Giuseppe De Palma

Exhaled breath condensate as a suitable matrix to assess lung dose and effects in workers exposed to cobalt and tungsten.

The aim of the present study was to investigate whether exhaled breath condensate (EBC), a fluid formed by cooling exhaled air, can be used as a suitable matrix to assess target tissue dose and effects of inhaled cobalt and tungsten, using EBC malondialdehyde (MDA) as a biomarker of pulmonary oxidative stress. Thirty-three workers exposed to Co and W in workshops producing either diamond tools or hard-metal mechanical parts participated in this study. Two EBC and urinary samples were collected: one before and one at the end of the work shift. Controls were selected among nonexposed workers. Co, W, and MDA in EBC were analyzed with analytical methods based on mass spectrometric reference techniques. In the EBC from controls, Co was detectable at ultratrace levels, whereas W was undetectable. In exposed workers, EBC Co ranged from a few to several hundred nanomoles per liter. Corresponding W levels ranged from undetectable to several tens of nanomoles per liter. A parallel trend was observed for much higher urinary levels. Both Co and W in biological media were higher at the end of the work shift in comparison with preexposure values. In EBC, MDA levels were increased depending on Co concentration and were enhanced by coexposure to W. Such a correlation between EBC MDA and both Co and W levels was not observed with urinary concentration of either element. These results suggest the potential usefulness of EBC to complete and integrate biomonitoring and health surveillance procedures among workers exposed to mixtures of transition elements and hard metals.

CBX7 is a tumor suppressor in mice and humans

The CBX7 gene encodes a polycomb group protein that is known to be downregulated in many types of human cancers, although the role of this protein in carcinogenesis remains unclear. To shed light on this issue, we generated mice null for Cbx7. Mouse embryonic fibroblasts derived from these mice had a higher growth rate and reduced susceptibility to senescence compared with their WT counterparts. This was associated with upregulated expression of multiple cell cycle components, including cyclin E, which is known to play a key role in lung carcinogenesis in humans. Adult Cbx7-KO mice developed liver and lung adenomas and carcinomas. In in vivo and in vitro experiments, we demonstrated that CBX7 bound to the CCNE1 promoter in a complex that included HDAC2 and negatively regulated CCNE1 expression. Finally, we found that the lack of CBX7 protein expression in human lung carcinomas correlated with CCNE1 overexpression. These data suggest that CBX7 is a tumor suppressor and that its loss plays a key role in the pathogenesis of cancer.

Case-control study of interactions between genetic and environmental factors in Parkinson's disease

Several environmental risk factors and some allelic variants of polymorphic drug-metabolising enzymes have been associated with sporadic Parkinson’s disease. No study has to date explored the possible interaction between individual susceptibility and exposure to chemical pollutants. We genotyped enzymes expressed by the human brain and involved in oxidative stress through the generation of free radicals (phase I enzymes) or involved in scavenging (phase II enzymes). Candidate genes were characterised by a genetic polymorphism that leads to lessened or abolished enzyme activity, and by fixed expression or homozygous allelic deletion (a theoretical correspondence between genotype and phenotype). Cytochrome P-450 2D6 (CYP2D6) is a non-inducible phase I enzyme involved in the biotransformation of various chemicals, including tetrahydroisoquinolines. Alleles CYP2D6*3 and CYP2D6*4 account for about 90% of the poor metaboliser autosomal recessive disorder, which has been associated with Parkinson’s disease. A meta-analysis of available studies showed an overall risk of borderline significance. Glutathione S-transferases (GSTs) are inducible phase II enzymes involved in the scavenging of many electrophilic reactive intermediates. Homozygous deletion of GSTM1 and GSTT1 loci affects, respectively, about 50% and 25% of white people (genotypes GSTM1*0 and GSTT1*0). We recruited 100 consecutive outpatients at the Institute of Neurology, University of Parma, with Parkinson’s disease (59 men, 41 women) aged 66·6 (SD 9·7) years, fulfilling the diagnostic criteria established by the UK Parkinson’s Disease Society Brain Bank. The mean age at the onset of Parkinson’s disease was 58·6 (9·7) years (mean duration of the disease 7·9 [4·6] years) and 15% of patients had a positive family history of the disease. We enrolled 200 controls (118 men, 82 women) aged 64·2 (9·2) years from outpatient specialist centres (nephrology clinic and taken on the same day as her baby’s and tested in parallel with her previous sera remained VZV IgG antibody positive and VZV IgM antibody negative. A month later, in February 1997, at another hospital in Surrey, the day after the birth of a healthy baby girl at term, birth weight 4·2 kg, her 2 –year-old sister developed chickenpox. The mother had had chickenpox 7 years previously (aged 24 years) and this was confirmed serologically by testing her stored antenatal booking serum (16 weeks’ gestation) which was VZV IgG antibody positive and VZV IgM antibody negative. Serological tests were not done on the infant at this time. Mother and baby were discharged home 2 days after delivery, with the baby well and breastfeeding. At 16 days of age the baby developed chickenpox. On day 4 of her illness the baby was seen in hospital and started on oral acyclovir. The typical rash involved the face and head, with a few lesions on the trunk and limbs. Serum from the baby at this time was VZV IgG positive and VZV IgM negative. A swab from the chickenpox lesions yielded VZV from routine tissue culture. Again, no illness or rash occurred in the mother and a repeat serum tested in parallel with her previous serum remained VZV IgG antibody positive and VZV IgM antibody negative. Although VZV was not isolated from the first baby the diagnosis was not in doubt. The strength of IgG reactivity of the mother and baby serum samples in the assay used are shown in the table. The first mother gave a test/positive control net absorbance ratio (T/P) of 1·2 (cutoff 0·8) for both early sera while that of the baby on day 4 of illness was significantly less at 0·46 (equivocal level). The 17 day postdelivery sample of the mother showed a boosted IgG response. The T/P ratios of the second mother/baby case were almost identical for the mother’s booking serum and the baby’s blood at 2·2 and 2·1, respectively—ie, both more than twice the reactivity of the positive control. In both babies the VZV IgG antibody detected (both at 4 days of illness) was likely to be entirely maternal and not their own. It is generally accepted that passively acquired maternal antibody protects neonates from varicella even in low-birthweight infants with neonatal titres of VZV IgG antibody usually matching maternal levels. We can only speculate that the reduced IgG titre in the first baby compared with his mother—possibly a result of being bottle fed—was a factor in his apparently failed immunity and more florid rash. Cell mediated immunity (CMI) was not studied in either baby. Non-specific, non-antibody-dependent cellmediated mechanisms, such as natural killer cells, are known to have a role in controlling the extent of disease due to primary varicella but cannot necessarily prevent infection in the first place. Although our two cases were of moderate severity, and both were treated with acyclovir, a defect in CMI might have been expected to have caused serious protracted disease which did not occur. There have been two previous reports of neonatal

Predictors of gefitinib outcomes in advanced non-small cell lung cancer (NSCLC): study of a comprehensive panel of molecular markers.

A number of different clinical characteristics and molecular markers related to epidermal growth factor receptor (EGFR) activation have been reported to singly correlate with therapeutic activity of EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). This study was designed to evaluate the predictive value on gefitinib outcomes of a comprehensive panel of molecular parameters in advanced NSCLC patients. EGFR and K-ras mutations were detected by direct sequencing on tumor DNA from paraffin embedded samples. EGFR and HER2 gene copy number was assessed by FISH. EGFR protein expression was quantified by immunohistochemistry. EGFR gene intron 1 polymorphism was assessed on genomic DNA isolated from venous whole blood samples. Ninety-one patients were prospectively enrolled and the overall gefitinib response rate was 18.7% (2 complete and 15 partial responses). Sex (p=0.005), non-smoking status (p=0.010), skin toxicity (p=0.020), EGFR gene mutations (p<0.001) and EGFR FISH positivity (p=0.016) were found to be associated with gefitinib response. K-ras mutation was detected in only seven non-responder patients. The median overall survival was of 10 months. Only non-smoking status and EGFR intron 1 polymorphism showed a statistically significant correlation with survival (p=0.031 and 0.044, respectively). In conclusion, we have confirmed the role of EGFR gene mutation as predictor of response to EGFR TKIs. Moreover, EGFR gene copy number and, potentially, also EGFR intron 1 polymorphism could aid in better prediction of EGFR TKI responsiveness in advanced NSCLC.

Biological monitoring of low benzene exposure in Italian traffic policemen.

A comparative evaluation of urinary biomarkers was carried out to characterize benzene exposure in a group of 100 traffic policemen of the city of Parma (Italy). All subjects were monitored once, in two consecutive days characterized by similar climatic conditions but preceded by two windy days. Benzene ambient concentration measured by municipal air monitoring stations was 1 microg/m(3) (Day 1) and 2 microg/m3 (Day 2). Personal exposure to ambient concentrations of benzene, toluene, ethylbenzene and xylene (BTEX) was assessed by using Radiello((R)) passive-diffusive samplers in a subgroup of 24 workers. Benzene metabolites, t,t-muconic acid (t,t-MA) and S-phenylmercapturic acid (S-PMA) were determined by isotopic dilution liquid chromatography-tandem mass spectrometry on spot urine samples collected at the end of the shift. Urinary benzene (U-B) was determined by solid-phase microextraction gas chromatography-mass spectrometry. Airborne benzene concentration expressed as median [and interquartile range] was 6.07 [0.28-9.53] microg/m(3), as assessed by personal sampling. Urinary concentrations of biomarkers in the whole group were 41.8 [34.1-89.8] microg/g creatinine for t,t-MA, 0.67 [0.23-1.32] microg/g creatinine for S-PMA, and 0.16 [0.13-0.26] microg/l for U-B. Smokers eliminated significantly higher concentrations of unchanged BTEX and benzene metabolites than non-smokers (p < 0.05). When traffic policemen were distinguished into indoor (n=31) and outdoor workers, no significant differences were observed for either airborne benzene or urinary biomarkers. Significantly lower concentrations of S-PMA and U-B were determined in samples collected at Day 1 as compared to Day 2 (p < 0.0001 and p = 0.003, respectively) suggesting that these biomarkers are enough sensitive and specific to detect changes in airborne benzene concentration even at few microg/m(3).

Triple negative breast cancer: looking for the missing link between biology and treatments

The so called “Triple Negative Breast Cancer” (TNBC) represents approximately 15-20% of breast cancers. This acronym simply means that the tumour does not express oestrogen receptor (ER) and progesterone receptor (PR) and does not exhibit amplification of the human epidermal growth factor receptor 2 (HER2) gene. Despite this unambiguous definition, TNBCs are an heterogeneous group of tumours with just one common clinical feature: a distinctly aggressive nature with higher rates of relapse and shorter overall survival in the metastatic setting compared with other subtypes of breast cancer. Because of the absence of well-defined molecular targets, cytotoxic chemotherapy is currently the only treatment option for TNBC. In the last decades, the use of more aggressive chemotherapy has produced a clear improvement of the prognosis in women with TNBC, but this approach results in an unacceptable deterioration in the quality of life, also if some support therapies try to relieve patients from distress. In addition, there is the general belief that it is impossible to further improve the prognosis of TNBC patients with chemotherapy alone. In view of that, there is a feverish search for new “clever drugs” able both to rescue chemo-resistant, and to reduce the burden of chemotherapy in chemo-responsive TNBC patients. A major obstacle to identifying actionable targets in TNBC is the vast disease heterogeneity both inter-tumour and intra-tumour and years of study have failed to demonstrate a single unifying alteration that is targetable in TNBC. TNBC is considered the subtype that best benefits from the neoadjuvant model, since the strong correlation between pathological Complete Response and long-term Disease-Free-Survival in these patients. In this review, we discuss the recent discoveries that have furthered our understanding of TNBC, with a focus on the subtyping of TNBC. We also explore the implications of these discoveries for future treatments and highlight the need for a completely different type of clinical trials.

Curcumin Inhibits Tumor Growth and Angiogenesis in an Orthotopic Mouse Model of Human Pancreatic Cancer

Pancreatic cancer is a malignant neoplasm originating from transformed cells arising in tissues forming the pancreas. The best chemotherapeutic agent used to treat pancreatic cancer is the gemcitabine. However, gemcitabine treatment is associated with many side effects. Thus novel strategies involving less toxic agents for treatment of pancreatic cancer are necessary. Curcumin is one such agent that inhibits the proliferation and angiogenesis of a wide variety of tumor cells, through the modulation of many cell signalling pathways. In this study, we investigated whether curcumin plays antitumor effects in MIA PaCa-2 cells. In vitro studies showed that curcumin inhibits the proliferation and enhances apoptosis of MIA PaCa-2 cells. To test whether the antitumor activity of curcumin is also observed in vivo, we generated an orthotopic mouse model of pancreatic cancer by injection of MIA PaCa-2 cells in nude mice. We placed mice on diet containing curcumin at 0.6% for 6 weeks. In these treated mice tumors were smaller with respect to controls and showed a downregulation of the transcription nuclear factor NF-κB and NF-κB-regulated gene products. Overall, our data indicate that curcumin has a great potential in treatment of human pancreatic cancer through the modulation of NF-κB pathway.

Creatine as a compatible osmolyte in muscle cells exposed to hypertonic stress.

Exposure of C2C12 muscle cells to hypertonic stress induced an increase in cell content of creatine transporter mRNA and of creatine transport activity, which peaked after about 24 h incubation at 0.45 osmol (kg H2O)−1. This induction of transport activity was prevented by addition of either cycloheximide, to inhibit protein synthesis, or of actinomycin D, to inhibit RNA synthesis. Creatine uptake by these cells is largely Na+ dependent and kinetic analysis revealed that its increase under hypertonic conditions resulted from an increase in Vmax of the Na+‐dependent component, with no significant change in the Km value of about 75 μmol l−1. Quantitative real‐time PCR revealed a more than threefold increase in the expression of creatine transporter mRNA in cells exposed to hypertonicity. Creatine supplementation significantly enhanced survival of C2C12 cells incubated under hypertonic conditions and its effect was similar to that obtained with the well known compatible osmolytes, betaine, taurine and myo‐inositol. This effect seemed not to be linked to the energy status of the C2C12 cells because hypertonic incubation caused a decrease in their ATP content, with or without the addition of creatine at 20 mmol l−1 to the medium. This induction of creatine transport activity by hypertonicity is not confined to muscle cells: a similar induction was shown in porcine endothelial cells.

B Cells Contribute to the Antitumor Activity of CpG-Oligodeoxynucleotide in a Mouse Model of Metastatic Lung Carcinoma

RATIONALE CpG-oligodeoxynucleotide (CpG-ODN; CpG), a Toll-like receptor-9 ligand, has been widely studied as a potential antitumor adjuvant. Toll-like receptor-9 is highly expressed on lung carcinoma tissues and some immune cells, such as plasmacytoid dendritic cells and B cells. OBJECTIVES The aim of our study was to elucidate the effect of CpG on B cells in a mouse model of lung carcinoma. METHODS C57Bl/6j, B cell-deficient, and Nude mice were intravenously implanted with the lung metastatic B16-F10 melanoma cells and killed 3 and 7 days after CpG administration. MEASUREMENTS AND MAIN RESULTS Administration of CpG increased lung tumor growth in B16-F10-implanted C57BL/6J mice. The genetic absence of B cells strongly facilitated CpG-induced tumor progression. In contrast, the adoptive transfer of CpG-activated B cells induced tumor arrest, associated with a reduced suppressive immune environment due to the lower recruitment of regulatory T cells, myeloid-derived suppressor cells, and CD8(+) regulatory T cells along with the reduced expression of suppressive cytokines such as IL-10 and transforming growth factor-β. Furthermore, concomitant with higher production of IFN-γ, the apoptosis rate in the lungs of mice adoptively transferred with CpG-activated B cells was increased. Depletion of mature CD20(+) B cells increased the lung tumor burden in CpG-treated C57BL/6J mice and nude mice. Moreover, nude mice had the same lung tumor burden as B cell-deficient mice when mature CD20(+) B cells were depleted. CONCLUSIONS Our data demonstrate the protective antitumor activity of CpG-activated B cells and shed light on CpG as an antitumor adjuvant for lung cancer therapy.

Lack of Correlation Between Metallic Elements Analyzed in Hair by ICP-MS and Autism

A cross-sectional case–control study was carried out to evaluate the concentrations of metallic elements in the hair of 44 children with diagnosis of autism and 61 age-balanced controls. Unadjusted comparisons showed higher concentrations of molybdenum, lithium and selenium in autistic children. Logistic regression analysis confirmed the role of risk factor for male gender and showed a slight association with molybdenum concentrations. Unconventional chelation and vitamin-mineral supplementation were ineffective on elemental hair concentrations. A meta-analysis including the present and previous similar studies excluded any association of autism with hair concentrations of mercury, cadmium, selenium, lithium and copper. A slight association was found for lead only, but it was very weak, as strictly dependent on the worst data from one study.