Combining a microphysiological system of three organ equivalents and transcriptomics to assess toxicological endpoints for cosmetic ingredients.

Animal testing for cosmetic ingredients and final products has been banned in Europe and is gaining legal force worldwide. However, the need for reliable testing methodologies remains for safety assessment of cosmetic ingredients. While new approach methodologies exist for many toxicological endpoints, some complex ones lack appropriate testing methods. Microphysiological systems (MPSs) have emerged as a promising tool to address this gap in pre-clinical testing, offering higher predictivity compared to animal models due to the phylogenetic distance between humans and animals. Moreover, they provide a more physiological approach than traditional in vitro testing by mimicking interconnections between different culture compartments as seen in complex organisms. This study presents a three-organ microfluidic MPS comprising skin, liver, and intestine equivalents. Combining this model with gene expression analysis, we evaluated toxicological endpoints of chemicals, demonstrating its potential for diverse applications. Our findings highlight the MPS model as a reliable and ethical method to be applied in an integrated approach for safety assessment in the cosmetic industry. It offers a promising strategy to evaluate toxicological endpoints for cosmetic ingredients and other chemicals, supporting the elimination of animal testing while ensuring consumer safety.

Obesity-Linked PPARγ Ser273 Phosphorylation Promotes Beneficial Effects on the Liver, despite Reduced Insulin Sensitivity in Mice.

Since the removal of thiazolidinediones (TZDs) from the market, researchers have been exploring alternative anti-diabetic drugs that target PPARγ without causing adverse effects while promoting insulin sensitization by blocking serine 273 phosphorylation (Ser273 or S273). Nonetheless, the underlying mechanisms of the relationship between insulin resistance and S273 phosphorylation are still largely unknown, except for the involvement of growth differentiation factor (GDF3) regulation in the process. To further investigate potential pathways, we generated a whole organism knockin mouse line with a single S273A mutation (KI) that blocks the occurrence of its phosphorylation. Our observations of KI mice on different diets and feeding schedules revealed that they were hyperglycemic, hypoinsulinemic, presented more body fat at weaning, and presented an altered plasma and hepatic lipid profile, distinctive liver morphology and gene expression. These results suggest that total blockage of S273 phosphorylation may have unforeseen effects that, in addition to promoting insulin sensitivity, could lead to metabolic disturbances, particularly in the liver. Therefore, our findings demonstrate both the beneficial and detrimental effects of PPAR S273 phosphorylation and suggest selective modulation of this post translational modification is a viable strategy to treat type 2 diabetes.

Green tea extract increases adiponectin and PPAR α levels to improve hepatic steatosis.

We postulated that Green tea (GT) improvements in non-alcoholic fatty liver disease (NAFLD) are dependent on adiponectin action in the liver. Male wild-type and adiponectin knockout (adipoKO) mice were induced to obesity for 8 weeks with a high-fat diet and then treated with GT for the last 12 weeks of the experimental protocol. Glucose and insulin tolerance tests, indirect calorimetry, histologic analysis of liver sections, and quantification of mRNA of hepatic genes related to glucose or fatty acid metabolism were performed. In vitro, we assessed the mechanism by which GT catechins act to improve hepatic steatosis by measuring lipid accumulation, and transcript levels of lipogenic genes in HepG2 cells treated with GT in the presence of a PPAR antagonist. Additionally, we performed a PPAR transactivation assay in 293T cells to test if catechins could activate PPARs. Different from wild-type mice, adipoKO animals treated with GT and fed a HFD gain body weight and fat mass, that were associated with a decrease in energy expenditure, were insulin resistant, and had no improvements in hepatic steatosis. Increased lipid levels were associated with no modulation of PPARα levels in the liver of adipoKO mice treated with GT. In vitro, we demonstrated GT catechins act to reduce hepatic steatosis in a PPARα-dependent manner, and especially epigallocatechin and epicatechin can indirectly activate PPARα, although it seems they are not direct ligands. By providing the mechanisms by which GT catechins act in the liver to improve steatosis, our data contribute to the discovery of novel therapeutic agents in the management of NAFLD.

Mass spectrometry-based proteomics of 3D cell culture: A useful tool to validate culture of spheroids and organoids.

Worldwide obesity, defined as abnormal or excessive fat accumulation that may result in different comorbidities, is considered a pandemic condition that has nearly tripled in the last 45 years. Most studies on obesity use animal models or adipocyte monolayer cell culture to investigate adipose tissue. However, besides monolayer cell culture approaches do not fully recapitulate the physiology of living organisms, there is a growing need to reduce or replace animals in research. In this context, the development of 3D self-organized structures has provided models that better reproduce the in vitro aspects of the in vivo physiology in comparison to traditional monolayer cell culture. Besides, recent advances in omics technologies have allowed us to characterize these cultures at the proteome, metabolome, transcription factor, DNA-binding and transcriptomic levels. These two combined approaches, 3D culture and omics, have provided more realistic data about determined conditions. Thereby, here we focused on the development of an obesity study pipeline including proteomic analysis to validate adipocyte-derived spheroids. Through the combination of collected mass spectrometry data from differentiated 3T3-L1 spheroids and from murine white adipose tissue (WAT), we identified 1732 proteins in both samples. By using a comprehensive proteomic analysis, we observed that the in vitro 3D culture of differentiated adipocytes shares important molecular pathways with the WAT, including expression of proteins involved in central metabolic process of the adipose tissue. Together, our results show a combination of an orthogonal method and an image-based analysis that constitutes a useful pipeline to be applied in 3D adipocyte culture.

GQ-130, a novel analogue of thiazolidinedione, improves obesity-induced metabolic alterations in rats: Evidence for the involvement of PPARß/δ pathway.

The present investigation aimed to characterize the effect of a short-time treatment with a new thiazolidinedione (TZD) derivative, GQ-130, on metabolic alterations in rats fed a high-fat diet (HFD). We investigated whether metabolic alterations induced by GQ-130 were mediated though a mechanism that involves PPARß/δ transactivation. Potential binding and transactivation of PPARα, PPARß/δ or PPARγ by GQ-130 were examined through cell transactivation, 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence quenching assays and thermal shift assay. For in vivo experiments, male 8-week-old Wistar rats were divided into three groups fed for 6 weeks with: (a) a standard rat chow (14% fat) (control group), (b) a HFD (57.8% fat) alone (HFD group), or (c) a HFD associated with an oral treatment with GQ-130 (10 mg/kg/d) during the last week (HFD-GQ group). In 293T cells, unlike rosiglitazone, GQ-130 did not cause significant transactivation of PPARγ but was able to activate PPARß/δ by 153.9 folds in comparison with control values (DMSO). Surprisingly, ANS fluorescence quenching assay reveals that GQ-130 does not bind directly to PPARß/δ binding site, a finding that was further corroborated by thermal shift assay which evaluates the thermal stability of PPARß/δ in the presence of GQ-130. Compared to the control group, rats of the HFD group showed obesity, increased systolic blood pressure (SBP), insulin resistance, impaired glucose intolerance, hyperglycaemia, and dyslipidaemia. GQ-130 treatment abolished the increased SBP and improved all metabolic dysfunctions observed in the HFD group. Oral treatment with GQ-130 was effective in improving HFD-induced metabolic alterations probably through a mechanism that involves PPARß/δ activation.

Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness.

BACKGROUND: Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. OBJECTIVE: The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. METHODS: A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. RESULTS: Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. CONCLUSION: This study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness.

Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness

BACKGROUND:Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases) and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. OBJECTIVE: The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. METHODS: A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. RESULTS: Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years) were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% ...