Dietary inulin decreases circulating ceramides by suppressing neutral sphingomyelinase expression and activity in mice.

Elevated circulating levels of ceramides (Cers) are associated with increased risk of cardiometabolic diseases, and Cers may play a causative role in metabolic dysfunction that precedes cardiac events, such as mortality as a result of coronary artery disease. Although the mechanisms involved are likely complex, these associations suggest that lowering circulating Cer levels could be protective against cardiovascular diseases. Conversely, dietary fibers, such as inulin, have been reported to promote cardiovascular and metabolic health. However, the mechanisms involved in these protective processes also are not well understood. We studied the effects of inulin on lipid metabolism with a model of atherosclerosis in LDL receptor-deficient mice using lipidomics and transcriptomics. Plasma and tissues were collected at 10 days and/or 12 weeks after feeding mice an atherogenic diet supplemented with inulin or cellulose (control). Compared with controls, inulin-fed mice displayed a decreased C16:0/C24:0 plasma Cer ratio and lower levels of circulating Cers associated with VLDL and LDL. Liver transcriptomic analysis revealed that Smpd3, a gene that encodes neutral SMase (NSMase), was downregulated by 2-fold in inulin-fed mice. Hepatic NSMase activity was 3-fold lower in inulin-fed mice than in controls. Furthermore, liver redox status and compositions of phosphatidylserine and FFA species, the major factors that determine NSMase activity, were also modified by inulin. Taken together, these results showed that, in mice, inulin can decrease plasma Cer levels through reductions in NSMase expression and activity, suggesting a mechanism by which fiber could reduce cardiometabolic disease risk.

LPA receptor 4 deficiency attenuates experimental atherosclerosis.

The widely expressed lysophosphatidic acid (LPA) selective receptor 4 (LPAR4) contributes to vascular development in mice and zebrafish. LPAR4 regulates endothelial permeability, lymphocyte migration, and hematopoiesis, which could contribute to atherosclerosis. We investigated the role of LPAR4 in experimental atherosclerosis elicited by adeno-associated virus expressing PCSK9 to lower LDL receptor levels. After 20 weeks on a Western diet, cholesterol levels and lipoprotein distribution were similar in WT male and Lpar4Y/- mice (P = 0.94). The atherosclerotic lesion area in the proximal aorta and arch was ∼25% smaller in Lpar4Y/- mice (P = 0.009), and less atherosclerosis was detected in Lpar4Y/- mice at any given plasma cholesterol. Neutral lipid accumulation in aortic root sections occupied ∼40% less area in Lpar4Y/- mice (P = 0.001), and CD68 expression was ∼25% lower (P = 0.045). No difference in α-smooth muscle actin staining was observed. Bone marrow-derived macrophages isolated from Lpar4Y/- mice displayed significantly increased upregulation of the M2 marker Arg1 in response to LPA compared with WT cells. In aortic root sections from Lpar4Y/- mice, heightened M2 "repair" macrophage marker expression was detected by CD206 staining (P = 0.03). These results suggest that LPAR4 may regulate the recruitment of specific sets of macrophages or their phenotypic switching in a manner that could influence the development of atherosclerosis.

Effects of diet and hyperlipidemia on levels and distribution of circulating lysophosphatidic acid.

Lysophosphatidic acids (LPAs) are bioactive radyl hydrocarbon-substituted derivatives of glycerol 3-phosphate. LPA metabolism and signaling are implicated in heritable risk of coronary artery disease. Genetic and pharmacological inhibition of these processes attenuate experimental atherosclerosis. LPA accumulates in atheromas, which may be a consequence of association with LDLs. The source, regulation, and biological activity of LDL-associated LPA are unknown. We examined the effects of experimental hyperlipidemia on the levels and distribution of circulating LPA in mice. The majority of plasma LPA was associated with albumin in plasma from wild-type mice fed normal chow. LDL-associated LPA was increased in plasma from high-fat Western diet-fed mice that are genetically prone to hyperlipidemia (LDL receptor knockout or activated proprotein convertase subtilisin/kexin type 9-overexpressing C57Bl6). Adipose-specific deficiency of the ENPP2 gene encoding the LPA-generating secreted lysophospholipase D, autotaxin (ATX), attenuated these Western diet-dependent increases in LPA. ATX-dependent increases in LDL-associated LPA were observed in isolated incubated plasma. ATX acted directly on LDL-associated lysophospholipid substrates in vitro. LDL from all human subjects examined contained LPA and was decreased by lipid-lowering drug therapies. Human and mouse plasma therefore contains a diet-sensitive LDL-associated LPA pool that might contribute to the cardiovascular disease-promoting effects of LPA.

The effect of the EP3 antagonist DG-041 on male mice with diet-induced obesity.

BACKGROUND/AIMS: The prostaglandin E2 (PGE2) EP3 receptor has a multifaceted role in metabolism. Drugs targeting EP3 have been proposed as therapeutics for diabetes; however, studies utilizing global EP3 knockout mice suggest that EP3 blockade increases obesity and insulin resistance. The present studies attempt to determine the effect of acute EP3 antagonist treatment on the diabetic phenotype. METHODS: DG-041 was confirmed to be a high affinity antagonist at the mouse EP3 receptor by competition radioligand binding and by blockade of EP3-mediated responses. DG-041 pharmacokinetic studies were performed to determine the most efficacious route of administration. Male C57BL/6 × BALB/c (CB6F1) mice were fed diets containing 10%, 45%, or 60% calories from fat to induce obesity. Changes to the metabolic phenotype in these mice were evaluated after one week treatment with DG-041. RESULTS: Subcutaneous injections of DG-041 at 20 mg/kg blocked the sulprostone-evoked rise in mean arterial pressure confirming the efficacy of this administration regime. Seven day treatment with DG-041 had minimal effect on body composition or glycemic control. DG-041 administration caused a reduction in skeletal muscle triglyceride content while showing a trend toward increased hepatic triglycerides. CONCLUSION: Short term EP3 administration of DG-041 produced effective blockade of the EP3 receptor and decreased skeletal muscle triglyceride content but had no significant effects on the diabetic phenotype.

Diabetes prevalence in NZO females depends on estrogen action on liver fat content.

In humans and rodents, risk of metabolic syndrome is sexually dimorphic, with an increased incidence in males. Additionally, the protective role of female gonadal hormones is ostensible, as prevalence of type 2 diabetes mellitus (T2DM) increases after menopause. Here, we investigated the influence of estrogen (E2) on the onset of T2DM in female New Zealand obese (NZO) mice. Diabetes prevalence (defined as blood glucose levels >16.6 mmol/l) of NZO females on high-fat diet (60 kcal% fat) in week 22 was 43%. This was markedly dependent on liver fat content in week 10, as detected by computed tomography. Only mice with a liver fat content >9% in week 10 plus glucose levels >10 mmol/l in week 9 developed hyperglycemia by week 22. In addition, at 11 wk, diacylglycerols were elevated in livers of diabetes-prone mice compared with controls. Hepatic expression profiles obtained from diabetes-prone and -resistant mice at 11 wk revealed increased abundance of two transcripts in diabetes-prone mice: Mogat1, which catalyzes the synthesis of diacylglycerols from monoacylglycerol and fatty acyl-CoA, and the fatty acid transporter Cd36. E2 treatment of diabetes-prone mice for 10 wk prevented any further increase in liver fat content and reduced diacylglycerols and the abundance of Mogat1 and Cd36, leading to a reduction of diabetes prevalence and an improved glucose tolerance compared with untreated mice. Our data indicate that early elevation of hepatic Cd36 and Mogat1 associates with increased production and accumulation of triglycerides and diacylglycerols, presumably resulting in reduced hepatic insulin sensitivity and leading to later onset of T2DM.

Effect of lifestyle-based lipid lowering interventions on the relationship between circulating levels of per-and polyfluoroalkyl substances and serum cholesterol.

Exposure to certain per-and polyfluoroalkyl substances (PFAS) has been shown to be positively associated with total and/or low-density lipoprotein cholesterol. Examining this association in lipid lowering interventions may provide additional evidence linking PFAS to cardiovascular risk. We examined the relationship of 6 PFAS with cholesterol in a 6-month lifestyle-based intervention. We quantitated PFAS in 350 individuals at baseline and post intervention and examined associations of PFAS with cholesterol before and after intervention. Food frequency questionnaires and GIS analyses were used to investigate PFAS hotspots and possible exposure routes. Cholesterol significantly decreased following intervention and in parallel, PFOS, PFOA, PFHxS, and PFHpA significantly decreased. PFOS was positively correlated with total cholesterol only post-intervention. We observed that PFOS was distributed among both non-albumin and albumin lipoprotein fractions pre-intervention, but entirely in albumin fraction post-intervention. Our results indicate that lipid-lowering via lifestyle modification may impact on circulating levels or distribution of PFAS.

Lipid phosphate phosphatase 3 in smooth muscle cells regulates angiotensin II-induced abdominal aortic aneurysm formation.

Genetic variants that regulate lipid phosphate phosphatase 3 (LPP3) expression are risk factors for the development of atherosclerotic cardiovascular disease. LPP3 is dynamically upregulated in the context of vascular inflammation with particularly heightened expression in smooth muscle cells (SMC), however, the impact of LPP3 on vascular pathology is not fully understood. We investigated the role of LPP3 and lysophospholipid signaling in a well-defined model of pathologic aortic injury and observed Angiotensin II (Ang II) increases expression of PLPP3 in SMCs through nuclear factor kappa B (NF-κB) signaling Plpp3 global reduction (Plpp3+/-) or SMC-specific deletion (SM22-Δ) protects hyperlipidemic mice from AngII-mediated aneurysm formation. LPP3 expression regulates SMC differentiation state and lowering LPP3 levels promotes a fibroblast-like phenotype. Decreased inactivation of bioactive lysophosphatidic acid (LPA) in settings of LPP3 deficiency may underlie these phenotypes because deletion of LPA receptor 4 in mice promotes early aortic dilation and rupture in response to AngII. LPP3 expression and LPA signaling influence SMC and vessel wall responses that are important for aortic dissection and aneurysm formation. These findings could have important implications for therapeutics targeting LPA metabolism and signaling in ongoing clinical trials.

Universal adhesives applied to deep dentin with different bonding treatments.

Eighth-generation adhesives may be applied with total etch, selective-etch or self-conditioning, and serve as primers for non-dental substrates. AIM: To determine the bonding characteristics of universal adhesives applied to the deep pulp wall with different strategies, by means of shear bond strength and laser microscopy. MATERIALS AND METHOD: Cavities 4 mm deep and maximum width were carved in 36 extracted molars. Nine groups were formed according to dental substrate treatment and adhesives, as follows: Total-etch: group 1-Monobond 7 self-etch, group 2-One coat 7 universal, and group 3-Single bond universal; Adamantine etch: group 4-Monobond 7 self-etch, group 5-One coat 7 universal, and group 6-Single bond universal; Self-conditioning: group 7-Monobond 7 self-etch, group 8-One coat 7 universal, and group 9-Single bond universal. Molars were filled following the manufacturer's instructions. Three specimens per group (27 altogether) were used to determine shear bond strength using a universal testing machine, while layer thicknesses were measured on the remaining specimens using microscope images and Olympus LEXT 3D Software. Analysis of variance was used to compare data. RESULTS: Mean (standard deviation) bond strength in megapascals (MPa) was: group 1: 7.06±3.01; group 2: 10.74±4.36; group 3: 8.20±3.92; group 4: 7.41±2.23; group 5: 6.84±1.50; group 6: 5.86±2.10; group 7: 5.83±1.94; group 8: 7.14±2.37; group 9: 8.06±3.51. Bond strength was higher (p=0.049) for total-etch (8.61±3.96) than for selective etch (6.71±1.98) and self-conditioning (6.91±2.68). No significant difference was found among the three adhesives (p=0.205). Adhesive layer in micrometers (µm) was total-etch 8.71±4.93, selective etch 5.49±1.70 and self-conditioning 6.27±3.01, with no significant difference. CONCLUSIONS: There were significant differences among bonding strategies, with the highest values for total-etch. No significant difference was observed between self-conditioning and selective etch. No significant difference was found among the adhesives, which all behaved similarly. The greatest adhesive layer thicknesses were recorded in the total-etch group, with no significant difference among the various adhesive approaches.

Los adhesivos universales de octava generación pueden ser aplicados con diferentes estrategias de unión: grabado total, grabado selectivo o autoacondicionamiento. Además, imprimen sustratos no dentales. OBJETIVO: Determinar las caracteristicas de unión de adhesivos universales con diferentes estrategias en pared pulpar profunda mediante resistencia adhesiva al corte y microscopía laser. Materiales y Método: En 36 molares se tallaron cavidades de 4 mm de profundidad y ancho máximo. Se dividieron en 9 grupos según tratamientos y adhesivos. Grabado total: grupo 1-Monobond 7 self-etching, grupo 2-One coat 7 universal y grupo 3-Single bond universal; Grabado selectivo: grupo 4-Monobond 7 self-etching; grupo 5-One coat 7 universal y grupo 6-Single bond universal y Autoacondicionamiento: grupo 7-Monobond 7 self-etching; grupo 8-One coat 7 universal y grupo 9-Single bond universal. Las obturaciones se realizaron siguiendo las instrucciones del fabricante. La resistencia adhesiva al corte se determinó utilizando una máquina de ensayo universal sobre 27 especímenes mientras que los restantes fueron empleados para evaluar los espesores de la capa generado sobre imágenes obtenidas con microscopía y con el software Olympus LEXT 3D. Se ultilizó análisis de varianza. RESULTADOS: Resistencia adhesiva en megapascal (MPa) media (desviación estándar): grupo 1: 7,06±3,01; grupo 2: 10,74±4,36; grupo 3: 8,20±3,92; grupo 4: 7,41±2,23; grupo 5: 6,84±1,50; grupo 6: 5,86±2,10; grupo 7: 5,83±1,94; grupo 8: 7,14±2,37; grupo 9: 8,06±3,51. Grabado total (8,61±3,96) registró los valores mayores (p=0,049) en comparación a grabado selectivo (6,71±1,98) y autoacondicionamiento (6,91±2,68). Los adhesivos no tuvieron diferencias significativas (p=0,205). Capa adhesiva en µm: Grabado total (8,71±4,93); grabado selectivo (5,49±1,70) y autoacondicionamiento (6,27±3,01) sin diferencias significativas (p=0,073). CONCLUSIONES: Las estrategias de unión mostraron diferencias significativas; los valores más altos se obtuvieron con grabado total y entre autoacondicionamiento y grabado selectivo no hubo significancia. Los adhesivos evidenciaron comportamientos similares sin registrar diferencias significativas.

Molecular identification of the HLA-DRB1-DQB1 for diagnosis and follow-up of acute leukemias.

We analyzed a group of 45 Brazilian individuals, 30 with acute myeloid leukemia (AML), 15 with acute lymphoid leukemia (ALL) and 100 healthy controls to assess genetic factor risk and HLA association contribution to the disease. Patient rates were compared with age and sex-matched control groups by directly typing the HLA-DRB1/3/4/5 and -DQB1 loci by PCR analysis. We observed significantly increased allelic distribution of HLA-DRB107 in AML patients and of HLA-DRB103 in ALL patients, which suggests that individuals in both groups are susceptible to the disease. We also found significantly decreased allelic distribution of HLA-DQB104 in AML patients and of HLA-DRB104 and -DQB103 in ALL patients, which suggests protection against the disease. We further found increased HLA-DRB107 and -DQB102 haplotypes in AML patients, which suggests susceptibility to the disease and decreased HLA-DRB104 and -DQB103 haplotypes in ALL patients, which also suggests protection against the disease. Future studies with larger and/or multicentric samples will be required for better comprehension of the HLA role in acute leukemia pathogenesis.

Roles for lysophosphatidic acid signaling in vascular development and disease.

The bioactive lipid lysophosphatidic acid (LPA) is emerging as an important mediator of inflammation in cardiovascular diseases. Produced in large part by the secreted lysophospholipase D autotaxin (ATX), LPA acts on a series of G protein-coupled receptors and may have action on atypical receptors such as RAGE to exert potent effects on vascular cells, including the promotion of foam cell formation and phenotypic modulation of smooth muscle cells. The signaling effects of LPA can be terminated by integral membrane lipid phosphate phosphatases (LPP) that hydrolyze the lipid to receptor inactive products. Human genetic variants in PLPP3, that predict lower levels of LPP3, associate with risk for premature coronary artery disease, and reductions of LPP3 expression in mice promote the development of experimental atherosclerosis and enhance inflammation in the atherosclerotic lesions. Recent evidence also supports a role for ATX, and potentially LPP3, in calcific aortic stenosis. In summary, LPA may be a relevant inflammatory mediator in atherosclerotic cardiovascular disease and heightened LPA signaling may explain the cardiovascular disease risk effect of PLPP3 variants.

Adipose-derived autotaxin regulates inflammation and steatosis associated with diet-induced obesity.

Autotaxin (ATX) is a secreted enzyme that generates the bioactive lipid lysophosphatidic acid (LPA). We generated mice with global inducible post-natal inactivation or adipose-specific loss of the Enpp2 gene encoding ATX. The animals are phenotypically unremarkable and exhibit differences in adipocyte size and adipose tissue expression of inflammatory genes after high fat feeding without gross differences in fat distribution or body mass. Surprisingly, both models of Enpp2- deficiency exhibited marked protection from high fat diet-induced hepatic steatosis. This phenotype was not associated with differences in dietary fat absorption but may be accounted for by differences in hepatic expression of genes involved in de novo synthesis of triglycerides. These findings suggest that pharmacological inhibition of ATX might be protective against hepatic steatosis.

Measurement of Lysophosphatidic Acid and Sphingosine-1-Phosphate by Liquid Chromatography-Coupled Electrospray Ionization Tandem Mass Spectrometry.

Lysophosphatidic acids and sphingosine-1-phosphate are bioactive lipids that regulate diverse cellular and physiological processes through actions that are largely mediated by cell surface receptors. The roles played by these lipids in multiple disease processes make the enzymes and receptors involved in their synthesis, inactivation, and signaling attractive targets for pharmacological therapies. In this chapter we describe methods for sensitive accurate quantitation of LPA and S1P levels in biological fluids using liquid chromatography-coupled electrospray ionization tandem mass spectrometry.

Genetic predisposition to obesity affects behavioural traits including food reward and anxiety-like behaviour in rats.

Here we sought to define behavioural traits linked to anxiety, reward, and exploration in different strains of rats commonly used in obesity research. We hypothesized that genetic variance may contribute not only to their metabolic phenotype (that is well documented) but also to the expression of these behavioural traits. Rat strains that differ in their susceptibility to develop an obese phenotype (Sprague-Dawley, Obese Prone, Obese Resistant, and Zucker rats) were exposed to a number of behavioural tests starting at the age of 8 weeks. We found a similar phenotype in the obesity susceptible models, Obese Prone and Zucker rats, with a lower locomotor activity, exploratory activity, and higher level of anxiety-like behaviour in comparison to the leaner Obese Resistant strain. We did not find evidence that rat strains with a genetic predisposition to obesity differed in their ability to experience reward from chocolate (in a condition place preference task). However, Zucker rats show higher motivated behaviour for sucrose compared to Obese Resistant rats when the effort required to obtain palatable food is relatively low. Together our data demonstrate that rat strains that differ in their genetic predisposition to develop obesity also differ in their performance in behavioural tests linked to anxiety, exploration, and reward and that these differences are independent of body weight. We conclude that genetic variations which determine body weight and the aforementioned behaviours co-exist but that future studies are required to identify whether (and which) common genes are involved.

Universal adhesives applied to deep dentin with different bonding treatments / Adhesivos universales aplicados a dentina profunda con diferentes tratamientos de unión

ABSTRACT Eighth-generation adhesives may be applied with total etch, selective-etch or self-conditioning, and serve as primers for non-dental substrates. Aim: To determine the bonding characteristics of universal adhesives applied to the deep pulp wall with different strategies, by means of shear bond strength and laser microscopy. Materials and Method: Cavities 4 mm deep and maximum width were carved in 36 extracted molars. Nine groups were formed according to dental substrate treatment and adhesives, as follows: Total-etch: group 1-Monobond 7 self-etch, group 2-One coat 7 universal, and group 3-Single bond universal; Adamantine etch: group 4-Monobond 7 self-etch, group 5-One coat 7 universal, and group 6-Single bond universal; Self-conditioning: group 7-Monobond 7 self-etch, group 8-One coat 7 universal, and group 9-Single bond universal. Molars were filled following the manufacturer's instructions. Three specimens per group (27 altogether) were used to determine shear bond strength using a universal testing machine, while layer thicknesses were measured on the remaining specimens using microscope images and Olympus LEXT 3D Software. Analysis of variance was used to compare data. Results: Mean (standard deviation) bond strength in megapascals (MPa) was: group 1: 7.06±3.01; group 2: 10.74±4.36; group 3: 8.20±3.92; group 4: 7.41±2.23; group 5: 6.84±1.50; group 6: 5.86±2.10; group 7: 5.83±1.94; group 8: 7.14±2.37; group 9: 8.06±3.51. Bond strength was higher (p=0.049) for total-etch (8.61±3.96) than for selective etch (6.71±1.98) and self-conditioning (6.91±2.68). No significant difference was found among the three adhesives (p=0.205). Adhesive layer in micrometers (μm) was total-etch 8.71±4.93, selective etch 5.49±1.70 and self-conditioning 6.27±3.01, with no significant difference. Conclusions: There were significant differences among bonding strategies, with the highest values for total-etch. No significant difference was observed between self-conditioning and selective etch. No significant difference was found among the adhesives, which all behaved similarly. The greatest adhesive layer thicknesses were recorded in the total-etch group, with no significant difference among the various adhesive approaches.

RESUMEN Los adhesivos universales de octava generación pueden ser aplicados con diferentes estrategias de unión: grabado total, grabado selectivo o autoacondicionamiento. Además, imprimen sustratos no dentales. Objetivo: Determinar las caracteristicas de unión de adhesivos universales con diferentes estrategias en pared pulpar profunda mediante resistencia adhesiva al corte y microscopía laser. Materiales y Método: En 36 molares se tallaron cavidades de 4 mm de profundidad y ancho máximo. Se dividieron en 9 grupos según tratamientos y adhesivos. Grabado total: grupo 1-Monobond 7 self-etching, grupo 2-One coat 7 universal y grupo 3-Single bond universal; Grabado selectivo: grupo 4-Monobond 7 self-etching; grupo 5-One coat 7 universal y grupo 6-Single bond universal y Autoacondicionamiento: grupo 7-Monobond 7 self-etching; grupo 8-One coat 7 universal y grupo 9-Single bond universal. Las obturaciones se realizaron siguiendo las instrucciones del fabricante. La resistencia adhesiva al corte se determinó utilizando una máquina de ensayo universal sobre 27 especímenes mientras que los restantes fueron empleados para evaluar los espesores de la capa generado sobre imágenes obtenidas con microscopía y con el software Olympus LEXT 3D. Se ultilizó análisis de varianza. Resultados: Resistencia adhesiva en megapascal (MPa) media (desviación estándar): grupo 1: 7,06±3,01; grupo 2: 10,74±4,36; grupo 3: 8,20±3,92; grupo 4: 7,41±2,23; grupo 5: 6,84±1,50; grupo 6: 5,86±2,10; grupo 7: 5,83±1,94; grupo 8: 7,14±2,37; grupo 9: 8,06±3,51. Grabado total (8,61±3,96) registró los valores mayores (p=0,049) en comparación a grabado selectivo (6,71±1,98) y autoacondicionamiento (6,91±2,68). Los adhesivos no tuvieron diferencias significativas (p=0,205). Capa adhesiva en μm: Grabado total (8,71±4,93); grabado selectivo (5,49±1,70) y autoacondicionamiento (6,27±3,01) sin diferencias significativas (p=0,073). Conclusiones: Las estrategias de unión mostraron diferencias significativas; los valores más altos se obtuvieron con grabado total y entre autoacondicionamiento y grabado selectivo no hubo significancia. Los adhesivos evidenciaron comportamientos similares sin registrar diferencias significativas. Los mayores espesores de capa fueron con grabado total sin diferencias significativas entre las técnicas.

Model for human skin reconstructed in vitro composed of associated dermis and epidermis.

CONTEXT AND OBJECTIVE: The technique of obtaining human skin with dermis and epidermis reconstructed from cells isolated from patients can enable autologous skin grafting on patients with few donor sites. It also enables in vitro trials on chemicals and drugs. The objective of this work was to demonstrate a method for obtaining human skin composed of associated dermis and epidermis, reconstructed in vitro. DESIGN AND SETTING: Experimental laboratory study, in the Skin Cell Culture Laboratory of Faculdade de Ciências Médicas, Universidade Estadual de Campinas. METHODS: Cells from human fibroblast cultures are injected into bovine collagen type I matrix and kept immersed in specific culturing medium for fibroblasts. This enables human dermis reconstruction in vitro. On this, by culturing human keratinocytes and melanocytes, differentiated epidermis is formed, leading to the creation of human skin composed of associated dermis and epidermis, reconstructed in vitro. RESULTS: We showed that human skin composed of associated dermis and epidermis can be successfully reconstructed in vitro. It is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with cells and extracellular matrix organized in parallel to multilayer epidermis. CONCLUSIONS: It is possible to obtain completely differentiated human skin composed of associated dermis and epidermis, reconstructed in vitro, from injection of human fibroblasts into bovine collagen type I matrix and culturing of human keratinocytes and melanocytes on this matrix.

Regulation of arterial reactivity by concurrent signaling through the E-prostanoid receptor 3 and angiotensin receptor 1.

Prostaglandin E2 (PGE2), a cyclooxygenase metabolite that generally acts as a systemic vasodepressor, has been shown to have vasopressor effects under certain physiologic conditions. Previous studies have demonstrated that PGE2 receptor signaling modulates angiotensin II (Ang II)-induced hypertension, but the interaction of these two systems in the regulation of vascular reactivity is incompletely characterized. We hypothesized that Ang II, a principal effector of the renin-angiotensin-aldosterone system, potentiates PGE2-mediated vasoconstriction. Here we demonstrate that pre-treatment of arterial rings with 1nM Ang II potentiated PGE2-evoked constriction in a concentration dependent manner (AUC-Ang II 2.778±2.091, AUC+Ang II 22.830±8.560, ***P<0.001). Using genetic deletion models and pharmacological antagonists, we demonstrate that this potentiation effect is mediated via concurrent signaling between the angiotensin II receptor 1 (AT1) and the PGE2 E-prostanoid receptor 3 (EP3) in the mouse femoral artery. EP3 receptor-mediated vasoconstriction is shown to be dependent on extracellular calcium in combination with proline-rich tyrosine kinase 2 (Pyk2) and Rho-kinase. Thus, our findings reveal a novel mechanism through which Ang II and PGE2 regulate peripheral vascular reactivity.

Association between psoriasis vulgaris and MHC-DRB, -DQB genes as a contribution to disease diagnosis.

We analyzed 100 control individuals and 60 patients with psoriasis vulgaris from the population of Campinas, Brazil. Typification of class II HLA alleles (HLA-DRB1-5 and -DQB1) was carried out through the DNA/PCR/SSP at medium and high resolution. DNA was extracted through a salting-out procedure: 13 DRB1 alleles, 3 DRB3 alleles, 1 DRB4 allele, 2 DRB5 alleles, and 5 DQB1 alleles were identified at a medium resolution using the PCR/SSP, and 45 DRB1 alleles were identified at a high resolution in analyzed patients. Results showed associations with psoriasis vulgaris: positive associations HLA-DRB3*02 (p < 0.05, chi(2) = 5.10, RR = 2.14); HLA-DRB1*0102 alleles (p < 0.05, RR = 5.44). Negative associations were found for HLA-DRB4*01 (chi(2) = 3.23, RR = 0.55) and HLA-DRB1*1302 alleles (p < 0.05, RR = 0.23). The haplotypes revealed positive association for HLA-DRB1*0102/DQB1*05 (p < 0.05, RR = 5.44) and HLA-DRB1*0701/DQB1*03 alleles (p < 0.02, RR = 9.00). These findings suggest a possible association of the DRB1 allele with the group of patients showing an early onset of the illness, as well as an association with haplotypes HLA-DRB1*0102/DQB1*05 and HLA-DRB1*0701/DQB1*03.

Compositional Variation and Bioactivity of the Leaf Essential Oil of Montanoa guatemalensis from Monteverde, Costa Rica: A Preliminary Investigation.

BACKGROUND: Montanoa guatemalensis is a small to medium-sized tree in the Asteraceae that grows in Central America from Mexico south through Costa Rica. There have been no previous investigations on the essential oil of this tree. METHODS: The leaf essential oils of M. guatemalensis were obtained from different individual trees growing in Monteverde, Costa Rica, in two different years, and were analyzed by gas chromatography-mass spectrometry. RESULTS: The leaf oils from 2008 were rich in sesquiterpenoids, dominated by α-selinene, ß-selinene, and cyclocolorenone, with lesser amounts of the monoterpenes α-pinene and limonene. In contrast, the samples from 2009 showed no α- or ß-selinene, but large concentrations of trans-muurola-4(14),5-diene, ß-cadinene, and cyclocolorenone, along with greater concentrations of α-pinene and limonene. The leaf oils were screened for cytotoxic and antimicrobial activities and did show selective cytotoxic activity on MDA-MB-231 breast tumor cells. CONCLUSION: M. guatemalensis leaf oil, rich in cyclocolorenone, α-selinene, and ß-selinene, showed selective in vitro cytotoxic activity to MDA-MB-231 cells. The plant may be a good source of cyclocolorenone.

Human leukocyte antigen (HLA) and single nucleotide polymorphisms (SNPs) tumor necrosis factor (TNF)-alpha -238 and -308 as genetic markers of susceptibility to psoriasis and severity of the disease in a long-term follow-up Brazilian study.

BACKGROUND: The strongest genetic marker for psoriasis is Cw*06. Polymorphisms in the tumor necrosis factor (TNF)-alpha promoter region, especially replacement of guanine with adenine in positions -238 and -308 are related to higher TNF-alpha production and higher risk for psoriasis in Caucasoid populations, not found in Asians. We performed a case-control study of 69 patients with psoriasis type I and 70 controls, characterized clinical progression along 10-years of follow-up in mild or severe disease and determined HLA class I, II, and TNF single nucleotide polymorphisms (SNPs) -238 and -308 polymorphisms to demonstrate whether these polymorphisms may be genetic risk for susceptibility to psoriasis or severity of the disease in Brazilians. METHODS: Polymorphisms were identified using PCR/SSP. Alleles, genotypes, and haplotypes frequencies were compared using Fisher's test. RESULTS: More severe disease was found in male patients. It may be suggested that alleles B*37, Cw*06, Cw*12, and DRB1*07 were associated with severe disease course, while B*57 with mild disease. No statistical difference was found between the patients and controls regarding polymorphisms frequencies in TNF SNPs. This study pointed to a higher TNF-238 G/G genotype frequency (OR: 3.21; CI: 1.06­9.71; P = 0.04) in the group with severe disease. CONCLUSIONS: Polymorphisms in the TNF-alpha SNPs do not seem to be a more important genetic risk factor for psoriasis than the already known Cw*06 in Brazilian patients, but these markers may be related to clinical manifestations.

Undifferentiated spondyloarthropathies in Brazilians: importance of HLA-B27 and the B7-CREG alleles in characterization and disease progression.

OBJECTIVE: To analyze the profile of the HLA-B27 and B7 cross-reactive group (CREG) alleles and the role of these markers in disease characterization and progression in patients with undifferentiated spondyloarthropathies (uSpA). METHODS: A total of 80 patients with a diagnosis of uSpA (40 HLA-B27 positive and 40 HLA-B27 negative) were prospectively studied for 2 years. The control group consisted of 66 HLA-B27 positive and 112 HLA-B27 negative individuals without a history of seronegative SpA. HLA-B alleles were typed at low (B7-CREG alleles, i.e., B*7, B*54, B*55, B*56, B*40, B*42) or high resolution (B*27 alleles) using polymerase chain reaction-amplified DNA hybridized with sequence-specific oligonucleotide probes. RESULTS: HLA-B*2705 was the most frequent allele, observed in 92.5% of the patients and in 77% of the controls, followed by the HLA-B*2702, observed in 5% of the patients and in 12% of the controls. HLA-B*2704 was observed in only one patient (2.5%), and was absent in the control population. HLA-B*2703 (6%) and HLA-B*2707 (5%) alleles were observed only in controls. No associations between HLA-B*27 alleles or B7-CREG alleles and any specific manifestation of uSpA were observed. HLA-B27 positive patients more frequently presented juvenile onset SpA (p = 0.002) and progression to ankylosing spondylitis (AS) (p = 0.03) than did HLA-B27 negative patients. The B7-CREG alleles were observed in 5% of the HLA-B27 positive uSpA group, in 25% of the HLA-B27 negative uSpA group, in 7% of the HLA-B27 positive controls, and in 13% of the HLA-B27 negative controls; a significant association was observed between the presence of the B7-CREG and the HLA-B27 negative uSpA group (p = 0.012). CONCLUSION: The frequency of the HLA-B*2705 allele among the B27 positive uSpA patients of this series was closely similar to that reported for patients with ankylosing spondylitis (AS). The presence of HLA-B*27 alleles was associated with the progression to AS, and the presence of B7-CREG was associated with uSpA in the HLA-B27 negative group.