Role of IL-13Rα2 in modulating IL-13-induced MUC5AC and ciliary changes in healthy and CRSwNP mucosa.

BACKGROUND: The IL-13 receptor α2 (IL-13Rα2) is a receptor for IL-13 which has conflicting roles in mediating IL-13 responses in the lower airway, with little known about its impact on upper airway diseases. We sought to investigate the expression of IL-13 receptors, IL-13Rα1 and IL-13Rα2, in chronically inflamed nasal epithelium, and explore IL-13-induced signaling pathways in an in vitro model of human nasal epithelial cells (hNECs). METHODS: The protein and mRNA expression levels of IL-13 and its receptors in nasal biopsies of patients with nasal polyps (NP) and healthy controls were evaluated. We investigated goblet cell stimulation with mucus hypersecretion induced by IL-13 (10 ng/mL, 72 hours) treatment in hNECs using a pseudostratified epithelium in air-liquid interface (ALI) culture. RESULTS: There were significant increases in IL-13, IL-13Rα1, and IL-13Rα2 mRNA and protein levels in NP epithelium with healthy controls as baseline. MUC5AC mRNA positively correlated with IL-13Rα2 (r = .5886, P = .002) but not with IL-13Rα1 in primary hNECs. IL-13 treatment resulted in a significant increase in mRNA and protein levels of IL-13Rα2 only in hNECs. IL-13 treatment induced an activation of extracellular signal-regulated kinases (ERK)1/2 and an upregulation of C-JUN, where the IL-13-induced effects on hNECs could be attenuated by ERK1/2 inhibitor (50 µmol/L) or dexamethasone (10-4 -10-7  mol/L) treatment. CONCLUSIONS: IL-13Rα2 has a potential role in IL-13-induced MUC5AC and ciliary changes through ERK1/2 signal pathway in the nasal epithelium. IL-13Rα2 may contribute to airway inflammation and aberrant remodeling which are the main pathological features of CRSwNP.

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces.

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.

Antibiograms, Resistance Genes, Class I Integrons and PFGE profiles of Zoonotic Salmonella in Malaysia.

Salmonella infections occur worldwide, in both developed and developing countries, and a major contributor to morbidity and economic costs. A total of 32 Salmonella isolates isolated from dogs (n=15/162), cats (n=1/126) and snakes (n=16/42) in the Klang valley, Peninsular Malaysia during 2012-2013, were used in this study and 6 serovars were identified. The isolates were then characterized for their susceptibility to commonly used antimicrobial agents using the standard disk diffusion method. The presence of relevant resistance genes and class 1 integrons were investigated by using PCR. Pulsed-field gel electrophoresis (PFGE) was carried out to determine the genetic diversity of these Salmonella strains. Higher resistance rates were observed for tetracycline (40.6%), nalidixic acid (21.9%), sulphamethazole-trimethoprim (18.7%), ampicillin (18.7%) followed by chloramphenicol (15.6%), streptomycin (6.25%), enrofloxacin (12.5%), cephalexin (6.25%), cephalothin (6.25%) and amoxicillin-clavulanic acid (3.12%). Nine percent (3/32) presented a single type of resistance, 6% (2/32) showed resistance to two classes of antimicrobials and 34% (11/32) were multidrugresistant (MDR) (resistant to 3 or more antimicrobials). Analysis of the carriage of resistance genes in the isolates revealed that seven (blaTEM-1, strA, strB, sulII, dfrhI, tetA, and cmlA) out of 10 resistance genes were present. Classes 1 integrons were present in 68.75% (11/16) of the resistance strains. PFGE analysis showed that the strains were very diverse and certain PFGE pattern clusters correlated well with antimicrobial resistance phenotypes. In conclusion, high rates of multidrug resistance were found among the dogs Salmonella strains.

Bacteria, viruses, yeasts and protozoans associated with diarrheal disease in Singapore.

Labile toxin producing enterotoxic E. coli (ETEC) were the commonest pathogen isolated from diarrheal stools of hospitalized children (21%) and adults (26%) in Singapore. Salmonellas ranked a close second in children (19%). Other bacterial pathogens were isolated from less than 5% of subjects. Blastocystis hominis was detected in 4.3% of diarrheal stools when a simple sedimentation technique was used. Cryptosporidium was not detected at all. An analysis of yeast counts in smears of diarrheal and non-diarrheal stools suggested they were etiologically associated with at least 6% of diarrhea in children and 19% in adults. Testing for rotaviruses by Latex agglutination and for adenovirus by electronmicroscopy showed an association with 6 per cent and 3 per cent diarrhea respectively. The study highlighted a need for: case control studies on ETEC and B. hominis; studies on the epidemiology of diarrhea by yeasts; establishing the true incidence of adenovirus diarrhea; studies on the prevalence and seasonality of rotavirus infection in Singapore.

Comparison of Pseudomonas pseudomallei from humans, animals, soil and water by restriction endonuclease analysis.

Pseudomonas pseudomallei isolates from 62 human, 17 animal, 3 soil and 3 water samples were examined by genomic DNA digestion with PstI. Five major (RE I, II, III, IV, V) reproducible restriction patterns were observed, with most (56/62) of the human isolates displaying RE I (30/62), II (5/62), III (15/62), IV (4/62), V (2/62), and the animal (16/17), soil (2/3), water (3/3) isolates showing predominantly RE II profiles. Six human and one soil isolates showed patterns different from those of RE I to V. Restriction endonuclease analysis may be applied in epidemiological studies of melioidosis.

Counterimmunoelectrophoresis in the rapid diagnosis of bacterial meningitis.

Cerebrospinal fluid from patients with clinically diagnosed meningitis was tested for meningococcal, pneumococcal, streptococcal Group B and Haemophilus influenzae antigens by counterimmunoelectrophoresis. Antigens were rapidly identified and the results compared favourably with that of bacteriological culture. In the case of pneumococcal meningitis counterimmunoelectrophoresis proved to be more sensitive than culture. The procedure was shown to be sensitive, specific, rapid and easily performed.

A clinically viable index for quantifying denture plaque.

A denture plaque index using the dye disclosing method was evaluated in 24 patients with Type II denture stomatitis and 17 control subjects with healthy palatal mucosa. Patients with denture stomatitis had statistically significantly higher plaque scores than did controls, indicating a quantitative increase in denture plaque in patients with denture stomatitis. When this plaque index was tested by two examiners for intraexaminer and interexaminer reproducibility, it yielded a 92% to 96% reproducibility. The plaque index used in this study seems to satisfy the criteria for an ideal clinical index: It is simple, reliable, reproducible, and economical and can be carried out in the shortest possible time in a clinical setting.

Clonal growth of Blastocystis hominis in soft agar with sodium thioglycollate.

The present report describes a method for establishment of colonies of Blastocystis hominis from single cells in soft agar. The percentage of colony-forming efficiency (% CFE = number of colonies grown/number of cells inoculated x 100) for the cultures was greatly improved by the addition of sodium thioglycollate. Five human Blastocystis isolates chosen for this study showed no apparent variation in colonial morphology. Isolated colonies were also successfully grown in liquid medium, providing a means of obtaining large numbers of B. hominis cells that had arisen from a single clone.

Sudden unexplained death syndrome--a new manifestation in melioidosis?

The indirect haemagglutination (IHA) test using sensitized turkey erythrocytes and the indirect immunofluorescence assay (IgM-IFA) was confirmed to be sensitive in the detection of a recent or current Pseudomonas pseudomallei infection in 19 culture-confirmed Singapore melioidosis patients. All were found to have antibody titres from 4 to 32768 in the IHA test and 10 to 320 in the IgM-IFA test. When these tests were employed on sera from 16 immigrant Thai construction workers who died of sudden unexplained death syndrome (SUDS) and 73 healthy Thai fellow workers, 93.8% and 68.8% of SUDS cases had IHA titre of greater than or equal to 4 and IgM-IFA titre of greater than or equal to 10 respectively, in contrast to 39.7% and 12.3% found among healthy Thai workers. These data indicate that at the time of death, most of the SUDS patients had an active infection with P. pseudomallei, possibly resulting from reactivation of a latent infection. The aetiological role of P. pseudomallei as the major cause of SUDS is discussed.