The impact of cytokines on the expression of drug transporters,cytochrome P450 enzymes and chemokine receptors in human PBMC

Background and purpose:

The function of transporters in peripheral blood mononuclear cells (PBMC) has been characterized, but less is known about cytochrome P450 (CYP) enzyme function in these cells. Given that cytokines are dysregulated in many diseases, the purpose of this work was to assess the impact of cytokines on the expression of CYPs, transporters and chemokine receptors in PBMC.

Experimental approach:

Human PBMC were incubated with cytokines for 48 h. ATP-binding cassette (ABC)B1, ABCC1, ABCC2, CYP2B6, CYP3A4, CXCR4 and CCR5 expression were measured by quantitative polymerase chain reaction and flow cytometry at 0, 4, 8, 24 and 48 h. Enzyme activity was assessed using fluorescent probes.

Key results:

We show here functional activity of CYP3A4 and CYP2B6 in PBMC. Furthermore, cytokines had a significant impact on the mRNA and protein expression of all proteins. For example, interleukin-2 (IL-2) had a marked impact on ABCB1 mRNA (% control 4745 ± 11961) and protein (% control 200 ± 57). Increases in drug efflux transporter expression, in response to cytokines, resulted in reduced cellular accumulation of digoxin [decrease of 17% and 26% for IL-2 and interferon-γ (IFNγ) respectively] and saquinavir (decrease of 28% and 30% for IL-2 and IFNγ respectively). The degree to which drug transporter and chemokine receptor expression changed in response to cytokines was positively correlated (e.g. ABCB1 and CXCR4, r² = 0.545).

Conclusions and implications:

These data have important implications for diseases in which cytokines are dysregulated and for which pharmacological intervention targets immune cells.     

Database searching for structural identification of metabolites in complex biofluids for mass spectrometry-based metabonomics

MS and HPLC are commonly used for compound characterization and obtaining structural information; in the field of metabonomics, these two analytical techniques are often combined to characterize unknown endogenous or exogenous metabolites present in complex biological samples. Since the structures of a majority of these metabolites are not actually identified, the result of most metabonomic studies is a list of m/z values and retention times. However, without knowing actual structures, the biological significance of these 'features' cannot be determined. The process of identifying the structures of unknown compounds can be time intensive, costly and frequently requires the use of multiple orthogonal analytical techniques - this laborious procedure seems insurmountable for the long lists of unknowns that must be identified for each study. In addition, the limited sample volume and the extremely low concentration of most endogenous analytes frequently make purification and identification by other instrumentation nearly impossible. This review is intended to explore the problems and progress with current tools that are available for MS-based structure identification for both endogenous and exogenous metabolites.     

The prevalence of misidentification syndromes in neurodegenerative diseases

Although misidentification syndromes (MISs) have been often described in Alzheimer disease (AD), the prevalence of these phenomena in different neurodegenerative diseases has not been systematically studied. Three hundred ninety-two individuals with probable AD, 119 patients with the behavioral variety of frontotemporal dementia (FTD-bv), 101 patients with primary progressive aphasia, 24 subjects with semantic dementia, 18 subjects with corticobasal degeneration, 8 patients with progressive supranuclear palsy, 36 individuals with probable Lewy body dementia (DLB), and 26 subjects with Parkinson disease (PD) were the participants of this study. On the basis of a semistructured interview with both patients and their reliable caregivers, MIS was identified in 15.8% of cases with AD, 16.6% of patients with DLB, and in 8.3% of individuals with semantic dementia. The most frequent form of MIS was Capgras delusions, often accompanied by reduplication of place, phantom border phenomenon, or both. Although MIS typically appears in later stages of the disease, it can also occur surprisingly early in patients with AD. None of the patients with FTD-bv, primary progressive aphasia, corticobasal degeneration/supranuclear palsy, or PD developed MIS. Thus, our findings suggest that MISs are characteristic of AD and DLB, and tend to exclude FTD/Pick complex and PD.     

Quantification of atherosclerotic coronary plaque components by submillimeter computed tomography

Background: Although several investigations have shown that multi-detecor row computed tomography (MDCT) of the coronary arteries can detect noncalcified atherosclerotic plaque, it has remained unresolved if the method also determines features of a rupture-prone plaque. We set out to correlate the size of atherosclerotic plaque components with cardiac MDCT with histology. Methods and results: In 30 autopsy cases, hearts were isolated, coronary arteries filled with contrast agent, and depicted with a clinical 16-row detector CT with a slice thickness of 0.63 mm. Transections of the three main coronary arteries were reconstructed and compared with histopathologic sections using light microscopy. MDCT measurements of total plaque area (r = 0.73, P < 0.0001) and calcified plaque area (r = 0.83, P < 0.0001) correlated well with histopathology, while measurements of non-calcified plaque area (r = 0.53, P < 0.0001) and lipid core size (r = 0.43; P < 0.0001) correlated less well. MDCT overestimated all plaque areas except lipid core size, which was underestimated. Conclusions: Coronary CT provides an accurate and reproducible method for the quantitative assessment of total plaque and calcified plaque areas. However, the method is less accurate for the quantification of non-calcified plaque area and lipid core size, which is ascribed to limited spatial and contrast resolution. With the present technique, the detection of vulnerable plaques by MDCT remains uncertain.     

Robust anti-arrhythmic efficacy of verapamil and flunarizine against dofetilide-induced TdP arrhythmias is based upon a shared and a different mode of action

BACKGROUND AND PURPOSE

The high predisposition to Torsade de Pointes (TdP) in dogs with chronic AV-block (CAVB) is well documented. The anti-arrhythmic efficacy and mode of action of Ca²⁺ channel antagonists, flunarizine and verapamil against TdP were investigated.

EXPERIMENTAL APPROACH

Mongrel dogs with CAVB were selected based on the inducibility of TdP with dofetilide. The effects of flunarizine and verapamil were assessed after TdP and in different experiments to prevent dofetilide-induced TdP. Electrocardiogram and ventricular monophasic action potentials were recorded. Electrophysiological parameters and short-term variability of repolarization (STV) were determined. In vitro, flunarizine and verapamil were added to determine their effect on (i) dofetilide-induced early after depolarizations (EADs) in canine ventricular myocytes (VM); (ii) diastolic Ca²⁺ sparks in RyR2^R4496+/+ mouse myocytes; and (iii) peak and late I_Na in SCN5A-HEK 293 cells.

KEY RESULTS

Dofetilide increased STV prior to TdP and in VM prior to EADs. Both flunarizine and verapamil completely suppressed TdP and reversed STV to baseline values. Complete prevention of TdP was achieved with both drugs, accompanied by the prevention of an increase in STV. Suppression of EADs was confirmed after flunarizine. Only flunarizine blocked late I_Na. Ca²⁺ sparks were reduced with verapamil.

CONCLUSIONS AND IMPLICATIONS

Robust anti-arrhythmic efficacy was seen with both Ca²⁺ channel antagonists. Their divergent electrophysiological actions may be related to different additional effects of the two drugs.     

Quantification of functional deficit in Alzheimer's disease using a computer-assisted mapping program for99mTc-HMPAO SPECT

A computer-assisted mapping program was developed to determine changes of cerebral perfusion in normal and pathological aging using single photon emission computer tomography (SPECT) and^99mTc-hexamethylpropyleneamine oxime (HMPAO). The software program outlined the cortex on 14 adjacent brain slices, and superimposed a ring of 12 regions of interest on each slice. Regional/global and regional/cerebellar relative flow values were calculated in 27 patients with clinically diagnosed Alzheimer's disease (AD) (mean age 71 years, SD 7.6) and in 10 normal controls (mean age 73.7 years, SD 7.3). The Dementia Rating Scale (DRS) was used to assess mental status in all subjects. Multiple regression analysis demonstrated a significant correlation between relative flow values and the DRS score. Regional/cerebellar (R=0.88,p<0.0001) relative flow values were a better indicator of cortical impairment than regional/global relative flow values (R=0.68,p=0.003). Of the brain regions of interest, the left parietal flow values correlated best with the DRS score (r=0.83,p<0.0001), a cutoff value of 77 accurately classifing 80% of the normals and 100% of the patients diagnosed as having Alzheimer's disease. The data show that computer-assisted mapping of SPECT can provide semiquantitative flow values with high diagnostic accuracy.