Altered expression of CD1d molecules and lipid accumulation in the human hepatoma cell line HepG2 after iron loading.

Iron overload in the liver may occur in clinical conditions such as hemochromatosis and nonalcoholic steatohepatitis, and may lead to the deterioration of the normal liver architecture by mechanisms not well understood. Although a relationship between the expression of ICAM-1, and classical major histocompatibility complex (MHC) class I molecules, and iron overload has been reported, no relationship has been identified between iron overload and the expression of unconventional MHC class I molecules. Herein, we report that parameters of iron metabolism were regulated in a coordinated-fashion in a human hepatoma cell line (HepG2 cells) after iron loading, leading to increased cellular oxidative stress and growth retardation. Iron loading of HepG2 cells resulted in increased expression of Nor3.2-reactive CD1d molecules at the plasma membrane. Expression of classical MHC class I and II molecules, ICAM-1 and the epithelial CD8 ligand, gp180 was not significantly affected by iron. Considering that intracellular lipids regulate expression of CD1d at the cell surface, we examined parameters of lipid metabolism in iron-loaded HepG2 cells. Interestingly, increased expression of CD1d molecules by iron-loaded HepG2 cells was associated with increased phosphatidylserine expression in the outer leaflet of the plasma membrane and the presence of many intracellular lipid droplets. These data describe a new relationship between iron loading, lipid accumulation and altered expression of CD1d, an unconventional MHC class I molecule reported to monitor intracellular and plasma membrane lipid metabolism, in the human hepatoma cell line HepG2.This work was funded by the Inova Foundation for Medical Research/The American Portuguese Biomedical Research Fund (APBRF, USA). The authors would like to thank L. Mayer and K. Uchida for providing antibodies, A. do Vale, F. Pisarra and M.T. Silva for help and comments on TEM, and M. Santos and R. Hultcrantz for critical reading of the manuscript. We also thank M. de Sousa for mentoring this work. M.C. and C.F.P. were supported by a fellowship from Inova/APBRF. E.M.C. was partially supported by EU grant (QLG1-CT-1999–00665)

Hepatocytes and IL-15: A Favorable Microenvironment for T Cell Survival and CD8+ T Cell Differentiation

Human intrahepatic lymphocytes are enriched in CD1d-unrestricted T cells coexpressing NKR. Although the origin of this population remains controversial, it is possible to speculate that the hepatic microenvironment, namely epithelial cells or the cytokine milieu, may play a role in its shaping. IL-15 is constitutively expressed in the liver and has a key role in activation and survival of innate and tissue-associated immune cells. In this in vitro study, we examined whether hepatocyte cell lines and/or IL-15 could play a role in the generation of NK-like T cells. The results show that both HepG2 cells and a human immortalized hepatocyte cell line increase survival and drive basal proliferation of T cells. In addition, IL-15 was capable of inducing Ag-independent up-regulation of NKR, including NKG2A, Ig-like receptors, and de novo expression of CD56 and NKp46 in CD8(+)CD56(-) T cells. In conclusion, our study suggests that hepatocytes and IL-15 create a favorable microenvironment for T cells to growth and survive. It can be proposed that the increased percentage of intrahepatic nonclassical NKT cells could be in part due to a local CD8(+) T cell differentiation.This work was supported by grants from the American Portuguese Biomedical Research Fund (Inova Grant) and from ISCSN-CESPU (Grants CESPU 1F/05/2005 and CESPU 2F/03/2006). M.P.C. was supported by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/24396/2005)

Biological effects of thermal water-associated hydrogen sulfide on human airways and associated immune cells: Implications for respiratory diseases

Natural mineral (thermal) waters have been used for centuries as treatment for various diseases. However, the scientific background of such therapeutic action is mostly empiric and based on knowledge acquired over time. Among the various types of natural mineral waters, sulfurous thermal waters (STWs) are the most common type in the center of Portugal. STWs are characterized by high pH, poor mineralization, and the presence of several ions and salts, such as bicarbonate, sodium, fluoride, silica, and carbonate. Furthermore, these waters are indicated as a good option for the treatment of various illnesses, namely respiratory diseases (e.g., allergic rhinitis, asthma, and chronic obstructive pulmonary disease). From the sulfide species present in these waters, hydrogen sulfide (H2S) stands out due to its abundance. In healthy conditions, H2S-related enzymes (e.g., cystathionine β-synthase and cystathionine γ-lyase) are expressed in human lungs, where they have mucolytic, antioxidant, anti-inflammatory, and antibacterial roles, thus contributing to airway epithelium homeostasis. These roles occur mainly through S-sulfhydration, a post-translational modification through which H2S is able to change the activity of several targets, such as ion channels, second messengers, proteins, among others. However, in respiratory diseases the metabolism of H2S is altered, which seems to contribute somehow to the respiratory deterioration. Moreover, H2S has been regarded as a good biomarker of airway dysfunction and severity, and can be measured in serum, sputum, and exhaled air. Hence, in this review we will recapitulate the effects of STWs on lung epithelial-immune crosstalk through the action of its main component, H2S

Establishment of a Taqman-based approach to monitor Fusarium spp. Airbone spores.

Aerobiological studies provide important information about the biological particles present in the air. Monitoring the presence of airborne fungal spores can help farmers to prevent the onset of fungal diseases that may affect both quantity and quality of crops. The fungi Fusarium spp. are among the most important phytopathogenic fungal communities with high impact at regional level by affecting important cultures such as almond, tomato, maize and cereals. The establishment of an approach that would enable farmers to early react upon the possibility of a Fusarium spp. infection will lead to a better control of the diseases associated to those fungi. Currently, to monitor Fusarium spp. airborne spores it is followed the Hirst-type methodology, which is based on spore’s identification and quantification by optical microscope; a hard and time consuming process due to the spore’s small size and colorless wall. In this context, the development of an alternative methodology that enable to get accurate and reliable results in a faster way will be of high interest. A Taqman specific assay for Fusarium spp. detection and quantification was previously established for a different purpose [1] and was here applied as a molecular-based tool to detect airborne Fusarium spp. spores. To collect the biological particles from the atmosphere a Burkard 7-Day Volumetric Spore Trap, and the Hirst associated methodology, was used as the methodology recommended by the European Aerobiology Society (EAS) and International Association for Aerobiology (IAA) [2]. As proof-of-concept, the analysis was focused on samples weekly collect, from 1st October - 31st December 2018 (14 weeks) at the station of Portuguese Aerobiology Network (RPA – SPAIC) (38° 34’ N; 7° 54’ W). Genomic DNA (gDNA) was extracted from collected biological particles adhered to the melinex tape following the CTAB protocol [3] with some modifications. Considering the results achieved, we consider the Taqman-specific assay as an alternative methodology for monitoring Fusarium spp. airborne spores

A study of some hepatic immunological markers, iron load and virus genotype in chronic hepatitis C.

BACKGROUND/AIMS: Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype. METHODS: Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, beta(2)m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases. RESULTS: A negative correlation was found between the number of CD8(+) cells and fibrosis. CD8(+) cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and beta(2)m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68(+) cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients. CONCLUSIONS: In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.We thank Dr Graça Porto for critical review of the manuscript. This study was supported by the EU QLG1-CT-1999-00665 project, the Calouste Gulbenkian Foundation/FCT Project on Hemochromatosis (Portugal) and the INNOVA Foundation/APBRF (USA)

Clinical improvement following therapy for periodontitis: Association with a decrease in IL-1 and IL-6

Although a number of inflammatory cytokines have been shown to be associated with periodontal pathogenesis, it is important to investigate further whether these biomarkers are associated with the degree of success in nonsurgical treatment of chronic periodontitis. The aim of the present study was to quantify the total levels of interleukin (IL)‑1α, ‑1β, ‑6, ‑10 and tumour necrosis factor (TNF)‑α in gingival crevicular fluid (GCF) of chronic periodontitis patients prior to and following nonsurgical periodontal therapy. In total, 52 GCF samples from disease sites of patients with chronic periodontitis, prior to and following periodontal therapy, and ten non‑disease sites from non‑periodontitis subjects, were collected and cytokine concentrations were determined using a multiplex method. Periodontal parameters, including bleeding on probing, probing pocket depth and the clinical attachment level, in all the sites were recorded. Untreated disease sites exhibited higher cytokine levels in the GCF when compared with the non‑disease sites. Nonsurgical periodontal therapy resulted in a statistically significant decrease in the total levels of IL‑1α, ‑1β and ‑6 in the GCF, but not in IL‑10 or TNF‑α. The results support the hypothesis that proinflammatory cytokines, including IL‑1α, IL‑1β and IL‑6, are likely to be involved in the pathogenesis of periodontitis and are good markers to evaluate the success of nonsurgical therapy in disease sites of patients with periodontitis.The study was supported by a grant from the Cooperativa de Ensino Superior, Politécnico e Universitário (CESPU), Portugal (no. 06‑GCD‑CICS‑09)

Treatment and reutilization of effluents: one Mediterranean project

The problematic of effluent treatment from olive oil industry as been the subject of an European Commission funded project (INCO-MED programme): “Mediterranean Usage of Biotechnological Treated effluent Water”. The potential that effluent offers to increase the availability of water, in mediterranean regions, was the final goal of the project, co-ordinated by INETI with partners from EU and MPC. In the project different systems for the treatment of this effluent had been studied: reactors systems (Intensive type) based on the jet-loop principle (JACTO) and an anaerobic UASB hybrid type reactor technology; lagoons (extensive type) for municipal wastewater treatment were also applied. The aerobic JACTO system demonstrated high unit capacity for biological conversion and operation at different loadings, allowing the removal of the pollutant organic load and the toxicity associated with this effluent. The use of this type of reactor for pre-treatment of OOWW prior to disposal on a lagoon system was tested at FSS (Morocco). Use of fungi as a pre-treatment was tested by UNITUS (Italy), EBC (Turkey) and CBS (Tunisia). In this way the effluent could be “improved” as demonstrated in the case of anaerobic digestion and biogas production (CBS). Effluent improvement and enrichment with phosphate was also tested by UNITUS. Analytical monitoring methodologies were developed at IA (Spain) and treated effluents were tested for a number of agricultural applications in different countries. The different alternatives studied will be analysed and compared taking account of technological and socio-economical criteria in relation with the project objectives