Size-switchable polymer-based nanomedicines in the advanced therapy of rheumatoid arthritis.

Chronic inflammatory diseases such as rheumatoid arthritis represent a substantial socio-economic impact and have a high prevalence in the modern world. Nano-sized polymer therapeutics have shown suitable characteristics for becoming the next generation of anti-inflammatory nanomedicines. Here, we present biocompatible and stimuli-sensitive N-(2-hydroxypropyl)methacrylamide based polymer conjugates with the anti-inflammatory drug dexamethasone (DEX), which has been tailored for prolonged blood circulation, enhanced inflammatory site accumulation, site-specific drug release and subsequent elimination of the carrier via urine excretion. The hydrodynamic size of novel polymer-DEX nanomedicine was adjusted to prolong its blood circulation whilst maintaining the renal excretability of the polymer carrier after drug release in inflamed tissue. The therapeutic efficacy of the studied polymer nanomedicines was evaluated in a model of dissipated chronic arthritis, i.e. collagen II-induced arthritis, in mice. The pH-sensitive drug attachment enabled enhanced blood circulation with minimal systemic drug release, as well as rapid drug activation in affected joints. Importantly, unlike free DEX, the polymer nanomedicines were able to diminish joint inflammation and arthritis-induced bone damage - even at a reduced dosing regimen - as evaluated by micro computed tomography (micro-CT).

Ultrasound and Doppler micro-imaging in a model of rheumatoid arthritis in mice.

OBJECTIVES: The evaluation of joints in arthritis using conventional ultrasonography is not really feasible in mice because of the small size of the animal. However, compared with classical analysis (clinical and histological examination) it is a non-invasive method that allows follow-up of the same animal throughout the whole experiment. Moreover, power Doppler allows the study of blood flow that reflects inflammatory activity within the synovium of arthritic joints. Our aim was to determine whether ultrasonography analysis could accurately detect arthritis lesions in a mouse model of rheumatoid arthritis, namely collagen-induced arthritis. METHODS: Collagen-induced arthritis was induced in 28 mice by immunising with collagen type II. Every week for 8 weeks, ultrasonography and Doppler analysis were performed on knees and ankles of all mice using the ultrasound biomicroscope (UBM), which is particularly dedicated to studying the mouse. Clinical and histological evaluations were performed as usual. RESULTS: We established a semiquantitative analysis by setting an UBM scoring. UBM grades were correlated to clinical and histological scores of arthritis. Vascularisation within the synovium could be estimated by power Doppler analysis and a semiquantitative vascularisation scale was established, which allowed us to show a good correlation between vascularisation scores and histological or clinical scores of arthritis. CONCLUSIONS: This is one of the first studies that shows it is possible to visualise a selected set of joints in a small animal using UBM analysis. It provides new perspectives in evaluating experimental models of rheumatoid arthritis and other joint diseases.

[High-resolution ultrasound imaging of the mouse]. / L'échographie haute résolution de la souris.

Small-animal ultrasound imaging has been made possible using high-resolution imaging devices. The spatial resolution is therefore sufficient to accurately measure anatomical parameters in mice. This paper reviews some of the main applications of high-resolution ultrasound imaging of the mouse and highlights what could be the forthcoming advances.

Activated oncogenes in human skin tumors from a repair-deficient syndrome, xeroderma pigmentosum.

The recessive autosomal hereditary disease, xeroderma pigmentosum (XP), is characterized by a high incidence of tumors in sun-exposed skin. The defect in early steps of excision repair of XP cells leads to hypermutability towards UV-mimicking agents. DNA from eight XP tumors were screened for activated transforming genes using 3T3 transfection. In two skin tumors isolated from a XP child, an activated N-ras oncogene was detected. Synthetic oligonucleotide probes were used to characterize the mutation in the ras gene. Both tumors were found to be mutated in the 61st N-ras codon from gln to his. The mutation was accompanied by an increase in the level of N-ras specific mRNA and in one transformant, by the alteration of the p21 protein. In the same tumors, c-myc amplification and over transcription, and Ha-ras gene rearrangement and amplification were also detected. Analysis of other XP tumors with eleven different oncogene probes revealed an amplification of the Ha-ras gene in 6 out of 10 cases. The normal skin fibroblasts from XP patients show normal pattern levels of N-ras, c-myc and Ha-ras sequences. The hypothesis is proposed that the presence of several oncogene alterations in the same tumor could be due to the high amount of UV-induced DNA lesions found in the exposed skin cells, in the absence of efficient repair.

A posteriori respiratory gating in contrast ultrasound for assessment of hepatic perfusion.

An original strategy is proposed to minimize the impact of respiratory motion on hepatic contrast-enhanced ultrasound studies. It is based on the a posteriori triggering of dynamic image sequences. It was tested on perfusion studies acquired with a high temporal resolution (8 images s-1) to enable parametric imaging. A respiratory component was first estimated by independent component analysis. The estimation of the local minima and maxima of this curve enabled us to select two subsets of frames, corresponding to the end-of-inspiration plane and to the end-of-expiration plane. Both subsets were simultaneously analysed using factor analysis of medical image sequences. This method identified the main contrast uptake kinetics and their associated localizations. The global strategy was validated firstly on a simulated study and then applied to 11 patients' studies. In both cases, the frame selection was judged relevant and a necessary preliminary step before applying methods of parametric imaging. In conclusion, the a posteriori gating method that is proposed is a first step towards local quantification of hepatic contrast-enhanced ultrasound studies.

The pde2 gene of Saccharomyces cerevisiae is allelic to rca1 and encodes a phosphodiesterase which protects the cell from extracellular cAMP.

The high affinity cAMP phosphodiesterase, encoded by PDE2, is an important component of the cAMP-dependent protein kinase signaling system in Saccharomyces cerevisiae. An unexpected phenotype of pde2 mutants is sensitivity to external cAMP. This trait has been found independently for rca1 mutants and has been used to monitor the effects of cAMP on several biological processes. We demonstrate here that RCA1 is identical to PDE2. Further analysis of the phenotype of pde2 deletions reveal that exogenously added cAMP results in an increase in the internal level of cAMP. This increase slows down the rate of cell division by increasing the length of the G1 phase of the cell cycle and leads to increased cell volume. Also, cells with a disrupted PDE2 gene previously arrested by nutrient starvation rapidly lose thermotolerance when incubated with exogenous cAMP. From these observations we propose that a role of the PDE2-encoded phosphodiesterase may be to help insulate the internal cAMP pools from the external environment. This protective role might also be important in other eukaryotic organisms where cAMP is a key second messenger.

Distribution of ultraviolet-induced lesions in simian virus 40 DNA.

In order to analyze the molecular mechanisms of mutagenesis in mammalian cells, we devised an analytical assay using Simian Virus 40 as biological probe. To study the possible correlations between the distribution of the lesions on the treated DNA and the distribution of mutations, we have located and quantified the lesions induced by ultraviolet light (254 nm) on a SV40 DNA fragment. At a fluence of 2,000 J/m2, our results show that the formation frequency of thymine-thymine dimers (TT) is three to four times higher than the formation frequency of the other types of dimers (TC, CT, CC). On the other hand, the formation frequency of a dimer is influenced by the adjacent sequence. In particular, a pyrimidine in the 5' position of a thymine-thymine dimer enhances its formation frequency. At the dose used the formation frequency of the pyrimidine (6-4) pyrimidone photoproducts is twenty times less than the formation frequency of pyrimidine dimers. This paper shows the distribution of the major lesions induced by UV-light on a defined fragment of SV40 genome after UV irradiation. This work is necessary to get an insight into the molecular mechanisms of UV-mutagenesis.

In situ NADH laser fluorimetry of rat fast- and slow-twitch muscles during tetanus.

To investigate the variations of oxidation-reduction status of fast- and slow-twitch muscles during intense contractions, we performed in situ NADH laser fluorimetry during 25-s tetanus in extensor digitorum longus (EDL) and in soleus (SOL) muscles of eight Sprague-Dawley rats anesthetized with pentobarbital sodium. At base line the compensated NADH fluorescence (F0) was not significantly different between EDL and SOL. In EDL, tetanic stimulation induced an increase of F0, which rapidly reached a plateau that was 124% over the base-line value and stable until the end of the stimulation. In SOL, after an initial shouldering there was a continuous increase of F0 until the end of tetanus, reaching 275% of the base-line value. After the stimulation the initial rate of recovery was significantly faster in SOL than in EDL. We conclude that during and after intense contraction the variation of NADH content vs. time can be evaluated by in situ NADH laser fluorimetry in different muscle types. This nondestructive method can be helpful to differentiate in situ the various physiological or pathological oxidative capabilities of skeletal muscles.

From simian virus 40 to transient shuttle vectors in mutagenesis studies.

Exogenous DNA probes are frequently used to study mutagenesis in mammalian cells. Experimental protocols utilizing simian virus 40 (SV40) and transient shuttle vectors able to replicate in mammalian cells as well as in bacteria are described. The main interests and the limits of the 2 genetic assays are discussed from results obtained with both systems. Despite some minor discrepancies, results obtained are very similar using either method. The complementarity of the 2 assays will allow a better comprehension of the mechanisms by which mutations may arise in mammalian cells.

Kinetics of induction of sister-chromatid exchanges by X-rays through two cell cycles.

V79 Chinese hamster cells were exposed to X-rays at various times through the two cell cycles required to obtain harlequin-stained chromosomes. A two-fold SCE enhancement was found between the first and the second G1 phase when BrdUrd was incorporated during the first S phase only. This BrdUrd effect was not found when MNNG was used. Furthermore, the kinetics of SCE and aberrations were different, suggesting two separate mechanisms for their formation: SCE activity takes place when DNA damage occurs before the DNA replication, and aberration activity when the DNA damage occurs chiefly after the DNA replication.

SV40-based shuttle viruses.

We summarize in this paper the advantages of the shuttle virus system. These SV40-based vectors exhibit the unique properties of being packaged as SV40 pseudo-virions and of being able to infect host cells. Using these transient vectors, we show that their replication can be regulated in some monkey cell lines, in such a way that either low or very high amounts of plasmid DNA can be obtained. The stability of these infectious shuttle vectors in different conditions is analyzed by rescuing them in E. coli, using various gene mutation targets. Moreover, we describe a new series of vectors which can be produced as single-stranded DNA in bacteria. They allow the transfection of a plasmid genome into mammalian cells, as either single-stranded or double-stranded DNA.

[Laser fluorimetry of NADH]. / Fluorimétrie laser du NADH.

Laser fluorimetry of reduced nicotinamide adenine-dinucleotide (NADH) in situ is a new technique used for real-time studies of the degree of reduction of the first link in the mitochondrial respiration chain. We present here the first results obtained in animal experiments and in clinical exploration: In rats, coronary occlusion produces a substantial rise in mitochondrial NADH, followed by a fall below basal level during reperfusion. Tetanic contraction of slow-twitch muscles in rats results in a rise in NADH level higher than that produced by contraction of fast-twitch muscles. During coronary angiography, injection of the contrast medium induces a rise in NADH in patients with a significant degree of coronary stenosis. Intravenous nitroglycerin reduces both left ventricular end-diastolic pressure and NADH fluorescence concomitantly, due to preferential distribution of the coronary blood flow to sub-endocardial layers. Exercise-induced ischaemia results in a decrease of muscular NADH concentration in Mc Ardle syndrome, due to phosphorylase deficiency.

[The use of laser fluorometry of NADH in cardiology]. / Applications de la fluorimétrie laser du NADH en cardiologie.

In this paper the first results of experimental studies of myocardial energy metabolism conducted in situ by laser fluorimetry are described. With this method the myocardial oxygen uptake is determined by measuring the fluorescence of NADH (reduced nicotinamide adenine dinucleotide). The equipment required consists of pulsed laser beams in the ultraviolet (337 nm) and visible (586 nm) spectra, very thin catheters with a single optic fibre, and an original digital data processing technique. The first applications envisaged concern: 1. pharmacological studies, notably evaluation of the protective effect of drugs against ischaemia; 2. evaluation of revascularization procedures in emergency surgery, notably assessment of reversibility of the ischaemic lesions; 3. monitoring of myocardial protection during cardiac surgery.

[Study of myocardial metabolism of NADH by laser fluorimetry during cardiac catheterization]. / Exploration du métabolisme myocardique par fluorimétrie laser du NADH au cours du cathétérisme cardiaque.

Laser fluorimetry of reduced nicotinamide-adenine-dinucleotide (NADH) p6 a new technique used for in situ and real-time study of myocardial metabolism. We have evaluated its applicability to clinical situations in 5 patients undergoing haemodynamic exploration. An optic fibre was inserted in a catheter the end of which was positioned in the postero-diaphragmatic part of the left ventricle. The optic fibre was connected to a Cilas-Alcatel fluorimeter. Variations in fluorescence were studied during variations in left ventricular end-diastolic pressure (LVEDP) and during coronary arteriography. An increase in LVEDP resulted in a slight increase in NADH, but when the LVEDP was reduced by a nitroglycerin infusion, NADH fell significantly below baseline values in patients with coronary disease. This effect was most probably due to redistribution of the coronary blood flow from healthy territories to ischaemic territories. In patients without significant coronary stenosis, NADH was not modified by an injection of 10 ml of contrast medium into the right coronary artery. In contrast, in patients with severe stenosis NADH fluorescence significantly increased during the injection, reflecting the ischaemia or myocardial anoxia induced by the contrast medium. NADH laser fluorimetry therefore seems to be promising as a means of exploring myocardial metabolism during cardiac catheterization.

[In situ evaluation of tissue metabolism by laser fluorimetry]. / Evaluation in situ du métabolisme tissulaire par fluorimétrie laser.

Laser fluorimetry is a new technique which provides continuous information on tissue metabolism in situ and without destruction. For the moment, it is mainly applied to the study of changes in redox gradients in various organs, including the heart, brain, liver, kidney and skeletal muscle, in cases with imbalance between oxygen supply and oxygen consumption. Other metabolisms, such as that of the crystalline lens with incipient cataract, can also be investigated by this technique.

On the ability of ultrasound parametric perfusion imaging to predict the area of infarction in acute ischemic stroke.

PURPOSE: Cerebral perfusion deficits in acute ischemic stroke can be detected by means of transcranial harmonic imaging after ultrasound contrast agent bolus injection. We evaluated five different parameters of the bolus kinetics as parametric images and correlated areas of disturbed perfusion with the area of definite infarction. MATERIALS AND METHODS: Perfusion harmonic imaging after SonoVue bolus injection (BHI) was used to investigate 22 patients suffering from acute internal carotid artery infarction. For each subject, we calculated five different images based on the following parameters from the time-intensity curve in each pixel: pixelwise peak intensity (PPI), area under the curve (AUC), positive gradient (PG), time to peak (TTP), and a three factor image from the factor analysis of medical image sequences (FAMIS). The findings in the diencephalic imaging plane were compared with the definite area of infarction, as diagnosed by cranial CT. RESULTS: In predicting the definite area of infarction in follow-up CT, we found the following sensitivities and positive predictive values (PPV): PPI (100 %/95 %), AUC (100 %/90 %), FAMIS (89 %/89 %), PG (84 %/94 %) and TTP (47 %/100 %). The areas of disturbed perfusion in all five types of parametric images correlated significantly with the area of infarction in CT. Images from the FAMIS algorithm and PPI images showed the highest Spearman rank correlation with the area of definite infarction as displayed in CT (both r = 0.76, p < 0.001). Images from the other parameters correlated as follows: PG: r = 0.62 (p = 0.003), AUC: r = 0.53 (p = 0.014), TTP: r = 0.50 (p = 0.021). CONCLUSION: BHI can detect disturbed perfusion in acute hemispheric stroke. In their ability to predict the development of an infarction, intensity-based parameters and FAMIS were determined to have a high sensitivity, and TTP was found to have a high PPV and specificity.

Sequence effect on alkali-sensitive sites in UV-irradiated SV40 DNA.

Ultraviolet light at 254 nm induces various kinds of DNA damage. We have located and quantified the pyrimidine (6-4) pyrimidone photoproducts along three hundred and forty two nucleotides of SV40 DNA. The level of photoproduct induction varies greatly according to the position on the DNA, but unlike what happens with pyrimidine dimers, the very adjacent nucleotides do not play a major role in the frequency of formation. A new alkali-sensitive site has been found on the ACA sequence after UV irradiation. This complex lesion is insensitive to the T4 endonuclease V and the E. coli photolyase, and may be involved with mutagenesis.