Evaluation of the ROS Inhibiting Activity and Mitochondrial Targeting of Phenolic Compounds in Fibroblast Cells Model System and Enhancement of Efficiency by Natural Deep Eutectic Solvent (NADES) Formulation

Purpose

Many phenolics have already been tested for their antioxidant activities using in vitro methods. However, such assays do not consider the complexity of real cellular systems, and most of the phenolics characterized with such assays shows disappointing results when evaluated in cells. Accordingly, there is a need to develop effective screening methods.

Methods

Antioxidants were first evaluated by CAT assay and then, evaluated for their ability (i) to reduce the level of ROS using fluorescent probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria. Antioxidants were also formulated in NADES.

Results

Correlation was obtained when comparing CAT results with short term inhibition (2 h) in the fibroblast cells. On the contrary, it was difficult to anticipate ROS inhibiting efficiency at long term (24 h) from both the CAT assay and the short term inhibition measurements. Indeed, some molecules displayed activity rapidly but lost it over time. In contrast, other molecules were better for long term. The comparable efficiency at long term of Bis-Ethylhexyl Hydroxydimethoxy Benzylmalonate (Bis-EHBm) and decyl rosmarinate, prompted us to further investigate the potential mitochondrial targeting of the former. Using mitochondrial probes, our results confirmed its mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity.

Conclusions

Combinations of fast acting and slow acting molecules could be promising strategies to identify a performant antioxidant system. Bis-EHBm behaves as decyl rosmarinate with a confirmed mitochondrial location. Finally, the formulation of antioxidants in NADES could greatly improve their activity for ROS inhibition.

     

Genetic polymorphism of the human cytochrome P450 CYP4B1: evidence for a non-functional allelic variant

In the present study, we report the first systematic investigation of polymorphism in the human CYP4B1 gene. Using a strategy based on single-strand conformation polymorphism analysis of PCR products (PCR-SSCP), we analyzed the twelve exons of the gene, as well as their 5'- and 3'- proximal flanking sequences, in DNA samples from 190 French Caucasians. In addition to the wild-type CYP4B1* allele (CYP4B1*1), four variants, namely CYP4B1*2, *3, *4 and *5, were characterized. The CYP4B1*3, *4 and *5 alleles encode missense mutations Arg173Trp, Ser322Gly and Met331Ile, respectively. The fourth variant, CYP4B1*2, harbors three missense mutations (Met331Ile, Arg340Cys and Arg375Cys) and a double nucleotide deletion (AT881-882del) that causes a frameshift and premature stop codon in the second third of the coding sequence of the gene. This latter mutation can be assumed to lead to the synthesis of a severely truncated protein and, therefore, probably contributes to interindividual variability of CYP4B1 expression and enzymatic activity. In order to investigate the extent of the CYP4B1*2 allele in a large population, a rapid genotyping test, based on restriction analysis of PCR products, was developed and applied to 2082 French Caucasians. Forty-two subjects were found homozygous for the AT881-882 deletion, which suggests that about 2% of individuals should be unable to develop metabolic reactions mediated by CYP4B1. Given the relatively high frequency and the functional consequences of the CYP4B1*2 allele, associations between CYP4B1 polymorphism and certain pathological processes should be considered.     

Safety and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine in HIV-positive women in South Africa: A partially-blind randomised placebo-controlled study

In developing countries, risk of human papillomavirus (HPV) infection may be increased by the high prevalence of human immunodeficiency virus (HIV) infection. We evaluated the safety and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine in HIV-infected women in South Africa. Asymptomatic HIV-positive women aged 18–25 years (N = 120) were stratified by CD4⁺ T-cell count and randomised (1:1) to receive HPV-16/18 vaccine (Cervarix^®; GlaxoSmithKline Vaccines) or placebo (Al[OH]₃) at 0, 1 and 6 months (double-blind). HIV-negative women (N = 30) received HPV-16/18 vaccine (open label). Anti-HPV-16/18 antibody and CD4⁺ T-cell responses, CD4⁺ T-cell count, HIV viral load, HIV clinical stage and safety were evaluated for 12 months. The safety and reactogenicity profile of the HPV-16/18 vaccine was comparable in HIV-positive and HIV-negative women. Irrespective of baseline HPV status, all HIV-positive and HIV-negative women who received the HPV-16/18 vaccine were seropositive for both HPV-16 and HPV-18 after the second vaccine dose (month 2) and remained seropositive for both antigens at month 12. Anti-HPV-16/18 antibody titres at month 12 remained substantially above levels associated with natural infection. The HPV-16/18 vaccine induced sustained anti-HPV-16/18 CD4⁺ T-cell responses in both HIV-positive and HIV-negative women. No impact of baseline CD4⁺ T-cell count or HIV viral load was observed on the magnitude of the immune response in HIV-positive women. In HIV-positive women, CD4⁺ T-cell count, HIV viral load and HIV clinical stage were unaffected by HPV-16/18 vaccine administration. In conclusion, the HPV-16/18 AS04-adjuvanted vaccine appears immunogenic and well-tolerated in women with HIV infection.     

Protection of dopaminergic nigrostriatal afferents by GDNF delivered by microspheres in a rodent model of Parkinson's disease

The use of glial cell line-derived neurotrophic factor (GDNF) appears to be a promising strategy to promote survival and function of the nigrostriatal dopaminergic pathway damaged in Parkinson's disease (PD). However, effective intracerebral administration is required for optimal therapeutic benefit and tools to evaluate such therapies must be developed. A rodent model of PD was therefore developed using striatal injection of 6-hydroxydopamine (6-OHDA) with simultaneous implantation of GDNF-delivering microspheres. The effects of GDNF released from microspheres were assessed by classical methods such as amphetamine-induced rotating behavior and tyrosine hydroxylase (TH) immunoreactivity, as well as by quantitative autoradiography using PE2I, a dopamine transporter (DAT) radiotracer, which is also suitable for SPET imaging in humans. 6-OHDA-lesioned animals that received microspheres without GDNF were used as controls. During the first 3 weeks after simultaneous lesion and implantation, the amphetamine-induced rotating behavior of GDNF-treated rats was improved compared to controls and an increase in TH expression (+26%) was measured in the striatum 6 weeks after lesion. In accordance with these results, an increase in striatal PE2I-labeled DAT density was obtained (+17%) after 3 and 6 weeks of treatment. In conclusion, this study demonstrates the neuroprotective action of GDNF delivered by microspheres and suggests that PE2I may be an appropriate radiotracer for use in SPET scintigraphy to follow up treatment of PD in humans.     

Rapidly destructive osteoarthritis of the hip: MR imaging findings

OBJECTIVE: The purpose of our study was to use an artificial neural network to differentiate benign from malignant pulmonary nodules on high-resolution CT findings and to evaluate the effect of artificial neural network output on the performance of radiologists using receiver operating characteristic analysis. MATERIALS AND METHODS: We selected 155 cases with pulmonary nodules less than 3 cm (99 malignant nodules and 56 benign nodules). An artificial neural network was used to distinguish benign from malignant nodules on the basis of seven clinical parameters and 16 radiologic findings that were extracted by attending radiologists using subjective rating scales. In the observer test, 12 radiologists (four attending radiologists, four radiology fellows, and four radiology residents) were presented with high-resolution CT images, first without and then with the artificial neural network output. Observer performance was evaluated by means of receiver operating characteristic analysis using a continuous rating scale. RESULTS: The artificial neural network showed a high performance in differentiating benign from malignant pulmonary nodules (A(z) = 0.951). The average A(z) value for all radiologists increased by a statistically significant level, from 0.831 to 0.959, with the use of the artificial neural network output. CONCLUSION: Our computerized scheme using the artificial neural network can improve the diagnostic accuracy of radiologists who are differentiating benign from malignant pulmonary nodules on high-resolution CT.     

An integrative approach to in-vivo protein synthesis measurement: from whole tissue to specific proteins

PURPOSE OF REVIEW: In-vivo estimation of protein turnover by stable isotopes in animals and humans has provided much relevant information on metabolic regulation and alterations for decades. While it was first appreciated at the whole body level in the 1970s and 1980s, new approaches have allowed inter-organ or tissue protein turnover rates to be measured, notably the incorporation rate of a labelled amino acid in muscle. These technical improvements have recently been completed by new isolation methods for the study of protein synthesis rates in various muscle and hepatic protein fractions in different blood cells or tissues such as bone and skin. RECENT FINDINGS: This new insight into tissue protein synthesis opens the door for exploration of single proteins, which may be fully achievable in the near future through the combination of proteomics analysis and technical progress in mass spectrometry. This is, therefore, a new area in which not only quantitative but also qualitative changes in specific proteins will be considered for a fully integrative approach to assessing protein metabolism in physiology and disease. SUMMARY: To understand the mechanisms by which protein metabolism is altered during physiopathological situations, it is of importance to measure the effect on specific proteins rather than on the body as a whole. Procedures are currently under development with the aim of isolating individuals proteins and to measure their synthesis rates by isotopic methods. Such technical progress is needed to gain a better understanding of the regulation of protein metabolism in situations in which loss of body protein mass occurs.     

The in situ up-regulation of chondrocyte interleukin-1-converting enzyme and interleukin-18 levels in experimental osteoarthritis is mediated by nitric oxide

OBJECTIVE: To investigate in situ the relationship between 2 key mediators implicated in osteoarthritic (OA) cartilage: nitric oxide (NO) and interleukin-1-converting enzyme (ICE). Interleukin-18 (IL-18) was also studied and served as reference for the effects of ICE. METHODS: An OA model was created in dogs by sectioning (stab wound) the anterior cruciate ligament of the right stifle joint. Three experimental groups were studied: unoperated untreated dogs, operated untreated dogs (OA), and OA dogs treated with oral N-iminoethyl-L-lysine (L-NIL), a specific inhibitor of inducible nitric oxide synthase (iNOS) (10 mg/kg twice a day starting immediately after surgery). At 12 weeks after surgery, cartilage from the femoral condyles and tibial plateaus were processed for immunohistochemistry for ICE, IL-18, and protease inhibitor 9 (PI-9), a natural inhibitor of ICE, followed by morphometric analysis. Cartilage specimens from the femoral condyles of untreated OA dogs were dissected and incubated with specific inhibitors of different signaling pathways likely to be involved in the OA process: SB 202190 (10 microM; a p38 mitogen-activated protein kinase [MAPK] inhibitor), PD 98059 (100 microM; a MAPK kinase 1/2 [MEK-1/2] inhibitor), NS-398 (10 ng/ml; a specific cyclooxygenase 2 [COX-2] inhibitor), and L-NIL (50 microM). RESULTS: Both ICE and IL-18 were present in situ in the canine cartilage, with a significant increase in the level of these 2 proteins in OA cartilage. In contrast, the level of PI-9 was lower in OA than in normal cartilage (difference not statistically significant). Compared with untreated OA cartilage, oral treatment with L-NIL significantly decreased ICE and IL-18 levels in cartilage from the femoral condyles and tibial plateaus, to values similar to those in normal dogs. L-NIL also increased the PI-9 level in normal dogs compared with OA dogs, reaching statistical significance for femoral condyle cartilage. Interestingly, in vitro experiments demonstrated significant inhibition of ICE levels by p38, MEK-1/2, and COX-2 inhibitors, but not by the iNOS inhibitor. CONCLUSION: This study demonstrated that in situ in OA cartilage, the stimulation of chondrocytes by NO is at least partly responsible for the up-regulation of ICE and IL-18 synthesis while decreasing the level of the ICE inhibitor PI-9. The ICE level is controlled by the activation of at least 2 MAPK pathways, p38 and MEK-1/2. Interestingly, it appears that ICE synthesis is not regulated by the endogenous production of NO. These data highlight the role played by iNOS in regulating the synthesis of major catabolic factors involved in OA cartilage degradation.