Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1

Background

We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.

Methods

GC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.

Results

TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.

Conclusions

Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.     

Ants as biological indicators of human impact in mangroves of the southeastern coast of Bahia, Brazil

Mangroves are common in estuaries along the Atlantic coast of Brazil. Although plant diversity is low, this ecosystem supports a range of animals, offering some resources for non-aquatic organisms. Many insects live in mangroves and, between them, many ant species that are exclusively arboreous. Mangroves throughout the world suffer from high levels of human impact, and this is particularly true for southeastern Bahia, where land-uses include traditional crab and fish exploitation, urban development, refuse pollution, recreation, and timber extraction. The ants of 13 mangrove sites, representing a range of levels of human use, have been studied along 250 km of the southern Bahia littoral, between Itacaré and Porto Seguro. Ants were sampled both inside and on the periphery of the tidal zone, using entomological rainbow, baiting, collect of hollow branches and pit-fall. A total of 108 species have been collected, with the richest genera being Camponotus and Pseudomyrmex, and the most frequent belonging to the genera Azteca and Crematogaster. The ant community living on the periphery of mangrove areas is rather homogeneous regardless of the degree of environmental perturbation, but varies markedly with the disturbance inside the mangroves themselves. The evolution of richness of the both communities, mangrove and periphery, is negatively related to the human effects, even limited to the periphery. Ant communities therefore have the potential to be useful as biological indicators of ecological impacts of land-use in these mangrove systems.     

In situ filamentous communities from the Ediacaran (approx. 563 Ma) of Brazil

Precambrian filamentous microfossils are common and diverse. Nevertheless, their taxonomic assignment can be difficult owing to their overall simple shapes typically lacking in diagnostic features. Here, we report in situ communities of well-preserved, large filamentous impressions from the Ediacaran Itajaí Basin (ca 563 Ma) of Brazil. The filaments are uniserial (unbranched) and can reach up to 200 µm in width and up to 44 mm in length. They occur as both densely packed or sparsely populated surfaces, and typically show a consistent orientation. Although simple in shape, their preferred orientation suggests they were tethered to the seafloor, and their overall flexibility (e.g. bent, folded and twisted) supports a biological (rather than sedimentary) affinity. Biometric comparisons with modern filamentous groups further support their biological affinity, suggesting links with either large sulfide-oxidizing bacteria (SOB) or eukaryotes. Other morphological and palaeoecological characteristics further corroborates their similarities with modern large filamentous SOB. Their widespread occurrence and association with complex Ediacaran macrobiota (e.g. frondose organisms, Palaeopascichnus) suggest that they probably played an important role in the ecological dynamics of these early benthic communities by providing firm substrates for metazoans to inhabit. It is further hypothesized that the dynamic redox condition in the latest Ediacaran, with the non-continuous rise in oxygen concentration and periods of hypoxia, may have created ideal conditions for SOB to thrive.     

N-Acetylcysteine Prevents Spatial Memory Impairment Induced by Chronic Early Postnatal Glutaric Acid and Lipopolysaccharide in Rat Pups

Background and Aims

Glutaric aciduria type I (GA-I) is characterized by accumulation of glutaric acid (GA) and neurological symptoms, such as cognitive impairment. Although this disease is related to oxidative stress and inflammation, it is not known whether these processes facilitate the memory impairment. Our objective was to investigate the performance of rat pups chronically injected with GA and lipopolysaccharide (LPS) in spatial memory test, antioxidant defenses, cytokines levels, Na+, K+-ATPase activity, and hippocampal volume. We also evaluated the effect of N-acetylcysteine (NAC) on theses markers.

Methods

Rat pups were injected with GA (5umol g of body weight-1, subcutaneously; twice per day; from 5th to 28th day of life), and were supplemented with NAC (150mg/kg/day; intragastric gavage; for the same period). LPS (2mg/kg; E.coli 055 B5) or vehicle (saline 0.9%) was injected intraperitoneally, once per day, from 25th to 28th day of life. Oxidative stress and inflammatory biomarkers as well as hippocampal volume were assessed.

Results

GA caused spatial learning deficit in the Barnes maze and LPS potentiated this effect. GA and LPS increased TNF-α and IL-1β levels. The co-administration of these compounds potentiated the increase of IL-1β levels but not TNF-α levels in the hippocampus. GA and LPS increased TBARS (thiobarbituric acid-reactive substance) content, reduced antioxidant defenses and inhibited Na+, K+-ATPase activity. GA and LPS co-administration did not have additive effect on oxidative stress markers and Na+, K+ pump. The hippocampal volume did not change after GA or LPS administration. NAC protected against impairment of spatial learning and increase of cytokines levels. NAC Also protected against inhibition of Na+,K+-ATPase activity and oxidative markers.

Conclusions

These results suggest that inflammatory and oxidative markers may underlie at least in part of the neuropathology of GA-I in this model. Thus, NAC could represent a possible adjuvant therapy in treatment of children with GA-I.     

Increased Cytokine and Nitric Oxide Levels in Serum of Dogs Experimentally Infected with Rangelia vitalii

This study aimed to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NO_x) in serum of dogs experimentally infected with Rangelia vitalii. Twelve female mongrel dogs were divided into 2 groups; group A (uninfected controls) composed by healthy dogs (n=5) and group B consisting of dogs inoculated with R. vitalii (n=7). Animals were monitored by blood smear examinations, which showed intraerythrocytic forms of the parasite on day 5 post-infection (PI). Blood samples were collected through the jugular vein on days 0, 10, and 20 PI to determine the serum levels of IFN-γ, TNF-α, IL-1, IL-6, and NO_x. Cytokines were assessed by ELISA quantitative sandwich technique, and NO_x was measured by the modified Griess method. Cytokine levels (IFN-γ, TNF-α, IL-1, and IL-6) were increased (P<0.01) in serum of infected animals. Serum levels of NO_x were also increased on days 10 PI (P<0.01) and 20 PI (P<0.05) in infected animals. Therefore, the infection with R. vitalii causes an increase in proinflammatory cytokines and nitric oxide content. These alterations may be associated with host immune protection against the parasite.     

Diversity, Geographic Distribution and Conservation of Squirrel Monkeys, Saimiri (Primates, Cebidae), in the Floodplain Forests of Central Amazon

Eleven taxa of primates are found in the floodplains of the western portion of the central Brazilian Amazon, protected in part by the Mamirauá Sustainable Development Reserve. The taxonomy of the squirrel monkeys, the number of taxa, and their geographic distributions are still poorly understood. Here we investigate differentiation among the taxa of this genus in Mamirauá, examining their morphology and geographic distribution. We registered 333 points of occurrence of squirrel monkeys and examined 117 specimens deposited in scientific collections. The results of the morphological analyses were generally in good agreement with field observations. Together they indicate the existence of three taxa: Saimiri vanzolinii, Saimiri sciureus macrodon, and S. s. cassiquiarensis. The restricted range of Saimiri vanzolinii in the southeastern portion of the reserve covers an area of 870 km², and it is among the smallest of the distribution areas for any Neotropical primate species. Saimiri sciureus cassiquiarensis has a disjunct distribution, crossing the Japurá River to the right bank into the reserve in two places, and S. s. macrodon is the squirrel monkey ranging widely in the northwest of Mamirauá. There are three areas of parapatry: one between Saimiri vanzolinii and Saimiri sciureus macrodon and two between S. vanzolinii and S. s. cassiquiarensis. We recommend that anthropogenic changes in the region be monitored, and conservation measures be taken to protect these primates, especially considering the endemism and very restricted range of Saimiri vanzolinii and its consequent vulnerability to extinction.     

Unilateral ovarian absence in a Black‐headed Squirrel Monkey (Saimiri vanzolinii Ayres, 1985), a threatened neotropical primate species

Ovarian agenesis is an unusual anomaly with traumatic or congenital origin. In the present case report, we describe our findings in a senile S. vanzolinii female. As this neotropical primate species is listed as vulnerable, with limited geographic distribution in the Brazilian Amazonia, ovarian agenesis may be an important finding to be reported.     

Does Resource Availability Affect the Diet and Behavior of the Vulnerable Squirrel Monkey, <Emphasis Type="Italic">Saimiri vanzolinii</Emphasis>?

Seasonal changes in food availability influence the behavior and diet of primates, and an understanding of dietary changes is important for the development of informed conservation plans. Saimiri vanzolinii is a small-bodied, omnivorous squirrel monkey endemic to the floodplain forests in Central Amazonia. It has a restricted distribution of only ca. 870 km² and is classified as Vulnerable. We examined seasonal variation in this species’ diet. We collected phenological (presence and absence of ripe fruits from 205 tree species) and dietary and behavioral data for unhabituated groups of S. vanzolinii on nine 2-km transects in the floodplain forests of Central Amazonia for 2 years, encompassing the high-water and low-water periods. Fruit availability was higher, and the monkeys traveled less and fed more during the high-water than the low-water period. There were no seasonal differences between the high-water and low-water periods in the time spent feeding on fruits, flowers, or arthropods. S. vanzolinii spent more time consuming fruit than arthropods in the high-water period, but this was not the case in the low-water period. These findings suggest that food selection changed between seasons, and S. vanzolinii focused more on fruit in the low-water period, when fruit was less available, than in the high-water period, when fruit was more available. These results support the predictions of the ecological constraints model.