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Background Cancer and anti-cancer treatment (ACT) may be risk factors for severe SARS-CoV-2 infection and limited vaccine efficacy. Long–term longitudinal studies are needed to evaluate these risks. The Scottish COVID cancer immunity prevalence (SCCAMP) study characterizes the incidence and outcomes of SARS-CoV-2 infection and vaccination in patients with solid tumors undergoing ACT. This preliminary analysis includes 766 patients recruited since May 2020. Methods Patients with solid-organ cancers attending secondary care for active ACT consented to the collection of routine electronic health record data and serial blood samples over 12 months. Blood samples were tested for total SARS-CoV-2 antibody. Results A total of 766 participants were recruited between May 28, 2020 and October 31, 2021. Most received cytotoxic chemotherapy (79%). Among the participants, 48 (6.3%) were tested positive for SARS-CoV-2 by PCR. Infection rates were unaffected by ACT, largely aligning with the local population. Mortality proportion was not higher with a recent positive SARS-CoV-2 PCR (10.4% vs 10.6%). Multivariate analysis revealed lower infection rates in vaccinated patients regardless of chemotherapy (HR 0.307 [95% CI, 0.144-0.6548]) or immunotherapy (HR 0.314 [95% CI, 0.041-2.367]) treatment. A total of 96.3% of patients successfully raised SARS-CoV-2 antibodies after >2 vaccines. This was independent of the treatment type. Conclusion This is the largest on-going longitudinal real-world dataset of patients undergoing ACT during the early stages of the COVID-19 pandemic. This preliminary analysis demonstrates that patients with solid tumors undergoing ACT have high protection from SARS-CoV-2 infection following COVID-19 vaccination. The SCCAMP study will evaluate long–term COVID-19 antibody trends, focusing on specific ACTs and patient subgroups.

ObjectiveEmergency interim guidance from the British Society for Gastroenterology (BSG) states that a no-biopsy strategy is possible to diagnose coeliac disease (CD) in adults with elevated transglutaminase IgA antibody (TGA-IgA) levels. We aimed to determine if the suggested TGA-IgA ≥10× ULN is safe and robust in making the diagnosis in adult patients in Scotland. We also aimed to establish if any important co-diagnoses would be missed if no biopsy was performed.MethodAll positive coeliac serology results for patients aged >15 years in Scotland in 2016 (Grampian 2019) were accessed. Data were collected on demographics, TGA-IgA titres, D1 sampling, histology and macroscopic findings at upper and lower gastrointestinal (GI) endoscopy.Results1037/1429 patients with positive serology proceeded to biopsy, of which 796/1037 (76.8%) were diagnosed as CD. A total of 320/322 (99.37%) patients with TGA-IgA ≥10× ULN were diagnosed as CD giving the cut-off a positive predictive value of 99.38%. No significant co-pathology was found at endoscopy in these patients.ConclusionOur results show that a no-biopsy strategy using a cut-off of TGA-IgA ≥10× ULN is safe to diagnose CD and that no important pathology would be missed. The European Society for Paediatric Gastroenterology, Hepatology and Nutrition 2020 and BSG COVID-19 interim guidelines are applicable to adult patients in Scotland.

Serological assays are being used to monitor antibody responses in individuals who had SARS-CoV-2 infection and those who received a COVID-19 vaccine. We aimed to determine whether such assays can predict neutralising antibody titres as antibody levels wane and viral variants emerge.We measured antibody levels in serum samples from a cohort of 112 participants with SARS-CoV-2 infection using ten high-throughput serological tests and functional neutralisation assays. Serum samples were taken at baseline and at up to four subsequent visits. We assessed the effects of time and spike protein sequence variation on the performance and predictive value of the various assays. We did correlation analyses for individual timepoints using non-parametric Spearman correlation, and differences between timepoints were determined by use of a two-tailed Wilcoxon matched-pairs signed rank test.Neutralising antibody titres decreased over the first few months post-infection but stabilised thereafter, at about 30% of the level observed shortly after infection. Serological assays commonly used to measure antibodies against SARS-CoV-2 displayed a range of sensitivities that declined to varying extents over time. Quantitative measurements generated by serological assays based on the spike protein were better at predicting neutralising antibody titres than those based on nucleocapsid, but performance was variable, and manufacturer positivity thresholds were not able to predict the presence or absence of detectable neutralising activity. Although we observed some deterioration in correlation between serological measurements and functional neutralisation activity, some assays maintained an ability to predict neutralising titres, even against variants of concern.The ability of high-throughput serological assays to predict neutralising antibody titres is likely to be crucial for evaluation of immunity at the population scale. These data can facilitate the selection of the most suitable assays as surrogates of functional neutralising activity and suggest that such measurements might be useful in clinical practice.US National Institutes of Health and National Health Service Research Scotland BioResource.

Chronic liver disease (CLD) is a significant health problem affecting millions of people worldwide. In Scotland, CLD is a major cause of premature mortality. Liver function tests (LFTs) are a panel of frequently requested blood tests which may indicate liver disease. However, LFTs commonly contain at least one abnormal result, and abnormalities are rarely investigated to the extent recommended by national guidelines. The intelligent Liver Function Testing (iLFT) pathway is a novel, automated system designed to improve early diagnosis of liver disease. Initial abnormal LFT results trigger a cascade of reflexive testing to help identify the cause of any liver dysfunction. Algorithms combine these results with demographic and clinical data (such as patient age, body mass index, and alcohol intake) and fibrosis estimates to produce an electronic diagnosis and management plan. The pilot trial demonstrated that iLFT increased diagnosis of liver disease whilst remaining cost-effective. As such, iLFT has been fully operational across our region (NHS Tayside, Scotland) since August 2018. In the first year, iLFT generated over 2000 diagnoses from 1824 patient samples with an abnormality in the initial LFTs. The majority of these patients could be safely managed in primary care. iLFT allows maximal value to be obtained from liver blood tests across biochemistry, virology, immunology, and hematology with only minor changes to working practices. ‘Intelligent’, algorithm-led testing pathways break down the barrier between clinical and laboratory medicine and offer solutions to many of the challenges experienced in modern healthcare systems.

BackgroundSero-surveillance of SARS-CoV-2 is crucial to monitoring levels of population exposure and informing public health responses, but may be influenced by variability in performance between available assays.MethodsFive commercial immunoassays and a neutralising activity assay were used to detect antibodies to SARS-CoV-2 in routine primary care and paediatric samples collected during the first wave of the pandemic in NHS Lothian, Scotland as part of ongoing surveillance efforts. For each assay, sensitivity and specificity was calculated relative to consensus results and neutralising activity. Quantitative correlation was performed between serological and neutralising titres.ResultsSeroprevalence ranged from 3.4-7.3 % in primary care patients and 3-5.9 % in paediatric patients according to different immunoassays. Neutralising activity was detectable in 2.8 % and 1.3 % respectively. Relative assay performance changed depending on comparison to immunoassay consensus versus neutralising activity and qualititative versus quantitative agreement. Cross-reactivity with endemic seasonal coronaviruses was confirmed by neutralising assay in false positives for one immunoassay. Presence of false positives for another assay was found specifically in paediatric but not adult samples.ConclusionsFive serological assays show variable accuracy when applied to the general population, impacting seroprevalence estimates. Assay performance may also vary in detection of protective neutralising antibody levels. These aspects should be considered in assay selection and interpretation in epidemiological studies.

This work was supported by the Scottish Government. Objectives : Studies that measure the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (‘seroprevalence’) are essential to understand population exposure to SARS-CoV-2 among symptomatic and asymptomatic individuals. We aimed to measure seroprevalence in the Scottish population over the course of the COVID-19 pandemic – from before the first recorded case in Scotland through to the second pandemic wave. Study design : The study design of this study is serial cross sectional. Methods : We tested 41,477 residual samples retrieved from primary and antenatal care settings across Scotland for SARS-CoV-2 antibodies over a 12-month period from December 2019-December 2020 (before rollout of COVID-19 vaccination). Five-weekly rolling seroprevalence estimates were adjusted for the sensitivity and specificity of the assays and weighted to reference populations. Temporal trends in seroprevalence estimates and weekly SARS-CoV-2 notifications were compared. Results : Five-weekly rolling seroprevalence rates were 0% until the end of March, when they increased contemporaneously with the first pandemic wave. Seroprevalence rates remained stable through the summer (range: 3%–5%) during a period of social restrictions, after which they increased concurrently with the second wave, reaching 9.6% (95% confidence interval [CI]: 8.4%–10.8%) in the week beginning 28th December in 2020. Seroprevalence rates were lower in rural vs. urban areas (adjusted odds ratio [AOR]: 0.70, 95% CI: 0.61–0.79) and among individuals aged 20–39 years and 60 years and older (AOR: 0.74, 95% CI: 0.64–0.86; AOR: 0.80, 95% CI: 0.69–0.91, respectively) relative to those aged 0–19 years. Conclusions : After two waves of the COVID-19 pandemic, less than one in ten individuals in the Scottish population had antibodies to SARS-CoV-2. Seroprevalence may underestimate the true population exposure as a result of waning antibodies among individuals who were infected early in the first wave. Postprint

Background Thrombocytosis is often an incidental finding in primary care with a range of causes. Despite evidence of a strong association between thrombocytosis and malignancy, guidelines for investigating thrombocytosis in the absence of red flag symptoms remain unclear. A novel automated system of laboratory analysis, intelligent Liver Function Testing (iLFT), launched in Tayside in 2018 and has identified a patient group with thrombocytosis and abnormal liver test (LFT) results. This study analysed the outcome of these patients and investigated the use of thrombocytosis combined with LFTs in predicting risk of cancer. Methods and findings Between August 2018 and August 2020, 6792 patients underwent iLFT, with 246 found to have both thrombocytosis and at least one abnormal LFT. A random case-matched control group of 492 iLFT patients with normal platelet count and at least one abnormal LFT was created. 7.7% (95% CI 4.7–11.8%) of patients with thrombocytosis had cancer compared to 2.0% (1.0–3.7%) of controls. Patients Conclusions These findings suggest a substantial increased risk of cancer in patients with thrombocytosis and raised ALP. This could be developed as an adjunct to current investigation algorithms, highlighting high-risk patients and prompting further investigation (such as computed tomography scans) where indicated.

BackgroundSerological assays are being deployed to monitor antibody responses in SARS-CoV-2 convalescents and vaccine recipients. There is a need to determine whether such assays can predict immunity, as antibody levels wane and viral variants emerge.MethodsWe measured antibodies in a cohort of SARS-CoV-2 infected patients using several high-throughput serological tests and functional neutralization assays. The effects of time and spike protein sequence variation on the performance and predictive value of the various assays was assessed.FindingsNeutralizing antibody titers decreased over the first few months post-infection but stabilized thereafter, at about 30% of the level observed shortly after infection. Serological assays commonly used to measure antibodies against SARS-CoV-2 displayed a range of sensitivities that declined to varying extents over time. Quantitative measurements generated by serological assays based on the spike protein were better at predicting neutralizing antibody titers than assays based on nucleocapsid, but performance was variable and manufacturer positivity thresholds were not able to predict the presence or absence of detectable neutralizing activity. Even though there was some deterioration in correlation between serological measurements and functional neutralization activity, some assays maintained an ability to predict neutralizing titers, even against variants of concern.InterpretationThe ability of high throughput serological assays to predict neutralizing antibody titers is likely crucial for evaluation of immunity at the population scale. These data will facilitate the selection of the most suitable assays as surrogates of functional neutralizing activity and suggest that such measurements may have utility in clinical practice.

BackgroundCancer and systemic anti-cancer treatment (SACT) have been identified as possible risk factors for infection and related severe illness associated with SARS-CoV-2 virus as a consequence of immune suppression. The Scottish COVID CAncer iMmunity Prevalence (SCCAMP) study aims to characterise the incidence and outcomes of SARS-Cov-2 infection in patients undergoing active anti-cancer treatment during the COVID-19 pandemic and their antibody response following vaccination.Patients and MethodsEligible patients were those attending secondary care for active anti-cancer treatment for a solid tumour. Blood samples were taken for total SARS-CoV-2 antibody assay (Siemens) at baseline and after 1.5, 3, 6 and 12 months. Data on COVID-19 infection, vaccination, cancer type, treatment and outcome was obtained from routine electronic health records.ResultsThe study recruited 766 eligible participants between 28th May 2020 and 31st October 2021. The median age was 62.7 years, and 66.5% were female. Most received cytotoxic chemotherapy (79%), with the remaining 14% receiving immunotherapy and 7% receiving another form of anti-cancer therapy (radiotherapy, other systemic anti-cancer treatment). 48 (6.3%) tested positive for SARS-CoV-2 by PCR during the study period. The overall infection rate matched that of the age-matched local general population until May 2021, after which population levels appeared higher. Antibody testing detected additional evidence of infection prior to vaccination, taking the total number to 58 (7.6%). There was no significant difference in SARS-CoV-2 PCR positive test rates based on type of anti-cancer treatment. Mortality proportion was similar between those who died within 90 days of a positive SARS-CoV-2 PCR and those with no positive PCR (10.4% vs 10.6%). Death from all causes was lowest among vaccinated patients, and of the patients who had a positive SARS-CoV-2 PCR at any time, all of those who died during the study period were unvaccinated. Multivariate analysis correcting for age, gender, socioeconomic status, comorbidities and number of previous medications revealed that vaccination was associated with a significantly lower infection rate regardless of treatment with chemotherapy or immunotherapy with hazard ratios of 0.307 (95% CI 0.144-0.6548) or 0.314 (95% CI 0.041-2.367) in vaccinated patients respectively. Where antibody data was available, 96.3% of patients successfully raised SARS-CoV-2 antibodies at a time point after vaccination. This was unaffected by treatment type.ConclusionSCCAMP provides real-world evidence that patients with cancer undergoing SACT have a high antibody response and protection from SARS-CoV-2 infection following COVID-19 vaccination.Highlights-The SCCAMP dataset represents the largest longitudinal study of patients with cancer undergoing anti-cancer treatment during the COVID-19 pandemic-Rates of infection in the cancer cohort mirrored those of the local age adjusted population-Vaccination was effective in patients with cancer undergoing active treatment in terms of antibody response and SARS-CoV-2 PCR rates-Treatment type did not impact the rate of SARS-CoV-2 antibody response

Background Healthcare workers (HCWs) are believed to be at increased risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. It is not known to what extent the natural production of antibodies to SARS-CoV-2 is protective against re-infection. Methods A prospective observational study of HCWs in Scotland (UK) from May to September 2020 was performed. The Siemens SARS-CoV-2 total antibody assay was used to establish seroprevalence in this cohort. Controls, matched for age and sex to the general local population, were studied for comparison. New infections (up to 2 December 2020) post antibody testing were recorded to determine whether the presence of SARS-CoV-2 antibodies protects against re-infection. Results A total of 2063 health and social care workers were recruited for this study. At enrolment, 300 HCWs had a positive antibody test (14.5%). 11 out of 231 control sera tested positive (4.8%). HCWs therefore had an increased likelihood of a positive test (OR 3.4, 95% CI 1.85–6.16; p, In this study, healthcare workers were three times more likely to test positive for #SARSCoV2 than the general population. Almost all infected individuals developed an antibody response, and this was 85% effective in protecting against re-infection. https://bit.ly/3mLPUmk

Introduction Neutrophil extracellular traps (NETs) are a form of antimicrobial defence which have been implicated in multiple inflammatory diseases. This study investigated the role of NETs in bronchiectasis, a neutrophilic disease which lacks therapies that directly target neutrophilic inflammation, through a series of UK and international studies. Methods LC/MS proteomics was used to compare protein profiles in sputum between severe and mild bronchiectasis (20 vs 20 patients). Microbiome changes, using 16S rRNA sequencing, clinical characteristics and NET levels using a validated histone-elastase immunoassay were analysed. Results were validated in an independent European cohort. Proteomics was used to identify proteins associated with treatment response of acute exacerbations of bronchiectasis in 20 patients treated with intravenous antibiotics for 14 days. Two studies of long-term macrolide treatment, one in bronchiectasis and a post-hoc analysis of the AMAZES trial in asthma, investigated the effect of macrolide treatment on NETs. Results 96 proteins were differentially expressed in sputum between severe and mild bronchiectasis, with known NET proteins being the most abundant and discriminating. The relationship between NETs and associated proteins were validated in two independent cohorts, a UK cohort (n=175) and a European cohort (n=275). Sputum NETs were associated with BSI (p Conclusion NET-associated proteins are elevated in bronchiectasis sputum and are associated with disease severity, bacterial infection and mortality. Treatment response is linked to successfully reducing NET levels with intravenous antibiotic or macrolide therapies suggesting that NETs may be an important therapeutic target in bronchiectasis.

Introduction Liver Function Tests (LFTs) are abnormal in 20% of cases in primary care. In addition, mortality from chronic liver disease continues to rise. Intelligent liver function testing (iLFT) was designed to improve diagnosis of liver disease, the quality of further investigations, and optimise the time of referral to secondary care. This will reduce both mortality and cost to practitioners. Following a trial it was launched in Tayside, Scotland in August 2018. Referrers provide information on alcohol intake, BMI and comorbidities, and those with abnormal LFTs have reflex tests including non-invasive fibrosis scores, without further venepuncture. General Practitioners (GPs) are provided with management plans with a recommended outcome: secondary care referral for fibrosis assessment or treatment; primary care follow up of early liver disease; or when a diagnosis is unclear, staging information including referral criteria. Methods A retrospective analysis was performed of all patients who had iLFT requested by GPs in the first eighteen months (August 2018 – January 2020) of the live system, recording the outcome(s) for each request. Results 4194 requests were received for iLFT in the first eighteen months from launch. iLFT did not cascade in 1106 cases, either because all liver enzymes were normal, or because insufficient data was provided. There were 4012 iLFT outcomes. The most common outcome was isolated ALT elevation without fibrosis (n=884; 22%), followed by alcohol related liver disease without fibrosis (n=534, 13.3%) and non-alcoholic fatty liver disease with fibrosis (n=354; 8.8%). The frequency of each outcome is shown in figure 1. Secondary care referral was recommended in 951 (23.7%) cases; of which 732 (77%) were for fibrosis assessment only. The remaining 3061 (76.3%) outcomes recommended primary care management. The number of iLFT requests rose each month from launch, from 159 in November 2018 to 431 in December 2019, and these now make up over 3% of all LFT requests in Tayside. Conclusions iLFT safely and rapidly detects patients at risk of chronic liver disease using reflex fibrosis scores and autoimmune, virology and genetic tests. Secondary care referral is recommended in only 24% of cases, and thus 76% of patients can be managed in primary care. The number of iLFT requests is rising each month. GPs initially used iLFT to investigate patients known to have abnormal LFTs, resulting in high numbers of full test cascades. iLFT is now being used as the first test, so the number cascading is falling, but still improving the diagnosis of liver disease. This ensures patients who require secondary care are seen, whilst reducing unnecessary referrals.

Background: Healthcare workers (HCW) are believed to be at increased risk of SARS-CoV-2 infection. The extent of that increased risk compared to the general population and the groups most at risk have not been extensively studied. It is also not known to what extent the natural production of antibodies to SARS-CoV-2 is protective against re-infection. Methods: A prospective observational study of health and social care workers in NHS Tayside (Scotland, UK) from May to September 2020. The Siemens SARS-CoV-2 total antibody assay was used to establish seroprevalence in this cohort. Controls, matched for age and sex to the general Tayside population, were studied for comparison. New infections post antibody testing were recorded to determine if the presence of SARS-CoV-2 antibodies protect against re-infection. Results: A total of 2063 health and social care workers were recruited for this study. 300 HCW had a positive antibody test (14.5%). 11/231 control sera tested positive (4.8%). HCW therefore had an increased likelihood of a positive test (Odds ratio 3.4 95% CI 1.85-6.16, p

IntroductionHealthcare workers are believed to be at increased risk of SARS-CoV-2 infection. The extent of that increased risk compared to the general population and the groups most at risk have not been extensively studied.MethodsA prospective observational study of health and social care workers in NHS Tayside (Scotland, UK) from May to September 2020. The Siemens SARS-CoV-2 total antibody assay was used to establish seroprevalence in this cohort. Patients provided clinical information including demographics and workplace information. Controls, matched for age and sex to the general Tayside population, were studied for comparison.ResultsA total of 2062 health and social care workers were recruited for this study. The participants were predominantly female (81.7%) and 95.2% were white. 299 healthcare workers had a positive antibody test (14.5%). 11 out of 231 control sera tested positive (4.8%). Healthcare workers therefore had an increased likelihood of a positive test (odds ratio 3.4 95% CI 1.85-6.16, pConclusionIn this study, healthcare workers were three times more likely to test positive for SARS-CoV-2 than the general population. The seroprevalence data in different populations identified in this study will be useful to protect healthcare staff during future waves of the pandemic.

Summary Background Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because little is understood of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a method of host defence that has been implicated in multiple inflammatory diseases. We aimed to investigate the role of NETs in disease severity and treatment response in bronchiectasis. Methods In this observational study, we did a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. First, we used liquid chromatography-tandem mass spectrometry to identify proteomic biomarkers associated with disease severity, defined using the bronchiectasis severity index, in patients with bronchiectasis (n=40) in Dundee, UK. Second, we validated these biomarkers in two cohorts of patients with bronchiectasis, the first comprising 175 patients from the TAYBRIDGE study in the UK and the second comprising 275 patients from the BRIDGE cohort study from centres in Italy, Spain, and UK, using an immunoassay to measure NETs. Third, we investigated whether pathogenic bacteria had a role in NET concentrations in patients with severe bronchiectasis. In a separate study, we enrolled patients with acute exacerbations of bronchiectasis (n=20) in Dundee, treated with intravenous antibiotics for 14 days and proteomics were used to identify proteins associated with treatment response. Findings from this cohort were validated in an independent cohort of patients who were admitted to the same hospital (n=20). Fourth, to assess the potential use of macrolides to reduce NETs in patients with bronchiectasis, we examined two studies of long-term macrolide treatment, one in patients with bronchiectasis (n=52 from the UK) in which patients were given 250 mg of azithromycin three times a week for a year, and a post-hoc analysis of the Australian AMAZES trial in patients with asthma (n=47) who were given 500 mg of azithromycin 3 times per week for a year. Findings Sputum proteomics identified that NET-associated proteins were the most abundant and were the proteins most strongly associated with disease severity. This finding was validated in two observational cohorts, in which sputum NETs were associated with bronchiectasis severity index, quality of life, future risk of hospital admission, and mortality. In a subgroup of 20 patients with acute exacerbations, clinical response to intravenous antibiotic treatment was associated with successfully reducing NETs in sputum. Patients with Pseudomonas aeruginosa infection had a lessened proteomic and clinical response to intravenous antibiotic treatment compared with those without Pseudomonas infections, but responded to macrolide therapy. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. Interpretation We identified NETs as a key marker of disease severity and treatment response in bronchiectasis. These data support the concept of targeting neutrophilic inflammation with existing and novel therapies. Funding Scottish Government, British Lung Foundation, and European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC).

ObjectivesTo investigate longitudinal trajectory of SARS-CoV-2 neutralising antibodies and the performance of serological assays in diagnosing prior infection and predicting serum neutralisation titres with timeDesignRetrospective longitudinal analysis of a COVID19 case cohort.SettingNHS outpatient clinicsParticipantsIndividuals with RT-PCR diagnosed SARS-CoV-2 infection that did not require hospitalizationMain outcome measuresThe sensitivity with which prior infection was detected and quantitative antibody titres were assessed using four SARS-CoV-2 serologic assay platforms. Two platforms employed SARS-CoV-2 spike (S) based antigens and two employed nucleocapsid (N) based antigens. Serum neutralising antibody titres were measured using a validated pseudotyped virus SARS-CoV-2 neutralisation assay. The ability of the serological assays to predict neutralisation titres at various times after PCR diagnosis was assessed.ResultsThe three of the four serological assays had sensitivities of 95 to100% at 21-40 days post PCR-diagnosis, while a fourth assay had a lower sensitivity of 85%. The relative sensitivities of the assays changed with time and the sensitivity of one assay that had an initial sensitivity of >95% declined to 85% at 61-80 post PCR diagnosis, and to 71% at 81-100 days post diagnosis. Median antibody titres decreased in one serologic assay but were maintained over the observation period in other assays. The trajectories of median antibody titres measured in serologic assays over this time period were not dependent on whether the SARS-CoV-2 N or S proteins were used as antigen source. A broad range of SARS-CoV-2 neutralising titres were evident in individual sera, that decreased over time in the majority of participants; the median neutralisation titre in the cohort decreased by 45% over 4 weeks. Each of the serological assays gave quantitative measurements of antibody titres that correlated with SARS-CoV-2 neutralisation titres, but, the S-based serological assay measurements better predicted serum neutralisation potency. The strength of correlation between serologic assay results and neutralisation titres deteriorated with time and decreases in neutralisation titres in individual participants were not well predicted by changes in antibody titres measured using serologic assays.ConclusionsSARS-CoV-2 serologic assays differed in their comparative diagnostic performance over time. Different assays are more or less well suited for surveillance of populations for prior infection versus prediction of serum neutralisation potency. Continued monitoring of declining neutralisation titres during extended follow up should facilitate the establishment of appropriate serologic correlates of protection against SARS-CoV-2 reinfection.

Background: Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because of a lack of understanding of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a mode of host defence that has been implicated in multiple inflammatory diseases. Methods: We conducted a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. Nano LC/MS-MS was used to identify proteomic markers associated with disease severity, defined using the bronchiectasis severity index (BSI) (n=40). These were validated in two cohorts (n=175 and n=275) using a validated immunoassay to measure NETs. 20 patients with acute exacerbations of bronchiectasis were treated with intravenous antibiotics for 14 days and proteomics used to identify proteins associated with treatment response. Two studies of long-term macrolide treatment, one in bronchiectasis and a post-hoc analysis of the AMAZES trial in asthma, investigated the effect of macrolide treatment on NETs. Findings: 96 proteins were differentially expressed in sputum between severe and mild bronchiectasis. The most abundant and most discriminating were proteins known to be NET proteins. This was validated in two observational cohorts where sputum NETs were associated with BSI, quality of life, lung function, future risk of hospital admissions and mortality. Antibiotic treatment in 20 patients was associated with 23 significantly down-regulated and 16 up-regulated proteins, with the “neutrophil degranulation” pathway being most strongly implicated in response. Patients with Pseudomonas aeruginosa infection had a blunted proteomic and clinical response to treatment. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. Interpretation: NETs are identified as a key marker of disease severity and treatment response in bronchiectasis. Funding: Supported by the Scottish Government Chief Scientist Office (Senior Clinical Fellowship to JDC) and British Lung Foundation through the BLF Chair of Respiratory Research. Supported by the European Bronchiectasis Network (EMBARC), a European Respiratory Society Clinical Research Collaboration. Declaration of Interests: None to declare Ethics Approval Statement: The studies were approved by local research ethics committees and patients provided informed consent.

Background Objectives To determine the Adult incidence of IIM in the Kingdom of Fife, Scotland. The Fife population is 370,000 and constitutes 6.8% of the Scottish Population (5,425,000). 98% of the Fife population is Caucasian. We also sought to characterise the incident IIM cases in terms of subtype and autoantibody profile. Methods All newly diagnosed Adult cases (≥ 18 years) of IIM between 01.03.17 and 30.11.18 were recorded (11.75 year period). The cases were identified by a search of the Fife Rheumatic Diseases Unit Database using IIM-specific ICD-10 codes. These were cross-referenced with a prospective personal database of IIM cases established by the Lead Author (JMcL) on 26.02.17. In addition a search of all Fife patients enrolled in the UKMYONET study was also performed. All patients with ‘definite’ or ‘probable’ IIM by the Bohan & Peter 1977 criteria; Clinically defined Inclusion Body Myositis (IBM) by the ENMC 2011 criteria; or other specific criteria in the case of Clinically Amyopathic Dermatomyositis (CADM) and Immune-mediated Necrotising myopathy (IMNM) were included. Results 53 newly diagnosed Adult IIM cases were identified. 11 Dermatomyositis (DM) (3 NXP2; 2 Jo-1; 2 TIF1-G; 1 Mi-2; 3 Myositis-specific antibody (MSA) not tested). 4 Polymositis (PM) (1 Jo-1; 1 MDA5; 1 MSA/Myosits-associated antibody (MAA) negative; 1 MSA not tested). 12 Anti-synthetase syndrome (8 Jo-1; 3 PL7; 1 PL12) and 3 patients with Jo-1 isolated Interstitial lung disease (ILD). 5 Overlap Connective tissue disease (CTD) : 2 Systemic Lupus Erythematosus; 2 Systemic sclerosis; 1 Mixed CTD. 5 Cancer-associated myositis (CAM) : 2 lung (1 NXP2 DM & 1 SRP PM); 1 ovarian (DM, MSA not tested); 1 Breast (DM, TIF1-G); 1 Prostate (DM, Mi-2). 5 CADM (2 MDA5; 1 TIF1-G; 1 NXP2; 1 MSA not tested). 4 IMNM (2 HMGCR; 1 HMGCR overlap with IBM; 1 SRP overlap with IBM who developed bladder cancer Conclusion The incidence of IIM in Fife, Scotland is 4.5 cases per year which equates to an annual incidence of 12.2 cases per million population. This is comparable with the incidence data published for Salford in the UK (17.6). However the IIM subtypes differed from Salford in that Fife has a much lower incidence of PM. 40/49 (82%) of Fife patients who were tested were found to be MSA positive. Disclosure of Interests None declared

Background Neutrophil extracellular traps (NETs) have been observed in the airway in patients with chronic obstructive pulmonary disease (COPD), but their clinical and pathophysiologic implications have not been defined. Objective We sought to determine whether NETs are associated with disease severity in patients with COPD and how they are associated with microbiota composition and airway neutrophil function. Methods NET protein complexes (DNA-elastase and histone-elastase complexes), cell-free DNA, and neutrophil biomarkers were quantified in soluble sputum and serum from patients with COPD during periods of disease stability and during exacerbations and compared with clinical measures of disease severity and the sputum microbiome. Peripheral blood and airway neutrophil function were evaluated by means of flow cytometry ex vivo and experimentally after stimulation of NET formation. Results Sputum NET complexes were associated with the severity of COPD evaluated by using the composite Global Initiative for Obstructive Lung Disease scale (P, Graphical abstract

Bacteria belonging to the normal colonic microbiota are associated with the etiology of ulcerative colitis (UC). Although several mucosal species have been implicated in the disease process, the organisms and mechanisms involved are unknown. The aim of this investigation was to characterize mucosal biofilm communities over time and to determine the relationship of these bacteria to patient age and disease severity and duration. Multiple rectal biopsy specimens were taken from 33 patients with active UC over a period of 1 year. Real-time PCR was used to quantify mucosal bacteria in UC patients compared to 18 noninflammatory bowel disease controls, and the relationship between indicators of disease severity and bacterial colonization was evaluated by linear regression analysis. Significant differences were detected in bacterial populations on the UC mucosa and in the control group, which varied over the study period. High clinical activity indices (CAI) and sigmoidoscopy scores (SS) were associated with enterobacteria, desulfovibrios, type E Clostridium perfringens , and Enterococcus faecalis , whereas the reverse was true for Clostridium butyricum , Ruminococcus albus , and Eubacterium rectale . Lactobacillus and bifidobacterium numbers were linked with low CAI. Only E. rectale and Clostridium clostridioforme had a high age dependence. These findings demonstrated that longitudinal variations in mucosal bacterial populations occur in UC and that bacterial community structure is related to disease severity.

Rationale: Sputum neutrophil elastase and serum desmosine, which is a linked marker of endogenous elastin degradation, are possible biomarkers of disease severity and progression in bronchiectasis. This study aimed to determine the association of elastase activity and desmosine with exacerbations and lung function decline in bronchiectasis. Methods: This was a single-center prospective cohort study using the TAYBRIDGE (Tayside Bronchiectasis Registry Integrating Datasets, Genomics, and Enrolment into Clinical Trials) registry in Dundee, UK. A total of 433 patients with high-resolution computed tomography confirmed bronchiectasis provided blood samples for desmosine measurement, and 381 provided sputum for baseline elastase activity measurements using an activity-based immunosassay and fluorometric substrate assay. Candidate biomarkers were tested for their relationship with cross-sectional markers of disease severity, and with future exacerbations, mortality and lung function decline over. 3 years. Measurement and Main Results: Elastase activity in sputum was associated with the bronchiectasis severity index (r = 0.49; P < 0.0001) and was also correlated with the Medical Research Council dyspnea score (r = 0.34; P < 0.0001), FEV1% predicted (r = 0.33; P < 0.0001), and the radiological extent of bronchiectasis (r = 0.29; P < 0.0001). During a 3-year follow-up, elevated sputum elastase activity was associated with a higher frequency of exacerbations (P < 0.0001) but was not independently associated with mortality. Sputum elastase activity was independently associated with FEV1 decline (13 coefficient, -0.139; P = 0:001). Elastase showed good discrimination for severe exacerbations with an area under the curve of 0.75 (95% confidence interval [CI], 0.72-0.79) and all-cause mortality (area under the curve, 0.70; 95% CI, 0.67-0.73). Sputum elastase activity increased at exacerbations (P = 0.001) and was responsive to treatment with antibiotics. Desmosine was correlated with sputum elastase (r = 0.42; P < 0.0001) and was associated with risk of severe exacerbations (hazard ratio 2.7; 95% CI, 1.42-5.29; P = 0.003) but not lung function decline. Conclusions: Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.

BackgroundIn cystic fibrosis and bronchiectasis, genetic mannose binding lectin (MBL) deficiency is associated with increased exacerbations and earlier mortality; associations in COPD are less clear. Preclinical data suggest MBL interferes with phagocytosis of Haemophilus influenzae, a key COPD pathogen. We investigated whether MBL deficiency impacted on clinical outcomes or microbiota composition in COPD.MethodsPatients with COPD (n=1796) underwent MBL genotyping; linkage to health records identified exacerbations, lung function decline and mortality. A nested subcohort of 141 patients, followed for up to 6 months, was studied to test if MBL deficiency was associated with altered sputum microbiota, through 16S rRNA PCR and sequencing, or airway inflammation during stable and exacerbated COPD.FindingsPatients with MBL deficiency with COPD were significantly less likely to have severe exacerbations (incidence rate ratio (IRR) 0.66, 95% CI 0.48 to 0.90, p=0.009), or to have moderate or severe exacerbations (IRR 0.77, 95% CI 0.60 to 0.99, p=0.047). MBL deficiency did not affect rate of FEV1 decline or mortality. In the subcohort, patients with MBL deficiency had a more diverse lung microbiota (p=0.008), and were less likely to be colonised with Haemophilus spp. There were lower levels of airway inflammation in patients with MBL deficiency.InterpretationPatients with MBL deficient genotype with COPD have a lower risk of exacerbations and a more diverse lung microbiota. This is the first study to identify a genetic association with the lung microbiota in COPD.

Enteral feeding via a percutaneous endoscopic gastrostomy tube is required for nutritional support in patients with dysphagia. Enteral tube feeding bypasses the innate defence mechanisms in the upper gastrointestinal tract. This study examined the surface-associated microbial populations and immune response in the gastric and duodenal mucosae of eight enteral nutrition (EN) patients and ten controls. Real-time PCR and fluorescence in situ hybridization were employed to assess microbiota composition and mucosal pro-inflammatory cytokine expression. The results showed that EN patients had significantly higher levels of bacterial DNA in mucosal biopsies from the stomach and duodenum (Pα, IL-6 and tumour necrosis factor-α was significantly higher in gastric and small intestinal mucosae from patients fed normal diets in comparison with those receiving EN (P

Allergy is caused by an immune reaction that is out of all proportion to the antigenic stimuli. Classical allergy is a type I hypersensitivity reaction mediated by the interaction of mast cells (and eosinophils) coated with allergen-specific IgE and a cross-linking allergen. The physiological outcome is inflammation commonly displayed by urticaria, rhinitis, vomiting and diarrhoea, depending on the route of allergen entry. In extreme reactions anaphylactic shock can result that may lead to death. Chronic allergic responses most commonly present themselves as asthma and eczema. All these symptoms are the consequence of an imbalanced immune system making an unsuitable response to an environmental or food antigen. On bacterial colonisation of the colon after birth the appropriate microbiological stimuli is essential to redress the balance of the skewed T-helper 2 immune response present in the newborn. This normal interaction between baby and microbes is thought to be compromised in the Western world, with a reduction in bifidobacteria and an increase in clostridial species, particularly in bottle-fed infants. The use of probiotic therapy to prevent allergic disease has been demonstrated in two studies using a probiotic Lactobacillus rhamnosus GG in neonates. A long-term reduction in allergy has been shown in the test group, with lactobacillus reducing the incidence of atopic eczema. Management of allergy through probiotics has also been demonstrated in infants, using lactobacilli to control atopic eczema and cow's milk allergy. Unfortunately, these positive results have not been repeated in studies with older children and young adults.

The colonic epithelium provides an interface between the host and micro-organisms colonising the gastrointestinal tract. Molecular recognition of bacteria is facilitated through Toll-like receptors (TLR). The colonic epithelium expresses relatively high levels of mRNA for TLR3 and less for TLR2 and -4. Little is known of the expression patterns and mode of induction of expression for these pattern recognition receptors in human colon. The aim of this study was to investigate their localization in the gut and induction of expression in epithelial cell lines by mucosal bacteria. TLR2 and -4 were expressed only in crypt epithelial cells, expression was lost as the cells matured and moved towards the gut lumen. In contrast, TLR3 was only produced in mature epithelial cells. HT29 and CACO-2 had different levels of expression for TLR1-4. Co-culture of HT29 cells with different mucosal isolates showed that they were highly responsive to bacterial challenge, with up-regulation of mRNA for TLR1-4. In contrast, CACO-2 cells were refractive to bacterial challenge, showing little difference in mRNA levels. TLR3 was induced in HT29 only by Gram-positive commensals with up-regulation of both mRNA and protein and an enhancement of the antiviral immune response. This pattern of expression allows induction of responsiveness to bacteria only by the crypt epithelium so that tolerance to commensal organisms can be maintained. In contrast, mature columnar epithelium is able to respond to viral pathogens, which are not part of the normal gut commensal microbiota.

The etiology of ulcerative colitis (UC) is unknown, but evidence links it to bacteria belonging to the normal colonic microbiota. The aims of this study were to characterize bacteria colonizing the rectal epithelium, and to investigate whether significant differences existed in UC. Rectal biopsy specimens were obtained via endoscopy from 9 patients with active colitis and 10 patients without inflammatory bowel disease. Complex bacterial communities colonized the rectal mucosa in all subjects. Overall, 72 bacterial taxa (18 genera) were detected. Twenty species were common to both groups, but only differences in bifidobacteria were statistically significant (P = .005). Peptostreptococci were only detected in patients with UC. Microscopy showed that bacteria in mucosal biofilms often occurred in microcolonies. Interindividual variations in mucosal biofilms made it difficult to assign a role for specific bacteria in UC etiology. However, differences in bifidobacteria and peptostreptococci may implicate these organisms in this disease.

Background: Desulfovibrios produce sulphide, which is toxic to colonic epithelial cells. These bacteria have previously been linked to ulcerative colitis. Traditional methods of culturing these organisms are slow, and often unreliable, while molecular approaches are either non-quantitative or lack sensitivity. Aims: To develop a sensitive method for quantitating desulfovibrios in stools and biopsy tissue, and to investigate the effects of age and disease on these bacteria. Methods: Rectal biopsies were taken from 10 colitis patients and 10 healthy controls. Stool samples were obtained from 10 healthy infants (mean age 1.01 (0.18) years), 10 healthy young adults (26.7 (1.2) years), and 10 healthy elderly people (71.7 (1.2) years). Primers were designed and developed for analysing Desulfovibrio populations in the bowel using real time polymerase chain reaction (PCR). Results: The PCR primers were highly specific for desulfovibrios. Large numbers (approximately 106–107/g) occurred in biopsies in colitis patients and healthy subjects, and no disease related differences were observed. Measurements of mucosal desulfovibrios over 12 months showed marked changes in some patients. Infants (106–107/g) and elderly people (107–108/g) had significantly higher numbers of desulfovibrios in stools compared with young adults (105/g). Conclusions: Real time PCR analysis of desulfovibrios was an efficient and accurate method for studying these potentially harmful microorganisms. Desulfovibrios were ubiquitous in the bowel, irrespective of age. As rectal mucosae were heavily colonised in health and disease, if these bacteria play a role in colitis, some host defect, possibly in sulphide detoxication pathways or in bacterial antigen handling, is required for manifestations of pathogenicity.

Bacterial colonisers of the colon comprise several hundred bacterial species that live in a complex ecosystem. Study of this complex ecosystem has been carried out, until recently, by traditional culture techniques with biochemical methods to identify organisms. The development of molecular techniques to investigate ecological microbial communities has provided the microbiologist with a vast array of new techniques to investigate human intestinal microflora. Metagenomics, the science of biological diversity, combines the use of molecular biology and genetics to identify and characterise genetic material from complex microbial environments. The combination of metagenomics and subsequent quantitation of each identified species using molecular techniques allows the relatively rapid analysis of whole bacterial populations in human health and disease.

The bacterial species diversity of three colonic tissue samples from elderly people was investigated by sequence analysis of randomly cloned eubacterial 16S rDNA. The majority of sequences (87%) clustered within three bacterial groups: (1) Bacteroides; (2) low G+C content Gram-positives related to Clostridium coccoides (cluster XIVa); (3) Gram-positives related to Clostridium leptum (cluster IV). These groups have been shown to dominate the human faecal flora. Only 25% of sequences were closely related (97%) to current species type strains, and 28% were less than 97% related to any database entry. 19% of sequences were most closely related to recently isolated butyrate-producing bacteria belonging to clusters XIVa and IV, with a further 18% of the sequences most closely related to Ruminococcus obeum and Ruminococcus torques (members of cluster XIVa). These results provide the first molecular information on the microbial diversity present in human colonic samples.

The aetiology of ulcerative colitis (UC) is unknown, but there is evidence that bacteria are needed for initiation and maintenance of the disease. A number of organisms have been associated with UC, but evidence for a specific transmissible agent is weak. Despite this, there is a good case for mucosal bacterial involvement, either through pathogens colonizing the epithelial surface, by non-pathogenic commensal species occupying adhesion sites on the mucosa and preventing invasion by harmful bacteria, or by inappropriate host immune responses to members of the normal microflora. Since mucosal bacteria exist in close juxtaposition to host tissues, it might be expected that they interact to a greater extent with the immune and neuroendocrine systems than their luminal counterparts. For this reason, comparative bacteriological analyses were done on rectal biopsies from patients with active colitis, and individuals who had no inflammatory bowel disease. Complex bacterial communities colonized the rectal mucosa in all subjects and great interindividual variabilities in mucosal bacterial populations were observed in both groups. These organisms often occurred in microcolonies, which may have implications for UC, since it would result in higher localized concentrations of bacterial antigens, or toxins, than would be the case if the organisms were diffusely spread across the mucosa.

Background. Barrett’s esophagus (BE) is a complication of chronic gastroesophageal reflux disease, in which patients are at greatly increased risk of esophageal dysplasia and adenocarcinoma. Over the past 2 decades, there has been an increase in the incidence of both BE and adenocarcinoma; however, the involvement of microorganisms in BE is uncertain. The aim of this study was to characterize microbial communities in esophageal aspirate specimens and on distal esophageal mucosal samples from patients with BE. Methods. Biopsy and aspirate specimens were obtained by endoscopic examination from 7 patients with BE and 7 control subjects without BE. Samples were cultured under aerobic, anaerobic, and microaerophilic conditions for yeasts and bacteria, including Helicobacter pylori. Bacterial isolates were identified by 16S ribosomal RNA gene sequencing. Fluorescence microscopic examination was also used to determine the spatial localization of these organisms on mucosal surfaces. Significant colonization was detected in 6 patients with BE and in 4 control subjects. Results. Overall, 46 bacterial species belonging to 16 genera were detected, with 10 species being common in both groups. Both aspirate and biopsy samples from patients with BE contained complex populations of bacteria. Uniquely, high levels of Campylobacter species (Campylobacter concisus and Campylobacter rectus), which have been linked to enteritis, periodontal infections, and tumor formation in animals, were found in 4 (57%) of 7 patients with BE but in none of the control subjects. Microscopic examination revealed that bacteria on mucosal biofilms often occurred in microcolonies. Conclusions. The occurrence of nitrate-reducing Campylobacter species in patients with BE may suggest that there is a link in either the initiation, maintenance, or exacerbation of disease processes leading to adenocarcinoma formation. Barrett’s esophagus (BE) is a complication of chronic gastroesophageal reflux disease, in which squamous epithelial cells lining the distal esophagus undergo metaplastic changes, forming a columnar mucosa [1]. Patients with BE have a greatly increased risk of esophageal dysplasia and adenocarcinoma [2, 3]. From 6% to 12% of patients with gastroesophageal reflux disease develop BE [4], and those with BE develop esophageal adenocarcinoma at a rate of ∼1 case per 100 patient-years [4, 5]. Rates of carcinoma of the mid and distal stomach are decreasing; conversely, adenocarcinoma of the esophagus has been steadily increasing in

Ulcerative colitis (UC) is an acute and chronic inflammatory bowel disease of unknown aetiology, although bacterial species belonging to the normal colonic microbiota are known to be involved in its initiation and maintenance. Several organisms have been linked to the disease; however, mucosa-associated bacteria are more likely to be involved than their luminal counterparts, due to their close proximity to the host epithelium. Comparative bacteriological analyses were done on rectal biopsies to investigate differences in mucosal bacteria in patients with UC and healthy controls. Complex bacterial communities were found in both groups, with significant reductions in bifidobacterial numbers in UC, which suggested that they might have a protective role in the disease. Accordingly, a therapy for treating UC was designed, with the aim of modifying the mucosal microbiota to increase bifidobacterial colonisation and reduce inflammation. Ranges of mucosal and faecal bifidobacteria were tested for their substrate preferences and their abilities to survive under a variety of environmental conditions. A synbiotic comprising a probiotic (Bifidobacterium longum) isolated from healthy rectal mucosa combined with a prebiotic (oligofructose-enriched inulin – Synergy 1TM) was developed. The treatment was used in a randomised controlled trial involving eighteen patients with active UC, for a period of 1 month. Rectal biopsies were collected at the beginning and end of the study. Bacteriological analysis and transcription levels of epithelium-related immune markers were assessed. Results demonstrated that short-term synbiotic treatment resulted in increased bifidobacterial colonisation of the rectal mucosa and induced significant reductions in the expression of molecules that control inflammation in active UC.

Background and aims: Ulcerative colitis (UC) is an acute and chronic inflammatory disease of the large bowel with unknown aetiology. The immune response against normal commensal microorganisms is believed to drive inflammatory processes associated with UC. Therefore, modulation of bacterial communities on the gut mucosa, through the use of probiotics and prebiotics, may be used to modify the disease state. Methods: A synbiotic was developed for use in UC patients combining a probiotic, Bifidobacterium longum , isolated from healthy rectal epithelium, and a prebiotic (Synergy 1), a preferential inulin-oligofructose growth substrate for the probiotic strain. Treatment was employed in a double blinded randomised controlled trial using 18 patients with active UC for a period of one month. Clinical status was scored and rectal biopsies were collected before and after treatment, and transcription levels of epithelium related immune markers were measured. Results: Sigmoidoscopy scores (scale 0–6) were reduced in the test group (start 4.5 (1.4), end 3.1 (2.5)) compared with placebo (start 2.6 (2.1), end 3.2 (2.2)) (p = 0.06). mRNA levels for human beta defensins 2, 3, and 4, which are strongly upregulated in active UC, were significantly reduced in the test group after treatment (p = 0.016, 0.038, and 0.008, respectively). Tumour necrosis factor α and interleukin 1α, which are inflammatory cytokines that drive inflammation and induce defensin expression, were also significantly reduced after treatment (p = 0.018 and 0.023, respectively). Biopsies in the test group had reduced inflammation and regeneration of epithelial tissue. Conclusions: Short term synbiotic treatment of active UC resulted in improvement of the full clinical appearance of chronic inflammation in patients receiving this therapy.

Real-time PCR and northern hybridisations were used to quantify bacterial populations in the large gut of infants. PCR primers for rapid, sensitive, high throughput detection of bifidobacteria, bacteroides, sulphate-reducing bacteria and Enterococcus faecalis, based on analysis of 16S rRNA genes were used. Bacterial populations were analysed in faeces from 40 infants aged 0-6, 7-12 and 13-24 months. The effects of breast versus bottle feeding was also investigated. Real-time PCR indicated that bacteroides and desulfovibrio numbers increased markedly in the 7-12 and 13-24 month age groups, and that the reverse occurred with Ent. faecalis. With the exception of desulfovibrios, this was seen with northern hybridisations, which also showed increased colonisation by the Clostridium coccoides group and Faecalibacterium prausnitzii after 6 months. Both methodologies indicated increased bifidobacteria in breast-fed babies, and higher levels of desulfovibrios in bottle-fed children.

Migration-stimulating factor (MSF) is a 70-kDa motogenic protein previously reported to be expressed by fetal and cancer patient fibroblasts cultured in vitro and present in the serum of breast cancer patients. A 2.2-kb full-length MSF cDNA has been cloned and shown to be a truncated isoform of fibronectin generated from its primary gene transcript by a hitherto unrecognized intron read-through mechanism. MSF cDNA is identical to the 5' end of fibronectin cDNA, up to and including exon III-1a, and terminates in a novel 195-nucleotide 3' sequence. This MSF unique sequence is derived from the intron immediately downstream of exon III-1a in the fibronectin gene and is not found in any previously identified "full-length" fibronectin cDNA. MSF mRNA is 1000-fold less abundant than full-length fibronectin message in fetal fibroblasts and exhibits rapid biphasic decay kinetics previously associated with oncogenes and stress response molecules. MSF recombinant protein exhibits a potent and substratum-dependent motogenic activity, with half-maximal response manifest at 0.1-1.0 pg/ml. This activity is (a) mediated by the IGD amino acid motif; and (b) not expressed by (i.e., cryptic within) full-length fibronectin. In situ hybridization and immunohistochemistry confirm that MSF is expressed by tumor-associated fibroblasts and additionally indicate that it is also expressed by carcinoma cells and tumor-associated vascular endothelial cells. MSF, as a consequence of its potent bioactivities and expression by both stromal and carcinoma cell populations, is well placed to function as an epigenetic effector promoting cancer development.

Background and aims: The mucosa in ulcerative colitis (UC) is replete with antibody producing plasma B cells and polymorphonuclear leucocytes (PMN). This combination of effector cells requires a crosslinking antigen to evoke an antibody driven PMN inflammatory response via their Fc receptors. The stimulus for activation is thought to be commensal bacteria colonising the gut mucosa. The aim of this investigation was to compare the principal culturable bacterial populations on the rectal mucosa of UC patients, and to determine whether specific antibodies towards these bacteria can activate infiltrating PMN through opsonisation. This would provide an explanation for this chronic inflammatory condition. Methods: Bacteria colonising rectal tissue were characterised using chemotaxonomic techniques. Systemic antibody responses were measured against total antigens and surface antigens of these organisms in UC and Crohn’s disease (CD) patients, together with healthy controls. Antibody enhancement of the respiratory burst in PMN was also investigated, against a range of mucosal isolates. Results: Distinct differences were observed in some bacterial populations in UC biopsies, which were generally reflected in antibody responses towards these organisms. UC patients had higher IgG responses to surface antigens, primarily IgG1, whereas the response in CD was mainly IgG2. Antibodies from UC patients greatly enhanced the respiratory burst in PMN, in response to individual bacterial species. Conclusions: Changes in mucosal bacteria, and a switch from internal to surface antigen/antibody reactivity of a predominantly IgG1 type, leads to greater opsonisation of the respiratory burst in PMN, providing a mechanism for maintaining the inflammatory state in UC.

In humans, nonstarch polysaccharides (NSP), such as arabinoxylans (AX), are not digested in the upper gut and provide fermentable carbon sources for bacteria growing in the large bowel. Despite the ubiquity of AX in nature, the microbiologic and physiologic consequences of AX digestion in the gut are poorly understood. In this study, we investigated the breakdown of ferulic acid-cross-linked AX (AXF) and non-cross-linked AX in children's intestinal microbiotas, using starch as a readily fermentable polysaccharide for comparative purposes. The experiments were performed using pH-controlled fermentation vessels under anaerobic conditions. The results demonstrated that there was variation in the metabolism of these polysaccharides by colonic microbiotas. AX was always degraded more slowly than starch, while ferulic acid cross-linking reduced the rate of AX fermentation, as shown by fermentation product measurements. Starch digestion was associated with significant acetate and butyrate production, whereas AX breakdown resulted in increased propionate formation. In general, the presence of fermentable carbohydrate significantly increased the total anaerobe counts and eubacterial rRNA concentrations ( P < 0.01), while non-cross-linked AX digestion was principally associated with increased viable counts of Bacteroides fragilis group organisms, which was supported by increases in Bacteroides - Porphyromonas - Prevotella group rRNA ( P < 0.01). Starch was considerably more bifidogenic than AX in these fermentations. In conclusion, in this study we found that the effects of AX and AXF on the microbial ecology and metabolism of intestinal microbiotas are similar in children and adults.

Lipophilic immune stimulating complexes (ISCOMS) containing the Quil A adjuvant can induce local and systemic immune responses against orally delivered protein antigens. Here, we have examined the possibility that ISCOMS act by modulating local innate immune responses and antigen uptake in the intestine. Feeding ISCOMS to mice induced recruitment of dendritic cells (DCs), activated macrophages (mφ) and lymphocytes into the mesenteric lymph node (MLN), as well as recruitment of macrophages and B cells into the PP. Feeding ISCOMS also enhanced the absorption into circulation of a test feed of ovalbumin (OVA), with accelerated kinetics and increased peak levels. Serum taken 1 h after feeding a tolerogenic dose of OVA to mice given ISCOMS orally did not induce the tolerance of systemic delayed type hypersensitivity (DTH) and antibody responses found in mice receiving serum from donors fed OVA alone. Thus, ISCOMS may increase the immunogenicity of intestinal antigen via a combination of effects on antigen uptake and on local accessory cells.