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By using high-density oligonucleotide arrays, we profiled gene expression in reward-related brain regions of rats that developed escalated cocaine intake after extended access to cocaine (6 h per day). Rats allowed restricted daily access to cocaine (only 1 h) that displayed a stable level of cocaine intake and cocaine naive rats were used for controls. Four analysis methods were compared: Affymetrix microarray suite 4 and microarray suite 5, which use perfect-match-minus-mismatch models, and dchip and rma, which use perfect-match-only models to generate expression values. Results were validated by RT-PCR in individual animals from an independent replication of the experiment. A small number of genes was associated with escalated cocaine intake (ESC genes). Unexpectedly, of the brain regions examined [prefrontal cortex, nucleus accumbens, septum, lateral hypothalamus (LH), amygdala, and ventral tegmental area], the LH was the most transcriptionally responsive in escalation of cocaine intake. Most of the ESC genes identified are also expressed during synaptogenesis and synaptic plasticity and include genes that code for several presynaptic and postsynaptic proteins involved in neurotransmission. These results suggest that LH intrinsic circuitry undergoes a structural reorganization during escalation of cocaine use. This remodeling of LH circuitry could contribute to the chronic deficit in reward function that has been hypothesized to drive the transition to drug addiction. Results also support the value of using multiple analysis strategies to identify the most robust changes in gene expression and to compensate for the biases that affect each strategy.

Species identification represents a critical issue in food chain safety and quality control. Several procedures are available to detect animal proteins in cattle feed or to trace transgenic foods. The most effective approach is based on the use of DNA as a marker. Amplification of DNA provides rapid, sensitive and specific protocols. Several target genes can be used, but new insights come from the mitochondrial genome, which is naturally amplified in each cell and shows a remarkable resistance to degradation. These are key points when analysing complex matrices such as foods, animal feedstuff or environmental samples. Traceability is important to prevent BSE or to monitor novel foods, such as genetically modified organisms. Amplification is commonly performed, but it requires expertise and a molecular biology laboratory to perform restriction analysis, electrophoresis or gel staining for the visualisation of results. Hereby, we consider a strategy based on multiple nested amplification and reverse hybridisation assay that virtually requires only a thermocycler and a water bath. The protocol is rapid and simple and can simultaneously detect different species in a DNA sample. This promising approach allows microarray developments, opening up to further perspectives. An international application has been published under the patent cooperation treaty. Presently, a ban on feeding ruminants on cattle-derived proteins is in force in Europe and USA. The identification of metazoan traces in a sample is not only a mere preventive measure for BSE, but represents a possible screening system for monitoring biotechnology products and procedures, as well as a quality control strategy to assure consumer's rights.

Identification and molecular typing of environmental isolates by molecular techniques requires knowledge of the genetic characteristics of the microbe species being examined. The introduction of automated sequences has greatly speeded up the entire sequencing process as well as improved the accuracy of the collected information. Bioinformatics tools have become indispensable not only for setting up research studies, but also for storing, organizing and managing enormous quantities of sequencing data. Despite its great advantages, the use of bioinformatics is hindered by difficulties in learning how to use its software tools. The GenEnv database was developed to provide operators involved in biological risk management with a user-friendly tool for sequence analysis. Presently, there are over 20.000 sequence records, and over 9000 bacterial species represented in the database. The initial gene set comprises rDNA16S, rpoB, gyrB. The system allows sequence-driven microbe identification as well as the development of study protocols for research on specific microbe species. Nucleotide sequences are represented graphically. The GenEnv database was designed as a tool for public health operators but also offers wide prospects for scientific research.

In Italy controls on the hygienic quality of swimming pools are fixed by the Accordo tra il Ministero della Salute, le regioni e le province autonome di Trento e di Bolzano, come into force in 2003. In the present study swimming pools were investigated from the microbiological point of view on the basis of the new legislation. Contemporaneously, for the first time in Italy, analytical controls on the occurrence of the protozoa Cryptosporidium and Giardia were performed with both techniques of immunofluorescence and polymerase chain reaction. Results evidenced a good water quality when the bacterial parameters stated by law were considered. However in some swimming pools, the protozoa were also recovered. Factors affecting the use of molecular techniques on the analysis of environmental samples are also put into evidence.

Objective: To investigate an outbreak of Candida parapsilosis bloodstream infections (BSIs) involving three patients admitted to a pediatric oncology unit between April and June 2002., Methods: After the third case was documented, cultures were performed of the hands of all medical and paramedical staff members in the pediatric oncology unit and of environmental surfaces in the rooms occupied by the three patients. Electrophoretic karyotyping with pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction were used to assess the genetic relatedness among C. parapsilosis isolates., Results: The three cases of C. parapsilosis BSI were diagnosed based on blood cultures performed during a 38-day period. Evidence of prior C parapsilosis colonization of the gastrointestinal tract was present in only the first case. Each patient had an indwelling central venous catheter (CVC), which was promptly removed, and semiquantitative catheter tip cultures also revealed C. parapsilosis. None of the 30 environmental cultures performed was positive for C. parapsilosis, but the fungus was isolated from the hands of 6 of the 20 nurses tested. Both molecular typing methods revealed identical DNA fingerprinting patterns for all 13 patient isolates (7 from blood, 3 from CVC tips, and 3 from the gastrointestinal tract) and for 5 of the 6 recovered from the nurses' hands., Conclusions: These findings suggest the possibility of cross-infection with a single C. parapsilosis strain that was transmitted (probably during CVC dressing changes) by nurses whose hands were colonized with it. The role of previous gastrointestinal colonization in the first case cannot be excluded.

We investigated Legionella and Pseudomonas contamination of hot water in a cross-sectional multicentric survey in Italy. Chemical parameters (hardness, free chlorine, and trace elements) were determined. Legionella spp. were detected in 33 (22.6%) and Pseudomonas spp. in 56 (38.4%) of 146 samples. Some factors associated with Legionella contamination were heater type, tank distance and capacity, water plant age, and mineral content. Pseudomonas presence was influenced by water source, hardness, free chlorine, and temperature. Legionella contamination was associated with a centralized heater, distance from the heater point >10 m, and a water plant >10 years old. Furthermore, zinc levels of <100 microg/L and copper levels of >50 microg/L appeared to be protective against Legionella colonization [corrected]. Legionella species and serogroups were differently distributed according to heater type, water temperature, and free chlorine, suggesting that Legionella strains may have a different sensibility and resistance to environmental factors and different ecologic niches.

Genetically Modified Organisms (GMOs) are a fact of modern agriculture and a major field of discussion in biotechnology. As science incessantly achieves innovative and unexpected breakthroughs, new medical, political, ethical and religious debates arise over the production and consumption of transgenic organisms. Despite no described medical condition being directly associated with a diet including approved GM crops in large exposed populations such as 300,000,000 Americans and a billion Chinese, public opinion seems to look at this new technology with either growing concern or even disapproval. It is generally recognized that a high level of vigilance is necessary and highly desirable, but it should also be considered that GMOs are a promising new challenge for the III Millennium societies, with remarkable impact on many disciplines and fields related to biotechnology. To acquire a basic knowledge on GMO production, GM-food consumption, GMO interaction with humans and environment is of primary importance for risk assessment. It requires availability of clear data and results from rigorous experiments. This review will focus on public health risks related with a GMO-containing diet. The objective is to summarize state of the art research, provide fundamental technical information, point out problems and perspectives, and make available essential tools for further research. Are GMO based industries and GMO-derived foods safe to human health? Can we consider both social, ethical and public health issues by means of a constant and effective monitoring of the food chain and by a clear, informative labeling of the products? Which are the so far characterized or alleged hazards of GMOs? And, most importantly, are these hazards actual, potential or merely contrived? Several questions remain open; answers and solutions belong to science, to politics and to the personal opinion of each social subject.

Despite legionellosis surveillance is active in Italy since many years, the disease notification appears still undervalued. A multicentric survey was carried out among 5 big Italian hospitals. It examined 11,435 discharge forms (1999-2001), reporting pneumonia diagnosis. Legionellosis (II class of notify system for infectious disease) was studied among pneumonia diagnosis by discharge forms. According to the ICD9-CM, there's no specific code for legionellosis (this disease is included among "others gram-negative pneumonia"). So the presumed pneumonia imputable to Legionella spp were the 2.7% of the whole number of analyzed discharge forms. Besides, the data regarding the other pneumonia showed that the etiological agent was specified only in the 11.2% of the case. This situation could be rectified both introducing adequate discharge forms codes and promoting the etiological diagnosis during the hospital stay.

Management of genetic data needs the fulfillment of technical requirements, involving ethics, law, and social issues. Introduction of genetic information in preventive medicine implies complex consequences and specialized expertise. Genetic screenings represent a promising tool, but further advances are needed to provide their effective and appropriate use in the respect of the privacy. Application areas, tests validation procedures and availability of effective laboratory and qualified service networks are considered. The Authors present main laws and bioethics guidelines in the field.

A multicentric Italian investigation on legionnaires' disease is in course to clarify host factors as well as pathogen associated characteristics involved in the infection/disease. The main goal of the research plan is to account for some critical aspects concerning identification and prevention of legionellosis. To improve knowledge on factors associated with Legionella spp colonisation in hot waters, to detect cases and to characterize risk factors in subjects which develop pneumonia are specific objectives of the research programme. Preliminary results show that hot waters of houses and hotels are frequently contaminated (22.6% and 54.6%, respectively), mainly by L. pneumophila. Microbial concentrations were low in domestic waters (<1.000 ufc/l), but higher in samples from the hotels (geom. mean 1.85 x 10(3) ufc/l). Warming system, age of the plant, type of building were risk factors significantly associated with Legionella spp positivity. The active surveillance on patients affected by pneumonia with search for Legionella urinary antigen allowed the identification of 34 cases, 3 of which of nosocomial origin, corresponding to 4.2% of the screened pneumonia. After informed consent, 26 subjects were recruited for a case-control-study to clarify risk factors for the disease.

Ets genes are transcription factors expressed in different tissues. Biochemical pathways interfering with the intracellular calcium ion concentration are known to influence members of this gene family. Considering experimental observations and previous reports, we proposed a hypothetical model of interaction between EMF and Ets, based on possible interference in pathways involving calcium as a second messenger. This approach comprehend different hypothesis and may contribute to clarify EMF biological effects, providing an experimental model and a procedure based on gene expression analysis. Additional knowledge on biological mechanisms is required to support these promising results. Further experiments are in progress to assess the role of EMF in modulating gene expression and its biological meaning. Identification of early endpoints is needed for mechanism understanding and development of sensitive biomarkers for molecular epidemiology studies.

The authors report the early results of a case-control study carried out about the risk of legionellosis. During the first year, they have tested urine samples from patients with unknown pneumonia, using a legionella-sensitive test for the detection of its antigen. Out of 171 samples, 9 have turned out to be positive (5.3%). Lifestyles, predisposing factors, possible sources of infection and stressful events were investigated.

The relationship between Legionella and Pseudomonas spp contamination and mineral content of domestic hot waters was investigated in a cross-sectional Italian survey. Pseudomonas spp contamination was associated with elevated Ca, but lower Fe and free chlorine content in the water. Waters with Cu concentrations > 50 microg/L prevented Legionella spp colonisation (OR = 0.14, 95% CI = 0.02-1.13), whereas the risk of legionellae occurrence increased in waters with Mn levels > 3 microg/L (OR = 2.37, 95% CI = 1.06-5.30). Furthermore, Mn was positively associated with the risk of colonisation by eterotrophic bacteria growing at either 22 and 36 degrees C. Legionella species and serogroups were differently distributed according to Cu and Mn concentrations, suggesting that Legionella strains may have a different sensibility/resistance to trace elements. A specific action of Cu as decontamination factor is suggested and the consideration of Mn as a risk indicator for bacterial colonisation and biofilm presence is proposed.

Over the past 20 years, the interest of the scientific community was increasingly placed in the field of genetic epidemiology and molecular genetics of blood pressure control. This paper explores references related to essential hypertension, gene and genetic epidemiology indexed in the MedLine health science database during the period 1980-2001. A systematic literature search was performed using selected keywords, such as 'genetic', 'genome' or a combination of words. We considered the study heading and evaluated the time profile of published articles. A total number of 3116 publications was collected and analyzed. Allelic distribution for the most studied polymorphisms of the renin-angiotensin system in different world populations was reviewed and reported together with a detection of their frequency in Italy: essential hypertensive patients (n = 90), healthy unrelated subjects (n = 300). Molecular variants at angiotensinogen (M and T), angiotensin II type 1 receptor (A and C) and angiotensin-converting enzyme (D and I) genes were analyzed by amplified fragment length polymorphism. A significant association was detected by chi2 analysis for angiotensinogen and angiotensin II-type I receptor allele distribution in hypertensive patients, in accordance with previous reports. Genetic data and methods are contributing more and more to epidemiological studies of complex diseases, and their application is influenced by information availability and Genome Project results.

Left ventricular hypertrophy in patients with hypertension is a main clinical prognostic entity The aim of this study was to evaluate the association between mutations at genes of the renin-angiotensin system (RAS) and the development of left ventricular hypertrophy. Genetic polymorphism in angiotensinogen (AGT) and angiotensin Il-type 1 receptor (AT1R) genes was examined in a group of well-selected essential hypertensive caucasians with left ventricular involvement (n = 40) and a group of healthy unrelated caucasians (n = 150). Cardiac morphology and function were assessed by M-mode echocardiography. Molecular variants were analysed by amplified fragment length polymorphism. We observed a statistically significant difference both for AGT and AT1R genotype distribution in patients with left ventricular hypertrophy compared with controls (p<0.05). A 0.49 and 0.225 frequency was detected among cases for T and C mutant alleles at AGT and AT1R genes. Mutations in RAS genes are involved in the pathophysiology of target-organ damage in essential hypertension. Evaluation of molecular factors conferring a risk of developing heart involvement may lead to better identification of patient subgroups and more effective control of the clinical course.

We have previously shown that electromagnetic field (EMF) exposure induces ETS1 oncogene overexpression in different cell lines. In order to investigate in vivo EMF effects, BALB/c mice were exposed at different times to 50 MHz radiation, modulated (80%) at 16 Hz. The exposed and control animals were sacrificed and the spleen excised for rt-pcr and western blot analysis. We observed an increase in ETS1 mRNA and protein expression, but a decrease in ETS2 protein levels. Preliminary results from this experimental model show in vivo evidence of the effect of EMF on ETS oncogene expression.

Background: Blood pressure is a quantitative multifactorial trait influenced by environmental and genetic determinants. Although several candidate genes have been associated with the development of essential hypertension, the mechanisms of individual susceptibility still remain unclear. Knowledge on the distribution of genetic polymorphisms in different populations is fundamental for the assessment of the predictive value of genetic variation., Methods: We genotyped 300 healthy normotensive subjects from the Italian population for three polymorphisms, at the angiotensinogen (AGT, M and T), angiotensin II type 1 receptor (ATIR, A and C) and angiotensin-converting enzyme (ACE, D and I) genes. Polymorphisms were analyzed by polymerase chain reaction and restriction enzyme digestion. Statistical analysis was performed to verify the agreement with the Hardy-Weinberg equilibrium., Results: The observed allelic distribution was in accordance with estimates reported for Caucasian populations. Variant allelic frequencies were 0.36 for the T and C alleles at the AGT andAT1R locus and 0.47 for the I allele of the ACE gene. AT1R and ACE genotype frequencies were in Hardy-Weinberg equilibrium, while there was a deviation of the AGT genotypes from those predicted by the equation., Conclusions: The studied polymorphisms are largely distributed in the Italian population sample, with a frequency of homozygous subjects for mutant alleles ranging from 9 to 22%. Epidemiology of mutations in the genes involved in blood pressure regulation provides tools to evaluate susceptibility to hypertension.

Background/aims: Several epidemiological studies have shown the existence of other routes of transmission of the hepatitis C virus besides the parenteral one, but the mechanisms involved are not yet understood. The general aim of this study was to evaluate the prevalence of hepatitis C virus infection in family contacts of infected patients and to analyze the possible risk factors and alternative transmission routes., Methodology: One hundred and thirty-eight relatives of 45 patients (index cases) affected by C virus-related chronic hepatitis were studied. The relatives were 45 spouses, 89 children and 4 cohabitants who underwent detection of serum anti-HCV antibodies; the anti-HCV-positive subjects were tested for serum HCV-RNA. The index cases, all the spouses and only other infected relatives were tested for the presence of HCV-RNA in saliva, Results: Antibodies to hepatitis C virus were detected in 5.7% of the family members while 11.1% of the analyzed spouses were serum HCV-RNA-positive. HCV-RNA was found in 44% of the examined saliva and 39% of these were found serum HCV-RNA-negative. The prevalence of hepatitis C virus among household contacts, excluding cases with previous parenteral exposure, was 3.6%., Conclusions: The epidemiological data on the intrafamilial spread of hepatitis C virus may be underestimated owing to the existence of infected relatives serum-negative but saliva-positive for the presence of the virus. The whole of these observations suggests a possible role of biological fluids in intrafamilial spread of hepatitis C virus.

The use of antiblastic drugs has opened up new perspectives in improvement of therapy and life quality for cancer patients. The widespread clinical application of cytostatic drugs implies risks for exposed hospital personnel, due to genotoxic and toxic-reproductive effects. Biological monitoring is fundamental to identify individuals at risk but is limited by the long latency of chronic effects, absence of unique cellular targets and low sensitivity of available laboratory tests. The objective of this study was to investigate toxic mechanisms by a molecular biology approach, searching for biomarkers potentially useful in monitoring programs. The proposed experimental model consisted of cell line exposure to cyclophosphamide, an alkylating agent of wide clinical use. Cellular response has been investigated focusing on potential targets at RNA level, through reverse transcription polymerase chain reaction (RT-PCR) and differential display analysis. We studied the expression of several genes involved in differentiation, apoptosis and chemoresistance: ets1, bax, bcl-2, bag-1, bcl-X, mdr1 and mrp. Specific patterns of mRNA modulations were observed. Differential display analysis revealed candidate genes induced or repressed following exposure: their characterization is in progress. Besides improving the understanding of toxic mechanisms, identification of modulated molecular targets opens up new perspectives in exposure risk assessment, biomonitoring and preventive strategies at occupational level.

In hypertension both beta-blockers and calcium antagonists are drugs with proved efficacy. Because only half the patients respond to a single drug, even at full dosage, a second hypotensive agent is frequently required to obtain adequate blood pressure control. The combination of a dihydropyridine calcium antagonist and a beta-blocker can be justified by their different mechanisms of action. A randomised double blind parallel group study versus placebo was performed, in order to assess the efficacy of atenolol combined with amlodipine in the treatment of stage I-II essential hypertension not controlled by atenolol alone. Twenty-four-hour arterial blood pressure monitoring showed that amlodipine added to atenolol produced a statistically significant reduction of blood pressure values compared with placebo in patients whose blood pressure was not controlled by atenolol alone. Blood pressure circadian rhythm was unchanged. The reduction of side-effects, obtained by adding a dihydropyridine derivate to a beta-blocker, confirms the effectiveness of this combination.

We characterized a new human ATP-binding cassette (ABC) transporter gene that is highly expressed in the placenta. The gene, ABCP, produces two transcripts that differ at the 5' end and encode the same 655-amino acid protein. The predicted protein is closely related to the Drosophila white and yeast ADP1 genes and is a member of a subfamily that includes several multidrug resistance transporters. ABCP, white, and ADP1 all have a single ATP-binding domain at the NH2 terminus and a single COOH-terminal set of transmembrane segments. ABCP maps to human chromosome 4q22, between the markers D4S2462 and D4S1557, and the murine gene (Abcp) is located on chromosome 6 28-29 cM from the centromere. ABCP defines a new syntenic segment between human chromosome 4 and mouse chromosome 6. The abundant expression of this gene in the placenta suggests that the protein product has an important role in transport of specific molecule(s) into or out of this tissue.

Using differential display method, we have isolated and characterized a novel gene, N8, encoding an approximately 24 kDa protein. It is located on human chromosome 8q13 region. N8 gene is expressed at high levels in tumor derived cell lines from multiple cancers. It is also expressed at higher levels in lung tumors than normal lung tissue. N8 is also differentially expressed in fetal and adult tissues. In adult, N8 is expressed at high levels in brain, kidney, prostate, pancreas and intestine and at very low levels in lung, liver, hematopoietic cells and gonads. During murine embryonic development N8 is expressed in the epithelium of the intestine, stomach, olfactory epithelium, neuronal layers of retina, kidney and salivary gland. Taken together, these results suggest that N8 may play different roles during embryogenesis and in the adult animals.

The ETS1 gene encodes a sequence-specific transcription factor binding to purine-rich DNA sequences (-GGAA-) present in the transcriptional regulatory regions of many cellular and viral promoters/enhancers, including many lymphokine genes. The ETS1 gene is expressed at high levels in resting T cells and at very low levels after T cell activation, suggesting it may suppress the expression of genes induced during T cell activation. To find out if ETS1 regulates expression of the IL-2 gene, we have ectopically expressed antisense (AS) ETS1 in Jurkat T cells to block the formation of ETS1 proteins. AS ETS1 transfectants produce higher levels of IL-2 compared with sense ETS1 transfectants. Expression of ETS1 DNA binding domain in Jurkat T cells also decreased the production of IL-2. In AS ETS1 transfectants, IL-2 formation was completely inhibited by cyclosporin A and FK590. The IL-2 promoter linked to a chloramphenicol acetyl transferase reporter gene has high activity in AS ETS1 transfectants, indicating that increased IL-2 production seems to be a result of transcriptional induction. Taken together, these results suggest the possibility that ETS1 may act as a negative regulator of IL-2 gene transcription and provide a rational approach toward engineering the endogenous expression of IL-2 in T cells.

The epitope for E44 monoclonal antibody (mAb) was mapped using mutated ETS1 proteins lacking different carboxy-terminal regions and by the employment of synthetic oligopeptides spanning the epitope region. This epitope lies around Arg211 of the human ETS1 protein since substitution of Arg211 by Gln211 in the epitope region results in the loss of recognition of the mouse ETS1 protein by E44 mAb. Substitution of Leu214 by valine214 in the epitope region (as is found in the chicken ETS1 and viral Ets proteins) does not alter the capacity of the E44 mAb to recognize this antigen. Taken together, these results suggest that a specific ionic interaction is able to play a pivotal role in the recognition of the ETS1 protein by the E44 mAb.

We studied the correlation between dexamethasone (Dex) induced growth effects and modulation of epidermal growth factor receptor (EGFR) expression in OVCA 433 ovarian cancer cells. These cells express specific high and low affinity 125I-EGF binding sites and are growth stimulated by EGF. Dex exhibits mitoinhibitory effects by recruiting OVCA 433 cells in the G0-G1 phase of the cycle, but increases the number of both the high and the low affinity EGFR in a dose dependent manner. The maximal EGFR expression increase occurs after 24 h of Dex treatment consistently with Northern blot studies. The mitogenic activity of EGF in OVCA 433 cells is not affected by the presence of Dex. Moreover Dex growth inhibition occurs in JA1 cells, an ovarian cancer cell line which expresses unfunctional EGFR and which is unresponsive to EGF. Our results indicate that the Dex induced growth effects occur independently of EGFR expression.