Macrophage Toxicity Research Articles

Multiplex antimicrobial activities of the self-assembled amphiphilic polypeptide β nanofiber KF-5 against vaginal pathogens

BackgroundVaginal infections caused by multidrug-resistant pathogens such as Candida albicans and Gardnerella spp. represent a significant health challenge. Current treatments often fail because of resistance and toxicity. This study aimed to synthesize and characterize a novel amphiphilic polypeptide, KF-5, and evaluate its antibacterial and antifungal activities, biocompatibility, and potential mechanisms of action.ResultsThe KF-5 peptide was synthesized via solid-phase peptide synthesis and self-assembled into nanostructures with filamentous and hydrogel-like configurations. Characterization by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) confirmed the unique nanostructural properties of KF-5. KF-5 (125, 250, or 500 µg/ml) demonstrated potent antibacterial and antifungal activities, with significant inhibitory effects on drug-resistant Candida albicans and Gardnerella spp. (P < 0.05). In vitro assays revealed that 500 µg/ml KF-5 disrupted microbial cell membranes, increased membrane permeability, and induced lipid oxidation, leading to cell death (P < 0.05). Cytotoxicity tests revealed minimal toxicity in human vaginal epithelial cells, keratinocytes, and macrophages, with over 95% viability at high concentrations. Molecular dynamics simulations indicated that KF-5 interacts with phospholipid bilayers through electrostatic interactions, causing membrane disruption. In vivo studies using a mouse model of vaginal infection revealed that 0.5, 1, and 2 mg/ml KF-5 significantly reduced fungal burden and inflammation, and histological analysis confirmed the restoration of vaginal mucosal integrity (P < 0.01). Compared with conventional antifungal treatments such as miconazole, KF-5 exhibited superior efficacy (P < 0.01).ConclusionsKF-5 demonstrates significant potential as a safe and effective antimicrobial agent for treating vaginal infections. Its ability to disrupt microbial membranes while maintaining biocompatibility with human cells highlights its potential for clinical application. These findings provide a foundation for further development of KF-5 as a therapeutic option for combating drug-resistant infections.

Chemical screens for particle-induced macrophage death identifies kinase inhibitors of phagocytosis as targets for toxicity

BackgroundNanoparticles are increasingly being used in medicine, cosmetics, food, and manufacturing. However, potential toxicity may limit the use of newly engineered nanoparticles. Prior studies have identified particle characteristics that are predictive of toxicity, although the mechanisms responsible for toxicity remain largely unknown. Nanoparticle treatment in cell culture, combined with high-throughput chemical screen allows for systematic perturbations of thousands of molecular targets against potential pathways of toxicity. The resulting data matrix, called chemical compendium, can provide insights into the mechanism of toxicity as well as help classify nanoparticles based on toxicity pathway.ResultsWe performed unbiased screens of 1280 bioactive chemicals against a panel of four particles, searching for inhibitors of macrophage toxicity. Our hit compounds clustered upon inhibitors of kinases involved in phagocytosis, including focal adhesion kinase (FAK), with varying specificity depending on particles. Interestingly, known inhibitors of cell death including NLRP3 inflammasome inhibitor were unable to suppress particle-induced macrophage death for many of the particles. By searching for upstream receptors of kinases, we identified Cd11b as one of the receptors involved in recognizing a subset of particles. We subsequently used these hit compounds and antibodies to further characterize a larger panel of particles and identified hydrodynamic size as an important particle characteristic in Cd11b-mediated particle uptake and toxicity.ConclusionsOur chemical compendium and workflow can be expanded across cell types and assays to characterize the toxicity mechanism of newly engineered nanoparticles. The data in their current form can also be analyzed to help design future high-throughput screening for nanoparticle toxicity.Graphical abstract

Discovery of 8-hydroxy-2-quinoline carbaldehyde derivatives as inhibitors for M1 aminopeptidase of Leishmania donovani

M1 aminopeptidase is a metallopeptidase that plays a vital role in protein catabolism and has been identified as a validated drug target in various parasites; however, our understanding of this enzyme is restricted for leishmanial parasite. The present investigation involved the purification of Leishmania donovani M1 aminopeptidase (LdM1AP) to homogeneity by affinity chromatography. Purified LdM1AP was observed to be enzymatically active and displayed maximal activity in the presence of cobalt ions, whereas secondary structure analysis confirmed the dominance of α-helices. Intrinsic fluorescence and quenching studies of LdM1AP has revealed that tryptophan residues were predominantly concealed within the hydrophobic areas. The synthesized 8-hydroxy-2-quinoline carbaldehyde derivatives were screened, wherein HQ2 and HQ12 were found as potent inhibitors for LdM1AP that compete with the substrate and exhibit pharmacokinetic properties as well as no toxicity for macrophages. Moreover, structural insights of protein and ligand complexes demonstrated that lead compounds mostly interact via hydrophobic contacts into the substrate binding pocket of LdM1AP. Furthermore, lead compounds exhibited a greater affinity for LdM1AP compared to the substrate during in vitro and in silico studies. This report establishes the possibility of quinoline derivatives to target the LdM1AP activity and provide a platform to design the specific antileishmanial drugs.

Phenotypic and Functional Outcomes in Macrophages Exposed to an Environmentally Relevant Mixture of Organophosphate Esters in Vitro.

Organophosphate esters (OPEs) are flame retardants and plasticizers used in consumer products. OPEs are found ubiquitously throughout the environment with high concentrations in indoor house dust. Exposure to individual OPEs is associated with immune dysfunction, particularly in macrophages. However, OPEs exist as complex mixtures and the effects of environmentally relevant mixtures on the immune system have not been investigated. The objectives of this study were to evaluate the toxicity of an environmentally relevant mixture of OPEs that models Canadian house dust on macrophages using phenotypic and functional assessments in vitro. High-content live-cell fluorescent imaging for phenotypic biomarkers of toxicity in THP-1 macrophages treated with the OPE mixture was undertaken. We used confocal microscopy and cholesterol analysis to validate and expand on the observed OPE-induced lipid phenotype. Then, we used flow cytometry and live-cell imaging to conduct functional tests and uncover mechanisms of OPE-induced phagocytic suppression. Finally, we validated our THP-1 findings in human primary peripheral blood mononuclear cells (hPBMC) derived macrophages. Exposure to non-cytotoxic dilutions of the OPE mixture resulted in higher oxidative stress and disrupted lysosome and lipid homeostasis in THP-1 and primary macrophages. We further observed that phagocytosis of apoptotic cells in THP-1 and primary macrophages was lower in OPE-exposed cells vs. controls. In THP-1 macrophages, phagocytosis of both Gram-positive and Gram-negative bacteria was also lower in OPE-exposed cells vs. controls. Additionally, the OPE mixture altered the expression of phagocytic receptors linked to the recognition of phosphatidylserine and pathogen-associated molecular patterns. The results of this in vitro study suggested that exposure to an environmentally relevant mixture of OPEs resulted in higher lipid retention in macrophages and poor efferocytic response. These effects could translate to enhanced foam cell generation resulting in higher cardiovascular mortality. Furthermore, bacterial phagocytosis was lower in OPE-exposed macrophages in an in vitro setting, which may indicate the potential for reduced bacterial clearance in models of infections. Taken together, our data provide strong evidence that mixtures of OPEs can influence the biology of macrophages and offer new mechanistic insights into the impact of OPE mixtures on the immune system. https://doi.org/10.1289/EHP13869.

Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

The influence of cation exchange on the possible mechanism of erionite toxicity: A synchrotron-based micro-X-ray fluorescence study on THP-1-derived macrophages exposed to erionite-Na

Fibrous erionite is the only zeolite classified as Group 1 carcinogen by the International Agency for Research on Cancer (IARC). Carcinogenesis induced by erionite is thought to involve several factors as biopersistence, the iron role and cation exchange processes. To better understand these mechanisms, a detailed investigation at the micro scale was performed, collecting elemental information on iron and cation release and their distribution in biological systems by synchrotron micro-X-ray fluorescence mapping (SR-micro-XRF) and synchrotron micro-X-ray absorption spectroscopy (SR-micro-XANES) at the TwinMic beamline (Elettra synchrotron) and at the ID21 beamline of the European Synchrotron Radiation Facility (ESRF). By microscopy and chemical mapping, highly detailed maps of the chemical and morphological interaction of biological systems with fibres could be produced. In detail, THP-1 cell line derived macrophages, used as in vitro model, were analysed during erionite-Na phagocytosis at different time intervals, after single dose exposure. For comparison, cellular fluorescent probes were also used to evaluate the intracellular free sodium and calcium concentrations. Synchrotron analyses visualised the spatial distribution of both fibre and mineral particle associated metals during the phagocytosis, describing the mechanism of internalisation of erionite-Na and its accessory mineral phases. The intracellular distribution of metals and other cations was mapped to evaluate metal release, speciation changes and/or cation exchange during phagocytosis. The fluorescent probes complemented microchemical data clarifying, and confirming, the cation distribution observed in the SR-micro-XRF maps. The significant cytoplasmic calcium decrease, and the concomitant sodium increase, after the fibre phagocytosis seemed due to activation of plasma membrane cations exchangers triggered by the internalisation while, surprisingly, the ion-exchange capacity of erionite-Na could play a minor role in the disruption of the two cations intracellular homeostasis. These results help to elucidate the role of cations in the toxicity of erionite-treated THP-1 macrophages and add knowledge to its carcinogenicity process.

The role of transcriptional regulators in metal ion homeostasis of Mycobacterium tuberculosis.

Metal ions are essential trace elements for all living organisms and play critical catalytic, structural, and allosteric roles in many enzymes and transcription factors. Mycobacterium tuberculosis (MTB), as an intracellular pathogen, is usually found in host macrophages, where the bacterium can survive and replicate. One of the reasons why Tuberculosis (TB) is so difficult to eradicate is the continuous adaptation of its pathogen. It is capable of adapting to a wide range of harsh environmental stresses, including metal ion toxicity in the host macrophages. Altering the concentration of metal ions is the common host strategy to limit MTB replication and persistence. This review mainly focuses on transcriptional regulatory proteins in MTB that are involved in the regulation of metal ions such as iron, copper and zinc. The aim is to offer novel insights and strategies for screening targets for TB treatment, as well as for the development and design of new therapeutic interventions.

Potencial terapêutico da hesperidina metil chalcona no tratamento experimental da leishmaniose cutânea

ABSTRACT Drug repositioning seeks to discover new applications for a drug that has already been approved in the market, resulting in faster and lower cost solutions, generally used for diseases that receive little investment, such as cutaneous leishmaniasis. In this context, hesperidin, commercially approved as Daflon®, is a flavonoid that belongs to the chalcones group, a class that has antileishmanial potential. The present study evaluated the in vitro and in vivo antileishmanial activity of commercial hesperidin. Hesperidin (9 to 0.56 mg mL-1) was tested against promastigote and amastigote forms of four dermotropic species of Leishmania, namely L. (L.) amazonensis, L. (V.) guyanensis, L. (V.) braziliensis and L. (V.) naiffi. For the in vivo tests, hamsters were infected in the snout and the lesions were treated with intralesional hesperidin. The treatment effectiveness was assessed by measuring the total volume of the lesion on the snout and determining the parasitic load. The in vitro results showed moderate toxicity in murine macrophages, with higher efficacy in L. (L.) amazonensis when compared to the other species tested. The in vivo results showed that hesperidin was able to gradually reduce the size of lesions by L. (L.) amazonensis, although it did not induce clinical and parasitological cure. Thus, hesperidin showed potential in in vitro tests against L. (L.) amazonensis and further studies with new formulations and experimental treatment schemes should be carried out.

Understanding the pathogenesis of engineered stone-associated silicosis: The effect of particle chemistry on the lung cell response.

The resurgence of severe and progressive silicosis among engineered stone benchtop industry workers is a global health crisis. We investigated the link between the physico-chemical characteristics of engineered stone dust and lung cell responses to understand components that pose the greatest risk. Respirable dust from 50 resin-based engineered stones, 3 natural stones and 2 non-resin-based materials was generated and analysed for mineralogy, morphology, metals, resin, particle size and charge. Human alveolar epithelial cells and macrophages were exposed in vitro to dust and assessed for cytotoxicity and inflammation. Principal component analysis and stepwise linear regression were used to explore the relationship between engineered stone components and the cellular response. Cutting engineered stone generated fine particles of <600 nm. Crystalline silica was the main component with metal elements such as Ti, Cu, Co and Fe also present. There was some evidence to suggest differences in cytotoxicity (p = 0.061) and IL-6 (p = 0.084) between dust samples. However, IL-8 (CXCL8) and TNF-α levels in macrophages were clearly variable (p < 0.05). Quartz explained 11% of the variance (p = 0.019) in macrophage inflammation while Co and Al accounted for 32% of the variance (p < 0.001) in macrophage toxicity, suggesting that crystalline silica only partly explains the cell response. Two of the reduced-silica, non-engineered stone products induced considerable inflammation in macrophages. These data suggest that silica is not the only component of concern in these products, highlighting the caution required as alternative materials are produced in an effort to reduce disease risk.

Leishmanicidal and healing effects of 3β,6β,16β-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo.

Treatment of cutaneous leishmaniasis depends on drugs that potentially cause serious side effects and resistance. Thus, topical therapies are attractive alternatives to the drugs currently used. 3β, 6β, 16β-trihydroxylup-20 (29)-ene is a lupane triterpene isolated from Combretum leprosum Mart. leaves (CLF-1), with reports of in vitro antileishmanial effect against L. amazonensis and to promote lesion healing in animal model. Herein, we evaluated the in vitro and in vivo antileishmanial and healing effects of CLF-1 against L. braziliensis. CLF-1 treatment showed low toxicity in macrophages and significantly reduced parasite load in vitro. CLF-1 induced higher IL-12 and TNF-α production and more discrete IL-4 and IL-10 production. For in vivo evaluation, a CLF-1 cream formulation was prepared to treat hamsters infected with L. braziliensis. CLF-1 treatment was able to reduce parasite load of the infected skin and lymph node more efficiently than the conventional treatment. Histopathological analysis indicated a strong inflammatory response accompanied by an important healing response. Data from this study indicate that topical CLF-1 treatment was effective and non-toxic in L. braziliensis infected hamsters suggesting its potential for further development as a future therapeutic intervention.

An in vitro study of the antileishmanial and cytotoxic activity of extracts of Sclerolobium paniculatum (Fabaceae)

Leishmaniasis is a neglected endemic disease and the pharmacotherapy indicated is expensive, in addition to the unwanted effects generated by the drugs, resulting in low adherence to treatment. Therefore, the search for therapeutic alternatives includes the use of medicinal plants, such as Sclerolobium paniculatum Vogel, which has antioxidant, healing and antifungal properties. Therefore, the aim of this study was to evaluate the in vitro antileishmanial activity of the methanolic and hexanic extracts of S. paniculatum against the promastigote forms of Leishmania (L.) amazonensis and Leishmania (V.) guyanensis and their toxicity in J774 macrophages. As a result, the hexanic extract was considered active against L. guyanensis with an IC50 of 40 μg mL-1 over 48 hours, with no significant activity for L. amazonensis with an IC50 above 100 μg mL-1 over 24 and 48 hours. The methanolic extract showed moderate activity against L. guyanensis, with an IC50 of 82 μg mL-1 over 48 hours and an IC50 of 67 μg mL-1 over 48 hours against L. amazonensis, making it moderately active. Both extracts did not present cytotoxic profiles at any of the concentrations, being recommended for future research with other species of Leishmania sp. to optimize the treatment of cutaneous leishmaniasis.

Translational characterization of macrophage responses to stable and non-stable CoCrMo wear and corrosion debris generated in-situ for total hip replacement

Metal wear and corrosion debris remain a limiting factor for long-term durability of total hip replacement (THR). Common wear particle production techniques for research differ from the actual tribocorrosion processes at the implant site, potentially causing loss of valuable information. The aim of this study was to investigate reactions to freshly generated and time-stabilized particles and ions released from CoCrMo-alloy using a bio-tribometer, which mimics conditions of the periprosthetic environment.THP-1 macrophages were challenged with freshly produced or time-stabilized wear debris. Wear generation took place in a custom-built bio-tribometer inside a CO2 incubator operating with a reciprocating rotation of an Al2O3 ball against a CoCrMo disc. Two different electrochemical conditions with increasingly forced corrosion rates were tested: +0.45 V (passive domain) and +0.67 V (transition to transpassive domain). Cell viability, proinflammatory cytokines, electrochemical measurements and ICP-MS metal ion content analyses were performed.Cobalt/ chromium concentrations were 6.6/ 1.6 ppm in the passive domain and almost doubled to 11.4/ 3.0 ppm in the passive-transpassive domain. Under those electrochemical conditions, freshly produced and time-stabilized CoCrMo wear decreased cell viability to the same extent. Secretion of proinflammatory cytokines were not significantly different for freshly produced and time-stabilized debris.This study suggests that freshly generated and time-stabilized metal particles/ions cause similar toxicity and inflammatory reactions in macrophages, indicating that standard practices for generating wear debris are valid methods to evaluate wear particle disease. Other cell types, materials, and corrosion potentials need to be studied in the future to solidify the conclusion.

Aflatoxin B1 affects porcine alveolar macrophage growth through the calcium signaling pathway mediated by the ceRNA regulatory network.

Aflatoxin B1 (AFB1), one of the most prevalent contaminants in human and animal food, impairs the immune system, but information on the mechanisms of AFB1-mediated macrophage toxicity is still lacking. In this study, for the first time, we employed whole transcriptome sequencing technology to explore the molecular mechanism by which AFB1 affects the growth of porcine alveolar macrophages (PAM). We found that AFB1 exposure reduced the proliferative capacity of PAM and prevented cell cycle progression. Based on whole transcriptome analysis, RT-qPCR, ICC and RNAi, we verified the role and regulatory mechanism of the competing endogenous RNA (ceRNA) network in the process of AFB1 exposure affecting the growth of PAM. We found that AFB1 induced MSTRG.43,583, MSTRG.67,490, MSTRG.84,995, and MSTRG.89,935 to competitively bind miR-219a, miR-30b-3p, and miR-30c-1-3p, eliminating the inhibition of its target genes CACNA1S, RYR3, and PRKCG. This activated the calcium signaling pathway to regulate the growth of PAM. These results provide valuable information on the mechanism of AFB1 exposure induced impairment of macrophage function in humans and animals.

Exploring Novel Drug Combinations: The Therapeutic Potential of Selanyl Derivatives for Leishmania Treatment.

This work describes the design, synthesis, and biological activities of new selenoester derivatives and its homologs thioesters. Thirty-two compounds were developed following an economical synthetic route, achieving small molecules, with structural characteristics similar to those present in antileishmanial drugs such as miltefosine (MIL) and paromomycin (PMN). These compounds were tested in vitro against strains of Leishmania major (L. major) and Leishmania infantum (L. infantum). The L. infantum strain (causative agent of visceral leishmaniasis) exhibited the highest sensitivity. Thus, four selanylacetic acid derivatives (A4, A5, A6 and A8) presented IC50 values below 40 µM in this strain. These derivatives also demonstrated low toxicity and high selectivity in PMA-differentiated THP-1 macrophages. The A4-A6 and A8 derivatives were evaluated in order to determine their pharmacological behavior, using drug combination studies with the reference drugs amphotericin B (AMB), MIL and PMN. Compounds A6 and A8 presented a potent synergistic interaction with MIL, which is the only oral drug available for the treatment of visceral leishmaniasis. Therefore, compounds A6 and A8 present significant potential as therapeutic candidates for the treatment of leishmaniasis based on their remarkable leishmanicidal characteristics and pharmacological synergism.

In vitro Evaluation of the Potent Antileishmanial Activity of Ferula tabasensis Alone or in Combination with Shark Cartilage Extract Against the Standard Iranian Strain of Leishmania major (MRHO/IR/75/ER).

The available drugs for the treatment of leishmaniasis are highly toxic and extremely expensive, with low efficiency; therefore, the development of effective therapeutic compounds is essential. The present study aimed to explore the antileishmanial effects of ethyl acetate extract, methanol extract, and fractions 1-4 (F1-F4) of Ferula tabasensis, alone or in combination with shark cartilage extract (ShCE), on L. major in vitro. In this study, ethyl acetate, methanol, and n-hexane extracts were extracted from the aerial roots of F. tabasensis by the maceration method. The silica gel column chromatography was used to separate n-hexane extracts at varying polarities (F1-F4 fractions). Subsequently, the effects of extracts and fractions against promastigotes were assessed by the parasite counting method microscopic inhibition test and MTT assay. Besides, their effects on the infected macrophage cells and the number of amastigotes were investigated. Cytotoxicity was evaluated in non-infected J774A.1 macrophage cells. Finally, apoptosis induction of promastigotes, including infected and non-infected macrophages, was evaluated. The results indicated the highly potent activity of F. tabasensis extracts and F1-F4 fractions, alone or in combination with ShCE, against L. major promastigotes and amastigotes in a dose-dependent manner (P < 0.05). The F1 fraction and methanol extract showed markedly higher toxicity compared to the other extracts and fractions, with 50% inhibitory concentrations (IC50/72h) of 2.4 ± 0.29 and 2.9 ± 0.55 µg/mL against promastigotes and 1.79 ± 0.27 µg/mL and 1.39 ± 0.27 µg/mL against amastigotes (P < 0.001). Moreover, they had a high selectivity index (SI) due to the low toxicity of macrophages (P < 0.0001). The results of flow cytometry indicated that the percentages of apoptotic promastigote cells in contact with IC50 concentrations of F1 and methanol extract alone after 72 h were 43.83 and 43.93%, as well as 78.4%, and 65.45% for their combination with ShCE, respectively.Also, apoptosis of infected macrophages induced by F1 and methanol extracts was estimated at 68.5% and 83.7%, respectively. In this study, the F1 fraction and methanol extract of F. tabasensis showed potent efficacy against L. major, associated with low toxicity and apoptosis induction. Therefore, they can be promising therapeutic candidates in future animal and even human studies.

Integration of transcriptomics, proteomics, and metabolomics data for the detection of the human pathogenic Prototheca wickerhamii from a One Health perspective.

Prototheca species are the only microalgae known to cause opportunistic infections in vertebrates and humans. Most cases of protothecosis in humans are caused by Prototheca wickerhamii, but knowledge of the pathogenicity and biology of Prototheca is limited. Globally, the diagnostic rate of Prototheca species infection is much lower than the actual rate of P. wickerhamii. The precise mechanisms underlying the pathogenesis of Prototheca infections remain unclear. In this study, we identified a strain of P. wickerhamii with atypical colony morphology. To reveal the morphological differences between P. wickerhamii S1 (mucous) and the molecular basis of its pathogenicity, the transcriptomics, proteomics, and metabolomics of two pathogenic P. wickerhamii strains and one environmental strain were analysed. Interestingly, mannan endo-1,4-β-mannosidase was significantly downregulated in P. wickerhamii S1, which contributes to a thinner cell wall in S1 compared to strains with typical colony morphology, and the toxicity of macrophages is reduced. Metabolite analysis revealed that the mucoid appearance of P. wickerhamii S1 may have been caused by an increase in linoleic acid, glycerol, and other metabolites. There is still a need to better understand the ecology, aetiology, and pathogenesis of P. wickerhamii, and in particular, its transmission between humans, animals, and the environment, from a One Health perspective.

The dependence of particle size on cell toxicity for modern mining dust

Progressive massive pulmonary fibrosis among coal miners has unexpectedly increased. It would likely due to the greater generation of smaller rock and coal particles produced by powerful equipment used in modern mines. There is limited understanding of the relationship between micro- or nanoparticles with pulmonary toxicity. This study aims to determine whether the size and chemical characteristics of typical coal-mining dust contribute to cellular toxicity. Size range, surface features, morphology, and elemental composition of coal and rock dust from modern mines were characterized. Human macrophages and bronchial tracheal epithelial cells were exposed to mining dust of three sub- micrometer and micrometer size ranges at varying concentrations, then assessed for cell viability and inflammatory cytokine expression. Coal had smaller hydrodynamic size (180–3000 nm) compared to rock (495–2160 nm) in their separated size fractions, more hydrophobicity, less surface charge, and consisted of more known toxic trace elements (Si, Pt, Fe, Al, Co). Larger particle size had a negative association with in-vitro toxicity in macrophages (p < 0.05). Fine particle fraction, approximately 200 nm for coal and 500 nm for rock particles, explicitly induced stronger inflammatory reactions than their coarser counterparts. Future work will study additional toxicity endpoints to further elucidate the molecular mechanism causing pulmonary toxicity and determine a dose–response curve.

Exploration of seryl tRNA synthetase to identify potent inhibitors against leishmanial parasites

Aminoacyl-tRNA synthetases are crucial enzymes for cellular protein metabolism and have been considered as an attractive target for development of new antimicrobials. In the current study, seryl tRNA synthetase of Leishmania donovani (LdSerRS) and its mutants were purified and characterized through biochemical and structural methods. Purified LdSerRS was found to be enzymatically active and exhibited more alpha helices in secondary structure. The enzymatic activity of purified protein was observed as highest near physiological temperature and pH. Mutation in ATP binding residues (R295 and E297) demonstrated reduction in the affinity for cofactor with no significant deviation in secondary structure. In vitro inhibition studies with ureidosulfocoumarin derivatives helped to identify Comp 5l as a specific inhibitor for leishmanial SerRS that showed lesser potency towards purified HsSerRS. The identified compound presented competitive mode of inhibition for LdSerRS and also revealed druglikeness along with very low toxicity for human macrophages. Structural analysis of protein and ligand complex depicted the binding of Comp 5l into the cofactor binding site of LdSerRS with high affinity succeeded by validation employing molecular dynamics simulations. Altogether, our study presents a promising scaffold to explore small molecules to target the enzymatic activity of leishmanial SerRS to develop the specific therapeutics.

Mixed micelles and gels of a hydrophilic poloxamine (Tetronic 1307) and miltefosine: Structural characterization by small-angle neutron scattering and in vitro evaluation for the treatment of leishmaniasis

Hypothesis/background: Tetronic® is a family of four-armed amphiphilic block copolymers of polyethylene oxide (PEO) and polypropylene oxide (PPO) that self-aggregate to form micelles and hydrogels. Due to their temperature and pH-responsiveness, they are emerging as smart nanomaterials in the area of drug delivery. Here we propose the use of Tetronic 1307 (T1307) as a nanocarrier of miltefosine (MF), a zwitterionic alkylphospholipid highly active against leishmaniasis, one of the most threating neglected tropical diseases. Given the amphiphilic nature of the drug, both surfactants can combine to form mixed micelles, reducing the cytotoxicity of MF by lowering its dose and improving its internalization, hence its antileishmanial effect.Experiments: The structure of the T1307 micelles, MF micelles, mixed micelles and hydrogels, formed in buffered solution (pH = 7.4) at different concentrations has been investigated in-depth by a combination of small-angle neutron scattering (SANS), dynamic light scattering (DLS), fluorescence spectroscopy and nuclear magnetic resonance methods (1D, 2D NOESY, and diffusion NMR). The cytotoxicity of the aggregates in macrophages has been assessed, as well as the antileishmanial activity in both Leishmania major promastigotes and amastigotes.Findings: T1307 and MF combine into mixed aggregates over a wide range of temperatures and compositions, forming ellipsoidal core–shell mixed micelles. The shell is highly hydrated and comprises most of the PEO blocks, while the hydrophobic core contains the PO blocks and the MF along with a fraction of EO and water molecules, depending on the molar ratio in the mixture. The combination with T1307 amplified the leishmanicidal activity of the drug against both forms of the parasite and dramatically reduced drug cytotoxicity. T1307 micelles also showed a considerable leishmanicidal activity without exhibiting macrophage toxicity. These results support the use of T1307 as a MF carrier for the treatment of human and animal leishmaniasis, in its different clinical forms.

Low doses of 3-phenyl-lawsone or meglumine antimoniate delivery by tattooing route are successful in reducing parasite load in cutaneous lesions of Leishmania (Viannia) braziliensis-infected hamsters.

Current therapeutic ways adopted for the treatment of leishmaniasis are toxic and expensive including parasite resistance is a growing problem. Given this scenario, it is urgent to explore treatment alternatives for leishmaniasis. The aim of this study was to evaluate the effect of 3-phenyl-lawsone (3-PL) naphthoquinone on Leishmania (Viannia) braziliensis infection, both in vitro and in vivo, using two local routes of administration: subcutaneous (higher dose) and tattoo (lower dose). In vitro 3-PL showed low toxicity for macrophages (CC50 >3200 µM/48h) and activity against intracellular amastigotes (IC50 = 193 ± 19 µM/48h) and promastigotes (IC50 = 116 ± 26 µM/72h), in which induced increased ROS generation. Additionally, 3-PL up-regulated the production of cytokines such as tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6) and IL-10 in infected macrophages. However, the anti-amastigote action was independent of nitric oxide production. Treatment of hamsters infected with L. (V.) braziliensis from one week after infection with 3-PL by subcutaneous (25 µg/Kg) or tattooing (2.5 µg/Kg) route, during 3 weeks (3 times/week) or 2 weeks (2 times/week) significantly decreased the parasite load (p<0.001) in the lesion. The reduction of parasite load by 3-PL treatment was comparable to reference drug meglumine antimoniate administered by the same routes (subcutaneous 1mg/Kg and tattoo 0.1mg/Kg). In addition, treatment started from five weeks after infection with 3-PL per tattoo also decreased the parasite load. These results show the anti-leishmanial effect of 3-PL against L. (V.) braziliensis and its efficacy by subcutaneous (higher dose) and tattoo (lower dose) routes. In addition, this study shows that drug delivery by tattooing the lesion allows the use of lower doses than the conventional subcutaneous route, which may support the development of a new therapeutic strategy that can be adopted for leishmaniasis.