Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials.

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.

Multi-walled carbon nanotubes induce airway hyperresponsiveness in human bronchi by stimulating sensory C-fibers and increasing the release of neuronal acetylcholine.

OBJECTIVES: The potential of multi-walled carbon nanotubes (MWCNTs) in inducing airway hyperresponsiveness (AHR) was investigated in human airways. METHODS: Human isolated bronchi were exposed to MWCNTs and the contractility to electrical field stimulation (EFS) was measured. Neuronal acetylcholine (ACh) and cyclic adenosine monophosphate (cAMP) were quantified. Some tissues were desensitized by consecutive administrations of capsaicin. RESULTS: MWCNTs (100 ng/ml - 100 µg/ml) induced AHR (overall contractile tone vs. negative control: +83.43 ± 11.13%, P < 0.01). The potency was significantly (P < 0.05) greater when airways were stimulated at low frequency (EFS3Hz) then at medium-to-high frequencies (EFS10Hz and EFS25Hz) (delta potency: +2.13 ± 0.74 and +2.40 ± 0.65 logarithms, respectively). In capsaicin-desensitized airways, the AHR to MWCNTs 100 ng/ml was abolished. MWCNTs increased the release of ACh, an effect prevented by capsaicin-desensitization (-90.17 ± 18.59%, P < 0.05). MWCNTs did not alter the level of cAMP. CONCLUSION: MWCNTs administered at low concentrations elicit AHR in human airways by activating sensory C-fibers and, in turn, increasing the release of neuronal ACh. Our results suggest that work is required to understand the impact of MWCNTs in patients at risk of AHR, such as those suffering from chronic obstructive respiratory disorders.

Oxidative Stress Biomarkers in Urine of Metal Carpentry Workers Can Be Diagnostic for Occupational Exposure to Low Level of Welding Fumes from Associated Metals.

Urinary concentrations of 16 different exposure biomarkers to metals were determined at the beginning and at the end of a working shift on a group of workers in the metal carpentry industry. Five different oxidative stress biomarkers were also measured, such as the oxidation products of RNA and DNA metabolized and excreted in the urine. The results of workers exposed to metals were compared to those of a control group. The metal concentrations found in these workers were well below the occupational exposure limit values and exceeded the mean concentrations of the same metals in the urine of the control group by a factor of four at maximum. Barium (Ba), mercury (Hg), lead (Pb) and strontium (Sr) were correlated with the RNA oxidative stress biomarker, 8-oxo-7, 8-dihydroguanosine (8-oxoGuo), which was found able to discriminate exposed workers from controls with a high level of specificity and sensitivity. The power of this early diagnostic technique was assessed by means of the ROC curve. Ba, rubidium (Rb), Sr, tellurium (Te), and vanadium (V) were correlated with the level of the protein oxidation biomarker 3-Nitrotyrosine (3-NO2Tyr), and Ba, beryllium (Be), copper (Cu), and Rb with 5-methylcytidine (5-MeCyt), an epigenetic marker of RNA damage. These effect biomarkers can help in identifying those workers that can be defined as "occupationally exposed" even at low exposure levels, and they can provide information about the impact that such doses have on their health.

Cost-Effectiveness of Annual Screening for Tuberculosis among Italian Healthcare Workers: A Retrospective Study.

BACKGROUND: In the past few years, healthcare workers (HCWs) have been considered at higher risk for tuberculosis (TB) infection than the general population. On the other hand, recent studies have reported a low conversion rate among these workers. Recently, the Center for Disease Control (CDC) updated its recommendations, suggesting that an annual screening should not be performed in the absence of a documented exposure but only in workers with high-risk duties or with job tasks in settings at high risk of tuberculosis contagion (e.g., departments of infectious or pulmonary diseases). In fact, some studies showed that annual tuberculosis screening for all the HCWs was not cost-effective in countries with a low incidence of TB. In this study, we evaluated the conversion rate and the cost-effectiveness of two different tuberculosis screening strategies in a large population of Italian HCWs. METHODS: In our retrospective study, we reviewed data coming from a tuberculosis screening conducted on 1451 HCWs in a teaching hospital of Rome. All workers were evaluated annually by means of the Quantiferon test (QFT) for a five-year period. Then, the conversion rate was calculated. RESULTS: We found a cumulative conversion rate of 0.6%. Considering the cost of the QFT test (48.26 euros per person), the screening of the HCWs resulted in a high financial burden (38,902.90 euros per seroconversion). Only one seroconversion would have been missed by applying the CDC updated recommendations, with a relevant drop of the costs: 6756.40 euros per seroconversion, with a global save of 296,075.10 euros. CONCLUSION: The risk of TB conversion among our study population was extremely low and it was related to the risk classification of the setting. Giving these results, the annual tuberculosis screening appeared to not be cost effective. We conclude that a targeted screening would be a better alternative in HCWs with a higher risk of TB exposure.

Length-dependent toxicity of TiO2 nanofibers: mitigation via shortening.

Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO2 nanofibers (TiO2 NF) cause a dose-dependent decrease of cell viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO2 NF of industrial origin. Long TiO2 NF (L-TiO2 NF) were more cytotoxic than their shortened counterparts (S-TiO2 NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO2 NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO2 NF, S-TiO2 NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO2 NF but not to S-TiO2 NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO2 NF toxic effects.

Night Shift Working Is Associated With an Increased Risk of Thyroid Nodules.

OBJECTIVES: To study thyroid alterations in health care workers according to their working status. METHODS: We performed a retrospective study including 299 hospital employers who underwent in 2016 a periodic health surveillance checks in the Service of Occupational Medicine. According to the working status (rotating night-shift working [no. 160] vs day-working [no. 139]), we divided participant's clinical, anthropometric, and thyroid echographic characteristics. RESULTS: Respect to day workers, rotating night-shift workers were slightly older and more frequently male whereas had similar thyroid stimulating hormone, Ft3, Ft4 levels, and autoimmunity (anti-TPO levels more than 30). Univariate and multivariate regression analysis revealed that rotating night shift work is associated to a significantly increased number of thyroid nodules. CONCLUSIONS: This retrospective report suggests that the alteration in the molecular clocks typical of rotating night-shift workers harbors a higher risk of thyroid nodule development compared with diurnal workers. This novel result deserves replication in larger cohorts since thyroid nodules not rarely can represent thyroid cancers.

A novel approach for geographical risk mapping of morbidity and mortality rates: the case of Val D'Agri, Italy.

While associations between exposure to air pollutants and increased morbidity and mortality are well established, few rigorous studies on this issue are available. The aim of the current study is to implement a new approach to the spatial analysis of mortality and morbidity, based on testing for the presence of the same association in other areas of similar size. Additionally, we perform a case study in Val d'Agri (VA), an area of Basilicata Region, Southern Italy, where oil and natural gas extraction began in 1998. In order to examine the spatial distribution of morbidity and mortality in the region of interest, Hospital discharge (2001-2013) and mortality (2003-2014) rates for the main environment-related diseases were calculated. In addition, a comparison between the period 1980-1998 and the period 1999-2014 was performed for cardiovascular disease mortality. For the period under study, a neutral scenario emerged for cancer and respiratory diseases, where we found no differences in morbidity and mortality as compared to the national benchmark. In some cases significantly lower values (as compared to the nation-wide benchmark) were found. Conversely, a slight excess in morbidity and mortality (as compared to the nation-wide benchmark) emerged for cardiovascular diseases. Still, this excess was common to a number of municipalities in the surroundings of VA, and appeared to be already present in 1980. Higher rates of cardiovascular diseases, lower rates of neoplastic disorders no differences in mortality for respiratory causes (as compared to the nation-wide benchmark) were found in multiple areas of the region, and were therefore not specific to VA. In summary, our data do not support the hypothesis of a role of industrial activities related to oil extraction in VA in determining mortality and morbidity patterns and trends.

Risk of radiation-induced lens opacities among surgeons and interventional medical staff.

The main effect of ionizing radiation on the eyes is the onset of posterior cortical and subcapsular cataracts. Recent studies have raised questions about the mechanism of ocular damage and the threshold dose for the onset of such effects. Currently, operators may be exposed to ionizing radiation during surgical procedures. It has been estimated that urologists can be exposed to an annual dose close to or above 20 mSv/year. The aim of our study was to evaluate the frequency of cataracts in a group of professional radiological operators to verify their possible association with the radiation dose to the crystalline lens and the tasks performed. The records of 73 health workers exposed to ionizing radiation were reviewed. The average annual dose to the crystalline lens, the number of years of exposure, and the presence of radiation-compatible opacities were assessed for all operators. Lenticular opacities were observed in 16.4% of subjects. The presence of alterations was associated with exposure doses below 10 mSv and > 10 years' experience in fluoroscopically guided procedures. Based on our results, protection of the crystalline lens against exposure to ionizing radiation by means of goggles is recommended. In addition, examination of the lens via slit lamp examination is recommended for all operators involved in interventional procedures with the current levels of radiation exposure.

Case Report: Occupational therapy in a patient with an overgrowth syndrome that restricts movement.

Background: Overgrowth syndromes are a heterogeneous group of conditions characterized by excessive body growth - localized or generalized - commonly associated with various malformities and an increased oncological risk. Case report: Here we present the case of a 57-year old man, employed in an office, who suffers from an asymmetric overgrowth of the lower limbs. Currently the patient presents malformations of the lower left arm (hip, knee and ankle), evident on the articular and periarticular level, where there are diffuse exostoses. This case discusses the main occupational concerns relating to the patient's workspace at a high floor level that could create critical issues in the event of an emergency exodus. Given the impossibility of placing the patient in heavy manual activities, employment is limited to office activities. Adjustments were carried out at the patient's workstation, and thus the patient has been recognized as fit to work. Increased frequency of breaks were prescribed in order to allow the physiological alternation of postures. Conclusions: In cases of overgrowth syndromes, the exact identification of the limitations presented by the patient and observations about ambulatory functions must be carefully evaluated in order to modulate the work environment.

Nanomaterial exposure, toxicity, and impact on human health.

The use of engineered nanomaterials (ENM) has grown after the turn of the 21st century. Also, the production of ENM has globally grown, and exposure of workers especially via the lungs to ENM has increased. This review tackles with effects of ENM on workers' health because occupational environment is the main source of exposure to ENM. Assessment of exposure to ENM is demanding, and today there are no occupational exposure level (OEL) for ENM. This is partly due to challenges of such measurements, and in part to the unknown causality between ENM metrics and effects. There are also marked gaps in systematic knowledge on ENM hazards. Human health surveys of exposed workers, or human field studies have not identified specific effects of ENM linking them with a specific exposure. There is, however, a consensus that material characteristics such as size, and chemistry influence effects of ENM. Available data suggest that multiwalled carbon nanotubes (MWCNT) affect the immunological system and cause inflammation of the lungs, or signs of asthma whereas carbon nanofibers (CNF) may cause interstitial fibrosis. Metallic and metal oxide nanoparticles together with MWCNT induce genotoxicity, and a given type of MWCNT has been identified as a possible human carcinogen. Currently, lack of understanding of mechanisms of effects of ENM renders assessment of hazards and risks of ENM material-by-material a necessity. The so called "omics" approaches utilizing ENM-induced alterations in gene and protein expression may be useful in the development of a new paradigm for ENM hazard and risk assessment. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 signalling pathway.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-ß/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-ß-mediated signalling pathway. METHODS: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-ß production. Experiments with neutralizing anti-TGF-ß antibody, specific inhibitors of GSK-3ß and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test. RESULTS: Fully characterized MWCNT (mean length < 5 µm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-ß secretion, Akt activation and GSK-3ß inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear ß-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure. CONCLUSIONS: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-ß and its intracellular effectors Akt/GSK-3ß that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

[Risk of tuberculosis in healthcare workers: risk assessment and medical surveillance]. / Rischio tubercolosi in ambiente sanitario. Valutazione del rischio e sorveglianza sanitaria.

Tuberculosis screening is recommended for all health care workers. We evaluated the prevalence of latent tuberculosis infection among 939 hospital workers of Tor Vergata University teaching hospital in Rome, Italy, in the period 2007-2013, by using the QuantiFERON Gold In-Tube (QFT) test. The mean age of subjects tested was 31 years. The prevalence of positive subjects (cut-off 0.35 UI/ml) was 5.5% (46/939) and the mean age of those who tested positive was 39 years. The low rate of positivity may be partly related to the higher reliability of QFT in comparison to tuberculin skin testing.

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy.

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

Targeted nanodrugs for cancer therapy: prospects and challenges.

The recent advent of nanomedicine holds potential to revolutionize cancer therapy. This innovative discipline has paved the way for the emergence of a new class of drugs based on nanoengineered particles. These "nanodrugs" are designed to greatly enhance drug therapeutic indices. First-generation nanodrugs consisted of conventional anti-cancer drugs loaded into/onto nanoengineered particles (nanocarriers) devoid of targeting features (non-targeted nanodrugs). Non-targeted nanodrugs have provided the opportunity to carry large amounts of drugs, including poorly water-soluble and/or permeable drugs, to several types of tumors, improving the therapeutic index with respect to comparable free drugs. Although effective, the primary delivery mechanism of non-targeted nanodrugs was through passive tissue accumulation, due to pathophysiological differences between tumor-associated and healthy vessels, and through non-specific targeting of cell subsets, posing the danger of off-target binding and effects. Recently, the therapeutic indices of certain anti-cancer drugs were further improved by attaching targeting ligands to nanodrugs (targeted-nanodrugs). Targeted-nanodrugs selectively bind to cognate receptors expressed on target cells and enter cells more efficiently than non-targeted formulations. Although these advancements have been sufficiently beneficial to place targeted-nanodrugs into clinical development for use in cancer therapy, they also come at a price. The addition of ligands to drug-loaded nanocarriers often leads to additional synthesis steps and costs, and more complex biological performance relative to ligand-devoid nanodrugs. Here, we will discuss the benefits and challenges facing the addition of targeting features to nanodrugs for cancer therapy.

Chronic obstructive pulmonary disease (COPD) patients with osteoporotic vertebral compression fractures (OVCFs): improvement of pulmonary function after percutaneous vertebroplasty (VTP).

OBJECTIVES: To investigate the changes of respiratory function in patients affected by chronic obstructive pulmonary disease (COPD) with single dorsal osteoporotic vertebral compression fractures (OVCFs) treated with vertebroplasty (VTP). METHODS: Forty-five patients affected by COPD and single dorsal OVCF underwent VTP (29 men, 16 women; mean age 71.4 years, range 65-77 years). Inclusion criteria were magnetic resonance findings of bone marrow oedema, without intracanal bone fragments and refractory pain to medical treatment for at least 3 months. Osteoporosis was assessed by bone densitometry. Spirometry was performed before and after treatment. RESULTS: A significant VAS-score decrease was observed 1 week after VTP, with a subsequent decrease over time; vital capacity (VC) and forced vital capacity (FVC) improved over time, reaching a plateau at 3 months. Forced expiratory volume at 1 s (FEV1) did not significantly differ between the pre-VTP values and follow-up values. A significant correlation was observed between VAS-score values and VC, and VAS-score values and FVC. No significant correlation was observed between VAS-score values and FEV1 values. CONCLUSIONS: VTP improves restrictive ventilatory impairment in patients with moderate and severe COPD affected by single thoracic OVCFs. We recommend this treatment in the management of these patients. KEY POINTS: • Osteoporosis is a major comorbidity in chronic obstructive pulmonary disease (COPD) patients. • Pain due to osteoporotic vertebral compression fractures worsens respiratory failure in COPD. • Vertebroplasty improves ventilatory impairment in COPD patients with osteoporotic vertebral compression fractures.

The interaction of ACP1, ADA1, diabetes and gender in coronary artery disease.

INTRODUCTION: Previous separate studies have shown associations of coronary artery disease (CAD) with acid phosphatase locus 1 (ACP1) and adenosine deaminase locus 1 (ADA1) genetic polymorphisms. Because it is known that the 2 systems interact and have important immunologic and metabolic functions, these 2 genes were both examined in the same sets of subjects. METHOD: Two-hundred forty subjects with CAD, 156 subjects with cardiovascular diseases without CAD, 279 subjects with Non Insulin Dependent Diabetes Mellitus (NIDDM) without CAD and 771 consecutive healthy newborn infants have been studied. RESULTS: The association of ACP1 and ADA1 with CAD depends on sex and diabetes. In particular, the association between ADA1 and CAD is present in nondiabetic subjects only, and it is dependent on sex (males), whereas the association of CAD with ACP1 is present in diabetic subjects only, and it is dependent on sex (females). CONCLUSIONS: The fact that the association of ACP1 with CAD is evident only in diabetic subjects, whereas the association of ADA1 with CAD is evident only in nondiabetic subjects suggests an heterogeneity in the pathogenetic mechanisms leading to CAD. In addition, the association with sex that could be based on hormonal differences is in favor of heterogenity.

Isolated H. pylori duodenal colonization and idiopathic duodenal ulcers.

OBJECTIVE: To evaluate whether some duodenal ulcers (DU) classified as idiopathic according to standard criteria may be causally related to isolated duodenal colonization by H. pylori. METHODS: We studied consecutive ambulatory patients undergoing upper gastrointestinal endoscopy in a secondary care setting. Gastric and duodenal biopsies for diagnosing H. pylori infection were taken from all patients. Independently from the findings of duodenal biopsies, DU patients without gastric infection were classified as having idiopathic ulcers, and underwent urea C13 breath test and subsequent eradication therapy. Endoscopy was repeated 6 months after eradication treatment. RESULTS: Among 608 DU patients, 42 (6.9%) were classified as idiopathic: 24 (3.9%) were free from gastric and duodenal infection (group A) and 18 (3.0%) (group B) had isolated duodenal colonization. Urea C13 breath test was positive in one (4.2%) group A patient and in 3 (16.7%) group B patients. After eradication therapy, DU were detected in 14 out of 20 group A patients (70%) (four patients did not perform control endoscopy) and in 2 group B patients (11.1%): OR 18.66, 95% CI 3.23-107.82, P= 0.002. The difference was still detectable after multivariate analysis taking into account possible confounding factors: OR 15.79, 95% CI 2.48-100.53, P= 0.001. CONCLUSIONS: Isolated duodenal colonization by H. pylori is detectable in a substantial proportion of patients with so-called idiopathic DU, and eradication therapy is effective in these patients.

Helicobacter pylori and extragastric diseases--other Helicobacters.

The involvement of Helicobacter pylori in the pathogenesis of extragastric diseases continues to be an interesting topic in the field of Helicobacter-related pathology. Although conflicting findings have been reported for most of the disorders, a role of H. pylori seems to be important especially for the development of cardiovascular and hematologic disorders. Previously isolated human and animal Helicobacter sp. flexispira and "Helicobacter heilmannii" strains have been validated using polyphasic taxonomy. A novel enterohepatic Helicobacter has been isolated from mastomys and mice, adding to the list of helicobacters that colonize the liver. Genetic targets that may aid the classification of novel Helicobacter species have emerged. Animal models of Helicobacter-induced gastric and hepatobiliary diseases have offered insights to the mechanisms associated with premalignant transformation.

New site of modification of 23S rRNA associated with clarithromycin resistance of Helicobacter pylori clinical isolates.

Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 micro g/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.