Bioaccumulation and morphological traits in a multi-generation test with two Daphnia species exposed to lead.

Anthropic pressure negatively affects natural environments. Lead (Pb) is a non-essential highly toxic metal that is present in aquatic ecosystems. Two daphnid species from two different latitudes, the temperate Daphnia magna and the tropical Daphnia similis were used as test-organisms to evaluate a long-term Pb exposure. Both species were exposed for nine generations to a chronic concentration of Pb (50 µg/L) and the effects were explored, considering some endpoints not commonly used in toxicity tests: body burden of Pb and presence of granules in the dorsal region of neonates, hemoglobin contents, carapace deformation and morphology, production of males and ephippia (or dormant haploid egg), changes in the eggs' colour and eggs abortion. This multi-generation test was conducted under two food regimes, the usual (3 × 105 cells/mL) and the restricted (1.5 × 105 cells/mL) regime. On generation F6, Pb acclimated neonates were changed to a clean media for three generations, to evaluate exposure retrieval (recovery period). Negative and adverse effects occurred through generations, but no disparity was shown between D. magna and D. similis. The D. magna Pb accumulation showed different patterns regarding food regime. Bioaccumulation was faster under usual food, rapidly reaching a saturation point, whereas a gradual increase occurred under food restriction. A successful retrieval happened regarding Pb in D. magna, since no difference between control and recovering organisms was evidenced regarding their Pb body burdens. Generational Pb exposure led to carapace malformations, Pb aggregation in neonates' dorsal region, reddish extremities, production of males, ephippia (or dormant haploid egg), and aborted eggs, and changes in the eggs' colour (green and white). Food restriction also induced the production of males. Reddish extremities disappeared in recovering organisms and ephippia (or dormant haploid egg) did not occurred during the recovery period. Existent males revealed a shorter lifespan than females (under stress). D. magna and D. similis presented similar responses, for the endpoints analysed; however, it does not mean that this lack of sensitivity difference will be observed when other endpoints (e.g. survival, reproduction) are considered. Bioaccumulation of Pb and adverse effects occurred at the tested concentration of 50µg/L, although higher Pb levels are allowed in the environment as safe concentrations, as reported by the Brazilian legislation and the literature where effects are evidences above 400 µg/L of Pb. Pb effects on reproduction, respiration, malformation, and other adverse effects suggest that a chronic generational exposure can be harmful to both D. magna and D. similis, and that such chronic contaminated environments should not be disregarded when it comes to environmental monitoring.

RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal.

Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus.

Validation of the new Sepsis-3 definitions: proposal for improvement in early risk identification.

OBJECTIVES: Sepsis-3 definitions generated controversies regarding their general applicability. The Sepsis-3 Task Force outlined the need for validation with emphasis on the quick Sequential Organ Failure Assessment (qSOFA) score. This was done in a prospective cohort from a different healthcare setting. METHODS: Patients with infections and at least two signs of systemic inflammatory response syndrome (SIRS) were analysed. Sepsis was defined as total SOFA ≥2 outside the intensive care unit (ICU) or as an increase of ICU admission SOFA ≥2. The primary endpoints were the sensitivity of qSOFA outside the ICU and sepsis definition both outside and within the ICU to predict mortality. RESULTS: In all, 3346 infections outside the ICU and 1058 infections in the ICU were analysed. Outside the ICU, respective mortality with ≥2 SIRS and qSOFA ≥2 was 25.3% and 41.2% (p <0.0001); the sensitivities of qSOFA and of sepsis definition to predict death were 60.8% and 87.2%, respectively. This was 95.9% for sepsis definition in the ICU. The sensitivity of qSOFA and of ≥3 SIRS criteria for organ dysfunction outside the ICU was 48.7% and 72.5%, respectively (p <0.0001). Misclassification outside the ICU with the 1991 and Sepsis-3 definitions into stages of lower severity was 21.4% and 3.7%, respectively (p <0.0001) and 14.9% and 3.7%, respectively, in the ICU (p <0.0001). Adding arterial pH ≤7.30 to qSOFA increased sensitivity for prediction of death to 67.5% (p 0.004). CONCLUSIONS: Our analysis positively validated the use of SOFA score to predict unfavourable outcome and to limit misclassification into lower severity. However, qSOFA score had inadequate sensitivity for early risk assessment.

Development of a sensitive cost-effective capture ELISA for detection of murine monoclonal antibodies: correlation with SPR biosensor technology.

The development of antibodies for diagnostic and therapeutic applications in inflammatory diseases is a major focus for biotechnology and pharmaceutical companies. Production of monoclonal antibodies requires the development of fast, high-throughput methodologies for screening and selecting appropriate candidate antibodies for development. Capture (sandwich) enzyme linked immunosorbent assay (ELISA) provides a quick and reliable method that could be used for hybridoma screening of potential candidates accompanied with surface plasmon resonance (SPR) biosensor technology for identifying high affinity biomolecular interactions. A sensitive, cost-effective, robust and accurate capture ELISA for detection of murine monoclonal antibodies in culture supernatants was developed. This assay was optimized for high sensitivity and specificity with a capture anti-mouse polyclonal antibody. Using serial dilutions of a defined murine IgG antibody, a linear dose-response was observed between 2 and 1200 ng/ml antibody with a coefficient of determination r2 of 0.98. The detection limit of the assay was established as 2ng/ml (12.5pM). A similar concentration-dependent doseresponse was also observed using serial dilutions of antibody-containing supernatants from anti-alpha glycophorinproducing hybridomas (detection limit 1:2000). Specific capture of antibodies from supernatants in a similar setting was also confirmed using SPR biosensor technology and correlated well with the immunoassay results. The latter technology can be performed in order to provide quick screening results and kinetic analysis of antibody binding interactions aiming at identifying candidates with high affinity and specificity.

Procalcitonin as an early indicator of outcome in sepsis: a prospective observational study.

This study explores the role of procalcitonin (PCT) in predicting the outcome of sepsis. In a prospective multicentre observational investigation, blood was sampled within 24 h of onset of sepsis in 1156 hospitalised patients; 234 were in the intensive care unit (ICU) at the point of presentation of sepsis while 922 were not. PCT was estimated in serum by the ultrasensitive Kryptor assay in a double-blinded fashion. Among patients outside the ICU, mortality was 8% in those with PCT ≤0.12 ng/mL but 19.9% in those with PCT >0.12 ng/mL [P<0.0001, odds ratio (OR) for death: 2.606; 95% confidence interval (CI): 1.553-4.371]. Among patients whose sepsis presented in ICU, mortality was 25.6% in those with PCT ≤0.85 ng/mL but 45.3% in those with PCT >0.85 ng/mL (P=0.002; OR for death: 2.404; 95% CI: 1.385-4.171). It is concluded that PCT cut-off concentrations can contribute to predicting the outcome of sepsis and might be of particular value in identifying patients who would benefit from ICU admission.