RBL cells as models for in vitro studies of mast cells and basophils

Since their establishment in 1981, RBL-2H3 cells have been widely used as a mast cell (MC) model. Their ability to be easily grown in culture in large amounts, their responsiveness to FcεRI-mediated triggers and the fact that they can be genetically manipulated, have provided advantages over primary MCs, in particular for molecular studies relying on genetic screening. Furthermore, the ability to generate clones that stably express proteins of interest, for example, a human receptor, have marked the RBL cells as an attractive MC model for drug screening. Indeed, 3 RBL reporter cell lines (RS-ATL8, NFAT-DsRed, and NPY-mRFP) have been generated providing useful models for drug and allergen screening. Similarly, RBL cells stably expressing the human MrgprX2 receptor provide a unique paradigm for analyzing ligand interactions and signaling pathways of the unique human receptor. Finally, transient co-transfections of RBL cells allow functional genomic analyses of MC secretion by combining library screening with simultaneous expression of a reporter for exocytosis. RBL cells thus comprise powerful tools for the study of intracellular membrane trafficking and exocytosis and the detection of allergens, vaccine safety studies and diagnosis of allergic sensitization. Their recent uses as an investigative tool are reviewed here.     

Combined Haploidentical and Umbilical Cord Blood Allogeneic Stem Cell Transplantation for High-Risk Lymphoma and Chronic Lymphoblastic Leukemia

Limited studies have reported on outcomes for lymphoid malignancy patients receiving alternative donor allogeneic stem cell transplants. We have previously described combining CD34-selected haploidentical grafts with umbilical cord blood (haplo-cord) to accelerate neutrophil and platelet engraftment. Here, we examine the outcome of patients with lymphoid malignancies undergoing haplo-cord transplantation at the University of Chicago and Weill Cornell Medical College. We analyzed 42 lymphoma and chronic lymphoblastic leukemia (CLL) patients who underwent haplo-cord allogeneic stem cell transplantation. Patients underwent transplant for Hodgkin lymphoma (n?=?9, 21%), CLL (n?=?5, 12%) and non-Hodgkin lymphomas (n?=?28, 67%), including 13 T cell lymphomas. Twenty-four patients (52%) had 3 or more lines of therapies. Six (14%) and 1 (2%) patients had prior autologous and allogeneic stem cell transplant, respectively. At the time of transplant 12 patients (29%) were in complete remission, 18 had chemotherapy-sensitive disease, and 12 patients had chemotherapy-resistant disease. Seven (17%), 11 (26%), and 24 (57%) patients had low, intermediate, and high disease risk index before transplant. Comorbidity index was evenly distributed among 3 groups, with 13 (31%), 14 (33%), and 15 (36%) patients scoring 0, 1 to 2, and ≥3. Median age for the cohort was 49 years (range, 23 to 71). All patients received fludarabine/melphalan/antithymocyte globulin conditioning regimen and post-transplant graft-versus-host disease (GVHD) prophylaxis with tacrolimus and mycophenolate mofetil. The median time to neutrophil engraftment was 11 days (range, 9 to 60) and to platelet engraftment 19.5 days (range, 11 to 88). Cumulative incidence of nonrelapse mortality was 11.6% at 100 days and 19 % at one year. Cumulative incidence of relapse was 9.3% at 100 days and 19% at one year. With a median follow-up of survivors of 42 months, the 3-year rates of GVHD relapse free survival, progression-free survival, and overall survival were 53%, 62%, and 65%, respectively, for these patients. Only 8% of the survivors had chronic GVHD. In conclusion, haplo-cord transplantation offers a transplant alternative for patients with recurrent or refractory lymphoid malignancies who lack matching donors. Both neutrophil and platelet count recovery is rapid, nonrelapse mortality is limited, excellent disease control can be achieved, and the incidence of chronic GVHD is limited. Thus, haplo-cord achieves high rates of engraftment and encouraging results.     

Testing patients during seizures: A European consensus procedure developed by a joint taskforce of the ILAE – Commission on European Affairs and the European Epilepsy Monitoring Unit Association

There is currently no international consensus procedure for performing comprehensive periictal testing of patients in the epilepsy monitoring units (EMUs). Our primary goal was to develop a standardized procedure for managing and testing patients during and after seizures in EMUs. The secondary goal was to assess whether it could be implemented in clinical practice (feasibility). A taskforce was appointed by the International League Against Epilepsy (ILAE)—Commission on European Affairs and the European Epilepsy Monitoring Unit Association, to develop a standardized ictal testing battery (ITB) based on expert opinion and experience with various local testing protocols. ITB contains a comprehensive set of 10 items that evidence the clinically relevant semiologic features, and it is adaptive to the dynamics of the individual seizures. The feasibility of the ITB was prospectively evaluated on 250 seizures from 152 consecutive patients in 10 centers. ITB was successfully implemented in clinical practice in all 10 participating centers and was considered feasible in 93% of the tested seizures. ITB was not feasible for testing seizures of very short duration.     

Clinical and Epidemiological Significance of Carbapenem Resistance in Acinetobacter baumannii Infections

Carbapenems are considered the treatment of choice for Acinetobacter baumannii infections. Many facilities implement preventive measures toward only carbapenem-resistant A. baumannii (CRAB). However, the independent role of the carbapenem resistance determinant on patient outcomes remains controversial. In a 6-year analysis of adults with A. baumannii bloodstream infection (BSI), the outcomes of 149 CRAB isolates were compared to those of 91 patients with carbapenem-susceptible A. baumannii. In bivariable analyses, CRAB BSIs were significantly associated with worse outcomes and with a delay in the initiation of appropriate antimicrobial therapy (DAAT). However, in multivariable analyses, carbapenem resistance status was no longer associated with poor outcomes, while DAAT remained an independent predictor. The epidemiological significance of A. baumannii should not be determined by its resistance to carbapenems.     

A bioactive self-assembled membrane to promote angiogenesis

We report here on a bioactive hierarchically structured membrane formed by self-assembly. The membrane is formed with hyaluronic acid and peptide amphiphiles with binding affinity for heparin, and its hierarchical structure contains both an amorphous zone and a layer of fibrils oriented perpendicular to the membrane plane. The design of bioactivity is based on the potential ability to bind and slowly release heparin-binding growth factors. Human mesenchymal stem cells (hMSCs) seeded on these membranes attached and remained viable. Basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF) were incorporated within the membrane structure prior to self-assembly and released into media over a prolonged period of time (14 days). Using the chicken chorioallantoic membrane (CAM) assay, we also found that these membranes induced a significant and rapid enhancement of angiogenesis relative to controls.     

Neonatal stress modulates sickness behavior

The quality of the early environment, especially during the neonatal period, influences the development of individual differences in resistance to stress and illness in adulthood. A previous study demonstrated that neonatal stress augmented proinflammatory cytokine expression and viral replication in influenza virus-infected adult mice. The goal of the following study was to examine the lifelong effects of neonatal stress on the behavioral response to an immune challenge. Neonatal stress consisted of separating mouse pups from their dams (maternal separation, MSP) at critical points of their development. In the first study, pups were separated from the dam daily for 6 h between postnatal day 1 and 14. As adults, these mice were infected with influenza A/PR8 virus. In a second study, a similar paradigm of MSP was employed, and as adults mice were injected with lipopolysaccharide (LPS) (ip). In a third study pups were separated from the dam for 24 h on postnatal day 4 or 9. As adults, these mice received ip injections of LPS. In all three studies, changes in body weight, food and sweet solution consumption were examined following immune challenge. As previously described, activation of the immune system using influenza virus infection or LPS administration resulted in sickness behavior that consisted of body weight loss, anorexia and reduced consumption of a sweet solution. Furthermore, neonatal stress induced more rapid kinetics of sickness behavior and augmented several aspects of these symptoms. Together with previous studies, these findings suggest that neonatal stress disrupted the regulation of innate resistance to an immune challenge resulting in enhanced immunological and behavioral responses to immune activation. Thus, long lasting effects of early stress events may be the basis for individual differences in health and susceptibility to disease.     

Monotherapy of lamotrigine versus carbamazepine in patients with poststroke seizure

OBJECTIVES: The incidence of seizures is known to be high in the elderly. The most common cause of an unprovoked seizure in the elderly population is stroke. These patients require effective and well-tolerated antiepileptic treatment because they frequently experience other medical conditions and use other medications that can interact with the antiepileptic treatment. The aim of the study was to analyze the tolerability and efficacy of lamotrigine (LTG) versus sustained-release carbamazepine (CBZ) treatment in newly diagnosed symptomatic poststroke seizure. METHODS: Sixty-four patients with a first post episode of seizures were randomized in a 1:1 ratio to either LTG or CBZ treatment and were followed up prospectively for up to 12 months for efficacy and tolerability of the drugs. RESULTS: More patients in the LTG group were seizure-free (72%) versus those in the CBZ group (44%; P = 0.06), but the numbers did not reach statistical significance because of a relative small number of study patients. The number of patients who withdraw from the study because of adverse events was statistically significantly less in the LTG group (3%) compared with the CBZ group (31%; P = 0.02). CONCLUSIONS: The LTG treatment in poststroke seizures versus CBZ treatment is a relatively better-tolerated drug and can be acceptable as initial treatment in this specific group of patients.     

Effect of scanner type on the reproducibility of extracoronary measures of calcification: the multi-ethnic study of atherosclerosis

RATIONALE AND OBJECTIVES: Cardiac computed tomography (CT) has been used extensively to measure coronary artery calcification. However, extracoronary calcifications, such as aortic valve calcification (AVC), may have independent clinical significance as well. The ability to track calcification is dependent on the reproducibility of the original measurement, and the variability of extracoronary calcification measurements still is unknown. Accurate quantification of calcification of the aortic valve, mitral annulus (MAC), and thoracic aortic (TAC) may be possible by using cardiac CT. METHODS: A total of 1,729 randomly chosen participants (ages 45-84, 53% female, 28% African-American, 36% Caucasian, 11% Chinese, 25% Hispanic) of the Multi-Ethnic Study of Atherosclerosis underwent dual scanning by electron beam CT (EBT) or multidetector CT (MDCT) to assess coronary and extra-coronary calcifications. Two calcium measurement methods--Agatston score (AS) and volume score (VS)--were measured for each scan. Concordance for calcium positivity was assessed among all scans. Mean absolute and relative differences between calcium measures on scans 1 and 2, excluding cases for which both scans had a measure of zero, was modeled by using linear regression to compare variability between scanner types. A repeated measures analysis of variance test was used to compare variability across calcium measures, with mean percentage absolute difference as the outcome measure. RESULTS: Concordances for the presence of calcium between duplicate scans were high and similar for both EBT and MDCT. Concordance was high for all three extracoronary measures, with a kappa statistic of kappa = 0.94-0.96. For all three extracoronary sites, Bland-Altman plots demonstrated excellent agreement, with almost all measures falling within the boundaries of the 95% confidence limits of reproducibility. AVC interscan variability was approximately 8% for both AS and VS, with improved variability for EBT as compared with MDCT. Mitral annular calcification demonstrated slightly lower variability than AVC for both scanner types (approximately 6%), with no significant differences between MDCT and EBT. Of the three extracoronary sites, TAC had the highest variability (10%), with MDCT variability slightly lower than EBT variability (9.3 vs. 10.2%, respectively, P = NS). Agatson and volume scores for each of the three extracoronary sites were similar. CONCLUSIONS: Overall rescan measurement variabilities for extracoronary calcification are low and should not be an impediment to the use of this test for studying progression of extracoronary calcification over time.