Predominant Bacterial and Viral Otopathogens Identified Within the Respiratory Tract and Middle Ear of Urban Australian Children Experiencing Otitis Media Are Diversely Distributed.

Background: Otitis media (OM) is one of the most common infections in young children, arising from bacterial and/or viral infection of the middle ear. Globally, Streptococcus pneumoniae and non-typeable Haemophilus influenzae (NTHi) are the predominant bacterial otopathogens. Importantly, common upper respiratory viruses are increasingly recognized contributors to the polymicrobial pathogenesis of OM. This study aimed to identify predominant bacteria and viruses in the nasopharynx, adenoids and middle ears of peri-urban/urban South-East Queensland Australian children, with and without clinical history of chronic otitis media with effusion (COME) and/or recurrent acute otitis media (RAOM). Methods: Sixty children, 43 diagnosed with OM and 17 controls with no clinical history of OM from peri-urban/urban South-East Queensland community were recruited to the study. Respiratory tract bacterial and viral presence were examined within nasopharyngeal swabs (NPS), middle ear effusions (MEE) and adenoids, using real-time polymerase chain reaction (RT-PCR) and bacterial culture. Results: At least one otopathogen present was observed in all adenoid samples, 86.1% and 82.4% of NPS for children with and without OM, respectively, and 47.1% of the MEE from the children with OM. NTHi was the most commonly detected bacteria in both the OM and control cohorts within the adenoids (90.0% vs 93.8%), nasopharynx (67.4% vs 58.8%) respectively, and in the MEE (OM cohort 25.9%). Viruses were detected in all adenoid samples, 67.4% vs 47.1% of the NPS from the OM and control cohorts, respectively, and 37% of the MEE. Rhinovirus was the predominant virus identified in the adenoids (85.0% vs 68.8%) and nasopharynx (37.2% vs 41.2%) from the OM and control cohorts, respectively, and the MEE (19.8%). Conclusions: NTHi and rhinovirus are predominant otopathogens within the upper respiratory tract of children with and without OM from peri-urban and urban South-East Queensland, Australia. The presence of bacterial otopathogens within the middle ear is more predictive of concurrent URT infection than was observed for viruses, and the high otopathogen carriage within adenoid tissues confirms the complex polymicrobial environment in children, regardless of OM history.

Comparison of skin biopsy sample processing and storage methods on high dimensional immune gene expression using the Nanostring nCounter system.

BACKGROUND: Digital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System's ability to reproducibly quantify the expression of 730 immune genes from skin biopsies. METHODS: Healthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at - 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at - 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at - 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes. RESULTS: Except for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS < 2). There was a high degree of reproducibility in the expression of immune genes between all samples with the exception of saline, with the number of detected genes at counts < 100, between 100 and 1000 and > 10,000 similar across extraction protocols. CONCLUSIONS: A variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.

Probiotics, Anticipation Stress, and the Acute Immune Response to Night Shift.

Introduction: Sleep disturbance and sleep disruption are associated with chronic, low grade inflammation and may underpin a range of chronic diseases in night shift workers. Through modulation of the intestinal microbiota, probiotic supplements may moderate the effects of sleep disruption on the immune system. The aim of this study was to examine 14 days of daily probiotic supplementation on the acute response of acute phase proteins and immune markers to sleep disruption associated with night shift work (Australia and New Zealand Clinical Trials Registry: 12617001552370). Methods: Individuals (mean age 41 ± 11 yrs; 74% female) performing routine night shift were randomly assigned to a probiotic group (1 × 1010 colony forming units (CFU) Lactobacillus acidophilus DDS-1 or 1 × 1010 CFU Bifidobacterium animalis subsp. lactis UABla-12) or placebo (n= 29 per group). Participants undertook a 14-day supplementation period that coincided with a period of no night shifts followed by two consecutive night shifts. Blood samples were collected prior to the start of supplementation (V1), prior to commencing the first night shift (V2), after the first night shift (V3) and after the second night shift (V4). Serum was assessed for markers of stress (cortisol), acute phase response (C reactive protein (CRP), erythrocyte sedimentation rate, pentraxin), adhesion markers (serum E-selectin, mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), and serum cytokines (interleukin (IL)-1ra, IL-1ß, IL-6, tumor necrosis factor (TNF)-α, IL-10). Sleep quality was assessed with the Pittsburgh Sleep Quality Index (PSQI) and a Fitbit activity tracker. Results: The groups were well balanced on key markers and the probiotic strains were well tolerated. The 14-day supplementation period that coincided with typical night-day sleep-wake cycles leading up to night shift (V1 to V2) was associated with significant changes in the placebo group in the concentration of serum cortisol (p = 0.01), pentraxin (p = 0.001), MAdCAM-1 (p = 0.001), and IL-1ra (p=0.03). In contrast, probiotic supplementation moderated changes in these serum markers from V1 to V2. No significant interaction effects (time by group) were observed for the serum markers prior to and after night shift work following probiotic supplementation due to the substantial changes in the serum markers that occurred during the normal sleep period from V1 to V2. Conclusions: Probiotics may moderate the effects of anticipatory stress on the immune system in the lead up to night shift.

Paediatric and adult bronchiectasis: Vaccination in prevention and management.

Bronchiectasis has received increased attention recently, including an emphasis on preventing infective exacerbations that are associated with disease progression and lung function decline. While there are several bacteria and viruses associated with bronchiectasis, licensed vaccines are only currently available for Streptococcus pneumoniae, Haemophilus influenzae (H. influenzae protein D as a conjugate in a pneumococcal vaccine), Mycobacterium tuberculosis, Bordetella pertussis and influenza virus. The evidence for the efficacy and effectiveness of these vaccines in both preventing and managing bronchiectasis in children and adults is limited with the focus of most research being on other chronic lung disorders, such as chronic obstructive pulmonary diseases, asthma and cystic fibrosis. We review the existing evidence for these vaccines in bronchiectasis and highlight the existing gaps in knowledge. High-quality experimental and non-experimental studies using current state-of-the-art microbiological methods and validated, standardised case definitions are needed across the depth and breadth of the vaccine development pathway.

Distinct Gene Expression Patterns between Nasal Mucosal Cells and Blood Collected from Allergic Rhinitis Sufferers.

BACKGROUND: Investigations of gene expression in allergic rhinitis (AR) typically rely on invasive nasal biopsies (site of inflammation) or blood samples (systemic immunity) to obtain sufficient genetic material for analysis. New methodologies to circumvent the need for invasive sample collection offer promise to further the understanding of local immune mechanisms relevant in AR. METHODS: A within-subject design was employed to compare immune gene expression profiles obtained from nasal washing/brushing and whole blood samples collected during peak pollen season. Twelve adults (age: 46.3 ± 12.3 years) with more than a 2-year history of AR and a confirmed grass pollen allergy participated in the study. Gene expression analysis was performed using a panel of 760 immune genes with the NanoString nCounter platform on nasal lavage/brushing cell lysates and compared to RNA extracted from blood. RESULTS: A total of 355 genes were significantly differentially expressed between sample types (9.87 to -9.71 log2 fold change). The top 3 genes significantly upregulated in nasal lysate samples were Mucin 1 (MUC1), Tight Junction Protein 1 (TJP1), and Lipocalin-2 (LCN2). The top 3 genes significantly upregulated in blood samples were cluster of differentiation 3e (CD3E), FYN Proto-Oncogene Src Family Tyrosine Kinase (FYN) and cluster of differentiation 3d (CD3D). CONCLUSIONS: Overall, the blood and nasal lavage samples showed vastly distinct gene expression profiles and functional gene pathways which reflect their anatomical and functional origins. Evaluating immune gene expression of the nasal mucosa in addition to blood samples may be beneficial in understanding AR pathophysiology and response to allergen challenge.

Non-typeable Haemophilus Influenzae detection in the lower airways of patients with lung cancer and chronic obstructive pulmonary disease.

BACKGROUND: Chronic airway inflammation and hypersensitivity to bacterial infection may contribute to lung cancer pathogenesis. Previous studies have demonstrated that nontypeable Haemophilus influenzae (NTHi) is the most common colonizing bacteria in the lower airways of patients with COPD. The objective of this study was to determine the presence of NTHi and immunoglobulin concentrations in patients with lung cancer, COPD and controls. METHODS: Serum and bronchial wash samples were collected from patients undergoing diagnostic bronchoscopy. Total IgE, IgG and specific NTHi IgG were measured by enzyme linked immunosorbent assay. Bronchial wash samples were examined for the presence of NTHi via PCR. RESULTS: Out of the 60 patients: 20 had confirmed Lung Cancer, 27 had COPD only and 13 were used as Controls. NTHi was detected in the lower airways of all three groups (Lung Cancer 20%; COPD 22% and Controls 15%). Total IgE was highest in Lung Cancer subjects followed by COPD and control subjects (mean ± SD: 870 ± 944, 381 ± 442, 159 ± 115). Likewise total IgG was higher in Lung cancer (Mean ± SD: 6.99 ± 1.8) patients compared to COPD (Mean ± SD: 5.43 ± 2). CONCLUSIONS: The lack of difference in NTHi and specific antibodies between the three groups makes it less likely that NTHi has an important pathogenetic role in subjects with Lung Cancer. However the detection of higher IgE antibody in Lung Cancer subjects identifies a possible mechanism for carcinogenesis in these subjects and warrants further study.

Enteroendocrine and adipokine associations with type 2 diabetes: Phenotypic risk scoring approaches.

BACKGROUND AND AIM: The contribution of gut-derived factors to the mechanisms linking obesity and metabolic disease remains under-investigated. The aim of the current study was to examine the associations between glucagon and enteroendocrine signaling and type 2 diabetes (T2D) using a derived risk score approach. To compare the relative importance of the enteroendocrine system, associations between adipokine measures and T2D were also investigated. METHODS: A total of 130 individuals with T2D and 161 individuals without T2D were included in the study. Circulating concentrations of enteroendocrine (glucagon, ghrelin, glucagon-like peptide-1, and gastric inhibitory peptide) and adipokine mediators (adiponectin, leptin, resistin, visfatin, and adipsin) were measured. Standard scores (Z-scores) were determined for each measure and enteroendocrine risk scores (ERS) and adipokine risk scores (ARS) calculated based on summation of the component measures. Associations between both the ERS and ARS and T2D status were assessed using logistic regression models. RESULTS: The ERS was significantly associated with T2D status in an adjusted model (odds ratio: 1.36; 95% confidence interval [CI]: 1.08-1.72; P = 0.009). Associations between the ARS and T2D status were not independent of age, sex, and body mass index (odds ratio: 1.21; 95%CI: 0.99-1.47; P = 0.06). Quantification of risk across ERS tertiles revealed that individuals with an ERS in the upper tertile were 10 times more likely (CI: 3.23-32.73; P < 0.001) to have T2D. CONCLUSIONS: These data support an association between enteroendocrine signaling and T2D. Use of the ERS as a potential tool for classifying individuals with metabolic syndrome as high or low risk for T2D development is being considered.

Rubella antibodies in Australian immunoglobulin products.

Rubella antibodies are not routinely measured in immunoglobulin products and there is a lack of information on the titer in Australian products. To facilitate future studies of the effectiveness of passive immunisation for preventing rubella and congenital rubella syndrome, this study measured the concentration of rubella-specific antibodies in Australian intramuscular (IM) and intravenous (IV) human immunoglobulin products suitable for post-exposure prophylaxis using a chemiluminescent immunoassay. The GMT ± GSD for the IM product was 19 ± 1.2 IU/mg (2980 ± 1.2 IU/mL). The GMT ± GSD for the IV product was 12 ± 1.5 IU/mg (729 ± 1.5 IU/mL). At present, Australian guidelines recommend offering non-immune pregnant women exposed to rubella 20 mL of intramuscular immunoglobulin within 72 hours of exposure. This equates to 42,160 IU of rubella antibodies if the lowest titer obtained for the Australian IM product is considered. The same dose would be delivered by 176 mL of the Australian IV product at the lowest measured rubella-specific antibody titer.

Effect of the Streptococcus agalactiae Virulence Regulator CovR on the Pathogenesis of Urinary Tract Infection.

Background: Streptococcus agalactiae can cause urinary tract infection (UTI). The role of the S. agalactiae global virulence regulator, CovR, in UTI pathogenesis is unknown. Methods: We used murine and human bladder uroepithelial cell models of UTI and S. agalactiae mutants in covR and related factors, including ß-hemolysin/cytolysin (ß-h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI. Results: We found that covR-deficient serotype III S. agalactiae 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with covR-deficient S. agalactiae produced less proinflammatory cytokines than those infected with wild-type 874391. Acute cytotoxicity in uroepithelial cells triggered by covR-deficient but not wild-type 874391 was associated with significant caspase 3 activation. Mechanistically, covR mutation significantly altered the expression of several genes in S. agalactiae 874391 that encode key virulence factors, including ß-h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the ß-h/c, exerted significant effects on colonization of the murine urinary tract in vivo. Conclusions: S. agalactiae CovR promotes bladder infection and inflammation, as well as adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial, and influenced by virulence effects of CovR, HvgA, and capsule.

Otitis media.

Otitis media (OM) or middle ear inflammation is a spectrum of diseases, including acute otitis media (AOM), otitis media with effusion (OME; 'glue ear') and chronic suppurative otitis media (CSOM). OM is among the most common diseases in young children worldwide. Although OM may resolve spontaneously without complications, it can be associated with hearing loss and life-long sequelae. In developing countries, CSOM is a leading cause of hearing loss. OM can be of bacterial or viral origin; during 'colds', viruses can ascend through the Eustachian tube to the middle ear and pave the way for bacterial otopathogens that reside in the nasopharynx. Diagnosis depends on typical signs and symptoms, such as acute ear pain and bulging of the tympanic membrane (eardrum) for AOM and hearing loss for OME; diagnostic modalities include (pneumatic) otoscopy, tympanometry and audiometry. Symptomatic management of ear pain and fever is the mainstay of AOM treatment, reserving antibiotics for children with severe, persistent or recurrent infections. Management of OME largely consists of watchful waiting, with ventilation (tympanostomy) tubes primarily for children with chronic effusions and hearing loss, developmental delays or learning difficulties. The role of hearing aids to alleviate symptoms of hearing loss in the management of OME needs further study. Insertion of ventilation tubes and adenoidectomy are common operations for recurrent AOM to prevent recurrences, but their effectiveness is still debated. Despite reports of a decline in the incidence of OM over the past decade, attributed to the implementation of clinical guidelines that promote accurate diagnosis and judicious use of antibiotics and to pneumococcal conjugate vaccination, OM continues to be a leading cause for medical consultation, antibiotic prescription and surgery in high-income countries.

Pathogenesis of Streptococcus urinary tract infection depends on bacterial strain and ß-hemolysin/cytolysin that mediates cytotoxicity, cytokine synthesis, inflammation and virulence.

Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial ß-hemolysin/cytolysin (ß-H/C) because an ß-H/C-deficient UPSA 807 isogenic mutant, UPSA 807ΔcylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1ß, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of ß-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and ß-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder.

Discovery and Characterization of Human-Urine Utilization by Asymptomatic-Bacteriuria-Causing Streptococcus agalactiae.

Streptococcus agalactiae causes both symptomatic cystitis and asymptomatic bacteriuria (ABU); however, growth characteristics of S. agalactiae in human urine have not previously been reported. Here, we describe a phenotype of robust growth in human urine observed in ABU-causing S. agalactiae (ABSA) that was not seen among uropathogenic S. agalactiae (UPSA) strains isolated from patients with acute cystitis. In direct competition assays using pooled human urine inoculated with equal numbers of a prototype ABSA strain, designated ABSA 1014, and any one of several UPSA strains, measurement of the percentage of each strain recovered over time showed a markedly superior fitness of ABSA 1014 for urine growth. Comparative phenotype profiling of ABSA 1014 and UPSA strain 807, isolated from a patient with acute cystitis, using metabolic arrays of >2,500 substrates and conditions revealed unique and specific l-malic acid catabolism in ABSA 1014 that was absent in UPSA 807. Whole-genome sequencing also revealed divergence in malic enzyme-encoding genes between the strains predicted to impact the activity of the malate metabolic pathway. Comparative growth assays in urine comparing wild-type ABSA and gene-deficient mutants that were functionally inactivated for the malic enzyme metabolic pathway by targeted disruption of the maeE or maeK gene in ABSA demonstrated attenuated growth of the mutants in normal human urine as well as synthetic human urine containing malic acid. We conclude that some S. agalactiae strains can grow in human urine, and this relates in part to malic acid metabolism, which may affect the persistence or progression of S. agalactiae ABU.

Vaccination against respiratory Pseudomonas aeruginosa infection.

Respiratory infections caused by Pseudomonas aeruginosa are a major clinical problem globally, particularly for patients with chronic pulmonary disorders, such as those with cystic fibrosis (CF), non-CF bronchiectasis (nCFB) and severe chronic obstructive pulmonary disease (COPD). In addition, critically ill and immunocompromised patients are also at significant risk of P. aeruginosa infection. For almost half a century, research efforts have focused toward development of a vaccine against infections caused by P. aeruginosa, but a licensed vaccine is not yet available. Significant advances in identifying potential vaccine antigens have been made. Immunisations via both the mucosal and systemic routes have been trialled in animal models and their effectiveness in clearing acute infections demonstrated. The challenge for translation of this research to human applications remains, since P. aeruginosa infections in the human respiratory tract can present both as an acute or chronic infection. In addition, immunisation prior to infection may not be possible for many patients with CF, nCFB or COPD. Therefore, development of a therapeutic vaccine provides an alternative approach for treatment of chronic infection. Preliminary animal and human studies suggest that mucosal immunisation may be effective as a therapeutic vaccine against P. aeruginosa respiratory infections. Nevertheless, more research is needed to improve our understanding of the basic biology of P. aeruginosa and the mechanisms needed to upregulate the induction of host immune pathways to prevent infection. Recognition of variability in the host immune responses for a range of patient health conditions at risk from P. aeruginosa infection is also required to support development of a successful vaccine delivery strategy and vaccine. Activation of mucosal immune responses may provide improved efficacy of vaccination for P. aeruginosa during both acute exacerbations and chronic infection.

Peripheral blood natural killer (NK) cell function in healthy adults assessed using the target-induced NK loss (TINKL) assay.

In this technical note we provide data useful for the clinical application of the target-induced Natural Killer (NK) loss (TINKL) assay. The TINKL assay is a sensitive flow cytometry-based assay for measuring NK cell function. Loss of NK cells from the lymphocyte gate occurs following culture with K562 (the prototypic target cell for natural killing) and antibody-coated target cells (for antibody-dependent killing). By analysis of multiple samples of PBMC from single donors we document the intra-experimental variability and the inter-experimental variability of the assay. The intra-experimental coefficient of variation (CV) was on average 11% for natural killing and 3% for antibody-dependent killing, compared to 14% and 9% respectively for the inter-experimental variation. Analysis of a 123 normal healthy adults shows large variability in the functional capacity of NK cells in the population both for natural killing (CV 33%) and antibody-dependent killing (CV 27%).

Mucosal and systemic antibody responses to potential Pseudomonas aeruginosa vaccine protein antigens in young children with cystic fibrosis following colonization and infection.

Pseudomonas aeruginosa is an important prognostic determinant in cystic fibrosis (CF). Little is known however, about P. aeruginosa induced local mucosal and systemic immune responses. Twenty CF children were categorized according to their P. aeruginosa status: (1) chronic lower respiratory tract infection (LRTI), (2) prior successfully treated initial LRTI, (3) isolated upper respiratory tract (URT) colonization, and (4) no known URT colonization or previous LRTI. Their antibody responses, and those of six non-CF disease controls, in serum and bronchoalveolar lavage (BAL) fluid to potential P. aeruginosa vaccine antigens outer membrane protein F (OprF), outer membrane protein H (OprH), catalase A (KatA) and a whole killed cell (WKC) extract were evaluated. Outer membrane protein G (OprG) responses were also measured in blood. Natural exposure, colonization and infection resulted in detectable antibody levels in BAL and serum in all CF groups. Both chronically infected and URT colonized CF children had substantially elevated immunoglobulin A antibody levels in the BAL fluid and sera toward the WKC extract and OprF antigen compared with the other groups of CF children and non-CF controls. The serum levels of specific P. aeruginosa antibodies involving immunoglobulin G and M isotypes increased with chronic LRTI, especially antibody levels to KatA, OprH and WKC extract, which were substantially greater in chronically infected children compared with all other groups. In conclusion, natural exposure, URT colonization and LRTI with P. aeruginosa all induce substantial mucosal and systemic antibody responses to potential vaccine antigens with chronically infected CF children having the highest levels.

Genome-wide mapping of cystitis due to Streptococcus agalactiae and Escherichia coli in mice identifies a unique bladder transcriptome that signifies pathogen-specific antimicrobial defense against urinary tract infection.

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli; however, Gram-positive organisms, including Streptococcus agalactiae, or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection.

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10-deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

Epithelial cell coculture models for studying infectious diseases: benefits and limitations.

Countless in vitro cell culture models based on the use of epithelial cell types of single lineages have been characterized and have provided insight into the mechanisms of infection for various microbial pathogens. Diverse culture models based on disease-relevant mucosal epithelial cell types derived from gastrointestinal, genitourinary, and pulmonary organ systems have delineated many key host-pathogen interactions that underlie viral, parasitic, and bacterial disease pathogenesis. An alternative to single lineage epithelial cell monoculture, which offers more flexibility and can overcome some of the limitations of epithelial cell culture models based on only single cell types, is coculture of epithelial cells with other host cell types. Various coculture models have been described, which incorporate epithelial cell types in culture combination with a wide range of other cell types including neutrophils, eosinophils, monocytes, and lymphocytes. This paper will summarize current models of epithelial cell coculture and will discuss the benefits and limitations of epithelial cell coculture for studying host-pathogen dynamics in infectious diseases.

Haemophilus influenzae and smoking-related obstructive airways disease.

BACKGROUND: Intralumenal bacteria play a critical role in the pathogenesis of acute infective episodes and airway inflammation. Antigens from colonizing bacteria such as nontypeable Haemophilus influenzae (NTHi) may contribute to chronic lung disease through an immediate hypersensitivity response. The objective of this study was to determine the presence of specific NTHi-IgE antibodies in subjects with chronic bronchitis (CB) and COPD who had smoked. METHODS: Serum, sputum, and saliva samples were collected from subjects with CB and moderate-severe COPD and healthy aged-matched controls. Total IgE and specific NTHi IgE were measured by enzyme linked immmunosorbent assay. Throat swabs were examined for the presence of NTHi. RESULTS: THE RESULTS DEMONSTRATE THAT: i) specific NTHi IgE antibodies occur at a low level in healthy subjects; ii) those with both CB and moderate-severe COPD have elevated specific NTHi IgE antibody compared with healthy controls, with higher levels in those with most severe disease; iii) IgE levels are greater in those with moderate-severe COPD than in those with CB. They demonstrate specific NTHi IgE antibody is regularly found at higher than normal levels in COPD. CONCLUSION: The detection of IgE antibody to colonizing bacteria in all subjects with CB or moderate-severe COPD identifies a possible mechanism of bronchospasm in these subjects amenable to specific intervention therapy.

Developmental profiles of mucosal immunity in pre-school children.

This study investigated the effect of attending pre-school on mucosal immunity. Children 3.5 to 5 years of age who attended pre-school were observed for a 10 month period. Demographic information was collected on previous childcare experiences, the home environment and clinical information relating to the child and the family. A daily illness log was kept for each child. A multivariate longitudinal analysis of the relation between immunoglobulins in saliva and age, gender, childcare experience, pre-school exposure, number of siblings, environmental tobacco smoke (ETS), atopy and hospitalisation was conducted. There was a positive association of higher IgA levels with the winter season and with children being older than 4 years (P < .001), having attended childcare prior to commencing pre-school (P < .05), and having been exposed to ETS at home (P < .05). Lower IgA levels were associated with being atopic (P < .05). Higher IgG levels were associated with exposure to ETS (P < .001), while lower levels were associated to having atopy. Higher IgM levels were associated with previous childcare experience (P < .01) whilst having been hospitalised was associated with having low salivary IgM levels (P < .01). Lagged analyses demonstrated that immunological parameters were affected by the number of respiratory infections in the preceding 2 months.