Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Long-term reproductive effects in male rats prenatally exposed to sodium arsenite.

Arsenic is an environmental toxicant known to be a carcinogen and endocrine disruptor. Maternal exposure to arsenic has been associated with fetus malformation and reproductive disorders in male offspring. However, it is unclear the extent to which those effects remain during postnatal development and adulthood. Therefore, this study aimed to investigate the long-term effects of prenatal arsenic exposure on reproductive parameters of male offspring at peripubertal and adult periods. Pregnant female Wistar rats were exposed to 0 or 10 mg/L sodium arsenite in drinking water from gestational day 1 (GD 1) until GD 21 and male pups were analyzed at postnatal day 44 (PND 44) and PND 70. We observed that some reproductive parameters were affected differently by arsenic exposure at each age evaluated. The body and reproductive organs weights, as well as testicular and epididymal morphology were strongly affected in peripubertal animals and recovered at adult period. On the other hand, the antioxidant genes expression (SOD1, SOD2, CAT and GSTK1) and the endogenous antioxidant system were affected in the testes and epididymides from both peripubertal and adult rats. Finally, an impairment in daily sperm production and in sperm parameters was observed in adult animals. Taken together, our findings show that prenatal arsenic exposure affected reproductive parameters of peripubertal and adult male rats mainly due to oxidative stress. Collectively, those alterations may be affecting fertility potential of adult animals.

A Withanolide-rich Fraction of Athenaea velutina Induces Apoptosis and Cell Cycle Arrest in Melanoma B16F10 Cells.

Athenaea velutina is a promising Brazilian shrub with cytotoxic and antimigratory properties against cancer cells. However, the mechanism of induction of cancer cell death and the compounds involved remain unknown. To ascertain these bioactive compounds, bioassay-guided fractionation was performed, alongside the appropriate in vitro tests. A withanolide-rich fraction (FAv_5) from the dichloromethane extract increased cytotoxic activity by 1.5-fold (IC50 = 2.1 µg/mL). Fourteen withanolide steroids were tentatively identified for the first time for this species by mass spectrometry coupled to liquid chromatography (LC MS/MS), including withanolide A, aurelianolide A, and aurelianolide B. FAv_5 significantly decreased cell proliferation, migration, and invasion with a selectivity index greater than 8 for B16F10 cells. Furthermore, flow cytometry with annexin V fluorescein isothiocyanate/propidium iodide (V-FITC/PI) staining showed FAv_5 to promote cell cycle arrest at the G0/G1-phase as well as apoptotic cell death. Overall, these findings highlight A. velutina as a source of withanolide-steroids that inhibit cancer cell proliferation through apoptosis and cell cycle blockade mechanisms. Details on the geographic distribution of A. velutina and species conservation strategies have also been highlighted.

Antioxidant and anti-inflammatory activity of curcumin transdermal gel in an IL-10 knockout mouse model of inflammatory bowel disease.

Inflammatory bowel diseases are a group of inflammatory disorders of the gastrointestinal tract. Their prevalence is still low in Brazil, but the incidence is increasing annually. A variety of compounds present in Curcuma longa L., particularly curcumin, have been shown to reduce oxidative stress and aid in the prevention of associated diseases. This study aimed to assess the effect of curcumin transdermal gel on oxidative stress and intestinal inflammation in IL-10 knockout mice. Female mice were divided into four groups: a control group (C0) treated with vehicle and three experimental groups treated with transdermal gel containing 50 (C50), 75 (C75), and 100 (C100) mg curcumin kg-1 body weight. Colon malondialdehyde concentrations were lower in C50 and C75 groups. C100 treatment led to reduced catalase activity in the small intestine, whereas C50, C75, and C100 treatments resulted in decreased catalase activity in the colon. In contrast, superoxide dismutase activity increased in the small intestine of C50 and C75 mice and decreased in the colon of C50, C75, and C100 mice. Glutathione S-transferase activity increased in the small intestine and decreased in the colon of C75 animals. These findings suggest that curcumin transdermal gel exerts a protective effect against oxidative stress.

ß-hydroxy-ß-methylbutyrate supplementation benefits the effects of resistance training on body fat reduction via increased irisin expression in white adipose tissue.

The effects of resistance training (RT) associated with calcium ß-hydroxyß-methylbutyrate (CaHMB) supplementation on the body composition and gene expression of cytokines related to skeletal muscle hypertrophy and adipose tissue metabolism were studied in rats. Male Wistar rats were divided into four groups of 12 animals: sedentary control (SC); sedentary supplemented (SS); resistance training control (RTC) and resistance training supplemented (RTS). Rats from RTC and RTS groups were submitted to an RT programme and those from SS and RTS groups received 1 mL of CaHMB (320 mg kg-1 day-1) by gavage, for 8 weeks. We evaluated: body composition; plasma lipid profile; the gene expression of interleukin (IL)-6, IL-10, IL-15 and fibronectin type III domain-containing protein 5 (FNDC-5) in skeletal muscle, and IL-6, mitochondrial uncoupling protein 1 (UCP-1) in white adipose tissue (WAT); and the concentration of irisin in WAT. Compared to RTC alone, the combination of CaHMB with RT (RTS) further reduced abdominal circumference (5.3%), Lee index (2.4%), fat percentage (24.4%), plasma VLDL cholesterol (16.8%) and triglycerides (17%) and increased the gene expression of FNDC-5 (78.9%) and IL-6 (47.4%) in skeletal muscle and irisin concentration (26.9%) in WAT. Neither RT nor CaHMB affected the protein percentage or the gene expression of IL-6 and UCP-1 in WAT and IL-10, IL-15 in skeletal muscle. In conclusion, CaHMB supplementation increased the beneficial effects of RT on body fat reduction and was associated with muscular genic expression of IL-6 and FNDC-5 and irisin concentration in WAT, despite the lack of change in protein mass and maximal strength.

Concomitant Benznidazole and Suramin Chemotherapy in Mice Infected with a Virulent Strain of Trypanosoma cruzi.

Although suramin (Sur) is suggested as a potential drug candidate in the management of Chagas disease, this issue has not been objectively tested. In this study, we examined the applicability of concomitant treatment with benznidazole (Bz) and suramin in mice infected with a virulent strain of Trypanosoma cruzi. Eighty 12-week-old male C57BL/6 mice were equally randomized in eight groups: (i) noninfected mice (negative control) and mice infected with T. cruzi Y strain receiving (ii) no treatment (positive control), (iii) Bz, 100 mg/kg of body weight per day, (iv) Sur, 20 mg/kg/day, and (v to viii) Sur, 20 mg/kg/day, combined with Bz, 100, 50, 25, or 5 mg/kg/day. Bz was administered by gavage, and Sur was administered intraperitoneally. Sur dramatically increased the parasitemia, cardiac content of parasite DNA, inflammation, oxidative tissue damage, and mortality. In response to high parasitic load in cardiac tissue, Sur stimulated the immune system in a manner typical of the acute phase of Chagas disease, increasing tissue levels of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and inducing a preferential IgG2a anti-T. cruzi serum pattern. When Sur and Bz were combined, the infection severity was attenuated, showing a dose-dependent Bz response. Sur therapy had a more harmful effect on the host than on the parasite and reduced the efficacy of Bz against T. cruzi infection. Considering that Sur drastically reinforced the infection evolution, potentiating the inflammatory process and the severity of cardiac lesions, the in vivo findings contradicted the in vitro anti-T. cruzi potential described for this drug.

Trypanosoma cruzi infection and benznidazole therapy independently stimulate oxidative status and structural pathological remodeling of the liver tissue in mice.

This study used a murine model of Chagas disease to investigate the isolated and combined impact of Trypanosoma cruzi infection and benznidazole (BZ) therapy on liver structure and function. Male C57BL/6 mice were challenged with T. cruzi and BZ for 15 days. Serum levels of cytokines and hepatic enzymes, liver oxidative stress, morphology, collagen, and glycogen content were monitored. Separately, T. cruzi infection and BZ treatment resulted in a pro-oxidant status and hepatic reactive damage. Concurrently, both T. cruzi infection and BZ treatment induced upregulation of antioxidant enzymes and pathological reorganization of the liver parenchyma and stroma. T. cruzi infection increased serum levels of Th1 cytokines, which were reduced by BZ in both infected and non-infected animals. BZ also induced functional organ damage, increasing serum levels of liver enzymes. When combined, T. cruzi infection and BZ therapy elicited intense hepatic reactive damage that was not compensated by antioxidant enzymatic reaction, subsequently culminating in more severe morphofunctional hepatic injury. Taken together, these findings indicate that during specific treatment of Chagas disease, hepatic pathology may be a result of an interaction between BZ metabolism and specific mechanisms activated during the natural course of T. cruzi infection, rather than an isolated toxic effect of BZ on liver structure and function.

Dengue-1 envelope protein domain III produced in Pichia pastoris: potential use for serological diagnosis.

Dengue is a major international public health concern. There is no drug to treat dengue virus infections and a vaccine is yet to be licensed. The laboratory diagnosis of dengue virus infection has been greatly improved during the last decade; therefore, the main limiting factor is the production of recombinant viral antigens on a large scale. Domain III of dengue virus envelope protein contains multiplex conformation-dependent neutralizing epitopes, making it an attractive diagnostic candidate. In this work, we have demonstrated the expression of dengue virus type 1 envelope domain III protein (EDIII-D1) in methylotrophic yeast, Pichia pastoris GS115. The recombinant secreted protein (sEDIII-D1) was purified by affinity chromatography and characterized by SDS-PAGE. Purified protein was recognized in immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) with dengue-infected human serum samples. In conclusion, secreted expressions of domain III protein can be obtained in P. pastoris by methanol induction. This product has the potential to be used for the diagnosis of dengue infections.

Effect of antibiotics and low-crude protein diets on growth performance, health, immune response, and fecal microbiota of growing pigs.

This study aimed to investigate the effects of diets with and without antibiotics supplementation and diets with 18.5% and 13.0% crude protein (CP) on growth performance, carcass characteristics, disease incidence, fecal microbiota, immune response, and antioxidant capacity of growing pigs. One hundred and eighty pigs (59-day-old; 18.5 ±â€…2.5 kg) were distributed in a randomized complete block design in a 2 × 2 factorial arrangement, nine replicates, and five pigs per pen. The factors were CP (18.5% or 13.0%) and antibiotics (none or 100 mg/kg tiamulin + 506 mg/kg oxytetracycline). Medicated diets were fed from days 59 to 73. After that, all pigs were fed their respective CP diets from 73 to 87 days. Data were analyzed using the Mixed procedure in SAS version 9.4. From days 59 to 73, pigs fed antibiotics diets had higher (P < 0.05) average daily feed intake (ADFI), average daily weight gain (ADG), gain to feed ratio (G:F), compared to the diets without antibiotics. From days 73 to 87 (postmedicated period), any previous supplementation of antibiotics did not affect pig growth performance. Overall (days 59 to 87), pigs-fed antibiotics diets had higher (P < 0.05) G:F compared to pigs-fed diets without antibiotics. In all periods evaluated, pigs fed 18.5% CP diets had higher (P < 0.05) ADG and G:F compared to pigs fed 13.0% CP. Pigs fed the 13.0% CP diets had lower (P < 0.05) fecal score and diarrhea incidence than those fed 18.5% CP. Pigs fed 18.5% CP diets had improved (P < 0.05) loin area compared to pigs-fed diets with 13.0% CP. At 66 days of age, pigs-fed antibiotics diets had lower (P < 0.05) alpha diversity estimated with Shannon and Simpson compared to the pig-fed diets without antibiotics. At family level, pigs fed 18.5% CP diets had higher (P < 0.05) relative abundance of Streptococcaceae, and lower (P < 0.05) relative abundance of Clostridiaceae at days 66 and 87 compared with pigs fed 13.0% CP. Pigs-fed antibiotics diets had lower (P < 0.05) immunoglobulin G and protein carbonyl concentrations at day 66 compared to the pigs-fed diets without antibiotics. The reduction of dietary CP from 18.5% to 13.0% reduced the growth performance and loin muscle area of growing pigs, although it was effective to reduce diarrhea incidence. Antibiotics improved growth performance, lowered diarrhea incidence, improved components of the humoral immune response, and reduced microbiota diversity. However, in the postmedicated period, we found no residual effect on the general health of the animals, and considering the overall period, only G:F was improved by the use of antibiotics.

Dietary antibiotics have been used in pig farming practices to avoid health problems and improve animal growth performance. However, their use in production animals is considered a global health challenge, due to its association with selection of resistance in zoonotic bacteria. Another negative impact of pig farming that has gained attention is related to environmental pollution due to the excretion of nitrogenous compounds. Reducing dietary crude protein content has become a goal in the pig feed industry due to the limited availability and high cost of dietary protein sources, as well as the aim of enhancing gut health in pigs. Thus, the aim of this study was to investigate the effects of diets with and without antibiotics supplementation and diets with 18.5% and 13.0% crude protein for pigs. The reduction of dietary crude protein in this study reduced growth performance, although it was effective to reduce diarrhea incidence. Antibiotics improved growth performance, positively affected the overall health of animals, and reduced microbiota diversity. However, during the postmedicated period, we found no residual effect on the general health of the animals, and considering the overall period, only gain to feed ratio was improved by the use of antibiotics.

Characterization and optimization of ArtinM lectin expression in Escherichia coli.

BACKGROUND: ArtinM is a d-mannose-specific lectin from Artocarpus integrifolia seeds that induces neutrophil migration and activation, degranulation of mast cells, acceleration of wound healing, induction of interleukin-12 production by macrophages and dendritic cells, and protective T helper 1 immune response against Leishmania major, Leishmania amazonensis and Paracoccidioides brasiliensis infections. Considering the important biological properties of ArtinM and its therapeutic applicability, this study was designed to produce high-level expression of active recombinant ArtinM (rArtinM) in Escherichia coli system. RESULTS: The ArtinM coding region was inserted in pET29a(+) vector and expressed in E. coli BL21(DE3)-Codon Plus-RP. The conditions for overexpression of soluble ArtinM were optimized testing different parameters: temperatures (20, 25, 30 or 37°C) and shaking speeds (130, 200 or 220 rpm) during induction, concentrations of the induction agent IPTG (0.01-4 mM) and periods of induction (1-19 h). BL21-CodonPlus(DE3)-RP cells induced under the optimized conditions (incubation at 20°C, at a shaking speed of 130 rpm, induction with 0.4 mM IPTG for 19 h) resulted in the accumulation of large amounts of soluble rArtinM. The culture provided 22.4 mg/L of rArtinM, which activity was determined by its one-step purification through affinity chromatography on immobilized d-mannose and glycoarray analysis. Gel filtration showed that rArtinM is monomeric, contrasting with the tetrameric form of the plant native protein (jArtinM). The analysis of intact rArtinM by mass spectrometry revealed a 16,099.5 Da molecular mass, and the peptide mass fingerprint and esi-cid-ms/ms of amino acid sequences of peptides from a tryptic digest covered 41% of the total ArtinM amino acid sequence. In addition, circular dichroism and fluorescence spectroscopy of rArtinM indicated that its global fold comprises ß-sheet structure. CONCLUSIONS: Overall, the optimized process to express rArtinM in E. coli provided high amounts of soluble, correctly folded and active recombinant protein, compatible with large scale production of the lectin.

Effect of amino acid blend as alternative to antibiotics for growing pigs.

This study aimed to evaluate the effect of supplementing arginine (Arg) + glutamine (Gln) replacing antibiotics on performance, immune response, and antioxidant capacity of pigs in the growing phase. One hundred fifty 63-d-old pigs with initial body weight (BW) of 25.0 ± 1.46 kg were distributed in a randomized block design, with three treatments and ten replicates. The three diets were control; antibiotic, control + 100 mg/kg tiamulin and 506 mg/kg oxytetracycline; amino acid, control + 10 g/kg Arg and 2 g/kg Gln. Dietary treatments were fed from 63 to 77 d. Following the treatment period, all pigs were fed the control diet from 77 to 90 d. Data were analyzed using GLIMMIX and UNIVARIATE in SAS 9.4. From 63 to 70 d, pigs fed diets with antibiotics had improved (P < 0.05) average daily feed intake, average daily weight gain (ADG), gain to feed ratio (G:F), and 70-d BW compared to those fed control or amino acid diets. From 70 to 77 d, including antibiotics in the diet increased (P < 0.05) ADG and 77-d BW. From 77 to 90 d, pigs fed control or amino acid diets had greater (P < 0.05) ADG than those fed an antibiotic diet. From 63 to 90 d, although pig performance was not affected (P > 0.05), growth curve of pigs fed the antibiotic diets was different (P < 0.05) from those fed the control and amino acids diets. At 70 d, serum tumor necrosis factor-α and diamine oxidase (DAO) were lower (P < 0.05) in pigs fed the antibiotic diet than the control diet, and pigs fed the amino acid diet had intermediate results. Ferric reducing antioxidant power (FRAP) was lower (P < 0.05) in pigs fed the amino acid diet than the antibiotic diet, and pigs fed the control diet had intermediate results. Serum immunoglobulin A was lower (P < 0.05) in pigs fed the antibiotic diet. At 77 d, DAO and serum immunoglobulin G were lower (P < 0.05) in pigs fed the antibiotic diet. FRAP was lower (P < 0.05) in pigs fed the amino acid and control diets. Serum malondialdehyde was higher (P < 0.05) in pigs fed the amino acid diet than those fed the control diet, and pigs fed the antibiotic diet had intermediate results. At 90 d, antibiotics or amino acids did not affect (P > 0.05) serum parameters. Amino acid blend supplementation at the selected doses in this study did not positively affect growing pigs. Although from 63 to 77 d, antibiotics improved performance, when considering the overall study period, growing pigs did not benefit from a diet containing antibiotics.

Dietary antibiotics have been used in pig farming practices to avoid health problems, improving animal growth performance. However, antimicrobial resistance due to the use of antibiotics in farms is considered to be a global health challenge. Arginine and glutamine are amino acids with potential to improve gut health, immune function, and growth performance. Thus, the study aimed to evaluate the supplementation of those amino acids as an alternative to the use of dietary antibiotic for pigs. Moreover, after a 14-d treatment phase, we still monitor the pigs to evaluate the carryover effects of the antibiotics and amino acids. Amino acid supplementation at the selected doses in this study did not positively affect pigs. Although during the treatment phase, antibiotics improved performance, when considering the overall study period, pigs did not benefit from a diet containing antibiotics. Thus, antibiotics caused transient alterations in pig performance and should be further investigated, potentially guiding future research on its use and alternative technologies.

Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM.

We determined the prophylactic effect of both the d-mannose-binding lectin ArtinM extracted from the seeds of Artocarpus integrifolia (jackfruit) and its recombinant counterpart during the course of experimental paracoccidioidomycosis induced in BALB/c mice. Four experimental protocols of prophylaxis were employed to evaluate the most protective regimen of ArtinM administration. It was demonstrated that the best effect was obtained by administration of two ArtinM doses on days 10 and 3 before the challenge with Paracoccidioides brasiliensis. By following this protocol, the lungs of mice that received native or recombinant ArtinM exhibited reduced fungal burden and granuloma incidence. In addition, the protocol augmented contents of IL-12, IFN-gamma, TNF-alpha and NO. On the other hand, the control group consisting of untreated infected mice had higher pulmonary levels of IL-4 and IL-10. In conclusion, prophylaxis with ArtinM significantly reproduces the effect of its therapeutic administration, i.e, it confers resistance to P. brasiliensis infection in mouse models by promoting IL-12 production and favours Th1-immunity.

Therapeutic administration of KM+ lectin protects mice against Paracoccidioides brasiliensis infection via interleukin-12 production in a toll-like receptor 2-dependent mechanism.

KM(+) is a mannose-binding lectin from Artocarpus integrifolia that induces interleukin (IL)-12 production by macrophages and protective T helper 1 immune response against Leishmania major infection. In this study, we performed experiments to evaluate the therapeutic activity of jackfruit KM(+) (jfKM(+)) and its recombinant counterpart (rKM(+)) in experimental paracoccidioidomycosis. To this end, jfKM(+) or rKM(+) was administered to BALB/c mice 10 days after infection with Paracoccidiodes brasiliensis. Thirty days postinfection, lungs from the KM(+)-treated mice contained significantly fewer colony-forming units and little to no organized granulomas compared to the controls. In addition, lung homogenates from the KM(+)-treated mice presented higher levels of nitric oxide, IL-12, interferon-gamma, and tumor necrosis factor-alpha, whereas higher levels of IL-4 and IL-10 were detected in the control group. With mice deficient in IL-12, Toll-like receptor (TLR) 2, TLR4, or TLR adaptor molecule MyD88, we demonstrated that KM(+) led to protection against P. brasiliensis infection through IL-12 production, which was dependent on TLR2. These results demonstrated a beneficial effect of KM(+) on the severity of P. brasiliensis infection and may expand its potential use as a novel immunotherapeutic molecule.

BjussuSP-I: a new thrombin-like enzyme isolated from Bothrops jararacussu snake venom.

A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom.

The antimicrobial peptide HS-1 inhibits dengue virus infection.

Dengue virus (DENV) is an arbovirus that belongs to the Flaviviridae family. Studies reveal that peptides secreted by amphibians have many functions, such as antiviral and antimicrobial activities. As there is no antiviral drug effective against the DENV, the antiviral activity of a synthetic peptide called HS-1, derived from the secretion of the anuran Hypsiboas semilineatus, has been evaluated. The assays of neutralization in the Vero cells show a complete inhibition of infection of the serotypes 2 and 3. Furthermore, the direct action of peptides on the viral particle can be observed through atomic force microscopy. In vivo tests display 80% protection against the dengue-2 virus due to the presence of HS-1, which reveals its potential as an antiviral against the DENV.

Purinergic Antagonist Suramin Aggravates Myocarditis and Increases Mortality by Enhancing Parasitism, Inflammation, and Reactive Tissue Damage in Trypanosoma cruzi-Infected Mice.

Suramin (Sur) acts as an ecto-NTPDase inhibitor in Trypanosoma cruzi and a P2-purinoceptor antagonist in mammalian cells. Although the potent antitrypanosomal effect of Sur has been shown in vitro, limited evidence in vivo suggests that this drug can be dangerous to T. cruzi-infected hosts. Therefore, we investigated the dose-dependent effect of Sur-based chemotherapy in a murine model of Chagas disease. Seventy uninfected and T. cruzi-infected male C57BL/6 mice were randomized into five groups: SAL = uninfected; INF = infected; SR5, SR10, and SR20 = infected treated with 5, 10, or 20 mg/kg Sur. In addition to its effect on blood and heart parasitism, the impact of Sur-based chemotherapy on leucocytes myocardial infiltration, cytokine levels, antioxidant defenses, reactive tissue damage, and mortality was analyzed. Our results indicated that animals treated with 10 and 20 mg/kg Sur were disproportionally susceptible to T. cruzi, exhibiting increased parasitemia and cardiac parasitism (amastigote nests and parasite load (T. cruzi DNA)), intense protein, lipid and DNA oxidation, marked myocarditis, and mortality. Animals treated with Sur also exhibited reduced levels of nonprotein antioxidants. However, the upregulation of catalase, superoxide dismutase, and glutathione-S-transferase was insufficient to counteract reactive tissue damage and pathological myocardial remodeling. It is still poorly understood whether Sur exerts a negative impact on the purinergic signaling of T. cruzi-infected host cells. However, our findings clearly demonstrated that through enhanced parasitism, inflammation, and reactive tissue damage, Sur-based chemotherapy contributes to aggravating myocarditis and increasing mortality rates in T. cruzi-infected mice, contradicting the supposed relevance attributed to this drug for the treatment of Chagas disease.

Synergism of Plant Compound With Traditional Antimicrobials Against Streptococcus spp. Isolated From Bovine Mastitis.

Mastitis is an inflammation of the mammary gland that causes major losses in the dairy industry. Streptococcus spp. are among the main agents of this disease. Increased resistance to antibiotics is one of the causes of therapeutic failure. Plants, due to their broad chemodiversity, are an interesting source of new molecules with antibacterial activity. Using these compounds along with traditional antibiotics is a possible method for reversing resistance. The objective of this work was to determine the interactions between the activities of guttiferone-A and 7-epiclusianone, two active substances isolated from the fruits of Garcinia brasiliensis, and traditional antibiotics against Streptococcus spp. isolated from bovine mastitis and known to be resistant to them. First, the MIC for the antibiotics and bioactive compounds was determined, followed by their activities, alone and in combination. Then, their cytotoxicity was measured in bovine mammary epithelial cells. Finally, molecular docking simulations were performed to elucidate molecular details of the interactions between ß-lactamase and the compounds binding to it (clavulanic acid, ampicillin, 7-epiclusianone, and guttiferone-A). The bacterial isolates were resistant to ampicillin and gentamicin. Both antibiotics showed predominantly synergistic antibacterial activities in combination with guttiferone-A or 7-epiclusianone. These two active substances were not cytotoxic at synergistic concentrations and both showed strong binding to ß-lactamase, which may explain the reversal of ampicillin resistance. These substances are promising for the treatment of bovine mastitis.

Toxicity of squamocin on Aedes aegypti larvae, its predators and human cells.

BACKGROUND: The mosquito Aedes aegypti transmits a virus that causes diverse human diseases, and control of the vector is an important strategy to avoid disease propagation. Plants in the family Annonaceae are recognised as sources of molecules with uses in the medical and agriculture fields. Molecules of secondary metabolites of Annonaceae plants exhibit insecticidal potential against insect pests and vectors, especially acetogenins, showing high toxicity at low doses, which has encouraged research into producing new insecticide molecules. Herein, we identify an acetogenin from Annona mucosa seeds (chemical analysis) and provide the results of toxicity tests against larvae of A. aegypti (target insect) and its predators Culex bigoti and Toxorhynchites theobaldi (non-target insects) and cytotoxicity to human leukocytes. RESULTS: We identified squamocin (C37 H66 O7 ), a fatty acid with a bis-tetrahydrofuran ring. In A. aegypti, this compound caused behavioural disturbance before larval death and high mortality at low concentrations (LC50 = 0.01 µg mL-1 and LC90 = 0.11 µg mL-1 ). However, in predators and human leukocytes, squamocin showed no toxicity effect, indicating the selectivity of this molecule for non-target organisms. CONCLUSION: We identified squamocin from A. mucosa seeds, which exhibited lethal action against A. aegypti and showed selectivity for non-target insects and low cytotoxicity to human cells. © 2016 Society of Chemical Industry.

Orange juice modulates proinflammatory cytokines after high-fat saturated meal consumption.

We aimed to evaluate the postprandial secretion of inflammatory markers induced by SFA or MUFA high-fat meal consumption and whether orange juice intake could modulate this induction. This study included 55 healthy women (aged 20 to 40 years): 33 participants received an SFA high-fat meal (≈1000 kcal, 37.6% of energy intake (E) from SFA) and 22 participants received an MUFA high-fat meal (≈1000 kcal, 56.3% E from MUFA). Both interventions were accompanied by 500 ml of orange juice (test) or water (control). The plasma concentrations of inflammatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α) and CRP were determined before (fasting) and 2, 3 and 5 hours after the test meal intake. The SFA high-fat meal induced a significant increase in AUC values (for TNF-α, IL-12, IL-10, IL-6 and IL-2 adjusted for baseline concentrations) in comparison with MUFA high-fat meal intervention. The results were independent of the drink which accompanied the meal (water or orange juice). Both IL-4 and IL-17A AUC values were significantly increased after an SFA high-fat meal intake, accompanied by water, but not by orange juice. In addition, these values were higher in relation to MUFA high-fat meal interventions. Also, IL-17A significantly increased at 3 h after an SFA high-fat meal intake accompanied by water, but not by orange juice. Overall, our conclusions indicate an anti-inflammatory effect of MUFA compared to SFA high-fat meal intake, while orange juice intake was able to mitigate the subclinical increase of postprandial inflammation, induced by SFA high-fat meal consumption, for a particular biomarker (IL-17A).

A new TRAF-like protein from B. oleracea ssp. botrytis with lectin activity and its effect on macrophages.

Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H2O2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H2O2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal.