Conditioned polarized Caco-2 cell monolayers allow to discriminate for the ability of gut-derived microorganisms to modulate permeability and antigen-induced basophil degranulation.

BACKGROUND: Food allergy is a common allergic disorder--especially in early childhood. The avoidance of the allergenic food is the only available method to prevent further reactions in sensitized patients. A better understanding of the immunologic mechanisms involved in this reaction would help to develop therapeutic approaches applicable to the prevention of food allergy. OBJECTIVE: To establish a multi-cell in vitro model of sensitized intestinal epithelium that mimics the intestinal epithelial barrier to study the capacity of probiotic microorganisms to modulate permeability, translocation and immunoreactivity of ovalbumin (OVA) used as a model antigen. METHODS: Polarized Caco-2 cell monolayers were conditioned by basolateral basophils and used to examine apical to basolateral transport of OVA by ELISA. Activation of basophils with translocated OVA was measured by beta-hexosaminidase release assay. This experimental setting was used to assess how microorganisms added apically affected these parameters. Basolateral secretion of cytokine/chemokines by polarized Caco-2 cell monolayers was analysed by ELISA. RESULTS: Basophils loaded with OVA-specific IgE responded to OVA in a dose-dependent manner. OVA transported across polarized Caco-2 cell monolayers was found to trigger basolateral basophil activation. Microorganisms including lactobacilli and Escherichia coli increased transepithelial electrical resistance while promoting OVA passage capable to trigger basophil activation. Non-inflammatory levels of IL-8 and thymic stromal lymphopoietin were produced basolaterally by Caco-2 cells exposed to microorganisms. CONCLUSION: The complex model designed in here is adequate to learn about the consequence of the interaction between microorganisms and epithelial cells vis-a-vis the barrier function and antigen translocation, two parameters essential to mucosal homeostasis. It can further serve as a direct tool to search for microorganisms with anti-allergic and anti-inflammatory properties.

Two new species of Nomimoscolex (Cestoda: Proteocephalidea, Monticelliidae) from Gymnotus carapo (Pisces: Gymnotiformes) in Argentina

Nomimoscolex guillermoi n. sp. and N. dechambrieri n. sp. are described from the gymnotiform fish Gymnotus carapo from Argentina. The new species are placed into Nomimoscolex based on the cortical position of the vitelline follicles, and medullary position of the testes, ovary, and uterus. Both species were compared to the 13 species considered valid in the genus. The combination of features distinguishing N. guillermoi from N. dechambrieri is (1) the position of the vagina to cirrus pouch (anterior or posterior vs always anterior respectively), (2) the total number of testes (41-85 vs 108-130 respectively), (3) the distribution of the vitelline follicles (arranged in dorso-lateral and ventro-lateral bands vs lateral bands respectively), (4) the length of the uteroduct (ending 58% vs 35% from posterior margin of mature proglottis respectively), and (5) the presence of gland cells in the scolex (unicellular glands in the apical region and the external margin of suckers vs the presence of unicellular glands in the apex and other grouped in a cluster medially to the suckers respectively).

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human.

Cardiopulmonary support and extracorporeal membrane oxygenation for cardiac assist.

BACKGROUND: Use of cardiopulmonary bypass for emergency resuscitation is not new. In fact, John Gibbon proposed this concept for the treatment of severe pulmonary embolism in 1937. Significant progress has been made since, and two main concepts for cardiac assist based on cardiopulmonary bypass have emerged: cardiopulmonary support (CPS) and extracorporeal membrane oxygenation (ECMO). The objective of this review is to summarize the state of the art in these two technologies. METHODS: Configuration of CPS is now fairly standard. A mobile cart with relatively large wheels allowing for easy transportation carries a centrifugal pump, a back-up battery with a charger, an oxygen cylinder, and a small heating system. Percutaneous cannulation, pump-driven venous return, rapid availability, and transportability are the main characteristics of a CPS system. Cardiocirculatory arrest is a major predictor of mortality despite the use of CPS. In contrast, CPS appears to be a powerful tool for patients in cardiogenic shock before cardiocirculatory arrest, requiring some type of therapeutic procedures, especially repair of anatomically correctable problems or bridging to other mechanical circulatory support systems such as ventricular assist devices. CPS is in general not suitable for long-term applications because of the small-bore cannulas, resulting in significant pressure gradients and eventually hemolysis. RESULTS: In contrast, ECMO can be designed for longer-term circulatory support. This requires large-bore cannulas and specifically designed oxygenators. The latter are either plasma leakage resistent (true membranes) or relatively thrombo-resistant (heparin coated). Both technologies require oxygenator changeovers although the main reason for this is different (clotting for the former, plasma leakage for the latter). Likewise, the tubing within a roller pump has to be displaced and centrifugal pump heads have to be replaced over time. ECMO is certainly the first choice for a circulatory support system in the neonatal and pediatric age groups, where the other assist systems are too bulky. ECMO is also indicated for patients improving on CPS. Septic conditions are, in general, considered as contraindications for ECMO. CONCLUSIONS: Ease of availability and moderate cost of cardiopulmonary bypass-based cardiac support technologies have to be balanced against the significant immobilization of human resources, which is required to make them successful.

In vivo binding of the Cry11Bb toxin of Bacillus thuringiensis subsp. medellin to the midgut of mosquito larvae (Diptera: Culicidae)

Bacillus thuringiensis subsp. medellin produces numerous proteins among which 94 kDa known as Cry11Bb, has mosquitocidal activity. The mode of action of the Cry11 proteins has been described as similar to those of the Cry1 toxins, nevertheless, the mechanism of action is still not clear. In this study we investigated the in vivo binding of the Cry11Bb toxin to the midgut of the insect species Anopheles albimanus, Aedes aegypti, and Culex quinquefasciatus by immunohistochemical analysis. Spodoptera frugiperda was included as negative control. The Cry11Bb protein was detected on the apical microvilli of the midgut epithelial cells, mostly on the posterior midgut and gastric caeca of the three mosquito species. Additionally, the toxin was detected in the Malpighian tubules of An. albimanus, Ae. aegypti, Cx. quinquefasciatus, and in the basal membrane of the epithelial cells of Ae. aegypti midgut. No toxin accumulation was observed in the peritrophic membrane of any of the mosquito species studied. These results confirm that the primary site of action of the Cry11 toxins is the apical membrane of the midgut epithelial cells of mosquito larvae.

The in vitro cytopathology of a porcine and the simian (SA-11) strains of rotavirus

Rotaviruses have been implicated as the major causal agents of acute diarrhoea in mammals and fowls. Experimental rotavirus infection have been associated to a series of sub-cellular pathologic alterations leading to cell lysis which may represent key functions in the pathogenesis of the diarrhoeic disease. The current work describes the cytopathic changes in cultured MA-104 cells infected by a simian (SA-11) and a porcine (1154) rotavirus strains. Trypan blue exclusion staining showed increased cell permeability after infection by both strains, as demonstrated by cell viability. This effect was confirmed by the leakage of infected cells evaluated by chromium release. Nuclear fragmentation was observed by acridine orange and Wright staining but specific DNA cleavage was not detected. Ultrastructural changes, such as chromatin condensation, cytoplasm vacuolisation, and loss of intercellular contact were shown in infected cells for both strains. In situ terminal deoxynucleotidyl transferase (Tunel) assay did not show positive result. In conclusion, we demonstrated that both strains of rotavirus induced necrosis as the major degenerative effect.

Vasopressin-stimulated CFTR Cl- currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome.

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

Cytostatic lung perfusion results in heterogeneous spatial regional blood flow and drug distribution: evaluation of different cytostatic lung perfusion techniques in a porcine model.

OBJECTIVES: Comparison of doxorubicin uptake, leakage and spatial regional blood flow, and drug distribution was made for antegrade, retrograde, combined antegrade and retrograde isolated lung perfusion, and pulmonary artery infusion by endovascular inflow occlusion (blood flow occlusion), as opposed to intravenous administration in a porcine model. METHODS: White pigs underwent single-pass lung perfusion with doxorubicin (320 mug/mL), labeled 99mTc-microspheres, and Indian ink. Visual assessment of the ink distribution and perfusion scintigraphy of the perfused lung was performed. 99mTc activity and doxorubicin levels were measured by gamma counting and high-performance liquid chromatography on 15 tissue samples from each perfused lung at predetermined localizations. RESULTS: Overall doxorubicin uptake in the perfused lung was significantly higher (P = .001) and the plasma concentration was significantly lower (P < .0001) after all isolated lung perfusion techniques, compared with intravenous administration, without differences between them. Pulmonary artery infusion (blood flow occlusion) showed an equally high doxorubicin uptake in the perfused lung but a higher systemic leakage than surgical isolated lung perfusion (P < .0001). The geometric coefficients of variation of the doxorubicin lung tissue levels were 175%, 279%, 226%, and 151% for antegrade, retrograde, combined antegrade and retrograde isolated lung perfusion, and pulmonary artery infusion by endovascular inflow occlusion (blood flow occlusion), respectively, compared with 51% for intravenous administration (P = .09). 99mTc activity measurements of the samples paralleled the doxorubicin level measurements, indicating a trend to a more heterogeneous spatial regional blood flow and drug distribution after isolated lung perfusion and blood flow occlusion compared with intravenous administration. CONCLUSIONS: Cytostatic lung perfusion results in a high overall doxorubicin uptake, which is, however, heterogeneously distributed within the perfused lung.

The kidney during hibernation and arousal from hibernation. A natural model of organ preservation during cold ischaemia and reperfusion.

BACKGROUND: During hibernation the kidney is in a hypothermic condition where renal blood flow is minimal and urine production is much reduced. Periodical arousal from hibernation is associated with kidney reperfusion at increasing body temperature, and restored urine production rate. METHODS: To assess the degree of structural preservation during such extreme conditions, the kidney cortex was investigated by means of electron microscopy in the dormouse Muscardinus avellanarius during winter hibernation, arousal from hibernation and the summer active period. RESULTS: Results show that the fine structure of the kidney cortex is well preserved during hibernation. In the renal corpuscle, a sign of slight lesion was the focal presence of oedematous endothelial cells and/or podocytes. Proximal convoluted tubule cells showed fully preserved ultrastructure and polarity, and hypertrophic apical endocytic apparatus. Structural changes were associated with increased plasma electrolytes, creatinine and urea nitrogen, and proteinuria. During the process of arousal the fine structure of the kidney cortex was also well maintained. CONCLUSION: These results demonstrate that dormice are able to fully preserve kidney cortex structure under extreme conditions resembling e.g. severe ischaemia or hypothermic organ storage for transplantation, and reperfusion. Elucidation of the mechanisms involved in such a natural model of organ preservation could be relevant to human medicine.

Cardiac rotation and relaxation after anterolateral myocardial infarction.

BACKGROUND: Both systolic and diastolic dysfunction have been observed in patients with anterolateral myocardial infarction. Diastolic dysfunction is related to disturbances in relaxation and diastolic filling. OBJECTIVE: To analyse cardiac rotation, regional shortening and diastolic relaxation in patients with anterolateral infarction. METHODS: Cardiac rotation and relaxation in controls and patients with chronic anterolateral infarction were assessed by myocardial tagging. Myocardial tagging is based on magnetic resonance imaging and allows us to label specific myocardial regions for imaging cardiac motion (rotation, translation and radial displacement). A rectangular grid was placed on the myocardium (basal, equatorial and apical short-axis plane) of each of 18 patients with chronic anterolateral infarction and 13 controls. Cardiac rotation, change in area and shortening of circumference were determined in each case. RESULTS: The left ventricle in controls performs a systolic wringing motion with a clockwise rotation at the base and a counterclockwise rotation at the apex when viewed from the apex. During relaxation a rotational motion in the opposite direction (namely untwisting) can be observed. In patients with anterolateral infarction, there is less systolic rotation at the apex and diastolic untwisting is delayed and prolonged in comparison with controls. In the presence of a left ventricular aneurysm (n = 4) apical rotation is completely lost. There is less shortening of circumference in infarcted and remote regions. CONCLUSIONS: The wringing motion of the myocardium might be an important mechanism involved in maintaining normal cardiac function with minimal expenditure of energy. This mechanism no longer operates in patients with left ventricular aneurysms and operates significantly less than normal in those with anterolateral hypokinaesia. Diastolic untwisting is significantly delayed and prolonged in patients with anterolateral infarction, which could explain the occurrence of diastolic dysfunction in these patients.

Pintomyia (Pifanomyia) paleotownsendi, a new sand fly from the Miocene amber of Dominican Republic (Diptera: Psychodidae: Phlebotominae)

Phlebotominae includes some vector species, mainly that of leishmaniases, with a very old host-parasite relationship. Some species fossils of this subfamily have been recently described and this paper presents the description of a new sand fly Pintomyia (Pifanomyia) paleotownsendi sp. nov in amber. The gonostyle present four spines, being one apical, one external superior implanted close to the apical third, one external inferior in the middle of the structure and one internal implanted in the basal third. This disposition of the spines may separate the new species from others in the sub genus.

Description of Lutzomyia (Lutzomyia) falquetoi sp. nov. (Diptera: Psychodidae, Phlebotominae) a new species from the state of Espírito Santo, Brazil

Lutzomyia (Lutzomyia) falquetoi, sp. nov. (Diptera: Psychodidae, Phlebotominae) is described from the state of Espírito Santo, Brazil. This new species belongs to the series longipalpis and is easily distinguished from the other members of this taxon by the presence of five well-developed spines and a pre-apical spiniform bristle on the gonostyle as well as nine bristles on the basal tuft of the gonocoxite.

Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vaccine candidates of Plasmodium vivax

In previous immuno-epidemiological studies of the naturally acquired antibody responses to merozoite surface protein-1 (MSP-1) of Plasmodium vivax, we had evidence that the responses to distinct erythrocytic stage antigens could be differentially regulated. The present study was designed to compare the antibody response to three asexual erythrocytic stage antigens vaccine candidates of P. vivax. Recombinant proteins representing the 19 kDa C-terminal region of MSP-1(PvMSP19), apical membrane antigen n-1 ectodomain (PvAMA-1), and the region II of duffy binding protein (PvDBP-RII) were compared in their ability to bind to IgG antibodies of serum samples collected from 220 individuals from the state of Pará, in the North of Brazil. During patent infection with P. vivax, the frequency of individuals with IgG antibodies to PvMSP1(19), PvAMA-1, and PvDBP-RII were 95, 72.7, and 44.5% respectively. Although the frequency of responders to PvDBP-RII was lower, this frequency increased in individuals following multiple malarial infections. Individually, the specific antibody levels did not decline significantly nine months after treatment, except to PvMSP1(19). Our results further confirm a complex regulation of the immune response to distinct blood stage antigens. The reason for that is presently unknown but it may contribute to the high risk of re-infection in individuals living in the endemic areas.

Morphological study of the eggs and nymphs of Triatoma dimidiata (Latreille, 1811) observed by light and scanning electron microscopy (Hemiptera: Reduviidae: Triatominae)

Eggs and nymphs of Triatoma dimidiata were described using both light and scanning electron microscopy. The egg body and operculum have an exochorion formed by irregular juxtaposed polygonal cells; these cells are without sculpture and the majority of them are hexagonal in shape. The five instars of T. dimidiatacan be distinguished from each other by characteristics of the pre, meso and metanotum. The number of setiferous tubercles increases progressively among instars. The sulcus stridulatorium of 1st instar nymphs is amorphous, showing median parallel grooves; from the 2nd instar on the sulcus is, progressively, elongate, deep and posteriorly pointed with stretched parallel grooves. All instars have a trichobothrium on the apical 1/3 of segment II of the antenna. The opening of the Brindley's gland is on the mesopleura. Fifth instar nymphs have an apical ctenidium on the ventral surface of the fore tibia. Dorsal glabrous patches are found on the lateral 1/3 of abdomen. Bright oval patches are found on the ventral median line of the abdomen, from segment IV-VI; 1st instar nymphs lack these patches. Abdominal dorsal plates are present from the 1st-5th instars; the 1st instar also contains a rectangular plate in segment IX. From the 2nd instar on, variably-shaped plates are present on segments VII to IX. Morphometric data were also obtained and proved to be useful for distinguishing T. dimidiata instars.

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.