Inhibitory effect of proteinase K against dermatophyte biofilms: an alternative for increasing the antifungal effects of terbinafine and griseofulvin.

This study aimed to evaluate the effect of proteinase K on mature biofilms of dermatophytes, by assays of metabolic activity and biomass. In addition, the proteinase K-terbinafine and proteinase K-griseofulvin interactions against these biofilms were investigated by the checkerboard assay and scanning electron and confocal microscopy. The biofilms exposed to 32 µg ml-1 of proteinase K had lower metabolic activity and biomass, by 39% and 38%, respectively. Drug interactions were synergistic, with proteinase K reducing the minimum inhibitory concentration of antifungals against dermatophyte biofilms at a concentration of 32 µg ml-1 combined with 128-256 µg ml-1 of terbinafine and griseofulvin. Microscopic images showed a reduction in biofilms exposed to proteinase K, proteinase K-terbinafine and proteinase K-griseofulvin combinations. These findings demonstrate that proteinase K has activity against biofilms of dermatophytes, and the interactions of proteinase K with terbinafine and griseofulvin improve the activity of drugs against mature dermatophyte biofilms.

Emergence of an outbreak-associated Clostridium difficile variant with increased virulence.

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 strains are resistant to fluoroquinolones due to a mutation in gyrA, and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAPCR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAPCR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAPCR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.

Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans.

OBJECTIVE: Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. METHODOLOGY: Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. RESULTS: CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. CONCLUSION: This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries.

Contribution of adenosine A(2B) receptors in Clostridium difficile intoxication and infection.

Clostridium difficile toxins A (TcdA) and B (TcdB) induce a pronounced systemic and intestinal inflammatory response. A(2B) adenosine receptors (A(2B)ARs) are the predominant adenosine receptors in the intestinal epithelium. We investigated whether A(2B)ARs are upregulated in human intestinal cells by TcdA or TcdB and whether blockade of A(2B)ARs can ameliorate C. difficile TcdA-induced enteritis and alter the outcome of C. difficile infection (CDI). Adenosine receptor subtype (A(1), A(2A), A(2B), and A(3)) mRNAs were assayed in HCT-8 cells. Ileal loops from wild-type rabbits and mice and A(2B)AR(-/-) mice were treated with TcdA, with or without the selective A(2B)AR antagonist ATL692 or PSB1115. A murine model of CDI was used to determine the effect of A(2B)AR deletion or blockade with the orally available agent ATL801, on clinical outcome, histopathology and intestinal interleukin-6 (IL-6) expression from infection. TcdA and TcdB upregulated A(2B)AR gene expression in HCT-8 cells. ATL692 decreased TcdA-induced secretion and epithelial injury in rabbit ileum. Deletion of A(2B)ARs reduced secretion and histopathology in TcdA-challenged mouse ileum. Deletion or blockade of A(2B)ARs reduced histopathology, IL-6 expression, weight loss, diarrhea, and mortality in C. difficile-infected mice. A(2B)ARs mediate C. difficile toxin-induced enteritis and disease. Inhibition of A(2B)AR activation may be a potential strategy to limit morbidity and mortality from CDI.

Paraprobiotic Enterococcus faecalis EC-12 prevents the development of irinotecan-induced intestinal mucositis in mice.

AIMS: This study tested the protective effect of purified paraprobiotic Enterococcus faecalis (EC-12) and an E. faecalis-based formulation (Med LanS) on irinotecan-induced intestinal mucositis murine model. MAIN METHODS: C57BL/6 male mice received saline, irinotecan (75 mg/Kg, i.p.), EC-12 (0.3, 1, or 3 × 107 CFU/Kg, p.o.) + irinotecan or Med Lan-S (3 × 107 CFU/Kg, p.o.) + irinotecan. Body mass variation was assessed daily, and blood samples were collected for evaluating bacteremia and leukocyte count. The ileum was harvested for myeloperoxidase assay, histopathology, quantitative PCR, and immunofluorescence for macrophages (F4/80), TLR4, and IL-18 binding protein (IL-18BP). KEY FINDINGS: The best therapeutic strategy was EC-12 administration at 3 × 107 CFU/Kg, starting 1 week before irinotecan. EC-12 and Med Lan-S did not prevent the irinotecan-induced body mass loss or leukopenia but attenuated the neutrophil infiltration in the intestine and increased the villus/crypt ratio (P < 0.05). Additionally, EC-12 and Med Lan-S reduced the mRNA expression of Cldn-2, Ocln, and Tlr4 versus the irinotecan group (P < 0.05). Irinotecan also augmented the expression of Il-18, IL-18BP, the immunofluorescence of F4/80, and TLR4, while only EC-12 prevented the expression of all these markers. Remarkably, EC-12 and Med Lan inhibited the irinotecan-induced bacterial translocation to the blood. SIGNIFICANCE: Paraprobiotic E. faecalis EC-12 prevents the development of intestinal mucositis by downregulating the inflammatory response. Med Lan-S also protects from mucositis. Possibly, the complexity of the formulation accounts for an innate immune-driven protective mechanism.

Dual responsive dextran-graft-poly (N-isopropylacrylamide)/doxorubicin prodrug via Schiff base reaction.

Stimulus-responsive nanoparticles stand out in studies for cancer treatment since these systems can promote a selective release of the drug in tumor tissues and cells, minimizing the effects caused by conventional chemotherapy. Dextran-graft-poly (N-isopropylacrylamide) copolymers were synthesized via Schiff base formation. The synthesis of copolymers was confirmed by Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (NMR) and the analyses of dynamic light scattering (DLS) showed that the copolymers were thermal and pH dual-responsive. The chemotherapy drug doxorubicin (DOX) was conjugated to the copolymers via Schiff base formation, obtaining nanoparticles by self-assembling with size smaller than 130 nm. A higher percentage of doxorubicin was released at pH 5.0 (59.1 ± 2.1%) compared to physiological pH (34.9 ± 4.8%), confirming a pH-sensitive release profile. The in vitro cytotoxicity assay demonstrated that DOX-loaded nanoparticles can inhibit cancer cell proliferation and promote reduced cytotoxicity in non-tumor cells. The D45kP30k-DOX nanoparticles induced morphological changes in HCT-116 cells suggesting cell death and the cell uptake assay indicated that the nanoparticles can be internalized by endocytosis. Therefore, DOX-loaded nanoparticles exhibited potential as smart systems for cancer treatment.

Papain immobilization on heterofunctional membrane bacterial cellulose as a potential strategy for the debridement of skin wounds.

We combined the chemical and physical methods of papain immobilization through the aldehyde groups available on oxidized bacterial cellulose (OxBC) to provide high proteolytic activity for future applications as bioactive dressing. Bacterial cellulose (BC) was obtained by the fermentation of Komagataeibacter hansenii in Hestrin-Schramm medium for 5 days, followed by purification and oxidation using NaIO4. Surface response methodology was used to optimize papain immobilization (2%, w/v) for 24 h. The independent variables: pH (3-7) and temperature (5 to 45 °C) were investigated. The mathematically validated optimal conditions of 45 °C and pH 7 had a statistical effect on the immobilization yield (IY) of papain in OxBC (52.9%). These ideal conditions were also used for papain immobilization in BC (unoxidized). The IY of 9.1% was lower than that of OxBC. OxBC-Papain and BC-Papain were investigated using thermal analysis, confocal microscopy, and diffusion testing. The OxBC support exhibited a more interactive chemical structure than the BC support, and was capable of immobilizing papain by covalent bonds (-C-NHR) and adsorption (ion exchange), with 93.3% recovered activity, 49.4% immobilization efficiency, and better thermal stability. Papain immobilized to OxBC by adsorption displayed 53% widespread papain activity. The results indicate the potential of prolonged bioactivity in debrided chronic wounds.

Chitosan microparticles loaded with essential oils inhibit duo-biofilms of Candida albicans and Streptococcus mutans

Abstract Oral candidiasis is a common fungal infection that affects the oral mucosa, and happens when Candida albicans interacts with bacteria in the oral microbiota, such as Streptococcus mutans, causing severe early childhood caries. C. albicans and S. mutans mixed biofilms are challenging to treat with conventional antimicrobial therapies, thus, new anti-infective drugs are required. Objective This study aimed to test a drug delivery system based on chitosan microparticles loaded with geranium and lemongrass essential oils to inhibit C. albicans and S. mutans mixed biofilms. Methodology Chitosan microparticles loaded with essential oils (CM-EOs) were obtained by spray-drying. Susceptibility of planktonic were performed according CLSI at 4 to 2,048 µg/mL. Mixed biofilms were incubated at 37ºC for 48 h and exposed to CM-EOs at 256 to 4,096 µg/mL. The antimicrobial effect was evaluated using the MTT assay, with biofilm architectural changes analyzed by scanning electron microscopy. RAW 264.7 cell was used to evaluate compound cytotoxicity. Results CM-EOs had better planktonic activity against C. albicans than S. mutans. All samples reduced the metabolic activity of mixed C. albicans and S. mutans biofilms, with encapsulated oils showing better activity than raw chitosan or oils. The microparticles reduced the biofilm on the slides. The essential oils showed cytotoxic effects against RAW 264.7 cells, but encapsulation into chitosan microparticles decreased their toxicity. Conclusion This study demonstrates that chitosan loaded with essential oils may provide an alternative method for treating diseases caused by C. albicans and S. mutans mixed biofilm, such as dental caries.

Metabolic acidosis aggravates experimental acute kidney injury.

AIMS: Ischemia/reperfusion (I/R) injury and metabolic acidosis (MA) are two critical conditions that may simultaneously occur in clinical practice. The result of this combination can be harmful to the kidneys, but this issue has not been thoroughly investigated. The present study evaluated the influence of low systemic pH on various parameters of kidney function in rats that were subjected to an experimental model of renal I/R injury. MAIN METHODS: Metabolic acidosis was induced in male Wistar rats by ingesting ammonium chloride (NH4Cl) in tap water, beginning 2 days before ischemic insult and maintained during the entire study. Ischemia/reperfusion was induced by clamping both renal arteries for 45 min, followed by 48 h of reperfusion. Four groups were studied: control (subjected to sham surgery, n=8), I/R (n=8), metabolic acidosis (MA; 0.28 M NH4Cl solution and sham surgery, n=6), and MA+I/R (0.28 M NH4Cl solution plus I/R, n=9). KEY FINDINGS: Compared with I/R rats, MA+I/R rats exhibited higher mortality (50 vs. 11%, p=0.03), significant reductions of blood pH, plasma bicarbonate (pBic), and standard base excess (SBE), with a severe decline in the glomerular filtration rate and tubular function. Microscopic tubular injury signals were detected. Immunofluorescence revealed that the combination of MA and I/R markedly increased nuclear factor κB (NF-κB) and heme-oxygenase 1 (HO-1), but it did not interfere with the decrease in endothelial nitric oxide synthase (eNOS) expression that was caused by I/R injury. SIGNIFICANCE: Acute ischemic kidney injury is exacerbated by acidic conditions.

Evidences for a progressive microglial activation and increase in iNOS expression in rats submitted to a neurodevelopmental model of schizophrenia: reversal by clozapine.

Schizophrenia was proposed as a progressive neurodevelopmental disorder. In this regard herein we attempted to determine progressive inflammatory and oxidative alterations induced by a neonatal immune challenge and its possible reversal by clozapine administration. For this end, Wistar rats at postnatal day (PN) 5-7 were administered the viral mimetic polyriboinosinic-polyribocytidilic acid (polyI:C) or saline. A distinct group of animals additionally received the antipsychotic drug clozapine (25mg/kg) from PN60 to 74. At PN35 (periadolescence), 60 (adult) and 74 (adulthood) the animals were submitted to behavioral determinations of prepulse inhibition of the startle (PPI) and Y maze task for working memory evaluation. At PN35 and 74 the animals were sacrificed and the hippocampus (HC), prefrontal cortex (PFC) and striatum (ST) immunostained for Iba-1, a microglial marker, and inducible nitric oxide synthase (iNOS). At PN74 oxidative stress parameters, such as, reduced glutathione levels (GSH) and lipid peroxidation were determined. The results showed a progressive increase of microglial activation and iNOS immunostaining from PN35 to PN74 mainly in the CA2 and CA3 regions of the HC and in the ST. At PN74 neonatal challenge also induced an oxidative imbalance. These inflammatory alterations were accompanied by deficits in PPI and working memory only in adult life that were reversed by clozapine. Clozapine administration reversed microglial activation and iNOS increase, but not the alterations of oxidative stress parameters. Taken together these results give further evidences for a neuroprogressive etiology and course of schizophrenia and that clozapine may partly alleviate this process.

Cryptosporidium-malnutrition interactions: mucosal disruption, cytokines, and TLR signaling in a weaned murine model.

Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3-12 days, then were infected with 5 × 10(7) excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height-crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

Synthesis and antioxidant, anti-inflammatory and gastroprotector activities of anethole and related compounds.

Some derivatives of trans-anethole [1-methoxy-4-(1-propenyl)-benzene] (1) were synthesized, by introducing hydroxyl groups in the double bond of the propenyl moiety. Two types of reactions were performed: (i) oxymercuration/demercuration that formed two products, the mono-hydroxyl derivative, 1-hydroxy-1-(4-methoxyphenyl)-propane (2) and in lesser extent the dihydroxyl derivative, 1,2-dihydroxy-1-(4-methoxyphenyl)-propane (3) and (ii) epoxidation with m-chloroperbenzoic acid that also led to the formation of two products, the dihydroxyl derivative (3) and the correspondent m-chloro-benzoic acid mono-ester, 1-hydroxy-1(4-methoxyphenyl)-2-m-chlorobenzoyl-propane (4). The structures of these compounds were confirmed mainly by mass, IR, 1H and 13C NMR spectral data. The activity of anethole and hydroxylated derivatives was evaluated using antioxidant, anti-inflammatory and gastroprotector tests. Compounds (2) and (3) were more active antioxidant agents than (1) and (4). In the anti-inflammatory assay, anethole showed lower activity than hydroxylated derivatives. Anethole and in lesser extent its derivatives 2 and 4 showed significant gastroprotector activity. All tested compounds do not alter significantly the total number of white blood cells.