Gut Microbiome and Metabolic and Immune Indices in Males with or without Evidence of Metabolic Dysregulation.

Background: The contributions of the gut microbiota to obesity and metabolic disease represent a potentially modifiable factor that may explain variation in risk between individuals. This study aimed to explore relationships among microbial composition and imputed functional attributes, a range of soluble metabolic and immune indices, and gene expression markers in males with or without evidence of metabolic dysregulation (MetDys). Methods: This case-control study included healthy males (n=15; 41.9±11.7 years; body mass index [BMI], 22.9±1.2 kg/m2) and males with evidence of MetDys (n=14; 46.6±10.0 years; BMI, 35.1±3.3 kg/m2) who provided blood and faecal samples for assessment of a range of metabolic and immune markers and microbial composition using 16S rRNA gene sequencing. Metagenomic functions were imputed from microbial sequence data for analysis. Results: In addition to elevated values in a range of traditional metabolic, adipokine and inflammatory indices in the MetDys group, 23 immunomodulatory genes were significantly altered in the MetDys group. Overall microbial diversity did not differ between groups; however, a trend for a higher relative abundance of the Bacteroidetes (P=0.06) and a lower relative abundance of the Verrucomicrobia (P=0.09) phyla was noted in the MetDys group. Using both family- and genera-level classifications, a partial least square discriminant analysis revealed unique microbial signatures between the groups. Conclusion: These findings confirm the need for ongoing investigations in human clinical cohorts to further resolve the relationships between the gut microbiota and metabolic and immune markers and risk for metabolic disease.

Assessment of a Multispecies Probiotic Supplement for Relief of Seasonal Allergic Rhinitis: A Randomized Double-Blind Placebo-Controlled Trial.

Background: Early phase clinical research provided initial support for the use of a multispecies probiotic supplement to improve quality of life (QoL) in adults with seasonal allergic rhinitis (AR) and reduce the use of AR symptom relieving medication. This study aimed to confirm these early phase findings in a double-blind randomized placebo-controlled trial. Methods: Individuals, aged 18-65 years, with a minimum 2-year history of AR, moderate-to-severe AR symptoms, and a positive radio-allergosorbent test to Bermuda (Couch) Grass were randomized to receive either a multispecies probiotic supplement (total colony-forming units 4 × 109/day) or placebo twice daily for 8 weeks. A mini-rhinoconjunctivitis quality of life questionnaire (mRQLQ) scale was administered at screening, days 0, 28, and 56. The proportion of participants with a >0.7 improvement in mRQLQ was the primary outcome. Participants also completed a daily symptom and medication diary during the supplementation period. Results: There were 165 participants randomized, with 142 included in the primary outcome analysis. The percentage of participants meeting the threshold for a clinically meaningful reduction in the mRQLQ from days 0 to 56 was not significantly different between groups (61% vs. 62%, p = 0.90). However, 76 participants had a clinically meaningful improvement in QoL (decrease in mRQLQ >0.7) prior to the start of supplementation (screening to day 0). Conclusion: Changes in self-reported QoL and other disease severity metrics between screening and the start of supplementation limited the ability to discern an effect of supplementation and highlight the need for adaptive clinical trial designs in allergy research. Clinical Trial Registration: The trial was registered with the Australia and New Zealand Clinical Trials Registry (ACTRN12619001319167).

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.

Australian Aboriginal Otitis-Prone Children Produce High-Quality Serum IgG to Putative Nontypeable Haemophilus influenzae Vaccine Antigens at Lower Titres Compared to Non-Aboriginal Children.

Background: Nontypeable Haemophilus influenzae (NTHi) is the most common bacterial otopathogen associated with otitis media (OM). NTHi persists in biofilms within the middle ears of children with chronic and recurrent OM. Australian Aboriginal children suffer exceptionally high rates of chronic and recurrent OM compared to non-Aboriginal children. NTHi protein vaccines comprised of antigens associated with both adhesion and persistence in a biofilm are under development and could be beneficial for children with chronic and recurrent OM. Understanding the ontogeny of natural antibody development to these antigens provides insight into the value of vaccinating with particular antigens. Methods: An in-house multiplex fluorescent bead immunoassay was used to measure serum IgG titres and avidity for three putative vaccine antigens: recombinant soluble PilA (rsPilA), ChimV4, and outer membrane protein 26 (OMP26) in sera from Australian Aboriginal otitis-prone children (n=77), non-Aboriginal otitis-prone children (n=70) and non-otitis-prone children (n=36). Serum IgG titres were adjusted for age, and geometric mean concentrations (GMCs) were compared between groups using a univariate analysis model. Antibody avidity was calculated as a relative avidity index and compared between groups using ANOVA. Results: Australian Aboriginal otitis-prone children had lower serum IgG titres to rsPilA and ChimV4 than non-Aboriginal otitis-prone children (p<0.001), and non-otitis-prone children (p<0.020). No differences were observed between serum IgG titres from non-Aboriginal otitis-prone children and non-otitis-prone children. There were also no differences in the proportion of high avidity IgG specific for these antigens between these groups. Serum IgG titres to OMP26 were similar between all groups (p>0.670) although otitis-prone children had a higher proportion of high avidity antibodies to this antigen. Conclusions: Australian Aboriginal otitis-prone children had lower serum IgG titres to 2/3 major NTHi vaccine candidate antigens, suggesting these children are unable to develop persistent IgG responses due to repeated NTHi exposure. These reduced IgG titres may relate to earlier and more frequent exposure to diverse NTHi strains in Aboriginal children through carriage or infection. These data suggest that Aboriginal children may benefit from immunisation with vaccines containing these antigens to increase titres of protective antibodies.

The impacts of faecal subsampling on microbial compositional profiling.

OBJECTIVE: Despite the move to at-home, small-volume collection kits to facilitate large population-based studies of faecal microbial compositional profiling, there remains limited reporting on potential impacts of faecal subsampling approaches on compositional profiles. This study aimed to compare the microbial composition from faecal subsamples (< 5 g) collected from the beginning and end of a single bowel movement in ten otherwise healthy adults (6 female, 4 male; age: 24-55 years). Microbial composition was determined by V3-V4 16s rRNA sequencing and compared between subsamples. RESULTS: There were no significant differences in OTU count (p = 0.32) or Shannon diversity index (p = 0.29) between the subsamples. Comparison of relative abundance for identified taxa revealed very few differences between subsamples. At the lower levels of taxonomic classification differences in abundance of the Bacillales (p = 0.02) and the Eubacteriaceae family (p = 0.03), and the Eubacterium genera (p = 0.03) were noted. The observation of consistent microbial compositional profiles between faecal subsamples from the beginning and end of a single bowel movement is an important outcome for study designs employing this approach to faecal sample collection. These findings provide assurance that use of a faecal subsample for microbial composition profiling is generally representative of the gut luminal contents more broadly.

Adult allergic rhinitis sufferers have unique nasal mucosal and peripheral blood immune gene expression profiles: A case-control study.

BACKGROUND: Allergic rhinitis (AR) is a complex disease involving both mucosal and systemic immune compartments. Greater understanding of the immune networks underpinning AR pathophysiology may assist with further refining disease-specific biomarkers. OBJECTIVE: To compare immune gene expression profiles in nasal mucosa and peripheral blood samples between adults with AR and controls without AR. METHODS: This cross-sectional study included 45 adults with moderate-severe and persistent AR (37.6 ± 12.8 years; mean ± SD) and 24 adults without AR (36.6 ± 10.2). Gene expression analysis was performed using the NanoString nCounter PanCancer Immune profiling panel (n = 730 immune genes) in combination with the panel plus probe set (n = 30 allergy-related genes) with purified RNA from peripheral blood and cell lysates prepared from combined nasal lavage and nasal brushing. RESULTS: One hundred and thirteen genes were significantly differentially expressed in peripheral blood samples between groups (p < .05). In contrast, 14 genes were differentially expressed in nasal lysate samples between groups (p < .05). Upregulation of allergy-related genes in nasal mucosa samples in the AR group was observed. Namely, chemokines CCL17 and CCL26 are involved in the chemotaxis of key effector cells and TPSAB1 encodes tryptase, an inflammatory mediator released from activated mast cells and basophils. Six differentially expressed genes (DEGs) were in common between the nasal mucosa and blood samples. In addition, counts of specific DEGs in nasal mucosa samples were positively correlated with eosinophil and dust mite-specific immunoglobulin E (IgE) counts in blood. CONCLUSIONS AND CLINICAL RELEVANCE: Distinct gene expression profiles in blood and nasal mucosa samples were observed between AR sufferers and controls. The results of this study also provide evidence for a close interaction between the local site and systemic immunity. The genes identified in this study contribute to the current knowledge of AR pathophysiology and may serve as biomarkers to evaluate the effectiveness of treatment regimens, or as targets for drug discovery.

Nasal immune gene expression in response to azelastine and fluticasone propionate combination or monotherapy.

BACKGROUND: The combination of the antihistamine azelastine (AZE) with the corticosteroid fluticasone propionate (FP) in a single spray, has been reported to be significantly more effective at reducing allergic rhinitis (AR) symptoms than treatment with either corticosteroid or antihistamine monotherapy. However, the biological basis for enhanced symptom relief is not known. This study aimed to compare gene expression profiles (760 immune genes, performed with the NanoString nCounter) from peripheral blood and nasal brushing/lavage lysate samples in response to nasal spray treatment. METHODS: Moderate/severe persistent dust mite AR sufferers received either AZE (125 µg/spray) nasal spray (n = 16), FP (50 µg/spray) nasal spray (n = 14) or combination spray AZE/FP (125 µg AZE and 50 µg FP/spray) (n = 14) for 7 days, twice daily. Self-reported symptom questionnaires were completed daily for the study duration. Gene expression analysis (760 immune genes) was performed with the NanoString nCounter on purified RNA from peripheral blood and nasal brushing/lavage lysate samples. RESULTS: In nasal samples, 206 genes were significantly differentially expressed following FP treatment; 182 genes downregulated (-2.57 to -0.45 Log2 fold change [FC]), 24 genes upregulated (0.49-1.40 Log2 FC). In response to AZE/FP, only 16 genes were significantly differentially expressed; 10 genes downregulated (-1.53 to -0.58 Log2 FC), six genes upregulated (1.07-1.62 Log2 FC). Following AZE treatment only five genes were significantly differentially expressed; one gene downregulated (-1.68 Log2 FC), four genes upregulated (0.59-1.19 Log2 FC). Immune gene changes in peripheral blood samples following treatment were minimal. AR symptoms improved under all treatments, but improvements were less pronounced following AZE treatment. CONCLUSION: AZE/FP, FP, and AZE had diverse effects on immune gene expression profiles in nasal mucosa samples. The moderate number of genes modulated by AZE/FP indicates alternative pathways in reducing AR symptoms whilst avoiding extensive local immune suppression.

Innate Immunity in the Middle Ear Mucosa.

Otitis media (OM) encompasses a spectrum of clinical presentations ranging from the readily identifiable Acute OM (AOM), which is characterised by otalgia and fever, to chronic otitis media with effusion (COME) where impaired hearing due to middle ear effusion may be the only clinical symptom. Chronic suppurative OM (CSOM) presents as a more severe form of OM, involving perforation of the tympanic membrane. The pathogenesis of OM in these varied clinical presentations is unclear but activation of the innate inflammatory responses to viral and/or bacterial infection of the upper respiratory tract performs an integral role. This localised inflammatory response can persist even after pathogens are cleared from the middle ear, eustachian tubes and, in the case of respiratory viruses, even the nasal compartment. Children prone to OM may experience an over exuberant inflammatory response that underlies the development of chronic forms of OM and their sequelae, including hearing impairment. Treatments for chronic effusive forms of OM are limited, with current therapeutic guidelines recommending a "watch and wait" strategy rather than active treatment with antibiotics, corticosteroids or other anti-inflammatory drugs. Overall, there is a clear need for more targeted and effective treatments that either prevent or reduce the hyper-inflammatory response associated with chronic forms of OM. Improved treatment options rely upon an in-depth understanding of OM pathogenesis, particularly the role of the host innate immune response during acute OM. In this paper, we review the current literature regarding the innate immune response within the middle ear to bacterial and viral otopathogens alone, and as co-infections. This is an important consideration, as the role of respiratory viruses as primary pathogens in OM is not yet fully understood. Furthermore, increased reporting from PCR-based diagnostics, indicates that viral/bacterial co-infections in the middle ear are more common than bacterial infections alone. Increasingly, the mechanisms by which viral/bacterial co-infections may drive or maintain complex innate immune responses and inflammation during OM as a chronic response require investigation. Improved understanding of the pathogenesis of chronic OM, including host innate immune response within the middle ear is vital for development of improved diagnostic and treatment options for our children.

Differences in Pneumococcal and Haemophilus influenzae Natural Antibody Development in Papua New Guinean Children in the First Year of Life.

Background: Development of vaccines to prevent disease and death from Streptococcus pneumoniae, and nontypeable Haemophilus influenzae (NTHi), the main pathogens that cause otitis media, pneumonia, meningitis and sepsis, are a global priority. Children living in low and lower-middle income settings are at the highest risk of contracting and dying from these diseases. Improved vaccines with broader coverage are required. Data on the natural development of antibodies to putative vaccine antigens, especially in high-risk settings, can inform the rational selection of the best antigens for vaccine development. Methods: Serum IgG titres to four pneumococcal proteins (PspA1, PspA2, CbpA, and Ply) and five NTHi antigens (P4, P6, OMP26, rsPilA and ChimV4) were measured in sera collected from 101 Papua New Guinean children at 1, 4, 9, 10, 23 and 24 months of age using multiplexed bead-based immunoassays. Carriage density of S. pneumoniae and H. influenzae were assessed by quantitative PCR on genomic DNA extracted from nasopharyngeal swabs using species-specific primers and probes. All data were log-transformed for analysis using Student's unpaired t-tests with geometric mean titre (GMT) or density (GMD) calculated with 95% confidence intervals (CI). Results: Serum -pneumococcal protein-specific IgG titres followed a "U" shaped pattern, with a decrease in presumably maternally-derived IgG titres between 1 and 4 months of age and returning to similar levels as those measured at 1 month of age by 24 months of age. In contrast, NTHi protein-specific IgG titres steadily increased with age. There was no correlation between antibody titres and carriage density for either pathogen. Conclusion: This longitudinal study indicates that the waning of maternally- derived antibodies that is usually observed in infants, after infants does not occur for NTHi antigens in Papua New Guinean infants. Whether NTHi antigen IgG can be transferred maternally remains to be determined. Vaccines that are designed to specifically increase the presence of protective NTHi antibodies in the first few months of life may be most effective in reducing NTHi disease. Clinical Trial Registration: https://clinicaltrials.gov/, identifier NCT01619462.

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

[This corrects the article DOI: 10.3389/fimmu.2020.599547.].

Immunogenicity following revaccination or sequential vaccination with 23-valent pneumococcal polysaccharide vaccine (PPSV23) in older adults and those at increased risk of pneumococcal disease: a review of the literature.

Introduction: Immunogenicity studies evaluating sequential administration of pneumococcal conjugate vaccine (PCV) followed by 23-valent pneumococcal polysaccharide vaccine (PPSV23) or revaccination with PPSV23 have raised concerns that PPSV23 may not elicit higher antibody levels than those measured following PCV or first PPSV23 dose.Areas covered: Recent literature was evaluated for evidence of blunted immune response (hyporesponsiveness), focusing on studies using adequate intervals between doses in accordance with vaccination recommendations. In eight of nine studies that evaluated revaccination with PPSV23 at an interval of ≥5 years after the previous dose, immunoglobulin G geometric mean concentrations and/or opsonophagocytic assay geometric mean titers for most serotypes increased from pre- to post-repeat vaccination and were comparable between repeat and primary vaccination groups post-vaccination. In seven studies in which PPSV23 was administered after PCVs (8 weeks to 1 year apart), responses to PPSV23 were comparable to those seen after initial PCV dose for shared vaccine serotypes. Studies in which PCVs were administered after PPSV23 were not evaluated.Expert opinion: Published data suggest immune responses following repeat vaccination with PPSV23, or sequential PCV/PPSV23 vaccination, are robust, without evidence of hyporesponsiveness. PPSV23 vaccination of at-risk adults is essential to ensure broad protection against all 23 vaccine serotypes.

Efficacy and effectiveness of a 23-valent polysaccharide vaccine against invasive and noninvasive pneumococcal disease and related outcomes: a review of available evidence.

Introduction: Routine pneumococcal vaccination for adults aged ≥60 or ≥65 years and those with underlying at-risk and high-risk conditions is recommended in many countries. However, studies estimating the effectiveness of 23-valent pneumococcal polysaccharide vaccine (PPSV23) have revealed mixed results, partly due to variability in study design and endpoints used to assess outcomes.Areas covered: The authors conducted a literature review of independently randomized trials and real-world studies published from 2010 to 2020 that assessed the effectiveness and efficacy of PPSV23 against vaccine-type or any-serotype invasive and noninvasive pneumococcal disease in adults aged ≥60 years. The authors also evaluated differences in study design that may contribute to the heterogeneity of available evidence.Expert opinion: Policy decisions regarding the inclusion of vaccines into national immunization plans should consider study quality and limitations. This review shows that PPSV23 is effective against vaccine-type invasive pneumococcal disease and vaccine-type pneumococcal pneumonia and can lower the burden of vaccine-type pneumococcal pneumonia. PPSV23-conferred protection may be lower in adults aged ≥75 years, those with certain underlying conditions, and individuals who were vaccinated >5 years before disease onset. This is an important finding that supports the benefit of PPSV23 vaccination for older adults.

The Gut Microbiome of Adults with Allergic Rhinitis Is Characterised by Reduced Diversity and an Altered Abundance of Key Microbial Taxa Compared to Controls.

INTRODUCTION: Unique gut microbial colonisation patterns are associated with the onset of allergic disease in infants; however, there is insufficient evidence to determine if aberrant microbial composition patterns persist in adult allergic rhinitis (AR) sufferers. OBJECTIVE: To compare the gut microbiome composition between adult AR sufferers and controls. METHODS: Gut microbial composition in stool samples was compared between 57 adult AR sufferers (39.06 ± 13.29 years) and 23 controls (CG; 36.55 ± 10.51 years) via next-generation sequencing of the V3-V4 hypervariable regions of the 16S rRNA gene. Taxonomic classification and identity assignment was performed using a reference-based approach with the NCBI database of 16S rRNA gene sequences. RESULTS: Species richness determined via the Shannon index was significantly reduced in the AR cohort compared to the CG (4.35 ± 0.59 in AR vs. 4.65 ± 0.55 in CG, p = 0.037); trends for reductions in operational taxonomic unit (OTU) counts, inverse Simpson, and CHAO1 diversity indices were also noted. Bacteroidetes (p = 0.014) was significantly more abundant in the AR group than in the CG. In contrast, the Firmicutes phylum was significantly less abundant in the AR group than in the CG (p = 0.006). An increased abundance of Parabacteroides (p = 0.008) and a reduced abundance of Oxalobacter (p = 0.001) and Clostridiales (p = 0.005) were also observed in the AR cohort compared to the CG. CONCLUSION: Adult AR sufferers have a distinct gut microbiome profile, marked by a reduced microbial diversity and altered abundance of certain microbes compared to controls. The results of this study provide evidence that unique gut microbial patterns occur in AR sufferers in adulthood and warrant further examination in the form of mechanistic studies.

Fucoidan Supplementation Restores Fecal Lysozyme Concentrations in High-Performance Athletes: A Pilot Study.

Nutritional strategies to help promote immune competence are of particular interest for a range of population groups. This study aimed to assess the potential impacts of fucoidan, a seaweed-derived bioactive polysaccharide, on gut markers of immunity and inflammation. A group of professional team-sport athletes were selected for inclusion in the study given the recognized potential for intense physical activity to induce alterations in immune function. A retrospective analysis was performed on stored fecal samples which had been collected from professional team-sport athletes (n = 22) and healthy adults (n = 11) before and after seven days of supplementation with fucoidan (Fucus vesiculosus/Undaria pinnatifida extract, 1 g/d). Fecal concentrations of calprotectin, secretory immunoglobulin A (sIgA) and lysozyme were determined using enzyme-linked immunosorbent assays. The supplement was well tolerated by participants with no adverse events reported. At baseline, fecal lysozyme concentrations were ~73% higher in the healthy adults compared to the professional athletes (p = 0.001). For the professional athletes, a significant (~45%) increase in fecal lysozyme was observed following the supplementation period (p = 0.001). These data suggest that fucoidan supplementation may have the potential to promote the secretion of antimicrobial peptides in specific population groups and contribute to the regulation of mucosal immune health.

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.

Pharmacokinetic modeling to determine the minimum effective dose of disease-specific antibodies for preventing hepatitis A post-exposure.

BACKGROUND: The minimum effective dose of intramuscular polyvalent immune globulin for prevention of hepatitis A post-exposure is unknown. In Australia current dosing is according to weight category. METHODS: The peak concentration and decay of hepatitis A antibodies after intramuscular dosing of immune globulin in adults was modeled utilizing published parameters. Models simulated dosing according to current Australian guidelines, then adjusted the dose in clinically relevant increments to estimate the optimal dose of hepatitis A antibodies for post-exposure prophylaxis of nonimmune individuals. Optimal dosing assumed a target serum concentration of hepatitis A antibodies of the correlate of protection plus a 10% margin of error at an incubation period. The effect of weight on hepatitis A antibody concentration at an incubation period under current guidelines was examined by fixing weight in 5 kg increments. RESULTS: Current dosing guidelines in Australia may underdose people who weigh in excess of 85 kg. The optimal dose of hepatitis A-specific antibodies according to the model was 3.6, 2.5, and 1.9 IU/kg assuming 50%, 75% and 100% bioavailability respectively. CONCLUSIONS: For individuals in Australia recommended passive immunization as post-exposure prophylaxis and weighing in excess of 85 kg, conservative management would include dosing between 2.5 and 3.6 IU hepatitis A antibodies/kg.

Key viral immune genes and pathways identify elite athletes with URS.

PURPOSE: Habitual intense exercise may increase the incidence of upper respiratory symptoms (URS) in elite athletes. This study investigated whether immune gene expression could identify gene markers that discriminate athletes with a higher prevalence of URS. METHODS: This cross-sectional analysis of elite Australian athletes from various sports investigated whether athletes retrospectively reporting URS for two days or more in a month (n=38), had an altered immune gene expression profile compared with asymptomatic athletes (n=33). Peripheral blood samples were collected during Olympic selection events with corresponding URS data collected for the one-month period before sampling. Digital immune gene expression analysis was undertaken using the NanoString PanCancer Immune Profiling panel. RESULTS: Fifty immune genes were differentially expressed between the groups (p<0.05) and approximately 78% of these genes were more highly expressed in athletes reporting URS. Many of these genes were interferon-stimulated genes or genes involved in the Jak/Stat signalling pathway. Only interferon alpha inducible protein 27 (IFI27), an interferon stimulated gene involved in viral response, remained significantly higher in athletes reporting URS (log2 fold-difference=2.49, odds ratio 1.02 per unit increase; p<0.01) post-adjustment and discriminated athletes reporting URS from asymptomatic athletes with 78% accuracy. CONCLUSIONS: Expression of IFI27 could differentiate athletes reporting URS from asymptomatic athletes, a gene that is upregulated in the immune response to viral infection. Upregulation of viral signalling pathways provides novel information on the potential aetiology of URS in elite Olympic athletes.

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.

Immunological characterisation of truncated lipooligosaccharide-outer membrane protein based conjugate vaccine against Moraxella catarrhalis and nontypeable Haemophilus influenzae.

Moraxella catarrhalis and nontypeable Haemophilus influenzae are important bacterial causes of otitis media in children and respiratory diseases in adults. Lipooligosaccharide (LOS) from M. catarrhalis and outer membrane protein 26 (OMP26) from NTHi are major surface antigens identified as potential vaccine components against these organisms. We previously constructed M. catarrhalis in which LOS is truncated, but contains a structure common to the three known serotypes of M. catarrhalis. OMP26 is known to enhance clearance of NTHi following vaccination in animal models, so was chosen as the carrier protein. In this study, we conjugated wild-type and truncated M. catarrhalis detoxified-LOS to a recombinant modified OMP26, rOMP26VTAL. Vaccination of mice with these conjugates resulted in a significant increase in anti-LOS and anti-rOMP26VTAL IgG levels. Importantly, mouse antisera showed complement-mediated bactericidal activity against all M. catarrhalis serotype A and B strains and a NTHi strain tested. Serotypes A & B make up more than 90% of isolates. These data suggest that the LOS and OMP based conjugate can be used as vaccine components and require further investigation in animal models.

Recovery of Lactobacillus casei strain Shirota (LcS) from faeces with 14 days of fermented milk supplementation in healthy Australian adults.

BACKGROUND AND OBJECTIVES: A key measure for classifying bacteria as a probiotic is the ability to survive gastric transport and be recoverable in faeces. The aim of this study was to determine whether Lactobacillus casei strain Shirota (LcS) could be recovered in the faeces of healthy young Australian adults following ingestion of a fermented milk drink. METHODS AND STUDY DESIGN: A cohort of 25 healthy individuals (male/female: 14/11; age: 29.3±6.6 years; BMI: 25.3±2.7 kg/m2, mean±SD) ingested one 65 ml bottle of fermented milk containing 6.5×109 LcS live cells daily for 14 days. Participants provided a faecal sample at day 0, day 7 (mid-supplementation), day 14 (end of supplementation) and 14 days after cessation of the supplement (day 28) for assessment of the number of viable LcS via microbial culture on selective media with confirmation using a colony-direct polymerase chain reaction and species-specific primers. RESULTS: The supplement was well tolerated by participants. No LcS colonies were recovered from participants prior to ingestion of the fermented milk drink. All participants had recoverable LcS colonies at day 7 and day 14, with a mean recovery of 6.5±1.1 and 6.4±1.1 log10 CFU/g of faeces (mean±SD) at each time point respectively. LcS was detectable in only one sample at 14 days following the cessation of supplementation. CONCLUSIONS: Live LcS is recoverable in faeces from healthy Australian adults following daily ingestion of a fermented milk drink.