Autophagy: in: Cell Death

Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including cell organelles. Although the importance of autophagy for cell homeostasis and survival has long been appreciated, our understanding of how autophagy is regulated at a molecular level just recently evolved. The importance of autophagy for the quality control of proteins is underscored by the fact that many neurodegenerative and myodegenerative diseases are characterized by an increased but still insufficient autophagic activity. Similarly, if the cellular stress, leading to deoxyribonucleic acid (DNA) damage, mitochondrial damage and/or damaged proteins, does not result in sufficient autophagic repair mechanisms, cells seem to be prone to transform into tumour cells. Therefore, autophagy has multiple roles to play in the causation and prevention of human diseases.

Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats

Objective: Central to the process of osseointegration is the recruitment of mesenchymal progenitor cells to the healing site, their proliferation and differentiation to bone synthesising osteoblasts. The process is under the control of pro-inflammatory cytokines and growth factors. The aim of this study was to monitor these key stages of osseointegration and the signalling milieu during bone healing around implants placed in healthy and diabetic bone. Methods: Implants were placed into the sockets of incisors extracted from the mandibles of normal Wistar and diabetic Goto-Kakizaki rats. Mandibles 1-12 weeks post-insertion of the implant were examined by histochemistry and immunocytochemistry to localise the presence of Stro-1- positive mesenchymal progenitor cells, proliferating cellular nuclear antigen proliferative cells, osteopontin and osteocalcin, macrophages, pro-inflammatory cytokines interleukin (IL)-1 , IL-6, tumour necrosis factor (TNF)- and tumour growth factor (TGF)- 1. Image analysis provided a semi-quantification of positively expressing cells. Results: Histological staining identified a delay in the formation of mineralised bone around implants placed in diabetic animals. Within the diabetic bone, the migration of Stro-1 mesenchymal cells in the healing tissue appeared to be unaffected. However, in the diabetic healing bone, the onset of cell proliferation and osteoblast differentiation were delayed and subsequently prolonged compared with normal bone. Similar patterns of change were observed in diabetic bone for the presence of IL-1 , TNF- , macrophages and TGF- 1. Conclusion: The observed alterations in the extracellular presence of pro-inflammatory cytokines, macrophages and growth factors within diabetic tissues that correlate to changes in the signalling milieu, may affect the proliferation and differentiation of mesenchymal progenitor cells in the osseointegration process. To cite this article: Colombo JS, Balani D, Sloan AJ, St Crean J, Okazaki J, Waddington RJ. Delayed osteoblast differentiation and altered inflammatory response around implants placed in incisor sockets of type 2 diabetic rats Clin. Oral Impl. Res22, 2011; 578-586 doi: 10.1111/j.1600-0501.2010.01992.x.

Association of pharmacogenetic markers with premature discontinuation of first-line anti-HIV therapy: an observational cohort study

Poor tolerance and adverse drug reactions are main reasons for discontinuation of antiretroviral therapy (ART). Identifying predictors of ART discontinuation is a priority in HIV care.

ADME pharmacogenetics: investigation of the pharmacokinetics of the antiretroviral agent lopinavir coformulated with ritonavir

An ADME (absorption, distribution, metabolism and excretion)-pharmacogenetics association study may identify functional variants relevant to the pharmacokinetics of lopinavir co-formulated with ritonavir (LPV/r), a first-line anti-HIV agent.

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study

Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy.

Host and viral genetic correlates of clinical definitions of HIV-1 disease progression

Various patterns of HIV-1 disease progression are described in clinical practice and in research. There is a need to assess the specificity of commonly used definitions of long term non-progressor (LTNP) elite controllers (LTNP-EC), viremic controllers (LTNP-VC), and viremic non controllers (LTNP-NC), as well as of chronic progressors (P) and rapid progressors (RP).

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Biological applications of magnetic nanoparticles

In this review an overview about biological applications of magnetic colloidal nanoparticles will be given, which comprises their synthesis, characterization, and in vitro and in vivo applications. The potential future role of magnetic nanoparticles compared to other functional nanoparticles will be discussed by highlighting the possibility of integration with other nanostructures and with existing biotechnology as well as by pointing out the specific properties of magnetic colloids. Current limitations in the fabrication process and issues related with the outcome of the particles in the body will be also pointed out in order to address the remaining challenges for an extended application of magnetic nanoparticles in medicine.

Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

Are plasma levels valid surrogates for cellular concentrations of antiretroviral drugs in HIV-infected patients?

Total plasma concentrations are currently measured for therapeutic drug monitoring of HIV protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). However, the pharmacological target of antiretroviral drugs reside inside cells. To study the variability of their cellular accumulation, and to determine to which extent total plasma concentrations (TPC) correlate with cellular concentrations (CC), plasma and peripheral blood mononuclear cells (PBMCs) were simultaneously collected at single random times after drug intake from 133 HIV infected patients. TPC levels were analysed by high-performance liquid chromatography with ultraviolet detection and CC by LC-MS/MS from peripheral blood mononuclear cells. The best correlations between TPC and CC were observed for nelfinavir (NFV, slope=0.93, r=0.85), saquinavir (SQV, slope=0.76, r=0.80) and lopinavir (LPV, slope=0.87, r=0.63). By contrast, TPC of efavirenz (EFV) exhibited a moderate correlation with CC (slope=0.69, r=0.58), while no correlation was found for nevirapine (NVP, slope=-0.3, r=0.1). Interindividual variability in the CC/TPC ratio was lower for protease inhibitors (coefficients of variation 76%, 61%, and 80% for SQV, NFV and LPV, respectively) than for nonnucleoside reverse transcriptase inhibitors (coefficients of variation 101% and 318%, for EFV and NVP). As routine CC measurement raises practical difficulties, well-correlated plasma concentrations (ie, NFV, SQV and LPV) can probably be considered as appropriate surrogates for cellular drug exposure. For drugs such as EFV or NVP, there may be room for therapeutic drug monitoring improvement using either direct CC determination or other predictive factors such as genotyping of transporters or metabolizing enzyme genes.

Sirolimus- vs paclitaxel-eluting stents in de novo coronary artery lesions: the REALITY trial: a randomized controlled trial

CONTEXT: Compared with bare metal stents, sirolimus-eluting and paclitaxel-eluting stents have been shown to markedly improve angiographic and clinical outcomes after percutaneous coronary revascularization, but their performance in the treatment of de novo coronary lesions has not been compared in a prospective multicenter study. OBJECTIVE: To compare the safety and efficacy of sirolimus-eluting vs paclitaxel-eluting coronary stents. DESIGN: Prospective, randomized comparative trial (the REALITY trial) conducted between August 2003 and February 2004, with angiographic follow-up at 8 months and clinical follow-up at 12 months. SETTING: Ninety hospitals in Europe, Latin America, and Asia. PATIENTS: A total of 1386 patients (mean age, 62.6 years; 73.1% men; 28.0% with diabetes) with angina pectoris and 1 or 2 de novo lesions (2.25-3.00 mm in diameter) in native coronary arteries. INTERVENTION: Patients were randomly assigned in a 1:1 ratio to receive a sirolimus-eluting stent (n = 701) or a paclitaxel-eluting stent (n = 685). MAIN OUTCOME MEASURES: The primary end point was in-lesion binary restenosis (presence of a more than 50% luminal-diameter stenosis) at 8 months. Secondary end points included 1-year rates of target lesion and vessel revascularization and a composite end point of cardiac death, Q-wave or non-Q-wave myocardial infarction, coronary artery bypass graft surgery, or repeat target lesion revascularization. RESULTS: In-lesion binary restenosis at 8 months occurred in 86 patients (9.6%) with a sirolimus-eluting stent vs 95 (11.1%) with a paclitaxel-eluting stent (relative risk [RR], 0.84; 95% confidence interval [CI], 0.61-1.17; P = .31). For sirolimus- vs paclitaxel-eluting stents, respectively, the mean (SD) in-stent late loss was 0.09 (0.43) mm vs 0.31 (0.44) mm (difference, -0.22 mm; 95% CI, -0.26 to -0.18 mm; P<.001), mean (SD) in-stent diameter stenosis was 23.1% (16.6%) vs 26.7% (15.8%) (difference, -3.60%; 95% CI, -5.12% to -2.08%; P<.001), and the number of major adverse cardiac events at 1 year was 73 (10.7%) vs 76 (11.4%) (RR, 0.94; 95% CI, 0.69-1.27; P = .73). CONCLUSION: In this trial comparing sirolimus- and paclitaxel-eluting coronary stents, there were no differences in the rates of binary restenosis or major adverse cardiac events. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT00235092.

Does tenofovir influence efavirenz pharmacokinetics?

INTRODUCTION: A recent report described a possible interaction between tenofovir (TFV) and efavirenz (EFV). Patients developed neuropsychiatric manifestations upon introduction of TFV on a stable EFV-containing regimen. We evaluated the possibility of a pharmacokinetic interaction between TFV and EFV by assessing cross-sectional and longitudinal data in 169 individuals receiving EFV. RESULTS: EFV plasma area-under-the-curve (AUC) levels were comparable among individuals receiving (n=18) or not receiving TFV (n=151); 57,962 versus 52,293 ng*h/ml. However, under conditions of limited EFV metabolism, that is, the group of 23 individuals carrying two copies of CYP2B6 loss/diminished-function alleles, plasma AUC values were highest among individuals receiving TFV (n=5, 353,031 ng*h/ml), compared with those not receiving TFV (n=18, 180,689 ng*h/ml). Statistical analysis identified both a global, sixfold effect of CYP2B6 loss/diminished function (P < 0.0001) and a significant interaction between the number of loss/diminished-function alleles and the co-medication with TFV (P = 0.009). CONCLUSION: Although there is no clear evidence for a pharmacokinetic interaction between TFV and EFV, we cannot rule out an interaction between these drugs restricted to individuals who are slow EFV metabolizers.

Predictive value of known and novel alleles of CYP2B6 for efavirenz plasma concentrations in HIV-infected individuals

To assess the association of CYP2B6 allelic diversity with efavirenz (EFV) pharmacokinetics, we performed extensive genotyping of 15 relevant single nucleotide polymorphism in 169 study participants, and full resequencing of CYP2B6 in individuals with abnormal EFV plasma levels. Seventy-seven (45.5%) individuals carried a known (CYP2B6*6, *11, *15, or *18) or new loss/diminished-function alleles. Resequencing defined two new loss-of-function alleles: allele *27 (marked by 593T>C [M198T]), that results in 85% decrease in enzyme activity and allele *28 (marked by 1132C>T), that results in protein truncation at arginine 378. Median AUC levels were 188.5 microg h/ml for individuals homozygous for a loss/diminished-function allele, 58.6 microg h/ml for carriers, and 43.7 microg h/ml for noncarriers (P<0.0001). Individuals with a poor metabolizer genotype had a likelihood ratio of 35 (95% CI, 11-110) of presenting very high EFV plasma levels. CYP2B6 poor metabolizer genotypes explain to a large extent EFV pharmacokinetics and identify individuals at risk of extremely elevated EFV plasma levels.

Influence of ABCB1, ABCC1, ABCC2, and ABCG2 haplotypes on the cellular exposure of nelfinavir in vivo

OBJECTIVES: The human immunodeficiency virus protease inhibitor nelfinavir is substrate of polyspecific drug transporters encoded by ABCB1 (P-glycoprotein), ABCC1 (MRP1) and ABCC2 (MRP2), and an inhibitor of BCRP, encoded by ABCG2. Genetic polymorphism in these genes may be associated with changes in transport function. METHODS: A comprehensive evaluation of single nucleotide polymorphisms (39 SNPs in ABCB1, 7 in ABCC1, 27 in ABCC2, and 16 in ABCG2), and inferred haplotypes was done to assess possible associations of genetic variants with cellular exposure of nelfinavir in vivo. Analysis used peripheral mononuclear cells from individuals receiving nelfinavir (n=28). Key results were re-examined in a larger sample size (n=129) contributing data on plasma drug levels. RESULTS AND CONCLUSIONS: There was no significant association between cellular nelfinavir area under the curve (AUC) and SNPs or haplotypes at ABCC1, ABCC2, ABCG2. There was an association with cellular exposure for two loci in strong linkage disequilibrium: ABCB1 3435C>T; AUCTT>AUCCT>AUCCC (ratio 2.1, 1.4, 1, Ptrend=0.01), and intron 26 +80T>C; AUCCC> AUCCT > AUCTT (ratio 2.4, 1.3, 1, Ptrend=0.006). Haplotypic analysis using tagging SNPs did not improve the single SNP association values.

Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients

BACKGROUND: Efavirenz (EFV) and nevirapine (NVP) are metabolized by cytochrome P450 2B6 (CYP2B6). Allele 516 G>T (Gln172His) is associated with diminished activity of this isoenzyme, and may lead to differences in drug exposure. METHODS: We evaluated this allele as a pharmacogenetic marker of EFV and NVP pharmacokinetics and EFV toxicity in 167 participants receiving EFV and 59 receiving NVP recruited within the genetics project of the Swiss HIV Cohort Study. Drug concentrations were measured in plasma and in peripheral blood mononuclear cells (PBMCs) from the same sample. Neuropsychological toxicity of EFV (sleep disorders, mood disorders, fatigue) was assessed using a standardized questionnaire. RESULTS AND CONCLUSIONS: CYP2B6 516TT was associated with greater plasma and intracellular exposure to EFV, and greater plasma exposure to NVP. Intracellular drug concentration, and CYP2B6 genotype were predictors of EFV neuropsychological toxicity. CYP2B6 genotyping may be useful to complement an individualization strategy based on plasma drug determinations to increase the safety and tolerability of EFV.