The larval midgut of Anopheles, Aedes, and Toxorhynchites mosquitoes (Diptera, Culicidae): a comparative approach in morphophysiology and evolution.

The mosquito larval midgut is responsible for acquiring and storing most of the nutrients that will sustain the events of metamorphosis and the insect's adult life. Despite its importance, the basic biology of this larval organ is poorly understood. To help fill this gap, we carried out a comparative morphophysiological investigation of three larval midgut regions (gastric caeca, anterior midgut, and posterior midgut) of phylogenetically distant mosquitoes: Anopheles gambiae (Anopheles albimanus was occasionally used as an alternate), Aedes aegypti, and Toxorhynchites theobaldi. Larvae of Toxorhynchites mosquitoes are predacious, in contrast to the other two species, that are detritivorous. In this work, we show that the larval gut of the three species shares basic histological characteristics, but differ in other aspects. The lipid and carbohydrate metabolism of the An. gambiae larval midgut is different compared with that of Ae. aegypti and Tx. theobaldi. The gastric caecum is the most variable region, with differences probably related to the chemical composition of the diet. The peritrophic matrix is morphologically similar in the three species, and processes involved in the post-embryonic development of the organ, such as cell differentiation and proliferation, were also similar. FMRF-positive enteroendocrine cells are grouped in the posterior midgut of Tx. theobaldi, but individualized in An. gambiae and Ae. aegypti. We hypothesize that Tx. theobaldi larval predation is an ancestral condition in mosquito evolution.

Intestinal Transplant Immunology and Intestinal Graft Rejection: From Basic Mechanisms to Potential Biomarkers.

Intestinal transplantation (ITx) remains a lifesaving option for patients suffering from irreversible intestinal failure and complications from total parenteral nutrition. Since its inception, it became obvious that intestinal grafts are highly immunogenic, due to their high lymphoid load, the abundance in epithelial cells and constant exposure to external antigens and microbiota. This combination of factors and several redundant effector pathways makes ITx immunobiology unique. To this complex immunologic situation, which leads to the highest rate of rejection among solid organs (>40%), there is added the lack of reliable non-invasive biomarkers, which would allow for frequent, convenient and reliable rejection surveillance. Numerous assays, of which several were previously used in inflammatory bowel disease, have been tested after ITx, but none have shown sufficient sensibility and/or specificity to be used alone for diagnosing acute rejection. Herein, we review and integrate the mechanistic aspects of graft rejection with the current knowledge of ITx immunobiology and summarize the quest for a noninvasive biomarker of rejection.

Intestinal morphometry and cell density features in tropical farmed fish: A methodological approach.

The digestive tract of fish has many morphological adaptations related to habitat and nutrition. Intestinal biometry may reflect these adaptations. Here, we aimed to describe histometric patterns in farmed fish and their relationship with feeding by using a standardized protocol considering cell density by tissue area. Five juvenile specimens of each species (Pseudoplatystoma corruscans, Piaractus mesopotamicus, and Oreochromis niloticus) were used. O. niloticus possessed higher intestinal weight and length besides higher intestinal quotient and intestinal somatic index than the other species. The general histological composition was similar between species. However, P. corruscans showed differences in thickness between the anterior and posterior segments. O. niloticus had thinner serosa and muscularis layers than the other species. The cell density was distinct in both species and segments. Comparing the intestinal segments, O. niloticus displayed the lowest count of granulocytes. Goblet cell density was lower in P. mesopotamicus in all segments. However, the volume of these cells was higher in the anterior and middle anterior segments. Our data demonstrated that intestinal structural plasticity is associated with the difference in feeding habits. Here, we used quantitative standardized histometric criteria to understand the morphophysiological diversity of the fish digestive tract, and this technique can be applied in future studies to evaluate changes in the digestive tracts of vertebrates.

Pre-clinical evidence for the therapeutic effect of Pitaya (Hylocereus lemairei) on diabetic intestinal microenvironment.

Intestinal glucose absorption plays a central role in the regulation of glucose plasmatic; however, current clinical management does not target the gut for treating diabetes. This study evaluated the effects of peel and pulp aqueous extract from Hylocereus lemairei on human enterocytes under high glucose concentration. Anti-hyperglycemic and antiobesity activities in vitro were also evaluated. Extracts did not cause cytotoxicity at 1 to 500 µg/mL. Moreover, they were effective in attenuating oxidative stress (DCFH-DA assay) and inflammation (•ON production) caused by high glucose. Intestinal enzymes (α- glucosidase and pancreatic lipase) were inhibited by pulp and peel extracts (>60% and >95%, respectively). Extracts exhibited a redox capacity superior to ascorbic and chlorogenic acids, presenting high phenolic content, mainly anthocyanins. The main compounds for both extracts were chlorogenic acid and naringin, and peel stood both qualitatively and quantitatively. Data suggest red Pitaya has potential as a new medicine for diabetes.

Comparative analysis of the small and large intestines of Acará Geophagus brasiliensis (Quoy & Gaimard, 1824) (Pisces: Cichlidae).

Geophagus brasiliensis, popularly known as acará, is a common fish in lentic freshwater environments in South America. This species has a detritivorous-iliophagous or omnivorous feeding habit, with high food plasticity; however, there are no studies describing its intestinal tract histologically. Therefore, the present study analysed through histological and histochemical techniques the intestines of the acará. Adult specimens were collected with gillnets, anaesthetized and euthanized. Then, the fish were submitted to biometry and dissection to remove fragments of intestines. The samples were fixed in Bouin liquid for 12 hours and subjected to histological and histochemical techniques. Histologically, all samples of intestines were organized into four layers: mucosa, submucosa, muscular and serosa. The small intestine (foregut and midgut) was characterized by the presence of intestinal villi covered by simple prismatic epithelium with a striated border and goblet cells supported by the connective lamina propria. In the large intestine (hindgut), there was an absence of villi and an abundance of goblet cells. Positive reaction to Periodic Acid-Schiff (PAS) and Alcian Blue (AB) pH 2.5 reactions were detected in goblet cells, indicating the presence of mucosubstances. No lipids were detected in the intestine cells due to the negative reaction to the Sudan Black B. The results of the present study provide subsidies for a better understanding of the intestinal morphology of teleosts and provide valuable information for phylogenetic studies.

Mucosal Immunoregulatory Properties of Tsukamurella inchonensis to Reverse Experimental Food Allergy.

The intestinal mucosa is lined by epithelial cells, which are key cells to sustain gut homeostasis. Food allergy is an immune-mediated adverse reaction to food, likely due to defective regulatory circuits. Tsukamurella inchonensis is a non-pathogenic bacterium with immunomodulatory properties. We hypothesize that the anti-inflammatory effect of dead T. inchonensis on activated epithelial cells modulates milk allergy through the restoration of tolerance in a mouse model. Epithelial cells (Caco-2 and enterocytes from mouse gut) and macrophages were stimulated with T. inchonensis and induction of luciferase under the NF-κB promoter, ROS and cytokines production were studied. Balb/c mice were mucosally sensitized with cow´s milk proteins plus cholera toxin and orally challenged with the allergen to evidence hypersensitivity symptoms. After that, mice were orally administered with heat-killed T. inchonensis as treatment and then challenged with the allergen. The therapeutic efficacy was in vivo (clinical score and cutaneous test) and in vitro (serum specific antibodies and cytokines-ELISA, and cell analysis-flow cytometry) evaluated. Heat-killed T. inchonensis modulated the induction of pro-inflammatory chemokines, with an increase in anti-inflammatory cytokines by intestinal epithelial cells and by macrophages with decreased OX40L expression. In vivo, oral administration of T. inchonensis increased the frequency of lamina propria CD4+CD25+FoxP3+ T cells, and clinical signs were lower in T. inchonensis-treated mice compared with milk-sensitized animals. In vivo depletion of Tregs (anti-CD25) abrogated T. inchonensis immunomodulation. In conclusion, these bacteria suppressed the intestinal inflammatory immune response to reverse food allergy.

Morphological bases for intestinal paracellular absorption in bats and rodents.

Flying mammals present unique intestinal adaptations, such as lower intestinal surface area than nonflying mammals, and they compensate for this with higher paracellular absorption of glucose. There is no consensus about the mechanistic bases for this physiological phenomenon. The surface area of the small intestine is a key determinant of the absorptive capacity by both the transcellular and the paracellular pathways; thus, information about intestinal surface area and micro-anatomical structure can help explain differences among species in absorptive capacity. In order to elucidate a possible mechanism for the high paracellular nutrient absorption in bats, we performed a comparative analysis of intestinal villi architecture and enterocyte size and number in microchiropterans and rodents. We collected data from intestines of six bat species and five rodent species using hematoxylin and eosin staining and histological measurements. For the analysis we added measurements from published studies employing similar methodology, making in total a comparison of nine species each of rodents and bats. Bats presented shorter intestines than rodents. After correction for body size differences, bats had ~41% less nominal surface area (NSA) than rodents. Villous enhancement of surface area (SEF) was ~64% greater in bats than in rodents, mainly because of longer villi and a greater density of villi in bat intestines. Both taxa exhibited similar enterocyte diameter. Bats exceeded rodents by ~103% in enterocyte density per cm2 NSA, but they do not significantly differ in total number of enterocytes per whole animal. In addition, there is a correlation between SEF and clearance per cm2 NSA of L-arabinose, a nonactively transported paracellular probe. We infer that an increased enterocyte density per cm2 NSA corresponds to increased density of tight junctions per cm2 NSA, which provides a partial mechanistic explanation for understanding the high paracellular absorption observed in bats compared to nonflying mammals.

The reduction of oxidative stress by nanocomposite Fullerol decreases mucositis severity and reverts leukopenia induced by Irinotecan.

Irinotecan is a useful chemotherapeutic agent for the treatment of several solid tumors. However, this therapy is associated with side effects, including leukopenia and mucositis. Reactive oxygen species (ROS) activate inflammatory pathways and contribute to Irinotecan-induced mucositis. Fullerol is a nanocomposite with anti-oxidant properties that may reduce tissue damage after inflammatory stimuli. In this paper, the effects of Fullerol and mechanisms of protection were investigated in a model of Irinotecan-induced mucositis. Mucositis was induced by an injection of Irinotecan per 4 days in C57BL/6. Fullerol or a vehicle was injected every 12h. On day 7, the intestines were removed to evaluate histological changes, leukocyte influx, and the production of cytokines and ROS. Irinotecan therapy resulted in weight loss, an increased clinical score and intestinal injury. Treatment with Fullerol attenuated weight loss, decreased clinical score and intestinal damage. Irinotecan also induced increased ROS production in enterocytes, oxidative stress, IL-1ß production, neutrophil and eosinophil influx in the ileum. Fullerol treatment decreased production of ROS in the enterocytes, oxidative stress, IL-1ß production, neutrophil and eosinophil influx in the ileum. Irinotecan therapy also induced leukopenia in an ROS-dependent manner because leukopenia reverted in WT mice treated with Fullerol or Apocynin or in Gp91phox(-/-) mice. Mice treated with Irinotecan presented less melanoma tumor growth compared to the control group. Fullerol does not interfere in the anti-tumor action of Irinotecan. Fullerol has a great pharmacology potential to decreases the severity of mucositis and of leukopenia during chemotherapy treatment.

Histología del tubo digestivo de tres especies de pepino de mar Isostichopus badionotus, Isostichopussp.yStichopus hermanni (Aspidochirotida: Stichopodidae)

os pepinos de mar tienen un importante papel ecológico en el medio marino, ya que son capaces de alimentarse de la materia orgánica e inorgánica, con lo cual contribuyen a la oxigenación y la transferencia de energía en el ecosistema. En general, existe una falta de conocimiento de la morfología básica de especies nativas de pepino de mar y la función de los órganos vitales. El objetivo de este estudio fue describir la histología del tracto digestivo (DT) de tres especies de holotúridos de la bahía del Rodadero, Colombia. Treinta ejemplares de Isostichopus badionotus, Isostichopus sp. y Stichopus hermanni se capturaron y se sacrificaron por hipotermia. En el laboratorio se obtuvieron secciones del intestino anterior, medio y posterior y se fijaron en formalina (10 %) para su procesamiento histológico convencional. Además, algunas muestras fueron fijadas en glutaraldehído (3 %) para su inclusión en resinas y estudios en microscopía electrónica de alta resolución. Para las especies estudiadas, el TD es largo, lobulado, y se distribuye en la cavidad celómica; tiene al menos dos veces la longitud del cuerpo del pepino de mar. El TD presenta vellosidades revestidas por un epitelio ciliado columnar pseudoestratificado, que descansa sobre una membrana basal y una capa de fibras de colágeno. Se identificaron cuatro tipos de células: coelocmocitos, células cafes, enterocitos y células mucosas y las espículas fueron evidentes en todo el tejido del TD. La microscopía óptica mostró inclusiones alargadas de formación calcárea situadas esencialmente en el intestino grueso, "los cuerpos psamoma". Técnicas de microscopía de alta resolución y de microscopía electrónica mostraron células mucosas granulares cuya superficie apical contiene numerosas microvellosidades. La conformación tisular de los TD de I. badionotus, Isostichopus sp. y S. hermanni fueron similares. Se encontraron diferencias (p < 0.05) en el espesor del tejido submucosa intestinal de las especies estudiadas, que pueden estar ligadas a los hábitos específicos de alimentación de cada uno. La caracterización morfo-histológica del tracto digestivo del pepino de mar es una herramienta útil para entender su fisiología alimenticia.

ea cucumbers have an important ecological role in the marine environment because they are able to process organic and inorganic matter, which contributes to the oxygenation and energy transfer in the ecosystem.In general, there is a lack of knowledge on the basic morphology of native species of sea cucumber and the function of vital organs. The aim of this study was to describe the histology of the digestive tract (DT) of three species of holothuroids from Rodadero Bay, Colombia. Thirty specimens of Isostichopus badionotus, Isostichopus sp. and Stichopus hermanni were obtained and sacrificed by hypothermia. In the laboratory, sections of foregut, midgut and hindgut were obtained and fixed in formalin (10%) for later conventional histological processes; besides, some samples were fixed in glutaraldehyde (3%) for their inclusion in resins and studies in high resolution and electron microscopy. For the studied species, the DT is long, folded, and is distributed in the coelomic cavity; it has at least twice the length of the sea cucumber body. The DT presents villi lined by a columnar pseudostratified ciliated epithelium, which rests on a basement membrane and a layer of collagen fibers. Four types of cells were identified: coelocmocytes, brown cells, enterocytes and mucous cells, and the spicules were evident throughout the digestive tract tissue. Light microscopy showed elongated inclusions of calcareous formation located essentially in the hindgut, "the psamoma bodies". We observed granular mucous cells with an apical surface with numerous microvilli. The histology of the DT of I. badionotus, Isostichopus sp. and S. hermanni were found to be similar, but we found differences (p<0.05) in the thickness of the intestinal submucosa tissue, which can be tied to specific feeding habits of each species. Characterization of the morphohistology of the digestive tract of sea cucumber is a useful tool to understand their feeding physiology. Rev. Biol. Trop. 63 (4): 1021-1033. Epub 2015 December 01.

Intraepithelial lymphocytes in the duodenal mucosa of meat quails receiving diets containing or not containing probiotic and different levels of crude protein / Linfócitos intraepiteliais na mucosa do duodeno de codornas de corte recebendo dietas contendo ou não probiótico e diferentes níveis de proteína bruta / Linfocitos intraepiteliales en la mucosa duodenal de codornices de abate recibiendo dietas con o sin probiótico y diferentes niveles de proteína bruta

The aim of this study is to evaluate the effect of probiotic supplementation associated to different levels of crude protein (CP) on the count of intraepithelial lymphocytes (IELs) in the duodenum of meat quails. A total of 2304 quails were distributed in a completely randomized design in a 2 x 4 factorial scheme (with and without probiotic and four levels of CP - 15, 20, 25 and 30%), with two replicates per treatment in two experimental periods, in a total of 32 experimental units. At seven days old, two quails from each experimental unit were euthanized to harvest the duodenum segment. Semi-serial 7-µm histological sections were obtained, which were subsequently stained with hematoxylin-eosin to perform the IEL count. For the calculation, first 2500 epithelial cells were counted from the mucosa of each animal, and then the IELs present between these cells were counted, with the results expressed in amounts of IELs/100 epithelial cells. No differences were found in the IEL count among the treatments. Under the experimental conditions, it can be concluded that the use of probiotic associated to different levels of CP supplementation does not alter the IEL count in the duodenum of meat quails.

O objetivo deste trabalho foi avaliar o efeito da suplementação de probiótico associado com diferentes níveis de proteína bruta (PB) sobre a contagem de linfócitos intraepiteliais (LIEs) no duodeno de codornas de corte. Um total de 2304 codornas foram distribuídas em um delineamento inteiramente casualizado em esquema fatorial 2 x 4 (com e sem probiótico e quatro níveis de PB ­ 15, 20, 25 e 30%), com duas repetições por tratamento em dois períodos experimentais, totalizando 32 unidades experimentais. Aos sete dias de idade, duas codornas de cada unidade experimental foi eutanasiada para colheita do segmento do duodeno. Cortes histológicos semi-seriados de sete µm foram obtidos, os quais foram posteriormente corados com hematoxilina-eosina para realização da contagem de LIEs. Para o cálculo, primeiro foram contados 2500 células epiteliais da mucosa de cada animal, e então os LIEs presentes entre essas células foram contados e os resultados expressos como quantidade de LIEs /100 células epiteliais. Não foram encontradas diferenças entre os tratamentos na contagem de LIEs. Nas condições em que o experimento foi desenvolvido, conclui-se que o uso de probiótico associado a diferentes níveis de PB não altera a contagem de LIEs do duodeno de codornas de corte.

Esta investigación buscó evaluar el efecto de suplementos probióticos asociados a diferentes niveles de proteína bruta (PB) sobre el contaje de linfocitos intraepiteliales (LIEs) del duodeno de codornices de abate. Un total de 2304 codornices fueron distribuidas en un delineamiento enteramente casualizado en esquema factorial 2x4 (con y sin probiótico y cuatro niveles de PB ­ 15, 20, 25 y 30%), con dos repeticiones por tratamiento, en dos períodos experimentales, totalizando 32 unidades experimentales. A los siete días de edad, dos codornices de cada unidad experimental sufrieron eutanasia, para colecta de segmento del duodeno. Se obtuvo cortes histológicos semi seriados con sete µm, que fueron posteriormente colorados con hematoxilina-eosina para realización de contaje de LIEs. Para el cálculo, primeramente se contó 2500 células del epitelio de la túnica mucosa de cada animal, y en seguida los LIEs presentes entre esas células, siendo los resultados expresos en cantidad de LIEs/100 células epiteliales. No hubo diferencias en el contaje de LIEs en función de los tratamientos. En las condiciones en que el experimento se realizó, se puede concluir que el uso de probiótico asociado a diferentes niveles de suplementos de PB no altera el contaje de LIEs en el duodeno de codornices de abate.

A Comparison of mucosal surface area and villous histology in small intestines of the Brazilian free-tailed bat (Tadarida brasiliensis) and the mouse (Mus musculus).

Studies on birds have led to the hypothesis that increased intestinal absorption between enterocytes (paracellular) evolved as a compensation for smaller intestinal size in fliers, which was perhaps selected to minimize the mass of digesta carried. This hypothesis predicts that bats will also exhibit relatively reduced intestinal size and high paracellular absorption, compared with nonflying mammals. Published studies on three bat species indicate relatively high paracellular absorption. One mechanism for increasing paracellular absorption per cm2 small intestine (SI) is increased number of tight junctions (TJs) across which paracellular absorption occurs. To our knowledge, we provide the first comparative analysis of enterocyte size and number in flying and nonflying mammals. Intestines of insectivorous bats Tadarida brasiliensis were compared with Mus musculus using hematoxylin and eosin staining method. Bats had shorter and narrower SIs than mice, and after correction for body size difference by normalizing to mass3/4, the bats had 40% less nominal surface area than the mouse, as predicted. Villous enhancement of surface area was 90% greater in the bat than in the mouse, mainly because of longer villi and a greater density of villi in bat intestines. Bat and mouse were similar in enterocyte diameter. Bats exceeded mice by 54.4% in villous area per cm length SI and by 95% in number of enterocytes per cm2 of the nominal surface area of the SI. Therefore, an increased density of TJs per cm2 SI may be a mechanistic explanation that helps to understand the high paracellular absorption observed in bats compared to nonflying mammals.

Intraepithelial lymphocytes in the duodenal mucosa of meat quails receiving diets containing or not containing probiotic and different levels of crude protein / Linfócitos intraepiteliais na mucosa do duodeno de codornas de corte recebendo dietas contendo ou não probiótico e diferentes níveis de proteína bruta / Linfocitos intraepiteliales en la mucosa duodenal de codornices de abate recibiendo dietas con o sin probiótico y diferentes niveles de proteína bruta

The aim of this study is to evaluate the effect of probiotic supplementation associated to different levels of crude protein (CP) on the count of intraepithelial lymphocytes (IELs) in the duodenum of meat quails. A total of 2304 quails were distributed in a completely randomized design in a 2 x 4 factorial scheme (with and without probiotic and four levels of CP - 15, 20, 25 and 30%), with two replicates per treatment in two experimental periods, in a total of 32 experimental units. At seven days old, two quails from each experimental unit were euthanized to harvest the duodenum segment. Semi-serial 7-µm histological sections were obtained, which were subsequently stained with hematoxylin-eosin to perform the IEL count. For the calculation, first 2500 epithelial cells were counted from the mucosa of each animal, and then the IELs present between these cells were counted, with the results expressed in amounts of IELs/100 epithelial cells. No differences were found in the IEL count among the treatments. Under the experimental conditions, it can be concluded that the use of probiotic associated to different levels of CP supplementation does not alter the IEL count in the duodenum of meat quails.(AU)

O objetivo deste trabalho foi avaliar o efeito da suplementação de probiótico associado com diferentes níveis de proteína bruta (PB) sobre a contagem de linfócitos intraepiteliais (LIEs) no duodeno de codornas de corte. Um total de 2304 codornas foram distribuídas em um delineamento inteiramente casualizado em esquema fatorial 2 x 4 (com e sem probiótico e quatro níveis de PB ­ 15, 20, 25 e 30%), com duas repetições por tratamento em dois períodos experimentais, totalizando 32 unidades experimentais. Aos sete dias de idade, duas codornas de cada unidade experimental foi eutanasiada para colheita do segmento do duodeno. Cortes histológicos semi-seriados de sete µm foram obtidos, os quais foram posteriormente corados com hematoxilina-eosina para realização da contagem de LIEs. Para o cálculo, primeiro foram contados 2500 células epiteliais da mucosa de cada animal, e então os LIEs presentes entre essas células foram contados e os resultados expressos como quantidade de LIEs /100 células epiteliais. Não foram encontradas diferenças entre os tratamentos na contagem de LIEs. Nas condições em que o experimento foi desenvolvido, conclui-se que o uso de probiótico associado a diferentes níveis de PB não altera a contagem de LIEs do duodeno de codornas de corte.(AU)

Esta investigación buscó evaluar el efecto de suplementos probióticos asociados a diferentes niveles de proteína bruta (PB) sobre el contaje de linfocitos intraepiteliales (LIEs) del duodeno de codornices de abate. Un total de 2304 codornices fueron distribuidas en un delineamiento enteramente casualizado en esquema factorial 2x4 (con y sin probiótico y cuatro niveles de PB ­ 15, 20, 25 y 30%), con dos repeticiones por tratamiento, en dos períodos experimentales, totalizando 32 unidades experimentales. A los siete días de edad, dos codornices de cada unidad experimental sufrieron eutanasia, para colecta de segmento del duodeno. Se obtuvo cortes histológicos semi seriados con sete µm, que fueron posteriormente colorados con hematoxilina-eosina para realización de contaje de LIEs. Para el cálculo, primeramente se contó 2500 células del epitelio de la túnica mucosa de cada animal, y en seguida los LIEs presentes entre esas células, siendo los resultados expresos en cantidad de LIEs/100 células epiteliales. No hubo diferencias en el contaje de LIEs en función de los tratamientos. En las condiciones en que el experimento se realizó, se puede concluir que el uso de probiótico asociado a diferentes niveles de suplementos de PB no altera el contaje de LIEs en el duodeno de codornices de abate.(AU)

Identification of uncoupling protein 4 from the blood-sucking insect Rhodnius prolixus and its possible role on protection against oxidative stress.

Uncoupling proteins (UCPs) play a critical role in the control of the mitochondrial membrane potential (ΔΨm) due to their ability to dissipate the proton gradient, which results in the uncoupling of mitochondrial respiration from ATP production. Most reactive oxygen species generation in mitochondria occurs in complex III, due to an increase of semiquinone (Q(-)) half-life. When active, UCPs can account as a potential antioxidant system by decreasing ΔΨm and increasing mitochondrial respiration, thus reducing Q(-) life time. The hematophagous insect Rhodnius prolixus, a vector of Chagas disease, is exposed to a huge increase in oxidative stress after a blood meal because of the hydrolysis of hemoglobin and the release of the cytotoxic heme molecule. Although some protective mechanisms were already described for this insect and other hematophagous arthropods, the putative role of UCP proteins as antioxidants in this context has not been explored. In this report, two genes encoding UCP proteins (RpUcp4 and RpUcp5) were identified in the R. prolixus genome. RpUcp4 is the predominant transcript in most analyzed organs, and both mRNA and protein expression are upregulated (13- and 3-fold increase, respectively) in enterocytes the first day after the blood feeding. The increase in UCP4 expression is coincident with the decrease in hydrogen peroxide (H2O2) generation by midgut cells. Furthermore, in mitochondria isolated from enterocytes, the modulation of UCP activity by palmitic acid and GDP resulted in altered ΔΨm, as well as modulation of H2O2 generation rates. These results indicate that R. prolixus UCP4 may function in an antioxidation mechanism to protect the midgut cells against oxidative damage caused by blood digestion.

Immunohistochemical studies on duodenum, spleen and liver in mice: distribution of ferroportin and prohepcidin in an inflammation model / Estudios inmunohistoquímicos en duodeno, bazo e hígado de ratón: distribución de ferroportina y prohepcidina en un modelo de inflamación

Duodenum, spleen and liver have a crucial role in iron balance on the whole organism and are the major sites of Ferroportin (FPN) expression. Specific regulations between FPN and hepcidin are responsible for changes seen in physiopathological conditions such as inflammation. We studied in vivo effects of turpentine oil-induced acute inflammation on FPN expression, and its relation with prohepcidin and iron mobilization. Immunohistochemical procedures were performed using rabbit anti-mouse FPN and prohepcidin antibodies with goat-labeled polymer-HRP anti-rabbit (DAB) as secondary antibody. Plasma and tissular iron were also studied. Our results showed a notable expression and redistribution of duodenal FPN to basolateral membrane in turpentine-treated mice, compared with supranuclear and the weak basolateral expression observed in healthy mice. Red pulp macrophages of healthy mice showed FPN-hemosiderin co-localization, compared with turpentine-treated mice which showed lack of FPN. In liver of healthy mice, FPN was seen in Kupffer cells, whereas in turpentine-treated mice decreased. In addition, we observed an increment of hepatic pro-hepcidin with a significant hypoferremia. Our findings demonstrated that acute inflammation induced a differential distribution of FPN, showing a cell type specific response. In macrophages, increased hepatic prohepcidin induced degradation of FPN, resulting in hypoferremia. In enterocytes, the redistribution observed of duodenal FPN reflects a different regulation in this tissue. The observed response of the proteins studied may be part of a cyclical pattern of systemic effects of acute inflammation on mouse tissue.

El duodeno, bazo e hígado desempeñan un rol clave en el balance de Fe del organismo y son los mayores sitios de expresión de ferroportina (FPN). Regulaciones específicas entre FPN y hepcidina son las responsables de los cambios observados en condiciones fisiopatológicas como la inflamación. Nuestro objetivo fue estudiar los efectos in vivo de la inflamación aguda inducida con turpentina sobre la expresión de FPN y su relación con prohepcidina y la movilización de hierro. Los procedimientos inmunohistoquímicos fueron desarrollados utilizando anticuerpos anti FPN y prohepcidina de ratón, desarrollados en conejo y un polímero conjugado con anticuerpos secundarios anti conejo desarrollado en cabra (HRP-DAB). Se evaluaron los niveles de Fe plasmático y tisular. Nuestros resultados mostraron una clara expresión y redistribución de FPN duodenal hacia la membrana basolateral en ratones tratados con turpentina, con respecto a la expresión perinuclear y leve expresión basolateral observada en ratón sano. Macrófagos de la pulpa roja esplénica mostraron co-localización de FPN y hemosiderina, comparado con la ausencia de expresión en ratón tratado con turpentina. En hígado de ratón sano, se observó expresión de FPN en células de Kupffer, mientras que en ratón tratado con turpentina la expresión fue menos evidente. Además, observamos un aumento en la expresión de prohepcidina hepática con una hipoferremia significativa. Nuestros resultados demostraron que la inflamación aguda indujo una distribución diferencial de FPN, mostrando una respuesta específica del tipo celular. En macrófagos, el aumento de prohepcidina hepática indujo degradación de FPN, resultando en hipoferremia. En enterocitos, la redistribución observada de FPN duodenal, refleja una regulación diferente en este tejido. La respuesta observada de las proteínas estudiadas podría ser parte de un patrón cíclico de efectos sistémicos de la inflamación aguda en tejidos murinos.

Distinct Interaction of Two Atypical Enteropathogenic Escherichia coli Strains with Enterocytes In Vitro.

Typical and atypical Enteropathogenic Escherichia coli (EPEC) promote attaching-effacing lesions in intestinal cells but only typical EPEC carry the EPEC adherence factor plasmid. Atypical EPEC (aEPEC) are emerging agents of acute and persistent diarrhea worldwide. We aimed at comparing the ability of two aEPEC strains, 1711-4 (serotype O51:H40) and 3991-1 (serotype O non-typeable:non-motile) to invade, persist inside Caco-2 and T84 cells, and to induce IL-8 production. Typical EPEC strain E2348/69 was used for comparisons. The strains associated more significantly with T84 than with Caco-2 cells, with 3991-1 being the most adherent (P < 0.001). In contrast, aEPEC 1711-4 was significantly more invasive than the other strains in both cell lines, and was found within vacuoles near the basolateral cell surfaces. Strains persisted within both cell lines for at least 48 hours, but the persistence index was higher for 3991-1 in Caco-2 cells. IL-8 production was significantly higher from Caco-2 cells infected with 1711-4 for at least 48 hours (P < 0.001), and from T84 cells after 24 and 48 h than with the other strains (P = 0.001). We demonstrated that aEPEC are heterogeneous in various aspects of their interaction with enterocytes in vitro.

Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.