Role of anti-cyclic citrullinated peptide antibodies in discriminating patients with rheumatoid arthritis from patients with chronic hepatitis C infection-associated polyarticular involvement

This study was performed to assess the utility of anti-cyclic citrullinated peptide (anti-CCP) antibodies in distinguishing between patients with rheumatoid arthritis (RA) and patients with polyarticular involvement associated with chronic hepatitis C virus (HCV) infection. Serum anti-CCP antibodies and rheumatoid factor (RF) were evaluated in 30 patients with RA, 8 patients with chronic HCV infection and associated articular involvement and 31 patients with chronic HCV infection without any joint involvement. In addition, we retrospectively analysed sera collected at the time of first visit in 10 patients originally presenting with symmetric polyarthritis and HCV and subsequently developing well-established RA. Anti-CCP antibodies and RF were detected by commercial second-generation anti-CCP2 enzyme-linked immunosorbent assay and immunonephelometry respectively. Anti-CCP antibodies were detected in 23 of 30 (76.6%) patients with RA but not in patients with chronic HCV infection irrespective of the presence of articular involvement. Conversely, RF was detected in 27 of 30 (90%) patients with RA, 3 of 8 (37.5%) patients with HCV-related arthropathy and 3 of 31 (9.7%) patients with HCV infection without joint involvement. Finally, anti-CCP antibodies were retrospectively detected in 6 of 10 (60%) patients with RA and HCV. This indicates that anti-CCP antibodies can be useful in discriminating patients with RA from patients with HCV-associated arthropathy.     

Antioxidant and pro-oxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus

Bivalve molluscs, particularly mussels, are sensitive biomarkers of aquatic ecosystem pollution. The tannins, water-soluble plant polyphenols, may play an important role in this environment and, mainly as a consequence of interaction with pollutants, their toxicity may change. We studied three naturally occurring compounds, tannic acid, ellagic acid and gallic acid, for their ability to modulate DNA damage produced by these tannins alone and in the presence of the oxidative stress inducer H(2)O(2), in cells of the digestive gland of mussels (Unio tumidus). After the treatment of the cells with polyphenols at different concentrations (1, 5, 15, 30, 60, 80, 100, 120, 180, 240 microM) and with hydrogen peroxide in the range of 0.04 and 0.1mM, single-strand breaks (ssb) in DNA were investigated, using the comet assay. The ability of phenolic acids to decrease DNA damage through their antioxidant properties was also assessed. The results show that the phenols, which are known as antioxidative agents, could also act as pro-oxidants. They induced ssb in DNA of the digestive gland at concentrations higher that 10 microM, but lower doses (1 and 5 microM) did not contribute to the DNA damage. This study was also designed to evaluate the protective effect of these tannins against H(2)O(2)-mediated DNA damage in the cells. In this treatment, the two concentrations (1 and 5 microM) significantly decreased the amount of lesions induced by H(2)O(2) (0.04 and 0.1mM). In conclusion, our results demonstrate that antioxidative properties of tannins may change to pro-oxidative activities at the higher concentrations. This suggests that the biologic actions of these compounds may be rather complicated.     

De novo balanced translocation (2;10)(q24;q22) associated with mental retardation

We report a case of a reciprocal translocation between the long arms of the 2 and 10 chromosomes observed in a 14-year-old male with mild mental impairment, compulsive and obsessive behavior. The apparently balanced translocation was characterized by fluorescence in situ hybridization and the karyotype was 46, XY, t(2;10)(q24;q22). The way by balanced chromosomal translocations can lead to a disease phenotype are reviewed and discussed.     

Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis

Rhodamine 123 (RH-123) was used to monitor the membrane potential of mitochondria isolated from rat liver. Mitochondrial energization induces quenching of RH-123 fluorescence and the rate of fluorescence decay is proportional to the mitochondrial membrane potential. Exploiting the kinetics of RH-123 fluorescence quenching in the presence of succinate and ADP, when protons are both pumped out of the matrix driven by the respiratory chain complexes and allowed to diffuse back into the matrix through ATP synthase during ATP synthesis, we could obtain an overall quenching rate proportional to the steady-state membrane potential under state 3 condition. We measured the kinetics of fluorescence quenching by adding succinate and ADP in the absence and presence of oligomycin, which abolishes the ADP-driven potential decrease due to the back-flow of protons through the ATP synthase channel, F(0). As expected, the initial rate of quenching was significantly increased in the presence of oligomycin, and conversely preincubation with subsaturating concentrations of the uncoupler carbonyl cyanide p-trifluoro-metoxyphenilhydrazone (FCCP) induced a decreased rate of quenching. N,N'-dicyclohexylcarbodiimide (DCCD) behaved similarly to oligomycin in increasing the rate of quenching. These findings indicate that RH-123 fluorescence quenching kinetics give reliable and sensitive evaluation of mitochondrial membrane potential, complementing steady-state fluorescence measurements, and provide a mean to study proton flow from the mitochondrial intermembrane space to the matrix through the F(0) channel.     

Cardioprotective effect of ischemic preconditioning is preserved in food-restricted senescent rats

Ischemic preconditioning (PC) has been proposed as an endogenous form of protection against-ischemia reperfusion injury. We have shown that PC does not prevent postischemic dysfunction in the aging heart. This phenomenon could be due to the reduction of cardiac norepinephrine release, and it has also been previously demonstrated that age-related decrease of norepinephrine release from cardiac adrenergic nerves may be restored by caloric restriction. We investigated the effects on mechanical parameters of PC against 20 min of global ischemia followed by 40 min of reperfusion in isolated hearts from adult (6 mo) and "ad libitum"-fed and food-restricted senescent (24 mo) rats. Norepinephrine release in coronary effluent was determined by high-performance liquid chromatography. Final recovery of percent developed pressure was significantly improved after PC in adult hearts versus unconditioned controls (85.2 +/- 19% vs. 51.5 +/- 10%, P < 0.01). The effect of PC on developed pressure recovery was absent in ad libitum-fed rats, but it was restored in food-restricted senescent hearts (66.6 +/- 13% vs. 38.3 +/- 11%, P < 0.05). Accordingly, norepinephrine release significantly increased after PC in both adult and in food-restricted senescent hearts, and depletion of myocardial norepinephrine stores by reserpine abolished the PC effect in both adult and in food-restricted senescent hearts. We conclude that PC reduces postischemic dysfunction in the hearts from adult and food-restricted but not in ad libitum-fed senescent rats. Despite the possibility of multiple age-related mechanisms, the protection afforded by PC was correlated with increased norepinephrine release, and it was blocked by reserpine in both adult and food-restricted senescent hearts. Thus caloric restriction may restore PC in the aging heart probably via increased norepinephrine release.     

Rat carotid artery dilation by PTCA balloon catheter induces neointima formation in presence of IEL rupture

The best animal angioplasty model is the porcine model, which is expensive and not available in all laboratories. The aim of this study was to describe a new rat model of angioplasty. An injury was induced with the use of a standard percutaneous transluminal coronary angioplasty (PTCA) 1.5-mm balloon catheter. The neointimal tissue, arterial dimensions, and the injury index were assessed following angioplasty. Ki-67 expression was detected to evaluate cell turnover after balloon angioplasty. In contrast with the standard Clowes model, a significant neointimal formation was detected only in the presence of ruptured internal elastic lamina (IEL). A positive correlation between the percentage of ruptured IEL and the amount of neointimal tissue was also demonstrated. The percentage of IEL fracture correlates with the proliferation index by anti-Ki-67 immunolabeling 7 and 14 days after the angioplasty. Significant arterial negative remodeling was observed following PTCA balloon dilation. In conclusion, our inexpensive animal model of restenosis after angioplasty may have great relevance toward a better understanding of the mechanisms and toward assessment of new therapeutical strategies for this phenomenon.     

Chromatin of Trypanosoma cruzi: in situ analysis revealed its unusual structure and nuclear organization

Chromatin of Trypanosoma cruzi is known to be organized in classical nucleosomal filaments, but surprisingly, these filaments do not fold in visible chromosomes and the nuclear envelope is preserved during cell division. Our hypothesis about the role of chromatin structure in regulating gene expression and, more generally, cell functioning, pressed us to verify if chromatin organization is modulated during the parasite life-cycle. To this end, we analyzed in situ the fine structural organization of T. cruzi chromatin by means of an integrated biophysical approach, using differential scanning calorimetry and fluorescence microscopy. We observed that logarithmic forms exhibit a less condensed chromatin with respect to the stationary ones. Thermal analysis revealed that parasite chromatin is organized in three main levels of condensation, barring from the polynucleosomal filament till to superstructured fibers. Besides, the fluorescence images of nuclei showed a characteristic chromatin distribution, with defined domains localized near to the nuclear envelope. While in stationary parasites, these regions are highly condensed, in logarithmic forms they unfold by extending themselves toward the center of nucleus. These observations suggest that, in comparison with higher eukaryotes, in T. cruzi the nuclear envelope plays an unusual and pivotal role in interphase and in mitosis.     

Metabolic profile of NO-flurbiprofen (HCT1026) in rat brain and plasma: a LC-MS study

Male rats were given the nitrooxybutyl ester of flurbiprofen HCT1026 (15 mg/Kg) by oral administration and plasma and brain levels of the parent drug and its potential metabolites (HCT1027 and flurbiprofen) were determined at different times post-administration by a validated HPLC method (UV-DAD detection; LOQ: 0.13 nmoles/ml and 0.3 nmoles/g respectively in plasma and brain tissue). Structural confirmation of the analytes was achieved by MS monitoring of their de-protonated (negative ion mode) or cationized/protonated (positive ion mode) molecular ions and of the relative fragment ions obtained by collision-induced dissociation (CID) experiments. The results indicate that flurbiprofen is the only metabolite found at measurable levels in both plasma and brain, while HCT1026 or its de-nitrated metabolite HCT1027 were always below the limit of detection at all the observation times. The same was observed after administration of the higher dose of HCT1026 (100 mg/Kg, i.p.). In orally-treated animals the time-course of flurbiprofen formation strictly parallels that of NOx (nitrite/nitrate) in plasma but not in brain, where the levels were always in the range of the controls. These data indicate that the NO molecule is not released from the parent drug within the brain.     

A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.