Chemical inhibition of ß-glucocerebrosidase does not affect phagocytosis and early containment of Leishmania by murine macrophages.

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.

The GM2 ganglioside inhibits iNKT cell responses in a CD1d-dependent manner.

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid antigens presented on CD1d molecules at the surface of antigen-presenting cells. GM2 is a glycosphingolipid abundant in cellular membranes and known to bind CD1d molecules, but the functional consequences of this binding are not completely clarified. Herein, we analyzed the effect of GM2 in iNKT cell activation. We found that culturing antigen-presenting cells or total peripheral blood mononuclear cells with GM2 did not induce activation of human iNKT cells, implying that this lipid is not antigenic for human iNKT cells. To investigate if this lipid could inhibit iNKT cell activation, we simultaneously incubated antigen-presenting cells with GM2 and the iNKT cell antigen α-Galactosylceramide (α-GalCer) and used them to stimulate iNKT cells. We found that GM2 reduced human iNKT cell activation in a dose-dependent manner. An explanation for this effect could be a direct competition of GM2 with antigenic lipids for CD1d binding. This was demonstrated by the use of an antibody (L363) that stains mouse CD1d:α-GalCer complexes, as in the presence of GM2 the amount of CD1d:α-GalCer complexes are reduced. We further explored the consequences of chronic GM2 overload on human iNKT cells by analyzing iNKT cells in patients diagnosed with GM2 gangliosidoses. We found that pediatric patients present a higher frequency of circulating CD4+ iNKT cells and concomitant lower frequency of CD4-CD8- iNKTs. A lower percentage of iNKT cells expressing the NK marker CD161 was also observed in these patients. In contrast, in two adult patients studied, no differences on iNKT cell phenotype were observed. Altogether, this study uncovers a new role for GM2 in the modulation of iNKT cell activation, thus strengthening the central role of lipid metabolism in iNKT cell biology.

Psychosine variants as antigens for natural killer T cells.

Natural killer T (NKT) cells play a central role in the interface between innate and adaptive immunity, and alpha-galactosylceramide was recently shown to be an endogenous antigen for these cells. The source of alpha-galactosylceramide has not yet been determined; however, in vivo degradation of alpha-galactosylceramide involves generation of alpha-psychosine (alpha-galactosylsphingosine). Alpha-psychosine stimulates cytokine release from NKT cells and constitutes an endogenous antigen for these cells. Alpha-psychosine contains a single lipid chain, while most antigens for NKT cells have two lipid chains, and we have investigated if other glycolipids with one lipid chain, derived from know antigens for NKT cells, stimulate cytokine release from NKT cells. Only psychosine variants derived from the most potent NKT cell antigens cause stimulation, and this stimulation occurs in vitro as well as in vivo. Truncated forms of weak antigens for NKT cells are not stimulatory.

A multiproxy approach to evaluate biocidal treatments on biodeteriorated majolica glazed tiles.

The Fishing House located on the grounds of the Marquis of Pombal Palace, Oeiras, Portugal, was built in the 18th century. During this epoch, Portuguese gardens, such as the one surrounding the Fishing House, were commonly ornamented with glazed wall tile claddings. Currently, some of these outdoor tile panels are covered with dark colored biofilms, contributing to undesirable aesthetic changes and eventually inducing chemical and physical damage to the tile surfaces. Phylogenetic analyses revealed that the investigated biofilms are mainly composed of green algae, cyanobacteria and dematiaceous fungi. With the aim of mitigating biodeterioration, four different biocides (TiO2 nanoparticles, Biotin® T, Preventol® RI 80 and Albilex Biostat® ) were applied in situ to the glazed wall tiles. Their efficacy was monitored by visual examination, epifluorescence microscopy and DNA-based analysis. Significant changes in the microbial community composition were observed 4 months after treatment with Preventol® RI 80 and Biotin® T. Although the original community was inactivated after these treatments, an early stage of re-colonization was detected 6 months after the biocide application. TiO2 nanoparticles showed promising results due to their self-cleaning effect, causing the detachment of the biofilm from the tile surface, which remained clean 6 and even 24 months after biocide application. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

An integrated approach for assessing the bioreceptivity of glazed tiles to phototrophic microorganisms.

A laboratory-based methodology was designed to assess the bioreceptivity of glazed tiles. The experimental set-up consisted of multiple steps: manufacturing of pristine and artificially aged glazed tiles, enrichment of phototrophic microorganisms, inoculation of phototrophs on glazed tiles, incubation under optimal conditions and quantification of biomass. In addition, tile intrinsic properties were assessed to determine which material properties contributed to tile bioreceptivity. Biofilm growth and biomass were appraised by digital image analysis, colorimetry and chlorophyll a analysis. SEM, micro-Raman and micro-particle induced X-ray emission analyses were carried out to investigate the biodeteriorating potential of phototrophic microorganisms on the glazed tiles. This practical and multidisciplinary approach showed that the accelerated colonization conditions allowed different types of tile bioreceptivity to be distinguished and to be related to precise characteristics of the material. Aged tiles showed higher bioreceptivity than pristine tiles due to their higher capillarity and permeability. Moreover, biophysical deterioration caused by chasmoendolithic growth was observed on colonized tile surfaces.

Invariant Natural Killer T Cells are Reduced in Hereditary Hemochromatosis Patients.

PURPOSE: Invariant natural killer T (iNKT) cells are CD1d restricted-T cells that react to lipid antigens. iNKT cells were shown to be important in infection, autoimmunity and tumor surveillance. Alterations in the number and function of these cells were described in several pathological conditions including autoimmune and/or liver diseases. CD1d is critical for antigen presentation to iNKT cells, and its expression is increased in liver diseases. The liver is the major organ affected in Hereditary Hemochromatosis (HH), an autosomal recessive disorder caused by excessive iron absorption. Herein, we describe the study of iNKT cells of HH patients. METHODS: Twenty-eight HH patients and 24 control subjects from Santo António Hospital, Porto, were included in this study. Patient's iron biochemical parameters (serum transferrin saturation and ferritin levels) and the liver function marker alanine transaminase (ALT) were determined at the time of study. Peripheral blood iNKT cells were analyzed by flow cytometry using an anti-CD3 antibody and the CD1d tetramer loaded with PBS57. RESULTS: We found a decrease in the percentage and number of circulating iNKT cells from HH patients when compared with control population independently of age. iNKT cell defects were more pronounced in untreated patients, relating with serum ferritin and transferrin saturation levels. No correlation was found with ALT, a marker of active liver dysfunction. CONCLUSIONS: Altogether, our results demonstrate that HH patients have reduced numbers of iNKT cells and that these are influenced by iron overload.

Indoor air quality in Portuguese archives: a snapshot on exposure levels.

Indoor air quality recently entered legislation in Portugal. Several parameters must be evaluated and kept within limits in order to obtain a certification for air quality and energy consumption. Certification parameters were analyzed in two Portuguese archives in order to assess indoor air quality both for people attending or working on these premises and for maintenance of a written heritage that must be retained for future generations. Carbon monoxide (CO) and carbon dioxide (CO2), formaldehyde, and fungal counts were kept within stipulated limits. Relative humidity (RH), volatile organic compounds (VOC), particulate matter (PM10), and ozone (O3) showed values above legislated levels and justified the implementation of corrective measures. In terms of conservation, studies on the limit values are still needed, but according to the available international guidelines, some of the analyzed parameters such as PM10, O3, and RH were also above desirable values. Corrective measures were proposed to these institutions. Although this study was only of a short duration, it proved valuable in assessing potential eventual problems and constitutes the first Portuguese indoor air quality assessment taking into consideration both aspects of an archive such as human health and heritage safekeeping.

Bioreceptivity of building stones: a review.

In 1995, Guillitte defined bioreceptivity, a new term in ecology, as the ability of a material to be colonized by living organisms. Information about the bioreceptivity of stone is of great importance since it will help us to understand the material properties which influence the development of biological colonization in the built environment, and will also provide useful information as regards selecting stones for the conservation of heritage monuments and construction of new buildings. Studies of the bioreceptivity of stone materials are reviewed here with the aim of providing a clear set of conclusions on the topic. Definitions of bioreceptivity are given, stone bioreceptivity experiments are described, and finally the stone properties related to bioreceptivity are discussed. We suggest that a standardized laboratory protocol for evaluating stone bioreceptivity and definition of a stone bioreceptivity index are required to enable creation of a database on the primary bioreceptivity of stone materials.

Laboratory-induced endolithic growth in calcarenites: biodeteriorating potential assessment.

This study is aimed to assess the formation of photosynthetic biofilms on and within different natural stone materials, and to analyse their biogeophysical and biogeochemical deterioration potential. This was performed by means of artificial colonisation under laboratory conditions during 3 months. Monitoring of microbial development was performed by image analysis and biofilm biomass estimation by chlorophyll extraction technique. Microscopy investigations were carried out to study relationships between microorganisms and the mineral substrata. The model applied in this work corroborated a successful survival strategy inside endolithic microhabitat, using natural phototrophic biofilm cultivation, composed by cyanobacteria and algae, which increased intrinsic porosity by active mineral dissolution. We observed the presence of mineral-like iron derivatives (e.g. maghemite) around the cells and intracellularly and the precipitation of hausmannite, suggesting manganese transformations related to the biomineralisation.

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells.

Low CD8(+) T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8(+) T lymphocyte numbers. The A-A-T haplotype was associated with a severe clinical expression of HH and low CD8(+) T lymphocyte numbers, while the G-G-G haplotype was associated with a milder clinical expression of HH and high CD8(+) T lymphocyte numbers. As CD8(+) T lymphocytes are a very heterogeneous population, in this study we analysed the CD8(+) subpopulations of naive, central memory (T(CM)) and effector memory (T(EM)), and further subsets of CD8(+) T(EM) cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8(+) T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For T(EM) cells and the T(EM) CD27(-)CD28(-) subset no effect of age was observed in HH [R(2) = 0.001, not significant (n.s.) and R(2) = 0.01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R(2) = 0.09, P = 0.017 and R(2) = 0.22, P = 0.0005, respectively). Interestingly, patients homozygous for the A-A-T haplotype have lower numbers of CD8(+) T(EM) cells due especially to lower numbers of T(EM) CD27(-)CD28(-) (0.206 +/- 0.119 and 0.066 +/- 0.067 x 10(6) cells/ml, respectively) than patients carrying the G-G-G haplotype (0.358 +/- 0.195 and 0.246 +/- 0.202 x 10(6) cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8(+) T cells into the most mature phenotype.

Isolation of five Rubrobacter strains from biodeteriorated monuments.

In the last few years, the microbial colonisation of mural paintings in ancient monuments has been attracting the attention of microbiologists and conservators. The genus Rubrobacter is commonly found in biodeteriorated monuments, where it has been reported to cause rosy discolouration. However, to date, only three species of this genus have been isolated, all from thermophilic environments. In this paper, we studied three monuments: the Servilia and Postumio tombs in the Roman Necropolis of Carmona (Spain), and Vilar de Frades church (Portugal), in search of Rubrobacter strains. In all cases, biodeterioration and the formation of efflorescences were observed, and five Rubrobacter strains were isolated. These isolates showed different physiology and migration in denaturing gradient gel electrophoresis, suggesting they might represent new species within this genus. The isolates reproduced some biodeterioration processes in the laboratory and revealed their biomediation in crystal formation.

Human T cell responses to endogenously presented HLA-A*0201 restricted peptides of Simian virus 40 large T antigen.

Presence of the simian virus 40 (SV40) has recently been demonstrated in a relatively high percentage of human mesotheliomas and it is associated with the development of these malignancies in pleural cavities. Therefore, we have initiated a study to identify candidate peptides presented by the human HLA-A*0201 molecule for vaccination approaches against SV40 and monitoring of SV40 directed human immune responses. Initial screening of SV40 large T (Tag) domains required for transformation of cells for HLA-A*0201 binding motifs revealed ten possible binding peptides. Screening of these candidate peptides showed that seven of the ten peptides could bind and stabilize HLA-A*0201 molecules. In an in vitro immunization assay the two peptides with the highest binding affinity for HLA-A*0201, Tag aa 396-405 and aa 577-585, were tested for their ability to induce peptide specific cytotoxic T cells in two healthy donors. One donor developed cytotoxic T cells against Tag aa 396-405 and in T cell cultures of both donors Tag aa 577-585 specific T cells were initiated. The T cells against Tag aa 577-585 not only recognized and killed peptide pulsed cells, but, most importantly, SV40 transformed human mesothelial cells. This is the first demonstration of the induction of SV40 specific human cytotoxic T lymphocytes that recognize endogenously processed peptides from SV40. This peptide identification study opens the possibility to investigate immune responses against SV40 in mesothelioma patients and in individuals exposed to SV40.

Cellular immunity and immunotherapy against deoxyribonucleic acid virus-induced tumors.

An important role in the immune defense against deoxyribonucleic acid virus induced tumors is mediated by T-cells, as is evident from aetiological, animal model, and clinical data. In this review the most recent observations in this field are described for three prominent members of this family of viruses, namely human papillomavirus associated with human cervical cancer, human adenovirus associated with lung infections in humans and tumors in rodents, and simian virus 40 associated with rodent tumors and human mesothelioma, osteosarcoma and ependymoma.

Identification of peptides for immunotherapy of cancer. It is worth the effort.

As the nature of the T cell immune response is defined by T cell receptor recognition of small protein fragments, referred to as peptides, the identification of peptides would lead us to understanding and directing the T-cell-mediated immune response. Immunogenic peptides might be used for vaccination and activation of the immune reaction against cancer- and virus-infected cells. Additionally, the knowledge of immunogenic peptides was expected to lead to blocking of allergic reactions and autoimmune diseases. Based on these assumptions, the search for immunogenic peptides was started in mice and man in the mid-1980s. After a decade of peptide identification and testing in vitro and in vivo, this may be a proper time to evaluate the results from the peptide-related work and determine the possible applications of this knowledge for the next decade. In this review we discuss the identification of peptides, their use in murine models, as well as clinical data from peptide vaccinations or therapies. Potential hazards and limitations of peptide use in immunotherapy and other possible applications for peptides or peptide motifs in immunotherapy are evaluated.