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The flatwormSchistosoma mansoniis a blood fluke parasite that causes schistosomiasis, a debilitating disease that occurs throughout the developing world. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control schistosomiasis is through immunization with an antischistosomiasis vaccine combined with drug treatment. In the search for potential vaccine candidates, numerous tegument antigens have been assessed. As the major interface between parasite and mammalian host, the tegument plays crucial roles in the establishment and further course of schistosomiasis. Herein, we evaluated the potential of a GPI fraction, containing representative molecules located on the outer surface of adult worms, as vaccine candidate. Immunization of mice with GPI-anchored proteins induced a mixed Th1/Th2 type of immune response with production of IFN-γand TNF-α, and low levels of IL-5 into the supernatant of splenocyte cultures. The protection engendered by this vaccination protocol was confirmed by 42% reduction in worm burden, 45% reduction in eggs per gram of hepatic tissue, 29% reduction in the number of granulomas per area, and 53% reduction in the granuloma fibrosis. Taken together, the data herein support the potential of surface-exposed GPI-anchored antigens from theS. mansonitegument as vaccine candidate.

In human beings, the central vein of the adrenal glands has been considered by some authors to be one of the preferential sites of Trypanosoma cruzi settling in the chronic form of Chagas' disease. This appears to be due to the local corticoid-rich environment that may protect the parasite against the immunological defences of the host. An immunocytochemical study of the adrenal glands of 15 dogs was carried out for the detection of tissue parasitism in the acute and chronic phases of experimental T. cruzi infection. No amastigote nests or isolated amastigotes were detected in the adrenal parenchyma or adrenal central vein of the animals studied. Minimal nonspecific inflammatory changes were found in some glands of both infected and noninfected animals. The present results show that the adrenal glands of the dog are not injured and do not constitute a site of escape or reservoir of parasites in experimental Chagas' disease.

Background Melatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites. Methods The effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites. Results The areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae. Conclusions These results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.

Nitric oxide (NO) and reactive oxygen species (ROS) are key molecules in resistance to pathogens. Little is known about their role in pathogenesis of periapical lesions. To address this issue, we induced periapical lesions in mice lacking nitric oxide synthase (iNOS-/-) or phagocyte oxidase (PHOX-/-). iNOS-/- mice expressed higher levels of IL-1β, TNF-α, RANK, RANKL, and MCP-1 than C57BL/6 and PHOX-/-. Apical thickening of the periodontal ligament was also greater in iNOS-/- compared with other groups. Interestingly, ROS production did not interfere in periapical lesion progression, but seemed to be essential for the appearance of multinucleated TRAP-positive cells. Thus, periapical lesion progression in iNOS-/- was associated with an imbalance of pro-inflammatory cytokines (IL-1β and TNF-α), bone-resorptive modulators (RANK and RANKL), and MCP-1. We conclude that NO, but not ROS, controls progression of bone resorption in a murine experimental model of apical periodontitis.

In this study we evaluated the cardiac effects of a pharmaceutical formulation developed by including angiotensin (Ang)-(1-7) in hydroxypropyl β-cyclodextrin (HPβCD), in normal, infarcted, and isoproterenol-treated rats. Myocardial infarction was produced by left coronary artery occlusion. Isoproterenol (2 mg/kg, IP) was administered daily for 7 days. Oral administration of HPβCD/Ang-(1-7) started immediately before infarction or associated with the first dose of isoproterenol. After 7 days of treatment, the rats were euthanized, and the Langendorff technique was used to analyze cardiac function. In addition, heart function was chronically (15, 30, 50 days) analyzed by echocardiography. Cardiac sections were stained with hematoxylin/eosin and Masson trichrome to evaluate cardiac hypertrophy and damage, respectively. Pharmacokinetic studies showed that oral HPβCD/Ang-(1-7) administration significantly increased Ang-(1-7) on plasma whereas with the free peptide it was without effect. Oral administration of HPβCD/Ang-(1-7) (30 μg/kg) significantly reduced the deleterious effects induced by myocardial infarction on systolic and diastolic tension, ±dT/dt, perfusion pressure, and heart rate. Strikingly, a 50% reduction of the infarcted area was observed in HPβCD/Ang-(1-7)–treated rats. Furthermore, HPβCD/Ang-(1-7) attenuated the heart function impairment and cardiac remodeling induced by isoproterenol. In infarcted rats chronically treated with HPβCD/Ang-(1-7), the reduction of ejection fraction and fractional shorting and the increase in systolic and diastolic left ventricular volumes observed in infarcted rats were attenuated. Altogether, these findings further confirm the cardioprotective effects of Ang-(1-7). More importantly, our data indicate that the HPβCD/Ang-(1-7) is a feasible formulation for oral administration of Ang-(1-7), which can be used as a cardioprotective drug.

Metallothionein (MT) is a protein correlated with cellular differentiation and proliferation, as well as with the inhibition of apoptosis. The aims were to report and to compare the MT expression in odontogenic cysts and keratocystic odontogenic tumor (KOT); to correlate the MT with cellular proliferation; and to evaluate the influence of the inflammation in MT.Nine cases of radicular cyst (RC), nine dentigerous cyst (DC), four orthokeratinized odontogenic cyst (OOC), and eight KOT were submitted to immunohistochemistry using anti-MT and anti-Ki-67. Indexes of MT (IMT) and Ki-67 (IK) were obtained. Lesions were grouped according to inflammation: mild-to-moderate (group A) and intense (group B).IMT proved to be highest in RC (91%), followed by DC (89%), KOT (78%), and OOC (63%). IMT was inversely correlated with IK in KOT, and OCC, but was positively correlated with RC and DC. No differences in IMT and in IK could be observed between groups A and B.The higher IMT found in RC and DC compared to OCC and KOT, as well as the differences between the last ones, is possibly correlated with their different histopathological features and clinical behavior. In RC and DC, MT may play a role in cellular proliferation. However, it seems that MT is either less or is not related to proliferation in OOC and in KOT. Moreover, inflammation does not seem to alter IMT and IK.

The most important risk factor linked to the development of oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) is tobacco use. Tobacco contains carcinogens that influence the DNA repair, cell cycle control and may produce chromosomal aberrations. The loss or acquisition of one or more chromosomes is defined as aneuploidy.Aneuploidy was determined by means of the DNA-content included in cells obtained by exfoliative cytology and Feulgen's staining. The cells were collected from the clinically healthy lateral margin of the tongue of non-smokers without oral lesions, smokers without oral lesions, smokers with OL, and smokers with OSCC, using the CytoBrush(®). Each group was composed of 20 individuals. A Carl Zeiss image analyzer system and the KS300 software were used. Statistical analysis was performed with BioEstat(®) software.The mean percentage of aneuploid nuclei was statistically higher in the smokers (79.65%), smokers with OL (68.4%), and smokers with OSCC (93.65%), as compared to non-smokers (39.3%) (P0.05). A trend toward an increase in the aneuploidy of the smokers with OSCC group (P=0.02), as compared to the non-smoker group, could be observed. No significant difference could be observed as regards the mean percentage of aneuploid nuclei in relation to duration of tobacco use or the number of the cigarettes smoked.Tobacco use is responsible for an increased number of aneuploid nuclei in the oral epithelium.

BackgroundHuman amoebiasis is caused by the parasitic protozoanEntamoeba histolyticathat lives in the large intestine of hosts, where can produce asymptomatic colonization until severe invasive infections with blood diarrhea and spreading to other organs. The amoebic abscesses in liver are the most frequent form of amoebiasis outside intestine and still there are doubts about the pathogenic mechanisms involved in their formation. In this study we evaluated thein situbinding of antibodies, C3 and C9 complement components on trophozoites, in livers of hamsters infected withE. histolyticaorE. dispar. These parameters were correlated with the extension of the hepatic lesions observed in these animals and with trophozoites survivor.MethodsHamsters were inoculated intra-hepatically with 100,000 trophozoites ofE. histolyticaorE. disparstrain and necropsied 12, 24, 48, 72, 144 and 192 h after inoculation. Antibodies, C3 and C9 binding to trophozoites were detected by immunohistochemistry. The estimation of the necrosis area and the number of labeled trophozoites was performed using digital morphometry analysis.ResultsIn the liver sections of animals inoculated with the amoebas, the binding of antibodies toE. histolyticatrophozoites was significantly lower than toE. dispartrophozoites. Trophozoites ofE. disparwere also more frequently vacuolated and high labeled cellular debris observed in the lesions. Positive diffuse reaction to C3 complement component was more intense in livers of animals inoculated withE. histolyticaafter 24 and 72 h of infection. C3(+)and C9(+)trophozoites were detected in the vascular lumen, granulomas and inside and in the border of necrotic areas of both infected group animals. C3(+)and C9(+)trophozoite debris immunostaining was higher in livers ofE. disparthan in livers ofE. histolytica. A positive correlation between necrotic areas and number of C9(+)trophozoites was observed in animals inoculated withE. dispar.ConclusionMorphological and immunohistochemical results suggest that antibodies and complement are able to bind and destroy some trophozoites in the liver of experimentally infected hamsters, perhaps selecting the more resistant parasites which are responsible by progression of amoebic abscesses. The findings indicate thatE. histolyticapossesses an enhanced abilityin vivoto evade the immune responses compared toE. dispar, although it also causes experimental hepatic lesions.

SUMMARYProteins associated with the schistosome tegument are of great importance for the development of new intervention strategies since they may be exposed on the surface of the parasite. Herein, we have isolated a cDNA clone encoding for theSchistosoma mansoniSmIg and its recombinant protein was tested as a potential vaccine candidate. Initially, its amino acid sequence was analysed by bioinformatics and shown to possess an N-terminal signal peptide, a C-terminal transmembrane helix, 4 glycosylation sites, an immunoglobulin conserved domain and 73% similarity with a hypotheticalS. japonicumprotein of unknown function. SmIg was produced byE. colias a recombinant protein (rSmIg) and its protective effectiveness was evaluated againstS. mansoniinfection with 100 cercariae in a murine model. Mice immunized with rSmIg induced an immune response characterized by dominant IgG1 isotype and significant levels of IFN-γ, TNF-α, IL-10 and IL-4. Although immunogenic, the recombinant vaccine failed to induce worm burden reduction when compared to the infected control group. However, rSmIg-immunized mice had significant reductions of liver granuloma volume and fibrosis content by 31·8% and 49%, respectively. In conclusion, SmIg is a new tegument protein fromS. mansonithat plays an important role in reducing pathology induced by parasite infection.

Summary The aim of this work was to study alterations in the extracellular matrix of liver in dogs naturally infected with Leishmania (Leishmania) chagasi that are correlated with clinical aspects and with histological, parasitological and immunological findings. The study was carried out on 30 dogs, 10 uninfected (control group) and 20 infected. The infected animals were further divided into two groups: an asymptomatic group of 10 dogs without clinical signs of the disease; and a symptomatic group of 10 dogs with classical clinical signs. All thirty animals were mongrel dogs of undefined age, obtained from the municipality of Belo Horizonte, MG, metropolitan area. During necropsy, liver fragments were collected and fixed in 10% buffered formaldehyde for histological examination. Paraffined sections of the tissues were stained with haematoxylin-eosin, Gomori's ammoniacal silver stain for reticular fibres and strepto-avidin peroxidase for immunohistochemical detection of Leishmania amastigotes. Frozen tissue sections were stained by immunofluorescence for fibronectin (FN) and laminin (LN). Liver collagen deposition was significantly greater in the infected than the control animals and differed significantly between the symptomatic and asymptomatic dogs. There was a positive correlation between the parasite load and liver collagen deposition. The increased collagen deposition in infected animal livers may be associated with the parasite burden. Adhesive FN and LN fibres were significantly more highly expressed in the livers of symptomatic than of asymptomatic dogs. Our results demonstrate that canine visceral leishmaniasis causes fibrogenesis in liver, associated with the parasite load and degenerative processes.

The study aimed to evaluate the suitability of myeloperoxidase (MPO) content as a local indicator of chronic inflammation, using the periodontal disease model. Anesthetized adult male Holtzman rats had their second left maxilar molar tied by a thread for 11 days and were then killed. Blood samples and photographic images from histopathological inflamed and noninflamed (contralateral) neighboring gingivomucosal specimens were collected for cell counts and MPO level analysis. Diseased animals were also treated with pharmacological tools such as the anti-inflammatory drug celecoxib or the opioid morphine. Increased blood neutrophils and local cell numbers characterized diseased animals. However, local MPO content was increased in inflamed and noninflamed tissues from diseased animals. Celecoxib and morphine reduced blood neutrophils and bilateral MPO content, but only celecoxib reduced local cell numbers in diseased animals. It is concluded that MPO content is a good indicator of a systemic rather than a local inflammation in a chronic inflammatory condition.

Background and Objective: Periodontal disease is an inflammatory condition of tooth-supporting tissues. Arachidonic acid metabolites have been implicated in development of periodontal disease, especially those derived from the cyclo-oxygenase (COX) pathway. This study investigated the role of inhibitors of cyclo-oxygenases (COX-1 and COX-2) in a model of periodontal disease in rats. Material and Methods: A ligature was placed around the molar of rats. Losses of fiber attachment and of alveolar bone were measured morphometrically in histologically prepared sections. Infiltration of cells into gingival tissue surrounding the ligated tooth was also determined. Results: Systemic and local administration of non-selective and selective COX-2 inhibitors, preventively, resulted in significant reduction of the losses of fiber attachment and alveolar bone, as well as decreased leukocyte numbers in gingival tissue. Preventive selective inhibition of COX-1 was as effective as COX-2 inhibition in reducing local fiber attachment loss and cell migration, but did not prevent alveolar bone loss. Conclusion: Our results provide evidence for participation of COX-1 and COX-2 in early stages of periodontal disease in rats. Furthermore, local administration of COX inhibitors reduced the signs of periodontal disease to the same extent as systemic treatment. Therapeutic approaches incorporating locally delivered anti-inflammatory drugs could be of benefit for patients suffering from periodontal disease.

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions.

Schistosomiasis continues to be a significant public health problem that affects 200 million people worldwide. This is one of the most important parasitic diseases, and one whose effective control is unlikely in the absence of a vaccine. In this study, we have isolated a cDNA clone encoding the Schistosoma mansoni Sm21.6 protein that has 45% and 44% identity with Sm22.6 and Sj21.7 EF-hand containing antigens, respectively. Confocal microscopy analysis revealed that Sm21.6 is a membrane-associated protein localized on the S. mansoni adult worm. Mouse immunization with rSm21.6 induced a mixed Th1/Th2 cytokine profile and no protection against infection. However, vaccination with rSm21.6 reduced by 28% of liver granuloma numbers, 21% of granuloma area and 34% of fibrosis. Finally, rSm21.6 was recognized by sera from individuals resistant to reinfection compared with patients susceptible to reinfection and this molecule should be further studied as potential biomarker for disease resistance. In conclusion, Sm21.6 is a new tegument protein from S. mansoni that plays an important role in reducing pathology induced by parasite infection.

Background and Objective: Periodontal disease is a chronic inflammatory condition of the tooth supporting tissues, the periodontium. Opioids have been shown to account for the relief of various chronic and acute inflammatory conditions. The aim of the present study was to investigate the participation of peripheral opioid receptors in development of periodontal disease. Material and Methods: Morphine and selective agonists and antagonists of opioid receptors were used in an experimental model of ligature-induced periodontal disease in rats. To evaluate the development of disease, the loss of fiber attachment, alveolar bone and number of cells in periodontal tissues were assessed. Measurements of these indicators were obtained by morphometric analysis of histological sections of periodontal-diseased tissues stained with hematoxylin and eosin. Results: Local administration of either morphine or a selective κ-opioid agonist for three consecutive days from the onset of periodontal disease reduced the loss of periodontal tissues, without changing the number of leukocytes in inflamed periodontium. Nor-binaltorphimine, a selective κ-antagonist, reversed the beneficial effects of both morphine and the compound U-50,488 in this model. The use of either an agonist or an antagonist of δ-opioid receptors, however, did not affect disease progression. Conclusion: Our results showed that the beneficial effect of opioids in periodontal disease depended mainly on the activation of specific κ-opioid receptors located in the periphery. Activation of such receptors could be considered in the management of periodontal disease, since it would not present the classical central side-effects associated with opioid use.

Background Schistosomiasis continues to be a significant public health problem. This disease affects 200 million people worldwide and almost 800 million people are at risk of acquiring the infection. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of Schistosoma mansoni. Our group recently identified Sm29, another antigen that is present at the adult worm tegument surface. In this study, we investigated murine cellular immune responses to recombinant (r) Sm29 and tested this protein as a vaccine candidate. Methods and Findings We first show that Sm29 is located on the surface of adult worms and lung-stage schistosomula through confocal microscopy. Next, immunization of mice with rSm29 engendered 51%, 60% and 50% reduction in adult worm burdens, in intestinal eggs and in liver granuloma counts, respectively (p, Author Summary Schistosomiasis is the most important human helminth infection in terms of morbidity and mortality. Although the efforts to develop a vaccine against this disease have experienced failures, a new generation of surface antigens revealed by proteomic studies changed this scenario. Our group has characterized the protein Sm29 described previously as one of the most exposed and expressed antigens in the outer tegument of Schistosoma mansoni. Studies in patients living in endemic areas for schistosomiasis revealed high levels of IgG1 and IgG3 anti-Sm29 in resistant individuals. In this study, confocal microscope analysis showed Sm29 present in the surface of lung-stage schistosoluma and adult worms. Recombinant Sm29, when used as vaccine candidate, induced high levels of protection in mice. This protection was associated with a typical Th1 immune response and reduction of worm burden, liver granulomas and in intestinal eggs. Further, microarray analysis of worms recovered from vaccinated mice showed significant down-regulation of several genes encoding previously characterized vaccine candidates and/or molecules exposed on the surface, suggesting an immune evasion strategy of schistosomes under immune attack. These results demonstrated that Sm29 as one of the important antigens with potential to compose a vaccine against schistosomiasis.

Avaliou-se o efeito de ácidos graxos voláteis (AGV) sobre a integridade do epitélio no rúmen, no plano nasolabial, na epicera e no perioplum traseiro e dianteiro de bezerros e validou-se a feitura de biópsias tegumentares como indicadores de alterações morfológicas da mucosa ruminal. Dezessete bezerros, com sonda no rúmen, receberam infusões intra-ruminais de AGV ou salina, durante 37 dias. Aos 89 dias de vida, após o abate, foram colhidas amostras dos tecidos. Os AGV aumentaram a área de epitélio total e a área de células metabolicamente ativas no epitélio ruminal, embora o butirato não tenha induzido ao desenvolvimento papilar. A área de epitélio não queratinizado no plano nasolabial foi reduzida pela infusão de AGV. Butirato e lactato foram mais indutores de alterações patológicas no epitélio ruminal. Não foram observadas lesões histológicas nos epitélios do plano nasolabial, da epicera e do perioplum, mostrando que essas são conseqüências do efeito direto dos AGV sobre o epitélio ruminal. Os efeitos indireto e direto dos AGV sobre a morfologia dos tecidos epiteliais queratinizados não foram iguais. Biópsias tegumentares podem ter utilidade como indicadores de alterações morfológicas da mucosa ruminal. The effect of volatile fatty acids (VFA) on rumen wall, epidermis of nasolabial surface, perioplum, and epicera of calves was evaluated. The experiment also aimed to validate the procedure of tegument biopsies as indicators of ruminal mucosa alterations. Seventeen neonatal calves with foley catheters received intraruminal infusions of VFA or saline, during 37 days. At 89-day-old, the animals were slaughtered and tissue samples were collected from rumen, nasolabial surface, epicera, and perioplum from face and hindquarters. VFA infusion increased total epithelium area and metabolically active ruminal cell area; although butirate did not induce the papilar development. The effect of nasolabial surface VFA infusion was the opposite to that observed in the rumen. No histological lesion was observed on nasolabial surface, epicera, and perioplum, demonstrating that these are consequences of VFA direct effect on ruminal epithelium. Butyrate and lactate induced more alterations on the ruminal epithelium. Indirect and direct VFA effects on keratinized epithelium tissues morphology were not identical. Tegumentary biopsies may be useful as indicators of morphological alterations of ruminal mucosa.

The aim of this study was to evaluate the effects of AVE 0991 (AVE), a nonpeptide compound that mimics Ang-(1-7) actions, on cardiac remodeling. Heart hypertrophy and heart dysfunction were induced by isoproterenol (ISO) (2 mg/kg i.p./day for 7 days) in male Wistar rats. At the end of the 7-day period, the hearts were perfused according to the Langendorff method to evaluate cardiac function. The hearts, atria, and right and left ventricles wet weights were recorded, normalized for body weight and then expressed as muscle mass index (mg/g). In addition, serial sections from left ventricle were stained with hematoxylin-eosin for cell morphometry and with collagen-specific Masson's trichrome for detection of fibrosis. Immunofluorescence-labeling and confocal microscopy were used to investigate the distribution and deposition of collagen types I, III, VI, and fibronectin. AVE reduced the ISO-induced hypertrophy as quantified by myocyte diameter measurements (Control: 10.60+/-0.08 microm; ISO: 14.60+/-0.11 mum; ISO+AVE: 11.22+/-0.08 microm, n = 5). In addition, AVE markedly attenuated the increase of extracellular matrix proteins induced by ISO. AVE treatment also attenuated the decrease in systolic tension and +/-dT/dt and exacerbated the vasodilatation induced by ISO. These results show that AVE has a cardioprotective effect on ISO-induced cardiac remodeling.

Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica. This ameba can colonize the human intestine and persist as a commensal parasite, similar to Entamoeba dispar, an ameba considered to be non-pathogenic. The similarities between E. histolytica and E. dispar make the latter an attractive model for studies aimed at clarifying the pathogenesis of amebiasis. However, in addition to being an interesting experimental model, this relative of E. histolytica remains poorly understood. In the 1990, it was believed that E. dispar was unable to produce significant experimental lesions. This scenario began to change in 1996, when E. dispar strains were isolated from symptomatic patients in Brazil. These strains were able to produce liver and intestinal lesions that were occasionally indistinguishable from those produced by E. histolytica. These and other findings, such as the detection of E. dispar DNA sequences in samples from patients with amebic liver abscess, have revived the possibility that this species can produce lesions in humans. The present paper presents a series of studies on E. dispar that begin to reveal a new facet of this protozoan.

The nonpeptide AVE-0991, which has been reported as a selective ligand for the angiotensin-(1–7) [ANG-(1–7)] receptor Mas, has actions similar to those attributed to the cardioprotective product of the renin-angiotensin system, ANG-(1–7). In this study, we evaluated the cardiac effects of AVE-0991 in normal and infarcted male Wistar rats. Myocardial infarction was induced by left coronary artery ligation. At the end of the treatment, the Langendorff technique was used to analyze cardiac function. Left ventricle serial sections were dyed with Gomori trichrome stain to quantify the infarcted area. In normal hearts, AVE-0991 produced a significant decrease in perfusion pressure and an increase in systolic tension, rate of tension rise and fall (±dT/d t), and heart rate. These effects were completely blocked by the perfusion of the hearts with a solution containing the selective ANG-(1–7) antagonist A-779. NG-nitro-l-arginine methyl ester treatment abolished the AVE-0991-induced vasodilation in isolated hearts. AVE-0991 significantly attenuated the decrease in systolic tension (sham operated, 13.00 ± 1.02 g; infarction, 7.18 ± 0.66 g; AVE treated, 9.23 ± 1.05 g, n = 5), +dT/d t, −dT/d t, and heart rate induced by myocardial infarction. Infarction-induced vasoconstriction was completely prevented by AVE-0991 treatment. Furthermore, AVE-0991 significantly decreased the infarcted area (6.98 ± 1.01 vs. 3.94 ± 1.04 mm2 in AVE-treated rats). These data indicate that the compound AVE-0991 produces beneficial effects in isolated perfused rat hearts involving the ANG-(1–7) receptor Mas and the release of nitric oxide. In addition, our results indicate that AVE-0991 attenuates postischemic heart failure.

There is growing evidence for the participation of opioid receptors in the development of inflammation. The present study was designed to clarify the role played by opioid receptors in periodontal disease. Periodontal disease was induced by placing a sterile silk ligature around the cervix of the second maxillary tooth on day 0. Morphine was administered either systemically or locally before and after the onset of periodontal disease. The results showed that in both patterns, morphine treatment reduced fiber attachment and alveolar bone loss, without affecting the increased leukocyte count in the gingivae. Naltrexone, a specific opioid antagonist, reversed the inhibitory effects induced by morphine in diseased rats, while the increased number of inflammatory cells remained unaffected. These results point to a possible role for local opioids in experimental periodontal disease.

Metallothioneins (MT) are small proteins able to strongly bind and therefore neutralize heavy-metal, free-radical and other genotoxic compounds. A close relationship between MT and zinc-finger motif-dependent transcription factors has also been shown, with implications on cell proliferation and survival. Human malignant neoplasms (e.g. breast cancer) usually present worse prognosis with increased MT content. We therefore evaluated the immunohistochemical expression of MT in a group of 15 canine mammary gland tumors. There was no relationship between MT immunostaining and histological type or malignancy grading of the lesions. Comparison of MT immunolabelling with the overall survival of the animals revealed that MT overexpression was related to better prognosis, a contrasting finding with the human counter-part. We considered that differential MT gene expression could be responsible for this variation, as observed for some human neoplasms of distinct embryonic origin, but further investigation is required to elucidate this topic.

Dental pulp reactivity to various pro-inflammatory stimuli was independently evaluated in rats in terms of a vascular permeability increase and leukocyte recruitment. Substance P, calcitonin-gene related peptide (CGRP) and prostaglandin E(2) (in the picomol range) were applied to the exposed pulp from anesthetised animals and the plasma extravasation measured by the Evans blue content in the tissue following 10 min of administration. Leukocyte recruitment was evaluated morphometrically by counting the cell number present in serial sections of 1:3 4 microm pulp tissue 6 h after bacterial endotoxin (LPS; 0.06-1.2 microg/site) administration. Increase in pulp vascular permeability and cellular recruitment due to the injection of mentioned mediators in the skin or LPS in the peritoneal cavity were used as positive controls. Increase in vascular permeability in the pulp occurred in the same dose-range as observed in the skin, being CGRP the most active substance in both cases. However, it was necessary a higher dose of LPS (1.2 microg) to induce a similar cell recruitment in the pulp as that observed in the rat peritoneal cavity (0.3 microg). These data indicate that dental pulp reactivity presents the same pattern of increase in vascular permeability to other tissues in the rat, being CGRP the most potent mediator in this respect. In addition, they suggest the presence of CGRP receptors in the dental pulp. However, an adequate leukocyte recruitment response to bacterial endotoxin was not mounted, suggesting a deficiency in adhesion molecules in blood vessels in the rat dental pulp.

Lesions observed in chronic chagasic cardiopathy frequently produce electrocardiographic alterations and affect cardiac function. Through a computerized morphometrical analysis we quantified the areas occupied by cardiac muscle, connective and adipose tissues in the right atrium of dogs experimentally infected with Trypanosoma cruzi. All of the infected dogs showed chronic myocarditis with variable reduction levels of cardiac muscle, fibrosis and adipose tissue replacement. In the atrial myocardium of dogs infected with Be78 and Be62 cardiac muscle represented 34 and 50%, fibrosis 28 and 32% and adipose tissue 38 and 18%, respectively. The fibrosis observed was both diffuse and focal and mostly intrafascicular, either partially or completely interrupting the path of muscle bundles. Such histological alterations probably contributed to the appearance of electrocardiographic disturbances verified in 10 out 11 dogs which are also common in human chronic chagasic cardiopathy. Fibrosis was the most important microscopic occurrence found since it produces rearrangements of collagen fibers in relation to myocardiocytes which causes changes in anatomical physiognomy and mechanical behavior of the myocardium. These abnormalities can contribute to the appearance of cardiac malfunction, arrythmias and congestive cardiac insufficiency as observed in two of the analyzed dogs. Strain Be78 caused destruction of atrial cardiac muscle higher than that induced by strain Be62. As lesões observadas na cardiopatia chagásica crônica frequentemente produzem alterações eletrocardiográficas e afetam a função cardíaca. Através de uma análise morfométrica computadorizada nós quantificamos as áreas ocupadas por músculo cardíaco, tecido conjuntivo fibroso e tecido adiposo no átrio direito de cães experimentalmente infectados pelo Trypanosoma cruzi. Todos os cães infectados apresentaram miocardite crônica fibrosante com graus variáveis de redução de músculo cardíaco, fibrose e substituição por tecido adiposo. No miocárdio atrial dos cães infectados pelas cepas Be78 e Be62 foram observados 34 e 50% de músculo cardíaco, 28 e 32% de fibrose e, 38 e 18% de tecido adiposo, respectivamente. A fibrose observada era tanto difusa quanto focal e, principalmente intrafascicular interrompendo total ou parcialmente o percurso dos feixes musculares. Tais alterações histológicas provavelmente contribuiram para o surgimento dos distúrbios eletrocardiográficos verificados em 10 dos 11 cães estudados e que são comuns na cardiopatia chagásica crônica humana. De todos os achados microscópicos encontrados, a fibrose foi a mais importante por produzir rearranjos na fibras colágenas em relação aos miocardiócitos, modificando a fisionomia anatômica e o comportamento mecânico do miocárdio. Tais anormalidades estruturais podem contribuir para o surgimento à disfunção cardíaca, arritmias e à insuficiência cardíaca congestiva como verificado em dois cães analisados. A cepa Be78 produziu uma destruição de músculo cardíaco atrial estatisticamente superior à induzida pela cepa Be62.

This paper aimed to verify the influence of the inoculum source (blood or metacyclic trypomastigote) and the route of inoculation (intraperitoneal or conjunctival) on the course of T. cruzi infection in dogs, using comparatively the T. cruzi strains Berenice-62 and Berenice-78. All dogs inoculated intraperitoneally became infected independently of the T. cruzi strain and source of trypomastigotes used. High level of infectivity was also observed when metacyclic trypomastigotes of both strains were inoculated by conjunctival route. However, when blood trypomastigotes were inoculated by conjunctival route the percentages of infectivity were significantly lower in dogs inoculated with both strains. Parasitaemia was significantly higher in animals infected with metacyclic trypomastigotes via the conjunctival route independently of the T. cruzi strain used. All animals infected with Berenice-78 strain showed severe acute myocarditis. On the other hand, animals infected with Berenice-62 showed severe acute myocarditis only when infected with metacyclic trypomastigote, via the intraperitoneal route. The results suggest that the source of the inoculum and the route of inoculation remarkably influence the evolution of the infection for the T. cruzi in the vertebrate host even when the same strain of the parasite is used.

Background The parasitic flatworm Schistosoma mansoni is a blood fluke that causes schistosomiasis. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy to control disease is a combination of drug treatment and immunization with an anti-schistosome vaccine. Numerous antigens that are expressed at the interface between the parasite and the mammalian host have been assessed. Among the most promising molecules are the proteins present in the tegument and digestive tract of the parasite. Methodology/Principal Findings In this study, we evaluated the potential of Sm10.3, a member of the micro-exon gene 4 (MEG-4) family, for use as part of a recombinant vaccine. We confirmed by real-time PCR that Sm10.3 was expressed at all stages of the parasite life cycle. The localization of Sm10.3 on the surface and lumen of the esophageal and intestinal tract in adult worms and lung-stage schistosomula was confirmed by confocal microscopy. We also show preliminary evidence that rSm10.3 induces erythrocyte agglutination in vitro. Immunization of mice with rSm10.3 induced a mixed Th1/Th2-type response, as IFN-γ, TNF-α, and low levels of IL-5 were detected in the supernatant of cultured splenocytes. The protective effect conferred by vaccination with rSm10.3 was demonstrated by 25.5–32% reduction in the worm burden, 32.9–43.6% reduction in the number of eggs per gram of hepatic tissue, a 23.8% reduction in the number of granulomas, an 11.8% reduction in the area of the granulomas and a 39.8% reduction in granuloma fibrosis. Conclusions/Significance Our data suggest that Sm10.3 is a potential candidate for use in developing a multi-antigen vaccine to control schistosomiasis and provide the first evidence for a possible role for Sm10.3 in the blood feeding process., Author Summary Schistosomiasis mainly occurs in developing countries and is the most important human helminth infection in terms of global mortality. This parasitic disease affects more than 200 million people worldwide and causes more than 250,000 deaths per year. Current schistosomiasis control strategies are mainly based on chemotherapy, but many researchers believe that the best long-term strategy for controlling schistosomiasis is a combination of drug treatment and immunization with an anti-schistosome vaccine. Consequently, significant effort has been dedicated to developing and characterizing an anti-schistosome vaccine. Over the last five years, considerable data have been generated regarding the genomics, transcriptomics and proteomics of Schistosoma mansoni. In the present study, we characterize the Sm10.3 protein and evaluate its potential to protect against S. mansoni infection in a murine model. We demonstrate that Sm10.3 is primarily expressed during the stages of the parasite life cycle that involve infection and disease development in the human host. Sm10.3 is located on the surface of the digestive epithelia of adult female worms, an important host/parasite interface. Moreover, the vaccination of mice with rSm10.3 confers partial protection against S. mansoni. Taken together, our data suggest that Sm10.3 may be a useful component of a multi-antigen vaccine against schistosomiasis.

Heart autonomic innervation was studied in dogs during the acute phase of the experimental infection with the Berenice-78 strain of Trypanosoma cruzi. A glyoxylic acid-induced fluorescence method for catecholamines and a thiocholine method for demonstrating acetylcholinesterase activity showed the sympathetic and the parasympathetic nerve fibers, respectively. At day 34 of infection, moderate-to-intense rarefaction of both cholinergic and noradrenergic nerve fibers occurred in the atria of all animals coincident with moderate to intense myocarditis. In the ventricles, sympathetic denervation was clearly present only when the inflammatory processes were moderate to intense. Preliminary results on the chronic phase indicate that normal autonomic innervation coexists with an incipient chronic fibrosing myocarditis.

Qualitative and quantitative aspects of the superficial and profound cardiac plexus of dogs experimentally infected with Be-62 and Be-78 strains of Trypanosoma cruzi were studied. Animals were autopsied in the acute phase of infection. The inflammatory process, lesions and number of parasites were more intense and frequent in animals infected with the Be-78 strain than in those infected with Be-62. Despite this, no statistically significant differences could be found between the number of neuron bodies in the ganglia of infected and control dogs.Foi realizado estudo qualitativo e quantitativo dos plexos cardíacos superficiais e profundos em cães inoculados com o Trypanosoma cruzi das cepas Be-62 e Be-78 e sacrificados na fase aguda. O processo inflamatório, as lesões e o parasitismo dos plexos foram mais intensos e frequentes nos animais inoculados com a cepa Be-78 do que naqueles inoculados com a cepa Be- 62. Apesar deste fato, não foi verificada diferença estatisticamente significativa entre o número de corpos de neurônio por gânglio dos animais chagásicos e os controles.

Objective This study focused on comparing the expression levels of p16, Ki-67, and minichromosome maintenance 7 (MCM7) protein in normal and affected cervical epithelium to ascertain the biological significance of these markers in detecting progressive cervical disease. Methods A quantitative and based on-scanning-microscopy analysis of the three markers expression was performed in normal and cervical intraepithelial neoplasia (CIN) I, II, and III tissues. p16 area as well as p16, Ki-67, and MCM7 positive cells or nuclei were evaluated according to their distribution and extent through the cervical epithelium. Results A clear p16 over-expression was observed in all the dysplastic epithelium tissue samples. The quantitative analysis of p16 area as well as the number of p16 positive cells was able to better discriminate the CIN lesions grades than the usual semi-quantitative analysis. The average Ki-67 labeling indexes for the normal epithelium, CIN I, CIN II, and CIN III groups were 19.8%, 27.3%, 32.8%, and 37.1%, respectively, whereas the mean MCM7 labeling indexes for the correspondent grades were 27.0%, 30.4%, 50.5%, and 67.2%. The Ki-67 and MCM7 labeling indexes were closely correlated with the CIN histological grade, with higher labeling indexe values obtained from the more severe lesions (p

The sequence of hepatic necrotic-inflammatory events produced by Entamoeba dispar are originally described in this work. For the first time were described in details the experimental lesions produced by E. dispar, as well as the distribution of the trophozoites detected by the immunohistochemistry. Animals experimentally infected with E. dispar presented necrosis, thrombosis and chronic granulomatous inflammation. Immunoreactive products derived from trofozoites were observed close or associated with trophozoites, epithelioid cells, leucocytes and hepatocytes. Few are the articles on the literature about virulence of E. dispar, which is approximately 9 times more frequent than to E. histolytica. Variation in the virulence is, therefore expected and signalizing the need of the continuity of studies with E. dispar strains from different places in the world. Taking into account that E. dispar is a closely related species to E. histolytica, these studies could determine new elements involved with E. histolytica pathogenesis, helping us to understand better the disease.

Giardiasis is one of the most common parasitic diseases worldwide, and the disease is an important cause of diarrhoea and malabsorption in children and immunosuppressed individuals. However, there is no evidence that characterises malnutrition as an aggravating factor for this disease. We evaluated changes in villi structures to examine the association between malnutrition and Giardia lamblia infection.We used 32 gerbils, divided into 4 groups: Control (CT) and Control Infected (CTIn), which each received a 20% protein diet, Malnourished (MN) and Malnourished Infected (MNIn), which each received a 5% protein diet. Groups CTIn and MNIn were inoculated with 1×106 trophozoites of G. lamblia, while the remaining groups were mock infected. Seven days post-infection, all groups were sacrificed, and the proximal portions of the small intestines were collected for the analysis of villus height, mucus area and extent of Giardia infection.Gerbils fed with a low-protein diet had significantly lower body weights.Malnourished infected animals presented significantly increased production of mucus, suggesting a synergism occurs between malnutrition and Giardiasis, potentially to control the adhesion of Giardia in the mucosa.Villus height was significantly lower in group MNIn compared to CTIn. This work suggests that malnutrition contributes to severity of Giardiasis by decreasing the intestinal absorption capacity via shortening of the villi.

The influence of inflammation on the number of trophozoites and on the murine amoebic liver abscess area following infection with Entamoeba histolytica and E. dispar was evaluated. Immunohistochemistry and digital morphometry were used to identify and quantify the trophozoites, neutrophils, macrophages, and lesions. Positive correlation was observed between the number of trophozoites and inflammatory cells. A significant decrease in parasitism and inflammation in groups treated with dexamethasone was observed. The scarceness or absence of trophozoites in the treated groups suggest the importance of the inflammatory response in the production of amoebic hepatic abscesses in spite of the inherent virulence of the parasite being decisive in the establishment of the lesion.

Campomanesia lineatifolia Ruiz & Pav. (Myrtaceae) é uma espécie nativa comestível encontrada na Floresta Amazônica, comumente conhecida como gabiroba. No Brasil, espécies de Campomanesia são frequentemente usadas na medicina tradicional para desordens gastrintestinais. Estudos anteriores de nosso grupo de pesquisa demonstraram elevado teor de fenóis e alta atividade antioxidante para o extrato etanólico das folhas de C. lineatifolia, sugerindo relação de sua utilização etnofarmacológica como antidiarreico, antiúlcera gástrica e cicatrizante. O objetivo deste trabalho foi avaliar a fitoquímica, a atividade gastroprotetora, anti-inflamatória e anti-Helicobacter pylori de C. lineatifolia e contribuir para sua padronização químico-biológica. Para isso, foi desenvolvido método extrativo otimizado para obtenção de extrato enriquecido em fenólicos e método analítico por cromatografia líquida de ultra eficiência para avaliação qualitativa e quantitativa desse extrato. Na sequência, utilizou-se a cromatografia em contracorrente de alta velocidade e métodos espectroscópicos para isolar e caracterizar as substâncias químicas presentes no extrato otimizado. Análise por cromatografia gasosa-espectro de massas foi utilizada para identificação das substâncias químicas presentes no óleo essencial. Para avaliação das atividades gastroprotetora, anti-inflamatória e anti-H. pylori, foram realizados ensaios in vivo em modelos experimentais de úlcera gástrica e ensaios in vitro. Os resultados do planejamento extrativo permitiram selecionar o extrato etanólico obtido por sistema de refluxo como aquele enriquecido em fenólicos. Análises obtidas por cromatografia líquida-espectro de massas e ressonância magnética nuclear em 13C e 1H permitiram identificar 15 substâncias químicas no extrato etanólico otimizado, das quais 12 delas são inéditas para a espécie, sendo os flavonoides quercitrina e miricitrina as substâncias majoritárias, e a quercitrina selecionada como marcador para o desenvolvimento do método analítico. Já no óleo essencial, foram identificadas 8 substâncias químicas, representando um total de 98,09%, sendo o 3-hexen-1-ol (46,15%) e o α-cadinol (20,35%) os constituintes majoritários. Ensaio in vivo em modelo de úlcera gástrica induzida por etanol demonstrou que o extrato protegeu o estômago de camundongos, nas doses de 100 e 250 mg/Kg, e promoveu um aumento do muco gástrico. No modelo de úlcera gástrica induzida por indometacina, os animais tratados com o extrato apresentaram redução das citocinas inflamatórias CXCL-1 e IL-6, além da inibição da via do NF-κB no tecido gástrico. Ensaios in vitro em células THP-1 estimuladas por LPS mostraram que o extrato e substâncias isoladas, quercitrina e miricitrina, foram capazes de reduzir TNF-α e NF-κB. Ensaios in vitro de atividade anti-H. pylori demonstraram que o extrato etanólico foi capaz de inibir o crescimento bacteriano de cepas comerciais (MIC 0,49 μg/mL) e clínicas (MIC 125-250 μg/mL). Ainda, o óleo essencial também apresentou atividade anti-H. pylori, na menor concentração testada (MIC 0,6%) em cepas comerciais e isolados clínicos. Como conclusão, o extrato de C. lineatifolia foi otimizado e padronizado quimicamente e os ensaios in vitro e in vivo, sugerem uma relação mecanística da gastroproteção à atividade anti-inflamatória e estimuladora de muco protetor mucosal, além da atividade antibacteriana anti-H. pylori. Isto abre perspectivas para um fitoterápico como nova opção terapêutica para pacientes com úlcera gástrica, tendo em vista que os protocolos atuais de manejo clínico não contam com fármacos que apresentem estas atividades farmacológicas em associação. Campomanesia lineatifolia Ruiz and Pav. (Myrtaceae) is an edible native species found in the Amazon Rainforest, popularly known as gabiroba. In Brazil, Campomanesia species are traditionally used for medicinal purposes such as gastrointestinal disorders. Previous studies conducted by our research group demonstrated high phenolic content and high antioxidant activity for C. lineatifolia ethanolic extract, suggesting a relation with its ethnopharmacological use as antidiarrheal, gastric ulcer and healing. The objective of this study was to evaluate the phytochemistry, gastroprotective, anti-inflamatory, and anti-Helicobacter pylori activities of C. lineatifolia, and to contribute to its chemical-biological standardization. For this, an optimized extractive method was developed to obtain a phenolic-enriched extract, and an analytical method by ultra-performance liquid chromatography for qualitative and quantitative evaluation. Next, high-speed countercurrent chromatography and spectroscopic methods were respectively used to isolate and characterize chemical compounds in optimized extract. Gas chromatography-mass spectrometry analysis was used to identify the chemicals present in the essential oil. To evaluate the gastroprotective, anti-inflammatory and anti-H. pylori activities, in vivo assays were performed in experimental gastric ulcer models, and in vitro assays. Results of the extractive planning allowed selecting the ethanolic extract obtained by reflux system as the one enriched in phenolics. Analyzes obtained by liquid chromatography-mass spectrum and nuclear magnetic resonance in 13C and 1H allowed the identification of 15 chemical compounds in the optimized ethanolic extract, of which 12 were new for the species, with the flavonoids quercitrin and myricitrin being the major substances, thus being quercitrin selected as a marker for the development of the analytical method. In the essential oil analysis, 8 chemicals were identified, representing a total of 98.09%, with 3-hexen-1-ol (46.15%) and α-cadinol (20.35%) being the major constituents. In vivo assay in ethanol-induced ulcer model demonstrated that the extract protected the stomach of mice, at doses of 100 and 250 mg/Kg, and promoted an increase in gastric mucus. In indomethacin-induced ulcer model, mice treated with the extract showed a reduction of the inflammatory cytokines CXCL-1 and IL-6, and inhibition of the NF-κB pathway in gastric tissue. In vitro assays in LPS-stimulated THP-1 cells showed that the extract and isolated compounds, quercitrin and myricitrin, were able to reduce TNF-α and NF-κB. In vitro anti-H. pylori activity assays showed that the ethanolic extract was able to inhibit bacterial growth of commercial (0.49 μg/mL) and clinical (125-250 μg/mL) strains. Furthermore, the essential oil also showed anti-H. pylori activity at the lowest concentration tested (0.6%) in commercial and clinical isolated strains. In conclusion, the C. lineatifolia extract was chemically optimized and standardized, and the in vitro and in vivo assays suggest a mechanistic relationship between gastroprotection and anti-inflammatory activity, and mucosal protective mucus stimulator, in addition to anti-H. pylori activity. This opens perspectives for an herbal medicine as new therapeutic option for patients with gastric ulcers, considering that current clinical management protocols do not include drugs that have these pharmacological activities in association.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A forma tradicional de ensino já vem há muito sendo debatida. Novas abordagens metodológicas que incrementem a assimilação dos conteúdos são incentivadas. Nesse contexto articulamos e testamos uma nova metodologia de ensino-aprendizagem baseada na teoria da lei da difusão e inovação de Everett Rogers, cuja teoria até a presente data havia sido utilizada para o marketing. Tendo como pilares o construtivismo Piagetiano e sociointeracionismo Vygotskyano adaptados a aprendizagem Baseada em Problemas. O estudo foi realizado através da temática parasitoses, relevante e próxima à realidade da comunidade com 48 alunos do 1º ano do ensino médio numa escola estadual, na periferia de Belo Horizonte. A metodologia aplicada através da discussão de situações-problema em pequenos grupos de alunos, visando a interação e reorganização do conhecimento, aos quais foram atribuídas funções diferenciadas de acordo com seus múltiplos perfis dentro da proposta de Rogers. Os resultados mostraram maior integração e apropriação do conteúdo parasitoses entre os alunos com diferentes perfis. Houve envolvimento ativo dos alunos através da geração de questões e problemas investigativos por meio de coleta, análise e interpretação de dados, levando os educandos a formularem hipóteses e conclusões de forma reflexiva, ressaltando competências socioemocionais contextualizadas à temática. Diante do exposto, acreditamos que nossa metodologia seja reproduzível, podendo ser aplicada a qualquer área do conhecimento e faixa etária. A metodologia se mostrou eficaz, promovendo, além do entrosamento, horizontalização do conhecimento e assim o protagonismo e empoderamento dos alunos permitindo que o conhecimento estabelecido sobre as parasitoses contribuísse para profilaxia e manutenção da saúde geral do alunado. The traditional form of teaching has been debated for a long time. New methodological approaches that increase the assimilation of content are encouraged. In this context, we articulated and tested a new teaching-learning methodology based on the theory of the law of diffusion and innovation by Everett Rogers, whose theory until the present date had been used for marketing, having as pillars Piagetian constructivism and socio- interactionism adapted to learning Problem-based The study was carried out through the theme of parasites, relevant and close to the reality of the community with 48 students from the 1st year of high school in a state school, on the outskirts of Belo Horizonte. The methodology applied through the discussion of problem situations in small groups, aiming at the interaction and reorganization of knowledge, which were assigned different functions according to their multiple profiles within Rogers' proposal. The results showed a greater integration and appropriation of the parasitic content among students with different profiles. There was active involvement through the generation of investigative questions and problems through the collection, analysis and interpretation of data, leading the students to formulate hypotheses and conclusionsin a reflexive way, emphasizing socio- emotional competences contextualized to the theme. Through a dynamic and replicable methodology, easy to conduct, it can be used for any area of knowledge and age group. The methodology proved to be effective, promoting, in addition to the integration, horizontalization of knowledge and thus the protagonism and empowerment of students, allowing the knowledge established about parasites to contribute to prophylaxis and maintenance of the general health of students.

FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais Introdução: A maturação funcional e estrutural da pele é um potencial marcador para a datação da cronologia da gravidez. Ela é o resultado de um processo dinâmico, que começa durante a embriogênese e termina no primeiro ano de vida. Já é de conhecimento que existe uma associação entre o desenvolvimento da barreira protetora epidérmica e a sobrevida neonatal. Apesar da importância da análise morfométrica da pele para aplicações clínicas em neonatologia, pouco são os estudos a respeito da evolução cronológica destas medidas ao longo da gestação, principalmente pelas limitações na obtenção do espécime. Objetivo: Avaliar a relação entre a espessura e a quantidade de tecido conjuntivo fibroso da derme, áreas de glândulas, em pele de recém-nascido com a cronologia da gravidez. Metodologia: Estudo observacional transversal, no qual medidas da espessura da pele foram obtidas por meio de biopsias em 35 conceptos com idade gestacional entre 20 e 42 semanas gestacionais. Os critérios de inclusão foram: existência de ultrassonografia obstétrica precoce na gravidez antes de 14 semanas de idade gestacional (IG) e um intervalo máximo de 48 horas entre a morte e a biopsia de amostra de pele. As regiões biopsiadas foram: a pele do dorso da mão, sola do pé e periumbilical. As lâminas histológicas foram coradas por tricrômico de Gomori. As dimensões das camadas da pele foram mensuradas com o apoio do software de análise de imagens KS300. A análise estatística determinou dimensões das camadas da pele e a sua variabilidade, comparação entre regiões do corpo e calculou a correlação entre as características morfométricas da pele com a IG. Resultados: A espessura da pele foi semelhante no dorso da mão e na sola do pé: estrato córneo (p = 0,707), epiderme (p = 0,618) e derme (0,977). No entanto, a pele estava mais fina no abdômen 17 17 quando comparada à mão (p = 0,002; p

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As enteroparasitoses representam um problema de saúde mundial, notadamente nos países em desenvolvimento, e apresentam um alto percentual de morbidade. A ocorrência dessas doenças está ligada à falta ou precariedade de saneamento básico, ausência de cuidados de higiene pessoal e deficiência das informações sobre a prevenção, o contágio e o ciclo de vida dos parasitos. Tal situação demonstra a necessidade de identificar a ocorrência dessas parasitoses bem como propor estratégias eficientes como forma de intervenção na transmissão das mesmas. Nesse contexto, uma proposta pedagógica mais desafiadora, que estimule o protagonismo do aluno, torna-se uma ferramenta de grande importância, uma vez que permite a aquisição de conhecimento sobre as mais diversas parasitoses, propiciando uma mudança de comportamento do aluno de modo a torná-lo um disseminador dessas ideias. O objetivo desse trabalho foi utilizar o tema enteroparasitoses e sua prevenção para estimular os alunos a protagonizarem seu aprendizado sobre tal conteúdo. Participaram da pesquisa 33 alunos da E. E. Zeca Guida que cursavam o 1° e 2° anos do ensino médio que apresentaram a documentação exigida em consonância com os aspectos éticos. O conhecimento gerado através das ações pedagógicas elaboradas e executadas pelos discentes permitiu a fixação de conceitos relacionados às parasitoses intestinais tornando-os aptos a transmitirem informações sobre essas infecções aos membros da comunidade escolar. As estratégias de educação para promoção da saúde se mostram eficientes quando pautadas no estímulo ao protagonismo dos alunos, que poderão identificar situações em seu cotidiano que favorecem a incidência dessas infecções e assim buscarem medidas eficientes de controle e prevenção. The enteroparasites represent a worldwide health problem, especially in developing countries and present a high percentage of morbidity. The occurrence of these diseases is linked to the lack or precariousness of basic sanitation, lack of personal hygiene care and lack of information on prevention, contagion and life cycle of parasites. This situation demonstrates the need to identify the occurrence of these parasitoses as well as to propose efficient strategies as a form of intervention in the transmission of these parasites. In this context, a more challenging pedagogical proposal that stimulates the protagonism of the student, becomes a tool of great importance, since it allows the acquisition of knowledge about the most diverse parasitoses, providing a change of behavior of the student in order to make him a disseminator of these ideas. The objective of this study was to use the subject of enteroparasites and its prevention to stimulate the students to carry out their learning about such content. Thirty-three students from the public school Zeca Guida enrolled in the study who attended the first and second years of high school who presented the required documentation in line with ethical aspects. The knowledge generated through the pedagogical actions developed and carried out by the students allowed the establishment of concepts related to the intestinal parasitoses making them able to transmit information about these infections to the members of the school community. The strategies of education for health promotion are efficient when guided by the stimulus to the protagonism of the students, who can identify situations in their daily life that favor the incidence of these infections and thus seek efficient measures of control and prevention.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A giardíase, enteroparasitose cosmopolita causada pelo protozoário Giardia lamblia, se encontra dentre as doenças diarreicas mais recorrentes nos países desenvolvidos e, principalmente, nos países em desenvolvimento, sendo responsável pela ocorrência de síndromes má-digestivas e déficits físicos e cognitivos em crianças residentes. Dentre os grupos de vulnerabilidade na giardíase, incluem-se os indivíduos imunossuprimidos em decorrência de terapias cada vez mais agressivas, utilizadas para o tratamento de doenças auto-imunes ou transplantes de órgãos. Nessa população, a giardíase pode resultar em cronicidade e gravidade da infecção. No entanto, alguns indivíduos imunodeficientes podem permanecer assintomáticos, ainda que infectados pelo parasito durante longos períodos. Diante das controvérsias que envolvem a giardíase em hospedeiros imunossuprimidos, buscou neste trabalho contribuir para a elucidação do assunto, por meio da avaliação da permeabilidade intestinal e da análise histopatológica e morfométrica no intestino delgado de gerbils, experimentalmente imunossuprimidos e infectados por G. lamblia. Utilizou-se 35 gerbils com 4 a 6 semanas de idade, distribuídos em 4 grupos experimentais, a saber: (CT) Controle (n=8), (CTIn) Controle-infectado por G. lamblia (n=9), (IM) Imunossuprimido (n=9) e (IMIn) Imunossuprimido-infectado por G. lamblia (n=9). Os animais dos grupos IM e IMIn receberam, durante 25 dias, um tratamento com Dexametasona via intramuscular, para a indução de imunossupressão. No 11º dia experimental, os animais dos grupos CTIn e IMIn foram desafiados com trofozoítos de G. lamblia, e permaneceram infectados durante 14 dias. No 25º dia experimental, administrou-se nos gerbils de todos os grupos, uma solução contendo 99mTc-DTPA para a medição da permeabilidade intestinal. Após esse procedimento, realizou-se a eutanásia dos animais, seguida da coleta e do processamento histopatológico do intestino delgado e do baço. Os tecidos foram analisados por meio de histopatologia, morfometria digital e reação imuno-histoquímica para o parasitismo. As análises histopatológicas e morfométricas evidenciaram lesões significativas na mucosa duodenal do grupo CTIn, com vilosidades reduzidas de tamanho, criptas hiperplásicas, infiltrado inflamatório moderado na lâmina própria congruentes com o aumento da taxa de renovação epitelial do tecido. Por outro lado, no grupo IMIn, observou-se ausência de inflamação, criptas intestinais profundas, porém, com vilosidades longas, delgadas e íntegras. As células caliciformes no grupo IMIn se apresentaram hiperplásicas e culminaram em aumento na produção de muco intestinal. Observou-se ainda, no grupo IMIn aumento significativo no número de trofozoítos de G. lamblia imunomarcados no lúmen dos animais. Não foram observadas alterações na permeabilidade intestinal dos gerbils experimentados. Conclui-se, portanto, que a redução dos danos à mucosa intestinal observada nos animais imunossuprimidos, infere que a resposta imune do hospedeiro seja a principal responsável pelo surgimento das lesões durante a enterite produzida por G. lamblia, no modelo experimentado. A proteção da integridade intestinal, em animais imunossuprimidos, particularmente, demonstra importante ação do sistema imune inato, representado pelo muco, que se apresentou como um agente dificultador à adesão ao epitélio intestinal pelos trofozoítos de G. lamblia. O aumento do número de parasitos no lúmen intestinal dos gerbils do grupo IMIn, confirma a cronicidade observada em humanos imunossuprimidos acometidos por giardíase, suscitando os gerbils como bons modelos para o estudo de infecções por G. lamblia em hospedeiros imunossuprimidos. The giardiasis, cosmopolitan enteroparasitosis caused by the protozoan Giardia lamblia is among the most recurrent diarrheal diseases in developed countries and especially in developing countries, being responsible for poor digestion’s syndromes, physical and cognitive deficits in resident children. Among the groups of vulnerability in the management, include the main immunosuppressed, as a result of aggressive therapies used for the treatment of autoimmune diseases or organ transplants. In this population, giardiasis can result in chronicity and more severe infection. However, many immunodeficient individuals remain asymptomatic, although infected by the parasite for long periods. In view of this controversy, the aims of this study were to contribute to the elucidation of the subject through the evaluation of small intestinal permeability, histopathological and morphometric analysis in gerbils experimentally immunosuppressed and infected by G. lamblia. In this study were used 35 gerbils 4 to 6 weeks old distributed in 4 experimental groups: Control (CT) [n = 8], Control-infected by G. lamblia (CTIn) [n = 9], Immunosuppressed IM) [n = 9] and Immunosuppressed-infected by G. lamblia (IMIn) [n = 9]. The animals of the IM and IMIn groups received a treatment with Dexamethasone intramuscularly for 25 days for induction of immunosuppression. On the 11th experimental day, animals in the groups CTIn and IMIn were challenged with G. lamblia trophozoites remained infected for 14 days. On the 25th experimental day was administered on gerbils of all groups, a solution containing 99mTc-DTPA for evaluation of intestinal permeability. After this procedure, the euthanasia of the animals was produced, followed by collection and histopathological processing of the small intestine and spleen. The tissues were analyzed by histopathology, digital morphometry and immunohistochemistry for parasitism. Histopathological and morphometric analyzes revealed significant lesions in the duodenal mucosa of the CTIn group, with reduced villi size, hyperplastic crypts, moderate inflammatory infiltrate in the lamina propria and high rate of tissue epithelial renewal. On the other hand, in the IMIn group no inflammatory events were seen and villi were long, thin and intact. Goblet cells in the IMIn group were hyperplastic and culminated in increased production of intestinal mucus. Furthermore, in the IMI group, a significant increase in the number of G. lamblia trophozoites immunolabelled in the lumen of the animals was observed. No changes were observed in the intestinal permeability of the gerbils experienced. Thus, the reduction of intestinal damage in the intestinal mucosa of immunosuppressed animals, inferred an immune response of the host is one of the main responsible for the emergence of interactions during infection by G. lamblia in the experienced model. The protection of the intestinal gut in immunosuppressed animals, in particular, is an important action of the immune system, represented by mucus, which presents as an agent that makes it difficult for adhesion to the intestinal epithelium by G. lamblia trophozoites. The increase in the number of parasites in the gut of gerbils of the IMIn group confirms a chronicity observed in immunosuppressed men affected by giardiasis, such as gerbils as good models for the study of G. lamblia infections in immunosuppressed hosts.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais A colonização pela microbiota intestinal é um processo importante para o desenvolvimento do intestino, promovendo a maturação do sistema imune da mucosa, da fisiologia digestiva e do sistema nervoso entérico. Recentemente, tem sido observado que certos quadros psicopatológicos possam estar associados à disbiose da microbiota intestinal, indicando uma comunicação entre o intestino e o cérebro. Por meio de mecanismos humorais e não-humorais, a administração de probióticos geralmente é capaz de interferir positivamente na mucosa intestinal, modulando diretamente ou indiretamente a microbiota para um estado normal. Neste sentido, foi avaliado se a Weissella paramesenteroides WpK4 possuía efeitos benéficos nos contextos da colite ulcerativa e do estresse crônico. Nestes ensaios, foi observado que a ingestão da bactéria melhorou os aspectos de saúde dos camundongos com colite, reduzindo a perda de peso e a permeabilidade intestinal, além de ter melhorado os aspectos histopatológicos do cólon. Já nos camundongos estressados, o consumo da bactéria foi associado à redução dos comportamentos tipo-ansioso e tipo-depressivo. Em ambos os ensaios, o papel benéfico da W. paramesenteroides WpK4 foi relacionado à sua capacidade imunomodulatória, reduzindo respostas inflamatórias. Em um modelo animal mais complexo, camundongos machos e fêmeas com colite, estressados ou estressados com colite foram tratados com a bactéria ou com o Citalopram, buscando evidenciar os papéis destes agentes nos processos de regulação da permeabilidade intestinal, da resposta imunológica e da dinâmica de neurotransmissores. De uma forma geral, a WpK4 e o Citalopram exerceram seus papéis benéficos no cólon dos camundongos por meio da imunomodulação de MYLK, reduzindo a sua expressão de tal forma a promover menor permeabilidade intestinal. No hipocampo destes animais, o papel imunomodulador destes agentes parece ter determinado maior expressão de Bdnf e de p11, melhorando os processos cognitivos dos camundongos com colite, estressados e estressados com colite. Neste sentido, é possível afirmar que a W. paramesenteroides WpK4 e o Citalopram exerceram seus papéis no eixo intestino-cérebro por meio de suas atividades imunomodulatórias com consequências em diversas vias metabólicas.

A Entamoeba histolytica produz a amebíase, doença infecciosa que atinge o intestino humano, constituindo a 2ª maior causa de morte entre as doenças parasitárias. Considerada comensal, alguns autores têm reproduzido experimentalmente a colite e o abscesso hepático amebiano (AHA) utilizando Entamoeba dispar isolada de hospedeiros assintomáticos e sintomáticos. Das cepas de E. dispar disponíveis em nosso acervo, apenas a MCR foi estudada com maiores detalhes, tendo sido analisada a cinética das lesões hepáticas entre 12 horas e 8 dias após a infecção (DAI) e, mensurada as lesões. Ainda não foi avaliada a virulência das cepas de E. dispar de nosso acervo, através do ensaio de eritrofagocitose, muitas cepas ainda não foram testadas quanto à sua patogenicidade e é desconhecida a evolução do AHA após 12 DAI. Diante disso, foi objetivo deste estudo avaliar a virulência e a patogenicidade das cepas MCR, ACFN, ICS, ADO E VEJ de E. dispar após 3, 8, 12 e 16 dias de infecção. No ensaio in vitro, os trofozoítos interagiram com eritrócitos humanos, sendo então contadas as hemácias fagocitadas e os trofozoítos que fagocitaram. Quinze hamsters foram inoculados com 1x105 trofozoítos de cada cepa e, como controle, 1 hamster foi inoculado em cada grupo com a microbiota indígena. Cinco hamsters de cada grupo foram eutanasiados aos 3, 8, 12 e 16 DAI para necropsia, coleta do fígado e processamento para histopatologia e morfometria digital. Todas as cepas de E. dispar foram capazes de fagocitar eritrócitos, sem diferenças significativas entre elas, porém em quantidades inferiores em relação à E. histolytica. A cepa ACFN demonstrou ser patogênica no hamster, sendo capaz de produzir o AHA e, 100% de mortalidade em períodos superiores a 8 DAI. Já a MCR produziu 29% de mortalidade do 8º ao 16º DAI, enquanto as cepas ICS, VEJ e ADO não causaram mortalidade. O AHA foi observado em todos os grupos de hamsters, sendo mais frequente nos inoculados com as cepas ACFN e MCR. A ACFN produziu área de necrose significativamente superior à ADO no 8º DAI, enquanto a necrose foi gerada somente pela MCR no 12º DAI. A extensão da necrose estava associada ao intenso parasitismo (predominante no 8º DAI) e geralmente ao infiltrado inflamatório granulocitário, histiocitário ou misto, focal e/ou difuso (predominante geralmente no 12º DAI). No 12º DAI foram observados os últimos abscessos em atividade e os primeiros em reparo. A inflamação crônica granulomatosa foi mais frequente no 8º e 12º DAI, variando entre os grupos. A trombose e a hemorragia foram mais frequentes nos hamsters inoculados com a ACFN, enquanto os fenômenos degenerativos, com a MCR. A maioria dos hamsters no 16º DAI apresentou preservação da maior parte do parênquima hepático. A regeneração é o mecanismo de cura predominante na amebíase experimental produzida pela E. dispar. A cicatrização ocorre em pequenas áreas, a despeito das grandes extensões de tecido de granulação. Assim, com exceção da ACFN, todas as outras cepas foram capazes de produzir danos hepáticos autolimitantes. Entamoeba histolytica produces amebiasis, an infectious disease that affects the human intestine, making it the second largest cause of death among parasitic diseases. Considered commensal, some authors have experimentally reproduced colitis and amebic liver abscess (AHA) using Entamoeba dispar isolated from asymptomatic and symptomatic hosts. Of the strains of E. dispar available in our collection, only MCR was studied in greater detail, analyzing the kinetics of hepatic lesions between 12 hours and 8 days after infection (DAI) and, measuring the lesions. Virulence of the strains of E. dispar from our collection has not yet been evaluated through the erythrophagocitose assay. Many strains have not yet been tested for their pathogenicity and the evolution of AHA after 12 DAI is unknown. The objective of this study was to evaluate the virulence and pathogenicity of E. dispar strains MCR, ACFN, ICS, ADO and VEJ after 3, 8, 12 and 16 days of infection. In the in vitro assay, the trophozoites interacted with human erythrocytes, and the phagocytosed erythrocytes and trophozoites that phagocytosed were counted. Fifteen hamsters were inoculated with 1x105 trophozoites from each strain and, as a control, 1 hamster was inoculated in each group with the native microbiota. Five hamsters from each group were euthanized at 3, 8, 12 and 16 DAI for necropsy, liver collection and processing for histopathology and digital morphometry. All strains of E. dispar were able to phagocyte erythrocytes, with no significant differences between them, but in lesser amounts in relation to E. histolytica. The ACFN strain proved to be pathogenic in the hamster, being able to produce AHA and 100% mortality in periods superior to 8 DAI. On the other hand, MCR produced 29% mortality from the 8th to the 16th DAI, whereas the ICS, VEJ and ADO strains did not cause mortality. AHA was observed in all groups of hamsters, being more frequent in the inoculated with the strains ACFN and MCR. The ACFN produced a necrosis area significantly higher than ADO in the 8th DAI, while necrosis was generated only by the MCR in the 12th DAI. The extent of necrosis was associated with intense parasitism (predominant in the 8th ICD) and usually to the granulocytic, histiocytic or mixed, focal and / or diffuse inflammatory infiltrate (predominantly in the 12th). At the 12th DAI, the last active abscesses were observed and the first ones under repair. Chronic granulomatous inflammation was more frequent in the 8th and 12th DAI, varying between groups. Thrombosis and hemorrhage were more frequent in hamsters inoculated with ACFN, while degenerative phenomena, with the MCR. Most hamsters in the 16th DAI presented preservation of most of the liver parenchyma. Regeneration is the predominant healing mechanism in the experimental amoebiasis produced by E. dispar. Healing occurs in small areas, despite large extensions of granulation tissue. Thus, with the exception of ACFN, all other strains were able to produce self-limiting liver damage.

No presente estudo foi utilizado o modelo de oclusão bilateral da carótida comum BCCAo) durante 25 minutos em camundongos machos C57BL/6 selvagens com objetivo de investigarmos as alterações neurológicas e os aspectos patológicos e inflamatórios durante a fase aguda da isquemia global transitória no encéfalo. Observamos déficits neurológicos associados à alterações morfológicas e processos inflamatórios no sistema nervoso central (SNC) após 72 horas de reperfusão. Nossos resultados demonstraram que os níveis de TNF, IL-1, CXCL1 e CCL5 aumentaram após isquemia/reperfusão no grupo BCCAo em comparação com o grupo sham. A atividade das etaloproteinases MPP-1 e MMP-2 foi analisada na área de lesão porimunoistoquímica. Os dois marcadores aumentaram no grupo BCCAo, em comparação com o grupo Sham. Com o mesmo modelo de BCCAo em camundongos machos C57BL/6 selvagens e deficientes para o receptor CCR5 (CCR5-/-), investigarmos a participação do CCR5 após isquemia e reperfusão por 24 horas. Na deficiência de CCR5, observamos uma melhora significativa nos déficits neurológicos associados à diminuição da área infartada e das áreas necróticas e de neurônios isquêmicos. Os camundongos CCR5-/- BCCAo apresentaram níveis mais baixos de CXCL1 e níveis mais altos de BDNF, em comparação com camundongos C57BL/6 BCCAo. O presente estudo demonstrou a associação dos déficits neurológicos com alterações istopatológicas e a expressão de fatores pró-inflamatórios após isquemia cerebral e reperfusão. Além disso, nossos resultados sugerem uma neuroproteção potencial na ausência do receptor CCR5 durante a lesão por isquemia/reperfusão global. In the present study, we used the BCCAo model for 25 minutes in wild C57BL/6 male mice to investigate neurological, pathological and inflammatory patterns during the acute phase from transient global ischemia in the brain. We observed neurological changes associated with morphological changes and inflammatory processes in the CNS, 72 hours after a reperfusion. Our results demonstrate brain levels of TNF, IL-1, CXCL1, and CCL5 were increased post-ischemia compared with sham-operated group. The activity of MPP-1 and MMP-2 was analyzed in the injury area by immunohistochemistry. The two markers increased in the BCCA group compared to the sham group. With the same BCCAo model in wild C57BL/6 and CCR5 receptor deficiente (CCR5-/-) C57BL/6 male mice, we investigate the involvement effects of CCR5 after ischemia. In CCR5 deficiency, we observed a significant improvement in the neurological deficits associated with the decrease of the infarcted area. In addition, a decrease in necrotic areas and ischemic neurons has been reported. C57BL/6 (CCR5-/-) ischemic mice showed lower levels of CXCL1 and higher levels of BDNF compared to C57BL/6 ischemic mice. Together, our results suggest potential neuroprotection in the absence of the CCR5 receptor in global ischemia/reperfusion injury. With the same BCCAo model. The present study demonstrated the association of neurological deficits with histopathological changes and expression of proinflammatory factors after cerebral ischemia and subacute eperfusion. Moreover, our data suggest a potential neuroprotection in the absence of the CCR5 receptor during global ischemia/reperfusion injury

A amebíase é a protozoose mais grave que atinge o ser humano e a segunda maior causa de morte entre as doenças parasitárias, superada apenas pela malária. Considera-se que apenas a Entamoeba histolytica é capaz de penetrar nos tecidos e produzir amebíase invasiva. Contudo, alguns estudos têm demonstrado que a Entamoeba dispar é capaz de produzir lesões em modelos experimentais. Com o objetivo de determinar se as bactérias enteropatogênicas contribuem para aumentar a expressão gênica dos fatores de virulência amebianos, bem como de induzir lesões mais intensas, propusemos avaliar a influência da bactéria Salmonella enterica sorovar Typhimurium sobre a virulência da Entamoeba dispar e da Entamoeba histolytica em trofozoítos isolados da cultura in vitro e avaliar patologicamente a colite amebiana, em ratos infectados com trofozoítos amebianos e co-infectados com S. Typhimurium. As cepas MCR, ADO e VEJ de E. dispar e, EGGp e EGGa de E. histolytica foram cultivadas separadamente e, associadas ou não à bactéria S. Typhimurium. Em seguida foi realizada a extração do RNA dessas culturas para produção de cDNA dos fatores de virulência de E. histolytica e E.dispar e quantificada a expressão dos fatores de virulência. Para a indução de colite amebiana, 8 ratos wistar (Rattus norvegicus) por grupo, previamente submetidos a uma intubação intragástrica da S. Typhimurium, foram inoculados pela via intracecal com 5x105 trofozoítos. Como grupos controles, 40 ratos foram somente inoculados com trofozoítos amebianos das cepas de Entamoeba estudadas. Outros 16 ratos constituíram os grupos controles somente infectados com S. Typhimurium e controle não infectado. Todos os ratos foram sacrificados 7 dias após a infecção amebiana para a coleta do ceco, confecção de lâminas histológicas para análise histopatológica e morfométrica e, para a realização de reações imuno-histoquímicas anti IL-1á, IL4, COX-2 e MUC-2. Nosso estudo permitiu observar que a co-cultura de E. dispar com a enterobactéria S. Typhimurium foi capaz de aumentar a expressão dos fatores de virulência amebianos amebaporo A, cisteína proteinase-5 e lectina ligante de Galactose e N-Acetilgalactosamina. Ao contrário do observado por outros autores, as cepas de E. dispar por nós estudados foram capazes de expressar o fator de virulência amebiano cisteína proteinase-5. A co-cultura de E. histolytica com a enterobactéria S. Typhimurium foi capaz de aumentar a expressão do fator de virulência amebiano amebaporo A. Também verificamos que na maioria dos grupos analisados, a associação in vivo da E. dispar ou da E. histolytica com a S. Typhimurium foi capaz de aumentar a intensidade da colite amebiana ulcerativa, também acompanhada pelo aumento da expressão de mediadores pró-inflamatórios e da redução da expressão de IL-4. Além disso, na maioria dos grupos analisados, a associação in vivo da E. dispar ou da E. histolytica com a S. Typhimurium foi capaz de aumentar a expressão da mucina MUC2, em virtude da maior intensidade da agressão à mucosa colônica. Diante dos resultados obtidos, concluímos que a interação da S. Typhimurium com a E. dispar e a E. histolytica foi capaz aumentar a virulência destas espécies de amebas levando à colite ulcerativa amebiana de maior intensidade. Amebiasis is the most serious protozoa that hits the human intestine and is the second-leading cause of death among parasitic diseases, surpassed only by malaria. It is considered that only Entamoeba histolytica is able to penetrate tissues and produce invasive amebiasis. However, some studies have shown that Entamoeba dispar is capable of producing lesions in experimental models. In order to determine if the enteropathogenic bacteria contribute to increase the gene expression of amebic virulence factors, as well as to induce more intense lesions, we propose to evaluate the influence of Salmonella Enterica sorovar Typhimurium on the virulence of Entamoeba dispar and Entamoeba histolytica in trophozoites isolated from the in vitro culture and pathologically evaluate amoebic colitis in rats infected with amoebic trophozoites and co-infected with S. typhimurium. E. dispar, MCR, ADO and VEJ strains from E. dispar and, EGGp and EGGa from E. histolytica were cultured separately and associated or not with the S. Typhimurium bacterium. Then, the RNA extraction from these cultures was carried out to produce cDNA from the virulence factors of E. histolytica and E.dispar and quantified the expression of the virulence factors. For the induction of amoebic colitis, 8 wistar rats (Rattus norvegicus) per group, previously submitted to an intragastric intubation of S. Typhimurium, were inoculated by the intracecal route with 5x105 trophozoites. As control groups, 40 rats were only inoculated with amebian trophozoites from the Entamoeba strains studied. Another 16 rats were the control groups infected only with S. typhimurium and uninfected control. All rats were sacrificed 7 days after amebic infection to collect cecum, histological slides for histopathological and morphometric analysis, and for the immunohistochemical anti-IL1á, IL4, COX-2 and MUC-2 immunohistochemical reactions . Our study showed that the co-culture of E. dispar with the S. typhimurium enterobacterium was able to increase the expression of amebaporo A, cysteine proteinase-5 and Galactose-binding lectin and N-acetylgalactosamine virulence factors. Contrary to what was observed by other authors, E. dispar strains studied by us were able to express the amoebic virulence factor cysteine proteinase-5. The co-culture of E. histolytica with the S. typhimurium enterobacterium was able to increase the expression of the amebian virulence factor amebaporo A. We also found that in the majority of the groups analyzed, the in vivo association of E. dispar or E. histolytica with S. typhimurium was able to increase the intensity of ulcerative amebic colitis, also accompanied by increased expression of proinflammatory mediators and reduced IL-4 expression. In addition, in most of the analyzed groups, the in vivo association of E. dispar or E. histolytica with S. typhimurium was able to increase MUC-2 mucin expression, due to the greater intensity of aggression to the colonic mucosa. In view of the results obtained, we conclude that the interaction of S. Typhimurium with E. dispar and E. histolytica was able to increase the virulence of these species of amoebae leading to ulcerative amebic colitis of greater intensity.

Atualmente, o número de indivíduos obesos no mundo tem atingido proporções alarmantes, o que tem chamado muita atenção devido às alterações metabólicas, imunológicas e ainda, à maior susceptibilidade a doenças infecciosas que a obesidade pode desencadear. Nos últimos anos tem surgido estudos que buscam esclarecer qual o impacto da adiposidade também nas infecções parasitárias. Entretanto, ainda não existem estudos sobre a associação entre obesidade e giardíase, a parasitose mais frequente em países desenvolvidos e com elevada prevalência em países em desenvolvimento. Como a prevalência de ambas as doenças se sobrepõe em diversas áreas, é importante avaliar se esta parasitose seria influenciada pelas debilidades na resposta imunológica de hospedeiros obesos. Foram utilizados 42 gerbils, machos, com idade média de 25 semanas, distribuídos em 6 grupos, com 7 animais cada: Controle (CT), Controle Infectado por 1 semana (CTIN) e Controle Infectado por 2 semanas (XCTIN), recebendo dieta com 68%, 7% e 20% de calorias provenientes de carboidratos, lipídeos e proteínas, respectivamente; Obeso (OB), Obeso Infectado por 1 semana (OBIN) e Obeso Infectado por 2 semanas (XOBIN), com dieta contendo 42%, 37,5% e 20% de calorias provenientes de carboidratos, lipídeos e proteínas, respectivamente. Na 10ª semana de experimento, os animais dos grupos CTIN, XCTIN, OBIN e XOBIN foram infectados com Giardia lamblia, oralmente, permanecendo infectados por 7 ou 14 dias, conforme o grupo, quando todos animais foram eutanasiados e coletados sangue, intestino delgado, tecido adiposo, fígado, baço e conteúdo fecal. Os animais foram pesados semanalmente utilizando-se balança com escala. A avaliação bioquímica, as dosagens séricas de glicemia, proteínas totais, albumina, colesterol e triglicerídeos foram determinadas por ensaio enzimático. As dosagens de citocinas foram feitas por ELISA. A determinação dos lípides hepáticos e fecais foi realizada conforme descrito por Folch e colaboradores. A avaliação do estresse oxidativo baseou-se na dosagem da concentração de substâncias reativas ao ácido tiobarbitúrico, na dosagem de catalase e de superóxido dismutase. Para análise histológica, a porção proximal do intestino delgado, uma fração do fígado e do tecido adiposo epididimal foram coletadas, fixadas e destinadas à obtenção de cortes e coloração com Hematoxilina & Eosina. A altura das vilosidades, profundidade de criptas e área total ocupada por gordura foram calculadas através do software KS300 contido no analisador de imagens Carl Zeiss. Os resultados foram expressos em média±erro padrão, utilizando-se os testes de Shapiro-Wilk, para verificar a normalidade da amostra e a Análise de Variância, seguida do teste de Newman-Keuls ou de Dunn para comparação entre os grupos experimentais. Utilizou-se o programa Prisma versão 6.0, considerando-se um nível de significância de 5%. Observou-se que os gerbils obesos apresentaram maior peso corporal, dos pesos relativos do tecido adiposo e do fígado, dislipidemia, maior deposição hepática de gordura total, colesterol e triglicerídeos, além da alteração da produção de adipocinas, tornando-o bom modelo para o estudo desta patologia associada ou não à infecção parasitária. A infecção por G.lamblia nos animais controle acarretou em lesão nas vilosidades e criptas intestinais, aumento de inflamação na submucosa e maior expressão de citocinas no intestino, principalmente na primeira semana de infecção. Além disso, provocou maior inflamação no tecido adiposo e deposição de lípides no fígado que se reestabeleceram na semana seguinte. Em obesos, não houve aumento de IL-2, 5 e 6 suficientemente permanente para conter a infecção, causando maior lesão no intestino e mais estresse oxidativo no fígado. Os grupos OBIN e XOBIN apresentaram ainda maior excreção de lípides nas fezes e XOBIN menor peso e adiposidade. Gerbils são modelos efetivos para estudo da obesidade induzida pela dieta. Além disso, a obesidade induzida pela dieta ocasionou alterações imunológicas e morfométricas em vários tecidos dos gerbils que prejudicaram a contenção do processo infeccioso por G. lamblia. Nowadays, the number of obese people in the world has reached alarming proportions, which has brought so much attention because of the metabolic, immunological and the increased susceptibility to infectious diseases that obesity can trigger. In recent years, the number of studies that seek to clarify the impact of adiposity also in parasitic infections have been increasing. However, there are no studies on the association between obesity and giardiasis, the most frequent parasitic disease in developed countries with a high prevalence in developing countries. As the prevalence of both diseases overlap in many areas, it is important to evaluate if this parasitosis would be influenced by the weaknesses in the immune response of obese hosts. 42 gerbils, males with a mean age of 25 weeks were used, divided into 6 groups with 7 animals each: control (CT), Infected Control for 1 week (CTIN) and Infected Control for 2 weeks (XCTIN), receiving diet with 68%, 7% and 20% of calories from carbohydrates, lipids and proteins, respectively; Obese (OB), Obeso Infected for 1 week (OBIN) and obese Infected for 2 weeks (XOBIN) with diet containing 42%, 37.5% and 20% of calories from carbohydrates, lipids and proteins, respectively. In the 10th week of the experiment, the animals of the groups CTIN, XCTIN, OBIN and XOBIN were orally infected with Giardia lamblia, and remain infected for 7 or 14 days, according to the group, when all animals were euthanized and collected blood, small intestine, adipose tissue, liver, spleen and fecal contents. The animals were weighed weekly using scale. For biochemical evaluation, serum glucose, total protein, albumin, cholesterol and triglycerides were determined by enzymatic assay. Cytokines dosages were performed by ELISA. The determination of liver and fecal lipids was performed as described by Folch et al. The evaluation of oxidative stress was based on the determination of the concentration of thiobarbituric acid reactive substances and the dosage of catalase and superoxide dismutase. For histological analysis, the proximal portion of the small intestine, a fraction of liver and epididymal adipose tissue were collected, fixed and intended for obteaining sections and staining with Hematoxylin & Eosin. The villus height, crypt depth and the total area occupied by fatty liver were calculated using the KS300 software from the Carl Zeiss image analyzer. The results were expressed in mean ± standard error, using the Shapiro-Wilk test to verify the normality of the sample, and analysis of variance, followed by Newman-Keuls or a Dunn's test for comparisons between experimental groups. We used the Prism version 6.0, considering a 5% significance level. It was observed that obese gerbils showed higher body weight, relative weight of adipose tissue and liver, dyslipidemia, increased hepatic deposition of total fat, cholesterol and triglycerides in addition to changed adipokines production, making it a good experimental model for this disease with or without parasitic infection. G.lamblia infection in control animals resulted in intestinal villi and crypts injury, increase submucosal inflammation and increased expression of cytokines in the intestine, especially in the first week of infection. Furthermore, it caused increased inflammation in adipose tissue and lipid deposition in the liver that reestablished the following week. In obese, there was no permanent increase in IL-2, 5 and 6 sufficiently to contain the infection, causing greater damage in the intestine and more oxidative stress in the liver. The OOBIN and XOBIN groups still showed a higher excretion of lipids in feces and XOBIN lower weight and adiposity. Gerbils are effective models for the study of obesity induced by diet. In addition, the diet-induced obesity caused immunological and histological changes in various tissues of gerbils that impaired the containment of the G. lamblia infection.

intestinal e a displasia, sendo mais frequentes no grupo CagA3Ep. Os resultados mostraram que linhagens de H. pylori contendo maior número de sítios de fosforilação EPIYA-C são significativamente mais patogênicas do que as linhagens com apenas um sítio de fosforilação EPIYA-C, levando ao surgimento de gastrite crônica mais grave e ao desenvolvimento mais frequente de lesões pré-cancerosas.O Helicobacter pylori (H. pylori) é uma bactéria que infecta cronicamente a mucosa gástrica de mais da metade da população mundial e que está relacionada ao desenvolvimento de gastrite crônica, úlceras duodenal e gástrica, adenocarcinoma gástrico e linfoma de células B da zona marginal do tecido linfóide associado a mucosa (linfoma MALT). Há estudos em humanos demonstrando que a infecção por linhagens de H. pylori contendo mais de um sítio de fosforilação de CagA, está relacionada ao desenvolvimento de lesões gástricas mais graves. Diante da inexistência de estudos experimentais sobre o tema, foi objetivo deste trabalho, avaliar os efeitos da infecção com linhagens de H. pylori contendo diferentes números de sítios de fosforilação de CagA sobre a mucosa gástrica de gerbils, em relação às alterações macroscópicas e microscópicas, o número de células G e D e, a expressão das mucinas MUC1 e MUC5AC, em períodos distintos do processo infeccioso. Quarenta e oito gerbils fêmeas com três meses de idade foram divididos em três grupos experimentais: um grupo controle consistindo de animais não infectados, animais tratados com linhagem de H. pylori CagA-positiva contendo um segmento EPIYA-C (CagA1Ep), e animais inoculados com linhagem de H. pylori com três segmentos EPIYA-C (CagA3Ep). Cada grupo foi constituído de 16 animais; oito animais de cada grupo foram eutanasiados 45 dias após a infecção, e os oito animais restantes foram eutanasiados seis meses após a infecção. Aos quarenta e cinco dias após a infecção, houve um aumento progressivo e significativo no número de células inflamatórias e na área de lâmina própria dos grupos CagA1Ep e CagA3Ep em relação ao grupo controle, sendo esse aumento mais expressivo no grupo CagA3Ep. Neste período da infecção foi observada uma diminuição no número de células D no grupo CagA3EP. Também aos 45 dias foi verificada uma diminuição na área total das mucinas MUC1 e MUC5AC nos grupos CagA1Ep e CagA3Ep em relação ao grupo controle, sendo mais intensa no grupo CagA3Ep. Aos seis meses após a infecção, todas as alterações mencionadas aos 45 dias, foram bem mais intensas, ocorrendo um aumento progressivo e significativo do infiltrado inflamatório, assim como também na área de lâmina própria nos grupos de animais infectados, principalmente no grupo CagA3Ep. Neste mesmo grupo houve aumento do número de células G e redução do número de células D. Nos grupos de animais infectados foi observada uma diminuição significativa e progressiva na expressão das mucinas MUC1 e MUC5AC, sendo mais proeminente no grupo CagA3Ep. Aos seis meses de infecção também foram observadas atrofia, metaplasia Helicobacter pylori (H. pylori) is a bacterium that infects chronically the gastric mucosa in more than half of the world population and it is related to the development of chronic gastrits, duodenal and gastric ulcers, gastric adenocarcinoma and lymphoma cutaneous B, from the marginal zone of the lymphoid tissue associated to the mucosa (linfoma MALT). There are studies in humans demonstrating that the infection by strains of H. pylori containing more than one site of CagA phosphorylation, is related to the development of more severe gastric lesions. With the absence of experimental studies on the subject, the objective of this study was to evaluate the effects of infection with H. pylori strains containing different numbers of CagA phosphorylation sites on the gerbis Mongolian gastric mucosa, in relation to the macroscopic and microscopic changes, the number of D and G cells and the the expressiveness of and MUC5AC mucins in different periods of the infection process. 48 gerbis Mongolian 3-months-old females were divided in three experimental groups: one control group consisting of non-infected animals; animals treated with H. pylori positive lineage containing one segment EPIYA-C (CagA1Ep) and animals inoculated with H. pylori CagA lineage with three segments EPIYA-C (CagA3Ep). Each group was composed of 16 animals; 8 animals of each group were sacrificed 45 days post-infection and the 8 remaining animals were sacrificed 6 months post-infection. At 45 days post-infection there was a progressive and significant increase in the number of inflammatory cells and in the site of the typical slide of the CagA1Ep and CagA3Ep groups in relation to the control group, this increase being most significant in the CagA3Ep group. During this infection period it was observed a decrease in the number of D cells in the CagA3Ep. At 45 days it was also observed a decrease in the total site of MUCI and MUC5AC mucins in the groups CagA1Ep and CagA3Ep, in relation to the control group, this being more intense in the group CagA3Ep. 6 months post-infection all the changes mentioned at 45 days were much more intense, occurring a progressive and significant increase in the inflammatory infiltrate, as well as in the site of the typical slide in the infected groups, mainly in the CagA3Ep. In this same group there was na increase of G cells and a reduction in the number of D cells. It was observed a significant and progressive decrease in the expressiveness of MUCI and MUC5AC mucins in the group of infected animals, this being more prominent in CagA3Ep group. At 6 months of infection it was also observed atrophy, intestinal metaplasia and dysplasia this being more frequent in CagA3Ep group. The results showed that H. pylori lineages containing a greater number of phosphorylation sites are sifnificantly more pathogenic than strains with only one site of phosphorylation EPIYA-C, leading to the development of more severe chronic gastrits and frequently to the development of pre-cancerous lesions.

São poucas as descrições das alterações patológicas e parasitológicas relacionadas ao envolvimento do trato gastrointestinal (TGI) na leishmaniose visceral canina, sobretudo de forma sistemática. Assim, neste trabalho, objetivou-se um estudo sistemático, anatomopatológico e parasitológico do TGI de cães naturalmente infectados com Leishmania (Leishmania) infantum. Após confirmação sorológica (RIFI e ELISA) e parasitológica (esfregaço de medula óssea), os cães foram divididos clinicamente em três grupos: sintomáticos (n =6) quando a manifestação de pelo menos um sinal clínico sugestivo de leishmaniose visceral; assintomáticos (n=6) quando da ausência de sinais clínicos e o grupo controle não infectado (n=5). Os animais foram cedidos pelo centro de zoonoses de Ribeirão das Neves e Carandaí, MG, região metropolitana de Belo Horizonte e área não endêmica dadoença, consecutivamente. Os animais foram eutanasiados e necropsiados com coleta dos seguintes segmentos do TGI: esôfago, estômago, duodeno, jejuno, íleo, ceco, cólon e reto. Fragmentos de todos os segmentos citados foram coletados em triplicata e fixados: (1) formalina tamponada 10%, solução de Carnoys e Acetona absoluta. OS tecidos após fixação foram incluídos em resina de glicolmetacrilato(GMA) e parafina para as colorações rotineiras de histologia e reações de imunohistoquímicas. As amostras do TGI foram utilizadas para colorações (H&E) e especiais (azul de toluidina, Dominici e PAS). Após padronização da técnica de inclusão em resina de GMA foram realizados estudos qualitativos e quantitativos (morfométricos) utilizando microscopia optica. Os principais sinais clínicos da doença foram dermatites não esfoliativas e a onicogrifose. Na análisemicroscópica, em todos os animais infectados, observou-se um notável aumento da celularidade da lâmina própria e submucosa em todos os segmentos do TGI em comparação com os animais controles.Lâminas feitas de tecidos incluídos em GMA forneceu uma análise morfológica com qualidade superior quando comparada aos tecidos incluídos em parafina, por apresentar maiores detalhes celulares confirmando o predomínio das células mononucleadas, difusamente localizadas na lamina própria (mucosa) e submucosa, representadas pelos plasmócitos, linfócitos e macrófagos. Células polimorfonucleares como neutrófilos e eosinófilos eram raramente observadas em todos os casos estudados. Macrófagos com morfologia peculiar, núcleo grandes (em relação as outras células) com cromatina frouxa e citoplasma amplo foram visualizados, as vezes vacuolizados com grande numerode formas amastigotas de Leishmania. A imuno-histoquímica de tecidos em GMA não foi satisfatória como comparada com tecidos parafinados. Amastigotas eram encontradas em maior proporção no intestino grosso (ceco,colón e reto) e delgado (estômago, duodeno, e íleo). Não foram encontrados marcações de amastigotas em esôfago e jejuno dos cães infectados. A técnica de inclusão em GMA em tecido canino mostrou-se de grande importância para as avaliações histológicas das amostras de TGI canino, apresentando um melhor resultado , principalmente no que diz respeito as colorações especiais, principalmente quando avaliados os mastócitos pelo azul de toluidina. Observou-se uma redução dessas células no em todos os segmentos de cães infectados. Comprovou-se a atrofia do epitélio em alguns segmentos de cães infectados quando comparados aos controles. Assim, concluímos que a técnica histológica do GMA é uma excelente ferramenta histológica para estudos morfológicos de amostras de TGI canino que reintera a presença de uma inflação crônica difusa ao longo de todo o TGI, sem formação de granulomas, com redução do número de mastócitos e atrofia epitelial em ambos os cães assintomáticos e sintomáticos. There are few descriptions of pathological and parasitological changes related to the involvement of the gastrointestinal (GIT) tract in canine visceral leishmaniasis especially systematically studies. Thus, this work aimed to a systematic study, pathological and parasitological GIT of dogs naturally infected with Leishmania (Leishmania) infantum. After serological confirmation (IFAT and ELISA) and parasitological (bone marrow smear), the dogs were divided clinically into three groups : symptomatic (n = 6) when the expression of at least one clinical sign suggestive of visceral leishmaniasis; asymptomatic (n = 6) when the absence of clinical signs and the uninfected control group (n = 3). The animals were provided by zoonoses center of Ribeirão das Neves andCarandaí, MG, Belo Horizonte metropolitan and non-endemic area of the disease, consecutively . The animals were euthanized and necropsied to collect the following segments of the GIT: esophagus, stomach , duodenum , jejunum , ileum , cecum , colon and rectum. Fragments of all segments cited were collected in triplicate and determined: (1) 10 % buffered formalin, (2)Carnoy 's solution and (3) absolute acetone . After fixation tissues were embedded in glycol methacrylate resin (GMA) andparaffin for routine histology staining and immunohistochemical reactions. GIT samples were used for staining (H & E) and special staining (toluidine blue ,Dominici and PAS). After standardization ofinclusion in GMA resin qualitative and quantitative (morphometric) using optical microscopy studies were performed. The main clinical signs of disease were exfoliative dermatitis and onychogryphosis . On microscopic examination, in all infected dogs, there was a remarkable increase in cellularity of the lamina propria and submucosa in all segments of the GIT compared with control dogs. Blades made oftissues embedding in GMA provided a morphological analysis with superior quality when compared to paraffin-embedded tissues, due to the higher cellular detail confirming the predominance of mononuclear cells diffusely located in the lamina propria (mucosa) and submucosa , represented by plasma cells , lymphocytes and macrophages. Polymorphonuclear cells such as neutrophils and eosinophils were rarely observed in all cases studied. Macrophages with peculiar morphology, large core (relative to other cells) with loose chromatin and large cytoplasm were visualized, sometimes vacuolated with large number of Leishmania amastigotes. Immunohistochemistry on tissue GMA was not satisfactory as compared to paraffin embedded tissues. Amastigotes were found in greater proportion in the large intestine (caecum, colon and rectum) and small intestine (stomach, duodenum,and ileum) . No markings of amastigotes in the esophagus and jejunum of infected dogs were found. The technique of embedding in GMA in canine tissue proved to be of great importance for the histological evaluations of samples of canine GIT, with a better result , especially as regards the special staining , especially when evaluated mast cells with toluidine blue. There was a reduction in these cells in every segment of infected dogs . Proved the epithelial atrophy in some segments ofinfected dogs when compared to controls . Thus, we conclude that the GMA histological technique is an excellent tool for morphological studies Histological samples of canine GITconfirm the presence of a diffuse chronic inflation throughout the GIT tract without formation of granulomas , which reduces the number of mast cells and epithelial atrophy in both asymptomatic and symptomatic dogs

Este estudo objetivou avaliar o papel das enzimas-conversoras de angiotensina (ECAs) na osteoartrite (OA) de joelho. Para tal, primeiramente, foi realizada a padronização do modelo de OA - induzida com monoiodoaceto de sódio (MIA) em ratos, quanto aos parâmetros inflamatórios e histopatológicos. Para induzir a OA foi feita uma única injeção intra-articular no joelho esquerdo de 2mg de MIA dissolvido em salina estéril (~50L). Os animais controles (CTL) receberam apenas uma injeção de salina. As medidas de hiperalgesia e edema de joelho foram realizadas antes e após os tempos de 6, 12, 24, 48 e 72 horas, 7, 14, 21 e 28 dias. Foram utilizados 8 animais em cada tempo de avaliação, sendo 4 animais CTL e 4 animais MIA. Nesses tempos os animais foram eutanasiados e foi realizado o lavado articular, utilizado para a verificação da cinética de migração celular e análise de citocinas. O joelho foi retirado para processamento histopatológico. O papel das ECAs foi avaliado após 28 dias de indução da OA, utilizando os seguintes tratamentos por 8 semanas (n=7 por grupo): Salina, Captopril [inibidor da ECA (iECA), 30mg/kg/dia], Losartan [bloqueador do receptor AT1, 30mg/kg/dia], Aceturato de diminazeno (DIZE) (ativador da ECA2), 1mg/kg/dia] ou [Captopril, 30mg/kg/dia + L-NAME (inibidor da óxido nítrico sintase), 15mg/kg/dia]. Os mesmos parâmetros avaliados na padronização do modelo foram utilizados nos grupos de tratamento, acrescentando a mensuração da pressão arterial média (PAM) e imunohistoquímica para IL-10 e TNF-. Os resultados da padronização mostraram que a hiperalgesia foi significativamente maior nos animais MIA em todos os tempos de medição (6h - 28 dias). A cinética de migração celular evidenciou que o pico de leucócitos ocorre no tempo de 72h, sendo o macrófago a principal célula responsável por esse pico. A diferença significativa dos macrófagos manteve-se em todos os tempos avaliados, exceto na medida de 21 dias. Já os neutrófilos tiveram o pico no tempo de 24h, sendo que nesse tempo também houve um aumento significativo no nível de IL-10. Observamos diferença estatisticamente significativa em relação ao CTL nos tempos de 12h e 24h no que se refere ao edema de joelho. Também no tempo de 12h ocorreu o pico de IL-6. A perda de proteoglicanos iniciou a partir do 7º dia, mas foi a partir do 14º dia que ela se tornou significativa ficando ainda maior nas avaliações dos dias 21 e 28 pós-indução. A avaliação do tecido de granulação presente entre o osso subcondral e a cartilagem mostrou que a alteração na celularidade inicia-se com 72 horas, mas apenas no tempo de 21 dias que se torna significativa. Não houve correlação entre o infiltrado inflamatório no osso e na cartilagem e a perda de proteoglicanos. Além disso, na avaliação das alterações morfológicas da cartilagem observou-se desorientação dos condrons, formação de clusters e atrofia de condrócitos a partir do dia 14 e de fissuras verticais e hipocelularidade a partir do dia 21. No dia 28 todas estas alterações encontram-se mais pronunciadas. Com relação à avaliação das ECAs na OA, os resultados mostraram que o Captopril e o Losartan foram capazes de reduzir de forma significativa o número de leucócitos totais e macrófagos na cavidade articular do joelho dos animais MIA. Além disso, o efeito do Captopril foi parcialmente bloqueado pelo L-NAME. Também houve aumento da área marcada para IL-10 na cartilagem dos animais controle tratados com Captopril. Por último, o iECA promoveu um pequeno efeito condroprotetor na cartilagem. Já o perfil inflamatório dos animais MIA tratados com DIZE não alterou significativamente, sendo o resultado similar ao dos animais tratados com salina. O tratamento com DIZE reduziu a intensidade de marcação de IL-10 e TNF- na cartilagem, sinóvia e osso subcondral dos animais MIA. Entretanto, o uso do ativador da ECA2 não impediu a perda de cartilagem nos animais MIA. Na avaliação histopatológica após 12 semanas de indução da OA foi visto que as alterações osteoartríticas foram muito pronunciadas, uma vez que havia grande perda de cartilagem e de proteoglicanos, com deformação do osso subcondral e presença de tecido fibrocartilaginoso na articulação. Houve uma tendência de redução da PAM nos animais MIA, mesmo quando tratados com salina. Concluindo, a inibição da ECA possui efeito anti-inflamatório na OA de joelho e um pequeno efeito condroprotetor. Esses resultados foram, possivelmente, limitados pela dose de MIA usada para induzir a OA e pelo longo tempo pós-indução da OA para o início dos tratamentos. The aim of this study was to evaluate the role of angiotensin-converting enzymes (ACEs) in osteoarthritis (OA) of the knee. Firstly, the standardization of the OA model by sodium monoiodoaceto (MIA) in rats, in relation to inflammatory and pathological parameters, was performed. Male Wistar rats (n=4 per group) were submitted to intra articular injection of 2mg (~50L) of MIA in the knee joint in order to induce OA. Control animals were injected with saline alone (~50L). Paw hyperalgesia and swelling of the knee before and after the intervals of 6, 12, 24, 48 and 72 hours and 7, 14, 21 and 28 days were evaluated. After these periods, the animals were euthanized and the knee joint lavage was performed and used to analyze the kinetics of cell migration and cytokines. Also, the knee was collected for histological analysis. The role of the ACEs was evaluated 28 days after induction of OA using the following orally treatment for 8 weeks (n=7): saline; Captopril [ACE inhibitor, 30mg/kg/day]; Losartan [AT1 receptors antagonist, 30mg/kg/day]; [Diminazene aceturate (DIZE) (ACE2 activator, 1mg/kg/day) or [Captopril, 30mg/kg/day + L-NAME (nitric oxide synthase inhibitor), 15mg/kg/day]. The parameters evaluated in the treatment groups were similar to those performed in the standardization protocol added to the measurement of mean arterial pressure (MAP) and immunohistochemistry for IL-10 and TNF-. The results of the standardization of the experimental model showed that the hyperalgesia was significantly higher in MIA animals at all times points (6 hours to 28 days). The kinetics of cell migration revealed that the leukocyte peak occurred at the time of 72 hours and the macrophage was the main cell responsible for this peak. This difference remained at all times evaluated, except to 21 days. Neutrophils peak happened at 24h and, in this time point, there was also a significant increase in the level of IL-10. It was observed that the swelling of the knee occurred at the time of 12h and 24h and the peak of IL-6 happened at the time of 12h. The loss of proteoglycans started in the 7th day, becoming more significant in the days 14, 21 and 28 post-induction. The evaluation of the granulation tissue present between the subchondral bone and cartilage showed that the change in the cellularity began at 72 hours, but only at the time of 21 days it is statistically different. No significant correlation was viewed between the inflammatory infiltrate in the bone and cartilage and the loss of proteoglycans. Furthermore, the evaluation of the morphological changes in the cartilage revealed disorientation of condrons, clustering and atrophy of chondrocytes starting at the day 14 and vertical fissures and hypocellularity starting at the day 21. On the day 28, all these changes were more pronounced. Regarding to the role of the ACEs in OA, the results showed that Captopril and Losartan were able to significantly reduce the number of total leukocytes and macrophages in the knee joint cavity of the MIA animals. Also, the effect of Captopril was partially blocked by L-NAME. There was a marked increase in the area of IL-10 labeling in the cartilage of control animals treated with Captopril. In addition, ACE inhibition led to a small chondroprotective effect in cartilage. However, the inflammatory profile of the DIZE treated MIA animals did not change significantly. Treatment with DIZE reduced the intensity staining of IL-10 and TNF- in the cartilage, subchondral bone and synovium of MIA rats. Nevertheless, the ACE2 activator did not preserve the cartilage of MIA animals. After 12 weeks of the induction of OA, the histopathological analysis showed that the osteoarthritic changes were more pronounced with great loss of cartilage and proteoglycans, deformation of the subchondral bone and presence of fibrocartilaginous tissue in the joint. There was a trend of reduction in MAP in MIA animals, even when treated with saline. In conclusion, inhibition of ACE has anti-inflammatory effects in the knee of OA rats, as well as a small chondroprotective effect. Likely, these results were limited by the dose of MIA used to induce OA and by the duration of the post-induction period before the evaluation of the parameters.

Modelos pré-clínicos de artrite reumatoide (AR) em roedores são amplamente empregados em estudos sobre os mecanismos da doença, bem como na triagem de candidatos a fármacos antiartríticos. Semelhanças morfológicas com a doença humana e capacidade de prever a eficácia clínica são critérios importantes na escolha do modelo. A artrite induzida por adjuvante em ratos é um modelo experimental de AR com histórico comprovado de previsibilidade de eficácia de candidatos a fármacos antiartríticos. O esquema de administração do adjuvante completo de Freund (CFA; Mycobacterium tuberculosis, 1 mg/mL), constituído de duas injeções no primeiro dia (100 ìL por via intradérmica na base da cauda e 100 ìL por via intraplantar na pata direita) e uma injeção no segundo dia (100 ìL por via intradérmica na base da cauda) em ratos Holtzman fêmeas, induziu inflamação periarticular localizada na pata injetada, de início imediato e de duração prolongada (21 dias). O caráter monoartrítico do modelo permitiu a observação de alterações típicas da fase inicial da AR em humanos, como edema, alodínia mecânica, paniculite, sinovite, neoangiogênese e elevação da atividade da mieloperoxidase, sem ocorrência de doença generalizada que pudesse resultar em grave desconforto para os animais. O aumento de temperatura corporal foi observado apenas após as duas primeiras injeções de CFA. Tendo em vista a atividade da riboflavina e da tiamina em modelos experimentais de dor e inflamação agudas, foi avaliado o efeito induzido pelo tratamento prolongado com essas vitaminas sobre a alodínia e o edema no modelo experimental. Riboflavina (125, 250 ou 500 mg/kg) ou tiamina (150, 300 ou 600 mg/kg) foram administradas per os, duas vezes ao dia, ao longo do período de avaliação. Os resultados demonstraram que a riboflavina não reduziu significativamente o edema de pata e a alodínia mecânica. O tratamento prolongado dos animais com tiamina não inibiu o edema de pata, mas atenuou a alodínia mecânica de forma dose-dependente no 6º e no 12º dias de avaliação. Também foi investigado o efeito induzido pela associação de tiamina (600 mg/kg) ou riboflavina (500 mg/kg) com dexametasona (0,5 mg/kg, per os) sobre a alodínia mecânica e edema de pata induzidos pelas injeções de CFA. A alodínia mecânica não foi atenuada pelo tratamento com dexametasona e não foi observado efeito antialodínico induzido pela associação de riboflavina com dexametasona. A associação tiamina e dexametasona induziu efeito antiedematogênico de maior magnitude do que aquele induzido por tiamina ou dexametasona isoladamente. Concluindo, os resultados demonstraram que o modelo de monoartrite induzida por CFA em ratos Holtzman foi reprodutível, sendo observadas alterações inflamatórias como sinovite, paniculite, neoangiogênese, edema de pata e alodínia mecânica persistentes. Entre as vantagens de um modelo experimental de AR no qual os animais desenvolvem monoartrite, mas não poliartrite, inclui a possibilidade de identificar sinais característicos da doença em apenas uma articulação, com menor comprometimento das funções fisiológicas e sofrimento dos animais e maior probabilidade de observação de efeitos induzidos por candidatos a fármacos. Os resultados também indicam que a riboflavina e, principalmente, a tiamina apresentam potencial no tratamento de pacientes com AR, principalmente em associação com outros fármacos com indicação já estabelecida. Preclinical models of rheumatoid arthritis (RA) in rodents have been widely used to investigate the disease mechanisms and also to screen new drugs. Similarities with the human disease and predictive value for efficacy and safety are important criteria to choose a preclinical model of RA. Arthritis induced by adjuvant in rats is a preclinical model of RA with a well established predictive value for efficacy of antiarthritic drugs. Two injections of complete Freund adjuvante (CFA; Mycobacterium tuberculosis, 1 mg/mL) in the first day (one intradermal injection of 100 ìL into the tail base and one intraplantar injection of 100 ìL into the right paw) followed by a third injection in the second day (a intradermal injection of 100 ìL into the tail base) in female Holtzman rats induced an immediate and long lasting (21 days) localized periarticular inflammation.The monoarthritic model allowed to identify characteristic changes of the early phase of RA in humans, including edema, mechanical allodynia, panniculitis, synovitis, neoangiogenesis and increase of myeloperoxidase activity, without systemic disease that resulted in severe animal suffering. Body temperature increase was observed only after the second injection of CFA. As riboflavin and thiamine activities have been demonstrated in acute models of pain and inflammation, their effects on the mechanical allodynia and edema in the experimental model were investigated. Riboflavin (125, 250 or 500 mg/Kg) or thiamine (150, 300 or 600 mg/Kg) were administered per os, twice daily, throughout the experimental period. Riboflavin reduced neither paw edema nor mechanical allodynia. Thiamine did not change paw edema, but dose-dependently inhibited the mechanical allodynia on days 6 and 12 of the experimental period. The effects induced by the association of thiamine (600 mg/Kg) or riboflavin (500 mg/kg) with dexamethasone (0.5 mg/Kg, per os) on the mechanical allodynia and paw edema induced by the injections of CFA were also investigated. Mechanical allodynia was inhibited neither by dexamethasone nor by the association of riboflavin with dexamethasone. The association of thiamine with dexamethasone, when compared with each drug, induced a greater antiedematogenic effect. In conclusion, the results of the present study demonstrate that the model of monoarthritis induced by CFA in Holtzman rats is reproducible, allowing to identify RA typical inflammatory changes such as long lasting synovitis, panniculitis, neoangiogenesis, paw edema and mechanical allodynia. Advantages of an experimental model of RA in which the animals develop monoarthritis, but not polyarthritis, include the possibility of identifying characteristic signs of the disease in a single joint, with reduced impact over the physiological functions, minor animal suffering and greater probability of detecting effects induced by drug candidates. The results also indicate that riboflavin and mainly thiamine may be useful in the treatment of RA patients, especially in association with other drugs with established efficacy.