Comparison of coronal leakage of different root canal filling techniques: an ex vivo study.

The aim of this study was to compare the quality of the coronal seal, using an in vitro bacterial invasion test, of three different root canal filling systems. Twenty-seven freshly extracted mandibular premolars were selected and divided into three experimental groups (G1, G2 and G3 n=7) and two control groups (Ct+ and Ct- n=3). All teeth in the experimental groups were prepared using NiTi Mtwo rotating instruments and then the endodontic treatments were completed using the three-tested warm guttapercha root filling techniques: Microseal (G1), Thermafil (G2) and System B (G3). All root filling techniques were performed using the same endodontic sealer (Pulp Canal Sealer). Three teeth were instrumented and not filled, serving as positive controls (Ct+) and the last three teeth, with intact crowns and no endodontic treatment, served as negative controls (Ct-). All samples were mounted in a two-chamber apparatus and exposed to Enterococcus faecalis performing a bacterial infiltration test. All samples were observed for a maximum period of 60 days checking for turbidity of the BHI broth on a daily basis recording when contamination occurred. A quantitative evaluation of the bacterial CFU/ml was performed using the URO-QUICK™ system. On day 32 an overall value was recorded of contamination of 42.85% for group G1, 71.42% for G2 and 42.85% for G3; after 60 days, the final contamination result was 85.71% for group G1, and 100% for both G2 and G3 groups. Considering the number of contaminated samples at the end of the observation period, the three techniques showed no statistically significant differences. The study highlighted the bacterial permeability of gutta-percha/seal barrier, underlining the importance of an effective coronal restoration to ensure a durable seal after root canal treatment.

Ribavirin increases mitogen- and antigen-induced expression of CD40L on CD4+ T cells in vivo.

Here, CD40L expression and cytokine production have been analysed in peripheral blood cells from orthotopic liver transplantation (OLT) recipients treated with ribavirin for recurrent chronic hepatitis C. The study included 18 OLT recipients treated with ribavirin, eight control OLT recipients and 10 healthy controls. FACS analysis showed that baseline expression of CD40L was not different between ribavirin-treated patients and controls. In contrast, after stimulation with both HCV core antigen and phorbol myristate acetate (PMA) plus ionomycin (IO), the expression of CD40L on CD4 lymphocytes was significantly higher in the ribavirin group compared with controls. In the ribavirin group, the increased expression of CD40L significantly correlated with reduction of HCV RNA levels with respect to pretreatment values. Finally, ribavirin treatment was not associated with modification of PMA-IO-induced cytokine production by T lymphocytes and interleukin (IL)-1beta and tumour necrosis-alpha (TNF)-alpha production by CD40L-stimulated monocytes. In conclusion, these data indicate that ribavirin -upmodulates CD40L expression on CD4 T cells, a property which may account in part for its ability to enhance the antiviral activity of interferon-alpha in the treatment of chronic HCV infection.

PON1 L55M polymorphism is not a predictor of coronary atherosclerosis either alone or in combination with Q192R polymorphism in an Italian population.

BACKGROUND: The present study evaluated the role of the PON1 L55M polymorphism independently and in conjunction with the Q192R polymorphism on the risk of coronary atherosclerosis in an Italian population. MATERIALS AND METHODS: Three hundred and ninety-one subjects with significant coronary stenosis (> 50%) (coronary artery disease-positive; CAD+), 196 subjects with normal coronary arteries (< 10% stenosis) (CAD-) and 178 healthy controls were screened using a combination of polymerase chain reaction and restriction enzyme digestion. RESULTS: In the pooled population, the frequencies of L and M alleles were 0.63 and 0.37, respectively; the most common haplotypes were QQ/LM (24.2%) and QR/LL (21.8%) and a strong linkage disequilibrium between L/55 and R/192 alleles was observed (D' = -0.91; P < 0.0001). CAD+ subjects did not show any significant differences in the distribution of PON1-55 genotypes as compared to CAD- subjects and population controls (chi2 = 1.5, P = 0.8). After controlling for other risk factors, the low-concentration M allele was not associated with a significant change of CAD risk (OR 1.02; 95% CI 0.80-1.29; P = 0.87). Moreover, the L55M polymorphism did not show any interaction with other risk factors such as smoking, diabetes, hypertension, low levels of high-density lipoprotein (HDL) or high ratios of low-density to high-density lipoproteins. The combination of L55M with the Q192R polymorphism did not show any effect on CAD risk. However, a marginal decrease in myocardial infarction risk was detected when QQ/MM carriers (OR 0.51; 95% CI 0.26-0.99; P = 0.048), but not LL/RR carriers, were compared with subjects not homozygous for an L or R allele. CONCLUSIONS: These findings did not indicate a major effect of the PON1 L55M polymorphism, either alone or in combination with the Q192R polymorphism, on CAD risk. Additional studies are needed for a better evaluation of the role of the 55/192 PON1 genotypes in combination on myocardial infarction risk.

Treatment with ribavirin and interferon-alpha reduces interferon-gamma expression in patients with chronic hepatitis C.

Recent studies in vitro and in animals have suggested that ribavirin may potentiate the antihepatitis C virus (HCV) activity of interferon-alpha (IFN-alpha) by up-modulating the production of T cell-derived cytokines, such as interleukin (IL)-2 and IFN-gamma, which play a key role in the cellular immune response against HCV. To study the immune-modulatory mechanisms of ribavirin further, cytokine production by activated T cells and circulating cytokine levels were studied by FACS analysis and ELISA testing in 25 patients with chronic hepatitis C unresponsive to IFN-alpha, before and after treatment with either ribavirin plus IFN-alpha or IFN-alpha alone. After 16 weeks of treatment, both the expression of IFN-gamma by activated T cells and the blood levels of IFN-gamma, were significantly reduced with respect to pretreatment values in patients treated with ribavirin and IFN-alpha but not in those undergoing treatment with IFN-alpha alone. The expression of IFN-gamma was significantly lower in patients that gained normal ALT levels with respect to those that did not. No modification of the expression of IL-2, IL-4 and IL-10 was found before and after treatment in either group of patients. In conclusion, the results of this study do not support up-modulation of IFN-gamma and IL-2 production as the mechanism by which ribavirin potentiates IFN-alpha anti HCV activity. In addition, our findings suggest that ribavirin may exert an anti-inflammatory effect and may help reducing IFN-gamma-driven T cell activation and liver damage.

Lack of association of the common TaqIB polymorphism in the cholesteryl ester transfer protein gene with angiographically assessed coronary atherosclerosis.

The anti-atherogenic effect of cholesteryl ester transfer protein (CETP) genetic variants associated with lowered enzyme activity is controversial. Moreover, in a few studies, this effect has been evaluated in the presence of a certain risk factor constellation. We addressed this issue in a case-control study, where 415 subjects with angiographically documented coronary artery disease (CAD +), 397 subjects without CAD (in 215, CAD was excluded by coronarography (CAD-)), and 188 healthy population controls, were screened for the CETP TaqIB polymorphism. The prevalence of the low-activity TaqIB2 allele was 0.396 in CAD+, and 0.428 and 0.416 in CAD- and population controls, respectively (p = 0.40). Its presence was significantly associated with increased high-density lipoprotein cholesterol (HDL-C) in population controls (1.40 +/- 0.40 mmol/l in B1B1, 1.52 +/- 0.39 mmol/l in B1B2 and 1.58 + 0.46 mmol/l in B2B2; p < 0.03 for trend), but not in the other groups. The CETP TaqIB polymorphism accounted for < 1% of the HDL-C variance in the whole cohort (p = 0.048). After adjustment for other risk factors, the CETP TaqIB2 allele was found not to be associated with significant changes in CAD risk independently of an assumed either dominant (odds ratio (OR) 0.97; 95% confidence interval (CI) 0.66-1.44; p = 0.89) or recessive effect (OR 0.68; 95% CI 0.42-1.12; p = 0.13). The CETP TaqIB polymorphism did not show a significant interaction with other risk factors in influencing CAD risk. Our findings do not support the hypothesis that a genetic variant resulting in lowered CETP activity is associated with reduced risk of coronary atherosclerosis.

Unfavourable effects of colchicine in combination with interferon-alpha in the treatment of chronic hepatitis C.

BACKGROUND: The prognosis of chronic hepatitis depends on the progression of hepatic fibrosis. AIM: To investigate whether the antifibrotic drug colchicine, in combination with interferon-alpha has a role in the treatment of chronic hepatitis C. METHODS: Sixty-five HCV-RNA positive patients with chronic hepatitis were randomized to receive interferon-alpha, 6 MU t.i.w. for 6 months followed by 3 MU t.i.w. for further 6 months, with or without the adjunct of colchicine, 1 mg o.d., 6 days a week, for 3 years. We report an interim analysis after the first 18 months. RESULTS: Thirty-four patients received interferon-alpha and 31 received interferon-alpha and colchicine. The two groups were comparable for baseline data, including HCV-RNA levels, genotypes and histological grading/staging. Drop-outs and side-effects were similar. The proportion of patients who achieved alanine transaminase normalization or undetectable HCV-RNA at month 6 was higher in the interferon-alpha (68% and 47%, respectively) than in the interferon-alpha plus colchicine group (32% and 23%, P=0.004 and P=0. 04, respectively). End-of-treatment biochemical and virological response occurred in 41% and 29% of the interferon-alpha and 19% and 10% of the combination group, respectively (P=0.05 and P=0.05). Sustained biochemical response occurred in 26% of the interferon-alpha and 6% of the interferon-alpha plus colchicine group (P=0.03), corresponding percentages of sustained HCV-RNA loss being 21% and 3% (P=0.04). CONCLUSIONS: The combination of colchicine and interferon-alpha worsens the effectiveness of interferon-alpha alone in HCV chronic hepatitis. These alarming findings prompted us to interrupt the trial at this stage.

Haemochromatosis gene mutations and risk of coronary artery disease.

The identification of mutations in the haemochromatosis gene (HFE) (C282Y and H63D) provides the unique opportunity to test whether genetic variants that are associated with tissue iron accumulation may influence the risk of coronary atherosclerosis. To this aim the prevalence of C282Y and H63D mutations was determined in 174 patients with angiographically documented CAD (>50% stenosis) and history of MI, 187 healthy free-living individuals and 142 blood donors. C282Y and H63D mutations were not found to be more frequent in coronary patients as compared to controls. Moreover, these HFE variants were unrelated to the severity of coronary atherosclerosis. These findings did not provide evidence of an association between HFE mutations and the presence of coronary atherosclerosis or its major ischaemic complications, thus indicating that HFE mutations are poor genetic markers of coronary risk.

Low levels of hepatitis C virus RNA in blood of infected patients under maintenance haemodialysis with high-biocompatibility, high-permeability filters.

BACKGROUND: Patients undergoing maintenance haemodialysis are often infected with hepatitis C virus, yet the clinical course of liver disease is usually mild. We investigated whether the hepatitis C virus viral load is influenced by the haemodialytic procedure and the type of dialyser. METHODS: Hepatitis C virus-RNA levels were measured using a quantitative polymerase chain reaction assay in predialysis blood from 51 hepatitis C virus-infected patients dialysed with different membranes. Paired pre- and post-dialysis measurements were also obtained in 18 patients. RESULTS: Patients dialysed using cuprammonium-regenerated cellulose membranes with low (cuprofan) or intermediate (cellulose acetate or diacetate) biocompatibility had higher pre-dialysis hepatitis C virus-RNA levels (p<0.05] compared to those dialysed with synthetic high-biocompatibility, high-permeability polymeric membranes (polyacrylonitrile, polysulfone, polymethylmethacrylate or polycarbonate). Hepatitis C virus-RNA levels were unrelated to the duration of haemodialysis and the presence of abnormal transaminases. A significant reduction (p=0. 04) of serum hepatitis C virus-RNA levels was observed after a single haemodialysis, particularly in patients with high pre-dialysis viral load. Patients with low pre-dialysis hepatitis C virus-RNA levels (<2. 5 x 10(3) copies/ml) exhibited only minimal changes following the procedure. Four patients with medium-high basal viral load switched from a low-biocompatibility/low-permeability to a high-biocompatibility/high permeability filter had a marked reduction of viraemia after three weeks, in one case followed by a new increase after return to the original filter. CONCLUSIONS: Haemodialysis with high-biocompatibility/high-permeability filters in hepatitis C virus-infected patients is associated with low blood levels of hepatitis C virus-RNA. This finding may be of clinical relevance, especially in patients listed for kidney transplantation.

The gln-Arg192 polymorphism of human paraoxonase gene is not associated with coronary artery disease in italian patients.

Serum paraoxonase (PON) is an HDL-bound enzyme protecting LDL from oxidation. A common polymorphism of the paraoxonase gene (PON1) involving a Gln-to-Arg interchange at position 192 has been demonstrated to modulate PON activity toward paraoxon, a nonphysiological substrate; Arg192 (allele B) is associated with higher activity than Gln192 (allele A). This polymorphism has been proposed as a genetic marker of risk for coronary artery disease (CAD). However, the relationships between codon 192 PON1 genotypes, coronary atherosclerosis, and the occurrence of myocardial infarction (MI) are still controversial. PON1 genotypes were determined in 472 consecutive subjects (>40 years old) who underwent coronary angiography. CAD (>50% stenosis) was detected in 310 subjects (CAD+); 162 subjects with <10% stenosis served as controls (CAD-). We also evaluated 204 randomly selected individuals as population controls. PON1 genotypes were determined by PCR and AlwI restriction enzyme digestion. Frequencies of alleles A and B were 0. 70 and 0.30 in angiographically assessed subjects and 0.73 and 0.27 in population controls, respectively (chi2=2.0; P<0.3). Distribution of PON1 genotypes in CAD+ were not significantly different from those in CAD- (chi2=2.10; P<0.3). Similarly, no differences were observed in the subgroup of CAD+ with MI nor in that at higher oxidative risk (smokers and/or diabetics). After controlling for other coronary risk factors, no association was found between PON1 alleles and the presence of CAD. PON1 AA genotype was associated with reduced concentration of apolipoprotein B-containing triglyceride-rich lipoproteins. This study did not provide evidence of a significant association between codon 192 PON1 genotypes and coronary atherosclerosis in Italian patients. However, it did confirm that the PON1 low-activity allele is associated with a less atherogenic lipid profile.

The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins.

The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e., endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.

Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells.

Several polycations such as polylysine polymers are efficient transfection agents due to their capacity to bind DNA at physiological pH. By linking a ligand for a cell surface receptor to the polycation domain, a selective delivery of polyanionic compounds such as oligodeoxynucleotides (ODNs) without cell membrane-disruption can be achieved. Therefore, we aimed to develop this strategy to improve the uptake of oligomers in cancer cells. In particular, cationic polymers polylysine were covalently linked to a molecule of Folic Acid (FA) to deliver complexed ODNs in human melanoma (M-14) cells which express FA receptors. Since in these cells c-myc oncogene seems to play a crucial role in tumor growth, we used a c-myc antisense ODNs (15mer base antisense c-myc ODNs phosphorothioate) to inhibit its expression. The cellular uptake of the complexed ODNs was improved compared to the cellular uptake of free ODNs with a significant decrease in the intracellular c-myc protein level, resulting in a reduction of the growth rate and colony-forming capacity of the cells. No such effect was observed when ODNs in scrambled sequences were administered under the same experimental conditions. The efficacy of the uptake of the complex is receptor-related since a Transferrin-polylysine carrier produced no significant biological effects (in melanoma cells the Fe uptake is mediated by an oxidoreductase present in the cell membrane and not by Transferrin receptor pathway). Our results demonstrate that: a) By choosing the appropriate ligand for the membrane receptor present on the target cells, selective targeting of ODNs can be achieved. b) The uptake of the ODNs can be improved by receptor-mediated endocytosis. c) In a model system the complexed ODNs are capable of impairing gene product synthesis and function.