Long-term glucocorticoid infusion impairs epididymal adipocyte metabolism and maturation and affects miR-150-5p actions.

The most common form of hypercortisolism is iatrogenic Cushing's syndrome. Lipodystrophy and metabolic disorders can result from the use of exogenous glucocorticoids (GC). Adipocytes play an important role in the production of circulating exosomal microRNAs, and knockdown of Dicer promotes lipodystrophy. The aim of this study is to investigate the effect of GCs on epididymal fat and to assess their influence on circulating microRNAs associated with fat turnover. The data indicate that despite the reduction in adipocyte volume due to increased lipolysis and apoptosis, there is no difference in tissue mass, suggesting that epididymal fat pad, related to animal size, is not affected by GC treatment. Although high concentrations of GC have no direct effect on epididymal microRNA-150-5p expression, GC can induce epididymal adipocyte uptake of microRNA-150-5p, which regulates transcription factor Ppar gamma during adipocyte maturation. In addition, GC treatment increased lipolysis and decreased glucose-derived lipid and glycerol incorporation. In conclusion, the similar control and GC epididymal fat mass results from increased dense fibrogenic tissue and decreased adipocyte volume induced by the lipolytic effect of GC. These findings demonstrate the complexity of epididymal fat. They also highlight how this disease alters fat distribution. This study is the first in a series published by our laboratory showing the detailed mechanism of adipocyte turnover in this disease.

Sex-specific regulation of miR-22 and ERα in white adipose tissue of obese dam's female offspring impairs the early postnatal development of functional beige adipocytes in mice.

During inguinal adipose tissue (iWAT) ontogenesis, beige adipocytes spontaneously appear between postnatal 10 (P10) and P20 and their ablation impairs iWAT browning capacity in adulthood. Since maternal obesity has deleterious effects on offspring iWAT function, we aimed to investigate its effect in spontaneous iWAT browning in offspring. Female C57BL/6 J mice were fed a control or obesogenic diet six weeks before mating. Male and female offspring were euthanized at P10 and P20 or weaned at P21 and fed chow diet until P60. At P50, mice were treated with saline or CL316,243, a ß3-adrenoceptor agonist, for ten days. Maternal obesity induced insulin resistance at P60, and CL316,243 treatment effectively restored insulin sensitivity in male but not female offspring. This discrepancy occurred due to female offspring severe browning impairment. During development, the spontaneous iWAT browning and sympathetic nerve branching at P20 were severely impaired in female obese dam's offspring but occurred normally in males. Additionally, maternal obesity increased miR-22 expression in the iWAT of male and female offspring during development. ERα, a target and regulator of miR-22, was concomitantly upregulated in the male's iWAT. Next, we evaluated miR-22 knockout (KO) offspring at P10 and P20. The miR-22 deficiency does not affect spontaneous iWAT browning in females and, surprisingly, anticipates iWAT browning in males. In conclusion, maternal obesity impairs functional iWAT development in the offspring in a sex-specific way that seems to be driven by miR-22 levels and ERα signaling. This impacts adult browning capacity and glucose homeostasis, especially in female offspring.

Maternal obesity attenuates PPARγ nuclear migration impairing offspring adipogenesis.

Maternal obesity predisposes offspring to obesity in adulthood. Since the perinatal period is a critical window for adipose organogenesis, we evaluated if maternal obesity affects the perinatal offspring adipogenesis. Female mice were fed a standard diet (eutrophic dam, ED) or a high-fat diet supplemented with condensed milk (obese dam, OD) for 6 weeks before mating, and the diets were maintained until the end of the protocol. Inguinal adipose tissue of offspring at gestational day 16.5 (E16.5), postnatal day 0 (P0), and P2 was collected to analyze morphological and molecular features. In OD offspring, the number of preadipocytes increased at E16.5 and P0 compared to ED offspring. The cell cycle-related elements Ccnd1 and Ki67 were also upregulated in these groups. In parallel, lipid accumulation started at E16.5 in OD offspring, while ED offspring preadipocytes only accumulated lipids after P0. Peroxisome proliferator-activated receptor gamma (PPARγ) levels and activity were decreased in OD offspring due to impaired nuclear migration. Increased Hdac1 expression, which negatively regulates PPAR-responsive elements in the genome, was also detected. At P2, OD adipocytes presented abnormal features, including a clustered distribution and decreased expression of PPARγ target genes and Adbr3 and Slc2a4, which are highly expressed in mature functional adipocytes. The abnormal adipose tissue is one of the major factors promoting metabolic abnormalities in adulthood. This study demonstrates for the first time the morphological and molecular alterations induced by maternal obesity in vivo in the perinatal adipogenesis in murine inguinal adipose tissue.

Cold acclimation and pioglitazone combined increase thermogenic capacity of brown and white adipose tissues but this does not translate into higher energy expenditure in mice.

Cold acclimation and pharmacological peroxisome proliferator-activated receptor γ (PPARγ) activation have each earlier been shown to recruit brown adipose tissue (BAT) and beige adipocytes thermogenic machinery, enhancing uncoupling protein 1 (UCP1)-mediated thermogenic capacity. We here investigated whether cold acclimation and PPARγ agonism combined have additive effects in inducing brown and beige adipocytes UCP1 content and whether this translates into a higher thermogenic capacity and energy expenditure. C57BL/6J mice treated or not with pioglitazone (30 mg/kg/day) were maintained at 21°C or exposed to cold (7°C) for 15 days and evaluated for thermogenic capacity, energy expenditure and interscapular BAT (iBAT) and inguinal white adipose tissue (iWAT) mass, morphology, UCP1 content and gene expression, glucose uptake and oxygen consumption. Cold acclimation and PPARγ agonism combined synergistically increased iBAT and iWAT total UCP1 content and mRNA levels of the thermogenesis-related proteins PGC1a, CIDEA, FABP4, GYK, PPARa, LPL, GLUTs (GLUT1 in iBAT and GLUT4 in iWAT), and ATG when compared to cold and pioglitazone individually. This translated into a stronger increase in body temperature in response to the ß3-adrenergic agonist CL316,243 and iBAT and iWAT respiration induced by succinate and pyruvate in comparison to that seen in either cold-acclimated or pioglitazone-treated mice. However, basal energy expenditure, BAT glucose uptake and glucose tolerance were not increased above that seen in cold-acclimated untreated mice. In conclusion, cold acclimation and PPARγ agonism combined induced a robust increase in brown and beige adipocytes UCP1 content and thermogenic capacity, much higher than each treatment individually. However, our findings enforce the concept that increases in total UCP1 do not innately lead to higher energy expenditure.NEW & NOTEWORTHY Cold acclimation and PPARγ agonism combined markedly increase brown and white adipose tissue total UCP1 content and mRNA levels of thermogenesis-related proteins. Higher UCP1 protein levels did not result in higher energy expenditure. The high thermogenic capacity induced by PPARγ agonism in cold-exposed animals markedly increases animals' body temperature in response to the ß3-adrenergic agonist CL316,243.

VDAC1 regulates neuronal cell loss after retinal trauma injury by a mitochondria-independent pathway.

The voltage-dependent anion channel 1 (VDAC1) was first described as a mitochondrial porin that mediates the flux of metabolites and ions, thereby integrating both cell survival and death signals. In the nervous system, the functional roles of VDAC1 remain poorly understood. Herein, the rat retina was employed to study VDAC1. First, it was observed that even subtle changes in VDAC1 levels affect neuronal survival, inducing severe alterations in the retinal morphology. We next examined the regulation of VDAC1 after traumatic retinal injury. After mechanical trauma, SOD1 translocates towards the nucleus, which is insufficient to contain the consequences of oxidative stress, as determined by the evaluation of protein carbonylation. Using in vitro models of oxidative stress and mechanical injury in primary retinal cell cultures, it was possible to determine that inhibition of VDAC1 oligomerization by 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) rescues cell viability, impacting microglial cell activation. We next focused on the regulation of VDAC1 after retinal mechanical injury. VDAC1 was promptly upregulated 2 h after lesion in the plasma membrane and endoplasmic reticulum rather than in the mitochondria, and multimers of VDAC1 were assembled after lesion. DIDS intraocular application decreased apoptosis and prevented microglial polarization, which confirmed in vitro observations. Considering the role of microglia in neuroinflammation, multiplex evaluation of cytokines showed that DIDS application disorganized the inflammatory response 2 h after the lesion, matching the fast regulation of VDAC1. Taken together, data disclosed that fine regulation of VDAC1 influences neuronal survival, and pharmacological inhibition after trauma injury has neuroprotective effects. This protection may be attributed to the effects on VDAC1 abnormal accumulation in the plasma membrane, thereby controlling the activation of microglial cells. We concluded that VDAC1 is a putative therapeutic target in neuronal disorders since it integrates both death and survival cellular signaling.

Aerobic exercise training regulates serum extracellular vesicle miRNAs linked to obesity to promote their beneficial effects in mice.

There is a growing body of evidence that extracellular vesicles (EVs) and their cargo of RNA, DNA, and protein are released in the circulation with exercise and might mediate interorgan communication. C57BL6/J male mice were subjected to diet-induced obesity and aerobic training on a treadmill for 8 wk. The effect of aerobic training was evaluated in the liver, muscle, kidney, and white/brown adipose tissue. To provide new mechanistic insight, we profiled miRNA from serum EVs of obese and obese trained mice. We demonstrate that aerobic training changes the circulating EV miRNA profile of obese mice, including decreases in miR-122, miR-192, and miR-22 levels. Circulating miRNA levels were associated with miRNA levels in mouse liver white adipose tissue (WAT). In WAT, aerobically trained obese mice showed reduced adipocyte hypertrophy and increased the number of smaller adipocytes and the expression of Cebpa, Pparg, Fabp4 (adipogenesis markers), and ATP-citrate lyase enzyme activity. Importantly, miR-22 levels negatively correlated with the expression of adipogenesis and insulin sensitivity markers. In the liver, aerobic training reverted obesity-induced steatohepatitis, and steatosis score and Pparg expression were negatively correlated with miR-122 levels. The prometabolic effects of aerobic exercise in obesity possibly involve EV miRNAs, which might be involved in communication between liver and WAT. Our data provide significant evidence demonstrating that aerobic training exercise-induced EVs mediate the effect of exercise on adipose tissue metabolism.

MicroRNA miR-222 mediates pioglitazone beneficial effects on skeletal muscle of diet-induced obese mice.

Pioglitazone belongs to the class of drugs thiazolidinediones (TZDs) and is an oral hypoglycemic drug, used in the treatment of type 2 diabetes, which improves insulin sensitivity in target tissues. Adipose tissue is the main target of pioglitazone, a PPARg and PPARa agonist; however, studies also point to skeletal muscle as a target. Non-PPAR targets of TZDs have been described, thus we aimed to study the direct effects of pioglitazone on skeletal muscle and the possible role of microRNAs as targets of this drug. Pioglitazone treatment of obese mice increased insulin-mediated glucose transport as a result of increased fatty acid oxidation and mitochondrial activity. PPARg blockage by treatment with GW9662 nullified pioglitazone's effect on systemic and muscle insulin sensitivity and citrate synthase activity of obese mice. After eight weeks of high-fat diet, miR-221-3p expression in soleus muscle was similar among the groups and miR-23b-3p and miR-222-3p were up-regulated in obese mice compared to the control group, and treatment with pioglitazone was able to reverse this condition. In vitro studies in C2C12 cells suggest that inhibition of miR-222-3p protects C2C12 cells from insulin resistance and increased non-mitochondrial respiration induced by palmitate. Together, these data demonstrate a role of pioglitazone in the downregulation of microRNAs that is not dependent on PPARg. Moreover, miR-222 may be a novel PPARg-independent mechanism through which pioglitazone improves insulin sensitivity in skeletal muscle.

Adiponectin is required for pioglitazone-induced improvements in hepatic steatosis in mice fed a high-fat diet.

Pioglitazone has been used for the treatment of nonalcoholic fatty liver disease (NAFLD) related to diabetes. The role of adiponectin in pioglitazone-induced improvements in NAFLD was studied by using wild-type (adipoWT) and adiponectin knockout (adipoKO) mice. High-fat diet fed mice were insulin resistant, glucose intolerant and had increased hepatic lipid accumulation as evidenced by increased NAFLD activity score. Despite pioglitazone has improved insulin resistance in both genotypes, hepatic steatosis was only improved in adipoWT obese mice. Amelioration of NAFLD in adipoWT mice promoted by pioglitazone was associated with up-regulation of Pparg, Fgf21 and down-regulation of Pepck liver expression. On the other hand, resistance to pioglitazone treatment in adipoKO mice was associated with increased expression of miR-192 and Hsl, which was not followed by increased fatty acid oxidation. In conclusion, our data provides evidence that increased adiponectin production by pioglitazone is necessary for its beneficial action on NAFLD.

Fenofibrate reverses changes induced by high-fat diet on metabolism in mice muscle and visceral adipocytes.

The effect of fenofibrate on the metabolism of skeletal muscle and visceral white adipose tissue of diet-induced obese (DIO) mice was investigated. C57BL/6J male mice were fed either a control or high-fat diet for 8 weeks. Fenofibrate (50 mg/Kg BW, daily) was administered by oral gavage during the last two weeks of the experimental period. Insulin-stimulated glucose metabolism in soleus muscles, glucose tolerance test, insulin tolerance test, indirect calorimetry, lipolysis of visceral white adipose tissue, expression of miR-103-3p in adipose tissue, and miR-1a, miR-133a/b, miR-206, let7b-5p, miR-23b-3p, miR-29-3p, miR-143-3p in soleus muscle, genes related to glucose and fatty acid metabolism in adipose tissue and soleus muscle, and proteins (phospho-AMPKα2, Pgc1α, Cpt1b), intramuscular lipid staining, and activities of fatty acid oxidation enzymes in skeletal muscle were investigated. In DIO mice, fenofibrate prevented weight gain induced by HFD feeding by increasing energy expenditure; improved whole body glucose homeostasis, and in skeletal muscle, increased insulin dependent glucose uptake, miR-1a levels, reduced intramuscular lipid accumulation, and phospho-AMPKα2 levels. In visceral adipose tissue of obese mice, fenofibrate decreased basal lipolysis rate and visceral adipocytes hypertrophy, and induced the expression of Glut-4, Irs1, and Cav-1 mRNA and miR-103-3p suggesting a higher insulin sensitivity of the adipocytes. The evidence is presented herein that beneficial effects of fenofibrate on body weight, glucose homeostasis, and muscle metabolism might be related to its action in adipose tissue. Moreover, fenofibrate regulates miR-1a-3p in soleus and miR-103-3p in adipose tissue, suggesting these microRNAs might contribute to fenofibrate beneficial effects on metabolism.

Determining the Roles of Inositol Trisphosphate Receptors in Neurodegeneration: Interdisciplinary Perspectives on a Complex Topic.

It is well known that calcium (Ca2+) is involved in the triggering of neuronal death. Ca2+ cytosolic levels are regulated by Ca2+ release from internal stores located in organelles, such as the endoplasmic reticulum. Indeed, Ca2+ transit from distinct cell compartments follows complex dynamics that are mediated by specific receptors, notably inositol trisphosphate receptors (IP3Rs). Ca2+ release by IP3Rs plays essential roles in several neurological disorders; however, details of these processes are poorly understood. Moreover, recent studies have shown that subcellular location, molecular identity, and density of IP3Rs profoundly affect Ca2+ transit in neurons. Therefore, regulation of IP3R gene products in specific cellular vicinities seems to be crucial in a wide range of cellular processes from neuroprotection to neurodegeneration. In this regard, microRNAs seem to govern not only IP3Rs translation levels but also subcellular accumulation. Combining new data from molecular cell biology with mathematical modelling, we were able to summarize the state of the art on this topic. In addition to presenting how Ca2+ dynamics mediated by IP3R activation follow a stochastic regimen, we integrated a theoretical approach in an easy-to-apply, cell biology-coherent fashion. Following the presented premises and in contrast to previously tested hypotheses, Ca2+ released by IP3Rs may play different roles in specific neurological diseases, including Alzheimer's disease and Parkinson's disease.

Pilocarpine-induced seizures trigger differential regulation of microRNA-stability related genes in rat hippocampal neurons.

Epileptogenesis in the temporal lobe elicits regulation of gene expression and protein translation, leading to reorganization of neuronal networks. In this process, miRNAs were described as being regulated in a cell-specific manner, although mechanistics of miRNAs activity are poorly understood. The specificity of miRNAs on their target genes depends on their intracellular concentration, reflecting the balance of biosynthesis and degradation. Herein, we confirmed that pilocarpine application promptly (<30 min) induces status epilepticus (SE) as revealed by changes in rat electrocorticogram particularly in fast-beta range (21-30 Hz). SE simultaneously upregulated XRN2 and downregulated PAPD4 gene expression in the hippocampus, two genes related to miRNA degradation and stability, respectively. Moreover, SE decreased the number of XRN2-positive cells in the hilus, while reduced the number of PAPD4-positive cells in CA1. XRN2 and PAPD4 levels did not change in calretinin- and CamKII-positive cells, although it was possible to determine that PAPD4, but not XRN2, was upregulated in parvalbumin-positive cells, revealing that SE induction unbalances the accumulation of these functional-opposed proteins in inhibitory interneurons that directly innervate distinct domains of pyramidal cells. Therefore, we were able to disclose a possible mechanism underlying the differential regulation of miRNAs in specific neurons during epileptogenesis.

MicroRNAs in neuronal communication.

MicroRNAs (miRNAs) are short nucleotides sequences that regulate the expression of genes in different eukaryotic cell types. A tremendous amount of knowledge on miRNAs has rapidly accumulated over the last few years, revealing the growing interest in this field of research. On the other hand, clarifying the physiological regulation of gene expression in the central nervous system is important for establishing a reference for comparison to the diseased state. It is well known that the fine tuning of neuronal networks relies on intricate molecular mechanisms, such as the adjustment of the synaptic transmission. As determined by recent studies, regulation of neuronal interactions by miRNAs has critical consequences in the development, adaptation to ambient demands, and degeneration of the nervous system. In contrast, activation of synaptic receptors triggers downstream signaling cascades that generate a vast array of effects, which includes the regulation of novel genes involved in the control of the miRNA life cycle. In this review, we have examined the hot topics on miRNA gene-regulatory activities in the broad field of neuronal communication-related processes. Furthermore, in addition to indicating the newly described effect of miRNAs on the regulation of specific neurotransmitter systems, we have pointed out how these systems affect the expression, transport, and stability of miRNAs. Moreover, we discuss newly described and under-investigation mechanisms involving the intercellular transfer of miRNAs, aided by exosomes and gap junctions. Thus, in the current review, we were able to highlight recent findings related to miRNAs that indisputably contributed towards the understanding of the nervous system in health and disease.

A possible new mechanism for the control of miRNA expression in neurons.

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR, we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs "make sense" if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were concurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression.

Developmental and functional expression of miRNA-stability related genes in the nervous system.

In the nervous system, control of gene expression by microRNAs (miRNAs) has been investigated in fundamental processes, such as development and adaptation to ambient demands. The action of these short nucleotide sequences on specific genes depends on intracellular concentration, which in turn reflects the balance of biosynthesis and degradation. Whereas mechanisms underlying miRNA biogenesis has been investigated in recent studies, little is known about miRNA-stability related proteins. We first detected two genes in the retina that have been associated to miRNA stability, XRN2 and PAPD4. These genes are highly expressed during retinal development, however with distinct subcellular localization. We investigated whether these proteins are regulated during specific phases of the cell cycle. Combined analyses of nuclei position in neuroblastic layer and labeling using anti-cyclin D1 revealed that both proteins do not accumulate in S or M phases of the cell cycle, being poorly expressed in progenitor cells. Indeed, XRN2 and PAPD4 were observed mainly after neuronal differentiation, since low expression was also observed in astrocytes, endothelial and microglial cells. XRN2 and PAPD4 are expressed in a wide variety of neurons, including horizontal, amacrine and ganglion cells. To evaluate the functional role of both genes, we carried out experiments addressed to the retinal adaptation in response to different ambient light conditions. PAPD4 is upregulated after 3 and 24 hours of dark- adaptation, revealing that accumulation of this protein is governed by ambient light levels. Indeed, the fast and functional regulation of PAPD4 was not related to changes in gene expression, disclosing that control of protein levels occurs by post-transcriptional mechanisms. Furthermore, we were able to quantify changes in PAPD4 in specific amacrine cells after dark -adaptation, suggesting for circuitry-related roles in visual perception. In summary, in this study we first described the ontogenesis and functional expression of these two miRNA-stability related proteins in the retina.

A new and reliable guide for studies of neuronal loss based on focal lesions and combinations of in vivo and in vitro approaches.

In this study, we describe a simple and reliable method to study neuroprotective effects in living and organized neural tissue. This method, which was based on retinal explants for in vivo focal lesions, was conceived as a collection of modular procedures, which can be customized for particular demands. With this model, it is possible to combine immunohistochemistry with image data analysis to track the two- or three-dimensional redistribution of proteins as a time/space function of primary cell loss. At the same time, it is possible to finely control the exposure of the tissue to specific drugs and molecules. In order to illustrate the use of the proposed method, we tested the effects of two different nanotube compounds on retinal explant viability. Transcriptome analyses can be separately performed in the lesion focus and penumbra with laser capture microdissection followed by polymerase chain reaction analyses. In addition, other common experimental drawbacks, such as high individual variance, are eliminated. With intraocular injections, treatments can be verified in vivo, with one eye serving as the experimental tissue and the other serving as the control tissue. In summary, we describe a flexible and easy method, which can be useful in combination with a broad variety of recently developed neuroprotective strategies, to study neurodegeneration.

Prospección fitoquímica y actividad antiplasmódica del extracto hexánico de Montrichardia linifera (Arruda) Schott / Phytochemical prospection and antiplasmoidal activity of the hexane extract from Montrichardia linifera (Arruda) Schott

Introducción: los extractos de M. linifera presentaron alta toxicidad para Artemia salina, lo cual sugiere un alto potencial biológico para actividad antitumoral, antibacteriana, antifúngica y actividad contra el Trypanosoma cruzi. Objetivo: evaluar el potencial antimalárico del extracto hexánico obtenido de hojas de Montrichardia linifera (Arruda) Schott y conocer su perfil fitoquímico. Métodos: la prospección fitoquímica se realizó por el método de precipitación y la actividad antiplasmódica fue evaluada in vitro utilizando el clon W2 de Plasmodium falciparum. Resultados: la prospección fitoquímica sugirió solamente la presencia de esteroides. En relación con la actividad antiplasmódica, después de 24 h en la concentración de 100 µg/mL la inhibición del crecimiento parasitario fue de 50,5 por ciento y en las demás no fue observada una inhibición significativa. En 48 h, en la concentración de 100 µg/mL, la inhibición del crecimiento fue de 34,7 por ciento. Después de 72 h las concentraciones de 100 y 50 µg/mL presentaron hemólisis, que imposibilitó la determinación porcentual parasitada. Conclusiones: el extracto hexánico obtenido de las hojas de M. linifera tiene un bajo potencial antimalárico y mostró positivamente esteroides.

Introduction: Montrichardia linifera extracts showed high toxicity to Artemia salina, which indicated high biological potential for antitumoral, antibacterial, antifungal activities, and action against Trypanossoma cruzi. Objective: to evaluate the antimalarial potential of the hexane extract from the Montrichardia linifera (Arruda) Schott leaves and to discover its phytochemical profile. Methods: the phytochemical prospection was conducted by the precipitation method whereas the antiplasmodial activity was evaluated in vitro using the W2 Plasmodium falciparum clone. Results: the phytochemical prospection just suggested the presence of steroids. Regarding the antiplasmodial activity after 24 hours at 100 µg/mL concentration, the parasite growth inhibition was 50.5 percent but at other concentration ranges, significant inhibition was not observed. At 48 hours, the growth inhibition at 100 µg/mL concentrations was 34.7 percent. After 72 hours, hemolysis was observed at 100 µg/mL and 50 µg/mL concentrations, so the percentage determination of the parasites was not possible. Conclusions: the hexane extract obtained from the M. linifera leaves presented with low antimalarial potential and proved to be positive for steroids.

Brazilian Sepsis Epidemiological Study (BASES study).

INTRODUCTION: Consistent data about the incidence and outcome of sepsis in Latin American intensive care units (ICUs), including Brazil, are lacking. This study was designed to verify the actual incidence density and outcome of sepsis in Brazilian ICUs. We also assessed the association between the Consensus Conference criteria and outcome METHODS: This is a multicenter observational cohort study performed in five private and public, mixed ICUs from two different regions of Brazil. We prospectively followed 1383 adult patients consecutively admitted to those ICUs from May 2001 to January 2002, until their discharge, 28th day of stay, or death. For all patients we collected the following data at ICU admission: age, gender, hospital and ICU admission diagnosis, APACHE II score, and associated underlying diseases. During the following days, we looked for systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, and septic shock criteria, as well as recording the sequential organ failure assessment score. Infection was diagnosed according to CDC criteria for nosocomial infection, and for community-acquired infection, clinical, radiological and microbiological parameters were used. RESULTS: For the whole cohort, median age was 65.2 years (49-76), median length of stay was 2 days (1-6), and the overall 28-day mortality rate was 21.8%. Considering 1383 patients, the incidence density rates for sepsis, severe sepsis and septic shock were 61.4, 35.6 and 30.0 per 1000 patient-days, respectively. The mortality rate of patients with SIRS, sepsis, severe sepsis and septic shock increased progressively from 24.3% to 34.7%, 47.3% and 52.2%, respectively. For patients with SIRS without infection the mortality rate was 11.3%. The main source of infection was lung/respiratory tract. CONCLUSION: Our preliminary data suggest that sepsis is a major public health problem in Brazilian ICUs, with an incidence density about 57 per 1000 patient-days. Moreover, there was a close association between ACCP/SCCM categories and mortality rate.