The In Vitro Susceptibility of Biofilm Forming Medical Device Related Pathogens to Conventional Antibiotics

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) and kill kinetics were established for vancomycin, rifampicin, trimethoprim, gentamicin, and ciprofloxacin against the biofilm forming bacteria Staphylococcus epidermidis (ATCC 35984), Staphylococcus aureus (ATCC 29213), Methicillin Resistant Staphylococcus aureus (MRSA) (ATCC 43300), Pseudomonas aeruginosa (PAO1), and Escherichia coli (NCTC 8196). MICs and MBCs were determined via broth microdilution in 96-well plates. MBECs were studied using the Calgary Biofilm Device. Values obtained were used to investigate the kill kinetics of conventional antimicrobials against a range of planktonic and biofilm microorganisms over a period of 24 hours. Planktonic kill kinetics were determined at 4xMIC and biofilm kill kinetics at relative MBECs. Susceptibility of microorganisms varied depending on antibiotic selected and phenotypic form of bacteria. Gram-positive planktonic isolates were extremely susceptible to vancomycin (highest MBC: 7.81 mg L−1: methicillin sensitive and resistant S. aureus) but no MBEC value was obtained against all biofilm pathogens tested (up to 1000 mg L−1). Both gentamicin and ciprofloxacin displayed the broadest spectrum of activity with MIC and MBCs in the mg L−1 range against all planktonic isolates tested and MBEC values obtained against all but S. epidermidis (ATCC 35984) and MRSA (ATCC 43300).

SMAD inhibition attenuates epithelial to mesenchymal transition by primary keratinocytes in vitro

Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype and contribute directly to fibrotic disease. Recent studies support a role for EMT in cutaneous fibrotic diseases including scleroderma and hypertrophic scarring, though there is limited data on the cytokines and signalling mechanisms regulating cutaneous EMT. We investigated the ability of TGF-β and TNF-α, both over-expressed in cutaneous scleroderma and central mediators of EMT in other epithelial cell types, to induce EMT in primary keratinocytes and studied the signalling mechanisms regulating this process. TGF-β induced EMT in normal human epidermal keratinocytes (NHEK cells) and this process was enhanced by TNF-α. EMT was characterised by changes in morphology, proteome (down-regulation of E-cadherin and Zo-1, and up-regulation of vimentin and fibronectin), MMP secretion and COL1α1 mRNA expression. TGF-β and TNF-α in combination activated SMAD and p38 signalling in NHEK cells. P38 inhibition with SB203580 partially attenuated EMT, whereas SMAD inhibition using SB431542 significantly inhibited EMT and also reversed established EMT. These data highlight the retained plasticity of adult keratinocytes and support further studies of EMT in clinically relevant in vivo models of cutaneous fibrosis, and investigation of SMAD inhibition as a potential therapeutic intervention. This article is protected by copyright. All rights reserved.

Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

Effects of Undaria pinnatifida, Himanthalia elongata and Porphyra umbilicalis extracts on in vitro α-glucosidase activity and glucose diffusion

Background: Seaweeds are good sources of dietary fibre, which can influence glucose uptake and glycemic control.Objective: To investigate and compare the in vitro inhibitory activity of different extracts from Undaria pinnatifida (Wakame), Himanthalia elongata (Sea spaghetti) and Porphyra umbilicalis (Nori) on α-glucosidase activity and glucose diffusion.Methods: The in vitro effects chloroform-, ethanol- and water-soluble extracts of the three algae were assayed on α- glucosidase activity and glucose diffusion through membrane. Principal Components Analysis (PCA) was applied to identify patterns in the data and to discriminate which extract will show the most proper effect.Results: Only water extracts of Sea spaghetti possessed significant in vitro inhibitory effects on α-glucosidase activity (26.2% less mmol/L glucose production than control, p < 0.05) at 75 min. PCA distinguished Sea spaghetti effects, supporting that soluble fibre and polyphenols were involved. After 6 h, Ethanol-Sea spaghetti and water-Wakame extracts exerted the highest inhibitory effects on glucose diffusion (65.0% and 60.2% vs control, respectively). This extracts displayed the lowest slopes for glucose diffusion-time lineal adjustments (68.2% and 62.8% vs control, respectively).Conclusions: The seaweed hypoglycemic effects appear multi-faceted and not necessarily concatenated. According to present results, ethanol and water extracts of Sea spaghetti, and water extracts of Wakame could be useful for the development of functional foods with specific hypoglycemic properties.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Expression of toll-like receptors by human muscle cells in vitro and in vivo:TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Aflatoxin exposure is inversely associated with IGF1 and IGFBP3 levels in vitro and in Kenyan schoolchildren

SCOPE: This study explores the relationship between aflatoxin and the insulin-like growth factor (IGF) axis and its potential effect on child growth.

METHODS AND RESULTS: One hundred and ninety-nine Kenyan schoolchildren were studied for aflatoxin-albumin adduct (AF-alb), IGF1 and IGF-binding protein-3 (IGFBP3) levels using ELISA. AF-alb was inversely associated with IGF1 and IGFBP3 (p < 0.05). Both IGF1 and IGFBP3 were significantly associated with child height and weight (p < 0.01). Children in the highest tertile of AF-alb exposure (>198.5 pg/mg) were shorter than children in the lowest tertile (<74.5 pg/mg), after adjusting for confounders (p = 0.043). Path analysis suggested that IGF1 levels explained ∼16% of the impact of aflatoxin exposure on child height (p = 0.052). To further investigate this putative mechanistic pathway, HHL-16 liver cells (where HHL-16 is human hepatocyte line 16 cells) were treated with aflatoxin B1 (0.5, 5 and 20 μg/mL for 24-48 h). IGF1 and IGFBP3 gene expression measured by quantitative PCR and protein in culture media showed a significant down-regulation of IGF genes and reduced IGF protein levels.

CONCLUSION: Aflatoxin treatment resulted in a significant decrease in IGF gene and protein expression in vitro. IGF protein levels were also lower in children with the highest levels of AFB-alb adducts. The data suggest that aflatoxin-induced changes in IGF protein levels could contribute to growth impairment where aflatoxin exposure is high.

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.

Methodology/Principal Findings: We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.

Conclusions/Significance: Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro

Background: Fasciola spp. liver fluke cause pernicious disease in humans and animals. Whilst current control is unsustainable due to anthelmintic resistance, gene silencing (RNA interference, RNAi) has the potential to contribute to functional validation of new therapeutic targets. The susceptibility of juvenile Fasciola hepatica to double stranded (ds)RNA-induced RNAi has been reported. To exploit this we probe RNAi dynamics, penetrance and persistence with the aim of building a robust platform for reverse genetics in liver fluke. We describe development of standardised RNAi protocols for a commercially-available liver fluke strain (the US Pacific North West Wild Strain), validated via robust transcriptional silencing of seven virulence genes, with in-depth experimental optimisation of three: cathepsin L (FheCatL) and B (FheCatB) cysteine proteases, and a σ-class glutathione transferase (FheσGST).

Methodology/Principal Findings: Robust transcriptional silencing of targets in both F. hepatica and Fasciola gigantica juveniles is achievable following exposure to long (200–320 nt) dsRNAs or 27 nt short interfering (si)RNAs. Although juveniles are highly RNAi-susceptible, they display slower transcript and protein knockdown dynamics than those reported previously. Knockdown was detectable following as little as 4h exposure to trigger (target-dependent) and in all cases silencing persisted for ≥25 days following long dsRNA exposure. Combinatorial silencing of three targets by mixing multiple long dsRNAs was similarly efficient. Despite profound transcriptional suppression, we found a significant time-lag before the occurrence of protein suppression; FheσGST and FheCatL protein suppression were only detectable after 9 and 21 days, respectively.

Conclusions/Significance: In spite of marked variation in knockdown dynamics, we find that a transient exposure to long dsRNA or siRNA triggers robust RNAi penetrance and persistence in liver fluke NEJs supporting the development of multiple-throughput phenotypic screens for control target validation. RNAi persistence in fluke encourages in vivo studies on gene function using worms exposed to RNAi-triggers prior to infection.

In Vitro Release from Reverse Poloxamine/alpha-Cyclodextrin Matrices: Modelling and Comparison of Dissolution Profiles

Gels obtained by complexation of octablock star polyethylene oxide/polypropylene oxide copolymers (Tetronic 90R4) with -cyclodextrin (-CD) were evaluated as matrices for drug release. Both molecules are biocompatible so they can be potentially applied to drug delivery systems. Two different types of matrices of Tetronic 90R4 and -CD were evaluated: gels and tablets. These gels are capable to gelifying in situ and show sustained erosion kinetics in aqueous media. Tablets were prepared by freeze-drying and comprising the gels. Using these two different matrices, the release of two model molecules, L-tryptophan (Trp), and a protein, bovine serum albumin (BSA), was evaluated. The release profiles of these molecules from gels and tablets prove that they are suitable for sustained delivery. Mathematical models were applied to the release curves from tablets to elucidate the drug delivery mechanism. Good correlations were found for the fittings of the release curves to different equations. The results point that the release of Trp from different tablets is always governed by Fickian diffusion, whereas the release of BSA is governed by a combination of diffusion and tablet erosion. 

Lack of in vitro-in vivo correlation for a UC781-releasing vaginal ring in macaques

This study describes the preclinical development of a matrix-type silicone elastomer vaginal ring device designed to provide controlled release of UC781, a non-nucleoside re- verse transcriptase inhibitor. Testing of both human- and macaque-sized rings in a sink condition in vitro release model demonstrated continuous UC781 release in quantities consid- ered sufficient to maintain vaginal fluid concentrations at levels 82–860-fold higher than the in vitro IC50 (2.0 to 10.4 nM) and therefore potentially protect against mucosal trans- mission of HIV. The 100-mg UC781 rings were well tolerated in pig-tailed macaques, did not induce local inflammation as determined by cytokine analysis and maintained median con- centrations in vaginal fluids of UC781 in the range of 0.27 to 5.18 mM during the course of the 28-day study. Analysis of residual UC781 content in rings after completion of both the in vitro release and macaque pharmacokinetic studies revealed that 57 and 5 mg of UC781 was released, respectively. The pharmacokinetic analysis of a 100-mg UC781 vaginal ring in pig-tailed macaques showed poor in vivo–in vitro correlation, attributed to the very poor solubility of UC781 in vaginal fluid and resulting in a dissolution-controlled drug release mecha- nism rather than the expected diffusion-controlled mechanism.

An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays

Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01–100 μM) was cytotoxic in the H295R and LC models used; following 48 h incubation with 100 μM. Flow cytometry analysis showed that ENN B exposure (0.1–25 μM) led to an increased proportion of cells in the S phase at higher ENN B doses (>10 μM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156–15.6 μM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6 μM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100 μM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p < 0.05) by ENN B (10 μM) compared to the control. Genes HMGR, StAR, CYP11A, 3βHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10 μM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.