The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis

Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL–1 and IL–18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL–1β (405%), IL–18 (365%), COX–2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88^-/- and TLR2^-/- mice, the irinotecan-injected TLR9^-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL–18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2^-/- or TLR9^-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.     

Seasonal change in the proportion of bacterial and phytoplankton production along a salinity gradient in a shallow estuary

We intended to evaluate the relative contribution of primary production versus allochthonous carbon in the production of bacterial biomass in a mesotrophic estuary. Different spatial and temporal ranges were observed in the values of bacterioplankton biomass (31–273 g C l^–1) and production (0.1–16.0 g C l^–1 h^–1, 1.5–36.8 mg C m^–2 h^–1) as well as in phytoplankton abundance (50–1700 g C l^–1) and primary production (0.1–512.9 g C l^–1 h^–1, 1.5–512.9 mg C m^–2 h^–1). Bacterial specific growth rate (0.10–1.68 d^–1) during the year did not fluctuate as much as phytoplankton specific growth rate (0.02–0.74 d^–1). Along the salinity gradient and towards the inner estuary, bacterio- and phytoplankton biomass and production increased steadily both in the warm and cold seasons. The maximum geographical increase observed in these variables was 12 times more for the bacterial community and 8 times more for the phytoplankton community. The warm to cold season ratios of the biological variables varied geographically and according to these variables. The increase at the warm season achieved its maximum in the biomass production, particularly in the marine zone and at high tide (20 and 112 times higher in bacterial and phytoplankton production, respectively). The seasonal variation in specific growth rate was most noticeable in phytoplankton, with seasonal ratios of 3–26. The bacterial community of the marine zone responded positively – generating seasonal ratios of 1–13 in bacterial specific growth rate – to the strong warm season increment in phytoplankton growth rate in this zone. In the brackish water zone where even during the warm season allochthonous carbon accounted for 41% (on average) of the bacterial carbon demand, the seasonal ratio of bacterial specific growth rate varied from about 1 to 2. During the warm season, an average of 21% of the primary production was potentially sufficient to support the whole bacterial production. During the cold months, however, the total primary production would be either required or even insufficient to support bacterial production. The estuary turned then into a mostly heterotrophic system. However, the calculated annual production of biomass by bacterio- and phytoplankton in the whole ecosystem showed that auto- and heterotrophic production was balanced in this estuary.     

Effectiveness of sulfur with Acidithiobacillus and gypsum in chemical attributes of a Brazilian sodic soil

Gypsum and sulfur have been used as amendments for application in sodic and saline sodic soils, although gypsum is not effective in soil pH reduction. In this study the combined effects of elemental sulfur inoculated with Acidithiobacillus (S*) and gypsum (G) in chemical attributes of a Brazilian solodic soil was evaluated. The treatments consisted in addition of S* and G in various levels (0, 0.8, 1.6, 2.4, and 3.2 t ha⁻¹) and different mixing proportions (100:0, 75:25, 50:50, 25:75, and 100:0), acting during 15, 30, and 45 days. Sulfur inoculated with Acidithiobacillus (S*) markedly reduced soil pH in the leaching solution, especially when applied in the highest levels. Gypsum or sulfur applied individually was not satisfactory for soil reclamation. At 15 days of incubation Na⁺, Ca²⁺, and Mg²⁺ showed higher values in the leaching solution, and a marked decrease was observed in the leaching solution at 30 days. Reduction in soil electrical conductivity and in exchangeable Na⁺, Ca²⁺, and Mg²⁺ was observed and in a general way best results were achieved with S* : G in the ratio 50:50, using 2.4 and 3.2 t ha⁻¹. Sulfur with Acidithiobacillus was more effective than gypsum in decreasing soil pH, and sulfur applied with gypsum in the proportion 50:50 showed the best results in relation to exchangeable sodium and electrical conductivity and showed values below those used for classification as sodic soils.     

Biology of Triatoma vitticeps (Stal, 1859) under laboratory conditions (Hemiptera: Reduviidae: Triatominae). I. Evolutive cycle

Observations were made on the evolutive cycle of Triatoma vitticeps, held under laboratory conditions and fed weekly in mice. Of the 435 eggs obtained, from 4 virgin couples, 149 were purposed for the biological cycle study and 286 to evaluate their resistance to starvation, which shall be a second part of this work. Only 50 specimens reached the adult stage in a period of means (S) = 270 +/- 45 days. At the incubation time, the first and second instars were of less than a month for each, while the third, fourth and fifth instars requires approximately one, two and three months, respectively. The search for the first meal occurred clearly on the 3rd, 6th and 10th day. During all the stages, more than 50% of the specimens had only one blood-meal, except the fifth one, when two blood-meals were required. In relation to the time-lapse between the presenting of the blood-meal and the beginning of feeding, as well as the length of the blood-meal, it was observed that these increased gradually according to the stage. From the 423 blood-meals performed, 390 were not followed by defecation in the settled period of 10 min. Under this point of view, T. vitticeps seems to be a poor transmissor of T. cruzi. The experiment was carried out for 13 months and by this time the averages of minimum and maximum temperatures and the humidity were 25 +/- 2 degrees C - 28 +/- 2 degrees C and 80 +/- 2%, respectively. The material belongs to the triatomine colony held at the Oswaldo Cruz Institute, Department of Entomology.     

The effect of ribonuclease on rat-liver ribosomes

1. Rat-liver ribosomes lose about 50% of their amino acid-incorporating activity when preincubated with ribonuclease. 2. This preincubation results also in loss of about 50% of the original protein content and 75% of the RNA. 3. Ribosomes sedimented by ultracentrifugation, after preincubation with ribonuclease, show negligible contamination by crystalline enzyme. 4. Washing of ribosomes treated with ribonuclease releases further protein, restoring the original RNA/protein ratio. 5. The washed particle is again capable of promoting amino acid incorporation. 6. Examination of ribosomes treated with ribonuclease in the analytical ultracentrifuge reveals destruction of ribosomes, disappearance of dimers and a decrease in the sedimentation coefficient of monomers. 7. Washed ribosomes consist of even smaller particles with a sedimentation coefficient 60s.     

Evaluation of the anti-inflammatory and antinociceptive properties of p-cymene in mice

We attempted to identify the antinociceptive and anti-inflammatory actions of the monoterpene p-cymene. Firstly, behavioural screening was carried out to verify the influence of p-cymene [25, 50, and 100 mg/kg intraperitoneal (i.p.)] on the central nervous system (CNS) activity. The antinociceptive activity of p-cymene was evaluated by the acetic acid-induced writhing response, formalin, and hot-plate test, respectively. The leukocyte migration induced by injection of carrageenan was used to assess the anti-inflammatory activity. p-Cymene showed depressant activity on CNS after 4 h of treatment and also a possible action on the autonomous nervous system (ANS), mainly at the dose of 100 mg/kg (i.p.). It was found that p-cymene (50 and 100 mg/kg, i.p.) significantly (p < 0.05) reduced the writhing responses induced by acetic acid. p-Cymene also decreased the licking time in the first and second phase, respectively, of the formalin test. The results of the hot-plate test showed that all doses of p-cymene increased significantly the latency time of the response to the thermal stimulus in both licking and jumping parameters. In addition, there was a significantly (p < 0.05) decreased leukocyte migration at all doses of p-cymene. The experimental data demonstrate that p-cymene possesses antinociceptive and anti-inflammatory activities.     

Self-incompatibility in a distylous species of Rubiaceae: is there a single incompatibility response of the morphs?

Heterostyly is a genetically controlled floral polymorphism usually associated with an incompatibility system. This set of features is known to occur in several angiosperm families, but some aspects of its biology has not been well studied. The present study investigates cellular aspects of the pollen–pistil interaction after compatible and incompatible pollinations of Psychotria nuda, to increase our knowledge of heteromorphic self-incompatibility (HetSI). The use of bright field, fluorescence and transmission electron microscopy methods allowed us to demonstrate that pollen tubes behave differently after incompatible and compatible pollinations. Pollen tubes were particularly distinct after incompatible pollinations of L- and S-morph flowers. Relative to compatible pollen tubes, incompatible L-morph tubes had a drastic reduction in cellular contents, but no cell rupture. Incompatible S-morph tubes exhibited dense cytoplasm in apical regions, as well as in other regions, accompanied by a rupture of the apex. These results support the hypothesis that L- and S-morph flowers have different incompatibility mechanisms during HetSI.     

Synthesis of new glycyrrhetinic acid derived ring A azepanone, 29-urea and 29-hydroxamic acid derivatives as selective 11β-hydroxysteroid dehydrogenase 2 inhibitors

Glycyrrhetinic acid, the metabolite of the natural product glycyrrhizin, is a well known nonselective inhibitor of 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 and type 2. Whereas inhibition of 11β-HSD1 is currently under consideration for treatment of metabolic diseases, such as obesity and diabetes, 11β-HSD2 inhibitors may find therapeutic applications in chronic inflammatory diseases and certain forms of cancer. Recently, we published a series of hydroxamic acid derivatives of glycyrrhetinic acid showing high selectivity for 11β-HSD2. The most potent and selective compound is active against human 11β-HSD2 in the low nanomolar range with a 350-fold selectivity over human 11β-HSD1. Starting from the lead compounds glycyrrhetinic acid and the hydroxamic acid derivatives, novel triterpene type derivatives were synthesized and analyzed for their biological activity against overexpressed human 11β-HSD1 and 11β-HSD2 in cell lysates. Here we describe novel 29-urea- and 29-hydroxamic acid derivatives of glycyrrhetinic acid as well as derivatives with the Beckman rearrangement of the 3-oxime to a seven-membered ring, and the rearrangement of the C-ring from 11-keto-12-ene to 12-keto-9(11)-ene. The combination of modifications on different positions led to compounds comprising further improved selective inhibition of 11β-HSD2 in the lower nanomolar range with up to 3600-fold selectivity.     

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.     

Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv)

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75?h, typically between 140 and 160?g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5?% of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0?% boundary for longer periods of time when compared to a traditional proportional–integral–derivative algorithm, which tends to destabilize with increasing cell density.