Infection and disruption of placental multidrug resistance (MDR) transporters: Implications for fetal drug exposure.

P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP/ABCG2) are efflux multidrug resistance (MDR) transporters localized at the syncytiotrophoblast barrier of the placenta and protect the conceptus from drug and toxin exposure throughout pregnancy. Infection is an important modulator of MDR expression and function. This review comprehensively examines the effect of infection on the MDR transporters, P-gp and BCRP in the placenta. Infection PAMPs such as bacterial lipopolysaccharide (LPS) and viral polyinosinic-polycytidylic acid (poly I:C) and single-stranded (ss)RNA, as well as infection with Zika virus (ZIKV), Plasmodium berghei ANKA (modeling malaria in pregnancy - MiP) and polymicrobial infection of intrauterine tissues (chorioamnionitis) all modulate placental P-gp and BCRP at the levels of mRNA, protein and or function; with specific responses varying according to gestational age, trophoblast type and species (human vs. mice). Furthermore, we describe the expression and localization profile of Toll-like receptor (TLR) proteins of the innate immune system at the maternal-fetal interface, aiming to better understand how infective agents modulate placental MDR. We also highlight important gaps in the field and propose future research directions. We conclude that alterations in placental MDR expression and function induced by infective agents may not only alter the intrauterine biodistribution of important MDR substrates such as drugs, toxins, hormones, cytokines, chemokines and waste metabolites, but also impact normal placentation and adversely affect pregnancy outcome and maternal/neonatal health.

Mid-pregnancy poly(I:C) viral mimic disrupts placental ABC transporter expression and leads to long-term offspring motor and cognitive dysfunction.

Limited information is available about the effect of mid-pregnancy viral infections on the placental expression of efflux transporters and offspring behavior. We hypothesized that maternal exposure to polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA viral mimic, would impair placental cell turnover, the expression of selected ABC transporters and adult offspring behavior. C57BL/6 mice were administered poly(I:C) (10 mg/Kg;ip) or vehicle at gestational day (GD) 13.5 (mid-pregnancy). Dams were euthanized for blood collection 4 h after injection, fetal and placental collection at GD18.5 or allowed to deliver spontaneously at term. At GD 13.5, poly(I:C) induced an acute pro-inflammatory response characterized by an increase in maternal plasma levels of IL-6, CXCL-1 and CCL-2/MCP-1. At GD 18.5, poly(I:C) decreased cell proliferation/death in the labyrinthine and increased cell death in the junctional zones, characterizing a disruption of placental cell turnover. Abca1 and Abcg1 immunolabelling was decreased in the labyrinthine zone, whereas Abca1, Abcg1 and breast cancer resistance transporter (Bcrp) expression increased in the junctional zone. Moreover, adult offspring showed motor and cognitive impairments in the Rotarod and T-water maze tests. These results indicate that viral infection during mid-pregnancy may disrupt relevant placental efflux transporters, as well as placental cell turnover and offspring behavior in adult life.

Breast cancer resistance protein (Bcrp/Abcg2) is selectively modulated by lipopolysaccharide (LPS) in the mouse yolk sac.

Bacterial infection alters placental ABC transporters expression. These transporters provide fetal protection against circulating xenobiotics and environmental toxins present in maternal blood. We hypothesized that lipopolysaccharide (LPS-bacterial mimic) alters the yolk sac morphology and expression of key ABC transporters in a gestational-age dependent manner. Yolk sac samples from C57BL/6 mice were obtained at gestational ages (GD) 15.5 and GD18.5, 4 or 24 h after LPS exposure (150ug/kg; n = 8/group). Samples underwent morphometrical, qPCR and immunohistochemistry analysis. The volumetric proportions of the histological components of the yolk sac did not change in response to LPS. LPS increased Abcg2 expression at GD15.5, after 4 h of treatment (p < 0.05). No changes in Abca1, Abcb1a/b, Abcg1, Glut1, Snat1, Il-1ß, Ccl2 and Mif were observed. Il-6 and Cxcl1 were undetectable in the yolk sac throughout pregnancy. Abca1, breast cancer resistance protein (Bcrp, encoded by Abcg2) and P-glycoprotein (P-gp/ Abcb1a/b) were localized in the endodermal (uterine-facing) epithelium and to a lesser extent in the mesothelium (amnion-facing), whereas Abca1 was also localized to the endothelium of the yolk sac blood vessels. LPS increased the labeling area and intensity of Bcrp in the yolk sac's mesothelial cells at GD15.5 (4 h), whereas at GD18.5, the area of Bcrp labeling in the mesothelium (4 and 24 h) was decreased (p < 0.05). Bacterial infection has the potential to change yolk sac barrier function by affecting Bcrp and Abcg2 expression in a gestational-age dependent-manner. These changes may alter fetal exposure to xenobiotics and toxic substances present in the maternal circulation and in the uterine cavity.

Accuracy of a smartphone application for triage of skin lesions based on machine learning algorithms.

BACKGROUND: Machine learning algorithms achieve expert-level accuracy in skin lesion classification based on clinical images. However, it is not yet shown whether these algorithms could have high accuracy when embedded in a smartphone app, where image quality is lower and there is high variability in image taking scenarios by users. In the past, these applications were criticized due to lack of accuracy. OBJECTIVE: In this study, we evaluate the accuracy of the newest version of a smartphone application (SA) for risk assessment of skin lesions. METHODS: This SA uses a machine learning algorithm to compute a risk rating. The algorithm is trained on 131 873 images taken by 31 449 users in multiple countries between January 2016 and August 2018 and rated for risk by dermatologists. To evaluate the sensitivity of the algorithm, we use 285 histopathologically validated skin cancer cases (including 138 malignant melanomas), from two previously published clinical studies (195 cases) and from the SA user database (90 cases). We calculate the specificity on a separate set from the SA user database containing 6000 clinically validated benign cases. RESULTS: The algorithm scored a 95.1% (95% CI, 91.9-97.3%) sensitivity in detecting (pre)malignant conditions (93% for malignant melanoma and 97% for keratinocyte carcinomas and precursors). This level of sensitivity was achieved with a 78.3% (95% CI, 77.2-79.3%) specificity. CONCLUSIONS: This SA provides a high sensitivity to detect skin cancer; however, there is still room for improvement in terms of specificity. Future studies are needed to assess the impact of this SA on the health systems and its users.

Dysregulation of placental ABC transporters in a murine model of malaria-induced preterm labor.

Malaria in Pregnancy (MiP) is characterized by placental accumulation of Plasmodium-infected erythrocytes, intrauterine growth restriction (IUGR) and preterm delivery (PTD). Placental ATP-binding cassette (ABC) transporters mediate the efflux of nutrients, cytokines and xenobiotics. The expression and activity of these transporters are highly responsive to infection. We hypothesized that MiP would perturb the expression of placental ABC transporters, promoting PTD. Peripheral blood, spleens, livers and placentas of pregnant mice, infected with Plasmodium berghei ANKA on gestational day (GD) 13.5, were collected and analyzed on GD18.5. The primary consequences of human MiP, including IUGR, PTD (20%) and placental inflammation, were recapitulated in our mouse model. Electron microscopy revealed attenuated presence of labyrinthine microvilli and dilated spongiotrophoblasts -granular endoplasmic reticulum cisternae. Additionally, a decrease in placental Abca1 (ABCA1), Abcb1b (P-glycoprotein), Abcb9 and Abcg2 (BCRP) expression was observed in MiP mice. In conclusion, MiP associated with PTD impairs placental ABC transporters' expression, potentially modulating placental nutrient, environmental toxin and xenobiotic biodistribution within the fetal compartment, and may, at some degree, be involved with pregnancy outcome in MiP.

Zika virus impairs the development of blood vessels in a mouse model of congenital infection.

Zika virus (ZIKV) is associated with brain development abnormalities such as primary microcephaly, a severe reduction in brain growth. Here we demonstrated in vivo the impact of congenital ZIKV infection in blood vessel development, a crucial step in organogenesis. ZIKV was injected intravenously in the pregnant type 2 interferon (IFN)-deficient mouse at embryonic day (E) 12.5. The embryos were collected at E15.5 and postnatal day (P)2. Immunohistochemistry for cortical progenitors and neuronal markers at E15.5 showed the reduction of both populations as a result of ZIKV infection. Using confocal 3D imaging, we found that ZIKV infected brain sections displayed a reduction in the vasculature density and vessel branching compared to mocks at E15.5; altogether, cortical vessels presented a comparatively immature pattern in the infected tissue. These impaired vascular patterns were also apparent in the placenta and retina. Moreover, proteomic analysis has shown that angiogenesis proteins are deregulated in the infected brains compared to controls. At P2, the cortical size and brain weight were reduced in comparison to mock-infected animals. In sum, our results indicate that ZIKV impairs angiogenesis in addition to neurogenesis during development. The vasculature defects represent a limitation for general brain growth but also could regulate neurogenesis directly.

Gene expression of T3-regulated genes in a mouse model of the human thyroid hormone resistance.

AIMS: To understand how thyroid hormone (TH) regulates tissue-specific gene expression in patients with the syndrome of resistance to TH (RTHß), we used a mouse model that replicates the human RTHß, specifically the ∆337T mutation in the thyroid hormone receptor ß (THRß). MAIN METHODS: We investigated the expression of key TH target genes in the pituitary and liver of TRß∆337T and wild type THRß mice by qPCR before and after a T3 suppression test consisting of the administration of increasing concentrations of T3 to hypothyroid mice. KEY FINDINGS: Pituitary Tshb and Cga expression decreased and Gh expression increased in TRß∆337T mice after T3 suppression. The stimulation of positively regulated TH genes was heterogeneous in the liver. Levels of liver Me1 and Thsrp were elevated in TRß∆337T mice after T3 administration. Slc16a2 and Gpd2 did not respond to T3 stimulation in the liver of TRß∆337T mice whereas Dio1 response was lower than that observed in WT mice. Moreover, although Chdh and Upd1 genes were negatively regulated in the liver, the expression of these genes was elevated after T3 suppression. We did not observe significant changes in THRα expression in the liver and pituitary, while THRß levels were diminished in the pituitary and increased in the liver. SIGNIFICANCE: Using a model expressing a THRß unable to bind T3, we showed the expression pattern of liver negative and positive regulated genes by T3.

Clinical and immunological aspects and outcome of a Brazilian cohort of 414 patients with systemic lupus erythematosus (SLE): comparison between childhood-onset, adult-onset, and late-onset SLE.

The clinical expression of systemic lupus erythematosus (SLE) is influenced by genetic and environmental factors and therefore varies between ethnicities. Information on the epidemiology of SLE in Brazil is scarce and practically limited to studies conducted in socioeconomically developed regions (South and Southeast). The objective of this study was to describe the clinical and immunological aspects and outcome of a cohort of patients with SLE treated at a university hospital in northeastern Brazil and compare patterns related to age at onset: childhood (cSLE), adult (aSLE), and late (lSLE). A random sample of 414 records (women: 93.5%) were reviewed. The mean age at SLE onset and the mean disease duration were 28.9 ± 10.9 years and 10.2 ± 6.6 years, respectively. Most patients had aSLE (n = 338; 81.6%), followed by cSLE (n = 60; 14.5%) and lSLE (n = 16; 3.9%). The female/male ratio was 6.5:1 in cSLE and 16.8:1 in aSLE; in lSLE, all patients were female (p = 0.05). During follow-up, the cSLE group presented higher rates of nephritis (70% vs. 52.9% vs. 12.5%; p = 0.0001) and leuko/lymphopenia (61.7% vs. 43.8% vs. 56.2%; p = 0.02). No significant differences were found for anti-dsDNA, anti-Sm, and antiphospholipid antibodies. Treatment with immunosuppressants was significantly more common, and higher doses of prednisone were used, in cSLE. The prevalence of cardiovascular diseases were more frequent in lSLE (p = 0.03). No significant differences were found between the three groups with regard to mean damage accrual (SDI), remission, and mortality. Although cSLE presented higher rates of nephritis and leuko/lymphopenia, more frequent use of immunosuppressants and higher prednisone doses than aSLE and lSLE, the three groups did not differ significantly with regard to damage accrual, remission, and mortality.

ATP-binding cassette transporters in reproduction: a new frontier.

BACKGROUND: The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS: This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS: ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as 'gatekeepers' at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1. CONCLUSIONS: The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus.

Adubação fosfatada associada à cama de frango e sua influência na produtividade e no teor de flavonoides da Marcela (Achyrocline satureioides (Lam. ) DC. ) em duas épocas de colheita / Total biomass and flavonoid contents of Achyrocline satureioides in response to phosphate fertilizer and broiler litter at two successive harvests

RESUMO: Objetivou-se com este trabalho avaliar a produção de massa e o teor de flavonoides de plantas de marcela cultivadas com fósforo e cama de frango e colhidas em duas épocas. Estudou-se a combinação de doses de P2O5 (0, 100, 200 e 300 kg ha-1) na forma de superfosfato triplo, com e sem cama de frango (10 t ha-1), arranjados como fatorial 4x2, no delineamento blocos casualizados, com quatro repetições. Foram feitas duas colheitas das plantas, consideradas como parcelas subdivididas no tempo. A propagação foi indireta, com semeio em bandejas e transplante ao campo. As plantas foram colhidas aos 195 e 223 dias após o transplantio - DAT. A altura máxima das plantas foi de 1,14 m com uso da cama de frango e de 0,97 m sem cama, aos 195 dias após o transplantio. A produção máxima de massa fresca de parte aérea das plantas foi de 30,31 t ha-1 na colheita aos 223 DAT, enquanto a de massa seca foi de 11,38 t ha-1, ambas com o uso de 300 kg ha-1 de P2O5.A massa fresca de inflorescências foi maior (4,08 t ha-1) com adição de cama de frango ao solo e menor (3,49 t ha-1) sem cama de frango; em resposta às doses de fósforo, cresceram linearmente, sendo a máxima de 4,65 t ha-1 com uso de 300 kg ha-1 de P2O5, independente da época de colheita. A massa seca de inflorescências foi máxima de 2,38 t ha-1 com o uso de 300 kg ha-1 P2O5 na colheita aos 223 DAT. A maior produção de massa seca e o maior teor de flavonoides das inflorescências foram obtidos com o cultivo das plantas de marcela em solo com cama de frango (10 t ha-1) e com P2O5 (300 kg ha-1) na colheita aos 223 dias após o transplantio.

ABSTRACT: The aim of this experiment was to evaluate the biomass yield and flavonoid contents of Achyrocline satureioides grown under different levels of phosphate fertilizer in association with broiler litter, during two successive harvests. Four levels of P2O5 (0, 100, 200 and 300 kg ha-1), as triple superphosphate, and two levels of broiler litter (0 and 10 t ha-1) were arranged as a 4x2 factorial design in randomized blocks with four replications. Two harvests of plants, considered as split-plot, were made. Seeds were sown in polystyrene trays then transplanted to the experimental field and harvested at 195 and 223 days after transplanting (DAT). The maximum heights of the plants were 1.14 m under the addition of broiler litter and 0.97 m without it, at 195 days after transplanting. The highest yields of total fresh weight (30.31 ton ha-1) and dry weight (11.38 ton ha-1) were obtained at 223 DAT, both with the concentration of 300 kg ha-1 P2O5. The fresh weight of the inflorescences was greater under the addition of broiler litter (4.08 ton ha-1) than without it (3.49 ton ha-1). The plants grew linearly in response to the use of phosphate, and the maximum yield was 4.65 ton ha-1 under the concentration of 300 kg ha-1 P2O5, regardless of the harvest season. The dry weight of the inflorescences reached maximum yield (2.38 ton ha-1) at 223 DAT, in response to the use of 300 kg ha-1 P2O5. The highest yield of dry weight and the highest contents of flavonoids from the inflorescences were obtained when A. satureioides was grown under the concentration of P2O5 (300 kg ha-1) with the addition of broiler litter (10 ton ha-1) and harvested at 223 DAT.

Efeito do resfriamento evaporativo e do balanço eletrolítico sobre a lactação de porcas em condições de verão tropical / Effect of evaporative cooling and electrolyte balance on lactating sows in tropical summer conditions

O experimento foi realizado em uma granja comercial com 72 fêmeas suínas da genética DanBred(r) e objetivou avaliar os efeitos do resfriamento evaporativo e do balanço eletrolítico sobre o desempenho e os parâmetros de termorregulação de porcas lactantes no verão. O delineamento experimental foi blocos ao acaso com quatro tratamentos em disposição fatorial 2 x 2 e 18 repetições por tratamento. Os tratamentos foram dois sistemas de ventilação (ventilação natural ou resfriamento evaporativo) e duas dietas (balanço eletrolítico de 175mEqkg-1 ou balanço eletrolítico alto de 275mEqkg-1). Os valores médios encontrados para temperatura ambiente e umidade relativa do ar foram de 24,8ºC e 64,5%, respectivamente, sendo obtidos valores diários de 31,6°C para a temperatura ambiente máxima e de 19,8°C para a temperatura ambiente mínima. Não houve efeito (P>0,05) significativo do resfriamento evaporativo e do balanço eletrolítico sobre o consumo de ração, a condição corporal e o intervalo desmame-estro das porcas. O sistema de resfriamento evaporativo proporcionou redução (P<0,05) na frequência respiratória e temperatura superficial mensuradas nos períodos da manhã e da tarde e na temperatura retal à tarde e aumento (P<0,10) no peso dos leitões ao desmame. A dieta com balanço eletrolítico alto não influenciou essas variáveis. Concluiu-se que a utilização do sistema de resfriamento evaporativo contribuiu para reduzir os efeitos do calor sobre as variáveis relacionadas à termorregulação das porcas em lactação durante o verão e proporcionou aumento no peso dos leitões ao desmame. Em dieta com níveis nutricionais específicos para climas quentes, o balanço eletrolítico alto não minimizou os efeitos do estresse calórico.(AU)

The field trial was conducted in a commercial farm with 72 sows from DanBred(r) genetics and aimed to evaluate the effects of the evaporative cooling and electrolyte balance on the performance and thermoregulatory parameters of lactating sows during summer. The experimental design was randomized blocks with four treatments in a factorial arrangement 2 x 2 and 18 replicates per treatment. Treatments were two ventilation systems (natural ventilation or evaporative cooling) and two diets (electrolyte balance of 175mEqkg-1 or high electrolyte balance of 275mEqkg-1). The average values for temperature and relative humidity were 24.8°C and 64.5%, respectively, being obtained daily values of 31.6°C for the maximum temperature and of 19.8°C for the minimum temperature. There was no effect (P>0.05) of evaporative cooling and electrolyte balance on feed intake, body condition and weaning-to-estrus interval of sows. The evaporative cooling caused a significant reduction (P<0.05) in respiratory rate and surface temperature measurements in the morning and afternoon and evening rectal temperature and increase (P<0.1) in the weight of piglets at weaning. The dietary high electrolyte balance did not change these variables. It was concluded that the use of the evaporative cooling system helped to reduce the effects of heat on the variables related to body thermoregulation during the summer and provided an increase in weaning piglet weight. The utilization of a diet with specific nutrient levels for hot climates, the high electrolyte balance did not reduce the effects of heat stress in sows.(AU)

Effects of maternal nicotine exposure on thyroid hormone metabolism and function in adult rat progeny.

Postnatal nicotine exposure leads to obesity and hypothyroidism in adulthood. We studied the effects of maternal nicotine exposure during lactation on thyroid hormone (TH) metabolism and function in adult offspring. Lactating rats received implants of osmotic minipumps releasing nicotine (NIC, 6 mg/kg per day s.c.) or saline (control) from postnatal days 2 to 16. Offspring were killed at 180 days. We measured types 1 and 2 deiodinase activity and mRNA, mitochondrial α-glycerol-3-phosphate dehydrogenase (mGPD) activity, TH receptor (TR), uncoupling protein 1 (UCP1), hypothalamic TRH, pituitary TSH, and in vitro TRH-stimulated TSH secretion. Expression of deiodinase mRNAs followed the same profile as that of the enzymatic activity. NIC exposure caused lower 5'-D1 and mGPD activities; lower TRß1 content in liver as well as lower 5'-D1 activity in muscle; and higher 5'-D2 activity in brown adipose tissue (BAT), heart, and testis, which are in accordance with hypothyroidism. Although deiodinase activities were not changed in the hypothalamus, pituitary, and thyroid of NIC offspring, UCP1 expression was lower in BAT. Levels of both TRH and TSH were lower in offspring exposed to NIC, which presented higher basal in vitro TSH secretion, which was not increased in response to TRH. Thus, the hypothyroidism in NIC offspring at adulthood was caused, in part, by in vivo TRH-TSH suppression and lower sensitivity to TRH. Despite the hypothyroid status of peripheral tissues, these animals seem to develop an adaptive mechanism to preserve thyroxine to triiodothyronine conversion in central tissues.

Cost-effectiveness of prostate cancer screening: a simulation study based on ERSPC data.

BACKGROUND: The results of the European Randomized Study of Screening for Prostate Cancer (ERSPC) trial showed a statistically significant 29% prostate cancer mortality reduction for the men screened in the intervention arm and a 23% negative impact on the life-years gained because of quality of life. However, alternative prostate-specific antigen (PSA) screening strategies for the population may exist, optimizing the effects on mortality reduction, quality of life, overdiagnosis, and costs. METHODS: Based on data of the ERSPC trial, we predicted the numbers of prostate cancers diagnosed, prostate cancer deaths averted, life-years and quality-adjusted life-years (QALY) gained, and cost-effectiveness of 68 screening strategies starting at age 55 years, with a PSA threshold of 3, using microsimulation modeling. The screening strategies varied by age to stop screening and screening interval (one to 14 years or once in a lifetime screens), and therefore number of tests. RESULTS: Screening at short intervals of three years or less was more cost-effective than using longer intervals. Screening at ages 55 to 59 years with two-year intervals had an incremental cost-effectiveness ratio of $73000 per QALY gained and was considered optimal. With this strategy, lifetime prostate cancer mortality reduction was predicted as 13%, and 33% of the screen-detected cancers were overdiagnosed. When better quality of life for the post-treatment period could be achieved, an older age of 65 to 72 years for ending screening was obtained. CONCLUSION: Prostate cancer screening can be cost-effective when it is limited to two or three screens between ages 55 to 59 years. Screening above age 63 years is less cost-effective because of loss of QALYs because of overdiagnosis.

Trypanosoma cruzi uses macropinocytosis as an additional entry pathway into mammalian host cell.

Several intracellular pathogens are internalized by host cells via multiple endocytic pathways. It is no different with Trypanosoma cruzi. Evidences indicate that T. cruzi entry may occur by endocytosis/phagocytosis or by an active manner. Although macropinocytosis is largely considered an endocytic process where cells internalize only large amounts of solutes, several pathogens use this pathway to enter into host cells. To investigate whether T. cruzi entry into peritoneal macrophages and LLC-MK2 epithelial cells can be also mediated through a macropinocytosis-like process, we used several experimental strategies presently available to characterize macropinocytosis such as the use of different inhibitors. These macropinocytosis' inhibitors blocked internalization of T. cruzi by host cells. To further support this, immunofluorescence microscopy and scanning electron microscopy techniques were used. Field emission scanning electron microscopy revealed that after treatment, parasites remained attached to the external side of host cell plasma membrane. Proteins such as Rabankyrin 5, tyrosine kinases, Pak1 and actin microfilaments, which participate in macropinosome formation, were localized at T. cruzi entry sites. We also observed co-localization between the parasite and an endocytic fluid phase marker. All together, these results indicate that T. cruzi is able to use multiple mechanisms of penetration into host cell, including macropinocytosis.

Leishmanicidal effects of piperine, its derivatives, and analogues on Leishmania amazonensis.

Leishmaniasis is a tropical disease caused by protozoan parasites of the genus Leishmania which affects 12 million people worldwide. The discovery of drugs for the treatment of leishmaniasis is a pressing concern in global health programs. The aim of this study aim was to evaluate the leishmanicidal effect of piperine and its derivatives/analogues on Leishmania amazonensis. Our results showed that piperine and phenylamide are active against promastigotes and amastigotes in infected macrophages. Both drugs induced mitochondrial swelling, loose kinetoplast DNA, and led to loss of mitochondrial membrane potential. The promastigote cell cycle was also affected with an increase in the G1 phase cells and a decrease in the S-phase cells, respectively, after piperine and phenylamide treatment. Lipid analysis of promastigotes showed that piperine reduced triglyceride, diacylglycerol, and monoacylglycerol contents, whereas phenylamide only reduced diacylglycerol levels. Both drugs were deemed non toxic to macrophages at 50 µM as assessed by XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt), Trypan blue exclusion, and phagocytosis assays, whereas low toxicity was noted at concentrations higher than 150 µM. None of the drugs induced nitric oxide (NO) production. By contrast, piperine reduced NO production in activated macrophages. The isobologram analysis showed that piperine and phenylamide acted synergistically on the parasites suggesting that they affect different target mechanisms. These results indicate that piperine and its phenylamide analogue are candidates for development of drugs for cutaneous leishmaniasis treatment.

The Δ337T mutation on the TRß causes alterations in growth, adiposity, and hepatic glucose homeostasis in mice.

Mice bearing the genomic mutation Δ337T on the thyroid hormone receptor ß (TRß) gene present the classical signs of resistance to thyroid hormone (TH), with high serum TH and TSH. This mutant TR is unable to bind TH, remains constitutively bound to co-repressors, and has a dominant negative effect on normal TRs. In this study, we show that homozygous (TRßΔ337T) mice for this mutation have reduced body weight, length, and body fat content, despite augmented relative food intake and relative increase in serum leptin. TRßΔ337T mice exhibited normal glycemia and were more tolerant to an i.p. glucose load accompanied by reduced insulin secretion. Higher insulin sensitivity was observed after single insulin injection, when the TRßΔ337T mice developed a profound hypoglycemia. Impaired hepatic glucose production was confirmed by the reduction in glucose generation after pyruvate administration. In addition, hepatic glycogen content was lower in homozygous TRßΔ337T mice than in wild type. Collectively, the data suggest that TRßΔ337T mice have deficient hepatic glucose production, by reduced gluconeogenesis and lower glycogen deposits. Analysis of liver gluconeogenic gene expression showed a reduction in the mRNA of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme, and of peroxisome proliferator-activated receptor-γ coactivator 1α, a key transcriptional factor essential to gluconeogenesis. Reduction in both gene expressions is consistent with resistance to TH action via TRß, reproducing a hypothyroid phenotype. In conclusion, mice carrying the Δ337T-dominant negative mutation on the TRß are leaner, exhibit impaired hepatic glucose production, and are more sensitive to hypoglycemic effects of insulin.

Effect of triiodothyronine on adiponectin expression and leptin release by white adipose tissue of normal rats.

Previous studies have shown that alterations in thyroid status may lead to changes in serum leptin and adiponectin, both in humans and rodents. The mechanisms, especially for adiponectin, are unclear. In the present study, we investigated the effect of triiodothyronine (T3) on the expression of adiponectin mRNA and the release of leptin and adiponectin by white adipose tissue (WAT) explants obtained from epididymal (visceral) or inguinal (subcutaneous) depots from normal rats. We also analyzed the effects of other known regulators of adiponectin and leptin release, such as rosiglitazone and dexamethasone. T3 acted directly at rat WAT explants in a depot-specific manner and in a unique fashion to each hormone. T3 was able to inhibit leptin release only by epididymal explants, and to reduce adiponectin mRNA expression only in inguinal explants. However, T3 was incapable of modifying adiponectin release by both explants. Additionally, rosiglitazone exhibited an inhibitory effect on adiponectin release by both WAT explants, even though adiponectin mRNA was importantly upregulated only in inguinal explants. Rosiglitazone acted as an inhibitor of leptin release by both studied fat depots, while only epididymal explants responded to the stimulatory effect of dexamethasone on leptin release. Therefore, the present model of isolated rat white adipose tissue explants highlights the fact that the regulation of hormonal production by white adipose tissue depends on the type of depot and its anatomical location. In this context, our results show for the first time a potential inhibitory effect of T3 on adiponectin mRNA expression specifically on WAT from a subcutaneous depot.

Tetranychus urticae (Acari: Tetranychidae) on Gerbera jamesonii Bolus and Hook (Asteraceae).

Gerbera (Gerbera jamesonii Bolus and Hook,) is an ornamental Asteraceae of great commercial value, and pests can affect adversely its cultivation. More than 20 species of arthropods cause economic damage on gerbera, among them the two spotted mite, Tetranychus urticae Koch, 1836 (Acari: Tetranychidae), considered a key pest for this and other ornamental plants. In this work, some life-cycle aspects of T. urticae on gerbera, considered important for the knowledge of its population dynamics and for pest management programs, were studied. Mites were reared on 3-cm diameter arenas of gerbera leaf discs maintained on distilled water in Petri dishes, under laboratory conditions of 25 masculineC, 70 +/- 10% RU and 14-hour photophase, with only one egg left per arena, in a total of 262 arenas. Egg viability was 96.5% and 97.1% for unmated and mated females, respectively. Unmated females originated larvae which lived for 3.2 days and the stages of protonymph and deutonymph, 1.9 and 1.6 days, respectively; those from mated females lived 3.5 days and for protonymphs and deutonymphs, 2.0 and 1.6 days, respectively. Except for the duration of one generation (T), with similar values, 18.6 and 19.7 days, respectively for unmated and mated females, the net reproductive rate of increase (R masculine), the innate capacity to increase in number (r m) and the finite rate of growth (lambda) were different for mated and unmated females, respectively 11.5 and 24.6 for R0; 0.12 and 0.17 for r m and 1.13 and 1.19 for lambda.

Participation of macrophage membrane rafts in Trypanosoma cruzi invasion process.

Membrane rafts are small and dynamic regions enriched in sphingolipids, cholesterol, ganglioside GM1 and protein markers like flotillins, forming the flatter domains or caveolins, which are characterized as stable flask-shape invaginations. We explored whether membrane rafts participate in the entry of Trypanosoma cruzi's trypomastigotes into murine macrophages through transient depletion of macrophage membrane cholesterol with methyl-beta-cyclodextrin and treatment with filipin. These treatments led to a decrease in the trypomastigote invasion process. Macrophage pre incubated with increasing concentrations of cholera toxin B, that binds GM1, inhibited the adhesion and invasion of trypomastigote and amastigote forms. Immunofluorescence analysis demonstrated a colocalization of GM1, flotillin 1 and caveolin 1 in the T. cruzi parasitophorous vacuole. Taken together these data suggest that membrane rafts, including caveolae, are involved in the process of T. cruzi invasion of macrophages.