Stefano Caimi

Exosome Release and Low pH Belong to a Framework of Resistance of Human Melanoma Cells to Cisplatin

Intrinsic resistance to cytotoxic drugs has been a main issue in cancer therapy for decades. Microenvironmental acidity is a simple while highly efficient mechanism of chemoresistance, exploited through impairment of drug delivery. The latter is achieved by extracellular protonation and/or sequestration into acidic vesicles. This study investigates the importance of extracellular acidosis and nanovesicle (exosome) release in the resistance of human tumour cell to cisplatin (CisPt); in parallel to proton pump inhibitors (PPI) ability of interfering with these tumour cell features. The results showed that CisPt uptake by human tumour cells was markedly impaired by low pH conditions. Moreover, exosomes purified from supernatants of these cell cultures contained various amounts of CisPt, which correlated to the pH conditions of the culture medium. HPLC-Q-ICP-MS analysis revealed that exosome purified from tumour cell culture supernatants contained CisPt in its native form. PPI pre-treatment increased cellular uptake of CisPt, as compared to untreated cells, in an acidic-depend manner. Furthermore, it induced a clear inhibition of exosome release by tumour cells. Human tumours obtained from xenografts pretreated with PPI contained more CisPt as compared to tumours from xenografts treated with CisPt alone. Further analysis showed that in vivo PPI treatment induced a clear reduction in the plasmatic levels of tumour-derived exosomes which also contained lower level of CisPt. Altogether, these findings point to the identification of a double mechanism that human malignant melanoma use in resisting to a dreadful cellular poison such as cisplatin. This framework of resistance includes both low pH-dependent extracellular sequestration and an exosome-mediated elimination. Both mechanisms are markedly impaired by proton pump inhibition, leading to an increased CisPt-dependent cytotoxicity.

Exposure of rome city tram drivers to airborne platinum, rhodium, and palladium

Objective: To investigate the exposure of tram drivers of Rome to Pt, Rh, and Pd in total suspended particulate, PM10, and PM2.5 fractions. Methods: Sampling was performed on a tram running on about 15-km pathway characterized by a medium-high traffic car density. The analytical determination of metals was performed using inductively coupled plasma mass spectrometry. Results: Mean element values (in pg/m3) total suspended particulate, PM10, and PM2.5, were Pt, 20.3, 14.1, and 8.86, Rh, 3.99, 2.83, and 1.94, and Pd, 82.1, 58.0, and 15.2, respectively. Conclusions: Although the Pt, Rh and Pd levels in urban air were found to be still 8 to 11 orders of magnitude below the values thought to cause adverse effects on adult worker health, periodic environmental monitoring would be necessary to detect possible increments which could affect occupational exposure of tram drivers.

Planning and certification of new multielemental reference materials for research in antarctica

The adoption of the principles of Good Laboratory Practice (GLP) and the implementation of the ensuing procedures for quality assurance (QA) and quality control (QC) of analytical determinations are nowadays deemed essential to the achievement of sound, credible and comparable experimental information. This is certainly also applicable to research projects currently being carried out in the antarctic continent, in particular as regards investigations on global change phenomena. Worldwide investments made so far in this field do require in fact that reliability of data be optimized so as to allow for a harmonized assessment of ongoing trends. In this context the Italian National Programme for Research in Antarctica has recently launched a project for the preparation of new multielemental certified reference materials (CRMs) to be used for QC of analyses performed on environmentally significant materials. The suitability of CRMs to verify the accuracy of experimental measurements acknowledgedly depends, among others, on the degree of similarity of their matrix composition and analyte levels to those expected for real samples. From this standpoint three CRMs are planned, namely: i) marine sediment certified for Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sn and Zn; ii) ocean water certified for Cd, Cr, Cu, Fe, Hg, Mn, Ni and Pb; iii) krill certified for As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sn and Zn. Regarding the first two matrices, amounts of ca. 100 kg and ca. 1001, respectively, have been collected during the IX (1993–1994) antarctic expedition, while krill was taken in the course of the X (1994–1995) campaign. The pretreatment of sediment has been accomplished in terms of homogenization, stabilization and characterization of the preliminary ground mass. Approximately 500 bottles, each containing 75 g of material are now ready for distribution to interested laboratories involved in chemical measurements of antarctic matrices, whereas ocean water and krill will follow at a later stage. Finally, depending on circumstances, also the certification of organochlorine compounds in the same materials will be attempted.

Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model.

BackgroundThe HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated.MethodsE7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM). The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months.ResultsIn western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant.ConclusionsThis report shows that a particulate form of HPV16 E7 is able to induce, without adjuvant, an E7-specific tumour protection in C57BL/6 mice. The protective immunity is sustained by both humoral and cell-mediated immune responses. The E. coli-derived HPV16 E7 assembled in vitro into micro- and nanoparticles represents not only a good substrate for antigen-presenting cell uptake and processing, but also a cost-effective means for the production of a new generation of HPV subunit vaccines.

Release of Palladium from Biomechanical Prostheses in Body Fluids Can Induce or Support PD-Specific IFNγ T Cell Responses and the Clinical Setting of a Palladium Hypersensitivity

The increased use of Palladium (Pd) for biomedical applications, which has more than doubled in the last ten years, appears to be associated with an increased frequency of adverse reactions to Pd. The aim of this study is to investigate the relationship between the implant of a biomechanical apparatus containing Pd and the setting of a hypersensitivity to Pd by determining the levels of the metal released in biological fluids, assessing the effects of Pd on peripheral blood mononuclear cell (PBMC) cytokine production and exploring the clinical setting of skin sensitization. Of a total of 3,093 subjects examined in 2006, sensitization to Pd alone or in association with nickel (Ni) was observed in 1.6% and 13.03% of the individuals, respectively. Of these, a group of six subjects positive to Pd and negative to Ni at patch testing were selected on the basis of the oral clinical symptoms in order to measure both the levels of Pd in biological fluids and the degradation of the dental prostheses. Specific Pd measurements were carried out on salivary fluid, urine and serum samples by High Resolution Inductively Coupled Plasma-Mass Spectrometry. In addition, the degradation of the dental prostheses was assessed by both a “leaching test” and an analysis of the micro morphology of orthodontic prostheses. The induction of IFN-γ production by Pd was assessed in PBMC by the ELISpot assay. Skin sensitization to Pd was evaluated by patch testing and clinical examination. Ten healthy subjects were comparatively tested as controls. We found a specific induction of an IFN-γ response by Pd in PBMC collected from all the subjects positive to Pd at patch testing. On the contrary, control subjects did not show any response to Pd as assessed by IFN-γ ELISpot assay or by skin testing. Remarkably, the levels of Pd in all biological samples (saliva, sera, urine) were significantly higher in Pd-sensitized patients than in those collected from controls, reaching the highest concentrations in the urine. The leaching studies gave additional evidence that the dental appliances can release measurable levels of Pd in saliva. Oral clinical symptoms in patients with Pd dental prostheses were associated with measurable levels of Pd in the biological fluids, the induction of Pd-specific IFN-γ responses in PBMC and the clinical evidence of skin sensitization to Pd. These data suggest that dental appliances may represent an active source of Pd in the body, and this, in turn, can favour the clinical setting of a hypersensitivity to this metal.

Hair analysis and the early detection of imbalances in trace elements for members of expeditions in Antarctica

Abstract In the framework of the Human Biology and Medicine Project of the National Program for Research in Antarctica (Programma Nazionale di Ricerche in Antartide, PNRA) a study was undertaken to explore the suitability of trace element determination in human hair as a tool for the early detection of the impact of extreme environmental conditions on the health of members of the Italian and French expeditions. This study included three campaigns, namely, the 1995–1996 expedition to the Italian base of Terra Nova Bay, as well as the 1997–1998 and 1998–1999 expeditions to both the Italian base of Terra Nova Bay and the Italian–French base of Dome C. Sampling of hair of participants in the expeditions was carried out both prior to departure and at the end of the period spent at the bases. As regards storage, pre-treatment and analysis of hair samples they were performed in accordance with previously established procedures. Acid-assisted microwave digestion was adopted as the best approach for preparing solutions for subsequent analysis by inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry. In the first part of this study, only the elements Ca, Cu, Fe, Mg and Zn were selected, while at a later stage it was decided to also include Co, Cr, Mn, Mo and Ni. Experimental results were subjected to basic statistical analysis to detect possible significant differences in element concentrations that occurred during the sojourn at the bases, with a view to identify possible remedial action to minimize adverse consequences.

Distribution and elimination of palladium in rats after 90-day oral administration.

This study determined the distribution in internal organs and the elimination routes in rats after oral administration of potassium hexachloro-palladate. Forty male Wistar rats were exposed for 90 days to 0, 10, 100 and 250 ng/mL of the palladium (Pd) salt in drinking water. Samples of urine and feces were collected on days 1, 30, 60 and 90, while organs (kidney, liver, lung, spleen and bones) and blood were collected at the end of the experiment. Quantification method was based on the sector-field inductively coupled plasma mass spectrometry. Results indicated that Pd ions were rapidly eliminated from the body. The principal excretion was through the feces (650 ± 72.7 ng/g dry weight, at the Pd dose of 250 ng/mL), but at the higher dosing Pd was also eliminated through the urine (6.16 ± 1.91 ng/mL for the Pd intake of 250 ng/mL). A clear relationship between the Pd ingested dose and the Pd excretion amount was observed mainly in the feces. Absorbed Pd was mostly found in the kidney of rats (124.4 ± 23.0 ng/g dry weight, following the highest dose), while liver, lung, spleen and bones did not accumulate the metal. At the higher dosing, Pd content in the kidney raised proportionally with the Pd dose. Our findings may be useful to help in the understanding of the health impact of Pd dispersed in the environment as well as in identifying appropriate biological indices of Pd exposure.

Distribution and Elimination of Palladium in Male Wistar Rats Following 14-Day Oral Exposure in Drinking Water

The Pd tissue distribution and elimination in rats following oral exposure in drinking water of dipotassium hexachloropalladate at doses of 100 or 250 ng/ml for 14 d were determined. The sector field inductively coupled plasma mass spectrometry used for Pd quantification showed the adequate sensitivity (10 ng/l) and accuracy (96–105%), and all the more in consideration of the very low levels of Pd accumulated. Tissues were taken and analyzed after 14 d. The tissue containing the highest Pd concentration was the kidney (4 ng/g dry weight in controls and 75 ng/g dry weight at the maximum dose), with left and right kidneys showing a comparable accumulation. The Pd kidney levels rose, but not significantly, with the administered dose. None of the other organs (liver, lung, spleen, adrenal glands, and bones) appeared to accumulate Pd, even at the highest dose. At the 250-ng/ml dose, small amounts of Pd were found in serum (0.27 ng/ml vs. 0.19 ng/ml in controls), while they were higher in urine (1.2 ng/ml vs. 0.16 ng/ml in controls) and in feces (3,231 ng/g dry weight vs. 69 ng/g dry weight in controls). Feces were the main excretion route for Pd, with a significant linear correlation with exposed dose, which is likely due to low intestinal absorption of Pd.

Comparative study of marine sediment from antarctica by low-pressure discharge atomic emission spectrometry and inductively coupled plasma-based spectrometry

An alternative to conducting or non-conducting solid sample analysis, e.g., by ICP-AES or ICP-MS coupled with electrothermal volatilization or laser ablation, is offered by low-pressure discharge (LPD) AES sources, such as glow discharge or hollow cathode discharge AES techniques. This approach turned out to be particularly advantageous for the quantification of a number of major, minor and trace elements in Antarctic sediment sampled from the coastal region of the Ross sea nearby the Italian base at Terra Nova Bay. In order to ascertain the potential of LPD-AES in both respects, a number of elements, namely Al, Cr, Fe, Mn, Ni, Pb and Zn, were determined after mixing sediment aliquots with high-purity copper powder and obtaining disc pellets by pressing the resulting mixtures. The results obtained were in good agreement with those achieved using ICP-AES and ICP-MS after microwave-assisted digestion of the sediment. The concentration ranges (µg g–1) determined were Al 68410–70 500, Cr 40.1–41.9, Fe 19 210–23550, Mn 424–430, Ni 9.0–9.8, Pb 17.2–19.5 and Zn 51.8–53.2.

Role of Diet in Nickel Dermatitis

Nickel is the twenty-second most abundant element and the seventh most abundant transitional metal. It is an ubiquitous trace element and it is mostly adopted to manufacture stainless steel used to produce food processing equip- ment and containers. The metal allergy is often caused from Ni and females are affected more commonly than males. When Ni allergy develop it tends to persist life-long. Food is considered to be a major source of Ni exposure for the gen- eral population. Significant Ni concentration was found in the following foods: mixed nuts, dried fruits, tea bag, cocoa, chocolate, varieties of crisps, soy products, legumes and wheat flour. Moreover, cooking acidic food in stainless steel utensils may increase the Ni content. Nickel content in food may vary considerably due to the difference in Ni content of the soil. Nickel-sensitive person can develop dermatitis when in their diet is present the element. Then, careful selection of food with relatively low Ni concentration can bring a reduction in the total dietary intake of Ni per day. This can influence the outcome of the disease and can benefit the nickel-sensitive patients.