APC and KRAS mutations in distal colorectal polyps are related to smoking habits in men: results of a cross-sectional study

BACKGROUND: The purpose of this study was (a) to evaluate the association between cigarette smoking and the prevalence of distal colorectal polyps and adenocarcinoma and (b) to analyse genetic alterations representing different molecular pathways of the colorectal carcinogenesis. METHODS: A total of 623 asymptomatic male (mean age: 53 years; 50-65) car factory workers were included. Information on smoking habits and other lifestyle factors were collected followed by a 60 cm colonoscopy. APC and KRAS mutations and microsatellite status were determined in colorectal lesions (colorectal carcinoma (CRC), hyperplastic (HP) and adenomatous polyps (AP)). Data were analysed using unconditional multiple logistic regression models. RESULTS: Smokers had a higher prevalence of AP (OR 2.1; 95% CI 1.2-3.6; p<0.05) and HP (OR 5.4; 95% CI 2.6- 11.1; p<0.05). No differences in CRC were observed. There was a dose-response relationship with the number of cigarettes smoked. The risk of developing AP or HP decreased after smoking cessation, even among heavy smokers (≥20 packs/year). KRAS mutations were more prevalent among smokers AP (OR 5.6; 95% CI 1.6-20.4; p=0.007). There was a trend of positive association with APC mutations (OR 3.5; 95% CI 0.9-4.4; p=0.096). APC and KRAS mutations were found in 36% and 61% of the HP of smokers, but were absent in non-smokers (p=0.89 and 0.78, respectively). There were no differences in MSI between smokers and non-smokers. CONCLUSIONS: Cigarette smoking is associated with a higher risk of developing both HP and AP and a higher prevalence of mutations in APC and KRAS (AU)

Pig liver gene therapy by noninvasive interventionist catheterism.

The efficacy of noninvasive interventionist catheterism in large animals as an alternative to the hydrodynamic procedure, described for small animals, is evaluated. Basically, gene transfer is performed by implantation and fixation of a balloon catheter within the suprahepatic vein of anesthetized pigs, through the femoral vein. The catheter tip is identified by fluoroscopy, injecting a contrast solution that marks large or small hepatic territories. Animals were injected with a 100 ml pTG7101 plasmid solution (40 microg/ml), which contains the human alpha-1 antitrypsin gene, perfused at a rate of 7.5 ml/s and efficacy and toxicity of the procedure were evaluated. The results show: (i) the highest efficacy in protein production is reached when perfusion is limited to small areas of the liver; (ii) no relevant hepatic toxicity was observed; (iii) gene transfer is mainly located in the areas around the central vein, as seen in the immunohistochemical studies; (iv) the electron microscopy studies indicate that the areas with good transfection efficacy show the presence of abundant endocytic vesicles that may even fuse among themselves. These data suggest that retrovenous injection by noninvasive interventionist catheterism could become an efficient procedure for hepatic gene transfer with potential clinical applications.

Real-time quantification of human telomerase reverse transcriptase mRNA in the plasma of patients with prostate cancer.

The aim of this study was to evaluate the potential diagnostic value of quantitative analysis of human telomerase reverse transcriptase (hTERT) mRNA in plasma for noninvasive diagnosis of prostate cancer (PCa). Expression levels of hTERT were analyzed by real-time quantitative RT-PCR in 68 patients showing elevated prostate-specific antigen (PSA) levels and a control group of 44 healthy volunteers. Sensitivity and specificity were determined and compared to the corresponding PSA values. Median values for hTERT gene expression in the PCa patients (0.72 ng; range 0.01-12.86) were statistically significantly higher (P < 0.001) than in the control group (0.13 ng; 0.02-0.35). Patients with clinically confirmed prostatitis showed lower plasma hTERT expression than PCa patients (0.29; 0.01-66.07). At a cutoff value of 0.35 sensitivity and specificity for the diagnosis of PCa were 81% and 60%, respectively. We suggest that hTERT mRNA in plasma is a very specific and sensitive method that may aid to differentiate between malignant and nonmalignant prostate tissue and may be a useful marker (in combination with PSA) for early PCa diagnosis.

Incidence, risk factors and clinical course of thiopurine-induced liver injury in patients with inflammatory bowel disease.

BACKGROUND: The incidence of thiopurine-induced hepatotoxicity in patients with inflammatory bowel disease varies in different studies. AIMS: To assess the rate of thiopurine-induced liver toxicity in patients with inflammatory bowel disease; to determine the predictive factors and to characterize its clinical course and management. METHODS: A cohort of 161 patients was prospectively followed for a median of 271 days. Hepatotoxicity was established when alanine transaminase or alkaline phosphatase plasma levels were greater than twice the upper normal limit. RESULTS: Abnormal liver function was detected in 21 patients (13%; 95% CI: 7-18). Hepatotoxicity occurred in 16 patients (10%; 95% CI: 6-16) after a median of 85 days. In five cases, treatment was withdrawn due to hepatotoxicity. Use of corticosteroids was associated with hepatotoxicity (OR: 4.94; 95% CI: 1.01-23.98) with antitumour necrosis factor concomitant therapy showing a protective role (OR: 0.3; 95% CI: 0.1-3.1). gamma-Glutamyl transferase plasma levels at the onset of hepatotoxicity showed the best predictive value for treatment withdrawal (area under the receiver operating characteristic curve: 0.95). CONCLUSIONS: The incidence of hepatotoxicity in inflammatory bowel disease patients receiving thiopurines is relevant, mainly in patients co-treated with corticosteroids. gamma-Glutamyl transferase plasma level is a useful biomarker in therapy withdrawal prediction.

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.

The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experiments were performed employing the hAAT gene, and the efficacy and differential identification in mouse plasma of human transgene versus mouse gene was assessed by ELISA and proteomic procedures, respectively. By applying different experimental strategies such as cumulative dose-response efficacy, hemodynamic changes reflected by venous pressures, intravital microscopy, and morphological changes established by transmission electron microscopy, we found that: (a) cumulative multiple doses of transgene by hydrodynamic injection are efficient and well tolerated, resulting in therapeutic plasma levels of hAAT; (b) hydrodynamic injection mediates a transient inversion of intrahepatic blood flow, with circulatory stasis for a few minutes mainly in pericentral vein sinusoids; (c) transmission electron microscopy shows hydrodynamic injection to promote massive megafluid endocytic vesicles among hepatocytes around the central vein but not in hepatocytes around the periportal vein. We suggest that the mechanism of hydrodynamic liver gene transfer involves transient inversion of intrahepatic flow, sinusoidal blood stasis, and massive fluid endocytic vesicles in pericentral vein hepatocytes.

Real time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA in patients with colorectal cancer.

BACKGROUND: Increased telomerase activity can be found in almost 90% of colorectal tumours. We aim to describe the preliminary results for quantification in plasma of hTERT mRNA in colorectal cancer patients. MATERIALS AND METHODS: Fifty patients undergoing surgery for colorectal cancer and a control group of 50 healthy volunteers were prospectively studied. Pre-operative venous blood samples were taken from all cancer patients and volunteers. Plasma hTERT expression was determined from peripheral blood based on real-time quantitative RT-PCR (qRT-PCR) method normalized to the amount of RNA input using 18S rRNA gene expression. Plasma pre-operative CEA levels were also determined. RESULTS: Median values for normalized hTERT (hTERT(N)) gene expression were higher in colorectal cancer patients (11.62, range 0.23-47.67) than healthy volunteers (0.29, range 0.00-4.63) (P < 0.001). Individual data showed that 82% of colorectal cancer patients had hTERT(N) expression values superior to the maximum value observed in the control group. Sensitivity and specificity of the assay for colorectal cancer detection were 98% and 64%, respectively. No significant differences in hTERT(N) expression between gender or with age (P > 0.05). No significant correlation was found between hTERT(N) expression and CEA values (Spearman's rank test = 0.136, P = 0.348). CONCLUSIONS: These results show that detection of mRNA based on the qRT-PCR of the telomerase hTERT(N) gene in plasma clearly differentiates between healthy and colorectal cancer patients and that hTERT(N) can be detected and quantified in plasma. This opens up a new field as a noninvasive blood test for colorectal cancer diagnosis.

Long-term therapeutic levels of human alpha-1 antitrypsin in plasma after hydrodynamic injection of nonviral DNA.

The transfection efficacy of several vectors containing the full genomic hAAT gene with its natural promoter (pTG7101) and others containing the cDNA of hAAT gene driven by cytomegalovirus immediate-early promoter or the 0.5 kb upstream of hAAT gene sequence has been studied by hydrodynamic tail-vein injection (20 microg/mouse). pTG7101 (but not the other plasmids) results in therapeutic and stable concentration of hAAT in plasma. A dose-response study with this plasmid (0.3-320 microg/mouse) confirms that hAAT remains long-term stable in plasma, with therapeutic concentrations of hAAT (>0.9 mg/ml). The parameters of the dose-response curve were: R: 0.98, E(max) 3449.0+/- 279.7 microg/ml and EC(50) 1.2 x 10(12) plasmid-gene units. In addition, 4 months after transfection, the intrinsic efficacy of transgenic expression (amount of RNA/DNA) in mouse liver was 50-80% that normally expressed by the mouse gene. The important efficacy of nonviral genomic DNA opens a new avenue in the safety applications of human gene therapy.

Stability of PEI-DNA and DOTAP-DNA complexes: effect of alkaline pH, heparin and serum.

DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degrees C in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds.

Asialofetuin liposome-mediated human alpha1-antitrypsin gene transfer in vivo results in stationary long-term gene expression.

The development of nonviral vectors for in vivo gene delivery to hepatocytes is an interesting topic in view of their safety and tremendous gene therapy potential. Since cationic liposomes and liposome uptake by receptor-mediated mechanisms could offer advantages in the efficacy of liposome-mediated gene transfer, we studied the effect of liposome charge (anionic vs. cationic) and the covalently coupled asialofetuin ligand on the liposome surface in mediating human alpha1-antitrypsin (hAAT) gene transfer to mice in vivo. The changes in liposome charge were made by adding the following lipids to the backbone liposomes: anionic phosphatidylserine, cationic N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate or a lipopeptide synthesized from dipalmitoylphosphatidylethanolamine and covalently coupled to the cationic nuclear localization signal peptide. Two plasmids containing the hAAT gene were used: pTG7101, containing the complete genomic sequence of the human gene driven by the natural promoter, and p216, containing the human hAAT cDNA under the control of the CMV promoter. The results indicate that both untargeted anionic and cationic liposomes mediate plasma levels of hAAT that decline over time. However, asialofetuin liposomes increase the plasma levels of hAAT and can mediate long-term gene expression (>12 months) with stationary plasma levels of protein. Results from quantitative and qualitative reverse transcriptase polymerase chain reaction match those from protein plasma levels and confirm both the human origin of the message and the liver as source of the protein. The use of asialofetuin liposomes in hepatic gene therapy may both increase and prolong in vivo gene expression of hAAT and other clinically important genes.

Antitumor effect of B16 melanoma cells genetically modified with the angiogenesis inhibitor rnasin.

The growth of new blood vessels is an essential condition for the development of tumors with a diameter greater than 1-2 mm and also for their metastatic dissemination. RNasin, the placental ribonuclease inhibitor, is known to have antiangiogenic activity through the inhibition of angiogenin and basic fibroblast growth factor. Nevertheless, the administration of the recombinant form of a protein poses several limitations; as a result, we have studied the antitumor effect of RNasin in a murine gene therapy model. RNasin cDNA was subcloned into the pcDNA3 expression vector, and the resulting recombinant plasmid was used to transfect the B16 murine melanoma cell line. An RNasin inverted construction was used as control. Mice intravenously injected with clones expressing RNasin showed a significant inhibition of tumor metastatic progression with respect to control groups (P<.001) and survived longer (P<.001). Tissue sections from RNasin-expressing cell tumors showed a lower number of blood vessels when compared to tissue sections from mice lungs that had been inoculated with control cell lines. The results of these experiments show that the genetic modification of tumor cells with RNasin cDNA yields a significant antitumor effect, and suggest that this effect is at least partially the result of angiogenesis inhibition.

Histamine up-regulates phosphodiesterase 4 activity and reduces prostaglandin E2-inhibitory effects in human neutrophils.

OBJECTIVE: To investigate whether histamine produces up-regulation of phosphodiesterase (PDE) activity with functional consequences in human peripheral blood neutrophils. METHODS: PDE activity was studied by a radioisotopic method following anion-exchange chromatography. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of mRNA transcripts of PDE4 subtypes. Cyclic AMP (cAMP) levels were measured by enzyme-immunoassay, and superoxide generation by cytochrome c reduction. TREATMENT: Neutrophils were incubated for 4 h with histamine (1 microM). RESULTS: PDE4 was the only isoenzyme activity increased in treated neutrophils. Kinetic analysis showed a approximately 1.5-fold increase in Vmax without alteration of Km values. cAMP content in treated cells was higher than resting values (0.52+/-0.07 vs. 2.75+/-0.31 pmol/10(6) cells). RT-PCR showed increased expression of mRNA transcripts for PDE4B in histamine-treated cells. Functionally, up-regulation of PDE4 reduced the inhibition by prostaglandin E2 of zymosan-induced superoxide generation. CONCLUSION: Histamine up-regulates PDE4 activity and produces heterologous desensitisation of human neutrophils.

beta-Adrenoceptor stimulation up-regulates phosphodiesterase 4 activity and reduces prostaglandin E2-inhibitory effects in human neutrophils.

Human neutrophils were treated for 4 h with a combination of salbutamol (1 microM), a beta2-adrenoceptor agonist, and rolipram (30 microM), a selective phosphodiesterase 4 inhibitor, to investigate whether this treatment produces up-regulation of phosphodiesterase activity with functional consequences. Anion-exchange chromatography coupled with the use of selective activators and inhibitors demonstrated that a phosphodiesterase activity with characteristics of the isoenzyme type 4 was increased in drug-treated cells. Kinetic analysis showed a approximately 1.5-fold increase in Vmax without alteration of Km values. The augmented phosphodiesterase activity in drug-treated cells was abolished by actinomycin D. Cyclic AMP content in drug-treated cells was higher than resting values (27.28+/-2.79 pmol/10(6) cells vs. 0.34+/-0.03 pmol/10(6) cells). Reverse transcriptase-polymerase chain reaction showed increased expression of mRNA transcripts for PDE4B and PDE4A in drug-treated cells. Functionally, up-regulation of phosphodiesterase 4 reduced the inhibition by prostaglandin E2 of zymosan-induced superoxide generation.

Bronchodilator and anti-inflammatory activities of SCA40: studies in human isolated bronchus, human eosinophils, and in the guinea-pig in vivo.

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM-0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 against spontaneous tone (6.52 +/- 0.10) was greater than against tone raised by equieffective concentrations (approximately 70%) of histamine (5.76 +/- 0.06), leukotriene C4 (5.44 +/- 0.11), and acetylcholine (4.98 +/- 0.09). In the presence of cytochalasin B, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.5 microM) induced leukotriene C4 production in human eosinophils isolated in discontinuous metrizamide gradients. The production of leukotriene C4 was inhibited by SCA40 in a concentration-related fashion (-log IC50 = 6.04 +/- 0.20; n = 6). Rolipram, a selective PDE 4 inhibitor, was also effective (-log IC50 = 7.29 +/- 0.32) but the selective PDE 3 inhibitor SKF94120 was scarcely effective (< 10% inhibition for 10 microM). In ovalbumin sensitized guinea-pigs, SCA40 (1 mg kg(-1), i.p.) given 30 min before antigen challenge significantly inhibited the acute bronchoconstriction produced by aerosol antigen (5 mg ml(-1), 30 s) (antigen response was 185 +/- 13 and 91 +/- 21 cmH2O l(-1) s(-1) in control and SCA40-treated animals, respectively, P < 0.05). Pretreatment with SCA40 (1 mg kg(-1), i.p., 30 min pre- and 3 h post-antigen exposure) prevented airway hyperreactivity to histamine which developed 24 h after exposure of conscious guinea-pigs to aerosol antigen. Eosinophil lung accumulation that accompanied airway hyperreactivity was also inhibited by SCA40 (from 6.15 +/- 0.86 in control to 1.27 +/- 0.27 in treated animals; expressed as eosinophils x 10(6); P < 0.05). SCA40 (1 mg kg(-1), i.p.) also inhibited the microvascular leakage produced after inhaled antigen (5 mg ml(-1), 30 s) at all airway levels. The haemodynamic effects of SCA40 (1 mg kg(-1), i.p.) consisted of a rapid decrease (peak at 5 min) in mean arterial blood pressure (-39.4 +/- 2.4%) and tracheal mucosal blood flow (-13.5 +/- 2.0%) that slowly recovered with time. These data support previous work showing that PDE inhibition results in antispasmogenic and anti-inflammatory effects. SCA40 was effective in vitro and in vivo and these effects are probably related to its activity as a mixed PDE inhibitor.