Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.     

Molecular mechanisms of cell-cell signaling by the Spemann-Mangold organizer

We review how studies on the first Spemann-Mangold organizer marker, the homeobox gene goosecoid, led to the discovery of secreted factors that pattern the vertebrate embryo. Microinjection of goosecoid mRNA formed secondary axes and recruited neighboring cells. These non-cell autonomous effects are mediated in part by the expression of secreted factors such as chordin, cerberus and Frzb-1. Unexpectedly, many of the molecules secreted by the Spemann-Mangold organizer turned out to be antagonists that bind growth factors in the extracellular space and prevent them from binding to their receptors. The case of chordin is reviewed in detail, for this molecule has provided biochemical insights into how patterning by Spemann's organizer can be regulated by diffusion and proteolytic control. The study of the BMP-binding repeats of Chordin, which are present in many extracellular proteins, may provide a new paradigm for how cell-cell signaling is regulated in the extracellular space not only in embryos, but also in adult tissues.     

An antibody against SSEA-5 glycan on human pluripotent stem cells enables removal of teratoma-forming cells

An important risk in the clinical application of human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), is teratoma formation by residual undifferentiated cells. We raised a monoclonal antibody against hESCs, designated anti-stage-specific embryonic antigen (SSEA)-5, which binds a previously unidentified antigen highly and specifically expressed on hPSCs--the H type-1 glycan. Separation based on SSEA-5 expression through fluorescence-activated cell sorting (FACS) greatly reduced teratoma-formation potential of heterogeneously differentiated cultures. To ensure complete removal of teratoma-forming cells, we identified additional pluripotency surface markers (PSMs) exhibiting a large dynamic expression range during differentiation: CD9, CD30, CD50, CD90 and CD200. Immunohistochemistry studies of human fetal tissues and bioinformatics analysis of a microarray database revealed that concurrent expression of these markers is both common and specific to hPSCs. Immunodepletion with antibodies against SSEA-5 and two additional PSMs completely removed teratoma-formation potential from incompletely differentiated hESC cultures.     

Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.     

Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition

Real-Time PCR has been applied to quantify extraradical soil mycelium of the edible ectomycorrhizal fungus Lactarius deliciosus in an interspecific competition experiment under greenhouse conditions. Couples of Pinus pinea seedlings inoculated with either L. deliciosus, Rhizopogon roseolus, or non-inoculated (control) were transplanted into pots filled with two types of soil in all the possible combinations. Total DNA was extracted from soil samples at 3 and 6 months after transplantation to perform real-time PCR analysis. DNA extractions from soil mixed with known amounts of mycelium of L. deliciosus were used as standards. Six months after transplantation, the percentage of mycorrhizas of L. deliciosus and seedling growth were significantly affected by the soil type. Extraradical soil mycelium of L. deliciosus was positively correlated with the final percentage of mycorrhizas and significantly affected by the sampling time and soil depth. The competition effect of R. roseolus was not significant for any of the measured parameters, probably due to the sharp decrease of the mycorrhizal colonization by this fungus. We conclude that real-time PCR is a powerful technique for extraradical mycelium quantification in studies aimed at evaluating the persistence of introduced strains of L. deliciosus in field plantations.     

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4°C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.     

Tau enhances α-synuclein aggregation and toxicity in cellular models of synucleinopathy

Background

The simultaneous accumulation of different misfolded proteins in the central nervous system is a common feature in many neurodegenerative diseases. In most cases, co-occurrence of abnormal deposited proteins is observed in different brain regions and cell populations, but, in some instances, the proteins can be found in the same cellular aggregates. Co-occurrence of tau and α-synuclein (α-syn) aggregates has been described in neurodegenerative disorders with primary deposition of α-syn, such as Parkinson''s disease and dementia with Lewy bodies. Although it is known that tau and α-syn have pathological synergistic effects on their mutual fibrillization, the underlying biological effects remain unclear.

Methodology/Principal Findings

We used different cell models of synucleinopathy to investigate the effects of tau on α-syn aggregation. Using confocal microscopy and FRET–based techniques we observed that tau colocalized and interacted with α-syn aggregates. We also found that tau overexpression changed the pattern of α-syn aggregation, reducing the size and increasing the number of aggregates. This shift was accompanied by an increase in the levels of insoluble α-syn. Furthermore, co-transfection of tau increased secreted α-syn and cytotoxicity.

Conclusions/Significance

Our data suggest that tau enhances α-syn aggregation and toxicity and disrupts α-syn inclusion formation. This pathological synergistic effect between tau and α-syn may amplify the deleterious process and spread the damage in neurodegenerative diseases that show co-occurrence of both pathologies.     

Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors

Background  

The tear film is a thin layer of fluid that covers the ocular surface and is involved in lubrication and protection of the eye. Little is known about the protein composition of tear fluid but its deregulation is associated with disease states, such as diabetic dry eyes. This makes this body fluid an interesting candidate for in-depth proteomic analysis.