J774 Macrophage Cell Research Articles

Combining Killed Vaccine Candidate with Different Adjuvants to Determine Prophylactic Potential against Leishmaniasis.

Visceral Leishmaniasis is a serious public health problem caused by Leishmania species parasites. Approximately 500 thousand people get Visceral Leishmaniasis (VL) every year. An effective and reliable vaccine against the disease has still not been formulated. Choosing the right adjuvant is important to increase immunogenicity in vaccines prepared with total antigens. In this study, we investigate the ideal adjuvant for use in vaccine formulations against VL. For this purpose, Leishmania antigens (FTLA) obtained from L. infantum parasites by the freeze-thaw method and three different adjuvants (alum-saponin and calcium phosphate) were used. The effectiveness of the formulations was investigated in vitro by cell viability analysis and determination of nitric oxide and cytokine production abilities in J774 macrophage cells. According to the study results, it was determined that formulations prepared with calcium phosphate produced 72% more NO and approximately 7.2 times more IL-12 cytokine. The results obtained showed that calcium phosphate salts can be used as ideal adjuvants in vaccine research against leishmaniasis.

Synergistic antifungal effect of thiophene derivative as an inhibitor of fluconazole-resistant Candida spp. biofilms.

Candida species resistant to fluconazole have raised concern in the scientific medical community due to high mortality in patients with invasive disease. In developing countries, such as Brazil, fluconazole is the most commonly used antifungal, and alternative treatments are expensive or not readily available. Furthermore, the occurrence of biofilms is common, coupled with their inherent resistance to antifungal therapies and the host's immune system, these microbial communities have contributed to making infections caused by these yeasts an enormous clinical challenge. Therefore, there is an urgent need to develop alternative medicines, which surpass the effectiveness of already used therapies, but which are also effective against biofilms. Therefore, the present study aimed to describe for the first time the antifungal and antibiofilm action of the derivative 2-amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-isopropyl carboxylate (2AT) against clinical strains of Candida spp. resistant to fluconazole (FLZ). When determining the minimum inhibitory concentrations (MIC), it was found that the compound has antifungal action at concentrations of 100 to 200µg/mL, resulting in 100% inhibition of yeast cells. Its synergistic effect with the drug FLZ was also observed. The antibiofilm action of the compound in subinhibitory concentrations was detected, alone and in association with FLZ. Moreover, using scanning electron microscopy, it was observed that the compound 2AT in isolation was capable of causing significant ultrastructural changes in Candida. Additionally, it was also demonstrated that the compound 2AT acts by inducing characteristics compatible with apoptosis in these yeasts, such as chromatin condensation, when visualized by transmission electron microscopy, indicating the possible mechanism of action of this molecule. Furthermore, the compound did not exhibit toxicity in J774 macrophage cells up to a concentration of 4000µg/mL. In this study, we identify the 2AT derivative as a future alternative for invasive candidiasis therapy, in addition, we highlighted the promise of a strategy combined with fluconazole in combating Candida infections, especially in cases of resistant isolates.

Lipopolysaccharide pretreatment increases the sensitivity of the TRPV1 channel and promotes an anti-inflammatory phenotype of capsaicin-activated macrophages

BackgroundThe transient receptor potential vanilloid 1 (TRPV1) is well-established in neuronal function, yet its role in immune reactions remains enigmatic. The conflicting data on its inflammatory role, suggesting both pro-inflammatory and anti-inflammatory effects upon TRPV1 stimulation in immune cells, adds complexity. To unravel TRPV1 immunomodulatory mechanisms, we investigated how the TRPV1 agonist capsaicin influences lipopolysaccharide (LPS)-induced pro-inflammatory macrophage phenotypes.ResultsChanges in the surface molecules, cytokine production, and signaling cascades linked to the phenotype of M1 or M2 macrophages of the J774 macrophage cell line and bone marrow-derived macrophages, treated with capsaicin before or after the LPS-induced inflammatory reaction were determined. The functional capacity of macrophages was also assessed by infecting the stimulated macrophages with the intracellular parasite Leishmania mexicana.ConclusionOur findings reveal that TRPV1 activation yields distinct macrophage responses influenced by the inflammatory context. LPS pre-treatment followed by capsaicin activation prompted increased calcium influx, accompanied by a shift toward an anti-inflammatory M2b-like polarization state.

Anti-Toxoplasma In Vitro and In Vivo Activity of Pyrus boissieriana Arbutin-Rich Fraction.

Pyrus boissieriana is a rich source of arbutin and has been used in herbal medicine to treat infectious diseases. This study aimed to investigate the effect of the arbutin-rich fraction of Pyrus boissieriana aerial parts on Toxoplasma gondii In Vitro and In Vivo. An arbutin-rich fraction of P. boissieriana was prepared beforehand. Flow cytometry was used to evaluate the effect of different concentrations (1-512µg/ml) of the P. boissieriana arbutin-rich fraction on Toxoplasma tachyzoites (RH strain). The cytotoxicity of the concentrations on the macrophage J774 cell line was also investigated by MTT assay. For In Vivo investigation, 4-6-week-old female mice infected with the RH strain of T. gondii were treated with different doses (16, 32, 64, 256, and 512mg/kg) of the fraction using gavage. The highest and lowest lethality of the tachyzoites were 89.6% and 25.9% related to the concentrations of 512µg/ml and 1µg/ml, respectively, with an IC50 value of 18.1µg/ml ± 0.37. The cytotoxicity test showed an IC50 value of 984.3µg/ml ± 0.76 after 48h incubation. The mean survival of mice at the lowest treated dose (16mg/kg) was 6.6days, and it was 15days at the highest dose (512mg/kg). The concentrations of 512, 256, 128, and 64mg/kg of the fraction compared to the negative control (6.2days mean survival) significantly increased the survival time of mice (P < 0.001, P = 0.009, P = 0.018, and P = 0.021, respectively). The results showed that the arbutin-rich fraction of P. boissieriana is effective against T. gondii In Vitro and In Vivo and may be a reliable alternative to conventional treatment for toxoplasmosis, although further studies are necessary.

Evaluating leishmanicidal effects of Lucilia sericata products in combination with Apis mellifera honey using an in vitro model.

Leishmaniasis is a zoonotic disease caused by an intracellular parasite from the genus Leishmania. Lack of safe and effective drugs has increasingly promoted researches into new drugs of natural origin to cure the disease. The study, therefore, aimed to investigate the anti-leishmanial effects of Lucilia sericata larval excretion/secretion (ES) in combination with Apis mellifera honey as a synergist on Leishmania major using an in vitro model. Various concentrations of honey and larval ES fractions were tested against promastigotes and intracellular amastigotes of L. major using macrophage J774A.1 cell line. The inhibitory effects and cytotoxicity of ES plus honey were evaluated using direct counting method and MTT assay. To assess the effects of larval ES plus honey on the amastigote form, the rate of macrophage infection and the number of amastigotes per infected macrophage cell were estimated. The 50% inhibitory concentration (IC50) values were 21.66 μg/ml, 43.25 60 μg/ml, 52.58 μg/ml, and 70.38 μg/ml for crude ES plus honey, ES >10 kDa plus honey, ES <10 kDa plus honey, and honey alone, respectively. The IC50 for positive control (glucantime) was 27.03 μg/ml. There was a significant difference between viability percentages of promastigotes exposed to different doses of applied treatments compared to the negative control (p≤ 0.0001). Microscopic examination of amastigote forms revealed that dosages applied at 150 to 300 μg/ml significantly reduced the rate of macrophage infection and the number of amastigotes per infected macrophage cell. Different doses of larval products plus honey did not show a significant toxic effect agaist macrophage J774 cells. The larval ES fractions of L. sericata in combination with A. mellifera honey acted synergistically against L. major.

Set up and validation of a sensitive method to quantify prostaglandins, prostaglandin-glycerol esters and prostaglandin-ethanolamides, as well as their respective precursors

Arachidonic acid-derived prostaglandins are widely studied for their role in inflammation. However, besides arachidonic acid, other arachidonic moiety-containing lipids can be metabolized by COX-2. Indeed, the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide, AEA) can follow the same biochemical pathways than arachidonic acid leading to the formation of prostaglandin-glycerol esters (PG-G) and prostaglandin-ethanolamides (or prostamides, PG-EA), respectively.The data reported so far support the interest of these bioactive lipids in inflammatory conditions. However, there is only a handful of methods described for their quantification in biological matrices. Moreover, given the shared biochemical pathways for arachidonic acid, 2-AG and AEA, a method allowing for the quantification of these precursors and the corresponding prostaglandin derivatives appears as largely needed. Thus, we report here the development and validation of a single run UPLC-MS/MS quantification method allowing the quantification of these endocannabinoids-derived mediators together with the classical prostaglandin. Moreover, we applied the method to the quantification of these lipids in vitro (using lipopolysaccharides-activated J774 macrophage cells) and in vivo in several tissues from DSS-induced colitis mice. This femtomole-range method should improve the understanding of the interaction between these lipid mediators and inflammation.

Scavenger receptors mediate increased uptake of irradiated T.gondii extracts by J774 macrophages.

ABSTRACT PurposeProtein extracts developed increased immunogenicity without the aid of adjuvants after gamma irradiation. Gamma irradiation of snake venom increased antivenin production by detoxification and enhanced immunity, probably due preferential uptake of irradiated venoms by macrophage scavenger receptors. We studied this uptake of irradiated soluble Toxoplasma gondii extract (STag) by the J774 macrophage cell line similar to antigen presenting cells. Material and Methods: We labeled STag by biosynthesis in living tachyzoites with radioactive amino acids before purification and irradiation or by adding labels as biotin or fluorescein in stored STag, for quantitative studies or subcellular distribution visualization. Results: There was enhanced binding and uptake of irradiated STag into the cells compared to non-irradiated STag. Using fluorescein labelled antigens and morphological assays, we confirmed that cells avidly ingested both native and irradiated proteins but native STag were digested after ingestion while irradiated proteins remained in the cell, suggesting diverse intracytoplasmic pathways. Native or irradiated STag present the same in vitro sensitivity to three types of peptidases. Inhibitors of scavenger receptors (SRs) such as Dextran sulfate (SR-A1 blocker) or Probucol (SR-B blocker) affect the specific uptake of irradiated antigens, suggesting its association with enhanced immunity. Conclusions: Our data suggests that cell SRs recognize irradiated proteins, mainly SRs for oxidized proteins, leading to antigen uptake by an intracytoplasmic pathway with fewer peptidases that prolongs presentation to nascent major histocompatibility complex I or II and enhances immunity by better antigen presentation.

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.

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.

Absolute Configuration and Antileishmanial Activity of (-)-Cyclocolorenone Isolated from Duguetia lanceolata (Annonaceae).

The fractionation of the n-hexane phase of the EtOH extract from the leaves of Duguetia lanceolata (Annonaceae) led to the identification of the sesquiterpene (-)-cyclocolorenone. Chemical characterization, including determination of the absolute stereochemistry, and in vitro evaluation of antileishmanial activity of the sesquiterpene (-)-cyclocolorenone, isolated from D. lanceolata, were carried out. (-)-Cyclocolorenone was isolated from D. lanceolata leaves using different chromatographic steps and its structure was defined by analysis of NMR and ESI-HRMS data. Additionally, the absolute configuration of (-)-cyclocolorenone was ambiguously assigned by means of vibrational circular dichroism (VCD). Antileishmanial activity of (-)-cyclocolorenone was evaluated on promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. The integrity of the cell membrane of L. (L.) amazonensis was analyzed using the SYTOX green probe. (-)-(1R,6S,7R,10R)-Cyclocolorenone displayed activity against promastigotes and amastigotes forms of L. (L.) amazonensis with IC50 of 4.54 and 28.44 µM, respectively. Furthermore, this compound was non-toxic in J774 macrophage cells (CC50 > 458.71 µM) with a selectivity index > 100 (promastigotes) and > 32.2 (amastigotes). Additionally, (-)-cyclocolorenone was observed to target the parasite cell membrane. Obtained data suggested that (-)-cyclocolorenone, in which absolute configuration was determined, can be considered as a scaffold for the development of new drugs for the treatment of leishmaniasis.

Farklı Antijen Hazırlama Metotları İle Hazırlanan İnsan Meme Kanseri Hücre Lizatlarının Adjuvan Kombinasyonları İle İmmünostimülan/Sitotoksik Aktivitelerinin İncelenmesi

In last decades, immunotherapies became more and more significant as a cancer treatment method. Many in vivo and in vitro studies have been conducted in the literature in order to determine the appropriate diagnostic and treatment methods for cancer. During the development of therapeutic vaccines, determination of vaccine concentration which is the beginning step of in vivo studies, is critical for the healthy progression of the studies. Breast cancer which occurs by uncontrolled proliferation as a result of exposure of healthy cells in breast tissue to factors such as DNA damage, UV, radiation, carcinogens and, various chemicals has a huge portion among the cancer types. In this study, for the first time, antigens generated by two different antigen preparation methods (autoclaving, freezing-thawing) using AU-565 / MCF-7 cell lines. Then, antigens derived from human breast cancer cell lines were applied alone and with different adjuvants (Polyoxidonium, Freund’s) to the J774 macrophage cell culture system and the L929 fibroblast cell line in order to investigate immunostimulant activity and cytotoxicity via Griess reagent and MTT test respectively. At the end of the 48th hour, the cell viability well will be examined and the most appropriate antigen-adjuvant combination and its concentration for vaccine preparation were determined. Among the antigen preparation methods, it was determined that the freeze-thaw method was more effective than the autoclave method, and the antigens prepared by the freeze-thaw method from the AU-565 and MCF-7 cell lines showed the highest immunostimulatory activity at a concentration of 160 µg/ml. Among the adjuvants, the immunostimulant effect of Freund's adjuvant was found to be higher. In the light of these results, the optimal vaccine concentration against breast cancer and the viability analysis of these concentrations were determined. All in all, it is thought that antibodies obtained from in vivo studies using the most appropriate antigen preparation method and the most appropriate vaccine concentration can be used in the diagnosis and treatment of breast cancer.

A Genomic Island of Vibrio cholerae Encodes a Three-Component Cytotoxin with Monomer and Protomer Forms Structurally Similar to Alpha-Pore-Forming Toxins.

Alpha-pore-forming toxins (α-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for α-PFTs, the membrane-spanning channel they form is composed of hydrophobic α-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent α-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an α-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential α-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the α-PFT family in the soluble state, and we modeled their protomer state based on the α-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are α-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells. IMPORTANCE The bacterium Vibrio cholerae causes gastrointestinal, wound, and skin infections. The motility-associated killing factor A (MakA) was recently shown to be cytotoxic against colon, prostate, and other cancer cells. However, at the outset of this study, the capacity of MakA to damage cells in combination with other Mak proteins encoded in the same operon had not been elucidated. We determined the structures of three Mak proteins and established that they are structurally related to the α-PFTs. Compared to MakA alone, the combination of all three toxins was more potent specifically in mouse macrophages. This study highlights the idea that the Mak toxins are selectively cytotoxic and thus may function as a tripartite toxin with cell type specificity.

Cannabinoids inhibit oxLDL internalization in J774 macrophage cell line

Cannabinoids inhibit oxLDL internalization in J774 macrophage cell line

Dereplication and Quantification of Major Compounds of Convolvulus arvensis L. Extracts and Assessment of Their Effect on LPS-Activated J774 Macrophages.

Convolvulus arvensis is used in Pakistani traditional medicine to treat inflammation-related disorders. Its anti-inflammatory potential was evaluated on hexane, dichloromethane, ethyl acetate, methanol, and aqueous extracts of whole plant on pro-inflammatory mediators in LPS-activated murine macrophage J774 cells at the non-cytotoxic concentration of 50 µg/mL. Ethyl acetate (ARE) and methanol (ARM) extracts significantly decreased mRNA levels of IL-6, TNF-α, MCP-1, COX-2, and iNOS. Furthermore, both extracts dose dependently decreased IL-6, TNF-α, and MCP-1 secretion. Forty-five compounds were putatively identified in ARE and ARM by dereplication (using HPLC-UV-HRMSn analysis and molecular networking), most of them are reported for the first time in C. arvensis, as for example, nineteen phenolic derivatives. Rutin, kaempferol-3-O-rutinoside, chlorogenic acid, 3,5-di-O-caffeoylquinic acid, N-trans-p-coumaroyl-tyramine, and N-trans-feruloyl-tyramine were main constituents identified and quantified by HPLC-PDA in ARE and ARM. Furthermore, chlorogenic acid, tyramine derivatives, and the mixture of the six identified major compounds significantly decreased IL-6 secretion by LPS-activated J774 cells. The activity of N-trans-p-coumaroyl-tyramine is shown here for the first time. Our results indicate that ARE, ARM and major constituents significantly inhibited the expression of pro-inflammatory mediators, which supports the use of this plant to treat inflammatory diseases.

Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios.

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

Abstract MP30: Excess HDL Free Cholesterol Bioavailability Drives Free Cholesterol Accretion Into Macrophages And Erythrocytes In Scarb1 -/- Mice

Aim: In humans, very high plasma HDL-cholesterol concentrations are associated with increased all cause- and atherosclerotic cardiovascular disease (ASCVD)-mortality. The HDL receptor-deficient mouse (Scarb1 -/- ), a robust model of this phenotype, is characterized by high free cholesterol (FC) bioavailability due to too many HDL particles that are FC-rich. Clinically, plasma LDL and HDL are quantified according to total cholesterol content, the sum of FC and esterified cholesterol, which likely contribute to ASCVD pathophysiology differently. A Western diet induces ASCVD in Scarb1 -/- mice, despite an attendant increase in HDL. We tested the hypothesis that high HDL-FC bioavailability contributes to ASCVD in Scarb1 -/- mice by increasing FC flux into macrophage cells, erythrocytes and other major tissues. Methods: Influx of HDL-FC and efflux of macrophage FC were determined between WT and Scarb1 -/- HDL and J774 macrophage cells. HDL of both genotypes were radiolabelled with [ 3 H]FC, injected into autologous mice, and the rates of plasma clearance and erythrocyte uptake were determined. Results: The magnitude of FC transfer from Scarb1 -/- HDL to LDL is greater than that from WT HDL; APOB-containing lipoproteins from Scarb1 -/- vs. WT mice are FC-enriched due likely to greater HDL-FC transfer. While macrophage efflux to HDL of Scarb1 -/- vs. WT HDL was not different, FC influx from Scarb1 -/- vs. WT HDL to macrophages was three-fold greater, a net effect that increased the FC burden of macrophages. In vivo studies showed that compared to WT mice, in Scarb1 -/- mice, autologous HDL-FC cleared more slowly and more FC transferred to erythrocytes. We compared the FC, CE, PL, and TG contents of all major tissues and determined that FC accretion by some tissues is higher among Scarb1 -/- vs. WT mice whereas in other tissues FC homeostasis is maintained. Lastly, we determined that the tissue compositions and plasma FC clearance kinetics varied according to sex, particularly among Scarb1 -/- mice. Conclusions: These findings are relevant to pathologies specific to Scarb1 -/- mice and to the evolving model of the role of HDL-FC in RCT. They provide a rationale for human studies to determine the utility of HDL-FC bioavailability as a risk factor for ASCVD and other pathologies.

In vitro effect of artemether-loaded nanostructured lipid carrier (NLC) on Leishmania infantum.

Visceral leishmaniasis (VL) is an acute and deadly form of leishmaniasis, caused by Leishmania infantum parasite. Due to the toxicity and side effects of conventional treatment options, such as glucantime and other pentavalent drugs, finding novel drugs with fewer adverse effects is required. Artemether (ART), is one of the derivatives of artemisinin, which was shown to be effective in treating malaria and more recently, leishmaniasis. In this fundamental-applied research, we compared the effect of ART and nanostructure loaded with artemether (NLC-ART) on Leishmania infantum promastigotes and amastigotes, at different concentrations (2.5-5-10-25-50-100μg/ml) using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method after 24 and 48h of treatment. Inhibitory concentration (IC50) values (μg/ml) of promastigote and amastigote of L. infantum to ART/ NLC-ART, after 48h of treatment, were found to be 37.12 / 32.1 and 16.43 / 15.42, respectively. Moreover, we found that (NLC-ART), had the lowest cytotoxicity against the J774 macrophage cell line. Conclusion: The NLC-ART can be a good candidate for the treatment of visceral leishmaniasis.

Nanostructured lipid carrier-mediated lung targeted drug delivery system to enhance the safety and bioavailability of clofazimine

Tuberculosis (TB) disease is caused due to the infection of Mycobacterium tuberculosis bacilli which reside in alveolar macrophages (AMs). Clofazimine (CLF) has been reinstated clinically for the treatment of TB. However, major challenge of using CLF is its severe side-effects after oral administration. The present research was aimed to establish the safety and enhance the bioavailability of CLF by loading it into nanostructured lipid carriers (CLF-NLCs) and mannosylated NLCs (M-CLF-NLCs) to selectively target the drug toward AMs. The safety of CLF-NLCs and M-CLF-NLCs was evaluated by in vitro hemocompatibility studies, cell viability studies on macrophage J774 cell lines, and in vivo acute inhalation toxicity studies. The bioavailability was estimated by single-dose pharmacokinetics and biodistribution studies. Hemocompatibility studies showed normal RBCs count and least hemolysis of 0.23 ± 0.081% for M-CLF-NLCs treated group. Cell viability studies revealed greater safety of NLCs than CLF-drug dispersion in the concentration range of 2.5–25 μg/ml. In vivo acute toxicity studies revealed no physiological or behavioral changes and no mortality recorded over 14 days period. In pharmacokinetic studies, a maximum concentration of the drug (C max) of 35.44 ± 0.34 μg/g from M-CLF-NLCs after 48 h and longer residence time in lung tissues observed due to its sustained release and mannose receptor-mediated endocytosis. M-CLF-NLCs showed a maximum AUC0–∞ value of 2691.83 h μg/ml in lungs that indicated twofold greater bioavailability as compared to CLF-drug dispersion. Thus, mannosylated NLCs can be used as promising carriers for the safe and effective delivery of CLF via inhalation route for the management of TB disease.

E17241 as a Novel ABCA1 (ATP-Binding Cassette Transporter A1) Upregulator Ameliorates Atherosclerosis in Mice.

[Figure: see text].

The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses

ABSTRACT Host engulfment protein ELMO1 generates intestinal inflammation following internalization of enteric bacteria. In Shigella, bacterial effector IpgB1 interacts with ELMO1 and promotes bacterial invasion. IpgB1 belongs to the WxxxE effector family, a motif found in several effectors of enteric pathogens. Here, we have studied the role of WxxxE effectors, with emphasis on Salmonella SifA and whether it interacts with ELMO1 to regulate inflammation. In-silico-analysis of WxxxE effectors was performed using BLAST search and Clustal W program. The interaction of ELMO1 with SifA was assessed by GST pulldown assay and co-immunoprecipitation. ELMO1 knockout mice, and ELMO1-depleted murine macrophage J774 cell lines were challenged with WT and SifA mutant Salmonella. Bacterial effectors containing the WxxxE motif were transfected in WT and ELMO1-depleted J774 cells to assess the inflammatory cytokines. ELMO1 generates differential pro-inflammatory cytokines between pathogenic and nonpathogenic bacteria. WxxxE motif is present in pathogens and in the TIR domain of host proteins. The C-terminal part of ELMO1 interacts with SifA where WxxxE motif is important for interaction. ELMO1–SifA interaction affects bacterial colonization, dissemination, and inflammatory cytokines in vivo. Moreover, ELMO1–SifA interaction increases TNF-α and IL-6 production from the macrophage cell line and is associated with enhanced Rac1 activity. ELMO1 also interacts with WxxxE effectors IpgB1, IpgB2, and Map and induces inflammation after challenge with microbes or microbial ligands. ELMO1 generates a differential response through interaction with the WxxxE motif, which is absent in commensals. ELMO1-WxxxE interaction plays a role in bacterial pathogenesis and induction of inflammatory response.