La catedral i Girona: l' entrada del complex episcopal dins murs entre els segles X-XI

Fins a la darreria del segle VIII, el complex episcopal gironí se situava fora murs, al voltant de l'esglésiamartirial de Sant Feliu; a principis del segle IX, el complex es desdoblà amb la consagració d'una segona església episcopal, Santa Maria, dins murs, raó per la qual la seu fou coneguda, al llarg d'aquella centúria, amb la doble advocació de Santa Maria i Sant Feliu. Al llarg del segle X, s'afermà la preeminència de Santa Maria fins al punt que, en l'últim terç del segle. Sant Feliu ja no és esmentada com a seu episcopal, encara que hi continuà vinculada com a una església dependent, sense personalitat ni administració pròpia. Des de final del segle, el procés s'accelerà amb la construcció d'una nova església catedral a Santa Maria, que complementà l'aixecament d'un nou complex episcopal dins murs, en gran part culminat a mitjan segle XI i format per xenodoquium, catedral, palau episcopal i conjunt canonical, a banda de noves residències de canonges i altres clergues en el seu entorn. Aquests fets significaren la consolidació de Santa Maria com a seu única, però també la profunda remodelació urbanística d'un sector significatiu de la ciutat, prop d'un 20% del total, expressió del nou domini del bisbe i dels clergues sobre la 'seva ciutat'

Dengue-2 infection and the induction of apoptosis in human primary monocytes

Monocytes/macrophages are important targets for dengue virus (DENV) replication; they induce inflammatory mediators and are sources of viral dissemination in the initial phase of the disease. Apoptosis is an active process of cellular destruction genetically regulated, in which a complex enzymatic pathway is activated and may be trigged by many viral infections. Since the mechanisms of apoptotic induction in DENV-infected target cells are not yet defined, we investigated the virus-cell interaction using a model of primary human monocyte infection with DENV-2 with the aim of identifying apoptotic markers. Cultures analyzed by flow cytometry and confocal microscopy yielded DENV antigen positive cells with rates that peaked at the second day post infection (p.i.), decayed afterwards and produced the apoptosis-related cytokines TNF-α and IL-10. Phosphatidylserine, an early marker for apoptosis, was increased at the cell surface and the Fas death receptor was upregulated at the second day p.i. at significantly higher rates in DENV infected cell cultures than controls. However, no detectable changes were observed in the expression of the anti-apoptotic protein Bcl-2 in infected cultures. Our data support virus modulation of extrinsic apoptotic factors in the in vitro model of human monocyte DENV-2 infection. DENV may be interfering in activation and death mechanisms by inducing apoptosis in target cells.

A new approach for potential drug target discovery through in silico metabolic pathway analysis using Trypanosoma cruzi genome information

The current drug options for the treatment of chronic Chagas disease have not been sufficient and high hopes have been placed on the use of genomic data from the human parasite Trypanosoma cruzi to identify new drug targets and develop appropriate treatments for both acute and chronic Chagas disease. However, the lack of a complete assembly of the genomic sequence and the presence of many predicted proteins with unknown or unsure functions has hampered our complete view of the parasite's metabolic pathways. Moreover, pinpointing new drug targets has proven to be more complex than anticipated and has revealed large holes in our understanding of metabolic pathways and their integrated regulation, not only for this parasite, but for many other similar pathogens. Using an in silicocomparative study on pathway annotation and searching for analogous and specific enzymes, we have been able to predict a considerable number of additional enzymatic functions in T. cruzi. Here we focus on the energetic pathways, such as glycolysis, the pentose phosphate shunt, the Krebs cycle and lipid metabolism. We point out many enzymes that are analogous to those of the human host, which could be potential new therapeutic targets.

The vacuolar transporter chaperone (VTC) complex is required for microautophagy.

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.

A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

Structural organization of Trypanosoma cruzi

Since the initial description of Trypanosoma cruzi by Carlos Chagas in 1909, several research groups have used different microscopic techniques to obtain detailed information about the various developmental stages found in the life cycle of this intracellular parasite. This review describes the present knowledge on the organization of the most important structures and organelles found in the protozoan, such as the cell surface, flagellum, cytoskeleton, kinetoplast-mitochondrion complex, glycosome, acidocalcisome, contractile vacuole, lipid inclusions, the secretory pathway, endocytic pathway and the nucleus.

Usefulness of PCR-based assays to assess drug efficacy in Chagas disease chemotherapy: value and limitations

One major goal of research on Chagas disease is the development of effective chemotherapy to eliminate the infection from individuals who have not yet developed cardiac and/or digestive disease manifestations. Cure evaluation is the more complex aspect of its treatment, often leading to diverse and controversial results. The absence of reliable methods or a diagnostic gold standard to assess etiologic treatment efficacy still constitutes a major challenge. In an effort to develop more sensitive tools, polymerase chain reaction (PCR)-based assays were introduced to detect low amounts of Trypanosoma cruzi DNA in blood samples from chagasic patients, thus improving the diagnosis and follow-up evaluation after chemotherapy. In this article, I review the main problems concerning drug efficacy and criteria used for cure estimation in treated chagasic patients, and the work conducted by different groups on developing PCR methodologies to monitor treatment outcome of congenital infections as well as recent and late chronic T. cruzi infections.

Cellular and genetic mechanisms involved in the generation of protective and pathogenic immune responses in human Chagas disease

Perhaps one of the most intriguing aspects of human Chagas disease is the complex network of events that underlie the generation of protective versus pathogenic immune responses during the chronic phase of the disease. While most individuals do not develop patent disease, a large percentage may develop severe forms that eventually lead to death. Although many efforts have been devoted to deciphering these mechanisms, there is still much to be learned before we can fully understand the pathogenesis of Chagas disease. It is clear that the host's immune response is decisive in this process. While characteristics of the parasite influence the immune response, it is becoming evident that the host genetic background plays a fundamental role in the establishment of pathogenic versus protective responses. The involvement of three complex organisms, host, parasite and vector, is certainly one of the key aspects that calls for multidisciplinary approaches towards the understanding of Chagas disease. We believe that now, one hundred years after the discovery of Chagas disease, it is imperative to continue with highly interactive research in order to elucidate the immune response associated with disease evolution, which will be essential in designing prophylactic or therapeutic interventions.

Innate immunity and regulatory T-cells in human Chagas disease: what must be understood?

There is a general consensus that during chronic Trypanosoma cruzi infection, the host immune system induces complex processes to ensure the control of parasite growth while preserving the potential to mount and maintain a life-long controlled humoral and cellular immune response against the invading pathogen. This review summarises evidence in an attempt to elucidate "what must be understood" to further clarify the role of innate immunity in the development/maintenance of clinical Chagas disease and the impact of etiological treatment on host immunity, highlighting the contributions of the innate immunity and regulatory T (Treg) cells. Recently, increasing focus on innate immunity suggest that chronic T. cruzi infection may cause morbidity when innate effector functions, or the down-regulation of adaptive regulatory mechanisms are lacking. In this context, stable asymptomatic host-parasite interactions seem to be influenced by the effector/regulatory balance with the participation of macrophages, natural killer (NK) and CD8+ T cells in parallel with the establishment of regulatory mechanisms mediated by NKT and Treg cells. Moreover, a balanced innate immune activation state, apart from Treg cells, may play a role in controlling the adverse events triggered by the massive antigen release induced by trypanosomicidal agents during Chagas disease etiological treatment.

α-N-acetylglucosamine-linked O-glycans of sialoglycoproteins (Tc-mucins) from Trypanosoma cruzi Colombiana strain

Trypanosoma cruzi sialoglycoproteins (Tc-mucins) are mucin-like molecules linked to a parasite membrane via a glycosylphosphatidylinositol anchor. We previously determined the structures of Tc-mucin O-glycan domains from several T. cruzi strains and observed significant differences among them. We now report the amino acid content and structure of Tc-mucin O-glycan chains from T. cruzi Colombiana, a strain resistant to common trypanocidal drugs. Amino acid analysis demonstrated the predominance of threonine residues (42%) and helped to identify the O-glycans as belonging to a Tc-mucin family that contain a ²-galactofuranose (²-Galf) residue attached to an α-N-acetylglucosamine (α-GlcNAc) O-4, with the most complex glycan, a pentasaccharide-GlcNAc-ol with a branched trigalactopyranose chain, on the GlcNAc O-6. The presence of ²-Galf on O-glycans from T. cruzi Colombiana mucins supports the use of glycosylation as a phylogenetic marker for the classification of Colombiana in the T. cruzi I group.

Tissue and serum immune response in chronic hepatitis C with mild histological lesions

The immunopathogenesis of chronic hepatitis C virus (HCV) infection is a matter of great controversy and has been suggested to involve a complex balance between cytokines with pro and anti-inflammatory activity. We investigated the expression of inflammatory cells and cytokines in the liver and serum of 51 chronically HCV infected patients and compared them to data from two sets of normal controls: 51 healthy blood donors and 33 liver biopsies of healthy liver donors. We also assessed the relationship between selected cytokines and cell populations in hepatic compartments and the disease stage. Compared with controls, hepatitis C patients had a greater expression of portal TNF-α, TGF-β and CD4+ and acinar IFN-γ, TNF-α, IL-1β and IL-4, as well as a higher serum concentration of IL-2, IL-10 and TGF-β. Significant positive correlations were found between portal CD4+ and TNF-α, portal CD8+ and TGF-β, portal CD45+RO and TNF-α, acinar CD45+RO and IFN-γ and acinar CD57+ and TGF-β. In conclusion, we have shown that (i) in this sample of predominantly mild disease, the immune response was associated with a pro-inflammatory response pattern, (ii) CD4+ T-lymphocytes played a major role in orchestrating the immune response and (iii) these events primarily took place in the portal space.

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

Alpha 1,2-mannosidases from glycosyl hydrolase family 47 participate in N-glycan biosynthesis. In filamentous fungi and mammalian cells, α1,2-mannosidases are present in the endoplasmic reticulum (ER) and Golgi complex and are required to generate complex N-glycans. However, lower eukaryotes such Saccharomyces cerevisiae contain only one α1,2-mannosidase in the lumen of the ER and synthesise high-mannose N-glycans. Little is known about the N-glycan structure and the enzyme machinery involved in the synthesis of these oligosaccharides in the dimorphic fungus Sporothrix schenckii. Here, a membrane-bound α-mannosidase from S. schenckii was solubilised using a high-temperature procedure and purified by conventional methods of protein isolation. Analytical zymograms revealed a polypeptide of 75 kDa to be responsible for enzyme activity and this purified protein was recognised by anti-α1,2-mannosidase antibodies. The enzyme hydrolysed Man9GlcNAc2 into Man8GlcNAc2 isomer B and was inhibited preferentially by 1-deoxymannojirimycin. This α1,2-mannosidase was localised in the ER, with the catalytic domain within the lumen of this compartment. These properties are consistent with an ER-localised α1,2-mannosidase of glycosyl hydrolase family 47. Our results also suggested that in contrast to other filamentous fungi, S. schenckii lacks Golgi α1,2-mannosidases and therefore, the processing of N-glycans by α1,2-mannosidases is similar to that present in lower eukaryotes.

Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.

Genetic-morphometric variation in Culex quinquefasciatus from Brazil and La Plata, Argentina

Variation among natural populations of Culex (Culex) quinquefasciatus Say is associated with different vectorial capacities. The species Cx. quinquefasciatus is present in the equatorial, tropical and subtropical zones in the Brazilian territory, with intermediate forms between Cx. quinquefasciatus and Culex pipiens occurring in regions of latitudes around 33°-35°S. Herein, we studied geographically distinct populations of Cx. quinquefasciatus by genetic characterization and analysis of intra-specific wing morphometrics. After morphological analysis, molecular characterization of Cx. quinquefasciatus and intermediate forms was performed by polymerase chain reaction of the polymorphic nuclear region of the second intron of the acetylcholinesterase locus. Additionally, the morphology of adult female wings collected from six locations was analyzed. Wing centroid sizes were significantly different between some geographical pairs. Mean values of R2/R2+3 differed significantly after pairwise comparisons. The overall wing shape represented by morphometric characters could be divided into two main groupings. Our data suggest that Brazilian samples are morphologically and genetically distinct from the Argentinean samples and also indicated a morphological distinction between northern and southern populations of Brazilian Cx. quinquefasciatus. We suggest that wing morphology may be used for preliminary assessment of population structure of Cx. quinquefasciatusin Brazil.