Animal model of corneal ectasia in rabbits by intrastromal injection of type II collagenase.

INTRODUCTION: Collagenase II has been used to induce experimental keratoconus in animal models. However, its effect when administered by intrastromal injection has not been studied, so the purpose of this study was to study the effects of intrastromal injection of collagenase II on corneal surface and corneal morphology. METHODS: Six New Zealand rabbits were used, collagenase II was administered by intrastromal injection (5µL of 2.5mg/mL) in the right eyes and balanced salt solution in the left eyes. Keratometry was performed to evaluate curvature alteration, also at day 7 corneas were obtained and Hematoxylin-Eosin staining was performed to examine morphologic changes. Likewise, changes in type I collagen expression were investigated by Sirius Red staining and semiquantitative PCR. RESULTS: K1, K2 and Km presented differences in the means with statistically significant changes. The morphological changes that were demonstrated were degradation and irregular arrangement of the corneal stroma, increase in the cellular density of keratocytes and slight cellular infiltration. Finally, it was demonstrated that there is greater expression of type I collagen fibers in the experimental group as opposed to the controls and the thickness of the fibers also increased due to the action of collagenase II, however, in terms of genetics there were no changes in the expression of type I collagen at molecular level between the control and experimental groups. CONCLUSIONS: Collagenase II administered by intrastromal injection is able to induce changes in the corneal surface and stroma, being able to simulate a model of keratoconus.

Sodium Selenite Diminished the Regulatory T Cell Differentiation In Vitro.

Sodium selenite modulates the activity of lymphocytes. It negatively regulates the suppressive activity of cells and increases the immune response. In this study, we evaluated whether the regulatory T cell differentiation was modulated by sodium selenite. The percentages of CD4+CD25+Foxp3+, CD4+CD25+, and CD4+CTLA-4+ cells in CD4+ T cells cultures stimulated with IL-2 and TGF-ß in the presence or absence of selenium, in the form of sodium selenite (2.0×10-6M), were evaluated by flow cytometry. The mRNA expression of TET2/3 enzymes and IL-10 was analyzed by RT-qPCR and the levels of IL-10 were measured by an ELISA. We observed a decrease in CD4+CD25+Foxp3+ and CD4+CTLA-4+ cells in presence of selenium. However, normal percentages were reached again after selenium removal. An increase in CD4+CTL4-4+ cells was detected in selenium-primed cell cultures in absence of IL-2 and TGF-ß. In addition, we observed a decrease in TET3 in presence of selenium. Finally, we observed an augment in IL-10 transcription and protein levels and relative expression of TET2 in cultures exposed to selenium. We suggest that selenium reversibly affects the regulatory T cell differentiation in vitro. Likewise, selenium may modulate Treg percentages promoting optimal immune responses and, at the same time, the expression of specific suppressor molecules.

Liver cirrhosis reversion is improved in hamsters with a neurointermediate pituitary lobectomy.

Regulating mechanisms of fibrosis is an important goal in the treatment of fibrosis and liver cirrhosis. The role of arginine vasopressin (AVP) in promoting fibrosis in several organs has been well documented. However, the result of an AVP deficiency during liver fibrosis has not been reported. We herein study the effects of an AVP deficiency, which was induced by neurointermediate pituitary lobectomy (NIL), on liver cirrhosis and liver cirrhosis reversion. Hamsters were intact (control) or underwent CCl4-induced cirrhosis, the latter animals divided into four groups: Cirrhotic, NIL-cirrhotic, Cirrhotic-reversion (R) and NIL-cirrhotic-R. Liver function, liver histopathology (including the fibrosis area and collagen types) and liver expression of MMP-13 and TIMP-2 were assessed. Results show that the AVP deficiency decreased the levels of alkaline phosphatase in serum and the expression of type I collagen and TIMP-2, and increased type III collagen deposition, MMP-13 expression and the size of regeneration nodules in NIL-cirrhotic and NIL-cirrhotic-R animals. A significantly greater recovery was found in the NIL-cirrhotic-R than the Cirrhotic-R group. We conclude that an AVP deficiency participates importantly in hamster liver regeneration by: 1) prompting the fibroblasts to produce type III collagen deposit, 2) influencing the activity of AP from bile duct cells, and 3) inhibiting TIMP-2 expression while favoring the fibrolytic activity of MMP-13.

A study on parasites in Mexican rheumatic disease patients.

Blastocystis hominis is a common human parasite with infection rates up to 50% in developing countries, and giardiasis is the commonest intestinal one in Mexico. No doubt, various parasites as Giardia lamblia and Entamoeba histolytica can cause rheumatic diseases. This study coproparasitoscopic analysis evaluated the cysts by B. hominis, G. lamblia, E. hartmani, E. coli and E. histolytica in Mexican rheumatic disease patients. Also, ELISA was used to detect E. histolytica, Ascaris lumbricoides, Toxocara canis, and Trichinella spiralis in Mexican patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Thirty-six patients (24 with AS and 12 with RA) and 77 healthy control individuals were enrolled in this study. The frequencies of protozoan cysts were comparable in rheumatic disease patients (AS and RA) and healthy control donors (33 and 25 vs. 26%, respectively; p > 0.05). The frequency of antibodies to T. canis was significantly higher in AS patients than in healthy control donors (16 vs. 2.6%, respectively; p = 0.027), whereas no differences were observed for the prevalence of antibodies for the other parasites (E. histolytica, A. lumbricoides and T. spiralis) (p > 0.05). This information indicates the need to intensify educational efforts for the prevention of parasite infections associated with AS disease that cannot be controlled only by drugs.

CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis.

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.

Delayed lung maturation of foetus of diabetic mother rats develop with a diminish, but without changes in the proportion of type I and II pneumocytes, and decreased expression of protein D-associated surfactant factor.

Newborn children of diabetic mothers have an increased morbidity and mortality because of respiratory distress syndrome. We study lung histogenesis during intrauterine development of offspring of diabetic Sprague-Dawley rats at 18, 19 and 21 days of gestation (DG). Pregnant rats were grouped into diabetic (streptozotocin-induced), citrate, and control groups; five female and five male offspring were selected randomly from each group at 18, 19 and 21 DG, and a biopsy of the lung was taken and processed in paraffin for histological examination. The biopsy for the transmission electron microscopy (TEM) analysis was taken at 21 days. A delay in alveolization of the offspring at 18, 19 and 21 days of the diabetic group was observed, which was confirmed at TEM level, and also less quantity of protein D associated to surfactant in diabetic group was detected (P < 0.001). The foetuses of the diabetic group presented a delay in lung histogenesis and in differentiation of the type II pneumocytes cells, but conserved the proportion with a decrease in 50% of pneumocytes, accompanied by a diminish of protein D associated to surfactant factor.

Ultrastructural analysis and identification of membrane proteins in the free-living amoeba Difflugia corona

Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.(AU)

Ultrastructural analysis and identification of membrane proteins in the free-living amoeba Difflugia corona

Syntaxin-1 and 25-kDa Synaptosome-associated Protein (SNAP-25) are present in the plasma membrane of several different secretory cell types and are involved in the exocytosis process. In this work, the free-living amoeba Difflugia corona was studied in relation to ultrastructure, structural membrane proteins, and proteins such as Syntaxin-1 and SNAP-25. Our results obtained by scanning electron microscopy in the amoeba without its theca, showed many membrane projections and several pore-like structures. Using immunocytochemistry, we found structural proteins Syntaxin-1 and SNAP-25.

Entamoeba histolytica: production of nitric oxide and in situ activity of NADPH diaphorase in amebic liver abscess of hamsters.

Entamoeba histolytica trophozoites were inoculated into the liver of hamsters and serum nitrate/nitrite levels [expressed as nitric oxide (NO) production] were determined at different times during amebic liver abscess (ALA) development. We also tested the effects of NO synthase (NOS) inhibitors such as N(G)-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, and dexamethasone during ALA production. Since NOS activity has been correlated with expression of reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) in tissues, we performed histochemistry studies to determine the activity of the latter in livers infected with E. histolytica trophozoites. Production of NO in serum was directly proportional to the size of ALAs, and NOS inhibitors caused low levels of NO and smaller ALAs. Our data suggest that NO does not have any lytic effect on E. histolytica trophozoites and is therefore incapable of providing protection against the amebic liver infection. In addition, NADPHd activity was detected histochemically in hepatocytes and inflammatory cells associated with focal necrosis containing trophozoites. The positive reactivity observed in these parasites may be attributable to a close biochemical similarity of NADPHd to the NADPH:flavin oxidoreductase described in E. histolytica by other investigators.

Induction of humoral immune response to Salmonella typhimurium in mouse Peyer's patches.

The mechanisms of immune response generation and regulation at the intestinal level are not well known, mainly due to the lack of suitable and reproducible methods to measure local immune responses. The Cunningham direct and indirect hemolytic plaque assay for the quantification of antibody producing cells against Salmonella in Peyer's patches of mice orally infected with Salmonella typhimurium was used. After infection IgM and IgG producing cells were determined on days 6, 9, 12 and 19. Specific antibody producing cells appeared after bacterial invasion of Peyer's patches, nine days after infection. At this time, there were more antibody producing cells in the distal part of the intestine, which correlated with a higher infection of these Peyer's patches as detected by bacterial culture. After day nine, the number of plaque forming cells was similar in both parts of the intestine. The peak of response was on day 12 and diminished on day 19. The number of IgM and IgG producing cells was similar in all days analyzed. Histological analysis of Peyer's patches of infected mice showed inflammation with disorganization and tumefaction.

[Prevalence of dental caries and rampant caries in a preschool population]. / Estudio sobre prevalencia de caries dental y caries rampante en población preescolar.

The results from a study on prevalence of ordinary dental caries and rampant caries in a children population are presented. The study was carried out at Dentistry Out patient Service in the Children Hospital "San Juan de Aragón" of the Federal District Department. The DMF index was obtained and its was proposed another one, in order to classify the different degrees of rampant caries in function of dental damage. An interview was applied to the fathers of the children of the study group in order to determinate hygiene oral habits, eating and familiar antecedents that could influence in the process of the ordinary and rampant caries and to compare between them. Serum and saliva were analyzed to determinate levels of albumin and IgM, IgG and IgA; there were also others factors tested that could indicate caries development.

[Mast cells in Peyer's patches in the mouse]. / Mastocitos en placa de Peyer de ratón.

Mast Cells participate in immediate and late hypersensitivity activities, immunoregulation and inflammation. Recently two groups of them have been detected on the basis to its morphology, content of granules and function: a) Those who are localized in connective tissue and b) Those of the intestinal mucosa. Those of the intestinal mucosa are T dependent and proliferate in parasitic as well as in intestinal hypersensitivity conditions to diverse antigens, in turn, Peyer's Patches (PP) are considered antigen catcher and initiators of intestinal immune responses; these are subject to the influence of diverse substances, several of which are within mast cells (for example: histamine, prostaglandins, etc); for which in this work we investigate the morphology relation between PP and mast cells. Balb/c mice small intestine segments with muscular layer and PP of proximal, middle and distal levels where studied; were histologically processed, toluidine blues stained and mast cells counted in different intestinal layers (PP underlying zone, rest of the muscular, corium and submucosa. Data were analyzed by the paired double T test for mean differences. A greater Quantity of mast cells were observed at the marginal zone of the PP in comparison to the rest of the muscular layer, submucosa and corium. The abundance of mast cells in relation to the PP possibly indicates its modulatory influence on the function of lymphoid cells of the PP.