Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H- vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Establishment and characterization of an Epstein-Barr virus transformed cell line with strong phagocytic activity

An immunoglobulin M (IgM)-positive cell line, Ms 28, apparently spontaneously transformed by Epstein-Barr virus (EBV) was established from peripheral blood cells of a patient with immature myeloblastic leukemia. It has been characterized according to phenotype, cytochemistry, and membrane antigen pattern. The cell line expresses lymphoid markers like CD 19, CD 22, and CD 30 and synthesizes and secretes IgM. Monocyte markers CD 11c, CD 14, and CD 15 are absent. Neither interleukin-1 (IL-1), nor tumor necrosis factor (TNF-alpha) are produced. But Ms 28 cells show strong phagocytic activity and engulf Latex particles and sheep RBCs (SRBCs) that need not to be opsonized. The phagocytic activity can be inhibited by chloroquine. Both phagocytosis and EBV nuclear-antigen (EBNA) expression can be observed in one and the same cell. Ms 28 cells might be useful to study immunologic activities like antigen processing and presentation.     

Reduced response of Cystathionine Beta-Synthase (CBS) to S-Adenosylmethionine (SAM): Identification and functional analysis of CBS gene mutations in Homocystinuria patients

A reduced response of cystathionine beta-synthase (CBS) to its allosteric activator S-adenosylmethionine (SAM) has been reported to be a cause of CBS dysfunction in homocystinuria patients. In this work we performed a retrospective analysis of fibroblast data from 62 homocystinuria patients and found that 13 of them presented a disturbed SAM activation. Their genotypic background was identified and the corresponding CBS mutant proteins were produced in E. coli. Nine distinct mutations were detected in 22 independent alleles: the novel mutations p.K269del, p.P427L, p.S500L and p.L540Q; and the previously described mutations p.P49L, p.C165Rfs*2, p.I278T, p.R336H and p.D444N. Expression levels and residual enzyme activities, determined in the soluble fraction of E. coli lysates, strongly correlated with the localization of the affected amino acid residue. C-terminal mutations lead to activities in the range of the wild-type CBS and to oligomeric forms migrating faster than tetramers, suggesting an abnormal conformation that might be responsible for the lack of SAM activation. Mutations in the catalytic core were associated with low protein expression levels, decreased enzyme activities and a higher content of high molecular mass forms. Furthermore, the absence of SAM activation found in the patients’ fibroblasts was confirmed for all but one of the characterized recombinant proteins (p.P49L). Our study experimentally supports a deficient regulation of CBS by SAM as a frequently found mechanism in CBS deficiency, which should be considered not only as a valuable diagnostic tool but also as a potential target for the development of new therapeutic approaches in classical homocystinuria.     

Antagonism of the interleukin 4 receptor α promotes TH1-signalling among T cells from patients with atopic dermatitis after stimulation

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease. Molecular characterization of AD shows an underlying inflammation with tissue infiltration of T helper (T_H) 2 cells and increased IL-4 and IL-13. The multifaceted roles of IL-4 and IL-13 in allergic disease development make IL-4Rα an attractive target for treatment strategies, and a neutralizing monoclonal antibody which antagonizes the effects of both IL-4 and IL-13 by blocking the interaction site found in the IL-4 receptor subunit α (IL-4Rα) has been successfully used to treat patients with moderate-to-severe AD. To elucidate the effects of IL-4Rα blockade on the cellular level, we used flow cytometry to examine cytokine production after antigen stimulation in human T cells from patients with AD (n = 12) and healthy controls (n = 6). The cells were stimulated with and without a neutralizing monoclonal antibody against IL-4Rα. Our results indicate that blocking IL-4Rα prohibits IL-4 signalling and IL-13 signalling and thereby T_H2 differentiation followed by an upregulation of interferon-γ-producing cells.     

The influence of vascular risk factors on cognitive decline in patients with Alzheimer's Disease

Introduction

The influence of vascular risk factors (VRFs) on the rate of cognitive decline in patients with established dementia is unclear. This study aims to examine the association between VRFs and the rate of cognitive decline in patients with Alzheimer's disease (AD).

Methods

Data were obtained from patients visiting a memory clinic between 2004 and 2012. VRFs were determined at baseline and included hypertension, hypercholesterolemia, diabetes mellitus, overweight and smoking. Continuous values of blood pressure, total cholesterol, glucose level and body mass index were also obtained. Mini-Mental State Exam (MMSE) scores were obtained at baseline and during follow-up visits. The association between VRFs and the annual change in MMSE scores was analysed with a multivariable linear mixed model adjusted for age, sex and the aforementioned VRFs.

Results

From 174 patients (mean age 78.3 years), with a follow-up time up to 5.8 years (mean 1.1 year), in total 447 MMSE scores were obtained. The multivariable analyses showed an association between age as well as systolic blood pressure and a decline in annual rates of change in MMSE scores of −0.05 (95% confidence interval (CI): −0.09 to 0.00) and −0.01 (CI: −0.03 to 0.00), respectively. For all other VRFs, including sex, patients did not show a significant difference.

Conclusion

This study did not find an association between preventable vascular risk factors and cognitive decline in patients with AD, except for systolic blood pressure. As the association between systolic blood pressure and decline in MMSE was small, clinical relevance may be limited.     

Ventricular Performance After Surgery for a Congenital Heart Defect as Assessed Using Advanced Echocardiography: From Doppler Flow to 3D Echocardiography and Speckle-Tracking Strain Imaging

A varying degree of impairment of ventricular performance is observed over the long-term after surgery for a congenital heart defect (CHD). Impaired ventricular performance has been shown to be of prognostic value for increased risk of cardiovascular events in adult CHD patients. This emphasizes the importance of delineating the timing and cause of this postoperative impairment. Impairment of ventricular performance could develop over time as a consequence of residua, sequelae and complications of the CHD or surgical procedure. Yet, impaired ventricular performance has also been observed immediately after surgery and can persist and/or worsen over time. This postoperative impairment of ventricular performance is the focus of this review. This article provides an overview of echocardiographic techniques currently used to assess ventricular performance. Furthermore, we review current literature describing ventricular performance, as assessed using echocardiography, after correction of a CHD. In general, a decrease in ventricular performance is observed directly after surgery for CHD’s. Subsequent follow-up of ventricular performance is characterized by a varying degree of postoperative recovery. A consistent observation is the persistent impairment of right-ventricular performance after repair in several different subgroups of CHD patients ranging from ventricular septal defect repair to surgery for Tetralogy of Fallot.     

The Ariadne principles: how to handle multimorbidity in primary care consultations

Multimorbidity is a health issue mostly dealt with in primary care practice. As a result of their generalist and patient-centered approach, long-lasting relationships with patients, and responsibility for continuity and coordination of care, family physicians are particularly well placed to manage patients with multimorbidity. However, conflicts arising from the application of multiple disease oriented guidelines and the burden of diseases and treatments often make consultations challenging. To provide orientation in decision making in multimorbidity during primary care consultations, we developed guiding principles and named them after the Greek mythological figure Ariadne. For this purpose, we convened a two-day expert workshop accompanied by an international symposium in October 2012 in Frankfurt, Germany. Against the background of the current state of knowledge presented and discussed at the symposium, 19 experts from North America, Europe, and Australia identified the key issues of concern in the management of multimorbidity in primary care in panel and small group sessions and agreed upon making use of formal and informal consensus methods. The proposed preliminary principles were refined during a multistage feedback process and discussed using a case example. The sharing of realistic treatment goals by physicians and patients is at the core of the Ariadne principles. These result from i) a thorough interaction assessment of the patient’s conditions, treatments, constitution, and context; ii) the prioritization of health problems that take into account the patient’s preferences – his or her most and least desired outcomes; and iii) individualized management realizes the best options of care in diagnostics, treatment, and prevention to achieve the goals. Goal attainment is followed-up in accordance with a re-assessment in planned visits. The occurrence of new or changed conditions, such as an increase in severity, or a changed context may trigger the (re-)start of the process. Further work is needed on the implementation of the formulated principles, but they were recognized and appreciated as important by family physicians and primary care researchers.Please see related article: http://www.biomedcentral.com/1741-7015/12/222.