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A fit middle aged man presented to the emergency department with headache, myalgia, vomiting, fever and rigours. He was hypotensive with mottled peripheries, tachycardic and dyspnoeic. The only significant medical history noted was an emergency splenectomy 30 years previously following a road traffic accident. The patient had been on prophylactic antibiotics initially and was vaccinated in line with recommendations at the time following splenectomy with no significant health issues in the intervening years. The patient was treated empirically for septic shock and meningitis based on presentation and admitted to the intensive care unit for pressor support and subsequently required intubation and ventilation. Investigations revealed bilateral pneumonia. Streptococcal pneumoniae urinary antigen and serum S. pneumoniae PCR were positive supporting a diagnosis of invasive pneumococcal infection. A lumbar puncture was negative for meningitis. Distal mottling affecting all limbs progressed with resultant bilateral upper limb digit and below knee amputation. The patient subsequently required extensive rehabilitation. Following a prolonged tertiary and rehabilitation hospital admission, the patient made an exceptional recovery and was discharged home with ongoing appropriate support and home adaptation.

A 77-year-old Lithuanian man presented to our institution with recurrent episodes of periorbital cellulitis, submandibular swelling and sialadenitis. Investigations revealed a positive QuantiFERON, raised inflammatory markers and normal autoimmune screen. Cross-sectional imaging showed no signs of occult malignancy, and work-up for mycobacterial infection including imaging and bronchoalveolar lavage did not show active tuberculosis. During hospitalisation, the patient developed fevers of unknown origin, which were investigated with a positron emission tomography (PET) scan and a bone marrow aspiration, without evidence of occult infection or malignancy. Serum IgG4 level was three times the upper limit of normal. The patient responded well to oral steroids but relapsed after completing a slow taper. Serum IgG4 level was three times the upper limit of normal. He had an American College of Rheumatology/European League Against Rheumatism score of 20, in conjunction with involvement of orbital and salivary tissue. Therefore, IgG4-related disease was considered the most likely diagnosis, despite prominent fevers, which are among the exclusion criteria for this diagnosis. After a multidisciplinary review including rheumatology and ophthalmology, the patient was commenced on maintenance methotrexate with remission of symptoms.

Background It is suggested that airway fungi, in particular Aspergillus may impinge on clinical phenotype in asthma. Indeed, the term severe asthma with fungal sensitization (SAFS) has been coined. We aimed to ascertain whether the presence of fungi, in particular Aspergillus fumigatus, in the airway correlated with asthma severity and control. Furthermore, we aimed to determine whether traditional markers of Aspergillus sensitization related to the presence of Aspergillus within the airway. Methods Sixty-nine patients characterized by asthma severity (GINA) and level of control (ACQ-7) underwent bronchoscopy and bronchoalveolar lavage (BAL). Serum was assessed for A fumigatus-specific IgE and total IgE. Galactomannan and relevant cytokine levels were assessed in serum, plasma and BAL. BAL was analyzed for the presence of A fumigatus. Results In BAL, fungi were visible by microscopy in 70% and present by qPCR in 86% of patients, while A fumigatus was detectable by qPCR in 46%. Plasma and BAL IL-4, IL-6, IL-10, IL-13 and TNF-α correlated with BAL fungal presence, while plasma IL-17 correlated with BAL fungal presence. Aspergillus positive BAL correlated with increased plasma and BAL IL-6 and BAL IL-13. There was no relationship between fungal airway presence and steroid dose, asthma severity or control. The presence of Aspergillus within the airway did not relate to serum IgE positivity for Aspergillus. Conclusions Fungi were present in a large proportion of our asthmatic patients' airways, but their presence was not predicted by traditional markers of sensitization, nor did it appear to be related to measures of disease severity or control.

Long Term Disruption of Cytokine Signalling Networks are Evident Following SARS-CoV-2 InfectionSinead Ahearn-Ford1, Nonhlanhla Lunjani1, Brian McSharry1,2, John MacSharry1,2,3, Liam Fanning1,3, Gerard Murphy4, Cormac Everard4, Aoife Barry4, Aimee McGreal4, Sultan Mohamed al Lawati4, Susan Lapthorne4, Colin Sherlock4, Anna McKeogh4, Arthur Jackson4, Eamonn Faller4, Mary Horgan3,4, Corinna Sadlier4, Liam O’Mahony1,2,3*1APC Microbiome Ireland, University College Cork, Cork, Ireland2School of Microbiology, University College Cork, Cork, Ireland3Department of Medicine, University College Cork, Cork, Ireland4 Department of Infectious Diseases, Cork University Hospital, Cork, Ireland*Corresponding author – liam.omahony@ucc.ieTo the Editor,The current pandemic caused by the SARS-CoV-2 virus has so far infected more than 130 million people worldwide, resulting in approximately 3 million deaths. While the current clinical and public health priorities are designed to limit severe acute and fatal episodes of the disease, and to quickly roll out vaccines to the general population, it has become apparent that there may also be significant detrimental long-term effects following SARS-CoV-2 infection that impact daily functioning and quality of life1. The mechanisms underpinning the post-acute sequelae of SARS-CoV-2 infection’s long-lasting symptoms can include direct effects of the infection (e.g. endothelial damage, lung fibrosis) or indirect effects associated with changes in the microbiome or abnormalities in inflammatory and immune signalling pathways stimulated by the infection2,3.In order to examine the potential long-term immune changes that occur following elimination of the primary infection, we examined the levels of 52 cytokines and growth factors (using MSD multiplex kits) in the serum of patients that attended follow-up post-COVID infection clinics at Cork University Hospital, Cork, Ireland (The Clinical Research Ethics Committee of the Cork Teaching Hospitals approved this study and all patients provided informed consent). All patients had been hospitalised for PCR-proven SARS-CoV-2 infection (median in-patient stay of 5.5 days, range 1 day to 24 days) during the first wave of the pandemic in Ireland (March-May 2020). 38 serum samples were obtained from 24 patients (median age 53.5 years, 11 female) at 3-9 months following hospital discharge. Clinical severity ranged from mild to critical during hospitalisation and the most common symptoms at follow-up clinics were fatigue and/or dyspnoea (supplementary Table S1). Sera obtained prior to the pandemic from 29 healthy volunteers (median age 43.2 years, 14 female) were analysed in parallel.Of the 52 analytes measured, 19 were significantly elevated in post-COVID patient sera compared to healthy controls (supplementary Table S2). These 19 mediators are illustrated as dot plots in Figure 1 and Figure 2. One group of mediators, c-reactive protein (CRP), serum amyloid A (SAA), Interleukin-1 receptor antagonist (IL-1RA), IL-6, IL-8, IL-15, IL-16, monocyte chemotactic protein (MCP)-1 and MCP-4, can be broadly categorised as being associated with ongoing inflammatory responses (Figure 1a)4. These mediators remained as elevated in samples taken 6-9 months following hospital discharge as those levels observed 3-6 months following discharge (p

Background & Aims: Options for the prevention of short-bowel syndromeâassociated liver disease (SBS-ALDs) are limited and often ineffective. The farnesoid X receptor (FXR) is a newly emerging pharmaceutical target and FXR agonists have been shown to ameliorate cholestasis and metabolic disorders. The aim of this study was to assess the efficacy of obeticholic acid (OCA) treatment in preventing SBS-ALDs. Methods: Piglets underwent 75% small-bowel resection (SBS) or sham surgery (sham) and were assigned to either a daily dose of OCA (2.4 mg/kg/day) or were untreated. Clinical measures included weight gain and stool studies. Histologic features were assessed. Ultraperformance liquid chromatography tandem mass spectrometry was used to determine bile acid composition in end point bile and portal serum samples. Gene expression of key FXR targets was assessed in intestinal and hepatic tissues via quantitative polymerase chain reaction. Results: OCA-treated SBS piglets showed decreased stool fat and altered liver histology when compared with nontreated SBS piglets. OCA prevented SBS-associated taurine depletion, however, further analysis of bile and portal serum samples indicated that OCA did not prevent SBS-associated alterations in bile acid composition. The expression of FXR target genes involved in bile acid transport and synthesis increased within the liver of SBS piglets after OCA administration whereas, paradoxically, intestinal expression of FXR target genes were decreased by OCA administration. Conclusions: Administration of OCA in SBS reduced fat malabsorption and altered bile acid composition, but did not prevent the development of SBS-ALDs. We postulate that extensive small resection impacts the ability of the remnant intestine to respond to FXR activation. Keywords: Short-Bowel Syndrome, Liver Disease, Intestinal FailureâAssociated Liver Disease, Obeticholic Acid, Bile Acids, Farnesoid X Receptor

Background Many people with asthma remain suboptimally controlled despite current treatments. Reasons include comorbidities that could aggravate asthma, including gastroesophageal reflux. We aimed to investigate whether aspiration occurs in patients with asthma and, if so, does it correlate with asthma control? Methods Patients had Asthma Control Questionnaire 7 (ACQ-7), fractional exhaled nitric oxide, and spirometry performed to characterize their level of asthma control. Barium swallow with provocation was performed to assess for predisposition to aspiration. Patients underwent bronchoscopic investigation, with BAL pepsin measured as a marker of aspiration. Results Seventy-eight patients stratified by disease severity (Global Initiative for Asthma) into mild (35.8%), moderate (21.7%) and severe (42.3%) were studied. Pepsin was detectable in BAL in 46/78 (58.9%). There were no differences between pepsin levels in patients with different disease severity. Furthermore, no significant associations were seen between pepsin level and measures of asthma control, FEV 1 , ACQ-7 or exacerbation frequency. Similarly no associations were found with adjustments for smoking history, BMI, proton pump inhibitor use, eosinophil count or IgE. When stratified into eosinophilic or neutrophilic asthmatic populations on the basis of BAL, there was no relationship to detected pepsin concentrations. A positive barium swallow (seen in 33/60 patients) did not correlate with BAL pepsin level and we found no significant association between barium swallow result and ACQ-7, Global Initiative for Asthma, exacerbation frequency or FEV 1 using either univariate or multivariate analyses. Conclusions This study suggests that the importance of aspiration on current asthma symptom control and exacerbation rate may be overstated. However, this study did not address the role of aspiration and future risk of exacerbation.

Background and Aim Short bowel syndrome (SBS) is primarily characterised by malabsorption and malnutrition resulting from loss of intestinal absorptive area following massive small bowel resection (SBR). Bile acids and the gut microbiota are functionally-linked within the gut-liver axis, however SBS-associated disturbances within the gut-liver axis remain largely unexplored. The aim of this study was to characterize the evolution of bile acid alterations within the gut-liver axis at both short- and long-term time points and to relate these changes to alterations in colonic bacterial composition. Methods Four week-old piglets were assigned to 75% small bowel resection (SBR), sham operation or non-operation control (NOC) groups. High throughput sequencing was employed to determine bacterial abundance in colonic content and ultra-performance liquid chromatography used to determine the bile acid concentration of gall bladder, portal serum and faecal samples. Results Bile acid complexity and relative abundance are altered in the SBS piglet model at two weeks post-SBR, and these changes persisted at six weeks post-SBR. Our examination of the microbial profile revealed an early and persistent loss in bacteria belonging to the Clostridiales order. Conclusions This study provides evidence of an early and persistent disturbance of the bile acid profile throughout the entero-hepatic circulation with an increase in the proportion of primary bile acids and a decrease in secondary bile acids following small bowel resection. These changes were associated with a loss of bacteria belonging to the Clostridiales order consistent with a disturbance in the bile-microbial axis following SBR.

Background: Molecular methods have shown that the airways harbour a distinct microbiome. Respiratory research is beginning to investigate the role of the host-microorganism interaction in both healthy and diseased lungs. Our aim was to characterise the lower airway microbiota in a cohort of well-defined adult asthmatics. Methods: 87 patients, clinically stratified by asthma severity (GINA) and level of control (ACQ-7) underwent bronchoscopy with BAL and endobronchial biopsies. Cell differentials, microscopy, DNA extraction and qPCR were performed to determine the presence and identity of microbes. Results: Microscopic evaluation demonstrated the presence of microbes in BAL, adherent to the epithelium of biopsies and demonstrated numerous intracellular microbial species within BAL macrophages. Overall qPCR revealed the presence of bacteria such as Firmicutes (69%), Proteobacteria (Pseudomonas (30%), Escherichia (35%), Bordetella (47%)), Prevotella (72%), Veillonella (85%), and the atypicals M. pneumoniae (54%) and C. pneumoniae (63%) in the BAL. Patients with severe asthma had higher levels of S. aureus (P Conclusions: The presence of distinct microbial species within the airway is associated with increased neutrophils, eosinophils and asthma severity. These findings suggest a complex interplay between microbial communities present in asthma and disease phenotype.

Background: Respiratory research is beginning to investigate the effects of host-microorganism interactions in the airway. Our aim was to determine if Aspergillus was detectable in the lower airways in a cohort of well-defined asthmatics and if so whether there was a relationship between its presence and asthma severity and level of disease control. Methods: 80 patients, stratified by asthma severity (GINA) and level of control (ACQ-7) were prospectively recruited. Blood serum was assessed for Aspergillus precipitins and IgE level and patients underwent bronchoscopy with bronchoalveolar lavage (BAL). Microscopy, cell differentials, DNA extraction and qPCR were performed on BAL to determine presence of Aspergillus. Results: Microscopic evaluation demonstrated the presence of Aspergillus in BAL. FungiQuant qPCR detected fungal presence in 84% of patient BAL. BAL qPCR revealed Aspergillus detectable in >46% of patients (19% mild, 10% moderate, 18% severe asthma GINA). Asthma control as determined by ACQ-7 was not associated with BAL aspergillus levels. Aspergillus levels in BAL correlated with BAL macrophage counts (%) (P Conclusions: Our study has demonstrated the presence of Aspergillus within the airways of almost half of our asthmatic cohort. Despite this, we were unable to find any association between the presence of Aspergillus and measures of either asthma severity or control. Further studies are now required to assess whether or not the presence of Aspergillus within the asthmatic airway has a pathological role in the disease.

Background: Asthma is generally well controlled but there is a subpopulation that remains uncontrolled. Recent research has shown that the airway is no longer sterile and has its own microbiome. The aim of our study was to characterise the lower airway microbiota in a cohort of well-defined asthmatics of varying disease severity and by doing so potentially identify alternate therapy strategies. Methods: We recruited 76 patients, stratified by asthma severity to our study (36% GINA 1 and 2; 22% GINA 3 and 42% GINA 4,5). All patients had a detailed clinical evaluation including ACQ-7, spirometry, and eosinophil levels prior to proceeding to bronchoscopy with bronchoalveolar lavage (BAL). Cell differential was performed on BAL, which was further evaluated for the presence of microbes using quantitative polymerase chain reaction (qPCR). Results: Microscopic evaluation of patient BAL demonstrated BAL macrophages (95%) containing numerous microbial species. qPCR demonstrated the presence of bacterial DNA in 71.8% of patients. Analysis found a significant difference between 16sDNA when patients were categorized as having BAL neutrophilia (>3%), (p Conclusions: Our results suggest that BAL neutrophilia is associated with the presence of microbes as well as markers of inflammation. These findings may prove a useful tool in future therapeutic strategies.

Following small bowel resection (SBR), the luminal environment is altered, which contributes to clinical manifestations of short bowel syndrome (SBS) including malabsorption, mucosal inflammation and bacterial overgrowth. However, the impact of SBR on the colon has not been well-defined. The aims of this study were to characterize the colonic microbiota following SBR and to assess the impact of SBR on mucosal inflammation in the colon.Analysis of the colonic microbiota demonstrated that there was a significant level of dysbiosis both two and six weeks post-SBR, particularly in the phylum Firmicutes, coupled with a decrease in overall bacterial diversity in the colon. This decrease in diversity was associated with an increase in colonic inflammation six weeks post-surgery.Female (4-week old) piglets (5-6/group) received a 75% SBR, a transection (sham) or no surgery. Compositional analysis of the colonic microbiota was performed by high-throughput sequencing, two- and six-weeks post-surgery. The gene expression of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8, IL-18 and tumor necrosis factor (TNF)-α in the colonic mucosa was assessed by qRT-PCR and the number of macrophages and percentage inducible nitric oxide synthase (iNOS) staining in the colonic epithelium were quantified by immunohistochemistry.SBR significantly decreased the diversity of the colonic microbiota and this was associated with an increase in colonic mucosal inflammation. This study supports the hypothesis that SBR has a significant impact on the colon and that this may play an important role in defining clinical outcome.

Different rates of bacterial translocation across the gut mucosa have been reported but few studies have examined translocation of commensals at the level of the gut epithelial microfold (M) cell. We used an in vitro M-cell model to quantify translocation and determine the transcriptional response of M cells to various commensal bacteria. The transport kinetics and gene expression profile of M cells in response to different bacterial strains, namely Lactobacillus salivarius, Escherichia coli and Bacteroides fragilis, was assessed. Bacterial strains translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency whereas L. salivarius translocated with less efficiency. Microarray analysis of the M cell response showed both common and differential gene expression changes between the bacterial strains. In the presence of bacteria, but not control beads, up-regulated genes were mainly involved in transcription regulation whereas pro-inflammatory and stress response genes were primarily up-regulated by E. coli and B. fragilis, but not L. salivarius nor beads. Translocation of bacteria and M-cell gene expression responses were confirmed in murine M cells following bacterial challenge in vivo. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.

Summary Citrobacter rodentium is a murine pathogen that transiently colonizes the lumen of the large intestine. C. rodentium induces colitis, but the relative importance and temporal induction of the T helper type 17 (Th17) and regulatory T cell (Treg) pathways in protection from the infection and inflammation have not been assessed. Our aim was to investigate the key immunological signalling events associated with successful clearance of C. rodentium. Mice were challenged with luminescent-tagged C. rodentium and killed at days 3 (early infection), 10 (peak infection) and 21 (late infection) post-infection. Bioluminescent imaging and bacterial culture determined levels of C. rodentium. Distal colon mRNA expression of interleukin (IL)-17, IL-6, IL-1β, tumour necrosis factor (TNF)-α, forkhead box P3 (FoxP3) and ghrelin were assessed using real-time polymerase chain reaction. Results were compared with age-matched non-infected mice. Low levels of C. rodentium were found at day 3, high levels at day 10, with clearance from the majority of the mice by day 21. In the distal colon, there was up-regulation of TNF-α and FoxP3 throughout the study and increases in IL-6 and IL-17 during the peak and late stages of infection. Ghrelin expression was increased at the peak and late stages of infection. This study has characterized changes to the T helper cell pathways, following the course of C. rodentium infection in mice. There were significant immunological changes, with up-regulation of the Th17 and Treg pathways in the distal colon and an increase in ghrelin expression compared with non-infected control mice. These changes may play a role in the pathology and clearance of C. rodentium.

Short bowel syndrome (SBS) is primarily characterized by malabsorption and malnutrition, resulting from loss of intestinal absorptive area following massive small bowel resection (SBR). Bile acids and the gut microbiota are functionally linked within the gut-liver axis; however, SBS-associated disturbances within the gut-liver axis remain largely unexplored. The aim of this study was to characterize the evolution of bile acid alterations within the gut-liver axis at both short-term and long-term time points and to relate these changes to alterations in colonic bacterial composition.Four-week-old piglets were assigned to 75% SBR, sham-operation or non-operation control groups. High throughput sequencing was employed to determine bacterial abundance in colonic content and ultra-performance liquid chromatography used to determine the bile acid concentration of gall bladder, portal serum, and fecal samples.Bile acid complexity and relative abundance are altered in the SBS piglet model at two weeks post-SBR, and these changes persisted at six weeks post-SBR. Our examination of the microbial profile revealed an early and persistent loss in bacteria belonging to the Clostridiales order.This study provides evidence of an early and persistent disturbance of the bile acid profile throughout the entero-hepatic circulation with an increase in the proportion of primary bile acids and a decrease in secondary bile acids following SBR. These changes were associated with a loss of bacteria belonging to the Clostridiales order consistent with a disturbance in the bile-microbial axis following SBR.

Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1β, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells. We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.

Background & Aims Despite the mortality associated with liver disease observed in patients with short bowel syndrome (SBS), mechanisms underlying the development of SBS-associated liver disease (SBS-ALD) are poorly understood. This study examines the impact of bacterially-mediated bile acid (BA) dysmetabolism on farnesoid X receptor (FXR) signalling pathways and clinical outcome in a piglet model of SBS-ALD. Methods 4-week old piglets underwent 75% small bowel resection (SBR) or sham operation. Liver histology and hepatic inflammatory gene expression were examined. Abundance of BA biotransforming bacteria was determined and metabolomic studies detailed the alterations in BA composition of stool, portal serum and bile samples. Gene expression of intestinal and hepatic FXR target genes and small heterodimer partner (SHP) transrepression targets were assessed. Results Histological evidence of SBS-ALD included liver bile duct proliferation, hepatocyte ballooning and fibrosis. Inflammatory gene expression was increased. Microbiota changes included a 10-fold decrease in Clostridium and a two-fold decrease in Bacteroides in SBS-ALD piglets. BA composition was altered and reflected a primary BA dominant composition. Intestinal and hepatic regulation of BA synthesis was characterised by a blunted intestinal FXR activation response and a failure of SHP to repress key hepatic targets. Conclusions We propose a pathological scenario in which microbial dysbiosis following SBR results in significant BA dysmetabolism and consequent outcomes including steatorrhoea, persistent diarrhoea and liver damage. Furthermore alterations in BA composition may have contributed to the observed disturbance in FXR-mediated signalling pathways. These findings provide an insight into the complex mechanisms mediating the development of liver disease in patients with SBS.

OBJECTIVES: Salmonella-induced damage to the small intestine may decrease the villi-associated enzyme activity, causing malabsorption of nutrients and diarrhea, and thus contribute to the symptoms of infection. The objective of this study was to determine the mechanism by which different doses and durations of Salmonella infection and lipopolysaccharide (LPS) affect brush border enzyme activity in the mouse, and to determine if the probiotic Bifidobacterium longum subspecies infantis 35624 could attenuate the intestinal damage. METHODS: BALB/c mice were challenged with Salmonella enterica serovar Typhimurium UK1 at various doses (102–108 colony-forming unit (CFU)) and durations (106 CFU for 1–6 days). Mice were also treated with B. longum subsp. infantis 35624 for 2 weeks before and during a 6-day S. Typhimurium challenge (106 CFU), or before injection of LPS. The small intestine was assessed for morphological changes, mRNA expression of cytokines, and activity of the brush border enzymes sucrase–isomaltase, maltase, and alkaline phosphatase. RESULTS: S. Typhimurium infection significantly reduced the activity of all brush border enzymes in a dose- and time-dependent manner (P