J774 Cells Research Articles

Inflammatory stimulus worsens the effects of UV-A exposure on J774 cells.

UV-A radiation affects skin homeostasis by promoting oxidative distress. Endogenous photosensitizers in the dermis and epidermis of human skin absorb UV-A radiation forming excited states (singlet and triplet) and reactive oxygen species (ROS) producing oxidized compounds that trigger biological responses. The activation of NF-kB induces the expression of pro-inflammatory cytokines and can intensify the generation of ROS. However, there is no studies evaluating the cross talks between inflammatory stimulus and UV-A exposure on the levels of redox misbalance and inflammation. In here, we evaluated the effects of UV-A exposure on J774 macrophage cells previously challenged with LPS in terms of oxidative distress, release of pro-inflammatory cytokines, and activation of regulated cell death pathways. Our results showed that LPS potentiates the dose-dependent UV-A-induced oxidative distress and cytokine release, in addition to amplifying the regulated (autophagy and apoptosis) and non-regulated (necrosis) mechanisms of cell death, indicating that a previous inflammatory stimulus potentiates UV-A-induced cell damage. We discuss these results in terms of the current-available skin care strategies.

Effects of Vigna angularis extract and its active compound hemiphloin against atopic dermatitis-like skin inflammation

Vigna angularis is an edible crop and herbal medicine that is known to have antipyretic, anti-inflammatory, and anti-edema effects. Many studies have been conducted on the 95% ethanol extract of V. angularis, but there is little research on the 70% ethanol extract and hemiphloin, which is a new indicator component of the 70% ethanol extract of V. angularis. To investigate the in vitro anti-atopic effect and verify the mechanism action of 70% ethanol extract of V. angularis (VAE), TNF-α/IFN-γ-induced HaCaT keratinocytes were used. The VAE treatment alleviated TNF-α/IFN-γ-induced IL-1β, IL-6, IL-8, CCL17/TARC, and CCL22/MDC gene expressions and productions. VAE also inhibited the phosphorylation of MAPKs, including p38, ERK, JNK, STAT1, and NF-κB in TNF-α/IFN-γ-induced HaCaT cells. 2,4-dinitochlorobenzene (DNCB)-induced skin inflammation mice model, and HaCaT keratinocytes were used. In the DNCB-induced mouse model, VAE treatment alleviated ear thicknesses and IgE levels. Furthermore, VAE decreased IL-1β, IL-6, IL-8, CCL17/TARC, and CCL22/MDC gene expressions of DNCB-applied ear tissue. Additionally, we investigated the anti-atopic and anti-inflammatory effects of hemiphloin using TNF-α/IFN-γ-induced HaCaT keratinocytes and LPS-induced J774 macrophages. Treatment hemiphloin decreased gene expressions and productions of IL-1β, IL-6, IL-8, CCL17/TARC, and CCL22/MDC in TNF-α/IFN-γ-induced HaCaT cells. The phosphorylations of p38, ERK, STAT1, and NF-κB were inhibited by hemiphloin in TNF-α/IFN-γ-induced HaCaT cells. Finally, hemiphloin showed anti-inflammatory activities in LPS-induced J774 cells. It decreased LPS-induced NO productions and iNOS and COX-2 expressions. Treatment of hemiphloin also inhibited LPS-induced TNF-α, IL-1β, and IL-6 gene expressions. These results suggest that VAE is an anti-inflammatory agent for inflammatory skin diseases and that hemiphloin could be a therapeutic candidate for inflammatory skin diseases.

Leishmania amazonensis infection impairs VLA-4 clustering and adhesion complex assembly at the adhesion site of J774 cells.

Cutaneous leishmaniasis is an infectious disease that may lead to a single or multiple disseminated cutaneous lesions. The mechanisms involved in Leishmania dissemination to different areas of the skin and the internal organs remain poorly understood. Evidence shows that Very Late Antigen-4 (VLA-4)-dependent phagocyte adhesion is impaired by Leishmania infection, which may be related to the mechanisms of parasite dissemination. We investigated factors potentially associated with decreased VLA-4-mediated adhesion in Leishmania-infected macrophages, including lipid raft-mediated VLA-4 mobilization along the cellular membrane, integrin cluster formation at the cell base (adhesion site), and focal adhesion complex assembly. Phagocytes treated with Methyl-β-Cyclodextrin (MβCD) demonstrated reduced adhesion, similarly to Leishmania amazonensis-infected J774 cells. Infected and MβCD-treated macrophages presented decreased VLA-4 mobilization to the adhesion plane, as well as reduced integrin clustering. Leishmania amazonensis-infected cells exhibited talin depletion, as well as a decreased mobilization of adhesion complex proteins, such as talin and viculin, which were associated with lower VLA-4 concentrations at the adhesion site and limited cell-spreading. Our results suggest that Leishmania infection may modulate the firm adhesion phase of the cell-spreading process, which could contribute to the bloodstream dissemination of infected cells.

Quercetin nano phytosome: as a novel anti-leishmania and anti-malarial natural product.

Leishmaniasis is a vector-borne disease that affects several populations worldwide with the clinical manifestations in skin, mucous membranes, and internal organs and there are not any effective and available vaccines and conventional treatments are highly toxic. Quercetin is a kind of flavonoid with different biological effects including free radical scavenging and anti-microbial activity and this study is aimed to assess the anti-leishmania and anti-malarial effects of quercetin loaded phytosome and quercetin alone. In this experimental study, the in vitro activity of above drugs were measured using microscopically examinations and for evaluation the anti-leishmanial efficacy, the size of lesions were measured. Moreover the cytotoxicity of the treatments was evaluated on WI38 and J774 cell lines. Our results indicated that quercetin loaded phytosome and quercetin alone have acceptable anti-parasitic activity mostly at concentration of 400µg/ml on both P. falciparium and L. major. The results of cytotoxicity revealed that the mentioned drugs have no effects on human cell lines and also have no hemolytic activity. The drug of choice for the treatment of leishmaniasis, in addition to killing the parasite, should not have a toxic effect on human cells and our results indicated that quercetin can be a valuable candidate for treatment of different kinds of leishmaniasis.

Evaluation of Ex Vivo Cultivation Potentials of Trypanosoma cruzi, Leishmania tropica ve Toxoplasma gondii Parasites in J774, Vero and HeLa Cell Lines

Three obligate intracellular protozoan parasite species, namely Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii, causative agents of Chagas disease, Leishmaniasis and toxoplasmosis, respectively, which are responsible for significant morbidity and mortality and reside in macrophage cells, affect more than half of the world's population in connection with socio-economic and geographical factors and also causes neglected parasitic diseases of increasing importance. This study aimed to evaluate the ex vivo cultivation potential of T.cruzi, L.tropica and T.gondii parasites in J774, Vero and HeLa cells and to reproduce in a short time and in large amounts without losing their virulence properties. Ex vivo experimental models were created by infecting J774, Vero and HeLa cell lines confluently produced in cell culture flasks with T.cruzi, L.tropica and T.gondii parasites. In ex vivo cultivation, one passage was applied for seven days and three times in a row. Cells removed from the surface after each passage were plated on eight-well chamber slides. Giemsa stained slides were prepared and infection rates were evaluated by light microscopic examination. At the end of the study, it was observed that all three cell lines could be infected with T.cruzi, L.tropica and T.gondii parasites, and infection rates increased in all cell lines after consecutive passages. As a result of ex vivo cultivation, the best cell lines from which T.cruzi and L.tropica strains grew, were J774, Vero and HeLa, and HeLa, J774 and Vero cell lines for T.gondii strain, respectively (p<0.05). Trypanosoma cruzi, L.tropica and T.gondii parasites were successfully grown in J774, Vero and HeLa cell lines by ex vivo culture method in a short time and in large amounts without losing their virulence properties. Cell lines with the best ex vivo cultivation potential for T.cruzi and L.tropica parasites were J774, Vero and HeLa, respectively, while HeLa, J774 and Vero for T.gondii. It is thought that the data obtained in this regard will contribute to many studies on the development of vaccines, drugs and new diagnostic kits.

Molecular Docking of Bacterial Protein Modulators and Pharmacotherapeutics of Carica papaya Leaves as a Promising Therapy for Sepsis: Synchronising In Silico and In Vitro Studies

Sepsis is a serious health concern globally, which necessitates understanding the root cause of infection for the prevention of proliferation inside the host's body. Phytochemicals present in plants exhibit antibacterial and anti-proliferative properties stipulated for sepsis treatment. The aim of the study was to determine the potential role of Carica papaya leaf extract for sepsis treatment in silico and in vitro. We selected two phytochemical compounds, carpaine and quercetin, and docked them with bacterial proteins, heat shock protein (PDB ID: 4PO2), surfactant protein D (PDB ID: 1PW9), and lactobacillus bacterial protein (PDB ID: 4MKS) against imipenem and cyclophosphamide. Quercetin showed the strongest interaction with 1PW9 and 4MKS proteins. The leaves were extracted using ethanol, methanol, and water through Soxhlet extraction. Total flavonoid content, DPPH assay, HPTLC, and FTIR were performed. In vitro cytotoxicity of ethanol extract was screened via MTT assay on the J774 cell line. Ethanol extract (EE) possessed the maximum number of phytocomponents, the highest amount of flavonoid content, and the maximum antioxidant activity compared to other extracts. FTIR analysis confirmed the presence of N-H, O-H, C-H, C=O, C=C, and C-Cl functional groups in ethanol extract. Cell viability was highest (100%) at 25 µg/mL of EE. The present study demonstrated that the papaya leaves possessed antibacterial and cytotoxic activity against sepsis infection.

ApoJ/Clusterin concentrations are determinants of cerebrospinal fluid cholesterol efflux capacity and reduced levels are associated with Alzheimer’s disease

BackgroundAlzheimer’s disease (AD) shares risk factors with cardiovascular disease (CVD) and dysregulated cholesterol metabolism is a mechanism common to both diseases. Cholesterol efflux capacity (CEC) is an ex vivo metric of plasma high-density lipoprotein (HDL) function and inversely predicts incident CVD independently of other risk factors. Cholesterol pools in the central nervous system (CNS) are largely separate from those in blood, and CNS cholesterol excess may promote neurodegeneration. CEC of cerebrospinal fluid (CSF) may be a useful measure of CNS cholesterol trafficking. We hypothesized that subjects with AD and mild cognitive impairment (MCI) would have reduced CSF CEC compared with Cognitively Normal (CN) and that CSF apolipoproteins apoA-I, apoJ, and apoE might have associations with CSF CEC.MethodsWe retrieved CSF and same-day ethylenediaminetetraacetic acid (EDTA) plasma from 108 subjects (40 AD; 18 MCI; and 50 CN) from the Center for Neurodegenerative Disease Research biobank at the Perelman School of Medicine, University of Pennsylvania. For CSF CEC assays, we used N9 mouse microglial cells and SH-SY5Y human neuroblastoma cells, and the corresponding plasma assay used J774 cells. Cells were labeled with [3H]-cholesterol for 24 h, had ABCA1 expression upregulated for 6 h, were exposed to 33 μl of CSF, and then were incubated for 2.5 h. CEC was quantified as percent [3H]-cholesterol counts in medium of total counts medium+cells, normalized to a pool sample. ApoA-I, ApoJ, ApoE, and cholesterol were also measured in CSF.ResultsWe found that CSF CEC was significantly lower in MCI compared with controls and was poorly correlated with plasma CEC. CSF levels of ApoJ/Clusterin were also significantly lower in MCI and were significantly associated with CSF CEC. While CSF ApoA-I was also associated with CSF CEC, CSF ApoE had no association with CSF CEC. CSF CEC is significantly and positively associated with CSF Aβ. Taken together, ApoJ/Clusterin may be an important determinant of CSF CEC, which in turn could mitigate risk of MCI and AD risk by promoting cellular efflux of cholesterol or other lipids. In contrast, CSF ApoE does not appear to play a role in determining CSF CEC.

In Vitro Bioaccessibility and Anti-Inflammatory Activity of a Chemically Characterized Allium cepa L. Extract Rich in Quercetin Derivatives Optimized by the Design of Experiments.

Allium cepa L. is a highly consumed garden crop rich in biologically active phenolic and organosulfur compounds. This study aimed to assess the in vitro bioaccessibility and anti-inflammatory effect of a chemically characterized A. cepa extract rich in quercetin and its derivatives. Different varieties of A. cepa were studied; based on the highest total phenolic content, the "Golden" variety was selected. Its extracts, obtained from the tunicate bulb, tunic, and bulb, were subjected to determination of quercetin and its derivatives with LC-MS analysis and based on the highest total quercetin content, the tunic extract was utilized for further experiments. The extraction method was optimized through a design of experiment (DoE) method via full factorial design, which showed that 40% ethanol and 1 g tunic/20 mL solvent are the best extraction conditions. HPLC analysis of the optimized tunic extract identified 14 flavonols, including 10 quercetin derivatives. As far as in vitro bioaccessibility was concerned, the increases in some quercetin derivatives following the gastro-duodenal digestion process support the bioaccessibility of these bioactive compounds. Moreover, the extract significantly inhibited the production of PGE2 in stimulated J774 cell lines, while no effects of the tunic extract were observed against the release of IL-1β, TNF-α, and nitrites. The study provided insights into the optimized extraction conditions to obtain an A. cepa tunic extract rich in bioavailable quercetin derivatives with significant anti-inflammatory effects against PGE2.

Human apoA-I[Lys107del] mutation affects lipid surface behavior of apoA-I and its ability to form large nascent HDL

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant apoA-I[K107del] adsorbed to surfaces of POPC-coated triolein drops at faster rates, remodeled the surfaces to a greater extent, and was ejected from the surfaces at higher surface pressures on compression of the lipid drops. These properties may drive increased binding of apoA-I[K107del] to and its better retention on large triglyceride-rich lipoproteins, thereby increasing the variant’s content on these lipoproteins. While K107del did not affect apoA-I capacity to promote ABCA1-mediated cholesterol efflux from J774 cells, it impaired the biogenesis of large nascent HDL particles resulting in the formation of predominantly smaller nascent HDL. Size-exclusion chromatography of spontaneously reconstituted 1,2-dimyristoylphosphatidylcholine-apoA-I complexes showed that apoA-I[K107del] had a hampered ability to form larger complexes but formed efficiently smaller-sized complexes. CD analysis revealed a reduced ability of apoA-I[K107del] to increase α-helical structure on binding to 1,2-dimyristoylphosphatidylcholine or in the presence of trifluoroethanol. This property may hinder the formation of large apoA-I[K107del]-containing discoidal and spherical HDL but not smaller HDL. Both factors, the increased content of apoA-I[K107del] on triglyceride-rich lipoproteins and the impaired ability of the variant to stabilize large HDL particles resulting in reduced lipid:protein ratios in HDL, may contribute to normal plasma apoA-I levels along with low HDL-C and increased risk for CVD.

Sichuan dark tea improves lipid metabolism and prevents aortic lipid deposition in diet-induced atherosclerosis model rats.

Sichuan dark tea (ST), Zangcha, is a traditional fermented Chinese tea found in Sichuan and Tibet and claimed for beneficial effects against lifestyle-related metabolic disorders. We examined the effects of ST on lipid metabolism and atherosclerosis. Sichuan dark tea was given to fat-rich diet-induced atherosclerosis model rats in comparison with dark-fermented Chinese Pu-erh tea (PT) and Japanese green tea (GT). After 8 weeks of feeding, ST and PT induced an increase in high-density lipoprotein (HDL)-cholesterol and a decrease in glucose, and ST decreased triglyceride in plasma. ST also induced low pH in the cecum. There was no significant change in their body weight among the fat-feeding groups but a decrease was found in the visceral fat and liver weight in the ST group. Accordingly, ST reduced lipid deposition in the aorta in comparison with PT and GT. ST increased mRNA of LXRα, PPARα, PPARγ, and ABCA1 in the rat liver. The extract of ST stimulated the AMPK pathway to increase the expression of ABCA1 in J774 cells and increased expression of lipoprotein lipase and hormone-sensitive lipase in 3T3L1 cells, consistent with its anti-atherogenic effects in rats. High-performance liquid chromatography analysis showed unique spectra of original specific compounds of caffeine and catechins in each tea extract, but none of them was likely responsible for these effects. Sichuan dark tea increases plasma HDL and reduces plasma triglyceride to decrease atherosclerosis through AMPK activation. Further study is required to identify specific components for the effects of this tea preparation.

The potential effect of silver nanoparticles synthesized with Coffea arabica green seeds on Leishmania major proliferation, cytotoxicity activity, and cytokines expression level.

The goal of this study was to analyze the antileishmanial and antibacterial activity of Coffea arabica green seed biosynthesize silver nanoparticles (C. arabica AgNPs), as well as cytotoxicity and cytokine gene expression. UV-vis spectroscopy, FTIR, and FESEM methods used to examine the C. arabica AgNPs. MTT test was used to assess the antileishmanial and cytotoxicity effects. The gene expression level was assessed in NPs-treated J774 cells by qPCR. The synthesized C. arabica AgNPs were in the size range of 20-70nm, through FESEM pictures. The IC50 values of the NPs were 65. 4 and 47.70μg/mL against promastigotes and amastigotes of Leishmania major, but these values were 580.1 and 171.1μg/mL for Glucantime® as the control drug. C. arabica AgNPs represented a significant increase in IL-12P40, as a Th1 cytokine, in comparison to Glucantime® at high concentrations (P < 0.01), whilst IL-10 expression level showed a significant reduction between NPs-treated and Glucantime®-treated macrophages at 250-1000μg/mL concentrations (P < 0.001). Moreover, the NPs were cytotoxic on cancer cell lines of Hek293, MCF7, and A172 with the CC50 values of 437.2, 116.8, and 72.9µg/mL, respectively. It showed a significant effect of these NPs against A172 (P < 0.001). Also, the lowest MIC values of the NPs were obtained for Bacillus subtilis and Staphylococcus aureus (204µg/mL). According to the antileishmanial, anticancer, and antibacterial activity of these NPs, it can considered a bio-agent drug in the future in endemic countries.

NCOA4 Regulates Iron Recycling and Responds to Hepcidin Activity and Lipopolysaccharide in Macrophages.

Macrophages, via erythrophagocytosis, recycle iron from effete erythrocytes to newly developing red blood cells. Conversion of potentially cytotoxic levels of iron from its heme into nonheme form during iron recycling is safely accomplished via coordinated regulations of cellular iron transport and homeostasis. Herein, we demonstrate the roles and regulation of NCOA4 (nuclear receptor coactivator 4)-mediated ferritinophagy in macrophages after erythrophagocytosis using the mouse macrophage cell line J774 cells. Ferritin in J774 cells increased with the rise of nonheme iron by erythrocyte ingestion and declined when total cellular iron contents subsequently decreased. NCOA4, a selective autophagic cargo receptor for ferritin, was responsible for the control of cellular ferritin and total iron contents at the later stage of erythrophagocytosis. A hepcidin analog, which limits the flux of iron through iron-recycling by inhibiting iron export at the plasma membrane, repressed NCOA4 expression and led to accumulation of ferritin in the mouse macrophages. Transcriptome analyses revealed a functional association of immune response with NCOA4-dependent gene expressions, and we confirmed repression of Ncoa4 by lipopolysaccharide (LPS) in J774 cells and the spleen of mice. Collectively, our studies indicate that NCOA4 facilitates cellular ferritin turnover and the release of iron by macrophages after erythrophagocytosis and functions as a regulatory target for molecular signals of systemic iron overload and inflammation. These identify macrophage NCOA4 as a potential therapeutic target for disorders of systemic iron dysregulation, including anemia of inflammation and hemochromatosis.

Electrochemical Molecule Trap-Based Sensing of Low-Abundance Enzymes in One Living Cell.

Measuring the activity of low-abundance enzymes, down to a few molecules in one living cell, is important but challenging to elucidate their biological function. Here, an electrochemical molecule trap is established at the tip of a nanopipette with an electrochemical detector, in which the diffusion of the molecules away from the electrochemical detector is prevented by electro-osmotic flow (EOF). Accordingly, a limited amount of enzymes is trapped to continuously catalyze the conversion of the substrate to generate a sufficient amount of the byproduct hydrogen peroxide for electrochemical measurements. The resistive pulse sensing of the enzymes in single liposomes validates the detection sensitivity down to 15 molecules. Using this ultrasensitive electrochemical strategy, the activity of 60 sphingomyelinase molecules inside single unstimulated living J774 cells is measured, which was hardly detected by previous methods. The established electrochemical molecule trap-based sensing approach opens the door toward single-molecule electrochemical detection in one living cell. This success will solve the long-standing problem regarding the study of the activity of low-abundance proteins in cells in their native physiological state and greatly enhance the understanding of the roles of proteins in cellular behavior.

Ponciri Fructus Immaturus ethanol extract attenuates septic shock through inhibition of the STAT1 signaling pathway.

Sepsis is a systemic inflammatory disease to infections and results in tissue damage and multiple organ failure. Ponciri Fructus Immaturus (PFI) is widely used in traditional medicine for allergic inflammation and gastrointestinal disorders. However, the effect of PFI on sepsis is still unknown. This study investigated the anti-inflammatory and antiseptic effects of PFI ethanol extract (PFIE) in LPS-stimulated J774 macrophages and mice with CLP- or LPS-induced sepsis, respectively. PFIE attenuates the LPS-induced production of the proinflammatory mediator NO by inhibiting the expression of iNOS in J774 cells. Real-time RT-PCR data and ELISA showed that the mRNA and protein levels of TNF-α, IL-1β, and IL-6 increased in LPS-stimulated J774 cells. However, this induction was significantly suppressed in PFIE pre-treated J774 cells. We also found that PFIE administration increased the survival rate of mice with LPS- and CLP-induced sepsis. Decreased serum levels of AST, ALT, and CK were observed after administration of PFIE, which was associated with reduced production of proinflammatory factors, such as NO, TNF-α, IL-1β, and IL-6. Moreover, PFIE suppressed the phosphorylation and nuclear translocation of STAT1 in LPS-stimulated J774 cells, suggesting that PFIE can inhibit LPS- and CLP-induced septic shock by suppressing the STAT1 activation. These findings provide the potential therapeutic relevance of PFIE in treating acute inflammatory disease.

Antimicrobial activity: potential of Spondias purpurea (Anacardiaceae) against bacterial and fungal species.

Introduction. Plants have been used as medicines for centuries to treat human diseases. Studies with plants are extremely important for the development of future drugs that can benefit the human population.Hypothesis/Gap Statement. With the emergence of pathogens resistant to antimicrobial agents, there is an urgent need to direct research towards the discovery of new antimicrobials.Aim. In this study, Spondias purpurea L. (Anacardiaceae) was evaluated for its antimicrobial activity, antioxidant activity and cytotoxicity.Methodology. Antimicrobial activity was evaluated by the MIC using the 96-well plate microdilution technique of ethanolic, hexanic and dicloromethanic extracts of dried S. purpurea leaves against bacteria, yeast and filamentous fungi. The antioxidant activity of extracts was evaluated by the 2,2-diphenyl-1-picrylhydrazine (DPPH) method. To evaluate the safety of extracts, a cytotoxicity study against HaCat, J774 and HepG2 cells was performed.Results. The extracts had no activity against the bacteria at the maximum concentration of 5.0 mg ml-1, but showed fungistatic action against Candida species and dermatophytes. The ethanolic extract showed 88 % antioxidant activity and showed no significant cytotoxicity against the previously mentioned cells.Conclusion. This study showed that the 100 % ethanolic (EtOH) extract was favourable for antifungal and antioxidant activities and did not present significant cytotoxicity against the three studied cell lines, indicating that S. purpurea leaves are promising for the development of new antimicrobials.

Wharton Jelly Derived Mesenchymal Stem Cell's Exosomes Demonstrate Significant Antileishmanial and Wound Healing Effects in Combination with Aloe-Emodin: An in Vitro Study

The aim of the present study was to explore the antileishmanial performance and wound healing effect of exosomes isolated from Wharton Jelly derived mesenchymal stem cells (WJ-MSCs) in combination with aloe-emodin. MSCs obtained from Wharton Jelly were characterized by flow cytometry. Exosomes were isolated from cultivated stem cells by ultacentrifugation method. Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and flow cytometry were used for characterization of obtained exosomes. The cytotoxicities of characterized exosomes and aloe-emodin at different concentrations were investigated on L929 and J774 cell lines. Non-toxic concentrations of each agent were combined and their inhibitory efficacies on L.major promastigotes and amastigotes were investigated by different techniques such as MTT, parasite count and measurements of infection index. Finally, wound healing activities of combinations were examined on in vitro artifical wound model and compared with the use of exosomes alone. According to outcome of flow cytometic analysis, vesicles isolated from WJ-MSCs highly expressed the markers such as CD63 special for exosome profile. SEM and NTA results demonstrated that derived exosomes possessed dimensions between 150 to 200 nanometers and elicited the cup-shape specific to exosomes. Combinations including non-toxic dosages of exosomes and aloe-emodin demonstrated superior antileishmanial effectivenesses both on promastigotes and amastigotes in contrast to use of exosome alone since they lead to inhibition of promastigotes and amastigotes for 4 and 10-folds in comparison to control, respectively. Additionally, combinations elicited more rapidly and effective in vitro wound-healing performance in contrast to use of exosome alone. At the end of 24 h incubation application of combinations gave rise to wound closure at a rate of 72 %, while in the control group 52 % of wound area has not been healed, yet. These results reflect that mentioned combination has great potential to be used in treatment of cutaneus leishmaniasis (CL) since they have magnificient capacity to inhibit Leishmania parasites while enhancing wound healing.

Prediction of Antileishmanial Compounds: General Model, Preparation, and Evaluation of 2-Acylpyrrole Derivatives.

In this work, the SOFT.PTML tool has been used to pre-process a ChEMBL dataset of pre-clinical assays of antileishmanial compound candidates. A comparative study of different ML algorithms, such as logistic regression (LOGR), support vector machine (SVM), and random forests (RF), has shown that the IFPTML-LOGR model presents excellent values of specificity and sensitivity (81-98%) in training and validation series. The use of this software has been illustrated with a practical case study focused on a series of 28 derivatives of 2-acylpyrroles 5a,b, obtained through a Pd(II)-catalyzed C-H radical acylation of pyrroles. Their in vitro leishmanicidal activity against visceral (L. donovani) and cutaneous (L. amazonensis) leishmaniasis was evaluated finding that compounds 5bc (IC50 = 30.87 μM, SI > 10.17) and 5bd (IC50 = 16.87 μM, SI > 10.67) were approximately 6-fold more selective than the drug of reference (miltefosine) in in vitro assays against L. amazonensis promastigotes. In addition, most of the compounds showed low cytotoxicity, CC50 > 100 μg/mL in J774 cells. Interestingly, the IFPMTL-LOGR model predicts correctly the relative biological activity of these series of acylpyrroles. A computational high-throughput screening (cHTS) study of 2-acylpyrroles 5a,b has been performed calculating >20,700 activity scores vs a large space of 647 assays involving multiple Leishmania species, cell lines, and potential target proteins. Overall, the study demonstrates that the SOFT.PTML all-in-one strategy is useful to obtain IFPTML models in a friendly interface making the work easier and faster than before. The present work also points to 2-acylpyrroles as new lead compounds worthy of further optimization as antileishmanial hits.

Hyaluronic acid-entecavir conjugates-core/lipid-shell nanohybrids for efficient macrophage uptake and hepatotropic prospects

Drug covalently bound to polymers had formed, lately, platforms with great promise in drug delivery. These drug polymer conjugates (DPC) boosted drug loading and controlled medicine release with targeting ability. Herein, the ability of entecavir (E) conjugated to hyaluronic acid (HA) forming the core of vitamin E coated lipid nanohybrids (EE-HA LPH), to target Kupffer cells and hepatocyte had been proved. The drug was associated to HA with efficiency of 93.48 ± 3.14 % and nanohybrids loading of 22.02 ± 2.3 %. DiI labelled lipidic nanohybrids improved the macrophage uptake in J774 cells with a 21 day hepatocytes retention post intramuscular injection. Finally, in vivo biocompatibility and safety with respect to body weight, organs indices and histopathological alterations were demonstrated. Coating with vitamin E and conjugation of E to HA (a CD44 ligand), could give grounds for prospective application for vectored nano-platform in hepatitis B.

Cytotoxic screening and in vitro effect of sodium chlorite against Leishmania major promastigotes.

Cutaneous leishmaniasis (CL) is one of the most important parasitic diseases in the world. Despite the existence of many therapeutic strategies, the treatment of this infection still faces problems. Sodium chlorite as an antimicrobial agent has been shown to have acceptable tissue regenerative and wound healing properties. Therefore, the present study aimed to analyze the in vitro effects of different concentrations of sodium chlorite on Leishmania major promastigotes and macrophage cells. The inhibitory and toxicity effect of various concentrations (0.0035, -1.8mg/ml) of sodium chlorite on the standard Iranian strain of L. major promastigotes were evaluated via counting the cells and flow cytometry. Furthermore, cytotoxicity on promastigotes and J774 macrophage cell line were performed by MTT assay. The results of the inhibitory test demonstrated that sodium chlorite had dose-dependent, anti-leishmanial activities. The half-maximal inhibitory concentration (IC50) for promastigotes and J774 cells by cytotoxicity test was detected at 0.17mg/ml and 0.08mg/ml after 48h respectively. Flow cytometry results showed that 27.34% death of promastigotes was observed in 0.0035mg/ml of sodium chlorite and 78.12% in 1.8mg/ml. The results of the present study showed that sodium chlorite could be used as an effective treatment for CL, especially in cases resistant to treatment with pentavalent compounds. However, the toxicity of this substance in high concentrations should be considered in clinical setting.

Insights into IL-33 on inflammatory response during in vitro infection by Trypanosoma cruzi

Inflammatory and regulatory cytokines play an important role in the immunopathogenesis of Trypanosoma cruzi infection. Interleukin (IL)-33 is a member of the IL-1 superfamily of cytokines whose expression/production is upregulated following pro-inflammatory stimulation to alert the immune system in response to tissue stress or damage. The aim of this study was to evaluate the inflammatory profile induced in cultured J774 cells stimulated or not with IL-33 (10 ng/mL), with live parasites (1 × 106 metacyclic trypomastigote forms) and/or total antigen, TcAg (100 µg/mL) and with both, IL-33 and TcAg/T. cruzi. The cultures were evaluated at 24 h and 48 h after addition of the stimuli. For this, the supernatants were collected for the measurement of TNF, IL-17, CCL2, and IL-10 by ELISA and of nitrite by the Griess method. TNF, IL-17, and CCL2 concentrations were elevated in the presence of TcAg or live T. cruzi parasites at 24 h, and the addition of IL-33 potentiated these effects at 48 h. In addition, the T. cruzi-amastigote forms reduced in those infected J774 cells stimulated with IL-33 at 48 h. In conclusion, the IL-33 elevated the production of the TNF, IL-17, and CCL2 in cultured J774 cells stimulated with T. cruzi and/or its antigen and reduced the intracellular parasites, providing impetus to new investigations on its potential actions on the parasite-induced inflammation.