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A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFβ/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.

Background The long-term consequences of COVID-19 remain unclear. There is concern a proportion of patients will progress to develop pulmonary fibrosis. We aimed to assess the temporal change in CXR infiltrates in a cohort of patients following hospitalisation for COVID-19. Methods We conducted a single-centre prospective cohort study of patients admitted to University Hospital Southampton with confirmed SARS-CoV2 infection between 20th March and 3rd June 2020. Patients were approached for standard-of-care follow-up 12-weeks after hospitalisation. Inpatient and follow-up CXRs were scored by the assessing clinician for extent of pulmonary infiltrates; 0–4 per lung (Nil = 0, 75% = 4). Results 101 patients with paired CXRs were included. Demographics: 53% male with a median (IQR) age 53.0 (45–63) years and length of stay 9 (5–17.5) days. The median CXR follow-up interval was 82 (77–86) days with median baseline and follow-up CXR scores of 4.0 (3–5) and 0.0 (0–1) respectively. 32% of patients had persistent CXR abnormality at 12-weeks. In multivariate analysis length of stay (LOS), smoking-status and obesity were identified as independent risk factors for persistent CXR abnormality. Serum LDH was significantly higher at baseline and at follow-up in patients with CXR abnormalities compared to those with resolution. A 5-point composite risk score (1-point each; LOS ≥ 15 days, Level 2/3 admission, LDH > 750 U/L, obesity and smoking-status) strongly predicted risk of persistent radiograph abnormality (0.81). Conclusion Persistent CXR abnormality 12-weeks post COVID-19 was common in this cohort. LOS, obesity, increased serum LDH, and smoking-status were risk factors for radiograph abnormality. These findings require further prospective validation.

[No Abstract Available], NIHR Southampton Biomedical Research Centre; Wellcome Trust [209553/Z/17/Z]; NIHR UCLH Biomedical Research Centre, UK, J.M. Wallis receives a research fellowship from the NIHR Southampton Biomedical Research Centre. This research was funded in whole or in part by the Wellcome Trust (209553/Z/17/Z). For this project, J. Jacob and E. Gudmundsson were also supported by the NIHR UCLH Biomedical Research Centre, UK. Funding information for this article has been deposited with the Crossref Funder Registry.

Background The impact of COVID-19 on physical and mental health and employment after hospitalisation with acute disease is not well understood. The aim of this study was to determine the effects of COVID-19-related hospitalisation on health and employment, to identify factors associated with recovery, and to describe recovery phenotypes. Methods The Post-hospitalisation COVID-19 study (PHOSP-COVID) is a multicentre, long-term follow-up study of adults (aged ≥18 years) discharged from hospital in the UK with a clinical diagnosis of COVID-19, involving an assessment between 2 and 7 months after discharge, including detailed recording of symptoms, and physiological and biochemical testing. Multivariable logistic regression was done for the primary outcome of patient-perceived recovery, with age, sex, ethnicity, body-mass index, comorbidities, and severity of acute illness as covariates. A post-hoc cluster analysis of outcomes for breathlessness, fatigue, mental health, cognitive impairment, and physical performance was done using the clustering large applications k-medoids approach. The study is registered on the ISRCTN Registry (ISRCTN10980107). Findings We report findings for 1077 patients discharged from hospital between March 5 and Nov 30, 2020, who underwent assessment at a median of 5·9 months (IQR 4·9–6·5) after discharge. Participants had a mean age of 58 years (SD 13); 384 (36%) were female, 710 (69%) were of white ethnicity, 288 (27%) had received mechanical ventilation, and 540 (50%) had at least two comorbidities. At follow-up, only 239 (29%) of 830 participants felt fully recovered, 158 (20%) of 806 had a new disability (assessed by the Washington Group Short Set on Functioning), and 124 (19%) of 641 experienced a health-related change in occupation. Factors associated with not recovering were female sex, middle age (40–59 years), two or more comorbidities, and more severe acute illness. The magnitude of the persistent health burden was substantial but only weakly associated with the severity of acute illness. Four clusters were identified with different severities of mental and physical health impairment (n=767): very severe (131 patients, 17%), severe (159, 21%), moderate along with cognitive impairment (127, 17%), and mild (350, 46%). Of the outcomes used in the cluster analysis, all were closely related except for cognitive impairment. Three (3%) of 113 patients in the very severe cluster, nine (7%) of 129 in the severe cluster, 36 (36%) of 99 in the moderate cluster, and 114 (43%) of 267 in the mild cluster reported feeling fully recovered. Persistently elevated serum C-reactive protein was positively associated with cluster severity. Interpretation We identified factors related to not recovering after hospital admission with COVID-19 at 6 months after discharge (eg, female sex, middle age, two or more comorbidities, and more acute severe illness), and four different recovery phenotypes. The severity of physical and mental health impairments were closely related, whereas cognitive health impairments were independent. In clinical care, a proactive approach is needed across the acute severity spectrum, with interdisciplinary working, wide access to COVID-19 holistic clinical services, and the potential to stratify care. Funding UK Research and Innovation and National Institute for Health Research.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGFβ increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting ‘bone-type’ cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of IPF lung mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Maintaining adherence to treatment for tuberculosis (TB) is essential if the disease is to be eliminated. As part of formative research to develop an intervention to improve adherence, we documented the lived experiences of adults receiving anti-TB treatment (ATT) in three UK cities and examined how personal, social, and structural circumstances interacted to impact on individuals’ adherence to treatment. Using a topic guide that explored social circumstances and experiences of TB care, we conducted in-depth interviews with 18 adults (six women) who were being or had been treated for TB (patients) and four adults (all women) who were caring for a friend, relative, or partner being treated for TB (caregivers). We analysed transcripts using an adapted framework method that classified factors affecting adherence as personal, social, structural, health systems, or treatment-related. Eleven of 18 patients were born outside the UK (in South, Central, and East Asia, and Eastern and Southern Africa); among the seven who were UK-born, four were Black, Asian, or Minority Ethnic and three were White British. TB and its treatment were often disruptive: in addition to debilitating symptoms and side effects of ATT, participants faced job insecurity, unstable housing, stigma, social isolation, worsening mental health, and damaged relationships. Those who had a strong support network, stable employment, a routine that could easily be adapted, a trusting relationship with their TB team, and clear understanding of the need for treatment reported finding it easier to adhere to ATT. Changes in circumstances sometimes had dramatic effects on an individual’s ability to take ATT; participants described how the impact of certain acute events (e.g., the onset of side effects or fatigue, episodes of stigmatisation, loss of income) were amplified by their timing or through their interaction with other elements of the individual’s life. We suggest that the dynamic and fluctuating nature of these factors necessitates comprehensive and regular review of needs and potential problems, conducted before and during ATT; this, coupled with supportive measures that consider (and seek to mitigate) the influence of social and structural factors, may help improve adherence.

The symptom of breathlessness is well recognized as part of the presentation of a wide range of medical conditions. It can be a manifestation of a life-threatening emergency. In acute medical settings, the priority is to quickly recognize patients who are critically unwell and require emergency treatment. For these patients, rapid initial assessment and immediate treatment are essential. However, once symptoms have stabilized or in less acute settings, a more thorough assessment is required. Cough is a common respiratory symptom, often part of a symptom complex, which is troublesome for the patient. It is important to recognize worrying associated features to prompt further investigation. In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection emerged from a zoonotic source, resulting in many cases of infection, hospitalizations and deaths, and has since spread in a pandemic across every continent. A substantial percentage of patients with COVID-19 develop an acute respiratory illness requiring hospital care. Cough and acute breathlessness are two of the most prevalent symptoms in this infection; any patient presenting to an acute setting should currently be assumed to have this infection and immediately tested with a viral swab from the upper airway to guide management.

Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-β-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

BACKGROUND Tuberculosis (TB) kills more people than any other infection, and new diagnostic tests to identify active cases are required. We aimed to discover and verify novel markers for TB in nondepleted plasma. METHODS We applied an optimized quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation combined with high-resolution mass spectrometry to study nondepleted plasma of 11 patients with active TB compared with 10 healthy controls. Prioritized candidates were verified in independent UK (n = 118) and South African cohorts (n = 203). RESULTS We generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We analyzed the predominantly low–molecular weight subproteome, identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤ 1%, q ≤ 0.05). Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in 2 independent cohorts. Receiver operating characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP, and SAA1) exhibited discriminatory power in distinguishing TB from other respiratory diseases (AUC = 0.81). CONCLUSION We report the most comprehensive TB plasma proteome to date, identifying novel markers with verification in 2 independent cohorts, leading to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test. FUNDING Colciencias, Medical Research Council, Innovate UK, NIHR, Academy of Medical Sciences, Program for Advanced Research Capacities for AIDS, Wellcome Centre for Infectious Diseases Research., Analysis of a comprehensive tuberculosis plasma proteome identified a 5-protein biosignature with potential to improve active tuberculosis diagnosis.

Background: Altered collagen architecture with increased “bone-type” pyridinoline collagen cross-linking, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in Idiopathic Pulmonary Fibrosis (IPF). We recently identified that hypoxia inducible factor (HIF) pathway activation promotes induction of the bone-type collagen cross-linking enzymes lysyl hydroxylase 2 and lysyl oxidase-like 2, yet the consequences of this on collagen structure-function in lung fibrosis remain unknown. Methods: Using a long term 3D in vitro model of the fibroblastic focus we cultured primary lung fibroblasts from IPF donors without or with the HIF-stabilising compound IOX2. Hydroxyproline and mature pyridinium cross-links were measured by colorimetric assays, and collagen ultrastructure assessed by electron microscopy (EM). The biomechanical effects of HIF stabilisation were investigated by parallel plate compression testing. Results: IOX2 stabilised HIF within the 3D fibroblastic focus model, promoting HIF pathway activation to disproportionately induce collagen-modifying enzymes relative to collagen fibril synthesis. After 6 weeks of culture, mature pyridinium cross-links were significantly increased by IOX2 to levels we have previously observed in IPF tissue. Ultrastructural analysis of the collagen fibrils with EM identified that IOX2 significantly reduced fibril diameter to sizes comparable with collagen fibrils enzymatically extracted from IPF tissue. IOX2 induced a greater than 3-fold increase in tissue stiffness. Conclusions: HIF pathway activation is a core regulator of bone-type collagen fibrillogenesis and altered structure-function in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic scarring disease in which aging, environmental exposure(s) and genetic susceptibility have been implicated in disease pathogenesis, however, the causes and mechanisms of the progressive fibrotic cascade are still poorly understood. As epithelial–mesenchymal interactions are essential for normal wound healing, through human 2D and 3D in vitro studies, we tested the hypothesis that IPF fibroblasts (IPFFs) dysregulate alveolar epithelial homeostasis. Conditioned media from IPFFs exaggerated the wound-healing response of primary human Type II alveolar epithelial cells (AECs). Furthermore, AECs co-cultured with IPFFs exhibited irregular epithelialization compared with those co-cultured with control fibroblasts (NHLFs) or AECs alone, suggesting that epithelial homeostasis is dysregulated in IPF as a consequence of the abnormal secretory phenotype of IPFFs. Secretome analysis of IPFF conditioned media and functional studies identified the matricellular protein, SPARC, as a key mediator in the epithelial–mesenchymal paracrine signaling, with increased secretion of SPARC by IPFFs promoting persistent activation of alveolar epithelium via an integrin/focal adhesion/cellular-junction axis resulting in disruption of epithelial barrier integrity and increased macromolecular permeability. These findings suggest that in IPF fibroblast paracrine signaling promotes persistent alveolar epithelial activation, so preventing normal epithelial repair responses and restoration of tissue homeostasis. Furthermore, they identify SPARC-mediated paracrine signaling as a potential therapeutic target to promote the restoration of lung epithelial homoestasis in IPF patients.

Background: Altered collagen architecture, rather than collagen quantity, is a key determinant of abnormal tissue structure-function in idiopathic pulmonary fibrosis (IPF), with the collagen cross-linking enzymes lysyl hydroxylase 2 (LH2) and lysyl oxidase-like 2 (LOXL2) promoting pathological bone-type collagen crosslinking and increased tissue stiffness (Jones et al eLife. 2018;7:e36354). However, the upstream mechanisms regulating this process remain unknown. Objective: To determine the cellular provenance of LOXL2 and LH2 in IPF tissue and identify the mechanisms promoting their dysregulation. Methods: Cellular provenance of LH2 and LOXL2 was analysed using mRNA in-situ hybridisation. IPF lung fibroblasts were cultured under a range of conditions that have been associated with IPF pathogenesis and their effects on LH2 and LOXL2 induction assessed by RTqPCR and Western Blotting. Small-interfering RNAs (siRNA) were transfected into IPF fibroblasts to investigate signalling pathways of interest and their role in regulating LH2 and LOXL2 expression. Results: LH2 and LOXL2 were co-expressed within fibroblastic foci in IPF tissue. Hypoxia-inducible factor (HIF) pathways were the strongest inducers of both LOXL2 and LH2 in IPF fibroblasts. siRNA mediated knockdown of HIF-1α but not HIF-2α prevented LH2 induction, whilst knockdown of both HIF-1α and HIF-2α was required to prevent LOXL2 induction. Conclusions: Our results suggest that HIF pathway activation via LH2/LOXL2 may be a core regulator of bone-type collagen fibrillogenesis in IPF.

Background: Objectively structured clinical examinations (OSCEs) are a stressful experience for many health care students and professionals in training. Mock OSCEs have been shown to be beneficial for student OSCE preparation. However, due to their expense and administrative burden students may only get a few opportunities to partake in these. To address this gap in student preparation a series of regularly run totally peer led multi-role practice OSCEs (PrOSCEs) was developed. Methods: Fifteen PrOSCEs were run over five-months. A total of 32 second year medical students took part, all of whom were enrolled on the graduate-entry programme at the University of Southampton. In each PrOSCE, 18 participants rotated through the roles of 'student', 'examiner' and 'patient' in six simulated stations designed by their peers. Peer feedback was provided after each station. At the end of the series of PrOSCEs students were asked to fill in an anonymous online feedback survey to assess the usefulness of the PrOSCEs in exam preparation. Results: Twenty-two students responded to the survey. 100% of respondents deemed routine participation either 'very useful' or 'useful' in preparing for their exam. PrOSCEs were found to improve confidence (mean = 7.9/10, 95% CI 7.4-8.3), expected performance (mean = 7.5/10, 95% CI 6.8-8.2) and help guide revision (mean = 8.3/10, 95% CI 7.6-9.0). Self-perceived teaching performance and confidence in providing feedback was also positively associated with participation. The most beneficial roles were 'student' and 'station creator'. Free-text feedback suggests that the informal setting and regular practice were particularly beneficial. Conclusion: The peer-led nature of the PrOSCEs allows for a low cost, low administrative burden and easy to replicate adjunct or alternative to large scale mock OSCEs. In addition the multi-role aspect of this approach could enhance exam preparation and may also improve aptitude as a clinical teacher. Further studies are required to understand if repeated practice has beneficial implications on OSCE performance.

The contribution of epithelial-mesenchymal transition (EMT) to human lung fibrogenesis is controversial. Here we provide evidence that ZEB1-mediated EMT in human alveolar epithelial type II (ATII) cells contributes to the development of lung fibrosis by paracrine signalling to underlying fibroblasts. Activation of EGFR-RAS-ERK signalling in ATII cells induced EMT via ZEB1. ATII cells had extremely low extracellular matrix gene expression even after induction of EMT, however conditioned media from ATII cells undergoing RAS-induced EMT augmented TGFβ-induced profibrogenic responses in lung fibroblasts. This epithelial-mesenchymal crosstalk was controlled by ZEB1 via the expression of tissue plasminogen activator (tPA). In human fibrotic lung tissue, nuclear ZEB1 expression was detected in alveolar epithelium adjacent to sites of extracellular matrix (ECM) deposition, suggesting that ZEB1-mediated paracrine signalling has the potential to contribute to early fibrotic changes in the lung interstitium. Targeting this novel ZEB1 regulatory axis may be a viable strategy for the treatment of pulmonary fibrosis.

Sarcoidosis is a highly variable, systemic granulomatous disease of hitherto unknown aetiology. The GenPhenReSa (Genotype–Phenotype Relationship in Sarcoidosis) project represents a European multicentre study to investigate the influence of genotype on disease phenotypes in sarcoidosis.The baseline phenotype module of GenPhenReSa comprised 2163 Caucasian patients with sarcoidosis who were phenotyped at 31 study centres according to a standardised protocol.From this module, we found that patients with acute onset were mainly female, young and of Scadding type I or II. Female patients showed a significantly higher frequency of eye and skin involvement, and complained more of fatigue. Based on multidimensional correspondence analysis and subsequent cluster analysis, patients could be clearly stratified into five distinct, yet undescribed, subgroups according to predominant organ involvement: 1) abdominal organ involvement, 2) ocular–cardiac–cutaneous–central nervous system disease involvement, 3) musculoskeletal–cutaneous involvement, 4) pulmonary and intrathoracic lymph node involvement, and 5) extrapulmonary involvement.These five new clinical phenotypes will be useful to recruit homogenous cohorts in future biomedical studies.

We read with interest the recent article by Pan et alwhich highlighted that a substantial proportion of patients with pulmonary and extrapulmonary tuberculosis (TB) have false-negative interferon-gamma release assay (IGRA) results, showing that a negative IGRA result does not rule out active TB. Unfortunately the authors excluded indeterminate assay results from the analyses, despite those results posing a considerable dilemma for clinical management, as they confer no information about the patient's TB infection status

The impact of immunosuppression on interferon-γ release assays and novel cytokine biomarkers of TB infection, mycobacteria-specific IL-2, IP-10 and TNF-α responses was investigated in an ex vivo model. Cytokine responses in standard QuantiFERON-TB Gold in-Tube (QFT-GIT) assays were compared with duplicate assays containing dexamethasone or infliximab. Dexamethasone converted QFT-GIT results from positive to negative in 30% of participants. Antigen-stimulated interferon-γ, IL-2 and TNF-α responses were markedly reduced, but IP-10 responses were preserved. Infliximab caused QFT-GIT result conversion in up to 30% of participants and substantial reductions in all cytokine responses. Therefore, corticosteroids and anti-TNF-α agents significantly impair interferon-γ release assay performance. IP-10 may be a more robust TB biomarker than interferon-γ in patients receiving corticosteroids.

Cell biology differs between traditional cell culture and 3-dimensional (3-D) systems, and is modulated by the extracellular matrix. Experimentation in 3-D presents challenges, especially with virulent pathogens. Mycobacterium tuberculosis (Mtb) kills more humans than any other infection and is characterised by a spatially organised immune response and extracellular matrix remodelling. We developed a 3-D system incorporating virulent mycobacteria, primary human blood mononuclear cells and collagen–alginate matrix to dissect the host-pathogen interaction. Infection in 3-D led to greater cellular survival and permitted longitudinal analysis over 21 days. Key features of human tuberculosis develop, and extracellular matrix integrity favours the host over the pathogen. We optimised multiparameter readouts to study emerging therapeutic interventions: cytokine supplementation, host-directed therapy and immunoaugmentation. Each intervention modulates the host-pathogen interaction, but has both beneficial and harmful effects. This methodology has wide applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimes and vaccination approaches.

Results from 3,263 QuantiFERON-TB Gold in-tube (QFT-GIT) assays were analyzed to determine the impact of age on test performance. The proportion of indeterminate results was significantly higher in pediatric and elderly (9.1% and 7.4%, respectively) than in adult (2.6%; chi-square test, P < 0.0001) patients. A detailed analysis of indeterminate QFT-GIT assay results is presented.

The management of idiopathic pulmonary fibrosis (IPF) has been transformed by the recent approval of two anti-fibrotic drugs, nintedanib and pirfenidone. An increasing number of patients with IPF are receiving treatment with these novel therapies, and the risk of adverse events that may be associated with their use must be carefully evaluated. Areas covered: Safety data about nintedanib and pirfenidone is critically evaluated, including data from randomized clinical trials and post-marketing reports. Management strategies to minimize the occurrence of side effects are summarized. Expert opinion: The safety profile of the two anti-fibrotic drugs approved for clinical use in IPF patients appears to be comparable. Data from clinical trials and initial post-marketing surveillance indicate that most of the observed side effects are mild and easily manageable. However, approximately 1/5 of patients may discontinue treatment as a consequence of side effects. Careful patient counselling, and regular follow-up during therapy could reduce the rate of discontinuations. Ongoing post-marketing surveillance may further inform our understanding of the safety profile of these therapies.

Although optic neuritis is commonly associated with multiple sclerosis, patients with atypical optic neuritis require further investigations to exclude other associated conditions. We report a woman presenting with cough, fatigue, atypical optic neuritis with chiasmitis. She responded partially to corticosteroids and we subsequently found she had a ground-glass lung nodule. Follow-up CT scan of thorax at 12 months showed new parenchymal lung lesions that suggested schistosomiasis. Further questioning by a respiratory physician identified, in retrospect, a previous exposure history; serological testing confirmed schistosoma infection. She was treated with praziquantel and slowly improved clinically, with radiological improvement in the optic chiasm, regression of the parenchymal lung lesions but with the ground glass nodule unchanged. We diagnosed parainfectious optic neuritis associated with schistosomiasis, based upon exposure history, serological confirmation and radiological features, together with the response to treatment, and having excluded other causes of an atypical optic neuritis.

Background and objective: a disintegrin and metalloproteinase (ADAM) 33 is a susceptibility gene associated with inflammatory lung and skin diseases. It is selectively expressed in mesenchymal cells, and its metalloprotease activity has been linked to angiogenesis and tissue remodelling. A soluble form of ADAM33 (sADAM33) has been identified in the bronchoalveolar lavage fluid (BALF) of asthmatic patients, and its levels inversely correlate with lung function. Because of its association with inflammatory lung diseases, it was hypothesized that sADAM33 is elevated in BALF of patients with pulmonary sarcoidosis. Methods: after removal of Ig using Protein A/G and enrichment using Concanavalin A beads, sADAM33 was identified in BALF by Western blotting. A fluorescence resonance energy transfer peptide cleavage assay was used to assess ADAM33-like activity in BALF. Results: sADAM33 protein in BALF was detected as a 25 kDa fragment, and levels were significantly increased in samples from sarcoid patients when compared to healthy controls (P Conclusions: release of sADAM33 is increased in sarcoid and may be associated with abnormal lung function. sADAM33 may be a biomarker of lung tissue inflammation and remodelling in sarcoid

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Using an ex vivo alveolar macrophage model, the hypothesis that inhaled preparations of corticosteroids might have important anti-inflammatory effects on cells of the peripheral airway was tested. The tumour necrosis factor (TNF)-alpha-inducing potential of three glycolipid preparations from nonpathogenic (arabinofuranasyl lipoarabinomannan (LAM (Ara-LAM)) and virulent (mannase LAM (ManLAM)) mycobacteria and Gram-negative bacteria (lipopolysaccharide (LPS)), in primary alveolar macrophage preparations was investigated. A novel inhaled chlorofluorocarbon (CFC)-free preparation of beclomethasone dipropionate (hydrofluoroalkane 134a (HFA)-BDP) with increased peripheral lung deposition was investigated for its ability to modulate glycolipidinduced TNF-alpha production by human alveolar macrophages, in comparison with a CFC-containing preparation and placebo. Compared to the basal TNF-alpha bioactivity of 0.72 ng x mL-1 (geometric mean), the TNF-alpha bioactivity in the macrophage preparation increased following incubation with LPS (138 ng x mL-1, p

SUMMARYImmunization with existing BCG vaccines has failed to confer consistent protection against tuberculosis. One of the ways to improve the efficacy of BCG is by enhancing its ability to induce a type-1 T cell response. However, this approach carries the risk that enhanced immunoreactivity may exacerbate tissue pathology associated with vaccination. The aim of the present study was to determine whether use of a recombinant BCG expressing IFN-γ (BCG-IFN) would result in an alteration in the pattern of inflammation and local tissue fibrosis. A murine intravenous BCG infection model was used in which there was a time- and dose-dependent increase in the weight and number of granulomas in the liver. Infection was associated with increased inflammatory activity in the liver, as shown by the increase in expression of inducible nitric oxide synthase (iNOS) assessed by immunochemistry and by measurement of specific mRNA, and in fibrosis measured by hydroxyproline content of the liver and percentage of granuloma cells staining positively for type 1 procollagen. Infection with BCG-IFN resulted in a reduction in organ weight and bacterial load on day 21 compared with infection with control BCG transformed with vector alone (BCG-plasmid). By day 21, there was also a reduction in iNOS mRNA and iNOS+ cells in granulomas in mice infected with BCG-IFN compared with infection with BCG-plasmid, and a similar reduction in both total number of granulomas and liver hydroxyproline content. These results demonstrate that the granulomas in the areas of mycobacterial infection are active sites of both inflammation and fibrosis, and that the local expression of IFN-γ by the recombinant BCG results in more efficient bacterial clearance which is accompanied by a reduction in tissue pathology.

Overexuberant production of tumour necrosis factor-alpha (TNF-alpha) by macrophages and other cells is thought to contribute to the development of permanent lung damage in many inflammatory conditions. There is a need for an agent, without the side-effects of corticosteroids, which can reduce the production of TNF-alpha by macrophages activated by disease. This study evaluated the effect of thalidomide on lipopolysaccharide (LPS)-induced TNF-alpha production by human alveolar macrophages obtained from patients with tuberculosis and a group of other diseases associated with macrophage activation. Alveolar macrophages obtained by bronchoalveolar lavage from 31 patients (tuberculosis = 12, sarcoidosis = 3, lung cancer = 5, chronic bronchitis = 5, pneumonia = 6) were stimulated with LPS alone or LPS in combination with either thalidomide or dexamethasone. Cell-associated TNF-alpha, as measured by immunochemistry, and TNF-alpha released by macrophages, as assessed by ELISA, were markedly increased when cells were incubated with LPS (P < 0.05), and both were decreased following addition of thalidomide (P < 0.05) or dexamethasone (P < 0.05) to amounts similar to those observed when macrophages were incubated with medium alone. Similarly, TNF-alpha mRNA as measured by in situ hybridization was increased following incubation with LPS (P < 0.05), but this increase was prevented by addition of thalidomide (P < 0.05) or dexamethasone (P < 0.05). The ability of thalidomide to reduce LPS-induced TNF-alpha production by alveolar macrophages was the same when cells from patients with tuberculosis (a disease associated with TNF-alpha production) and cells from patients with the other conditions were compared. The ability of thalidomide to reduce TNF-alpha production by human alveolar macrophages from patients with active lung disease suggests that thalidomide and its analogues may have potential as drugs to reduce TNF-alpha production in disease.

A disintegrin and metalloproteinase (ADAM) 33 is a susceptibility gene associated with inflammatory lung and skin diseases. It is selectively expressed in mesenchymal cells, and its metalloprotease activity has been linked to angiogenesis and tissue remodelling. A soluble form of ADAM33 (sADAM33) has been identified in the bronchoalveolar lavage fluid (BALF) of asthmatic patients, and its levels inversely correlate with lung function. Because of its association with inflammatory lung diseases, it was hypothesized that sADAM33 is elevated in BALF of patients with pulmonary sarcoidosis.After removal of Ig using Protein A/G and enrichment using Concanavalin A beads, sADAM33 was identified in BALF by Western blotting. A fluorescence resonance energy transfer peptide cleavage assay was used to assess ADAM33-like activity in BALF.sADAM33 protein in BALF was detected as a 25 kDa fragment, and levels were significantly increased in samples from sarcoid patients when compared to healthy controls (P0.05). Levels of sADAM33 were inversely correlated with lung function (FVC%) (P0.05) and DL(CO) % predicted (P0.01). No difference in sADAM33 enzymatic activity was observed between healthy and sarcoid BALF samples.Release of sADAM33 is increased in sarcoid and may be associated with abnormal lung function. sADAM33 may be a biomarker of lung tissue inflammation and remodelling in sarcoid.

Tropical pulmonary eosinophilia (TPE), a disease originally described in India in 1940 (l), is one of the many PIE (pulmonary infiltrates with peripheral blood eosinophilia) syndromes. TPE is an interstitial lung disease thought to result from immunological hyper-responsiveness to the human lymphatic-dwelling filarial parasites Wuchereria bancrofti and Bvugia malayi, although other non-filarial helminth-induced PIE syndromes resembling TPE have been described recently (2). TPE is characterized by paroxysmal bouts of cough and wheezing, pulmonary infiltrates; with laboratory studies demonstrating extreme peripheral eosinophilia (>2500 cells per ~1) and high serum levels of total, as well as filaria-specific, IgE and IgG antibodies (3). TPE is especially prominent in India and South-east Asia, although isolated cases have been reported from endemic areas elsewhere. The prevalence of TPE in the U.K. is unclear. Case reports in the literature are sporadic and probably underplay its true incidence, especially as this disease may go unrecognized or be misdiagnosed (4). With a doubling in the number of travellers returning from all endemic areas in the past 5 years (5) and a lo-fold increase in immigration rates since 1987 (6), it is inevitable that the disease burden will rise. Of the approximately 90 million people infected worldwide with these parasitic filarial nematodes, less than 1% manifest with symptoms of TPE (7). However, it is not clear what determines that unusual response to filarial infection. TPE can readily be distinguished from other PIE syndromes by the patient’s history of residence in the tropics, by the presence of extraordinarily high levels of both serum IgE and antifilarial antibodies, and by the dramatic therapeutic response to the filaricide diethylcarbamazine (DEC). Patients’ symptoms may masquerade as asthma for many years; it is essential, therefore, to take a detailed travel history from patients with suspected disease so that correct treatment may be offered early.

Some common childhood infections appear to prevent the development of atopy and asthma. In someMycobacterium bovisBCG-vaccinated populations, strong delayed-type hypersensitivity responses to mycobacterial antigens are associated with a reduced risk of atopy. Although BCG exposure decreases allergen-induced lung eosinophilia in animal models, little attention has been given to the effect of immunity to BCG on responses against live pathogens. We used the murineCryptococcus neoformansinfection model to investigate whether prior BCG infection can alter such responses. The present study shows that persistent pulmonary BCG infection of C57BL/6 mice induced an increase in gamma interferon, a reduction in interleukin-5, and a decrease in lung eosinophilia during subsequentCryptococcusinfection. This effect was long lasting, depended on the presence of live bacteria, and required persistence of mycobacterial infection in the lung. Reduction of eosinophilia was less prominent after infection with a mutant BCG strain (ΔhspR), which was rapidly cleared from the lungs. These observations have important implications for the development of vaccines designed to prevent Th2-mediated disease and indicate that prior lung BCG vaccination can alter the pattern of subsequent host inflammation.

A 39-year-old Indian man was diagnosed with ulcerative colitis on colonic biopsy and started on mesalazine, prednisolone and azathioprine. However, the colitis remained active and required antitumour necrosis factor (TNF) treatment with infliximab. Prior to starting infliximab, his chest X-ray was normal and QuantiFERON interferon γ release assay for tuberculosis (TB) was negative. However, his wife had been treated for pulmonary TB 11 years previously when they were cohabiting. On attending for his third dose of infliximab, he was feverish and tachycardic, and was admitted for investigation. Chest X-ray on admission showed changes consistent with miliary TB, and thoracic CT confirmed extensive miliary nodules with supraclavicular and mediastinal lymphadenopathy. Abdominal CT showed multiple mesenteric lymph nodes. Subsequent bronchoalveolar lavage, neck lymph node aspirate and colonic biopsies all cultured Mycobacterium tuberculosis. In retrospect, a clear history of close household TB exposure should have precipitated consideration of TB chemoprophylaxis prior to anti-TNF treatment.

With a view to exploring the role of transforming growth factor β (TGF-β) during mycobacterial infection, recombinant clones of bacillus Calmette-Guérin (BCG) were engineered to express the natural antagonist of TGF-β, latency-activated peptide (LAP). Induction of TGF-β activity was reduced when macrophages were infected with BCG expressing the LAP construct (LAP-BCG). There was a significant reduction in the growth of LAP-BCG in comparison to that of control BCG following intravenous infection in a mouse model. The enhanced control of mycobacterial replication was associated with an increase in the production of gamma interferon by splenocytes challenged during the acute stage of infection but with a diminished recall response assessed after 13 weeks. Organ weight and hydroxyproline content, representing tissue pathology, were also lower in mice infected with LAP-BCG. The results are consistent with the hypothesis that TGF-β has a detrimental effect on mycobacterial immunity. While a reduction in TGF-β activity augments the initial response to BCG vaccination, early bacterial clearance may adversely affect the induction of a long-term memory response by LAP-BCG.

Objectives: since 1987 there has been an increase in tuberculosis notifications in the U.K., with this increase disproportionately affecting London. A recent national survey suggests that co-infection with HIV occurs in less than 5% of tuberculosis patients. This study asked if local co-infection rates in Inner London differed from the national results. Methods: 157 consecutive patients starting antituberculous chemotherapy were venesected 2 weeks into treatment. Anonymized blood samples were screened for antibodies for HIV-1 and HIV-2 by enzyme-linked immunosorbent assay (ELISA). Epidemiological data were collected on each patient which was also coded before HIV test results were known. Results: of 157 patients commencing antituberculous therapy, 39 patients (24.8%) were found to be co-infected with HIV-1. HIV-negative and positive patients were similar in terms of age and sex. When 98 patients giving their country of origin as other than Europe were considered there were 22 co-infected with HIV (22.4%). Of the 39 HIV-positive identified in this study, 37 were also identified by our voluntary HIV testing programme. Conclusions: this study has shown that there may be very different rates of co-infection at a local level in the U.K. The local variation may be missed by national surveys and diverse local testing procedures. Anonymous testing identified only two patients with tuberculosis and HIV infection who were not identified by our voluntary HIV testing programme and this suggests that offering HIV tests to patients with tuberculosis is largely taken up by those at risk of HIV infection. Surveillance studies of this type are important, in identifying marked local variation from the national pattern of HIV and Mycobacterium tuberculosis infection.

We describe a case of unusual leishmaniasis in a Sudanese man with a history of progressively enlarging granulomatous mediastinal lymphadenopathy, worsening hemoptysis, and an intense mucosal granulomatous inflammatory response in the large bronchi. Leishmania donovani DNA was detected in bronchial biopsies by polymerase chain reaction. This is a novel description of human leishmanial infection in an immunocompetent patient involving this anatomical site. The patient's condition improved clinically, spirometrically, and radiologically after a course of treatment with amphotericin B. The cell-mediated immune response was analyzed before, during, and after successful antileishmanial chemotherapy.