Antimicrobial effects of bacterial binding to a dialkylcarbamoyl chloride-coated wound dressing: an in vitro study.

OBJECTIVE: Wound dressings that inactivate or sequestrate microorganisms, such as those with a hydrophobic, bacteria-binding dialkylcarbamoyl chloride (DACC) surface, can reduce the risk of clinical infections. This 'passive' bioburden control, avoiding bacterial cell wall disruption with associated release of bacterial endotoxins aggravating inflammation, is advantageous in hard-to-heal wounds. Hence, the full scope of DACC dressings, including the potential impact of higher inoculum densities, increased protein load and different pH on antibacterial activity, needs to be evaluated. METHOD: The Japanese Industrial Standard (JIS) L 1902 challenge test was used to evaluate the antimicrobial activity of the DACC-coated dressing against several World Health Organization (WHO)-prioritised wound pathogens (e.g., meticillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, microorganisms with extended-spectrum beta-lactamases and Acinetobacter baumannii), the effect of repeated bacterial challenge in an adverse wound environment, and antimicrobial performance at wound-related pH. RESULTS: High antibacterial activity of the DACC-coated dressing against the WHO-prioritised bacteria strains by its irreversible binding and inhibition of growth of bound bacteria was confirmed using JIS L 1902. At increased inoculation densities, compared to standard conditions, the DACC-coated dressing still achieved strong-to-significant antibacterial effects. Augmenting the media protein content also affected antibacterial performance; a 0.5-1 log reduction in antibacterial activity was observed upon addition of 10% fetal calf serum. The pH did not influence antibacterial performance. The DACC-coated dressing also sustained antibacterial activity over subsequent reinfection steps. CONCLUSION: It can be assumed that the DACC-coated dressing exerts beneficial effects in controlling the wound bioburden, reducing the overall demand placed on antibiotics, without using antimicrobial substances.

Significant and rapid reduction of free endotoxin using a dialkylcarbamoyl chloride-coated wound dressing.

OBJECTIVE: Endotoxin causes inflammation and can impair wound healing. Conventional methods that reduce bioburden in wounds by killing microorganisms using antibiotics, topical antimicrobials or antimicrobial dressings may induce endotoxin release from Gram-negative bacteria. Another approach is to reduce bioburden by adsorbing microorganisms, without killing them, using dialkylcarbamoyl chloride (DACC)-coated wound dressings. This study evaluated the endotoxin-binding ability of a DACC-coated wound dressing (Sorbact Compress, Abigo Medical AB, Sweden) in vitro, including its effect on the level of natural endotoxin released from Gram-negative bacteria. METHOD: Different concentrations of purified Pseudomonas aeruginosa endotoxin and a DACC-coated dressing were incubated at 37°C for various durations. After incubation, the dressing was removed and endotoxin concentration in the solution was quantified using a Limulus amebocyte lysate (LAL) assay. The DACC-coated dressing was also incubated with Pseudomonas aeruginosa cells for one hour at 37°C. After incubation, the dressing and bacterial cells were removed and shed endotoxin remaining in the solution was quantified. RESULTS: Overnight incubation of the DACC-coated wound dressing with various concentrations of purified Pseudomonas aeruginosa endotoxin (96-11000 EU/ml) consistently and significantly reduced levels of free endotoxin by 93-99% (p<0.0001). A significant endotoxin reduction of 39% (p<0.001) was observed after five minutes. The DACC-coated dressing incubated with clinically relevant Pseudomonas aeruginosa cells also reduced shed endotoxin by >99.95% (p<0.0001). CONCLUSION: In this study, we showed that a DACC-coated wound dressing efficiently and rapidly binds both purified and shed endotoxin from Pseudomonas aeruginosa in vitro. This ability to remove both endotoxin and bacterial cells could promote the wound healing process.

Effect of a DACC-coated dressing on keratinocytes and fibroblasts in wound healing using an in vitro scratch model.

Wound dressings that exert an antimicrobial effect in order to prevent and treat wound infections can be harmful to the wound healing process. Dressings with hydrophobic coatings, however, have been suggested to both reduce the microbial load and promote the healing process. Therefore, the potential effects of a dialkylcarbamoyl chloride (DACC)-coated dressing on fibroblasts and keratinocytes in wound healing were studied using mechanical scratch wounding of confluent cell layers as an in vitro model. Additionally, gene expression analysis by qRT-PCR was used to elucidate the longitudinal effects of the DACC-coated dressing on cell responses, specifically inflammation, growth factor induction and collagen synthesis. DACC promoted cell viability, did not stick to the cell layers, and supported normal wound healing progression in vitro. In contrast, cells became attached to the uncoated reference material, which inhibited scratch closure. Moreover, DACC slightly induced KGF, VEGF, and GM-CSF expression in HaCaT cells and NHDF. Physiological COL1A1 and COL3A1 gene expression by NHDF was observed under DACC treatment with no observable effect on S100A7 and RNASE7 levels in HaCaT cells. Overall, the DACC coating was found to be safe and may positively influence the wound healing outcome. Graphical abstract.

Establishment of esophageal-like non-keratinized stratified epithelium using normal human bronchial epithelial cells.

Current experimental models of esophageal epithelium in vitro suffer from either poor differentiation or complicated culture systems. We have established a model to study stratified squamous epithelium in vitro, which is very similar to esophageal epithelium in vivo. A stratified squamous multilayer epithelium was formed by seeding primary normal human bronchial epithelial (NHBE) cells onto collagen- and fibronectin-coated trans-well inserts and then cultivating the cells under air-liquid interface (ALI) conditions in the presence of growth factors and low levels of all-trans-retinoic acid. Trans-epithelial electrical resistance (TEER) measurements revealed the presence of a tight barrier, previously only achievable with esophageal biopsies mounted in Ussing chambers. Molecular markers for desmosomes, cornified envelope, tight junctions, and mature esophageal epithelium were upregulated in the differentiating culture in parallel with functional properties, such as decreased permeability and acid resistance and restoration. Acid exposure resulted in a decrease in TEER, but following 1-h recovery the TEER values were fully restored. Treatment with all-trans-retinoic acid decreased TEER and inhibited the recovery after acid challenge. PPAR-delta agonist treatment increased TEER, and this temporary increase in TEER was consistent with an increase in involucrin mRNA. Global gene expression analysis showed that ALI-differentiated NHBE cells had expression profiles more similar to epithelial biopsies from the esophageal tissue of healthy volunteers than to any other cell line. With respect to morphology, molecular markers, barrier properties, and acid resistance, this model presents a new way to investigate barrier properties and the possible effects of different agents on human esophagus-like epithelium.

Down regulation of TRPC1 by shRNA reduces mechanosensitivity in mouse dorsal root ganglion neurons in vitro.

Mechanosensitivity is a crucial but poorly understood property of the sensory nervous system. Transient receptor potential (TRP) channels, which have been found to be responsible for the detection of other sensory stimuli such as temperature and pungent chemicals, have been suggested to also recognize stretch or pressure to cell membranes. TRPC1 is one candidate from studies in oocytes but evidence in native sensory neurons has been lacking. Therefore, we have measured an increase in intracellular Ca(2+) levels upon mechanical activation of native mouse dorsal root ganglion (DRG) neurons in culture using hypoosmolar buffer. Our results show that down regulation of TRPC1 with short hairpin RNA results in a 65% reduction of neurons with stretch activated responses. These results implicate a direct or indirect involvement of TRPC1 in the mechanosensitivity of DRG neurons.

Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes.

BACKGROUND & AIMS: Clostridium difficile toxin B (TxB) mediates acute inflammatory diarrhea characterized by neutrophil infiltration and intestinal mucosal injury. In a xenograft animal model, TxB was shown to induce interleukin (IL)-8 gene expression in human colonic epithelium. However, the precise mechanisms of this TxB response are unknown. The aim of this study was to investigate the TxB-mediated proinflammatory pathway in colonocytes. METHODS: The effect of TxB on epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) 1/2 signaling pathway and IL-8 gene expression was assessed in nontransformed human colonic epithelial NCM460 cells. TxB regulation of EGFR-ERK1/2 signaling pathways was determined using immunoblot analysis, confocal microscopy, and enzyme-linked immunosorbent assay, whereas IL-8 gene expression was measured by luciferase promoter assay. RESULTS: TxB activates EGFR and ERK1/2 phosphorylation with subsequent release of IL-8 from human colonocytes. Pretreatment with either the EGFR tyrosine kinase inhibitor, AG1478, or an EGFR-neutralizing antibody blocked both TxB-induced EGFR and ERK activation. By using neutralizing antibodies against known ligands of EGFR, we found that the activation of EGFR and ERK1/2 phosphorylation was mediated by transforming growth factor-alpha (TGF-alpha). Inhibition of matrix metalloproteinase (MMP) decreased TGF-alpha secretion and TxB-induced EGFR and ERK activation. Inhibition of MMP, EGFR, and ERK activation significantly decreased TxB-induced IL-8 expression. CONCLUSIONS: TxB signals acute proinflammatory responses in colonocytes by transactivation of the EGFR and activation of the ERK/MAP kinase pathway.

Follicular growth of a thyroid carcinoma cell line (KAT-4) with abnormal E-cadherin and impaired epithelial barrier.

Loss of the epithelial phenotype is a well-established phenomenon during progression of carcinomas to a more malignant state. In the present study, we describe a human thyroid tumor cell line (KAT-4), established from a poorly differentiated carcinoma, which displays exceptional features. In culture, the KAT-4 cells had a fast proliferation rate that was not restricted by high cell density, resulting in multilayered growth. Unexpectedly, the cells expressed normal levels of epithelial markers, e.g., cytokeratin, occludin, and E-cadherin, showed apical-basolateral polarization of the plasma membrane including microvilli and junction complexes, and formed intercellular lumens resembling thyroid follicles. Yet, when grown on filter, the cells were unable to establish a tight paracellular barrier. Moreover, E-cadherin expressed at the cell surface consisted of two peptides with abnormal size (135 and 95 kd, respectively) as compared to mature E-cadherin (120 kd) in nonneoplastic thyrocytes. Northern blot analysis and examination of immunoreactivity, glycosylation, and catenin binding suggested that E-cadherin was aberrant because of altered posttranscriptional processing. Thus, the KAT-4 thyroid carcinoma cell line has a unique phenotype, with maintained epithelial morphology despite dysfunctioning tight junctions, abnormal E-cadherin, and loss of contact-inhibited growth, that is not previously identified in other wild-type tumor cell lines.