed to the post-politicization of conservation as the interests of Parks Canada and private developers were brought into alignment in terms of increasing visitation and the revenue imperative. The Parks Canada Agency, under structural pressure, employed several post-political strategies to suture the space for dissensus and debate and orchestrate the appearance of consent for the private development and management of park services. Central to these strategies were multiple (sovereign, disciplinary, and neoliberal) environmentalities constructed at multiple scales by multiple actors (the federal government, the local parks department, and private sector interests) aimed at producing environmental subjects who understand and acquiesce to the idea that ‘there is no alternative’ to a privatized conservation practice. In response, opponents attempted to mobilize an alternative environmentality, combining a social democratic critique of neoliberalism with a Romantic vision of wilderness conservation. Although opponents enrolled a sizeable number of allies, they fell short of stabilizing a liberation environmentality as several underlying points of ‘agreement’ contributed to the stabilization of post-political discourse and practice, foreclosing alternative political economies of conservation.
Mineralogical study of clays from Dobrodo, Serbia, for use in ceramics
Authors: Milošević Maja , Dabić Predrag , Kovač Sabina , Kaluđerović Lazar , Logar Mihovil
Published: October 2019

This study focuses on the mineralogical characterization of four raw clay samples from Dobrodo deposit, Serbia. Several analytical methods were applied to determine the chemical and mineralogical composition, morphology and physical properties (colour, plasticity, specific surface area, particle size and cation-exchange capacity) of the clay samples. Kaolinite, smectite and illite are the predominant phases in all of the samples studied that contain between 60.2 and 87.1 wt.% of clay. Quartz, feldspars, paragonite and Ti- and Fe-bearing phases were also identified. The relatively high SiO2/Al2O3 mass ratio indicates abundant quartz. The cation-exchange capacity of the samples varied between low and moderately charged clay minerals (12–52 mmol 100 g–1) with specific surface area values ranging from 94 to 410 m2 g–1. The plasticity index values (11–23%) suggest low to moderate plasticity. Preliminary results show that most of the raw clay from Dobrodo deposit might be suitable for use in ceramic applications.
Transplantation of unrelated donor umbilical cord blood in 102 patients with malignant and nonmalignant diseases: influence of CD34 cell dose and HLA disparity on treatment-related mortality and survival
Authors: Wagner John E. , Barker Juliet N. , DeFor Todd E. , Baker K. Scott , Blazar Bruce R. , Eide Cindy , Goldman Anne , Kersey John , Krivit William , MacMillan Margaret L. , Orchard Paul J. , Peters Charles , Weisdorf Daniel J. , Ramsay Norma K. C. , Davies Stella M.
Published: October 2019

The potential benefits of unrelated donor marrow transplantation are offset by the immunologic complications of graft-versus-host disease (GVHD) and infection. Therefore, we used cryopreserved umbilical cord blood (UCB) as a strategy to reduce the risks of GVHD and treatment-related mortality (TRM) and improve survival. Data on 102 patients (median age 7.4 years) who received transplants between 1994 and 2001 for the treatment of malignant (n = 65; 68% were high-risk patients) and nonmalignant (n = 37) diseases were evaluated. Log-rank tests and Cox regression analyses were used to determine the effects of various demographic, graft-related, and treatment factors on engraftment, GVHD, TRM, relapse, and survival. As of October 15, 2001, the median follow-up was 2.7 years (range, 0.3-7.2). Incidences of neutrophil and platelet engraftment were 0.88 (CI, 0.81-0.95) and 0.65 (CI, 0.53-0.77), respectively. Notably, incidences of severe acute and chronic GVHD were 0.11 (CI, 0.05-0.17) and 0.10 (CI, 0.04-0.16), respectively. At 1 year after transplantation, proportions of TRM and survival were 0.30 (CI, 0.21-0.39) and 0.58 (CI, 0.48-0.68), respectively. In Cox regression analyses, CD34 cell dose was the one factor consistently identified as significantly associated with rate of engraftment, TRM, and survival. Despite the low incidence of GVHD, the proportion of patients with leukemia relapse at 2 years was 0.17 (CI, 0.00-0.38) and 0.45 (CI, 0.28-0.61) for patients with standard and high-risk disease, respectively. There is a high probability of survival in recipients of UCB grafts that are disparate in no more than 2 human leukocyte antigens (HLAs) when the grafts contain at least 1.7 × 105 CD34+ cells per kilogram of recipient's body weight. Therefore, graft selection should be based principally on CD34 cell dose when multiple UCB units exist with an HLA disparity of 2 or less.
Phase 3 study of the multidrug resistance modulator PSC-833 in previously untreated patients 60 years of age and older with acute myeloid leukemia: Cancer and Leukemia Group B Study 9720
Authors: Baer Maria R. , George Stephen L. , Dodge Richard K. , O'Loughlin Kieran L. , Minderman Hans , Caligiuri Michael A. , Anastasi John , Powell Bayard L. , Kolitz Jonathan E. , Schiffer Charles A. , Bloomfield Clara D. , Larson Richard A.
Published: October 2019

The Cancer and Leukemia Group B conducted a phase 3 trial of the P-glycoprotein modulator PSC-833 in untreated acute myeloid leukemia patients aged 60 years and older. Patients were randomized to 1 of 2 regimens, with doses determined in a prior phase 1 study, consisting of cytarabine 100 mg/m2/d by 7-day infusion, with daunorubicin 60 mg/m2 and etoposide 100 mg/m2 daily for 3 days (ADE), or daunorubicin 40 mg/m2 and etoposide 60 mg/m2 for 3 days with PSC-833, 2.8 mg/kg over 2 hours, and then 10 mg/kg/d by 3-day infusion (ADEP). The ADEP arm was closed after randomization of 120 patients (61 to ADE and 59 to ADEP) because of excessive early mortality. Rates of complete remission, nonresponse, and death were 46%, 34%, and 20% for ADE, versus 39%, 17%, and 44% for ADEP (P = .008). Nevertheless, disease-free survival (median 7 vs 8 months; P = .38) and overall survival (approximately 33% alive at 1 year) did not differ and were similar to historical results. Although the number of patients was limited, ADE patients whose pretreatment cells exhibited PSC-833–modulated dye efflux in vitro (n = 22) had worse outcomes than those without efflux (n = 11) (complete remission, nonresponse, and death rates of 41%, 41%, and 18%, compared with 91%, 9%, and 0%; P = .03), but with ADEP outcomes were nearly identical. Moreover, for patients with PSC-833–modulated efflux, median disease-free survival was 5 months with ADE and 14 months with ADEP (P = .07). Further modulation trials in older patients must await the design of less-toxic regimens.
Characterization of the binding site on heparan sulfate for macrophage inflammatory protein 1α
Authors: Stringer Sally E. , Forster Mark J. , Mulloy Barbara , Bishop Christopher R. , Graham Gerard J. , Gallagher John T.
Published: October 2019

The CC chemokine macrophage inflammatory protein 1α (MIP1α) is a key regulator of the proliferation and differentiation of hematopoietic progenitor cells. The activity of MIP1α appears to be modulated by its binding to heparan sulfate (HS) proteoglycans, ubiquitous components of the mammalian cell surface and extracellular matrix. In this study we show that HS has highest affinity for the dimeric form of MIP1α. The predominantly dimeric BB10010 MIP1α interacts with an 8.3-kDa sequence in the HS polysaccharide chain, which it protects from degradation by heparinase enzymes. The major structural motif of this HS fragment appears to consist of 2 sulfate-rich S-domains separated by a short central N-acetylated region. The optimum lengths of these S-domains seem to be 12 to 14 saccharides. We propose that this binding fragment may wrap around the MIP1α dimer in a horseshoe shape, facilitating the interaction of the S-domains with the heparin-binding domains on each monomer. Molecular modeling suggests that these S-domains are likely to interact with basic residues Arg 17, Arg 45, and Arg 47 and possibly with Lys 44 on MIP1α and that the interconnecting N-acetylated region is of sufficient length to allow the 2 S-domains to bind to these sites on opposite faces of the dimer. Elucidation of the structure of the HS-binding site for MIP1α may enable us to devise ways of enhancing its myeloprotective or peripheral blood stem cell mobilization properties, which can be used to improve cancer chemotherapy treatments.
A high-resolution allelotype of B-cell chronic lymphocytic leukemia (B-CLL)
Authors: Novak Urban , Oppliger Leibundgut Elisabeth , Hager Jörg , Mühlematter Dominique , Jotterand Martine , Besse Celine , Leupin Nicolas , Ratschiller Daniel , Papp Jeanette , Kearsey Gina , Aebi Stefan , Graber Hans , Jaggi Rolf , Lüthi Jean-Marc , Meyer-Monard Sandrine , Lathrop Mark , Tobler Andreas , Fey Martin F.
Published: October 2019

The most frequent chromosomal aberrations in B-cell chronic lymphocytic leukemia (B-CLL) are deletions on 13q, 11q, and 17p, and trisomy 12, all of which are of prognostic significance. Conventional cytogenetic analysis and fluorescence in situ hybridization (FISH) are used for their detection, but cytogenetic analysis is hampered by the low mitotic index of B-CLL cells, and FISH depends on accurate information about candidate regions. We used a set of 400 highly informative microsatellite markers covering all chromosomal arms (allelotyping) and automated polymerase chain reaction (PCR) protocols to screen 46 patients with typical B-CLL for chromosomal aberrations. For validation, we compared data with our conventional karyotype results and fine mapping with conventional single-site PCR. All clonal cytogenetic abnormalities potentially detectable by our microsatellite PCR (eg, del13q14 and trisomy 12) were picked up. Allelotyping revealed additional complex aberrations in patients with both normal and abnormal B-CLL karyotypes. Aberrations detectable in the samples with our microsatellite panel were found on almost all chromosomal arms. We detected new aberrant loci in typical B-CLL, such as allelic losses on 1q, 9q, and 22q in up to 25% of our patients, and allelic imbalances mirroring chromosomal duplications, amplifications, or aneuploidies on 2q, 10p, and 22q in up to 27% of our patients. We conclude that allelotyping with our battery of informative microsatellites is suitable for molecular screening of B-CLL. The technique is well suited for analyses in clinical trials, it provides a comprehensive view of genetic alterations, and it may identify new loci with candidate genes relevant in the molecular biology of B-CLL.
A Novel Mechanism of Endothelial Protein C Receptor Release, in Microparticulate Form, by Activated Protein C.
Authors: Perez-Casal Margarita , Fukudome Kenji , Toh Cheng Hock
Published: October 2019

Activated protein C (APC) administration is now used for treating patients with severe sepsis. We investigated its effect on primary, physiologically relevant cells and demonstrate a novel mechanism of endothelial protein C receptor (EPCR) release from the cell surface. Exposure of human umbilical vein endothelial cells or monocytes to APC (from physiological levels of 0.5 up to 100nM) resulted in the increasing release of EPCR-containing microparticles (EPCR-MP), as demonstrated by confocal microscopy. Further characterisation through flow cytometry showed a concomitant fall in EPCR levels from the cell surface. This release of EPCR could not be inhibited by the metalloproteinase inhibitors 1, 10-phenanthroline or Ro31-9790, unlike soluble EPCR (sEPCR) that is metalloproteinase cleaved at the cell surface following thrombin or pro- inflammatory cytokine stimulation. Western blotting confirmed the molecular weight of EPCR-MP to be identical to the full-length membrane form (49 kD) and different from sEPCR (45 kDa). APC was also bound to EPCR-MP and could be quantified by ELISA using EPCR capture and APC detection by chromogenic substrate, S2366. Using an initial factor Va incubation step followed by a prothrombinase assay, the APC bound to EPCR-MP could significantly reduce thrombin generation. This was abrogated in the presence of excess α1-antitrypsin, an APC inhibitor. By contrast, APC bound to sEPCR could no longer inactivate factor Va. Further characterisation showed the APC induction of EPCR-MP to be time dependent with increasing release over 24 hours, as quantified by ELISA. The phenomenon also required the active site of APC. Neither protein C, heat-inactivated or D-Phe-Pro-Arg-chloromethylketone-blocked APC could induce EPCR-MP formation. Co-incubation with hirudin (6mM) did not alter the APC effect and excluded any role of contaminating thrombin. This novel observation provides new insights into the consequences of APC therapy in the septic patient as well as demonstrating for the first time that there can be 2 circulating forms of EPCR. Unlike sEPCR however, EPCR-MP can facilitate and potentially disseminate the anticoagulant activity of bound APC.
The Effect of Grp78 Inhibition on Tissue Factor Gene Expression, Procoagulant Activity, and Factor Xa Generation.
Authors: Bhattacharjee Gourab , Ahamed Jasimuddin , Pedersen Brian , El-Sheikh Amr , Liu Cheng , Mackman Nigel , Edgington Thomas S.
Published: October 2019

In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.
TAT/HDL Induces the Phospholylation of Cofilin in the Process of Platelet Production of Megakaryocytes.
Authors: Ishida Yoji , Kowata Syugo , Murai Kazunori , Oyake Tatsuo , Ito Shigeki
Published: October 2019

In the previous ASH Meetings, we have reported that thrombin/antithrombin complex in association with high density lipoprotein (TAT/HDL) directly stimulated proplatelet formation (PPF) of human or murine megakaryocytes. In this study, we investigated the signal transduction pathways responsible for platelet production from megakaryocytes using human megakaryoblastic cell line (MEG).

TAT/HDL stimulated MEGs to induce the many platelet-like particles (PP) around the cells in the serum free culture during 12~24 hrs (Fig. 1). The frequency of MEGs with PP increased with the increase of TAT/HDL in a dose dependent manner (0 μg/ml: 1.8±0.5%, 50 μg/ml: 30.8±4.5%, 100 μg/ml: 46.5±7.8%, 200 μg/ml: 57.8±8.3%, mean±SD, n=4). The frequency of MEGs with PP, expressed as a pecentage of the control, was significantly decreased after the incubation with TAT/HDL(150 μg/ml) and Y27632, a specific inhibitor for Rho kinase(ROCK), (Y27632 10 μM: 61.3±7.9% of the control (p<0.01), 100 μM: 12.5±5.9% of the control (p<0.01), mean±SD, n=4). MEGs were incubated with TAT/HDL (150 μg/ml) in serum free condition for 5, 10, 15, 30, 60, 90, 120, 150 and 180 min. MEGs were havested, washed twice and lysed by the lysis buffer (50mM Tris/HCl, 150 mM NaCl. 0.5% Nonidet P-40 pH 6.8 with protease inhibitors’ cocktail) for the Western blot analysis. Activated Rho (GTP-bound state Rho), measured by the Western blot using Rhotekin-Rho binding domain (RBD) beads and anti-Rho antibody, increased at 90, 120 and 150 min after the incubation. ROCK was co-precipitated with activated Rho using Rhotekin-RBD beads and detected by the Western blot analysis using anti-ROCK antibody at 90, 120 and 150 min. LIM kinase was immunoprecipitated with ROCK/anti-ROCK antibody and protein G sepharose. Western blot analysis using anti-phosphothreonine antibody revealed that LIM-kinase was phospholylated at 90, 120 and 150 min after the incubation. Phospholylated cofilin (Ser3) expression was detected by the Western blot using anti-phosphocofilin antibody at 60 min, peaked at 120 min and tapered at 150 min after the incubation (Fig.2). The intensity of the phospholylated cofilin (Ser3) band decreased in the incubation of MEGs with TAT/HDL and Y27632 (100 μM) at each time. These results strongly suggested the platelet-like particles production from MEGs, stimulated by TAT/HDL, was involved in the pathway from Rho activation, ROCK activation, LIM kinase phospholylation and cofilin phospholylation, based on the evidence that phospholylated cofilin showed the inhibition of actin depolymerization and the stabilization of actin filaments.

Fig.1 (a) MEGs with no stimulation (b) MEGs with TAT/HDL (200 μg/ml) for 24h

Fig.2 Expression of phospholylated cofilin (Ser3).

Figure Figure Figure Figure
CHIR-12.12, an Antagonist Anti-CD40 Antibody, Exhibits Greater ADCC Than Rituximab Against a Variety of Malignant B Cells: Evaluation of FcγRIIIa Polymorphism and ADCC Response.
Authors: Luqman Mohammad , Tong Xia , Niu Xiaohong , Garcia Pablo , Faure Michel , Yabannavar Asha , Jallal Bahija
Published: October 2019

We have generated a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, using XenoMouse® mice (Abgenix, Inc.) and have previously demonstrated that it inhibits normal human B cell proliferation and survival and has potent ADCC against primary CLL and NHL cells. CHIR-12.12 and the anti-CD20 monoclonal antibody rituximab were compared for their relative ADCC activity against a variety of malignant human B-cell lines expressing both CD40 and CD20 antigens, including two lymphoma cell lines (Daudi, Namalwa), two multiple myeloma cell lines (ARH77, IM-9), a B-ALL cell line (CCRF-SB), and a B-CLL cell line (EHEB). All cell lines expressed both CD20 and CD40 antigens, and the number of cell surface CD20 molecules per cell were 2.6- to 30.8-fold higher than CD40. For all target cell lines, despite the greater number of CD20 receptors, CHIR-12.12 showed greater maximum cell lysis and a lower ED50 than rituximab. ADCC activity of rituximab is known to correlate with the FcγRIIIa genotype of the effector cells. The homozygous valine (V/V) or heterozygous valine/phenylalanine (V/F) polymorphisms at aa158 are associated with greater cell lysis than is the homozygous F/F polymorphism. The role of the FcγRIIIa aa158 genotype as it relates to CHIR-12.12 activity was explored using Daudi lymphoma target cells and effector NK cells purified from human donors expressing the three polymorphisms. CHIR-12.12 induced potent ADCC with NK cells of all three genotypes (ED50s of 4, 2, and 0.4 pM for F/F, V/F, and V/V, respectively). The rituximab ED50s were 53, 21, and 9 pM for F/F, V/F, and V/V, respectively. Comparison of affinity of the FcγRIIIa F and V alleles for CHIR-12.12 and rituximab using Biacore® analysis showed that CHIR-12.12 bound the F allele with a 4.6-fold higher affinity than rituximab (2.8 μM versus 13 μM, respectively). These data demonstrate that CHIR-12.12 is a more potent ADCC mediator than rituximab, even with human NK cells of the aa158 F/F genotype. CHIR-12.12 is currently in Phase I clinical trials for B-cell malignancies.
Regulation of Exon 2′/2 of Protein 4.1R by Coordinated Transcription and Splicing.
Authors: Huang Shu-Ching , Norton Stephanie M. , Liu Eva S. , AnYu Zhou , Guang Yang , Benz Edward J.
Published: October 2019

Alternative splicing events altering the 5′ end of the red blood cell protein 4.1 (4.1R) mRNA are critical for modulating the expression of both the 135 kD and 80 kD isoforms. Three mutually exclusive 5′ exons, 1A, 1B, and 1C, are transcribed from their respective promoter and exhibit differential splicing to exon 2′/2 splice acceptor sites. Exon 1A splices to the distal 3′ splice site (3′ ss) excluding exon 2′ and encoding for the 80 kD isoform. Exons 1B and 1C splice to the proximal 3′ ss, including exon 2′ in mature mRNA and encoding for the 135 kD form. We investigated the regulatory mechanism involved in the selection of the alternative 3′ ss, using 1A, 1B, and 1C minigene constructs containing intronic sequences downstream of each exon joined with intronic sequences upstream of exon 2′. We positioned either CMV or the respective native promoter upstream of the minigene and analyzed the spliced products for exon 2′ expression in mouse (MELC or C2C12) or human (HeLa or RD) cells. When under the control of the CMV promoter, all 1A, 1B, and 1C minigenes resulted in the inclusion of exon 2′. However, when under the control of its respective promoter, the minigene mimicked its endogenous splicing pattern, suggesting that promoter identity influences the alternative exon 2′ splicing decision. To confirm this hypothesis, we switched 1A and 1B promoters in their respective minigene constructs. Replacement of the 1A promoter by the 1B promoter resulted in the inclusion of exon 2′ in the 1A minigene. Conversely, replacement of the 1B promoter by the 1A promoter caused increased exclusion of exon 2′ in the 1B minigene, confirming that alternative splicing of exon 2′ is sensitive to the type of promoter. The current model on the modulation of alternative splicing by promoters suggests that the promoter might control alternative splicing via the regulation of polymerase II (pol II) elongation or processivity. To test whether the same mechanism applies to exon 2′/2 splicing, we treated 1A and 1B minigene-transfected cells with transcription elongation inhibitor 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB). Inhibition of transcription elongation did not affect splicing of the 1B minigene, but it enhanced exon 2′ inclusion in the 1A minigene. The distal 3′ ss is a stronger splice site than the proximal 3′ ss. A highly processive elongating pol II favors the simultaneous presentation of both sites to the splicing machinery, a situation in which the distal 3′ ss out-competes the proximal 3′ ss, resulting in exon 2′ exclusion. Conversely, a slow pol II processivity on the 1B promoter favors the selection of the proximal 3′ ss and inclusion of exon 2′. Taken together, our results show that the alternative splicing of exon 2′/2 is tightly coupled to promoter architecture. Inclusion or exclusion of exon 2′ is achieved through a coordinated action of transcription and splicing.
Identification of New Megakaryocytic Subpopulations in Mouse Bone Marrow.
Authors: Sogo Shinji , Matsumura-Takeda Kuniko , Isakari Yoshimasa , Harada Yasuo , Nishioka Kinue , Kawakami Takuma , Ono Toshihide , Taki Takao
Published: October 2019

Platelets (PLT) are produced from megakaryocytes (Mks) via proplatelet formation (PPF). However, the molecular mechanisms from Mks to PPF are not clearly elucidated, because the maturational steps of the Mks in bone marrow (BM) are not analyzed in detail. Until now, mouse Mks have been only isolated as acetylcholinesterase (AchE) positive cells and they are understood as well maturated population. In this study, we found the presence of different megakaryocytic subpopulations in BM by flowcytometry. To isolate the Mks, first we depleted lineage marker (CD4, CD8a, CD11b, B220, CD71, CD90, TER119, Gr-1, F4/80, 7/4) positive cells from BM cells of BALB/c mice. The analysis of the expression-pattern of CD41, CD45 and CD61 in the lineage negative (Lin−) cells showed the presence of two types of megakaryocytic subpopulations. By sorting, they were identified as Lin−CD41+/45+/61+ cells (AchE negative) and Lin−CD41++/45+/61++ cells (partially AchE positive), respectively. To assess the maturational stages of the subpopulations, each population was cultured with 10ng/mL of TPO followed by counting of PPF and PLT production. Both PPF and PLT production were observed in Lin−CD41+/45+/61+ cells later than those in Lin−CD41++/45+/61++ cells. On the other hand, CFU-Mk was scarcely detected in each subpopulation. The results indicate that both populations are the committed megakaryocytes and Lin−CD41+/45+/61+ cells are more immature population than Lin−CD41++/45+/61++ cells. Then to characterize these subpopulations in detail, gene expression profiling was performed against four-megakaryocytic lineage-populations, Lin−CD41−Thy1lowc-kit+ cells as stem/progenitor, Lin−CD41+/45+/61+ cells, Lin−CD41++/45+/61++ cells and PLT using GeneChipU74 or RT-PCR. These analyses revealed that many PLT-specific genes including gpIb/IX, P-selectin, thrombin-R and ADP-R were already expressed on Lin−CD41+/45+/61+ cells but less than Lin−CD41++/45+/61++ cells. Especially, beta-1 tubulin that is necessary for PPF was only expressed on Lin−CD41++/45+/61++ cells. On the contrary, the expression of c-kit gene was gradually decreasing from stem/progenitor fraction to PLT. In conclusion, we succeeded in the isolation of new subpopulations distinguishable between immature Mks and more matured Mks beginning to prepare PLT. The present finding can contribute to elucidate the molecular mechanisms during terminal maturation.
Hydroxyurea Treated Sickle Disorders Demonstrate Decreased Plasma Hb, Red Cell Vesicles and Thrombolysis.
Authors: Westerman Maxwell P. , Pizzey Arnold , Hirshman Jocelyn , Cerino Mario , Eze Ada , Lawrie Andrew , Purdie Gordon , Mackie Ian , Porter John
Published: October 2019

Free plasma Hb has been recently implicated in the genesis of pulmonary hypertension in sickle cell disorders (SCD) through a putative NO scavenging mechanism. We have recently reported that free plasma Hb is correlated with increased plasma red cell derived vesicles (RBVes) in sickle cell and thalassemia disorders (Westerman, Blood2004; 104:1671). Since high levels of HbF have been associated with decreased elevation of RBVes in children with SCD (Setty Blood2000;96:1119–1124), we hypothesized that patients treated with hydroxyurea (HU) might show a similar normalisation of RBVes. Given our finding of an association between RBVes and free plasma Hb, such treatment might also be predicted to correct increments in free plasma Hb. In this study we have examined whether sickle cell patients treated with HU show evidence of a decrease in free plasma Hb and RBVes compared to untreated SCD patients. RBVes, free plasma Hb, and markers of activation of thrombolysis were quantitated. 35 adult SCD patients, 27 in steady state, untransfused and not receiving HU and 7 patients on long term HU at a dose of 10–30 mg/kg/day. These parameters were also measured in 27 healthy control subjects without SCD. RBVes were significantly higher in untreated SCD (15.59±3.07 x 103/μL of blood) than in control subjects (2.13 ± 0.5)(p=0.001). In HU-treated SCD patients, vesicles were significantly lower (3.97±0.83)(p=0.0371). than in untreated patients and approached levels found in healthy controls. Free plasma Hb was significaly increased in untreated SCD (16.74±3.87) compared with healthy controls (1.77±0.2)(p=0.001); these levels were also corrected in HU treated SCD patients (13.42±4.48). The proportion of RBVes expressing phosphatadyl serine was also lower in HU-treated SCD (37.25± 9.79) than in untreated SCD (52.52±4.74). This was paralleled by a decrease in red cells expressing PS in HU treated patients (11.83± 3.95) compared with untreated patients (42.71±8.45)(p=0.011). This decrease in both RBVes and RBCs expressing PS in HU-treated patients might in principle decrease activation of coagulation. Indeed, plasma Ddimers, which are increased in untreated SCD (480 ±60.14) compared with healthy controls (117.43±38)(p=0.018). are normalised in HU treated patients (190±28.69). These data provide evidence of a link between RBVes and plasma Hb and suggest that PS expression on RBVes and red cells plays a role in the prothrombotic state seen in SCD. As HU treatment appears to correct plasma Hb, long term treatment may be associated with a decreased scavenging of NO and hence the risk of pulmonary hypertension. The correction of the prothrombotic tendency may also be beneficial in this regard. Prospective trials are indicated to confirm whether HU treated patients have evidence of decreased risk of pulmonary hypertension.
Immunogenicity of a BCR-ABL e1a2 p190 Translocation in a Balb/c Mouse Model.
Authors: Gomez-Nunez Marta , Pinilla-Ibarz Javier , Dao Tao , Zakhaleva Victoriya , Korontsvit Tatyana , May Rena J. , Scheinberg David A.
Published: October 2019

Intracellular processing of the breakpoint products of the bcr-abl fusion gene may generate novel peptides that, if capable of binding to HLA class I molecules, would be potential targets for a cytotoxic T lymphocyte (CTL) response. In humans, peptides derived from the e1a2 p190 breakpoint have generated peptide-specific MHC class II proliferative responses. Short peptides have shown binding to MHC class I molecules, although processing and presentation of endogenous p190 protein has not been shown. In a Kd Balb/c mouse model, we tested the hypothesis that single, key amino acids near the breakpoint could be the reason of lack of immunogenicity of the p190 breakpoint peptides. We synthesized a native peptide from the 190 breakpoint (AFHGDAEAL) as well as a synthetic mutated peptide (AYHGDAEAL), which showed excellent predicted binding on the BIMAS algorithm (1152 and 2880 respectively), although in vivo experiments did not show any specific CTL response. In order to assess if the lack of immunogenicity in vivo was due to the absence of binding to the MHC class I molecule rather than to poor TCR interactions, we designed a series of peptides where neutral amino acid, alanine, substitutions were introduced at different potential binding sites in the synthetic peptide: at position three (AYAGDAEAL), position four (AYHADAEAL), position five (AYHGAAEAL) and position seven (AYHGDAAAL). The binding of these altered peptides to H2 class I was assessed using a MHC stabilization assay on T2-Kd cells (TAP deficient cells). In spite of the good computer prediction for binding the MHC stabilization assay did not show evidence of binding of the native and synthetic peptides (<1.9 over the background). In contrast, alanine substitution in position five resulted in the best binding (3.5x over the background). Alanine substitutions in positions three, four and seven also showed improved binding to Kd molecules (2.6x, 3.1x and 3x over the background respectively). These results were confirmed by immunization in vivo with these altered peptides. Only the peptide with the alanine substitution in position five generated a immune response in a CD8 gamma-IFN Elispot assay. Since the altered peptides with the alanine substitutions in positions three, four and seven have shown binding to the Kd molecules, but not a CTL response, change of the aspartic acid in position five near to the fusion breakpoint allows appropriate presentation and recognition of the sequence by the TCR.
Autologous Hematopoietic Stem Cell Transplantation for Refractory Crohn’s Disease, the Summary of Phase I Trial.
Authors: Oyama Yu , Craig Robert , Traynor Ann , Statkute Laivyde , Burt Richard
Published: October 2019

Background &Aims

Crohn’s disease (CD) is an immunologically mediated inflammatory disease of the gastrointestinal tract. In theory, immune ablation followed by autologous hematopoietic stem cell transplantation (HSCT) can induce remission by reconstituting a new immune system.

Methods

We conducted a phase I HSCT study in 17 patients with severe CD. Candidates were less than 60 years old with a Crohn’s Disease Activity Index (CDAI) of 250–400 or Craig Crohn’s Severity Index (CCSI) of >17 despite conventional therapy including infliximab. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte colony-stimulating factor and CD34+ enriched. The immune ablative conditioning regimen consisted of 200mg/kg cyclophosphamide, and 90mg/kg equine antithymocyte globulin.

Results

There was no treatment related mortality. The procedure was well tolerated with anticipated cytopenias, neutropenic fever, and disease-related fever, diarrhea, anorexia, nausea, vomiting. The median days for neutrophil and platelet engraftment were 9 (range 7–11) and 9 (range 8–18, three patients never below 20,000), respectively. The median infused CD34+ and CD3+ cell counts were 4.83 x 106/kg (range 1.73–24.88) and 0.56 x 104/kg (range 0.0–3.09), respectively. Initial median CDAI and CCSI were 282 (range 101–358) and 24 (range 18–33). Symptoms and CDAI/CCSI improved quickly and dramatically (figure), and radiographic and colonoscopy findings improved gradually over months to years following HSCT. For patients with >6 months follow up, twelve out of fifteen patients entered sustained remission defined by a CDAI < 150 or reduction of a CCSI >10 after median follow-up of 29 months (range 11–48).

Conclusion

Autologous HSCT can be performed safely and has a marked durable salutary effect on CD activity. A randomized study {KISS study: C(K)rohn’s Immune Suppression vs. Stem Cell Transplant} comparing HSCT to standard therapy is now enrolling patients.

Figure Figure