Intestinal spirochetosis: first cases reported in Brazil and the use of immunohistochemistry as an aid in histopathological diagnosis

Colonization of the colon and rectum by intestinal spirochetes is detected for the first time in Brazil in 4 of 282 (1.41%) patients who had undergone sigmoidoscopy and/or colonoscopy with a histopathological diagnosis of chronic non specific-colitis. This frequency is probably understimated, since surgically obtained specimens were not considered in the present study. Histopathological diagnosis was performed using routine stains like hematoxylin-eosin which showed the typical, of 3-µm thick hematoxyphilic fringe on the brush border of the surface epithelium, and by silver stains like the Warthin-Starry stain. Immunohistochemical procedures using two, polyclonal, primary antibodies, one against Treponema pallidum and the other against Leptospira interrogans serovar copenhageni serogroup Icterohaemorrhagiae cross-reacted with spirochetal antigen/s producing a marked contrast of the fringe over the colonic epithelium, preserving the spiral-shaped morphology of the parasite. In one case with marked diarrhea, immunohistochemistry detected spirochetal antigen/s within a cell in an intestinal crypt, thus demonstrating that the infection can be more widely disseminated than suspected using routine stains. Immunohistochemical procedures, thus, greatly facilitate the histological diagnosis of intestinal spirochetosis and may contribute to a better understanding of the pathogenesis of the disease. Transmission and scanning electron microscopy performed in one case showed that the spirochete closely resembled the species designated as Brachyspira aalborgi.

Stability of the Transthyretin Molecule as a Key Factor in

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/ABeta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4- (3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

A 3-YEAR FOLLOW-UP OF A BRAZILIAN AIDS PATIENT WITH PROTRACTED DIARRHEA CAUSED BY Enterocytozoon bieneusi

Enterocytozoon bieneusi is the most prevalent microsporidian parasite that causes gastrointestinal infection in persons with AIDS. Microsporidia are increasingly recognized as important opportunistic pathogens all over the world but in Brazil only few cases have been reported due either to the non awareness of the clinical presentation of the disease or to difficulties in the laboratory diagnosis. We report a 3-year follow-up of a Brazilian HIV-positive patient in whom microsporidial spores were detected in stools and were identified as E. bieneusi using electron microscopy and PCR. The patient presented with chronic diarrhea, CD4 T-lymphocytes count below 100/mm3 and microsporidial spores were consistently detected in stools. Albendazole was given to the patient in several occasions with transient relief of the diarrhea, which reappeared as soon as the drug was discontinued. Nevertheless, a diarrhea-free period with weight gain up to 18 Kg occurred when a combination of nucleoside and protease inhibitors was initiated as part of the antiviral treatment.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.

Development of new biomaterials based on liquid crystalline phases of cellulose derivatives

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Engenharia Biomédica

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N2-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.

Development of 2-oxazoline-based hydrogels and porous polyester microparticles in scCO2

Thesis submitted to Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa in partial fulfillment of the requirements for the obtention of the degree of Master of Science in Biotechnology

Clinical and diagnostic aspects of intestinal microsporidiosis in HIV-infected patients with chronic diarrhea in Rio de Janeiro, Brazil

The objectives of this study were to determine both the prevalence of microsporidial intestinal infection and the clinical outcome of the disease in a cohort of 40 HIV-infected patients presenting with chronic diarrhea in Rio de Janeiro, Brazil. Each patient, after clinical evaluation, had stools and intestinal fragments examined for viral, bacterial and parasitic pathogens. Microsporidia were found in 11 patients (27.5%) either in stools or in duodenal or ileal biopsies. Microsporidial spores were found more frequently in stools than in biopsy fragments. Samples examined using transmission electron microscopy (n=3) or polymerase chain reaction (n=6) confirmed Enterocytozoon bieneusi as the causative agent. Microsporidia were the only potential enteric pathogens found in 5 of the 11 patients. Other pathogens were also detected in the intestinal tract of 21 patients, but diarrhea remained unexplained in 8. We concluded that microsporidial infection is frequently found in HIV infected persons in Rio de Janeiro, and it seems to be a marker of advanced stage of AIDS.

Whipple's disease. Report of five cases with different clinical features

Whipple's disease (WD) is a rare systemic disease of infectious etiology which involves the small intestine but can virtually affect any organ. We present here five cases (four males and one female) ranging in age from 20 to 59 years. All patients had intestinal involvement associated or not with clinical manifestations linked to this organ. Vegetation in the tricuspid valve was observed in one patient, suggesting endocarditis caused by Tropheryma whippelii, with disappearance of the echocardiographic alterations after treatment. In one of the male patients the initial clinical manifestation was serologically negative spondylitis, with no diarrhea occurring at any time during follow-up. Ocular involvement associated with intestinal malabsorption and significant weight loss were observed in one case. In the other two cases, diarrhea was the major clinical manifestation. All patients were diagnosed by histological examination of the jejunal mucosa and, when indicated, of extraintestinal tissues by light and electron microscopy. After antibiotic treatment, full remission of symptoms occurred in all cases. A control examination of the intestinal mucosa performed after twelve months of treatment with sulfamethoxazole-trimethoprim revealed the disappearance of T. whippelii in four patients. The remaining patient was lost to follow-up.

Biological and genetic characteristics of uropathogenic Escherichia coli strains

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Development of electrospun Ion jelly fibers® for drug delivery

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Mycoplasma pneumoniae and Chlamydia pneumoniae in calcified nodules of aortic stenotic valves

Aortic Valve Stenosis (AVS) has been explained as an atherosclerotic process of the valve as they often exhibit inflammatory changes with infiltration of macrophages, T lymphocytes and lipid infiltration. The present study investigated whether the bacteria Chlamydia pneumoniae (CP) and Mycoplasma pneumoniae (MP), detected previously in atherosclerotic plaques, are also present in AVS. Ten valves surgically removed from patients with AVS were analyzed by immunohistochemistry, in situ hybridization, and electron microscopy. The mean and standard deviation of the percentage areas occupied by CP antigens and MP - DNA were respectively 6.21 +/- 5.41 and 2.27 +/- 2.06 in calcified foci; 2.8 +/- 3.33 and 1.78+/- 3.63 in surrounding fibrotic areas, and 0.21 +/- 0.17 and 0.12 +/- 0.13 in less injured parts of the valve. There was higher amount of CP and MP in the calcified foci and in the surrounded fibrosis than in more preserved valvular regions. In conclusion, the fact that there were greater amounts of CP and MP in calcification foci of AVS favors the hypothesis that AS is not an inevitable degenerative process due to aging, but rather that it may be a response to the presence of these bacteria, similarly to the morphology detected in atherosclerosis damage.

Smart Plastic Antibody Material for Hemoglobin Tailored by Silica Surface Imprinting and with Charged Binding Sites: Its use as Ionophore in Potentiometric Transduction

JORNADAS DE ELECTROQUÍMICA E INOVAÇÃO 2013

Isolation of Fonsecaea pedrosoi from thorns of Mimosa pudica, a probable natural source of chromoblastomycosis

We report the isolation of Fonsecaea pedrosoi from thorns of the plant Mimosa pudica L. at the place of infection identified by one of our patients. Clinical diagnosis of chromoblastomycosis was established by direct microscopic examination and cultures from the patient's lesion. The same species was isolated from the patient and from the plant. Scanning electron microscopy of the surface of the thorns showed the characteristic conidial arrangement of F. pedrosoi. These data indicate that M. pudica could be a natural source of infection for the fungus F. pedrosoi.

Some ultrastructural superficial changes in house fly (Diptera: Muscidae) and blow fly (Diptera: Calliphoridae) larvae induced by eucalyptol oil

The ultrastructural superficial changes in third instar house fly (Musca domestica) and blow fly (Chrysomya megacephala) induced by eucalyptol oil were observed using scanning electron microscopy. Dipped in 0.902 g/ml eucalyptol for 30 sec, the larvae integument of both species showed significant aberrant appearance of the body surface, particularly swelling integument, bleb formation, partial breach and deformation of spines.