Enteric Neuronal Damage,Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection

We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11^th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatory-induced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, the architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon.     

The structures of polysaccharides and glycolipids of Aspergillus fumigatus grown in the presence of human serum

A study was made of polysaccharides and glycosphingolipids isolated from Aspergillus fumigatus grown in media supplemented with human serum from healthy donors. Fractionation of Cetavlon-precipitated polysaccharides on Sephacryl S-400 gave rise to an excluded fraction (Fraction I) with molecular weight of >400 kDa and an included peak (Fraction II) with an average molecular weight of 30–80 kDa. Fraction I comprises about 5% of total polysaccharide and was identified as a glycogen-like molecule. Its structure was deduced from methylation data, treatment with amyloglucosidase, a red-brown coloration produced with an iodine solution and by ¹H and ¹³C-NMR spectroscopy. It was previously suggested that higher amounts of glycogen-like polysaccharide (20%) were present in A. fumigatus grown in serum-free medium. Fraction II was identified as a galactomannan and was the main polysaccharide of A. fumigatus grown in serum-supplemented medium. Its structure was elucidated mainly by ¹³C-NMR spectroscopy combined with partial acetolysis and methylation analysis. The ¹³C-NMR spectrum of the galactomannan showed a much greater complexity in the -d-galf and -d-manp C-1 regions, than was evident for galactomannan from serum-free cultures previously described, reflecting differences in the glycosylation pattern, stimulated in serum-supplemented medium.No differences in A. fumigatus glycosphingolipid could be detected between serum-containing and serum-free growth conditions.Our results demonstrate that the change in polysaccharide structure is a more specific response to the altered growth conditions and not merely a symptom of more general changes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Trypanosoma cruzi: acute and long-term infection in the vertebrate host can modify the response to benznidazole

We analyzed the influence of Trypanosoma cruzi maintenance in different hosts (dog and mouse) on its susceptibility to benznidazole treatment. Five T. cruzi stocks were isolated from dogs inoculated with Be-62 or Be-78 strain (both sensitive to benznidazole) 2-10 years ago, and the benznidazole sensitivity was then determined using the mouse as experimental model. The different T. cruzi stocks obtained from long-term infected dogs showed 50-90% drug resistance right after isolation. However, maintenance of these T. cruzi stocks in mice, by successive blood passages (2.5 years), led to either a decrease or stability of the drug resistance pattern and an increase in parasite virulence. We also demonstrated the effectiveness of the induction of parasitemia reactivation by cyclophosphamide immunosuppression in the evaluation of the response to the specific drug treatment.     

Diagnoses of and illustrated key to the species of <Emphasis Type="Italic">Ixodes</Emphasis> Latreille, 1795 (Acari: Ixodidae) from Brazil

The current Brazilian Ixodes fauna is composed of the following eight species: I. amarali Fonseca, 1935; I. aragaoi Fonseca, 1935; I. auritulus Neumann, 1904; I. fuscipes Koch, 1844; I. loricatus Neumann, 1899; I. luciae Sénevet, 1940; I. paranaensis Barros-Battesti, Arzua, Pichorim &; Keirans, 2003; and I. schulzei Aragão &; Fonseca, 1951. Further studies are needed to establish the taxonomic status of I. serrafreirei Amorim, Gazeta, Bossi &; Linhares, 2003, a recently proposed species based solely on the nymphal stage. We present an up-to-date key to adults of the currently valid Brazilian species of Ixodes based on scanning electron microscopy. The relationships between Brazilian and other Neotropical Ixodes are also discussed.     

Characterization of Polymeric Solutions as Injectable Vehicles for Hydroxyapatite Microspheres

A polymeric solution and a reinforcement phase can work as an injectable material to fill up bone defects. However, the properties of the solution should be suitable to enable the transport of that extra phase. Additionally, the use of biocompatible materials is a requirement for tissue regeneration. Thus, we intended to optimize a biocompatible polymeric solution able to carry hydroxyapatite microspheres into bone defects using an orthopedic injectable device. To achieve that goal, polymers usually regarded as biocompatible were selected, namely sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and Na-alginate (ALG). The rheological properties of the polymeric solutions at different concentrations were assessed by viscosimetry before and after moist heat sterilization. In order to correlate rheological properties with injectability, solutions were tested using an orthopedic device applied for minimal invasive surgeries. Among the three polymers, ALG solutions presented the most suitable properties for our goal and a non-sterile ALG 6% solution was successfully used to perform preliminary injection tests of hydroxyapatite microspheres. Sterile ALG 7.25% solution was found to closely match non-sterile ALG 6% properties and it was selected as the optimal vehicle. Finally, sterile ALG 7.25% physical stability was studied at different temperatures over a 3-month period. It was observed that its rheological properties presented minor changes when stored at 25°C or at 4°C.     

Genotoxic effects of white fluorescent light on human lymphocytes in vitro

Sources of light beams such as white fluorescent light, are present in our daily life to meet the needs of life in the modern world. This study was conducted with the objective of determining the possible genotoxic, cytotoxic and aneugenic effects caused by this agent in different stages of the cell cycle (G0/early G1, S, and late G2), using different cytogenetic parameters (sister chromatid exchanges--SCE, chromosome aberrations--CA, and detection of aneugenic effects) in lymphocytes from temporary cultures of human peripheral blood. WFL showed a genotoxic effect in vitro, expressed by an increase in the frequency of SCE's, regardless of the cell cycle stage. However, no increase in the frequency of CAs was observed. In addition, disturbances in cell cycle kinetics and chromosomal segregation were also observed. Taken together, such data may contribute to a better understanding and a different management in the use of phototherapy for some pathological conditions.     

Trypanosoma cruzi: immunoglobulin isotype profiles during the acute phase of canine experimental infection with metacyclic or blood trypomastigotes

A detailed investigation has been carried out about the serological profiles of groups of dogs experimentally infected with metacyclic (MT) or blood (BT) trypomastigotes of Berenice-78 Trypanosoma cruzi strain. Peripheral blood was collected from infected dogs and uninfected controls, weekly during 35 days following the acute phase of infection, and immunoglobulin profiles were determined by ELISA. Dogs infected with BT exhibited unaltered levels of IgG2, increases in IgM, IgE, IgA, IgG and IgG1. In contrast, dogs infected with MT presented unaltered levels of IgE and IgG1 and an increase in IgM, IgA, IgG and IgG2 levels. Compared with the MT group, animals infected with BT showed significant increases in IgM on days 7, 14 and 28, in IgA on days 7, 14 and 21, in IgE on days 7 and 14, in IgG on days 14 and 28, and in IgG1 on days 7, 14 and 21. Parasitemia levels of the infected animals were measured over the same time period. No correlations were found between the immunoglobulin profiles and the parasitemia levels. The results demonstrated that the inoculum source (BT or MT) influence the immunoglobulin isotype profile that may drive distinct outcome of acute canine Chagas disease.     

Variation in susceptibility to benznidazole in isolates derived from Trypanosoma cruzi parental strains.

In this work, the susceptibility to benznidazole of two parental Trypanosoma cruzi strains, Colombian and Berenice-78, was compared to isolates obtained from dogs infected with these strains for several years. In order to evaluate the susceptibility to benznidazole two groups of mice were infected with one of five distinct populations isolated from dogs as well as the two parental strains of T. cruzi. The first group was treated with benznidazole during the acute phase and the second remained untreated controls. The animals were considered cured when parasitological and serological tests remained persistently negative. Mice infected with the Colombian strain and its isolates Colombian (A and B) did not cure after treatment. On the other hand, all animals infected with Berenice-78 were cured by benznidazole treatment. However, 100%, 50% and 70% of cure rates were observed in animals infected with the isolates Berenice-78 B, C and D, respectively. No significant differences were observed in serological profile of infected control groups, with all animals presenting high antibody levels. However, the ELISA test showed differences in serological patterns between mice inoculated with the different T. cruzi isolates and treated with benznidazole. This variability was dependent on the T. cruzi population used and seemed to be associated with the level of resistance to benznidazole.     

Stroma cell-derived factor 1alpha mediates desensitization of human neutrophil respiratory burst in synovial fluid from rheumatoid arthritic patients

Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1alpha (SDF1alpha) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1alpha in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1alpha failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1alpha-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1alpha actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1alpha beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1alpha promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.