Gallic acid triphenylphosphonium derivatives TPP+-C10 and TPP+-C12 inhibit mitochondrial function in Candida albicans exerting antifungal and antibiofilm effects.

AIMS: To evaluate the antifungal and antibiofilm activity of gallic acid derivatives TPP+-C10 and TPP+-C12 and their effects on mitochondrial function on two Candida albicans reference strains (ATCC 90029 and ATCC 10231). METHODS AND RESULTS: First, we determined minimal inhibitory concentration (MIC) using a microdilution assay. Both compounds exerted antifungal effects, and their MICs ranged from 3.9 to 13 µM, with no statistically significant differences between them (P > 0.05, t-test). These concentrations served as references for following assays. Subsequently, we measured oxygen consumption with a Clark electrode. Our observations revealed that both drugs inhibited oxygen consumption in both strains with TPP+-C12 exerting a more pronounced inhibitory effect. We then employed flow cytometry with TMRE as a probe to assess mitochondrial membrane potential. For each strain assayed, the compounds induced a decay in transmembrane potential by 75%-90% compared to the control condition (P < 0.05, ANOVA). Then, we measured ATP levels using a commercial kit. TPP+-C12 showed a 50% decrease of ATP content (P < 0.05 ANOVA), while TPP+-C10 exhibited a less pronounced effect. Finally, we assessed the antibiofilm effect using the MTT reduction assay. Both compounds were effective, but TPP+-C12 displayed a greater potency, requiring a lower concentration to inhibit 50% of biofilms viability (P < 0.05, t-test). CONCLUSIONS: Derivatives of gallic acid linked to a TPP+ group exert antifungal and antibiofilm activity through impairment of mitochondrial function in C. albicans.

Indomethacin Induces Spermidine/Spermine-N1-Acetyltransferase-1 via the Nucleolin-CDK1 Axis and Synergizes with the Polyamine Oxidase Inhibitor Methoctramine in Lung Cancer Cells.

Indomethacin is a non-selective NSAID used against pain and inflammation. Although cyclooxygenase (COX) inhibition is considered indomethacin's primary action mechanism, COX-independent ways are associated with beneficial effects in cancer. In colon cancer cells, the activation of the peroxisome proliferator-activated receptor-γ (PPAR-γ) is related to the increase in spermidine/spermine-N1-acetyltransferase-1 (SSAT-1), a key enzyme for polyamine degradation, and related to cell cycle arrest. Indomethacin increases the SSAT-1 levels in lung cancer cells; however, the mechanism relying on the SSAT-1 increase is unclear. Thus, we asked for the influence of the PPAR-γ on the SSAT-1 expression in two lung cancer cell lines: H1299 and A549. We found that the inhibition of PPAR-γ with GW9662 did not revert the increase in SSAT-1 induced by indomethacin. Because the mRNA of SSAT-1 suffers a pre-translation retention step by nucleolin, a nucleolar protein, we explored the relationship between indomethacin and the upstream translation regulators of SSAT-1. We found that indomethacin decreases the nucleolin levels and the cyclin-dependent kinase 1 (CDK1) levels, which phosphorylates nucleolin in mitosis. Overexpression of nucleolin partially reverts the effect of indomethacin over cell viability and SSAT-1 levels. On the other hand, Casein Kinase, known for phosphorylating nucleolin during interphase, is not modified by indomethacin. SSAT-1 exerts its antiproliferative effect by acetylating polyamines, a process reverted by the polyamine oxidase (PAOX). Recently, methoctramine was described as the most specific inhibitor of PAOX. Thus, we asked if methoctramine could increase the effect of indomethacin. We found that, when combined, indomethacin and methoctramine have a synergistic effect against NSCLC cells in vitro. These results suggest that indomethacin increases the SSAT-1 levels by reducing the CDK1-nucleolin regulatory axis, and the PAOX inhibition with methoctramine could improve the antiproliferative effect of indomethacin.

Minireview: functional roles of tissue kallikrein, kinins, and kallikrein-related peptidases in lung cancer.

Despite campaigns and improvements in detection and treatment, lung cancer continues to increase worldwide and represents a major public health problem. One approach to treating patients suffering from lung cancer is to target surface receptors overexpressed on tumor cells, such as GPCR-family kinin receptors, and proteases that control tumor progression, such as kallikrein-related peptidases (KLKs). These proteases have been visualized in recent years due to their contribution to the progression of cancers, such as prostate and ovarian cancer, facilitating the invasive and metastatic capacity of tumor cells in these tissues. In fact, KLK3 is the specific prostate antigen, the only tissue-specific biomarker used to diagnose this malignancy. In lung cancer to date, evidence indicates that KLK5, KLK6, KLK8, KLK11, and KLK14 are the major peptidases regulated and involved in its progression. The expression levels of KLKs in this neoplasm are modulated by the secretome of the different cell types present in the tumor microenvironment, the cancer subtype and the tumor stage, among others. Considering the multiple functions of kinin receptors and KLKs, this review highlights their roles, even considering the SARS-CoV-2 effects. Since lung cancer is often diagnosed in advanced stages, our efforts should focus on early diagnosis, validating for example specific KLKs, especially in high-risk populations such as smokers and people exposed to carcinogenic fumes, oil fields, and contaminated workplaces, unexplored fields to investigate. Furthermore, their modulation could be considered as a promising approach in lung cancer therapeutics.

Mind the Curve: Dose-Response Fitting Biases the Synergy Scores across Software Used for Chemotherapy Combination Studies.

Drug combinations are increasingly studied in the field of anticancer agents. Mathematical models, such as Loewe, Bliss, and HSA, are used to interpret drug combinations, while informatics tools help cancer researchers identify the most effective combinations. However, the different algorithms each software uses lead to results that do not always correlate. This study compared the performance of Combenefit (Ver. 2.021) and SynergyFinder (Ver. 3.6) in analyzing drug synergy by studying combinations involving non-steroidal analgesics (celecoxib and indomethacin) and antitumor drugs (carboplatin, gemcitabine, and vinorelbine) on two canine mammary tumor cell lines. The drugs were characterized, their optimal concentration-response ranges were determined, and nine concentrations of each drug were used to make combination matrices. Viability data were analyzed under the HSA, Loewe, and Bliss models. Celecoxib-based combinations showed the most consistent synergistic effect among software and reference models. Combination heatmaps revealed that Combenefit gave stronger synergy signals, while SynergyFinder produced better concentration-response fitting. When the average values of the combination matrices were compared, some combinations shifted from synergistic to antagonistic due to differences in the curve fitting. We also used a simulated dataset to normalize each software's synergy scores, finding that Combenefit tends to increase the distance between synergistic and antagonistic combinations. We conclude that concentration-response data fitting biases the direction of the combination (synergistic or antagonistic). In contrast, the scoring from each software increases the differences among synergistic or antagonistic combinations in Combenefit when compared to SynergyFinder. We strongly recommend using multiple reference models and reporting complete data analysis for synergy claiming in combination studies.

Bovine tumor necrosis factor-alpha Increases IL-6, IL-8, and PGE2 in bovine fibroblast-like synoviocytes by metabolic reprogramming.

Lameness is a common condition in dairy cattle caused by infectious or noninfectious agents. Joint lesions are the second most common cause of lameness and can be diagnosed in association with the presentation of digit injuries. Fibroblast-like synoviocyte (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the expression of proinflammatory mediators. Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine involved in cyclooxygenase 2 (COX-2) and proinflammatory cytokine expression in FLS. Previously, TNF-α was demonstrated to increase hypoxia-inducible Factor 1 (HIF-1), a transcription factor that rewires cellular metabolism and increases the expression of interleukin (IL)-6 in bovine FLS (bFLS). Despite this, the proinflammatory effects of TNF-α in bFLS on metabolic reprogramming have been poorly studied. We hypothesized that TNF-α increases glycolysis and in this way controls the expression of IL-6, IL-8, and COX-2 in bFLS. Results first, gas chromatography/mass spectrometry (GC/MS)-based untargeted metabolomics revealed that bTNF-α altered the metabolism of bFLS, increasing glucose, isoleucine, leucine, methionine, valine, tyrosine, and lysine and decreasing malate, fumarate, α-ketoglutarate, stearate, palmitate, laurate, aspartate, and alanine. In addition, metabolic flux analysis using D-glucose-13C6 demonstrated an increase of pyruvate and a reduction in malate and citrate levels, suggesting a decreased flux toward the tricarboxylic acid cycle after bTNF-α stimulation. However, bTNF-α increased lactate dehydrogenase subunit A (LDHA), IL-6, IL-8, IL-1ß and COX-2 expression, which was dependent on glycolysis and the PI3K/Akt pathway. The use of FX11 and dichloroacetate (DCA), an inhibitor of LDHA and pyruvate dehydrogenase kinase (PDK) respectively, partially reduced the expression of IL-6. Our results suggest that bTNF-α induces metabolic reprogramming that favors glycolysis in bFLS and increases IL-6, IL-8, IL-1ß and COX-2/PGE2.

Anti-protozoal activity and metabolomic analyses of Cichorium intybus L. against Trypanosoma cruzi.

Chagas disease, caused by the protozoa Trypanosoma cruzi, is a potentially life-threatening parasitic zoonosis infecting 6-7 million people worldwide, mainly in Latin America. Due to the limited numbers of drugs available against this neglected disease and their frequent adverse effects, novel anti-chagasic agents are urgently needed. Cichorium intybus L. (chicory) is a bioactive plant with potent activity against parasitic nematodes, but its effects on protozoans are poorly known and no studies have explored its trypanocidal potential. Here, we investigated the activity of C. intybus against extracellular and intracellular stages of T. cruzi, including the prediction of trypanocidal compounds by metabolomic analyses and bioactivity-based molecular networking. Purified C. intybus extracts were prepared from leaves and roots of five C. intybus cultivars (cv. 'Benulite', 'Goldine', 'Larigot', 'Maestoso' and 'Spadona'). All C. intybus extracts induced concentration-dependent effects against T. cruzi trypomastigotes. C. intybus leaf extracts had higher trypanocidal selectivity and lower cytotoxicity on mammalian cells than root extracts. The leaf extract of C. intybus cv. Goldine also significantly reduced the number of mammalian cells infected with T. cruzi amastigotes. Metabolomic and bioactivity-based molecular networking analyses revealed 11 compounds in C. intybus leaves strongly linked with activity against trypomastigotes, including the sesquiterpene lactone lactucin, and flavonoid- and fatty acid-derivatives. Furthermore, seven distinct C. intybus molecules (including two sesquiterpene lactone-derivatives) were predicted to be involved in reducing the number of mammalian cells infected with amastigotes. This is the first report of the anti-protozoal activity of C. intybus against trypanosomatid parasites and expands our understanding of the anti-parasitic effects of this plant and its bioactive metabolites. Further studies to elucidate the anti-protozoal compound(s) in C. intybus and their mode(s) of action will improve our knowledge of using this bioactive plant as a promising source of novel broad-spectrum anti-parasitic compounds with associated health benefits and biomedical potential.

Origanum vulgare L. essential oil inhibits virulence patterns of Candida spp. and potentiates the effects of fluconazole and nystatin in vitro.

BACKGROUND: Recurrence and resistance of Candida spp. infections is associated with the ability of these microorganisms to present several virulence patterns such as morphogenesis, adhesion, and biofilm formation. In the search for agents with antivirulence activity, essential oils could represent a strategy to act against biofilms and to potentiate antifungal drugs. OBJECTIVE: To evaluate the antivirulence effect of Origanum vulgare L. essential oil (O-EO) against Candida spp. and to potentiate the effect of fluconazole and nystatin. METHODS: The effect of O-EO was evaluated on ATCC reference strains of C. albicans and non-albicans Candida species. Minimum inhibitory concentration (MIC) was determined through broth microdilution assay. Adhesion to microplates was determined by crystal violet (CV) assay. An adapted scratch assay in 24-well was used to determine the effect of essential oil on biofilms proliferation. Viability of biofilms was evaluated by MTT reduction assay and through a checkerboard assay we determined if O-EO could act synergistically with fluconazole and nystatin. RESULTS: MIC for C. albicans ATCC-90029 and ATCC-10231 was 0.01 mg/L and 0.97 mg/L, respectively. For non-albicans Candida strains MIC values were 2.6 mg/L for C. dubliniensis ATCC-CD36 and 5.3 mg/L for C. krusei ATCC-6258. By using these concentrations, O-EO inhibited morphogenesis, adhesion, and proliferation at least by 50% for the strains assayed. In formed biofilms O-EO decreased viability in ATCC 90029 and ATCC 10231 strains (IC50 7.4 and 2.8 mg/L respectively). Finally, we show that O-EO interacted synergistically with fluconazole and nystatin. CONCLUSIONS: This study demonstrate that O-EO could be considered to improve the antifungal treatment against Candida spp.

Chemical Characterization of Lavandula dentata Essential Oil Cultivated in Chile and Its Antibiofilm Effect against Candida albicans.

Candida albicans is the most common human fungal pathogen, and with the increase in resistance rates worldwide, it is necessary to search for new pharmacological alternatives. Lavandula dentata L. essential oil is recognized as having antimicrobial properties. However, its effect against fungal biofilms has been poorly described. C. albicans-related infections involve the development of biofilms, which are highly resistant to conventional antifungals. In this work, we evaluated the antibiofilm effect of L. dentata L. essential oil against C. albicans. First, we characterized the essential oil by gas chromatography-mass spectrometry. The antifungal effect on C. albicans reference strains was evaluated by a disk diffusion assay and the minimal inhibitory concentration was obtained through a microdilution assay. The effect of the essential oil on the adhesion ability of C. albicans was determined through a crystal violet assay, and morphogenesis inhibition was assessed by light microscopy. The effect of the essential oil on the microarchitecture of biofilms was evaluated through scanning electron microscopy. Finally, the antibiofilm effect was evaluated through an adapted biofilm scratch assay and XTT viability assay. The main constituent of the essential oil was the monoterpenoid eucalyptol (60%). The essential oil presented minimal inhibitory concentrations of 156 and 130 µg/mL against two strains assayed. This minimal inhibitory concentration inhibited adhesion, morphogenesis, biofilm formation, altered microarchitecture, and decreased the viability of established biofilms formed on abiotic surfaces for both strains assayed. This study demonstrates that the essential oil from L. dentata could be a promising treatment against C. albicans biofilms.

In vitro and in vivo activity of voriconazole and benznidazole combination on trypanosoma cruzi infection models.

Combination therapy has been proposed as an ideal strategy to reduce drug toxicity and improve treatment efficacy in Chagas disease. Previously, we demonstrated potent in vivo anti-Trypanosoma cruzi activity of voriconazole. In this work, we aimed to study the synergistic effect of voriconazole (VCZ) and benznidazole (BZ) both in vitro and in vivo models of T. cruzi infection using the Tulahuen strain. Combining VCZ and BZ at fixed concentrations, the inhibitory concentration 50% (IC50) on amastigotes was lower than the obtained IC50 for BZ alone and the Fractional Inhibitory Concentration Index (∑FIC) suggested an in vitro additive effect on T. cruzi amastigotes inhibition at concentrations devoid of cytotoxic effects. Treatment response in the in vivo model was evaluated by comparing behavior and physical aspects, parasitemia and mortality of mice infected with Tulahuen strain. VCZ and BZ treatments alone or in combination were well tolerated. All treated animals displayed significantly lower mean peak parasitemia and mortality compared to infected non-treated controls (p< 0.05). However, VCZ + BZ combination elicited no additional benefits over BZ monotherapy. VCZ efficacy was not enhanced by combination therapy with BZ at the doses studied, requiring further and astringent non-clinical studies to establish the VCZ efficacy and eventually moving forward to clinical trials.

Anthelmintic and metabolomic analyses of chicory (Cichorium intybus) identify an industrial by-product with potent in vitro antinematodal activity.

Chicory (Cichorium intybus) is a bioactive forage rich in sesquiterpene lactones (SLs) with reported in vitro and in vivo anthelmintic activity in livestock. However, the on-farm adoption of chicory as an anthelmintic crop is limited and may be facilitated by using standardised industrial chicory material. Chicory root pulp is a by-product obtained from industrial chicory roots after inulin extraction and can potentially retain SLs. However, SL content and associated anthelmintic activity of chicory root pulp have not been investigated. Here, we evaluated the anthelmintic activity of SL-enriched extracts from chicory root pulp and forage chicory, and used untargeted metabolomics and molecular networking to identify potential anthelmintic molecules. Six different sources of chicory material were used: fresh chicory root pulp (from industrial chicory roots C. intybus var. sativum; "Root Pulp"), fresh leaves from chicory cv. Spadona (sampled on four occasions) and fresh leaves from chicory cv. Choice. The resulting extracts were tested for anthelmintic activity against the free-living nematode Caenorhabditis elegans and the pig nematode Ascaris suum. The cytotoxicity of the chicory extracts was evaluated on mammalian (Vero) cells. In the C. elegans assays, the Root Pulp was the most potent extract and induced paralysis in >95% of worms exposed to >250  µg extract/mL (EC50 = 64.2 µg/mL). In the A. suum assays, the Root Pulp was also the most potent chicory extract to inhibit worm motility (EC50 = 87.6  µg/mL), followed closely by two of the Spadona leaf extracts (EC50 = 89.8  µg/mL and 112.2  µg/mL) The Root Pulp extract had the lowest cytotoxicity of all tested extracts towards mammalian cells, with a selectivity index of 5.37. Untargeted metabolomics revealed that chicory Root Pulp had a markedly different chemical profile in comparison with forage chicory extracts. Molecular networking confirmed several SLs and SL-derivatives mainly present in chicory root pulp, that may be responsible of its potent anti-parasitic activity. Bioactivity-based molecular networking of chicory root pulp and the most potent forage chicory extracts revealed a high predicted anthelmintic score for the guaianolide SL 11,13-dihydro-lactucopicrin. In conclusion, chicory root pulp showed potent and selective in vitro anthelmintic activity against C. elegans and A. suum, with low cytotoxicity in mammalian cells. The promising anthelmintic activity of chicory root pulp should be confirmed in vivo to further explore the potential of this agro-industrial by-product as a nutraceutical anthelmintic for livestock and as novel source of anti-parasitic compounds.

Tamoxifen in horses: pharmacokinetics and safety study.

BACKGROUND: Tamoxifen (TAM), a selective modulator of estrogen receptors (SERMs) has been recently explored as a therapeutic option for the oral treatment of airway inflammation in the horse. The objective of this work was to establish pharmacokinetic parameters of TAM and its main metabolites in equines, as well as to determine its clinical safety in short-term treatments. RESULTS: We determined TAM and its three main metabolites (4-OH tamoxifen, endoxifen, and N-desmethyl tamoxifen) in plasma after single administration of 0.25 mg/kg in healthy adult horses (n = 12). A maximum concentration of TAM was achieved 3 h after the oral administration (4.65 pg/mL ± 1.69); 4-OH tamoxifen was the metabolite that reached the highest concentration (78 pg/mL ± 70), followed by N-desmethyl tamoxifen (0.43 pg / mL ± 0.48), and finally endoxifen (0.17 pg/mL ± 0.17). All metabolites showed peak concentration 2 h after oral administration of the drug. Oral TAM bioavailability was 13,15% ± 4,18, with a steady state volume of distribution of 7831 ± 2922 (L/kg). Elimination half-life was 15.40 ± 5.80 h, and clearance was 5876 ± 699 (mL/kg/min). Clinical safety of TAM was determined over a 7-day course of treatment (0.25 mg/kg, orally q 24 h, n = 20). No adverse effects were observed through clinical examination, blood hematology, serum biochemistry, ophthalmological and reproductive examinations. Endometrial edema observed in some mares was attributed to normal cyclic activity. CONCLUSIONS: Tamoxifen has moderate oral bioavailability and a large volume of distribution, with three main metabolites in horses. Additionally, oral TAM administration over a 7-day treatment period demonstrated to be clinically safe, without adverse effects on clinical, hematological or serum biochemical parameters. These data could contribute to the continued research into this drug's potential for the treatment of different inflammatory conditions in equine species.

Reconsidering the Role of Cyclooxygenase Inhibition in the Chemotherapeutic Value of NO-Releasing Aspirins for Lung Cancer.

Nitric oxide-releasing aspirins (NO-aspirins) are aspirin derivatives that are safer than the parent drug in the gastrointestinal context and have shown superior cytotoxic effects in several cancer models. Despite the rationale for their design, the influence of nitric oxide (NO•) on the effects of NO-aspirins has been queried. Moreover, different isomers exhibit varying antitumor activity, apparently related to their ability to release NO•. Here, we investigated the effects and mode of action of NO-aspirins in non-small-cell lung cancer (NSCLC) cells, comparing two isomers, NCX4016 and NCX4040 (-meta and -para isomers, respectively). NCX4040 was more potent in decreasing NSCLC cell viability and migration and exhibited significant synergistic effects in combination with erlotinib (an epidermal growth factor receptor inhibitor) in erlotinib-resistant cells. We also studied the relationship among the effects of NO-aspirins, NO• release, and PGE2 levels. NCX4040 released more NO• and significantly decreased PGE2 synthesis relative to NCX4016; however, NO• scavenger treatment reversed the antiproliferative effects of NCX4016, but not those of NCX4040. By contrast, misoprostol (a PGE2 receptor agonist) significantly reversed the antiproliferative effect of NCX4040, but not those of NCX4016. Furthermore, misoprostol reversed the antimigratory effects of NCX4040. Overall, these results indicate that PGE2 inhibition is important in the mode of action of NO-aspirins.

Searching for Drug Synergy Against Cancer Through Polyamine Metabolism Impairment: Insight Into the Metabolic Effect of Indomethacin on Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is the most lethal and prevalent type of lung cancer. In almost all types of cancer, the levels of polyamines (putrescine, spermidine, and spermine) are increased, playing a pivotal role in tumor proliferation. Indomethacin, a non-steroidal anti-inflammatory drug, increases the abundance of an enzyme termed spermidine/spermine-N1-acetyltransferase (SSAT) encoded by the SAT1 gene. This enzyme is a key player in the export of polyamines from the cell. The aim of this study was to compare the effect of indomethacin on two NSCLC cell lines, and their combinatory potential with polyamine-inhibitor drugs in NSCLC cell lines. A549 and H1299 NSCLC cells were exposed to indomethacin and evaluations included SAT1 expression, SSAT levels, and the metabolic status of cells. Moreover, the difference in polyamine synthesis enzymes among these cell lines as well as the synergistic effect of indomethacin and chemical inhibitors of the polyamine pathway enzymes on cell viability were investigated. Indomethacin increased the expression of SAT1 and levels of SSAT in both cell lines. In A549 cells, it significantly reduced the levels of putrescine and spermidine. However, in H1299 cells, the impact of treatment on the polyamine pathway was insignificant. Also, the metabolic features upstream of the polyamine pathway (i.e., ornithine and methionine) were increased. In A549 cells, the increase of ornithine correlated with the increase of several metabolites involved in the urea cycle. Evaluation of the levels of the polyamine synthesis enzymes showed that ornithine decarboxylase is increased in A549 cells, whereas S-adenosylmethionine-decarboxylase and polyamine oxidase are increased in H1299 cells. This observation correlated with relative resistance to polyamine synthesis inhibitors eflornithine and SAM486 (inhibitors of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, respectively), and MDL72527 (inhibitor of polyamine oxidase and spermine oxidase). Finally, indomethacin demonstrated a synergistic effect with MDL72527 in A549 cells and SAM486 in H1299 cells. Collectively, these results indicate that indomethacin alters polyamine metabolism in NSCLC cells and enhances the effect of polyamine synthesis inhibitors, such as MDL72527 or SAM486. However, this effect varies depending on the basal metabolic fingerprint of each type of cancer cell.

Inflammatory and Pro-resolving Lipids in Trypanosomatid Infections: A Key to Understanding Parasite Control.

Pathogenic trypanosomatids (Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp.) are protozoan parasites that cause neglected diseases affecting millions of people in Africa, Asia, and the Americas. In the process of infection, trypanosomatids evade and survive the immune system attack, which can lead to a chronic inflammatory state that induces cumulative damage, often killing the host in the long term. The immune mediators involved in this process are not entirely understood. Most of the research on the immunologic control of protozoan infections has been focused on acute inflammation. Nevertheless, when this process is not terminated adequately, permanent damage to the inflamed tissue may ensue. Recently, a second process, called resolution of inflammation, has been proposed to be a pivotal process in the control of parasite burden and establishment of chronic infection. Resolution of inflammation is an active process that promotes the normal function of injured or infected tissues. Several mediators are involved in this process, including eicosanoid-derived lipids, cytokines such as transforming growth factor (TGF)-ß and interleukin (IL)-10, and other proteins such as Annexin-V. For example, during T. cruzi infection, pro-resolving lipids such as 15-epi-lipoxin-A4 and Resolvin D1 have been associated with a decrease in the inflammatory changes observed in experimental chronic heart disease, reducing inflammation and fibrosis, and increasing host survival. Furthermore, Resolvin D1 modulates the immune response in cells of patients with Chagas disease. In Leishmania spp. infections, pro-resolving mediators such as Annexin-V, lipoxins, and Resolvin D1 are related to the modulation of cutaneous manifestation of the disease. However, these mediators seem to have different roles in visceral or cutaneous leishmaniasis. Finally, although T. brucei infections are less well studied in terms of their relationship with inflammation, it has been found that arachidonic acid-derived lipids act as key regulators of the host immune response and parasite burden. Also, cytokines such as IL-10 and TGF-ß may be related to increased infection. Knowledge about the inflammation resolution process is necessary to understand the host-parasite interplay, but it also offers an interesting opportunity to improve the current therapies, aiming to reduce the detrimental state induced by chronic protozoan infections.

Antiparasitic activity of chicory (Cichorium intybus) and its natural bioactive compounds in livestock: a review.

Increasing drug resistance in gastrointestinal (GI) parasites of livestock and concerns about chemical residues in animal products and the environment are driving the development of alternative control strategies that are less reliant on the use of synthetic drugs. An increasingly investigated approach is the use of bioactive forages with antiparasitic properties as part of the animal's diet (nutraceuticals) or as potential sources of novel, natural parasiticides. Chicory (Cichorium intybus) is a multi-purpose crop and one of the most promising bioactive forages in temperate regions, and numerous in vivo trials have explored its potential against parasitic nematodes in livestock. However, it is unclear whether chicory can induce a direct and broad activity against various GI parasites in different livestock species, and the levels of chicory in the diet that are required to exert an efficient antiparasitic effect. Moreover, the mechanisms leading to the reported parasiticidal activity of chicory are still largely unknown, and its bioactive phytochemicals have only recently been investigated. In this review, we summarise the progress in the study of the antiparasitic activity of chicory and its natural bioactive compounds against GI parasites in livestock, through examination of the published literature. The available evidence indicates that feeding chicory can reduce faecal egg counts and/or worm burdens of abomasal nematodes, but not infections with intestinal worms, in ruminants. Highly chicory-rich diets (≥ 70% of chicory dry matter in the diet) may be necessary to directly affect abomasal parasitism. Chicory is known to synthesise several bioactive compounds with potential antiparasitic activity, but most research has been devoted to the role of sesquiterpene lactones (SL). Recent in vitro studies have confirmed direct and potent activity of SL-rich extracts from chicory against different GI helminths of livestock. Chicory SL have also been reported to exhibit antimalarial properties and its potential antiprotozoal activity in livestock remains to be evaluated. Furthermore, the detailed identification of the main antiparasitic metabolites of chicory and their pharmacokinetics need further confirmation. Research gaps and perspectives on the potential use of chicory as a nutraceutical forage and a source of bioactive compounds for parasite control in livestock are discussed.

Chronic Chagas cardiomyopathy: a therapeutic challenge and future strategies.

Infectious diseases are the main cause of acquired dilated cardiomyopathy. This group of disorders shares in common inflammatory cell infiltrate and myocardial remodeling. As part of its pathophysiology, there is coronary microvascular dysfunction, distinct from that observed in coronary artery disease. Chagas cardiomyopathy presents several vascular characteristics that are similar to those presented in other acquired cardiomyopathies. There is convincing evidence of the microvascular involvement and the inflammatory processes that lead to endothelial activation and ischemic damage. Current therapy for the Chagas disease is limited, and it is proposed to combine it with other pharmacological strategies that modify critical physiopathological aspects beneficial for the clinical course of the Chagas cardiomyopathy.

Evaluation of the Novel Antichagasic Activity of [1,2,3]Triazolo[1,5-a]pyridine Derivatives.

BACKGROUND: Trypanosoma cruzi is the causative agent of Chagas disease. This parasite is vulnerable to the effects of ROS as its main defense mechanism against exogenous agents trypanothione is also another weakness of the parasite that investigated related to the inhibition of enzymes belonging P450 system, mainly CYP51. In our group we have synthesized a series of triazoles known as [1,2,3]triazolo[1,5-a]pyridyl ketones, and pyridyl ketones. These families have shown interesting structural features due to the presence of electron withdrawing moieties attached to the main heterocycle (triazoles and/or pyridines) and are proposed as potential target in the parasite, by the presence of the carbonyl group being able to be reduced and form a free radical that could interact with molecular oxygen generating ROS in the parasite. Furthermore, the triazole ring and pyridines have been considered as potent inhibitors of sterol biosynthesis, the lock being part CYP51. RESULT: Our results showed that the series is capable of generating a stable radical species and generate ROS in the parasite. On the other hand these molecules are potent inhibitors of enzymes belonging to the complex P450. We have focused on the inhibition of ergosterol biosynthesis demonstrating that triazole/ pyridine families are able to affect this pathway being observed the accumulation of squalene and lanosterol.

Pentamidine antagonizes the benznidazole's effect in vitro, and lacks of synergy in vivo: Implications about the polyamine transport as an anti-Trypanosoma cruzi target.

Benznidazole is the first-line drug used in treating Chagas disease, which is caused by the parasite Trypanosoma cruzi (T. cruzi). However, benznidazole has limited efficacy and several adverse reactions. Pentamidine is an antiprotozoal drug used in the treatment of leishmaniasis and African trypanosomiasis. In T. cruzi, pentamidine blocks the transport of putrescine, a precursor of trypanothione, which constitutes an essential molecule in the resistance of T. cruzi to benznidazole. In the present study, we describe the effect of the combination of benznidazole and pentamidine on isolated parasites, mammalian cells and in mice infected with T. cruzi. In isolated trypomastigotes, we performed a dose-matrix scheme of combinations, where pentamidine antagonized the effect of benznidazole, mainly at concentrations below the EC50 of pentamidine. In T. cruzi-infected mammalian cells, pentamidine reversed the effect of benznidazole (measured by qPCR). In comparison, in infected BALB/c mice, pentamidine failed to get synergy with benznidazole, measured on mice survival, parasitemia and amastigote nest quantification. To further explain the in vitro antagonism, we explored whether pentamidine affects intracellular trypanothione levels, however, pentamidine produced no change in trypanothione concentrations. Finally, the T. cruzi polyamine permease (TcPAT12) was overexpressed in epimastigotes, showing that pentamidine has the same trypanocidal effect, independently of transporter expression levels. These results suggest that, in spite of the high potency in the putrescine transport blockade, TcPAT12 permease is not the main target of pentamidine, and could explain the lack of synergism between pentamidine and benznidazole.

Acute Chagas outbreaks: molecular and biological features of Trypanosoma cruzi isolates, and clinical aspects of acute cases in Santander, Colombia.

BACKGROUND: Outbreaks of acute Chagas disease associated with oral transmission are easily detected nowadays with trained health personnel in areas of low endemicity, or in which the vector transmission has been interrupted. Given the biological and genetic diversity of Trypanosoma cruzi, the high morbidity, mortality, and the observed therapeutic failure, new characteristics of these outbreaks need to be addressed at different levels, both in Trypanosoma cruzi as in patient response. The aim of this work was to evaluate the patient's features involved in six outbreaks of acute Chagas disease which occurred in Santander, Colombia, and the characteristics of Trypanosoma cruzi clones isolated from these patients, to establish the potential relationship between the etiologic agent features with host behavior. METHODS: The clinical, pathological and epidemiological aspects of outbreaks were analyzed. In addition, Trypanosoma cruzi clones were biologically characterized both in vitro and in vivo, and the susceptibility to the classical trypanocidal drugs nifurtimox and benznidazole was evaluated. Trypanosoma cruzi clones were genotyped by means of mini-exon intergenic spacer and cytochrome b genes sequencing. RESULTS: All clones were DTU I, and based on the mini-exon intergenic spacer, belong to two genotypes: G2 related with sub-urban, and G11 with rural outbreaks. Girón outbreak clones with higher susceptibility to drugs presented G2 genotype and C/T transition in Cyt b. The outbreaks affected mainly young population (±25.9 years), and the mortality rate was 10 %. The cardiac tissue showed intense inflammatory infiltrate, myocardial necrosis and abundant amastigote nests. However, although the gastrointestinal tissue was congestive, no inflammation or parasites were observed. CONCLUSIONS: Although all clones belong to DTU I, two intra-DTU genotypes were found with the sequencing of the mini-exon intergenic spacer, however there is no strict correlation between genetic groups, the cycles of the parasite or the clinical forms of the disease. Trypanosoma cruzi clones from Girón with higher sensitivity to nifurtimox presented a particular G2 genotype and C/T transition in Cyt b. When the diagnosis was early, the patients responded well to antichagasic treatment, which highlights the importance of diagnosis and treatment early to prevent fatal outcomes associated with these acute episodes.

Novel Gallate Triphenylphosphonium Derivatives with Potent Antichagasic Activity.

disease is one of the most neglected tropical diseases in the world, affecting nearly 15 million people, primarily in Latin America. Only two drugs are used for the treatment of this disease, nifurtimox and benznidazole. These drugs have limited efficacy and frequently induce adverse effects, limiting their usefulness. Consequently, new drugs must be found. In this study, we demonstrated the in vitro trypanocidal effects of a series of four gallic acid derivatives characterized by a gallate group linked to a triphenylphosphonium (TPP(+)) moiety (a delocalized cation) via a hydrocarbon chain of 8, 10, 11, or 12 atoms (TPP(+)-C8, TPP(+)-C10, TPP(+)-C11, and TPP(+)-C12, respectively). We analyzed parasite viability in isolated parasites (by MTT reduction and flow cytometry) and infected mammalian cells using T. cruzi Y strain trypomastigotes. Among the four derivatives, TPP(+)-C10 and TPP(+)-C12 were the most potent in both models, with EC50 values (in isolated parasites) of 1.0 ± 0.6 and 1.0 ± 0.7 µM, respectively, and were significantly more potent than nifurtimox (EC50 = 4.1 ± 0.6 µM). At 1 µM, TPP(+)-C10 and TPP(+)-C12 induced markers of cell death, such as phosphatidylserine exposure and propidium iodide permeabilization. In addition, at 1 µM, TPP(+)-C10 and TPP(+)-C12 significantly decreased the number of intracellular amastigotes (TPP(+)-C10: 24.3%, TPP(+)-C12: 19.0% of control measurements, as measured by DAPI staining) and the parasite's DNA load (C10: 10%, C12: 13% of control measurements, as measured by qPCR). Based on the previous mode of action described for these compounds in cancer cells, we explored their mitochondrial effects in isolated trypomastigotes. TPP(+)-C10 and TPP(+)-C12 were the most potent compounds, significantly altering mitochondrial membrane potential at 1 µM (measured by JC-1 fluorescence) and inducing mitochondrial transition pore opening at 5 µM. Taken together, these results indicate that the TPP(+)-C10 and TPP(+)-C12 derivatives of gallic acid are promising trypanocidal agents with mitochondrial activity.