Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms. .

Interações moleculares na adesão celular em suportes sólidos e o efeito de fotossensibilizadores porfirínicos / Molecular interactions in cell adhesion on solid substrates and the effect of porphyrinic photosensitizers

A adesão celular está ligada à formação e disseminação de metástases, a principal causa de óbito de pacientes diagnosticados com câncer. O objetivo deste trabalho foi investigar in vitro o efeito de fotossensibilizadores na adesão celular. Foram utilizadas porfirinas comerciais (PpIX, CPpI, TSPP, TMPyP e Zn(II)TMPyP) e um fotossensibilizador sintetizado através da ligação de poli-L-lisina à protoporfirina IX (PLLPpIX). A adesão celular foi estudada por RICM, técnica que permite quantificar a área de contato entre uma célula e um substrato por binarização das imagens digitais utilizando limiares apropriados. A técnica foi padronizada e revelou dois regimes de adesão celular: um limitado e outro não limitado pela quantidade de proteína de adesão adsorvida na superfície. Neste último foi observada lise celular. Todos os fotossensibilizadores estudados foram capazes de aumentar a adesão celular na ausência de irradiação comparados ao controle sem fotossensibilizador, o que não havia sido observado nos ensaios de resistência à tripsinização normalmente utilizados para estudar o efeito de fotossensibilizadores na adesão celular. Quanto maior a anfifilicidade do fotossensibilizador, maior foi o efeito na adesão, o que é explicado pela capacidade das moléculas em se intercalarem na membrana, mudando a sua rigidez. Este aumento da adesão no escuro correlaciona com a diminuição da migração segundo ensaios de ferida. A análise do padrão de expressão de integrinas na superfície celular revela que o aumento da adesão correlaciona com o aumento na expressão de αV. Quando os fotossensibilizadores estão concentrados na região perimembranar (1 minuto de incubação) e as células são irradiadas, há um aumento da adesão em relação ao controle sem fotossensibilizador, mas uma diminuição em relação ao controle tratado com o fotossensibilizador e não irradiado, o que implica que a PDT leva a uma diminuição da adesão celular e não a um aumento como reportado na literatura. Com 3h de incubação, PLLPpIX impede a adesão celular, enquanto PpIX praticamente não muda a adesão comparado ao controle não irradiado. Esta ausência do efeito da irradiação sugere que a PpIX afeta a adesão celular principalmente devido a sua intercalação na membrana e não devido à formação de espécies reativas. Com 3h de incubação os fotossensibilizadores não se encontram na membrana e, portanto, o efeito na adesão celular é indireto e também não está relacionado à diferenças na eficiência de internalização. O comportamento observado deve ter relação com diferenças de citolocalização. Outro processo que pode alterar a adesão celular é a oxidação das proteínas do soro fetal bovino. Como observado nos estudos de fotossensibilização de células, PLLPpIX foi capaz de impedir a adesão celular, diferentemente da PpIX. A maior eficiência da PLLPpIX foi associada a presença do polímero, o qual força por questões estéricas que a interação da PLLPpIX com a albumina, o componente majoritário do soro, fique restrita à superfície da proteína, deixando o fotossensibilizador disponível para interagir com o oxigênio molecular e gerar oxigênio singlete. Assim, a funcionalização com um polímero tornou a PpIX capaz de modular a adesão celular tanto agindo dentro da célula quanto na matriz extracelular

Cell adhesion is associated to the formation and spread of metastasis, the leading cause of death in cancer patients. The aim of this study was to investigate, in vitro, the effect of photosensitizers in cell adhesion. Five commercial porphyrins (PpIX, CPpI, TSPP, TMPyP e Zn(II)TMPyP) and Protoporphyrin IX covalently tethered to poli-L-lysine (PLLPpIX) were used. Cell adhesion was mainly studied by RICM, a technique that allows quantifying the contact area between a cell and a substrate for binarization of digital images using appropriate thresholds. The technique was standardized and disclosed two systems for cell adhesion: a system limited by the amount of adhesion proteina adsorbed on the surface and another one no limited, in which cell lysis was observed. All photosensitizers were able to enhance cell adhesion in the absence of irradiation compared to control without photosensitizer, which had not been observed in the trypsinization resistance tests usually used to study the effect of photosensitizers in cell adhesion. The greater the amphiphilicity of the photosensitizer, the greater was the effect on cell adhesion. This is explained by the ability of molecules to fit in the membrane, changing its tension. This increased adhesion correlates with the decrease in migration according to wound healing assays. Analysis of the integrin expression pattern on cell surface reveals that increased adhesion correlates with increased expression of alpha V. When photosensitizers are concentrated in the perimembranar region (1 minute of incubation) and cells are irradiated, there is an increase in adhesion when compared to control without photosensitizer, but a decrease relative to controls treated with the photosensitizer without irradiation, implying that PDT leads to a reduction of cell adhesion and not to an increase as reported in the literature. With 3h of incubation PLLPpIX prevents cell adhesion, while PpIX practically does not change the adhesion compared to dark control. This lack of effect of irradiation suggests that PpIX affects cell adhesion primarily because of its intercalation into the membrane and not due to the formation of reactive species. With 3h of incubation the photosensitizers are not on the membrane and therefore the effect on cell adhesion is indirect and it is not also related to differences in uptake efficiency. The observed behavior must be related to differences in subcellular localization arising from differences in molecular structure. Another process that can alter the cell adhesion is serum protein oxidation. As noted in the studies with cells, photosensitization of serum with PLLPpIX (but not with PpIX) was capable of preventing cell adhesion. The greater efficiency of PLLPpIX was associated with the presence of the polymer, which, by the steric hindrance, forces that interaction of PLLPpIX with albumin, the major serum component, is restricted to the protein surface, leaving the photosensitizer available to interact with molecular oxygen and generate singlet oxygen. Thus, the functionalization of a polymer has turned PpIX capable of modulating cell adhesion by acting both within and outside (in extracellular matrix) the cell

p53 and MDM2 protein expression in actinic cheilitis

Actinic cheilitis is a potentially malignant lip lesion caused by excessive and prolonged exposure to ultraviolet radiation, which can lead to histomorphological alterations indicative of abnormal cell differentiation. In this pathology, varying degrees of epithelial dysplasia may be found. There are few published studies regarding the p53 and MDM2 proteins in actinic cheilitis. Fifty-eight cases diagnosed with actinic cheilitis were histologically evaluated using Banóczy and Csiba (1976) parameters, and were subjected to immunohistochemical analysis using the streptavidin-biotin method in order to assess p53 and MDM2 protein expression. All studied cases expressed p53 proteins in basal and suprabasal layers. In the basal layer, the nuclei testing positive for p53 were stained intensely, while in the suprabasal layer, cells with slightly stained nuclei were predominant. All cases also tested positive for the MDM2 protein, but with varying degrees of nuclear expression and a predominance of slightly stained cells. A statistically significant correlation between the percentage of p53 and MDM2-positive cells was established, regardless of the degree of epithelial dysplasia. The expression of p53 and MDM2 proteins in actinic cheilitis can be an important indicator in lip carcinogenesis, regardless of the degree of epithelial dysplasia.

Differential gene expression profiles of human monocyte-derived antigen presenting cells in response to Penicillium marneffei: roles of DC-SIGN (CD209) in fungal cell uptake.

DNA microarray technology was used to determine the gene expression profile of human monocyte-derived macrophages (hMDMs) after stimulation by Penicillium marneffei yeast. The expression levels of 175 macrophage genes were found to be altered by a minimum of two-fold in magnitude following 4 hours of P. marneffei exposure. Among those, 41 genes were upregulated in activated hMDMs while 134 genes were downregulated. Real-time PCR and RT-PCR were performed to further examine gene expression associated with the inflammatory response. Increased levels of TNF-alpha and IL-1 beta gene expression in both hMDMs and human monocyte-derived dendritic cells (hMoDCs) were observed after stimulation by P. marneffei yeast. Furthermore, the genes encoding T-bet, IL-6 and ICAM-1 were also upregulated in hMDMs. Functional analysis of the adhesion of P. marneffei to dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209) was performed in hMoDCs since the microarray data revealed an increased expression of DC-SIGN in activated hMDMs. We found that DC-SIGN-Fc bound preferentially to P. marneffei yeast rather than to conidia. Moreover, an anti-DC-SIGN monoclonal antibody inhibited the binding of P. marneffei yeast to hMoDCs, but did not inhibit endocytosis of P. marneffei yeast. The mannose receptor, on the other hand, was important in both adhesion and phagocytosis. These results suggest that P. marneffei may exploit DC-SIGN as a receptor to facilitate the systemic spread of infection. Taken together, our study demonstrates the usefulness of microarray technology in generating valuable expression data to permit conventional immunologic investigations of host-fungal interactions.

Metastasis-suppressor KAI1/CD82 induces homotypic aggregation of human prostate cancer cells through Src-dependent pathway

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.