Airway and Systemic Immunoglobulin Profiling and Immune Response in Adult Asthma.

INTRODUCTION: Immunoglobulins play a vital role in host immune response and in the pathogenesis of conditions like asthma. Therapeutic agents such as monoclonal antibodies target specific elements of the asthmatic inflammatory cascade. Decisions to utilize these medications are often based on systemic inflammatory profiling without direct insight into the airway inflammatory profile. We sought to investigate the relationship between immunoglobulin and cytokine profiles in the airway and systemic immune compartments of adult asthmatics. METHODS: Blood sampling and bronchoscopy with bronchoalveolar lavage (BAL) were performed in 76 well-defined adult asthmatics. Antibody and cytokine profiles were measured in both BAL and serum using ELISA and quantibody arrays. RESULTS: There was no relationship between BAL and serum levels of IgE. This is of significance in an asthma population. For some analytes, correlation analysis was significant (P < 0.05) indicating representativeness of our cohort and experimental setup in those cases. Nevertheless, the predictive power (r2) of the BAL-to-serum comparisons was mostly low except for TNF-α (r2 = 0.73) when assuming a simple (linear) relationship. CONCLUSION: This study highlights the importance of sample site when investigating the roles of immunoglobulins and cytokines in disease pathogenesis and suggests that both localized and systemic immune responses are at play. The prescription of asthma monoclonal therapy is generally based on systemic evaluation of cytokine and immunoglobulin levels. Our research suggests that this approach may not fully reflect the pathophysiology of the disease and may provide insight into why some patients respond to these targeted therapies while others do not.

Importance of vaccination for disease prevention in post-splenectomy patients.

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.

Suspected IgG4-related disease of the submandibular salivary gland and periorbital soft tissue in a man with latent tuberculosis.

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.

Seroprevalence study of SARS-CoV-2 antibodies in healthcare workers following the first wave of the COVID-19 pandemic in a tertiary-level hospital in the south of Ireland.

OBJECTIVE: This study investigated seroprevalence of SARS-CoV-2-specific IgG antibodies, using the Abbott antinucleocapsid IgG chemiluminescent microparticle immunoassay (CMIA) assay, in five prespecified healthcare worker (HCW) subgroups following the first wave of the COVID-19 pandemic. SETTING: An 800-bed tertiary-level teaching hospital in the south of Ireland. PARTICIPANTS: Serum was collected for anti-SARS-CoV-2 nucleocapsid IgG using the Abbott ARCHITECT SARS-CoV-2 IgG CMIA qualitative assay, as per the manufacturer's specifications.The groups were as follows: (1) HCWs who had real-time PCR (RT-PCR) confirmed COVID-19 infection (>1-month postpositive RT-PCR); (2) HCWs identified as close contacts of persons with COVID-19 infection and who subsequently developed symptoms (virus not detected by RT-PCR on oropharyngeal/nasopharyngeal swab); (3) HCWs identified as close contacts of COVID-19 cases and who remained asymptomatic (not screened by RT-PCR); (4) HCWs not included in the aforementioned groups working in areas determined as high-risk clinical areas; and (5) HCWs not included in the aforementioned groups working in areas determined as low-risk clinical areas. RESULTS: Six of 404 (1.49%) HCWs not previously diagnosed with SARS-CoV-2 infection (groups 2-5) were seropositive for SARS-CoV-2 at the time of recruitment into the study.Out of the 99 participants in group 1, 72 had detectable IgG to SARS-CoV-2 on laboratory testing (73%). Antibody positivity correlated with shorter length of time between RT-PCR positivity and antibody testing.Quantification cycle value on RT-PCR was not found to be correlated with antibody positivity. CONCLUSIONS: Seroprevalence of SARS-CoV-2 antibodies in HCWs who had not previously tested RT-PCR positive for COVID-19 was low compared with similar studies.

The presence of Aspergillus fumigatus in asthmatic airways is not clearly related to clinical disease severity.

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.

Farnesoid X Receptor Agonist Treatment Alters Bile Acid Metabolism but Exacerbates Liver Damage in a Piglet Model of Short-Bowel Syndrome.

BACKGROUND & AIMS: Options for the prevention of short-bowel syndrome-associated 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.

The Potential Role of Aspiration in the Asthmatic Airway.

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, FEV1, 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 FEV1 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.

Unravelling the metabolic impact of SBS-associated microbial dysbiosis: Insights from the piglet short bowel syndrome model.

Liver disease is a major source of morbidity and mortality in children with short bowel syndrome (SBS). SBS-associated microbial dysbiosis has recently been implicated in the development of SBS-associated liver disease (SBS-ALD), however the pathological implications of this association have not been explored. In this study high-throughput sequencing of colonic content from the well-validated piglet SBS-ALD model was examined to determine alterations in microbial communities, and concurrent metabolic alterations identified in urine samples via targeted mass spectrometry approaches (GC-MS, LC-MS, FIA-MS) further uncovered impacts of microbial disturbance on metabolic outcomes in SBS-ALD. Multi-variate analyses were performed to elucidate contributing SBS-ALD microbe and metabolite panels and to identify microbe-metabolite interactions. A unique SBS-ALD microbe panel was clearest at the genus level, with discriminating bacteria predominantly from the Firmicutes and Bacteroidetes phyla. The SBS-ALD metabolome included important alterations in the microbial metabolism of amino acids and the mitochondrial metabolism of branched chain amino acids. Correlation analysis defined microbe-metabolite clustering patterns unique to SBS-ALD and identified a metabolite panel that correlates with dysbiosis of the gut microbiome in SBS.

Short bowel syndrome (SBS)-associated alterations within the gut-liver axis evolve early and persist long-term in the piglet model of short bowel syndrome.

BACKGROUND AND AIM: 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. METHODS: 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. 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 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.

Changes in the colon microbiota and intestinal cytokine gene expression following minimal intestinal surgery.

AIM: To investigate the impact of minor abdominal surgery on the caecal microbial population and on markers of gut inflammation. METHODS: Four week old piglets were randomly allocated to a no-surgery "control" group (n = 6) or a "transection surgery" group (n = 5). During the transection surgery procedure, a conventional midline incision of the lower abdominal wall was made and the small intestine was transected at a site 225 cm proximal to the ileocaecal valve, a 2 cm segment was removed and the intestine was re-anastomosed. Piglets received a polymeric infant formula diet throughout the study period and were sacrificed at two weeks post-surgery. Clinical outcomes including weight, stool consistency and presence of stool fat globules were monitored. High throughput DNA sequencing of colonic content was used to detect surgery-related disturbances in microbial composition at phylum, family and genus level. Diversity and richness estimates were calculated for the control and minor surgery groups. As disturbances in the gut microbial community are linked to inflammation we compared the gene expression of key inflammatory cytokines (TNF, IL1B, IL18, IL12, IL8, IL6 and IL10) in ileum, terminal ileum and colon mucosal extracts obtained from control and abdominal surgery groups at two weeks post-surgery. RESULTS: Changes in the relative abundance of bacterial species at family and genus level were confined to bacterial members of the Proteobacteria and Bacteroidetes phyla. Family level compositional shifts included a reduction in the relative abundance of Enterobacteriaceae (22.95 ± 5.27 vs 2.07 ± 0.72, P < 0.01), Bacteroidaceae (2.54 ± 0.56 vs 0.86 ± 0.43, P < 0.05) and Rhodospirillaceae (0.40 ± 0.14 vs 0.00 ± 0.00, P < 0.05) following transection surgery. Similarly, at the genus level, changes associated with transection surgery were restricted to members of the Proteobacteria and Bacteroidetes phyla and included decreased relative abundance of Enterobacteriaceae (29.20 ± 6.74 vs 2.88 ± 1.08, P < 0.01), Alistipes (4.82 ± 1.73 vs 0.18 ± 0.13, P < 0.05) and Thalassospira (0.53 ± 0.19 vs 0.00 ± 0.00, P < 0.05). Surgery-associated microbial dysbiosis was accompanied by increased gene expression of markers of inflammation. Within the ileum IL6 expression was decreased (4.46 ± 1.60 vs 0.24 ± 0.06, P < 0.05) following transection surgery. In the terminal ileum, gene expression of TNF was decreased (1.51 ± 0.13 vs 0.80 ± 0.16, P < 0.01) and IL18 (1.21 ± 0.18 vs 2.13 ± 0.24, P < 0.01), IL12 (1.04 ± 0.16 vs 1.82 ± 0.32, P < 0.05) and IL10 (1.04 ± 0.06 vs 1.43 ± 0.09, P < 0.01) gene expression increased following transection surgery. Within the colon, IL12 (0.72 ± 0.13 vs 1.78 ± 0.28, P < 0.01) and IL10 (0.98 ± 0.02 vs 1.95 ± 0.14, P < 0.01) gene expression were increased following transection surgery. CONCLUSION: This study suggests that minor abdominal surgery in infants, results in long-term alteration of the colonic microbial composition and persistent gastrointestinal inflammation.

Altered FXR signalling is associated with bile acid dysmetabolism in short bowel syndrome-associated liver disease.

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.

Concomitant exposure to ovalbumin and endotoxin augments airway inflammation but not airway hyperresponsiveness in a murine model of asthma.

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-1ß, 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.

Gut microbial diversity is reduced and is associated with colonic inflammation in a piglet model of short bowel syndrome.

BACKGROUND AND OBJECTIVES: 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. RESULTS: 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. METHODS: 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)-1ß, 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. CONCLUSIONS: 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.

Bifidobacterium infantis 35624 protects against salmonella-induced reductions in digestive enzyme activity in mice by attenuation of the host inflammatory response.

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 (10(2)-10(8) colony-forming unit (CFU)) and durations (10(6) 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 (10(6) 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<0.05). This also occurred following injection of LPS. Pre-treatment with B. longum subsp. infantis 35624 prevented weight loss, protected brush border enzyme activity, reduced the small intestinal damage, and inhibited the increase in interleukin (IL)-10 and IL-8 expression due to Salmonella challenge. CONCLUSIONS: Salmonella infection reduces the small intestinal brush border enzyme activity in mice, with the level of reduction and associated weight loss increasing with dose and duration of infection. B. longum subsp. infantis 35624 treatment attenuated the effect of Salmonella infection on brush border enzyme activity and weight loss, which may be due to modulation of the host immune response.

Differential intestinal M-cell gene expression response to gut commensals.

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.