Schistosoma mansoni Coinfection Attenuates Murine Toxoplasma gondii-Induced Crohn's-Like Ileitis by Preserving the Epithelial Barrier and Downregulating the Inflammatory Response.

Background and aims: Mice orally infected with T. gondii develop Crohn's disease (CD)-like enteritis associated with severe mucosal damage and a systemic inflammatory response, resulting in high morbidity and mortality. Previously, helminthic infections have shown therapeutic potential in experimental colitis. However, the role of S. mansoni in T. gondii-induced CD-like enteritis has not been elucidated. Our study investigated the mechanisms underlying T. gondii-induced ileitis and the potential therapeutic effect of S. mansoni coinfection. Methods: C57BL/6 mice were infected by subcutaneous injection of cercariae of the BH strain of S. mansoni, and 7-9 weeks later, they were orally infected with cysts of the ME49 strain of T. gondii. After euthanasia, the ileum was removed for histopathological analysis; staining for goblet cells; immunohistochemistry characterizing mononuclear cells, lysozyme expression, apoptotic cells, and intracellular pathway activation; and measuring gene expression levels by real-time PCR. Cytokine concentrations were measured in the serial serum samples and culture supernatants of the ileal explants, in addition to myeloperoxidase (MPO) activity. Results:T. gondii-monoinfected mice presented dense inflammatory cell infiltrates and ulcerations in the terminal ileum, with abundant cell extrusion, apoptotic bodies, and necrosis; these effects were absent in S. mansoni-infected or coinfected animals. Coinfection preserved goblet cells and Paneth cells, remarkably depleted in T. gondii-infected mice. Densities of CD4- and CD11b-positive cells were increased in T. gondii- compared to S. mansoni-infected mice and controls. MPO was significantly increased among T. gondii-mice, while attenuated in coinfected animals. In T. gondii-infected mice, the culture supernatants of the explants showed increased concentrations of TNF-alpha, IFN-gamma, and IL-17, and the ileal tissue revealed increased expression of the mRNA transcripts for IL-1 beta, NOS2, HMOX1, MMP3, and MMP9 and activation of NF-kappa B and p38 MAPK signaling, all of which were counterregulated by S. mansoni coinfection. Conclusion:S. mansoni coinfection attenuates T. gondii-induced ileitis by preserving mucosal integrity and downregulating the local inflammatory response based on the activation of NF-kappa B and MAPK. The protective function of prior S. mansoni infection suggests the involvement of innate immune mechanisms and supports a conceptually new approach to the treatment of chronic inflammatory diseases, including CD.

Sonic Hedgehog signaling and Gli-1 during embryonic chick myogenesis.

The Sonic Hedgehog signaling (Shh) pathway has been implicated in both proliferation of myoblast cells and terminal differentiation of muscle fibers, and contradictory results of these effects have been described. To clarify the role of Shh during myogenesis, we decided to study the effects of recombinant Shh and the distribution of Gli-1 during in vitro and in situ embryonic chick skeletal muscle differentiation at later stages of development. Gli-1 was found in small aggregates near the nucleus in mononucleated myoblasts and in multinucleated myotubes both in vitro and in situ chick muscle cells. Some Gli-1 aggregates colocalized with gamma-tubulin positive-centrosomes. Gli-1 was also found in striations and at the subsarcolemmal membrane in muscle fibers in situ. Recombinant Shh added to in vitro grown muscle cells induced the nuclear translocation of Gli-1, as well as an increase in the number of myoblasts and in the number of nuclei within myotubes. We suggest that Gli-1 aggregates observed in chick muscle cells near the nuclei of myoblasts and myotubes could be a storage site for the rapid cellular redistribution of Gli-1 upon specific signals during muscle differentiation.

Cynomolgus monkeys (Macaca fascicularis) experimentally and naturally infected with hepatitis E virus: The bone marrow as a possible new viral target.

Hepatitis E virus (HEV) transmission through infected blood and blood products has already been described. However, little is known about the bone marrow (BM) as source of HEV infection. Our study aimed to investigate the presence of HEV antigen (Ag) and histological changes in BM of cynomolgus monkeys (Macaca fascicularis) experimentally and naturally infected with HEV. Four cynomolgus monkeys with acute, and two with chronic hepatitis E ─ after immunosuppressive therapy with tacrolimus ─ were compared with one colony-bred animal naturally infected. Both, natural and experimental infections were characterized by anti-HEV IgG seroconversion detected by ELISA, and viral RNA isolation confirmed by RT-qPCR and qualitative nested RT-PCR. BM biopsies were collected from all animals, submitted to histology and indirect immunofluorescence techniques and observed, respectively, by light and confocal microscopy. The HEV Ag-fluorescent-labeled cells were detected from BM biopsies obtained from three monkeys with acute and one with chronic hepatitis E, and also from the naturally infected monkey. In the experimentally infected animals with acute hepatitis, HEV Ag detection occurred at 160 days post-infection, even after viral clearance in serum, feces, and liver. Double-stranded RNA, a replicative marker, was detected in BM cells from both acute and chronically infected animals. Major histological findings included vacuolization in mononuclear and endosteal cells, an absence of organized inflammatory infiltrates, and also some fields suggesting displasic focal BM disease. These findings support the hypothesis of BM cells as secondary target sites of HEV persistence. Further experimental studies should be carried out to confirm the assumption of HEV transmission through BM transplantation.

Hepatic microvascular dysfunction and increased advanced glycation end products are components of non-alcoholic fatty liver disease.

BACKGROUND: This study aimed to investigate the pathophysiology of hepatic microcirculatory dysfunction in non-alcoholic fatty liver disease (NAFLD). METHODS: In Wistar rats, NAFLD model was induced by 20 weeks of high-fat diet (HFD) feeding. Rolling and adhesion of leukocytes and tissue perfusion in hepatic microcirculation were examined using in vivo microscopic and laser speckle contrast imaging (LSCI), respectively. Oxidative stress and inflamatory parameters were analysed by TBARs, catalase enzyme activity, RT-PCR and ELISA. The participation of advanced glycation end-products (AGE) and its receptor RAGE was evaluated by the measurement of gene and protein expression of RAGE by RT-PCR and Western-blot, respectively and by liver and serum quantification of fluorescent AGEs. RESULTS: Wistar rats fed high-fat diet (HFD) showed increase in epididymal and abdominal fat content, systolic arterial blood pressure, fasting blood glucose levels, hepatic triglycerides and cholesterol, and impairment of glucose and insulin metabolisms. Liver histology confirmed the presence of steatosis and ultrasound analysis revealed increased liver size and parenchymal echogenicity in HFD-fed rats. HFD causes significant increases in leukocyte rolling and adhesion on hepatic microcirculation and decrease in liver microvascular blood flow. Liver tissue presented increase in oxidative stress and inflammtion. At 20 weeks, there was a significantly increase in AGE content in the liver and serum of HFD-fed rats and an increase in RAGE gene expression in the liver. CONCLUSION: The increase in liver AGE levels and microcirculatory disturbances could play a role in the pathogenesis of liver injury and are key components of NAFLD.

Combined therapy with metformin and insulin attenuates systemic and hepatic alterations in a model of high-fat diet-/streptozotocin-induced diabetes.

In this study we have explored the pathogenesis of the hepatic alterations which occur in diabetes and the modulation of these complications by the combination of metformin adjunct treatment and insulin monotherapy. For this purpose, diabetic rats were treated with insulin (DM + Ins) or metformin plus insulin (DM + Met + Ins). Biochemical and cardiometabolic parameters were analysed by spectrophotometry. Intravital microscopy was used to study the hepatic microcirculation. In the liver tissue, real-time PCR was used to analyse oxidative stress enzymes, inflammatory markers and receptors for advanced glycation end products (AGE) (RAGE) gene expression. Lipid peroxidation was assessed by thiobarbituric acid reactive species (TBARs) analyses. AGE deposition and RAGE protein expression were studied by fluorescence spectrophotometry and Western blot respectively. Body weight, %HbA1c , urea, total proteins and oxidative stress parameters were found to be similarly improved by insulin or Met + Ins treatments. On the other hand, Met + Ins treatment showed a more pronounced effect on fasting blood glucose level than insulin monotherapy. Fructosamine, uric acid, creatinine, albumin and amylase levels and daily insulin dose requirements were found to be only improved by the combined Met + Ins treatment. Liver, renal and pancreatic dysfunction markers were found to be more positively affected by metformin adjunct therapy when compared to insulin treatment. Liver microcirculation damage was found to be completely protected by Met + Ins treatment, while insulin monotherapy showed no effect. Our results suggest that oxidative stress, microcirculatory damage and glycated proteins could be involved in the aetiology of liver disease due to diabetes. Additionally, metformin adjunct treatment improved systemic and liver injury in induced diabetes and showed a more pronounced effect than insulin monotherapy.

In vitro Paracoccidioides brasiliensis biofilm and gene expression of adhesins and hydrolytic enzymes.

Paracoccidioides species are dimorphic fungi that initially infect the lungs but can also spread throughout the body. The spreading infection is most likely due to the formation of a biofilm that makes it difficult for the host to eliminate the infection. Biofilm formation is crucial for the development of infections and confines the pathogen to an extracellular matrix. Its presence is associated with antimicrobial resistance and avoidance of host defenses. This current study provides the first description of biofilm formation by Paracoccidioides brasiliensis (Pb18) and an analysis of gene expression, using real-time PCR, associated with 3 adhesins and 2 hydrolytic enzymes that could be associated with the virulence profile. Biofilm formation was analyzed using fluorescence microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Metabolic activity was determined using the XTT reduction assay. P. brasiliensis was able to form mature biofilm in 144 h with a thickness of 100 µm. The presence of a biofilm was found to be associated with an increase in the expression of adhesins and enzymes. GP43, enolase, GAPDH and aspartyl proteinase genes were over-expressed, whereas phospholipase was down-regulated in biofilm. The characterization of biofilm formed by P. brasiliensis may contribute to a better understanding of the pathogenesis of paracoccidioidomycosis as well as the search for new therapeutic alternatives; while improving the effectiveness of treatment.

Are fish paratenic natural hosts of the caiman haemoparasite Hepatozoon caimani?

The susceptibility of two fish and four mosquito species to the Caiman yacare haemoparasite Hepatozoon caimani was experimentally investigated. Mosquitoes belonging to four species (Aedes fluviatilis, Aedes albopictus, Aedes aegypti and Culex quinquefasciatus) were blood-fed on two naturally infected C. yacare from the Central-West Region of Brazil that exhibited distinct levels of parasitaemia: caimans A (11.05%) and B (1.25%). None of the engorged A. fluviatilis, A. albopictus or A. aegypti mosquitoes fed on caiman A survived for the duration of the sporogonic cycle; the great majority of the engorged mosquitoes died within 48 h of the blood meal. All A. aegypti fed on caiman B were negative, whereas 91.3% of dissected C. quinquefasciatus fed on the same caiman contained oocysts. Characid fish-Metynnis sp. and Astyanax sp.-were individually fed with C. quinquefasciatus females previously engorged (21-23 days) on caiman B. No parasite was found in the Astyanax fish. By contrast, 100% of the Metynnis fish depicted numerous cysts harbouring cystozoites identical to those of H. caimani, even more than 8 months after the ingestion of the infected mosquitoes. The cysts were located near the veins of the liver and, in some cases, close to the tunica intima of these vessels. No inflammatory reaction was observed. Gametocytes were observed in the blood smears of juvenile caimans that had ingested infected fish 9-12 weeks earlier. The potential role of fish as paratenic vertebrate hosts of H. caimani in nature is discussed.

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae.

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

Morphological and functional characterizations of Schwann cells stimulated with Mycobacterium leprae

Nerve damage, a characteristic of leprosy, is the cause of patient deformities and a consequence of Schwann cells (SC) infection by Mycobacterium leprae. Although function/dysfunction of SC in human diseases like leprosy is difficult to study, many in vitro models, including SC lines derived from rat and/or human Schwannomas, have been employed. ST88-14 is one of the cell lineages used by many researchers as a model for M. leprae/SC interaction. However, it is necessary to establish the values and limitations of the generated data on the effects of M. leprae in these SC. After evaluating the cell line phenotype in the present study, it is close to non-myelinating SC, making this lineage an ideal model for M. leprae/SC interaction. It was also observed that both M. leprae and PGL-1, a mycobacterial cell-wall component, induced low levels of apoptosis in ST88-14 by a mechanism independent of Bcl-2 family members.

Experimental leptospirosis in marmoset monkeys (Callithrix jacchus): a new model for studies of severe pulmonary leptospirosis.

Experimental infection of marmoset monkeys (Callithrix jacchus) with Leptospira interrogans serovar Copenhageni showed microscopic patterns of tissue reactions comparable to those seen in the severe forms of human leptospirosis, including intra-alveolar hemorrhage. The most impressive microscopic changes were seen in the lung and kidney of animals killed at days 6 and 12 after inoculation. There were extensive and irregular areas of hemorrhage predominating around main bronchial branches or diffusely spread to the pulmonary parenchyma, as well as severe tubulointerstitial nephritis. Antibody response detected by the microscopic agglutination test was quantitatively similar to those seen in humans and paralleled severity of tissue lesions. The distribution of leptospires or antigenic debris in infected tissues was observed by immunofluorescence and confocal laser scanning microscopy. Large numbers of typical leptospires were seen in the lumen of proximal renal tubules. Positive reactions showing antigenic debris were closely associated with sites of tissue damage.

Anopheles albitarsis embryogenesis: morphological identification of major events.

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180 degrees rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches.

Anopheles albitarsis embryogenesis: morphological identification of major events

Anopheles albitarsis embryogenesis was analyzed through confocal microscopy of clarified eggs. Using Drosophila melanogaster as reference system, the major morphogenetic events (blastoderm, gastrulation, germ band extension, germ band retraction, dorsal closure) were identified. The kinetics of early events is proportionally similar in both systems, but late movements (from germ band retraction on) progress slower in An. albitarsis. Major differences in An. albitarsis related to D. melanogaster were: (1) pole cells do not protrude from the blastoderm; (2) the mosquito embryo undergoes a 180º rotation movement, along its longitudinal axis; (3) the head remains individualized throughout embryogenesis; (4) extraembryonary membranes surround the whole embryo. A novel kind of malaria control is under development and is based on the use of genetically modified mosquitoes. Phenotypic analysis of the embryonic development of mutants will be imposed as part of the evaluation of effectiveness and risk of employment of this strategy in the field. In order to accomplish this, knowledge of the wild type embryo is a prerequisite. Morphological studies will also serve as basis for subsequent development biology approaches

Effects of schistosomal mansoni infection on Calomys callosus coelom-associated lymphomyeloid tissue (milky spots)

Calomys callosus Rengger, 1830 (Rodentia: Cricetidae) is a mouse-like South American wild rodent, which is permissive to Schistosoma mansoni infection. In this paper we studied the effect of schistosomal infection in C. callosus mesenteric and omental milky spots (MS), subsidiary foci of coelom-associated lymphomyeloid tissue (CALT), during the acute, transitional (acute to chronic), and chronic phases of the infection. MS were morphologically analyzed by historical methods, using brightfield and confocal laser scanning microscopies. The MS of infected animals were mainly of lymphomyelocytic (42 to 90 days) and lymphoplasmacytic (160 days of infection) types and showed frequent presence of lymphoid follicles with germinal centers, plasmacytogenesis and plasmacytosis, mastocytosis, megakaryopoiesis, erythropoiesis and less pronounced eosinopoiesis. These results indicate that MS are a preferencial site of germinal-center-dependent and independent plasmacytogenesis, and a bone narrow-like organ, committed with various cellular lineages. The consequence of a C. callosus MS reactivity for schistosomal infection is still unknown and is under investigation.

Histoarchitecture of schistosomal granuloma development and involution: morphogenetic and biomechanical approaches

The authors present morphogenetic and biomechanical approaches on the concept of the Schistosoma mansoni granulomas, considering them as organoid structures that depend on cellular adhesion and sorting, forming rearrangement into hierarchical concentric layers, creating tension-dependent structures, aiming to acquire round form, since this is the minimal energy form, in which opposing forces pull in equally from all directions and are in balance. From the morphogenetic point of view, the granulomas function as little organs, presenting maturative and involutional stages in their development with final disappearance (pre-granulomatous stages, subdivided in: weaky and/or initial reactive and exudative; granulomatous stages: exudative-productive, productive and involutional). A model for the development of granulomas was suggested according to the following stages: encapsulating, focal hystolysis, fiber production, orientation and compacting and involution and desintegration. The autors concluded that schistosomal granuloma is not a tangled web of individual cells and fibers, but an organized structure composed by host and parasite components, which is not formed to attack the miracidia, but functions as an hybrid interface between two different phylogenetic beings.

Morphological study of adult male worms of schistosoma mansoni Sambon, 1907 by confocal laser scanning microscopy

Aiming to detail data obtained through brightfield microscopy (BM) on reproductive, excretory and digestive system, specimens of Schistosoma mansoni eight weeks old, were recovered from SW mice stained with Langeron's carmine and analysed under a confocal laser scanning microscope CLSM 410 (Carl Weiss). The reproductive system presented a single and locate testis, with intercommunications between the lobes without efferent duct. Supernumerary testicular lobe was amorphus and isolated from the normal ones. Collecting tubules (excretory ducts), followed by the excretory bladder, opening to the external media through the excretory pore, were observed at the posterior extremity of the body. In the digestive tract, a cecal swelling was noted at the junction that originates the single cecum. It was concluded that through confocal laser scanning microscopy, new interpretations of morphological structures of S. mansoni worms could be achieved, modifying adopted and current descriptions. The gonad consists of a single lobed testis, similar to that observed in some trematode species. Moreover, the same specimens can be observed either by BM or CLSM, considering that the latter causes only focal and limited damage in tissue structures.