Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS) in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

Use of multiparameter flow cytometry to determine the effects of monoterpenoids and phenylpropanoids on membrane polarity and permeability in staphylococci and enterococci.

Monoterpenoids and phenylpropanoids are major components of many plant essential oils and are relatively simple, low-molecular-weight compounds with antimicrobial activity. This study used multiparameter flow cytometry to examine changes in membrane polarity and permeability in Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis following exposure to the monoterpenoids carvacrol, 1,8-cineole and terpinen-4-ol and the phenylpropanoids eugenol and cinnamaldehyde. Melaleuca alternifolia (tea tree) essential oil was also investigated. The fluorescent dyes DiOC(2)(3) (3,3'-diethyloxacarbocyanine oxide) and TO-PRO(®)-3 were used to assess membrane potential and permeability, respectively, following treatment with the minimum inhibitory concentration (MIC) of each test compound for 5 min and 30 min. Four subpopulations of cells were identified based on polarity and permeability. Eugenol treatment resulted in the greatest depolarisation and permeabilisation at 5 min, followed by carvacrol. Cinnamaldehyde, whilst having the lowest MICs (0.006-0.1%, v/v), did not induce changes in polarity or permeability at the MIC, and substantially higher concentrations were required to induce significant effects. At 30 min, treatment with all six compounds resulted in significant depolarisation (60.9-99.3% of cells), whereas fewer compounds (ranging from two to five per organism) resulted in significant permeabilisation. The extent of permeabilisation was always less than depolarisation, with overall means for all treatments of 46.1% and 89.4% of cells permeabilised and depolarised, respectively, at 30 min. These data demonstrate that several monoterpenoids and phenylpropanoids as well as tea tree oil alter membrane properties by decreasing polarity and increasing permeability in a time- and concentration-dependent manner.

Phenotypic and genotypic profile of human tympanic membrane derived cultured cells.

The human tympanic membrane (hTM), known more commonly as the eardrum, is a thin, multi-layered membrane that is unique in the body as it is suspended in air. When perforated, the hTM's primary function of sound-pressure transmission is compromised. For the purposes of TM reconstruction, we investigated the phenotype and genotype of cultured primary cells derived from hTM tissue explants, compared to epithelial (HaCaT cells) and mesenchymal (human dermal fibroblasts (HDF)) reference cells. Epithelium-specific ets-1 (ESE-1), E-cadherin, keratinocyte growth factor-1 (KGF-1/FGF-7), keratinocyte growth factor-2 (KGF-2/FGF10), fibroblast growth factor receptor 1 (FGFR1), variants of fibroblast growth factor receptor 2 (FGFR2), fibroblast surface protein (FSP), and vimentin proteins were used to assess the phenotypes of all cultured cells. Wholemount and paraffin-embedded hTM tissues were stained with ESE-1 and E-cadherin proteins to establish normal epithelial-specific expression patterns within the epithelial layers. Immunofluorescent (IF) cell staining of hTM epithelial cells (hTMk) demonstrated co-expression of both epithelial- and mesenchymal-specific proteins. Flow cytometry (FCM) analysis further demonstrated co-expression of these epithelial and mesenchymal-specific proteins, indicating the subcultured hTMk cells possessed a transitional phenotype. Gene transcript analysis of hTMk cells by reverse transcriptase polymerase chain reaction (RT-PCR) revealed a down regulation of ESE-1, E-cadherin, FGFR2, variant 1 and variant 2 (FGFR2v1 and FGFR2v2) between low and high passages, and up-regulation of KGF-1, KGF-2, and FGFR1. All results indicate a gradual shift in cell phenotype of hTMk-derived cells from epithelial to mesenchymal.

Cells of epithelial lineage are present in blood, engraft the bronchial epithelium, and are increased in human lung transplantation.

BACKGROUND: There is a growing expectation that cell-based therapies will prove effective for a wide range of conditions including lung diseases such as cystic fibrosis. The promise of these therapies will depend largely on effective delivery and engraftment. In this study, in the setting of human lung transplantation, we sought to determine whether exogenous epithelial cells are able to engraft the transplanted organ and if cells of a similar phenotype could be detected in peripheral blood. METHODS: Cells obtained from bronchial brushings and peripheral blood were analyzed via dual fluorescent in situ hybridization/fluorescent immunohistochemistry (FISH/IHC), short tandem repeat polymerase chain reaction (STR-PCR) and flow cytometry. RESULTS: In 2 of 3 gender-mismatched patients we observed limited (5.9% to 6.8% by STR-PCR and 3.5% to 4% by FISH/IHC) engraftment of the bronchial epithelium by exogenous epithelial cells. Engrafting cells were CD34(-) CD15(-) CD68(-) c-Kit(-), but expressed CXCR4 on the cell surface. Cells with a similar phenotype were also identified in peripheral blood. In 8 patients, at 2 to 66 months post-transplant, 0.57 +/- 0.17% of CD14(-) peripheral blood mononuclear cells were of epithelial lineage. Almost all were CD45(+) and most expressed CXCR4 on the cell membrane. Cells of epithelial lineage were also identified in peripheral blood in healthy individuals but in much lower numbers (0.08 +/- 0.01%, p < 0.05). CONCLUSIONS: Cells of epithelial lineage are detectable in peripheral blood and are able to engraft the bronchial epithelium in humans. Cell numbers are increased in lung transplantation.