Interaction of pneumococcal phase variation, host and pressure/gas composition: virulence expression of NanA, HylA, PspA and CbpA in simulated otitis media.

Streptococcus pneumoniae, a leading cause of otitis media (OM), adapts to the host environment and undergoes spontaneous intra-strain phase variations in colony morphology. Transparent (T) phase variants are more efficient in colonizing the nasopharynx while the opaque (O) phase variants exhibit greater virulence during systemic infections. We recently demonstrated that T phase variants exhibited a higher growth rate and greater epithelial adherence and destruction than did O phase variants during interactions with human middle ear (ME) epithelial cells. This study was to delineate the underlying molecular mechanisms. Human ME epithelial cells were preconditioned for 24 h under one of the three simulated ME gas/pressure conditions designed to reflect those for 1) normal ME, 2) ME with Eustachian tube obstruction (ETO) and 3) ME with tympanostomy tube (TT) placement; subsequently exposed to a dose (10(7) CFU/ml) of either T or O phase variants of S. pneumoniae (6A), and then incubated for 1h and 3 h. Gene expressions coding for pneumococcal NanA, HylA, PspA, and CbpA virulence factors in inoculum, epithelium-attached and free-floating bacteria were assayed using real-time PCR. Result showed significantly higher basal expression levels for NanA and HylA in T inoculums than in O inoculum. Furthermore, striking differences between the two phase variants were observed in the forms of the inocula, significantly higher expression levels for PspA in T inoculum, but for CbpA in O inoculum. The TT condition enhanced the molecular activities of NanA, HylA, and PspA virulence factors in epithelium-attached T phase variants and fluid-floating O variants, followed by the ETO condition. Our study suggests that the pneumococcal virulence of the phase variations are modulated by the pathological ME environment and host-pathogen interaction during the pathogenesis of OM.

Nasal secretion concentrations of IL-5, IL-6, and IL-10 in children with and without upper respiratory tract viruses.

OBJECTIVE: To determine if levels of interleukin (IL) 5, IL-6, and IL-10 or their ratios in nasal secretion are diagnostic of viral upper respiratory tract infections (vURTIs) and coldlike illnesses (CLIs) in children. DESIGN: Longitudinal study of children for vURTIs, CLIs, and concentrations and ratios of nasal cytokines. SETTING: Outpatient assessments of children. PARTICIPANTS: A total of 224 children, aged 1 to 9 years. MAIN OUTCOME MEASURES: Concentrations of IL-5, IL-6, and IL-10 in nasal secretions, vURTIs diagnosed by polymerase chain reaction (PCR) detection of upper respiratory tract viruses, and concurrent CLIs diagnosed by parents. RESULTS: Of 1269 secretion samples, 552 (43.5%) were collected during a vURTI (PCR findings positive for an assayed virus [PCR(+)]). A concurrent CLI was diagnosed for 34% of the PCR(+) samples and for 18% of the samples found to be negative by PCR analysis (PCR(-)). Cytokine concentrations and ratios were highly variable and skewed to the lower values. The significance of the cytokine concentrations and ratios as discriminators of groups defined by the presence or absence of virus and of subgroups defined by the presence or absence of a CLI was evaluated using receiver operating characteristic curves. All measures were significant discriminators of the PCR(+) vs PCR(-) groups, and most were significant discriminators of the paired CLI subgroups. The concentration of IL-6 and the IL-5/IL-6 ratio were the best discriminators across all groups and subgroups. However, the sensitivities and specificities of those discriminators at the best cutoff values were on the order of 0.7 for the most extreme pairwise comparison (PCR(+)CLI(+) vs PCR(-)CLI(-)) and lower for the other comparison groups. CONCLUSION: The low sensitivities and specificities for cytokine-based assignment of specimens to the paired groups and subgroups limit their usefulness for diagnosis of infection or illness.

The interleukin 6 -174 C/C genotype predicts greater rhinovirus illness.

BACKGROUND: In adults and children with respiratory syncytial virus (RSV) infection, a polymorphism in the interleukin 6 (IL-6) promoter at position -174 predicts illness magnitude. In addition, polymorphisms in the interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) genes are associated with immune responsiveness and the frequency of complications. Here, the effect of these polymorphisms on illness and seroconversion during infection with rhinovirus type 39 (RV39) was evaluated. METHODS: Seventy-two adults were genotyped for the selected polymorphisms, experimentally exposed to RV39, and followed to track infection, seroconversion, and symptoms and signs of illness. Regression analysis was used to determine whether these polymorphisms predicted seroconversion and illness magnitude in 57 infected subjects. RESULTS: The low-production IL-6 -174 phenotype (C/C genotype) was associated with greater symptom magnitudes, and the IFN-gamma phenotype +874 predicted the frequency of seroconversion. No relationship between the IL-10 or TNF-alpha polymorphisms and any measured outcome was documented. The concentration of IL-6 protein, as measured in nasal wash fluids from subjects, was positively correlated with symptom magnitude, but it was independent of the IL-6 -174 genotypes representing the high- and low-production phenotypes. CONCLUSIONS: These results document statistically significant associations between the IL-6 -174 and IFN-gamma +874 polymorphisms and specific responses to experimental RV39 infection. For the IL-6 -174 polymorphism, the results replicate those for experimental RSV infection.

Interaction of phase variation, host and pressure/gas composition: pneumococcal gene expression of PsaA, SpxB, Ply and LytA in simulated middle ear environments.

OBJECTIVE: Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variants are more efficient in colonizing the nasopharynx while opaque (O) variants exhibit greater virulence during systemic infections. This study was intended to delineate the underlying molecular mechanisms by which the predominant S. pneumoniae variant efficiently infects the middle ear (ME) mucosa. METHODS: Human ME epithelial cells were preconditioned for 24h under one of the three gas/pressure conditions designed to simulate those for (1) normal ME (NME), (2) ME with Eustachian tube obstruction (ETO) and (3) ME with tympanostomy tube placement (TT), and then were incubated with ∼ 10(7)CFU/ml of either T or O variants of S. pneumoniae (6A) for 3h. Relative expression levels of genes encoding virulence factors, PsaA (surface adhesion), SpxB (pyruvate oxidase), Ply (pneumolysin), and LytA (autolysin) were assessed separately in epithelium-attached and supernatant bacteria 3h post infection using real-time PCR. RESULTS: Basal levels of the virulence molecules in inocula were comparable between two variants. However, relative expression levels of the gene transcripts were significantly induced in epithelium-attached T variants 3h after infection. Comparing with NME and TT conditions, ETO environment produced the largest effect on the differential expression of the virulence genes in the infected ME epithelial cells between T (induced) and O (suppressed) phenotypic pneumococci. CONCLUSIONS: T variant is a predominant phenotype responsible for the pathogenesis of pneumococcal OM.

Interaction of pneumococcal phase variation and middle ear pressure/gas composition: an in vitro model of simulated otitis media.

Streptococcus pneumoniae, a leading cause of otitis media (OM), undergoes spontaneous intra-strain variations in colony morphology. Transparent (T) variant is more efficient in colonizing the nasopharynx while the opaque (O) variant exhibits greater virulence during systemic infections. We hypothesized that changes in middle ear (ME) gas pressure/composition during Eustachian tube (ET) dysfunction and the treatment of that dysfunction, e.g., tympanostomy tube (TT) insertion, play a role in selecting the S. pneumoniae variant that can efficiently colonize/infect the ME mucosa. Human ME epithelial cells were preconditioned for 24h under one of three conditions that simulated (1) normal ME, (2) ME with ET obstruction (ETO) and (3) ME with TT; subsequently exposed to a dose (approximately 10(7)CFU/ml) of either T or O variant of S. pneumoniae, and then incubated for 1h and 3h. Under the simulated ETO and TT conditions, T variant exhibited a higher growth rate and greater epithelial adherence and killing than did O variants. Attachment of T variant to epithelial cells was documented by scanning electron microscopy. These results suggest that the T variant is more highly adapted to various ME environments than the O variants.

Age-dependent differential expression of fibronectin variants in skin and airway mucosal wounds.

OBJECTIVE: To delineate age-dependent and tissue-specific molecular activities of the variant-inclusion fibronectin transcripts in fetal and postnatal skin and airway mucosal wounds during early events of the wound healing process. Fibronectin is involved in multiple steps of the wound healing process. The functional complexity of fibronectin is carried through its protein diversity, which is controlled in part by alternative RNA splicing, a coordinated transcription and RNA processing. From a rabbit model of airway mucosal wound healing, we isolated and cloned an RNA splicing factor, SRp20, that was coexpressed with Fn1 complementary DNA and suppressed in fetal wounds but induced in postnatal wounds. Previous studies revealed a link between the inclusion and/or exclusion of the alternatively spliced Fn1 variants (extra domain A [EDA], extra domain B [EDB], and a variable region [V]) and outcomes of wound repair. DESIGN: Skin and airway mucosal incisional wounds were made in fetal (gestational day 21-23), weanling (4-6 weeks), and adult (>6 months) rabbits. Tissues (nonwounded and wounded) were collected at 12 hours (all age groups), 24 hours, and 48 hours (weanling only) after wounding. The expression levels of the 3 Fn1 spliced domain (EDA, EDB, and V)-containing messenger RNA (mRNA) species were assessed by real-time polymerase chain reaction. RESULTS: Fn1 spliced variants were either suppressed or showed no change in fetal skin and airway mucosal wounds 12 hours after injury, whereas the spliced mRNAs were induced in postnatal wounds. The augmented molecular activities of Fn1 spliced variants were more prominent in airway mucosal wounds than in skin wounds. Furthermore, the EDA variant was dominantly selected in adult airway mucosal wounds (6-fold increase), which was strikingly different from the adult skin wounds (1-fold). CONCLUSION: Our study suggests that the age-dependent activation of Fn1-EDA mRNA may play a fundamental role in differentiating fetal wound regeneration from postnatal wound scar formation during the early events of airway mucosal wound healing.

Identification of a pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), and its co-expression with fibronectin in fetal and postnatal rabbit airway mucosal and skin wounds.

Fibronectin (FN) is a multi-functional, adhesion protein and involved in multi-steps of the wound healing process. Strong evidence suggests that FN protein diversity is controlled by alternative RNA splicing; a coordinated transcription and RNA processing that is development-, age-, and tissue/cell type-regulated. We previously demonstrated that fetal rabbit airway mucosal healing is regenerative and scarless. Expression, regulation, and biological function of the FN gene and various spliced forms in this model are unknown. Airway and skin incisional wounds were made in fetal (gestation days 21-23), weanling (4-6 weeks) and adult (>6 months) rabbits. Non-wounded and wounded tissues were collected at 12 h (all age groups), 24 h and 48 h (weanling only) post-wounding. Expression profiles were obtained using mRNA differential display and cDNAs of interest were cloned, sequenced and validated by real-time PCR. Here, we report two rabbit cDNAs that showed similar expression patterns after wounding. One encodes a rabbit fibronectin gene, Fn1, and another shares a high sequence homology to a human pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), coding for a RNA binding protein, SRp20. Both Fn1 and Sfrs3 mRNAs were suppressed in fetal wounds but induced in postnatal wounds 12 h post-wounding. The increased levels of both Fn1 and Sfrs3 transcripts were sustained up to 48 h in weanling airway mucosal wounds. The augmentations of the two genes in postnatal airway mucosal wounds were more prominent than that in skin wounds, indicating that the involvement of Sfrs3 and Fn1 genes in postnatal airway mucosal wounds is tissue-specific. Literature provides evidence that SRp20 is indeed involved in the alternative splicing of FN and that the embryonic FN variants reappear during adult wound healing. A connection between the enhanced molecular activity of Sfrs3 and the regulation of the FN gene expression through alternative splicing during the early events of postnatal airway mucosal wound repair was proposed.

Mucosal expression of genes encoding possible upstream regulators of Na+ transport during pneumococcal otitis media.

OBJECTIVE: Recently, we reported that gene transcripts encoding 3 Na+ transport proteins (pump, channel and exchanger) in the middle ear mucosa (MEM) were simultaneously suppressed at 12 and 48 h after Streptococcus pneumoniae (SP) challenge of rat middle ears. MATERIAL AND METHODS: From cDNA microarray screening of those specimens, several gene clusters, including Nos2 and the transcription factors Fos, Fosl1, Jun and Nfkb1, were identified as possible upstream regulators of Na+ transport protein expression. The altered expression of those genes in MEM was validated and quantified using real-time polymerase chain reaction and MEM protein expression for Atp1a1, Nos2 and Nfkb1 was studied using Western blot and/or immunohistochemistry assays. RESULTS: At both time-points. Atp1a1 mRNA and protein were decreased and Nos2 mRNA and protein were increased in MEM. While Nfkb1 protein was decreased at those times. the corresponding mRNA was increased at 12 h but decreased at 48 h. Gene expression for Fos was suppressed at both times, while that for Fosl1 and Jun was augmented at 12 h and suppressed at 48 h. Immunohistochemical study of specimens challenged with SP showed a swollen MEM with infiltration of inflammatory cells that stained positive for Nos2. CONCLUSION: Given the known activities of Nos2, these results can be interpreted as evidencing a transcriptional suppression of Na+ transport protein synthesis secondary to upregulated Nos2 expression during SP infection of the rat MEM. This proposed signaling pathway does not require the continuous upregulation of Nfkb1 or the other assayed transcription factors as early as 12 h after middle ear infection.

The effect of changes in ambient oxygen concentration on the bioelectric properties of middle ear mucosa.

The purpose of the present study was to compare the effect of 24 h of exposure to 7% O2 (normal middle ear physiological conditions) vs. 21% O2 (found in the middle ear after ventilation tube placement) on transepithelial Na+ absorption and Cl- secretion in cultured gerbil middle ear epithelial cell monolayers. Although no difference in apical Na+ absorption was identified, the UTP-induced stimulation of apical Cl- secretion in the presence of apical Na+ channel blockade with amiloride was significantly enhanced after exposure to 21% O2 compared with 7% O2 exposure. In the presence of a calcium-activated Cl- channel inhibitor, DIDS, UTP-induced stimulation of Cl- secretion after 21% O2 exposure was decreased, suggesting a role for calcium-activated Cl- channels in middle ear Cl- secretion in response to relative hyperoxia.