Kaurenoic acid nanocarriers regulates cytokine production and inhibit breast cancer cell migration.

Breast cancer is the type of cancer with the highest incidence in women around the world. Noteworthy, the triple-negative subtype affects 20% of the patients while presenting the highest death rate among subtypes. This is due to its aggressive phenotype and the capability of invading other tissues. In general, tumor-associated macrophages (TAM) and other immune cells, are responsible for maintaining a favorable tumor microenvironment for inflammation and metastasis by secreting several mediators such as pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α, chemokines like CCL2, and other proteins, as metalloproteinases of matrix (MMP). On the other hand, immunomodulatory agents can interfere in the immune response of TAM and change the disease prognosis. In this work, we prepared nanostructured lipid carriers containing kaurenoic acid (NLC-KA) to evaluate the effect on cytokine production in vitro of bone marrow-derived macrophages (BMDM) and the migratory process of 4 T1 breast cancer cells. NLC-KA prepared from a blend of natural lipids was shown to have approximately 90 nm in diameter with low polydispersity index. To test the effect on cytokine production in vitro in NLC-KA treated BMDM, ELISA assay was performed and pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α were quantified. The formulation reduced the secretion of IL-1ß and TNF-α cytokines while presenting no hemolytic activity. Noteworthy, an anti-migratory effect in 4 T1 breast cancer cells treated with NLC-KA was observed in scratch assays. Further, MMP9 and CCL2 gene expressions in both BMDM and 4 T1 treated cells confirmed that the mechanism of inhibition of migration is related to the blockade of this pathway by KA. Finally, cell invasion assays confirmed that NLC-KA treatment resulted in less invasiveness of 4 T1 cells than control, and it is independent of CCL2 stimulus or BMDM direct stimulus. Ultimately, NLC-KA was able to regulate the cytokine production in vitro and reduce the migration of 4 T1 breast cancer cells by decreasing MMP9 gene expression.

Photobiomodulation effects in metalloproteinases expression in zymosan-induced arthritis.

Matrix metalloproteinases (MMPs) play a crucial role in the degenerative course of rheumatic disorders. They are responsible for cartilage and other joint-associated tissues breakdown. Amid arthritis treatments, photobiostimulation (PBM), a non-thermal and non-invasive low-power laser application, appears to be an outstanding therapy alternative once it has succeeded in MMPs modulation. Thus, this study aimed to evaluate the PBM effects of low infrared laser (830 nm), testing two different energy densities (3 and 30 Jcm-2) in MMP-2, MMP-9, MMP-13, and MMP-14 as well as the inhibitor TIMP-2 expressions using zymosan-induced arthritis model. C57BL/6 mice were distributed into four groups (n = 8): zymosan-induced arthritis without treatment; zymosan-induced arthritis and dexamethasone-treated; zymosan-induced arthritis and PBM at energy density of 3 Jcm-2 treated; and zymosan-induced arthritis and PBM at energy density of 30 Jcm-2 treated. MMPs and TIMP-2 mRNA relative levels by qRT-PCR and proteins expression by immunohistochemical and Western blotting techniques were performed after PBM treatment in the inflamed joint. Our results demonstrated PBM could modulate both mRNA relative levels and proteins expression of the MMP-2, -9, -13, -14, and TIMP-2 in joint tissues, decreasing MMP-9 protein expression and increasing TIMP-2 protein expression. PBM promotes a better arthritis prognostic, modulating metalloproteinase and its inhibitor, especially MMP-9 and TIMP-2 protein expression that is important inflammatory markers. These findings may also corroborate that PBM may regulate MMPs expression using different pathways.

Short interfering RNA delivered by a hybrid nanoparticle targeting VEGF: Biodistribution and anti-tumor effect.

BACKGROUND: The use of RNA interference (iRNA) therapy has proved to be an interesting target therapy for the cancer treatment; however, siRNAs are unstable and quickly eliminated from the bloodstream. To face these barriers, the use of biocompatible and efficient nanocarriers emerges as an alternative to improve the success application of iRNA to the cancer, including breast cancer. RESULTS: A hybrid nanocarrier composed of calcium phosphate as the inorganic phase and a block copolymer containing polyanions as organic phase, named HNPs, was developed to deliver VEGF siRNA into metastatic breast cancer in mice. The particles presented a rounded shape by TEM images with average size measured by DLS suitable and biocompatible for biomedical applications. The XPS and EDS spectra confirmed the hybrid composition of the nanoparticles. Moreover, after intravenous administration, the particles accumulated mainly in the tumor site and kidneys, which demonstrates the tumor targeting accumulation through the Enhanced Permeability and Retention Effect (EPR). A significant decrease in size of the tumors treated with the nanoparticles containing siVEGF (HNPs-siVEGF) was observed and the reduction was related to enhanced tumor accumulation of siRNA as well as in vivo VEGF silencing at gene and protein levels. CONCLUSION: The hybrid system prepared was successful in promoting the RNAi effect in vivo with very low toxicity. GENERAL SIGNIFICANCE: This study shows the valuable development of a hybrid nanoparticle carrying VEGF siRNA, as well as their tumor targeting, accumulation and reduction in mice triple-negative breast cancer.

Pesquisa aponta que obesidade como fator de risco para Covid-19 fica mais evidente nos mais jovens

De acordo com os dados da pesquisa Covid-19 and Obesity: An Epidemiologic Analysis of the Brazilian Data (Covid-19 e obesidade: uma análise epidemiológica dos dados brasileiros), desenvolvida pela pesquisadora da Universidade Federal de Juiz de Fora (UFJF), Jacy Gameiro, o vírus potencializa que pacientes com obesidade cheguem a óbito mais novos que indivíduos sem a comorbidade. O estudo também mostra que o fator de risco está associado a um aumento na necessidade da internação nas Unidades de Terapia Intensivas (UTIs) e do uso do suporte ventilatório para quem está hospitalizado.

COVID-19 and Obesity: An Epidemiologic Analysis of the Brazilian Data.

Brazil has the second highest number of deaths due to COVID-19. Obesity has been associated with an important role in disease development and a worse prognosis. We aimed to explore epidemiological data from Brazil, discussing the potential relationships between obesity and COVID-19 severity in this country. We used a public database made available by the Ministry of Health of Brazil (182700 patients diagnosed with COVID-19). Descriptive statistics were used to characterize our database. Continuous data were expressed as median and analyzed by the nonparametric tests Mann-Whitney or one-sample Wilcoxon. The frequencies of categorical variables have been analyzed by chi-square tests of independence or goodness-of-fit. Among the number of deaths, 74% of patients were 60 years of age or older. Patients with obesity who died of COVID-19 were younger (59 years (IQR = 23)) than those without obesity (71 years (IQR = 20), P < 0.001, and η 2 = 0.0424). Women with obesity who died of COVID-19 were older than men (55 years (IQR = 25) vs. 50 (IQR = 22), P < 0.001, and η 2 = 0.0263). Furthermore, obesity increases the chances of needing intensive care unit (OR: 1.783, CI: 95%, and P < 0.001), needing ventilatory support (OR: 1.537, CI: 95%, and P < 0.001 and OR: 2.302, CI: 95%, and P < 0.001, for noninvasive and invasive, respectively), and death (OR: 1.411, CI: 95%, and P < 0.001) of patients hospitalized with COVID-19. Our analysis supports obesity as a significant risk factor for the development of more severe forms of COVID-19. The present study can direct a more effective prevention campaign and appropriate management of subjects with obesity.

The Weight of Obesity in Immunity from Influenza to COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged in December 2019 and rapidly outspread worldwide endangering human health. The coronavirus disease 2019 (COVID-19) manifests itself through a wide spectrum of symptoms that can evolve to severe presentations as pneumonia and several non-respiratory complications. Increased susceptibility to COVID-19 hospitalization and mortality have been linked to associated comorbidities as diabetes, hypertension, cardiovascular diseases and, recently, to obesity. Similarly, individuals living with obesity are at greater risk to develop clinical complications and to have poor prognosis in severe influenza pneumonia. Immune and metabolic dysfunctions associated with the increased susceptibility to influenza infection are linked to obesity-associated low-grade inflammation, compromised immune and endocrine systems, and to high cardiovascular risk. These preexisting conditions may favor virological persistence, amplify immunopathological responses and worsen hemodynamic instability in severe COVID-19 as well. In this review we highlight the main factors and the current state of the art on obesity as risk factor for influenza and COVID-19 hospitalization, severe respiratory manifestations, extrapulmonary complications and even death. Finally, immunoregulatory mechanisms of severe influenza pneumonia in individuals with obesity are addressed as likely factors involved in COVID-19 pathophysiology.

Peripheral leptin signaling persists in innate immune cells during diet-induced obesity.

Leptin is a pleiotropic adipokine that regulates immunometabolism centrally and peripherally. Obese individuals present increased levels of leptin in the blood and develop hypothalamic resistance to this adipokine. Here we investigated whether leptin effects on the periphery are maintained despite the hypothalamic resistance. We previously reported that leptin injection induces in vivo neutrophil migration and peritoneal macrophage activation in lean mice through TNF-α- and CXCL1-dependent mechanisms. However, leptin effects on leukocyte biology during obesity remain unclear. In this study, we investigated the in vivo responsiveness of leukocytes to i.p. injected leptin in mice with diet-induced obesity (DIO). After 14-16 wk, high-sucrose, high-fat diet (HFD)-fed mice showed hyperglycemia, hyperleptinemia, and dyslipidemia compared to normal-sucrose, normal-fat diet (ND). Exogenous leptin did not reduce food intake in DIO mice in contrast to control mice, indicating that DIO mice were centrally resistant to leptin. Regardless of the diet, we found increased levels of TNF-α and CXCL1 in the animals injected with leptin, alongside a pronounced neutrophil migration to the peritoneal cavity and enhanced biogenesis of lipid droplets in peritoneal macrophages. Supporting our in vivo results, data from ex vivo leptin stimulation experiments confirmed hypothalamic resistance in DIO mice, whereas bone marrow cells responded to leptin stimulation through mTOR signaling despite obesity. Altogether, our results show that leukocytes responded equally to leptin in ND- or HFD-fed mice. These results support a role for leptin in the innate immune response also in obesity, contributing to the inflammatory status that leads to the development of metabolic disease.

Semaphorin-3A-Related Reduction of Thymocyte Migration in Chemically Induced Diabetic Mice.

BACKGROUND: Previous work revealed the existence of a severe thymic atrophy with massive loss of immature CD4+CD8+ thymocytes in animals developing insulin-dependent diabetes, chemically induced by alloxan. Furthermore, the intrathymic expression of chemokines, such as CXCL12, is changed in these animals, suggesting that cell migration-related patterns may be altered. One molecular interaction involved in normal thymocyte migration is that mediated by soluble semaphorin-3A and its cognate receptor neuropilin-1. OBJECTIVES: We investigated herein the expression and role of semaphorin-3A in the migratory responses of thymocytes from alloxan-induced diabetic mice. We characterized semaphorin-3A and its receptor, neuropilin-1, in thymuses from control and diabetic mice as well as semaphorin-3A-dependent migration of developing thymocytes in both control and diabetic animals. METHODS: Diabetes was chemically induced after a single injection of alloxan in young adult BALB/c mice. Thymocytes were excised from control and diabetic individuals and subjected to cytofluorometry for simultaneous detection of semaphorin-3A or neuropilin-1 in CD4/CD8-defined subsets. Cell migration in response to semaphorin-3A was performed using cell migration transwell chambers. RESULTS: Confirming previous data, we observed a severe decrease in the total numbers of thymocytes in diabetic mice, which comprised alterations in both immature (double-negative subpopulations) and mature CD4/CD8-defined thymocyte subsets. These were accompanied by a decrease in the absolute numbers of semaphorin-3A-bearing thymocytes, comprising CD4-CD8-, CD4+CD8+, and CD4-CD8+ cells. Additionally, immature CD4-CD8- and CD4+CD8+ developing T cells exhibited a decrease in the membrane density of semaphorin-3A. The relative and absolute numbers of neuropilin-1-positive thymocytes were also decreased in diabetic mouse thymocytes compared to controls, as seen in CD4-CD8-, CD4+CD8+, and CD4-CD8+ cell subpopulations. Functionally, we observed a decrease in the chemorepulsive role of semaphorin-3A, as revealed by transwell migration chambers. Such an effect was seen in all immature and mature thymocyte subsets. CONCLUSIONS: Taken together, our data clearly unravel a disruption in the normal cell migration pattern of developing thymocytes following chemically induced insulin-dependent diabetes, as ascertained by the altered migratory response to sempahorin-3A. In conceptual terms, it is plausible to think that such disturbances in the migration pattern of thymocytes from these diabetic animals may exert an impact in the cell-mediated immune response of these mice.

High-fat diet-induced obesity worsens TH2 immune response and immunopathologic characteristics in murine model of eosinophilic oesophagitis.

BACKGROUND: Eosinophilic oesophagitis (EoE) is an emergent chronic immune-mediated disease of the oesophagus, which affects both children and adults. It is clinically characterized by dysphagia, food impaction and oesophageal eosinophilia. Epidemiological studies indicate that obesity can worsen allergic symptoms; however, its effect on EoE immunopathological response has not been evaluated yet. This study aimed to assess the effect of obesity on allergic inflammation and T helper-2 profile in an EoE experimental model. METHODS: Obesity was induced by high-fat feeding. After 7 weeks of diet, male BALB/c mice were subcutaneously sensitized and orally challenged with OVA. RESULTS: Obesity itself induced a significant mast cell and eosinophil accumulation in the oesophagus, trachea, gut and lung. After allergy induction, this number was higher, when compared to lean-allergic mice. Moreover, obese-allergic mice showed higher remodelling area, in the oesophagus, associated with higher IL-5 and TSLP mRNA expression. In contrast, FoxP3 and IL-10 were less expressed in comparison with lean-allergic mice. In addition, the amount of CD11c+ MHCII+ PDL1+ dendritic cells was reduced, while the number of CD11c+ MHCII+ CD80+ DCs and CD3+ CD4+ GATA3+ IL-4+ cells was increased in obese-allergic mice in the spleen and lymph nodes when compared to lean-allergic mice. CONCLUSION: Obesity aggravated the immune histopathological characteristics in the EoE experimental model, which was associated with the reduction in the regulatory profile, and the increased inflammatory cells influx, related to the TH 2 profile. Altogether, the data provide new knowledge about obesity as a risk factor, worsening EoE symptoms, and contribute for future treatment strategies for this specific profile.

4T1 Mammary Carcinoma Colonization of Metastatic Niches Is Accelerated by Obesity.

Breast cancer (BC) remains the leading cause of cancer-related deaths among women, and the chances to develop it are duplicated by obesity. Still, the impact of obesity during BC progression remains less understood. We investigated the role of obesity in tumor progression using the murine model of 4T1 mammary carcinoma in BALB/c female mice, previously high-fat-diet (HFD) fed. HFD induced obesity, metabolic impairment, and high serum and fat leptin levels. After injection of 4T1-cells, HFD-mice accelerated tumor progression and metastasis. 4T1-cells found within HFD-mice metastatic niches presented higher clonogenic potential. 4T1-cells treated in vitro with fat-conditioned medium derived from HFD-mice, increased migration capacity through CXCL12 and CCL25 gradients. In HFD-mice, the infiltration and activation of immune cells into tumor-sentinel lymph nodes was overall reduced, except for activated CD4+ T cells expressing low CD25 levels. Within the bone marrow, the levels of haematopoiesis-related IL-6 and TNF-α decreased after 4T1-cells injection in HFD-mice whereas increased in the controls, suggesting that upregulation of both cytokines, regardless of the tumor, is disrupted by obesity. Finally, the expression of genes for leptin, CXCR4, and CCR9 (receptors of CXCL12 and CCL25, respectively) was negatively correlated with the infiltration of CD8 T cells in human triple-negative BC tumors from obese patients compared to non-obese. Together, our data present early evidence of systemic networks triggered by obesity that promote BC progression to the metastatic niches. Targeting these pathways might be useful to prevent the rapid BC progression observed among obese patients.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

Modulation of immune response to induced-arthritis by low-level laser therapy.

As low-level laser therapy immune cells responses are not always clarified, this study aimed to evaluate cytokines and immune cells profile after low-level laser therapy (LLLT) on arthritis-induced model. Arthritis was induced in C57BL/6 mice divided into five groups: euthanized 5 hours after inflammation induction; untreated; dexamethasone treated; LLLT at 3 Jcm-2 ; LLLT at 30 Jcm-2 . Cytokine measurements by enzyme-linked immunosorbent assay and mRNA cytokine relative levels by real-time quantitative polymerase chain reaction were performed with arthritic ankle (IL-1ß, IL-6, TNF-α, IL-10 and TGF-ß). Macrophages, dendritic cells, natural killer cells, lymphocytes CD4+ , CD8+ , Treg and costimulatory proteins were quantified in proximal lymph node by flow cytometry. Data showed decrease in all cytokine levels after LLLT and alteration in mRNA relative levels, depending on the energy density used. LLLT was able to increase of immune cell populations analyzed in the lymph node as well as costimulatory proteins expression on macrophages and dendritic cells. Treg TCD4+ and TCD8+ population enrichment were observed in LLLT at 3 and 30 Jcm-2 groups, respectively. Furthermore, Treg TCD8+ cells expressing higher levels of CD25 were observed at LLLT at 30 Jcm-2 group. Our results indicate that LLLT could change the inflammatory course of arthritis, tending to accelerate its resolution through immune cells photobiostimulation.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.

Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.

Antileishmanial activity of a 4-hydrazinoquinoline derivative: Induction of autophagy and apoptosis-related processes and effectiveness in experimental cutaneous leishmaniasis.

Currently, available treatment options for leishmaniasis are limited and unsatisfactory. In a previous study, a quinoline derivative (AMQ-j), exhibited a strong effect against Leishmania amazonensis and its antileishmanial activity was preliminarily associated with mitochondrial dysfunction. The present study further explores the antileishmanial effect of this compound against L. amazonensis, as well as determines the main cellular processes involved in the death of the parasite. Moreover, this study evaluated the in vivo effect of the AMQ-j in BALB/c mice experimentally infected by L. amazonensis. The results showed that the compound AMQ-j induces a set of morphological and biochemical features that could correlate with both autophagy-related and apoptosis-like processes, indicating intense mitochondrial swelling, a collapse of the mitochondrial membrane potential, an abnormal chromatin condensation, an externalization of phosphatidylserine, an accumulation of lipid bodies, a disorganization of cell cycle, a formation of autophagic vacuoles, and an increase of acidic compartments. Treatment with AMQ-j through an intralesional route was effective in reducing the parasite burden and size of the lesion. No significant increase in the serum levels of hepatic or renal damage toxicity markers was observed. These findings contribute to the understanding of the mode of action of quinoline derivatives involved in the death of Leishmania parasites and encourage new studies in other experimental models of Leishmania infection.

Effect of 1,2,3-triazole salts, non-classical bioisosteres of miltefosine, on Leishmania amazonensis.

Here, we report the effect of new non-classical bioisosteres of miltefosine on Leishmania amazonensis. Fifteen compounds were synthesized and the compound dhmtAc, containing an acetate anion, a side chain of 10 carbon atoms linked to N-1 and a methyl group linked to N-3, showed high and selective biological activity against L. amazonensis. On the intracellular amastigotes, stages of the parasite related to human disease, the IC50 values were near or similar to the 1.0µM (0.9, 0.8 and 1.0µM on L. amazonensis-WT, and two transgenic L. amazonensis expressing GFP and RFP, respectively), being more active than miltefosine. Furthermore, dhmtAc did not show toxic effects on human erythrocytes and macrophages (CC50=115.9µM) being more destructive to the intracellular parasites (selectivity index>115). Promastigotes and intramacrophage amastigotes treated with dhmtAc showed low capacity for reversion of the effect of the compound. A study of the mechanism of action of this compound showed some features of metazoan apoptosis, including cell volume decreases, loss of mitochondrial membrane potential, ROS production, an increase in the intracellular lipid bodies, in situ labeling of DNA fragments by TUNEL labeling and phosphatidylserine exposure to the outerleaflet of the plasma membrane. In addition, the plasma membrane disruption, revealed by PI labeling, suggests cell death by necrosis. No increase in autophagic vacuoles formation in treated promastigotes was observed. Taken together, the data indicate that the bioisostere of miltefosine, dhmtAc, has promising antileishmanial activity that is mediated via apoptosis and necrosis.

Apoptosis induced by low-level laser in polymorphonuclear cells of acute joint inflammation: comparative analysis of two energy densities.

Anti-inflammatory property of low-level laser therapy (LLLT) has been widely described in literature, although action mechanisms are not always clarified. Thus, this study aimed to evaluate apoptosis mechanisms in the LLLT anti-inflammatory effects on the arthritis experimental model in vivo at two different energy densities (3 and 30 Jcm-2). Arthritis was induced in mice by zymosan solution, animals were distributed into five groups, and morphological analysis, immunocytochemistry and gene expressions for apoptotic proteins were performed. Data showed an anti-inflammatory effect, DNA fragmentation in polymorphonuclear (PMN) cells and alteration in gene expression of proteins related to apoptosis pathways after LLLT. p53 gene expression increased at both energy densities, Bcl2 gene expression increased at 3 Jcm-2, and Bcl2 tissue expression decreased at 30 Jcm-2. In addition, apoptosis was restricted to PMN cells. Results suggest that apoptosis in PMN cells comprise part of LLLT anti-inflammatory mechanisms by disbalance promotion between expression of pro-apoptotic (Bax and p53) and anti-apoptotic (Bcl-2) proteins, with pro-apoptotic gene expression selectively in PMN cells.

Topical application of the hexane fraction of Lacistema pubescens reduces skin inflammation and cytokine production in animal model.

OBJECTIVES: The aim of this study was to investigate the acute topical anti-inflammatory effect of the hexane fraction (HLP) of Lacistema pubescens in mice. METHODS: Ear oedema models induced by croton oil, arachidonic acid, phenol, histamine, ethyl phenyl propiolate and capsaicin. Histopathological analyses of ear tissue samples sensitized with croton oil were performed. Myeloperoxidase activity (MPO), the pro-inflammatory cytokine-inhibitory effect and dermatoxicity were also evaluated. KEY FINDINGS: HLP (1, 0.5 and 0.1 mg/ear) resulted in a substantial reduction in skin thickness or tissue weight on all models tested, except for capsaicin-induced ear oedema, similar to dexamethasone (0.1 mg/ear) and/or indomethacin (0.5 mg/ear). Histopathological analyses and neutrophil-mediated MPO activity confirmed the topical anti-inflammatory effect of HLP. In addition, HLP reduced IL-1ß, IL-6 and tumour necrosis factor-α cytokine levels. Sitosterol-rich fraction (SRF), obtained from HLP fractionation, reduced ear oedema on croton oil and phenol models at the same dose of dexamethasone (0.1 mg/ear). No dermotoxicity was observed. CONCLUSIONS: The mechanism of action of HLP was associated with the inhibition of several pro-inflammatory mediators, including cytokines, arachidonic acid metabolites and histamine, which suggested a glucocorticoid-like effect, reinforced by the presence of the steroid sitosterol. This is the first report on anti-inflammatory activity of L. pubescens leaves.

Pereskia aculeata Miller leaves present in vivo topical anti-inflammatory activity in models of acute and chronic dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Pereskia aculeata Miller (Cactaceae), known as Barbados gooseberry, are used in Brazilian traditional medicine as emollients and to treat skin wounds and inflammation. This study investigated the topical anti-inflammatory activity of the hexane fraction (HF) obtained from the methanol extract of the leaves of this species in models of acute and chronic ear dermatitis in mice. MATERIAL AND METHODS: Mice ear edema was induced by topical application of croton oil, arachidonic acid, capsaicin, ethyl-phenylpropiolate and phenol; and by subcutaneous injection of histamine. Ear biopsies were obtained to determine the levels of IL-1ß, IL-6 and TNF-α cytokines by ELISA assay. Histopathological analysis was also performed to evaluate the HF activity in croton oil multiple application test. In addition, acute dermal irritation/corrosion test in rats was accomplished. HF chemical characterization was performed by GC-MS analysis. RESULTS: HF intensively reduced the inflammatory process induced by all irritant agents used, except for arachidonic acid. This activity is related, at least in part, to the reduction of IL-6 and TNF-α cytokines levels. Moreover, when the glucocorticoid receptor antagonist mifepristone was used, HF failed to respond to the croton oil application.The results strongly suggested a glucocorticoid-like effect, which was reinforced by the presence of considerable amounts of sterol compounds identified in HF. The acute dermal irritaton/corrosion test showed no signs of toxicity. CONCLUSIONS: This study showed that the acute and chronic anti-inflammatory activity of P. aculeata leaves is very promising, and corroborates to better understand their ethnopharmacological applications.