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.

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.

[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.

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.

Analysis of trotter gait on the track by accelerometry and image analysis.

The aim of this study was to describe the correlation between the phases of the limb cycle of trotters on the track and specific points on the acceleration curves obtained from a new gait analysis system. We compared kinematic data obtained by video image analysis and 3-dimensional acceleration recordings made on 3 French trotters in training. They trotted on a race track at speeds of 8.33, 10 and 11.66 m/s, with a final stretch at maximum speed. Their locomotion was recorded with a synchronised video camera at a frame frequency of 200 Hz and with the Equimétrix gait analysis system. The gait variables were calculated using 3-dimension acceleration data recorded at the sternum (dorso-ventral, longitudinal and lateral axes) at a sampling rate of 100 Hz. Three phases of the stride were clearly identified on the dorsoventral acceleration signal: hoof-landing, midstance phase and toe-off. Braking and propulsion phases were identified on the corresponding longitudinal acceleration signal. The weight-bearing diagonal was identified by observing the lateral signal. The stride temporal variables (stride, stance, braking and propulsion durations for both diagonals), measured by video analysis and by acceleration signal analysis, were not significantly different (P>0.05). The identification of specific points on the acceleration pattern allowed an accurate temporal analysis of the stride. Potential applications could be the determination of locomotor factors related to racing performance or assessment of locomotor disorders at high speed.

The genomic structure of c14orf1 is conserved across eukarya.

We have recently cloned the gene C14orf1, which is strongly expressed in normal testis and in several cancer cell lines and tumors. This gene maps to 14q24.3 and is interrupted by four introns. Two of them are also represented in the open reading frame of Schizosaccharomyces pombe in the same phase. In Arabidopsis taliana only the first of the two introns was found, in the same phase as the corresponding ones in S. pombe and human. Disruption of the ortholog in Saccharomyces cerevisiae (Yer044c) led to a severe growth defect, and C14orf1 failed to complement mutant yeast when put under the control of the natural Yer044c promoter. Further studies are needed to understand the causes underlying the high degree of conservation of the C14orf1 genomic structure.

Functional cloning of the adenylate cyclase gene of Candida albicans in Saccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues of CHS6 and SAP185.

We have cloned a genomic fragment of Candida albicans by complementation of a Saccharomyces cerevisiae cyr1 mutant. This fragment contains the two-thirds C-terminal part of the adenylate cyclase CaCYR1. The complete gene has been sequenced from PCR fragments amplified from genomic DNA, and contains an ORF of 1690 amino acids closely related to other fungal adenylate cyclases. Adjacent to the adenylate cyclase gene, we have sequenced six other putative genes. CaCHS6, CaYNL191 and CaYJL098 are named on the basis of their close similarity with S. cerevisiae genes. ORFs CaYJL097a and CaYJL097b represent two repeated homologues of the S. cerevisiae YJL097w, which probably arose from an ancient duplication. The last one is a hypothetical ORF, CaYKR049, which presents only a very weak similarity with YKR049. The S. cerevisiae homologues of three of these genes are co-localized on chromosome X but with a different order and orientation.

Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor.

The CDC25 gene product from Saccharomyces cerevisiae, the prototype of the family of ras guanine nucleotide exchange factors, is expressed as a 180-kDa polypeptide, tightly bound to a membrane fraction. The ability to complement a cdc25 defect is located in the 3' part of the gene (codons 877-1589). Sequence analysis reveals only a short hydrophobic domain (residues 1459-1471) and no consensus sequence for post-translational acylation. The SH3 domain present in the N-terminal part of Cdc25p is not involved nor required for membrane localization, since the N-terminal part of Cdc25p did not fractionate with a membrane pellet. In contrast, the C-terminal part was attached to a 18000 g pellet after subcellular fractionation and immunoblotting. This subcellular localization was conserved in a ras1 ras2 double disruption mutant and in a ira2 disruption mutant. Immunofluorescence analysis showed a patchy staining, mainly at the periphery of the cells. These patches were quite distinct from actin patches by double immunolabeling. By analysing a set of truncated derivatives, the elements required for a particulate localization were restricted to residues 1441-1552.

[Yeast and the control of RAS by exchange factors]. / La levure et le contrôle de RAS par les facteurs d'échange.

Two isofunctional ras genes are present in the yeast Saccharomyces cerevisiae. Albeit their targets differ between mammals and yeast, they have conserved their regulators. The study of their positive regulators, guanine nucleotide exchange factors, have provided routes to the discovery of their regulatory elements in mammals. Ras are signal transducing proteins involved in the activation of the adenylate cyclase in yeast. They are activated by Cdc25p which has been shown to contain a Guanine Exchange Factor domain (GEF). SDC25, a gene partially homologous to CDC25, also contains a GEF domain but seems to be under a different regulation. It has been used to demonstrate the first guanine exchange activity on ras in vitro and was shown to be active by gene transfer in mammalian cells. Both Cdc25p and Sdc25p are associated to membrane and contain SH3 domains which are supposed to bind still unidentified proteins. Cdc25p is an unstable protein which contains a cyclin destruction box. Therefore activating effect on ras could be regulated by its level of expression. We have contributed to the isolation of a mammalian CDC25 homolog and we are analysing by directed mutagenesis key positions for ras activation of the human homolog HGRF55. That was performed by complementation analysis of yeast mutants as well as by use of two hybrid system. These approaches led us to the discovery of residues involved in ras interaction.

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.

Prenatal diagnosis in a subset of trichothiodystrophy patients defective in DNA repair.

Trichothiodystrophy (TTD) is an autosomal recessive disorder characterized by brittle hair with reduced sulphur content, and mental and physical retardation. Numerous additional clinical features may be present, producing a very heterogeneous syndrome. Many cases exhibit ichthyosis and photosensitivity. Cells from photosensitive TTD patients show reduced DNA repair levels similar to those found in xeroderma pigmentosum. TTD patients have a short life expectancy, and no treatment is known or envisaged. We report the prenatal diagnosis of TTD in two French families, based on DNA repair measurements in trophoblasts or amniotic cells, with later confirmation by microscopic analysis of the fetal hairs. Although the DNA repair defect was less marked in the fetal cells when compared with fibroblasts from the index case, measurement of DNA repair by unscheduled DNA synthesis provided unambiguous evidence of defective DNA repair in the fetal cells. This method is therefore a suitable prenatal diagnostic test for those TTD families in which a DNA repair defect has been identified.

In situ NADH laser fluorimetry during muscle contraction in humans.

The aim of the present study was to use nicotinamide adenine dinucleotide phosphate, reduced (NADH) fluorimetry, to investigate in situ NADH changes during muscle contraction in humans on an isokinetic dynamometer. Thirteen healthy male subjects each performed one maximal voluntary contraction (MVC) with the knee extensor muscle. The NADH muscle fluorescence was monitored by a double beam laser fluorimeter which uses an optical fibre, percutaneously inserted through a needle into the vastus lateral muscle, to guide the light. The NADH fluorescence was continuously measured at a wavelength of 337 nm. To estimate the haemodynamic artefact, blood backscattering was simultaneously determined at a wavelength of 586 nm. The fluorescence signal was recorded before, during and after contractions at 50% of MVC. The fibre was kept out of contact with the muscle during contractions at 100% of MVC and was only put into contact with it at the end of the contraction. At the onset of contractions at 50% of MVC, NADH fluorescence increased rapidly for 3 s and remained stable thereafter until exhaustion. After a muscle measurement had been made, the optical fibre was put successively into solutions of increasing NADH concentration to ascertain the relationship between the muscle fluorescence signal and the muscle NADH level. This procedure yielded estimated mean values for muscle NADH of 0.172 mmol.kg-1, SEM 0.028 and of 0.184 mmol.kg-1, SEM 0.027 after contractions at 50% and 100% of MVC, respectively, from a resting value of 0.087 mmol.kg-1, SEM 0.015. These results indicated that in situ laser fluorimetry could be used to evaluate NADH changes in humans during muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenic properties of a unique abasic site in mammalian cells.

The mutagenic properties of a true unique abasic site located opposite a guanine residue were studied. An oligonucleotide containing a chemically-produced abasic site was inserted into a shuttle vector able to replicate both in simian cells and in bacteria. Plasmid DNA was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Mutations were easily detected and sequenced. Results showed that opposite a guanine the abasic site was error free repaired or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at a frequency of approximately 1% in control host cells and at more than 3% in UV-pre-irradiated host cells. Adenine, cytosine or thymine were found to have been inserted opposite the abasic site. No preferential insertion for a particular base was observed in contrast to that reported in bacteria.

Mammalian cell processing of a unique uracil residue in simian virus 40 DNA.

The processing of a unique uracil in DNA has been studied in mammalian cells. A synthetic oligodeoxyribonucleotide carrying a potential Bgl II restriction site, where one base has been substituted with a uracil, was inserted in the early intron of SV40 genome. Various heteroduplexes were constructed in such a manner that the restitution of an active Bgl II restriction site corresponds in each case to the specific substitution of the uracil by one of the four bases normally present in the DNA. DNA cuts by this restriction enzyme in one or several constructed heteroduplexes immediately determine the type of base pair substitution produced at the site of the U residue. When the uracil is inserted opposite a purine it is fully repaired; when facing a guanine it is replaced by a cytosine and opposite an adenine it is replaced by a thymine. These results indicate the error-free repair of uracil when it appears in the cell with the usual mechanisms such as cytosine deamination or incorporation of dUTP in place of dTTP during replication. When the uracil is inserted opposite a pyrimidine no error free repair at all is detected for U:C or U:T mismatches. It appears, moreover, that in approximately 18% of the cases U:T mismatch leads to a C:G base pairing. In the majority of the U:pyrimidine mismatches, mutations occur in the vicinity of the uracil, including base substitutions and frameshifts by addition of one or several bases.

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.

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.

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.

Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum.

The distribution of spontaneous sister chromatid exchanges (SCEs) was studied in PHA-stimulated lymphocytes from 15 patients affected by xeroderma pigmentosum (XP). The study of unscheduled DNA synthesis (UDS) in twelve of these patients showed that seven were deficient and five proficient. The number of SCEs in XP patient cells was higher than in those of 19 controls, and the distributions of SCEs per cell were significantly different. However, the results varied when XP patients were considered in relation to their UDS: the group of XP patients with proficient UDS did not differ, whereas the group of XP patients with deficient UDS was very significantly different from controls. The group not tested for UDS was similar to the deficient UDS group. The possible relationship between the increase of SCEs and the type of DNA repair defect is discussed.

Enhanced deoxyribonuclease activity in human transformed cells and in Bloom's syndrome cells.

Human hereditary diseases such as xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome are characterized by a proneness for developing cancer associated with abnormalities in the processing of DNA damage. The molecular defects responsible for predisposing human tissues to cancer are still not well understood, despite the fact that a considerable amount of work has already been done on this problem. In this paper, we show that in human tumor cell lines, in cells transformed by DNA tumor viruses, and in cells derived from certain cancer-prone disorders, the level of activity of a 42-kDa deoxyribonuclease is many times higher than in diploid untransformed control cells. This suggests that this activity is linked to, or may play a role in, malignant transformation.