Schinus terebinthifolius Raddi (Brazilian pepper) leaves extract: in vitro and in vivo evidence of anti-inflammatory and antioxidant properties.

The aim of this work was to evaluate the anti-inflammatory and antioxidant effects of ethyl acetate extract obtained from the leaves of Brazilian peppertree Schinus terebinthifolius Raddi (EAELSt). Total phenols and flavonoids, chemical constituents, in vitro antioxidant activity (DPPH and lipoperoxidation assays), and cytotoxicity in L929 fibroblasts were determined. In vivo anti-inflammatory and antioxidant properties were evaluated using TPA-induced ear inflammation model in mice. Phenol and flavonoid contents were 19.2 ± 0.4 and 93.8 ± 5.2 of gallic acid or quercetin equivalents/g, respectively. LC-MS analysis identified 43 compounds, of which myricetin-O-pentoside and quercetin-O-rhamnoside were major peaks of chromatogram. Incubation with EAELSt decreased the amount of DPPH radical (EC50 of 54.5 ± 2.4 µg/mL) and lipoperoxidation at 200-500 µg/mL. The incubation with EAELSt did not change fibroblast viability up to 100 µg/mL. Topical treatment with EAELSt significantly reduced edema and myeloperoxidase activity at 0.3, 1, and 3 mg/ear when compared to the vehicle-treated group. In addition, EAELSt decreased IL-6 and TNF-α levels and increased IL-10 levels. Besides, it modulated markers of oxidative stress (reduced total hydroperoxides and increased sulfhydryl contents and ferrium reduction potential) and increased the activity of catalase and superoxide dismutase, without altering GPx activity.

Anti-proliferative and pro-apoptotic activity of glycosidic derivatives of lawsone in melanoma cancer cell.

BACKGROUND: Melanoma is a malignant cancer that affects melanocytes and is considered the most aggressive skin-type cancer. The prevalence for melanoma cancer for the last five year is about one million cases. The impact caused of this and other types of cancer, revel the importance of research into potential active compounds. The natural products are an important source of compounds with biological activity and research with natural products may enable the discovery of compounds with potential activity in tumor cells. METHODS: The Sulforhodamine B was used to determine cell density after treatment with lawsone derivatives. Apoptosis and necrosis were analyzed by flow cytometer. Morphological changes were observed by fluorescence using the Phalloidin/FITC and DAPI stains. The clonogenic and wound healing assays were used to analyze reduction of colonies formation and migratory capacity of melanoma cells, respectability. RESULTS: In pharmacological screening, seven compounds derived from lawsone were considered to have high cytotoxic activity (GI > 75%). Three compounds were selected to assess the inhibitory concentration for 50% of cells (IC50), and the compound 9, that has IC50 5.3 µM in melanoma cells, was selected for further analyses in this cell line. The clonogenic assay showed that the compound is capable of reducing the formation of melanoma colonies at 10.6 µM concentration. The compound induced apoptotic morphological changes in melanoma cells and increased by 50% the cells dying from apoptosis. Also, this compound reduced the migratory capacity of melanoma cells. CONCLUSIONS: The results of this study showed that the evaluated lawsone derivatives have potential activity on tumor cells. The compound 9 is capable of inducing cell death by apoptosis in melanoma cells (B16F10).

Seroprevalence of SARS-CoV-2 antibodies in the poorest region of Brazil: results from a population-based study.

Population-based seroprevalence studies on coronavirus disease 2019 (COVID-19) in low- and middle-income countries are lacking. We investigated the seroprevalence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies in Sergipe state, Northeast Brazil, using rapid IgM-IgG antibody test and fluorescence immunoassay. The seroprevalence was 9.3% (95% CI 8.5-10.1), 10.2% (95% CI 9.2-11.3) for women and 7.9% (IC 95% 6.8-9.1) for men (P = 0.004). We found a decline in the prevalence of SARS-CoV-2 antibodies according to age, but the differences were not statistically significant: 0-19 years (9.9%; 95% CI 7.8-12.5), 20-59 years (9.3%; 95% CI 8.4-10.3) and ≥60 years (9.0%; 95% CI 7.5-10.8) (P = 0.517). The metropolitan area had a higher seroprevalence (11.7%, 95% CI 10.3-13.2) than outside municipalities (8.0%, 95% CI 7.2-8.9) (P < 0.001). These findings highlight the importance of serosurveillance to estimate the real impact of the COVID-19 outbreak and thereby provide data to better understand the spread of the virus, as well as providing information to guide stay-at-home measures and other policies. In addition, these results may be useful as basic data to follow the progress of COVID-19 outbreak as social restriction initiatives start to be relaxed in Brazil.

Reduced cutaneous inflammation associated with antioxidant action after topical application of the aqueous extract of Annona muricata leaves.

Annona muricata L. is used in folk medicine for treatment of diseases related to inflammatory and oxidative processes. This study investigated the effect of the aqueous extract of A. muricata leaves (AEAM) on TPA-induced ear inflammation and antioxidant capacity, both in vitro and in vivo. The in vitro antioxidant capacity of AEAM was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP) and lipoperoxidation assays. Cytotoxicity and reactive oxygen species (ROS) release were evaluated in the L929 fibroblasts. Swiss mice were submitted to TPA application and were topically treated with AEAM (0.3, 1 or 3 mg/ear). After 6 h, inflammatory and oxidative parameters were evaluated. Quercetin 3-glucoside, rutin, chlorogenic acid, catechin and gallic acid were identified in AEAM. It also presented antioxidant activity in all in vitro assays used. Incubation with AEAM did not cause cell cytotoxicity but reduced ROS release from fibroblasts. Compared with the control group, treatment with AEAM significantly reduced ear oedema and mieloperoxidase activity in inflamed ears, as well as histological parameters of inflammation. These results were associated with the reduction of total hydroperoxides and modulation of catalase, but not superoxide dismutase activity. These findings show the anti-inflammatory effect of AEAM is associated with antioxidant capacity.

Assessment of phytotoxicity and impact on the enzymatic activity of soil microorganisms caused by veterinary antibiotics used in Brazilian farms.

This work aimed to evaluate the impact of veterinary antibiotics on biomass phytoproductivity and soil enzyme activity. The soil was sampled in the city of Camboriú (state of SC, Brazil). The soil enzyme activity was assessed through hydrolysis of fluorescein diacetate (FDA), while phytotoxicity was tested using Lactuca sativa (lettuce). Results showed that the most appropriate exposure time to assess the impact of antibiotics on soil microbiology was 24 h, while the incubation time of 3 h was the most appropriate for FDA hydrolysis. Ampicillin and Amoxicillin at the tested concentrations did not interfere with the enzyme activity of the soil microbiota, while Oxytetracycline and Neomycin showed a significant reduction in soil enzyme activity. For the dry and wet biomass of lettuce, 2% Colistin and 1% Ampicillin were the treatments that reduced lettuce biomass. Hence, the use of excessive antibiotics in animal production may lead to environmental impacts and, in the future, to public health problems.

Biochanin A attenuates zymosan-induced arthritis in mice similarly to 17-ß estradiol: an alternative to hormone replacement therapy?

OBJECTIVE AND DESIGN: Biochanin A (BCA), a phytoestrogen, has various pharmacological properties. This study was conducted to compare BCA's therapeutic property against 17-ß estradiol replacement therapy in zymosan-induced arthritis (ZIA) in mice. Additionally, we further investigated in vitro the anti-inflammatory action on neutrophils. TREATMENT: Ovariectomized (OVX) and non-OVX mice were pretreated with BCA (1, 3 and 9 mg/kg) or estrogen (50 µg/kg) for 14 days prior to ZIA. Neutrophils were pretreated with BCA (1, 10 and 100 µM) for 1 h prior to phorbol 12-myristate 13-acetate. METHODS: Anti-inflammatory effects of BCA were evaluated by cellular infiltrate, paw edema and cytokine measurement. In vitro, apoptosis was assessed by morphology and flow cytometry. Neutrophil extracellular traps (NET) were determined by fluorescent microscopy and DNA release. Statistical differences were determined by one- or two-way ANOVA. RESULTS: BCA inhibited neutrophil accumulation, paw edema and proinflammatory cytokine (TNF-α and IFN-γ) and increased anti-inflammatory cytokines (IL-4 and IL-10) in OVX and non-OVX mice, similar to 17-ß estradiol replacement therapy. In vitro, BCA increased apoptosis and consequently reduced NETs. CONCLUSION: BCA has a notable anti-inflammatory effect, similar to 17-ß estradiol, and is especially effective for treatment of ZIA. These results suggest that BCA may be promising for the treatment of postmenopausal arthritis.

DNA aptamers selection for carcinoembryonic antigen (CEA).

Carcinoembryonic antigen (CEA) is a glycoprotein antigen generally used for diagnosis, prognosis and treatment monitoring of several types of tumors, including colorectal cancer. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain aptamers specific to CEA for use as radiopharmaceuticals in colorectal cancer diagnosis. Five aptamers were selected through the Systematic Evolution of Ligands by EXponencial Enrichment (SELEX) and tested using T84 (CEA+) and Hela (CEA-) cells. Apta 3 and Apta 5 showed the best results presenting high specificity and affinity for T84 cells, with dissociation constants (Kd) of 60.4 ± 5.7 nM and 37.8 ± 5.8 nM, respectively. These results indicate that Apta 3 and Apta 5 are promising candidates for identifying tumor cells that overexpress CEA.

In situ photocrosslinkable formulation of nanocomposites based on multi-walled carbon nanotubes and formononetin for potential application in spinal cord injury treatment.

Carbon nanotubes (CN) have been studied to treat spinal cord injuries because of its electrical properties and nanometric dimensions. This work aims to develop a photopolymerizable hydrogel containing CN functionalized with an anti-inflammatory molecule to be used in situ on spinal cord injuries. The CN functionalization step was done using the drug (formononetin). The nanocomposites were characterized by morphological analysis, FTIR, Raman Spectroscopy, thermal analysis and cytotoxicity assays (MTT and HET-CAM). The nanocomposites were incorporated into gelatin methacryloyl hydrogel and exposed to UV light for photopolymerization. The volume of the formulation and the UV exposition time were also analyzed. The CN characterization showed that formononetin acted as a functionalization agent. The functionalized CN showed safe characteristics and can be incorporated in photocrosslinkable formulation. The UV exposition time for the formulation photopolymerization was compatible with the cell viability and also occurred in the injury site.

Ethanolic extract of Croton blanchetianus Ball induces mitochondrial defects in Leishmania amazonensis promastigotes.

Leishmaniasis is a neglected disease caused by Leishmania. Chemotherapy remains the mainstay for leishmaniasis control; however, available drugs fail to provide a parasitological cure, and are associated with high toxicity. Natural products are promising leads for the development of novel chemotherapeutics against leishmaniasis. This work investigated the leishmanicidal properties of ethanolic extract of Croton blanchetianus (EECb) on Leishmania infantum and Leishmania amazonensis, and found that EECb, rich in terpenic compounds, was active against promastigote and amastigote forms of both Leishmania species. Leishmania infantum promastigotes and amastigotes presented IC50 values of 208.6 and 8.8 µg/mL, respectively, whereas Leishmania amazonensis promastigotes and amastigotes presented IC50 values of 73.6 and 3.1 µg/mL, respectively. Promastigotes exposed to EECb (100 µg/mL) had their body cellular volume reduced and altered to a round shape, and the flagellum was duplicated, suggesting that EECb may interfere with the process of cytokinesis, which could be the cause of the decline in the parasite multiplication rate. Regarding possible EECb targets, a marked depolarization of the mitochondrial membrane potential was observed. No cytotoxic effects of EECb were observed in murine macrophages at concentrations below 60 µg/mL, and the CC50 obtained was 83.8 µg/mL. Thus, the present results indicated that EECb had effective and selective effects against Leishmania infantum and Leishmania amazonensis, and that these effects appeared to be mediated by mitochondrial dysfunction.

Antimicrobial activity of Lippia gracilis essential oils on the plant pathogen Xanthomonas campestris pv. campestris and their effect on membrane integrity.

Xanthomonas campestris pv.campestris (Xcc) is the causative agent of black rot, a disease that causes serious damage to plants from Brassicaceae family. However, there are no chemicals or biological agent commercially registered for the control of this disease. Thus, this study aimed to evaluate the antimicrobial activity and chemical composition of Lippia gracilis essential oils (EOs) on Xcc aiming its use as effective biological control. We also investigated the effect of EOs on the integrity of the bacterial cytoplasmic membrane. Chemical analysis by GC/MS showed that the major compounds of the seven EOs of L. gracilis are thymol or carvacrol. The seven genotypes showed inhibition of bacterial growth with MIC from 700 µg.ml-1 to 1000 µg.ml-1, with the genotype LGRA-106 (rich in Thymol) with higher antimicrobial activity. The MIC for thymol and carvacrol were 250 µg.ml-1. After exposure to LGRA-106 EO (2×, 1×, 1/2×, 1/4×, and 1/8 x MIC for 5 min, it was observed a decreased cell viability and increased pI fluorescence, which indicates damage to the cytoplasmic cell membrane. This study demonstrates that L. gracilis EOs have antimicrobial activity and have a potential to be used in the control of black rot. Furthermore this antimicrobial activity is due, at least in part, to bacterial cytoplasmic membrane damage.

Praziquantel-Solid Lipid Nanoparticles Produced by Supercritical Carbon Dioxide Extraction: Physicochemical Characterization, Release Profile, and Cytotoxicity.

Solid lipid nanoparticles (SLNs) can be produced by various methods, but most of them are difficult to scale up. Supercritical fluid (SCF) is an important tool to produce micro/nanoparticles with a narrow size distribution and high encapsulation efficiency. The aim of this work was to produce cetyl palmitate SLNs using SCF to be loaded with praziquantel (PZQ) as an insoluble model drug. The mean particle size (nm), polydispersity index (PdI), zeta potential, and encapsulation efficiency (EE) were determined on the freshly prepared samples, which were also subject of Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FTIR), drug release profile, and in vitro cytotoxicity analyses. PZQ-SLN exhibited a mean size of ~25 nm, PdI ~ 0.5, zeta potential ~-28 mV, and EE 88.37%. The DSC analysis demonstrated that SCF reduced the crystallinity of cetyl palmitate and favored the loading of PZQ into the lipid matrices. No chemical interaction between the PZQ and cetyl palmitate was revealed by FTIR analysis, while the release or PZQ from SLN followed the Weibull model. PZQ-SLN showed low cytotoxicity against fibroblasts cell lines. This study demonstrates that SCF may be a suitable scale-up procedure for the production of SLN, which have shown to be an appropriate carrier for PZQ.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil.

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.

Leishmanicidal Activity and Structure-Activity Relationships of Essential Oil Constituents.

Several constituents of essential oils have been shown to be active against pathogens such as bacteria, fungi, and protozoa. This study demonstrated the in vitro action of ten compounds present in essential oils against Leishmania amazonensis promastigotes. With the exception of p-cymene, all evaluated compounds presented leishmanicidal activity, exhibiting IC50 between 25.4 and 568.1 µg mL-1. Compounds with the best leishmanicidal activity presented a phenolic moiety (IC50 between 25.4 and 82.9 µg mL-1). Alicyclic alcohols ((-)-menthol and isoborneol) and ketones ((-)-carvone) promoted similar activity against the parasite (IC50 between 190.2 and 198.9 µg mL-1). Most of the compounds showed low cytotoxicity in L929 fibroblasts. Analysis of the structure-activity relationship of these compounds showed the importance of the phenolic structure for the biological action against the promastigote forms of the parasite.

Aptamers directly radiolabeled with technetium-99m as a potential agent capable of identifying carcinoembryonic antigen (CEA) in tumor cells T84.

Aptamers are small oligonucleotides that are selected to bind with high affinity and specificity to a target molecule. Aptamers are emerging as a new class of molecules for radiopharmaceutical development. In this study a new method to radiolabel aptamers with technetium-99m ((99m)Tc) was developed. Two aptamers (Apt3 and Apt3-amine) selected against the carcinoembryonic antigen (CEA) were used. Labeling was done by the direct method and the developed complex was subjected to quality control tests. Radiochemical purity and stability were monitored by Thin Layer Chromatography. Binding and specificity assays were carried out in the T84 cell line (CEA+) to evaluate tumor affinity and specificity after radiolabeling. Aptamers were successfully labeled with (99m)Tc in high radiochemical yields, showing in vitro stability in presence of plasma and cystein. In binding assays the radiolabeled aptamer Apt3-amine showed the highest affinity to T84 cells. When evaluated with HeLa cells (CEA-), lower uptake was observed, suggesting high specificity for this aptamer. These results suggest that the Apt3-amine aptamer directly labeled with (99m)Tc could be considered a promising agent capable of identifying the carcinoembryonic antigen (CEA) present in tumor cells.

Anti-migratory and cytotoxic effect of indole derivative in C6 glioma cells.

Gliomas are among the most common primary malignant brain tumors. Despite advances in cancer treatment, survival is very low, so the discovery of new therapeutic agents is essential. In this context, indole is an important source for the development of new bioactive molecules. A pharmacological screening of ten indole derivatives was carried out to evaluate the cytotoxic capacity against three tumor cell lines. After pharmacological screening, three compounds were selected, based on their high capacity to reduce cell proliferation, and their IC50 values were determined. Compound 9 exhibited the highest cytotoxic activity (IC50 = 0.4 µg/mL) in gliomas (C6 cell line), and were selected for further experiments. C6 cells were treated with compound 9 to evaluate cellular mechanisms such as colony formation and cell migration capacity and morphological alterations. Compound 9 decreased clone formation (0.4 and 0.8 µg/mL), and inhibited migration (0.2-0.8 µg/mL) in C6 cells. Morphological changes in cells treated with the compound 9 were also observed, such as chromatin condensation, and disorganization in cellular stress beams. Indole derivatives had a cytotoxic effect on tumor cells, and compound 9 showed the best anti-proliferative and anti-migratory activity in glioma cells.

CD4+ Th1 and Th17 responses and multifunctional CD8 T lymphocytes associated with cure or disease worsening in human visceral leishmaniasis.

Introduction: In VL, a proinflammatory phenotype is typically associated with enhanced phagocytosis and a Th1 mediated immune response resulting in infection control. In contrast, an anti-inflammatory phenotype, associated with a predominant regulatory response, typically enables intracellular multiplication of Leishmania parasites and disease progression. Methods: To investigate the impact of chemotherapy on Th2 and Th17 immune responses in patients with visceral leishmaniasis (VL), we assessed all combinations of intracellular expression of IFN-γ, IL-10, IL-4 and IL-17 in the CD4+ and CD8+ T cell populations of peripheral blood mononuclear cell (PBMC) samples from patients, after antigenic stimulation with Leishmania lysate, throughout treatment and follow-up. As increases in spleen and liver sizes and decreases in hematocrit, hemogloblin, erythrocytes, monocytes, leukocytes and platelets levels are strongly related to the disease, we studied the correlations between the frequencies of T cells producing the afore mentioned cytokines, individually and in combination, and these variables, as markers of disease or cure. Results: We found that the frequency of IFN-γ-producingCD4+ T cells increased until the end of chemotherapy with Glucantime® or AmBisome ®, while IL-10, IL-4 and IL-17-producing CD4+ T cells peaked on day 7 following the start of treatment. Although the frequency of CD4+IL-17+ cells decreased during treatment an increase was observed after clinical cure. The frequency of CD4+ T cells producing only IFN-γ or IL-17 correlated with blood monocytes levels. Frequencies of double-producers of IFN-γ and IL-10 or IL-4 correlated positively with eosinophils and platelets levels. Together, this suggest that IFN-γ drives the immune response towards Th1 at cure. In contrast, and associated with disease or Th2 response, the frequency of CD4+ IL-10+ cells correlated positively with spleen sizes and negatively with circulating monocyte levels, while the frequency of CD4+ producing both IL-4 and IL-10 correlated negatively with platelets levels. The frequency of CD8+ single-producers of IFN-γ increased from day 21 to 90 while that of single-producers of IL-10 peaked on day 7, of IL-4 on day 30 and of IL-17, on day 180. IFN-γ expression in CD8+ single- and double-producers of cytokines was indicative of an immune response associated with cure. In contrast, frequencies of CD8+ double-producers of IL-4 and IL-10, IL-4 and IL-17 and IL-10 and IL-17 and producers of three and four cytokines, were associated with disease and were low after the cure. Frequencies of CD8+ T cells producing IFN-γ alone or with IL-17 were positively correlated with platelets levels. In contrast, as markers of disease: 1) frequencies of single producers of IL-10 correlated negatively with leukocytes levels, 2) frequencies of double producers of IL-4 and IL-10 correlated negatively with platelet, leukocyte, lymphocyte and circulating monocyte levels, 3) frequencies of triple-producers of IFN-γ, IL-4 and IL-10 correlated negatively with platelet, leukocyte and neutrophil levels and 4) frequencies of producers of IFN-γ, IL-4, IL-10 and IL-17 simultaneously correlated positively with spleen size, and negatively with leukocyte and neutrophil levels. Discussion: Our results confirmed that the clinical improvement of VL patients correlates with the decrease of an IL-4 and IL-10 CD4+Th2 response, the recovery of CD4+ Th1 and Th17 responses and the frequency of CD8+ single-producers of IFN-γ and double producers of IFN-γ and IL-17.

Defective T-lymphocyte migration to muscles in dystrophin-deficient mice.

Duchenne muscular dystrophy (DMD), an X-linked recessive disorder affecting 1 in 3500 males, is caused by mutations in the dystrophin gene. DMD leads to degeneration of skeletal and cardiac muscles and to chronic inflammation. The mdx/mdx mouse has been widely used to study DMD; this model mimics most characteristics of the disease, including low numbers of T cells in damaged muscles. In this study, we aimed to assess migration of T cells to the heart and to identify any alterations in adhesion molecules that could possibly modulate this process. In 6-week-old mdx/mdx mice, blood leukocytes, including T cells, were CD62L(+), but by 12 weeks of age down-modulation was evident, with only approximately 40% of T cells retaining this molecule. Our in vitro and in vivo results point to a P2X7-dependent shedding of CD62L (with high levels in the serum), which in 12-week-old mdx/mdx mice reduces blood T cell competence to adhere to cardiac vessels in vitro and to reach cardiac tissue in vivo, even after Trypanosoma cruzi infection, a known inducer of lymphoid myocarditis. In mdx/mdx mice treated with Brilliant Blue G, a P2X7 blocker, these blood lymphocytes retained CD62L and were capable of migrating to the heart. These results provide new insights into the mechanisms of inflammatory infiltration and immune regulation in DMD.

Association between different soy lecithin-based extenders and freezing rates in ram semen cryopreservation.

The aim of the study was to evaluate the effect of the association between glycine-milk (GM) based extenders made with different concentrations of soy lecithin (SL) and freezing rates (FR) on semen quality after thawing. Pooled semen from rams (n = 12) were diluted in GM extenders with 20% egg yolk (EY-20%) or with different concentrations of SL: 0.5% (SL-0.5%), 1.0% (SL-1.0%), and 2.0% (SL-2.0%). The diluted semen (150 ×106 spermatozoa/0.25 mL) was frozen at three FR of - 10, - 20, and - 60 °C/min, and subsequently thawed and analyzed. Results revealed that EY-20% and SL-2.0% had better kinetic parameters, and showed higher proportions of viable, non-apoptotic, plasma-membrane-intact spermatozoa (A-/PI-) and non-capacitated spermatozoa (F), and had lower acrosome-reacted spermatozoa (AR) in the EY-20% and satisfactory values for SL-2.0% compared to SL-0.5% and SL-1.0% (P < 0.05). The FR at - 20 and - 60 °C/min maintained higher A-/PI- and viable spermatozoa compared to - 10 °C/min. The combination EY-20% and - 60 °C/min showed the highest A-/PI- and F (P < 0.05) and the lowest AR, and it did not differ from the combinations EY-20% at - 20 °C/min and SL-2.0% at - 20 °C/min (P > 0.05). In conclusion, the combination EY-20% and - 60 °C/min, showed the best cryoprotective effects on ram spermatozoa. Changes in spermatozoa after thawing were related to the use of the type of extender, the amounts of the same compound in the extender, and the freezing rates to which they were subjected.

Obesity in Severe COVID-19 Patients Has a Distinct Innate Immune Phenotype.

Obesity alters the capacity of effective immune responses in infections. To further address this phenomenon in the context of COVID-19, this study investigated how the immunophenotype of leukocytes was altered in individuals with obesity in severe COVID-19. This cross-sectional study enrolled 27 ICU COVID-19 patients (67% women, 56.33 ± 19.55 years) that were assigned to obese (BMI ≥ 30 kg/m2, n = 9) or non-obese (BMI < 30kg/m2, n = 18) groups. Monocytes, NK, and both Low-Density (LD) and High-Density (HD) neutrophils were isolated from peripheral blood samples, and surface receptors' frequency and expression patterns were analyzed by flow cytometry. Clinical status and biochemical data were additionally evaluated. The frequency of monocytes was negatively correlated with BMI, while NK cells and HD neutrophils were positively associated (p < 0.05). Patients with obesity showed a significant reduction of monocytes, and these cells expressed high levels of PD-L1 (p < 0.05). A higher frequency of NK cells and increased expression of TREM-1+ on HD neutrophils were detected in obese patients (p < 0.05). The expression of receptors related to antigen-presentation, phagocytosis, chemotaxis, inflammation and suppression were strongly correlated with clinical markers only in obese patients (p < 0.05). Collectively, these outcomes revealed that obesity differentially affected, and largely depressed, innate immune response in severe COVID-19.

Cytotoxic activity of essential oil from Leaves of Myrcia splendens against A549 Lung Cancer cells.

BACKGROUND: Plants of the Myrcia genus have been widely used in folk medicine to treat various diseases, including cancer. Myrcia splendens species has a diverse chemical constitution, but the biological activities of its essential oil have not been well investigated. In this study to out the chemistry characterization of essential oil (EO) from the leaves of the species M. splendens from Brazil and evaluate cytotoxic effect in A549 lung cancer cells. METHODS: M. splendens EO was obtained by hydrodistillation and analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). EO was isolated and evaluated for cellular viability in tumor cell lines by MTT assay. The evaluation of the formation of clones and the migratory capacity of the A549 cells treated with EO was done by the clonogenic assay and the wound healing assay. Morphological changes were observed in A549 cells by fluorescence using Phalloidin/FITC and DAPI. RESULTS: 22 compounds were identified in the chemical analysis of EO, corresponding to 88% of the sample. Major compounds were the sesquiterpenic hydrocarbons bicyclogermacrene (15.4%), germacrene D (8.9%) and E-caryophyllene (10.1%). The biological analysis of the EO showed high cytotoxic activity with an IC50 below 20 µg/ml in the THP-1, A549 and B16-F10 tumor cells. The treatment with EO reduced colony formation and inhibited the migratory capacity of A549 cells. Furthermore, apoptotic morphological changes in the nucleus and cytoplasm of A549 cells was observed after of treatment with EO. CONCLUSION: The findings of this study suggest that the M. splendens EO has cytotoxic compounds for the A549 lung cancer cells. Treatment with the EO decreased the colony formation and reduced the ability of lung cancer cells to migrate. Future studies may be used to isolate compounds from the EO for the study of lung cancer.