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Background The renin-angiotensin system (RAS) protects the circulation against sudden falls in systemic blood pressure via generation of angiotensin II (AII). Previously, we demonstrated that patients with anaphylaxis involving airway angioedema and cardiovascular collapse (AACVS) had significantly increased "I" gene polymorphisms of the angiotensin-converting-enzymes (ACE). This is associated with lower serum ACE and AII levels and was not seen in anaphylaxis without collapse nor atopics and healthy controls. Objectives To examine the angiotensinogen (AGT-M235T) and chymase gene (CMA-1 A1903G) polymorphisms in these original subjects. Method 122 patients with IgE-mediated anaphylaxis, 119 healthy controls and 52 atopics had polymorphisms of the AGT gene and chymase gene examined by polymerase chain reactions and gel electrophoresis. Their previous ACE genotypes were included for the analysis. Results AGT-MM genes (associated with low AGT levels) were significantly increased in anaphylaxis (Terr's classification). When combined with ACE, anaphylaxis showed increased MM/II gene pairing (p

Introduction: In cases of idiopathic pulmonary fibrosis, we have observed an elevation in mean red cell volume, serum gamma glutamyl transferase and peripheral monocyte counts, initially in a pilot study but also in new incident cases. These changes could not be explained by drug therapy, vitamin deficiency or other diseases. Method: We compared the peripheral blood abnormalities in 149 patients with lung fibrosis to 448 age and sex matched controls. We also examined the effect of cotrimoxazole treatment for 12 weeks on these abnormalities. From the pilot study of cotrimoxazole in lung fibrosis patients, the relationship of the peripheral blood monocyte count and serum cytokine transforming growth factor beta-1 was examined. Epstein Barr viral status was examined in a selection of patients in case it explained our observations. Results: The findings confirm the elevation in mean red cell volume, gamma glutamyl transferase and peripheral monocyte counts in patients compared with matched controls. Oral cotrimoxazole ameliorated these 3 blood abnormalities. Serological evidence of Epstein Barr viral infection was present in tested patients but active viral replication was absent. The monocyte count had a linear relationship with the serum transforming growth factor beta-1 levels, which increased by 600 pg/ml for every of 0.1 × 109/l increase in the monocyte count. Conclusion: These observations may reflect oxidative stress which was reduced by cotrimoxazole. A related sulphonamide “dapsone” is known to reduce oxidative stress through direct effects on neutrophil and monocyte function; similar effects may explain these findings and require a formal study.

Summary A diagnosis of idiopathic anaphylaxis following a detailed clinical assessment remains very challenging for patients and clinicians. Risk reduction strategies such as allergen avoidance are not possible. This study investigated whether the (ISAC) allergen array with 103 allergens would add diagnostic value in patients with idiopathic anaphylaxis. We extended the specific immunoglobulin (Ig)E testing in 110 patients with a diagnosis of idiopathic anaphylaxis from five UK specialist centres using ISAC arrays. These were divided into three groups: score I identified no new allergen sensitization beyond those known by previous assessment, score II identified new sensitizations which were not thought likely to explain the anaphylaxis and score III identified new sensitizations felt to have a high likelihood of being responsible for the anaphylaxis. A proportion (50%) of score III patients underwent clinical reassessment to substantiate the link to anaphylaxis in this group. The results show that 20% of the arrays were classified as score III with a high likelihood of identifying the cause of the anaphylaxis. A wide range of major allergens were identified, the most frequent being omega-5-gliadin and shrimp, together accounting for 45% of the previously unrecognized sensitizations. The ISAC array contributed to the diagnosis in 20% of patients with idiopathic anaphylaxis. It may offer additional information where a careful allergy history and follow-on testing have not revealed the cause of the anaphylaxis.

Background: Idiopathic pulmonary fibrosis (UIP/IPF) is increasing. The condition occurs both sporadically and in close blood relatives suggesting a genetic predisposition. Histology suggests that the fibrosis is linked to disordered apoptosis, but the causal agent is so far unrecognised. We previously published evidence of mannose binding lectin deficiency (MBL) in early onset disease and in those with a family history of UIP/IPF. MBL is part of the innate immune system with levels genetically determined. MBL activation is impaired if the mannose associated serine protease2 (MASP2) is severely deficient in the serum (levels 100 ng/ml) showed no differences between Healthy Controls, COPD, TB and sarcoid. UIP/IPF showed a significant increase in the frequency of severe deficiency. This was seen for early onset disease 55yrs (20% p=0.0001) and those with a family history (19% p=0.0143). The expected frequency for the Caucasian population is

Granulomas that occur in sarcoidosis are histologically identical to those found in a subset of Common Variable Immunodeficiency (CVID) patients where they are associated with a reduction in class switched memory B lymphocytes. The aim of this study was to investigate whether the abnormalities in peripheral blood lymphocyte populations associated with granulomatous variant CVID (gvCVID) are also present in individuals with sarcoidosis. We examined B lymphocyte populations using flow cytometry and found that the reduction of class switched memory (CSM: CD19+CD27+IgM-IgD-) and unswitched memory (CD19+CD27+IgM+IgD+) B cells in our sarcoidosis cohort was similar to that previously reported in gvCVID patients. The reduction of class switched memory B cells in sarcoidosis patients indicated a possible defect in the T cell repertoire as antibody class switching requires T cell help. We subsequently explored the peripheral blood T cell compartment of our sarcoidosis patients. The results identified a population of terminally differentiated effector CD8+ T cells (CCR7-CD45RA-CD127-CD27-CD28-) that were significantly expanded in the peripheral blood of sarcoidosis patients. Terminally differentiated effector CD8+ T cells have been defined as cytolytic, inflammatory cells with reduced replicative capacity. The discovery of abnormal peripheral blood B and T cells compartments in sarcoidosis may be of value in clinical diagnosis and could be relevant to the pathogenic process.

Background: Idiopathic pulmonary fibrosis (UIP/IPF) is increasingly recognized that siblings & close blood relatives suggesting a genetic predisposition. Serum mannose binding lectin levels (MBL) forms part of the innate immune system with deficiency produces an opsonisation and phagocytosis defect. Serum levels are genetically determined. Aims & method: We examined the serum MBL in healthy controls (HC), frequently exacerbating COPD, pulmonary TB & Sarcoidosis along with UIP/IPF patients including those with and without an affected family member. Results: Mean serum MBL levels were not statistically different in HC, COPD or TB. Sarcoid had statistically higher mean levels. Those with IPF onset at 55 yrs and no other affected relative. Chi squared analysis of these patterns showed no differences for HC, COPD & IPF>55 yrs. TB & Sarcoid had higher frequencies of normal MBL levels compared with HC (p=0.001 & 0.024 respectively). IPF

Summary: The technique of representation difference analysis PCR has been applied to find genes specific to Mycobacterium avium subsp. paratuberculosis. This generated a 671 bp fragment which was used to isolate a larger genetic element found in the enteric pathogens M. avium subsp. paratuberculosis and M. avium subsp. silvaticum but which was absent from the very closely related and relatively benign M. avium subsp. avium. This element, designated GS, is greater than 6·5 kbp in length and has a G+C content 9 mol% lower than other genes from this species. There is a previously uncharacterized insertion sequence associated with one end. The GS element encodes five ORFs in M. avium subsp. paratuberculosis and M. avium subsp. silvaticum, all of which have counterparts encoded in Mycobacterium tuberculosis. Database searches revealed homologues for these ORFs in a number of bacterial species, predominantly Gram-negative organisms, including a number of enteric pathogens. These homologous genes encode functions related to LPS or extracellular polysaccharide biosynthesis. This element has a number of features in common with pathogenicity islands such as its low G+C content, an association with a putative insertion sequence and a grouping of genes of related function with a possible link to virulence. No direct link to pathogenicity has been shown but GS may belong to a group of related ‘genetic islands’ and represents the first such element to be identified in mycobacteria.

The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. avium subsp. paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. avium subsp. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein. We have confirmed this unique targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcription-PCR. Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M. avium subsp. paratuberculosis. A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M. avium subsp. paratuberculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.

Background Idiopathic pulmonary fibrosis (IPF) is a serious progressive lung disease with likely environmental and genetic risk factors that are thought to contribute to the disease even though their exact nature is unknown. It is increasingly recognised that siblings and close blood relatives can develop the same condition suggesting an unknown genetic predisposition. Method We have examined the serum mannose binding lectin levels (MBL) in healthy controls (HC), frequently exacerbating COPD, pulmonary TB & Sarcoidosis along with IPF patients. Results Mean serum MBL levels were not statistically different in HC, COPD or TB using an unpaired t test. Cases with sarcoid had higher levels. Those with IPF onset at 50 years without a family history. Abstract S143 Table 1 shows means, SEM and p values, and the per cent of each patient group with normal >650, moderate 100–600 or severe deficiency levels Discussion Serum MBL forms part of the complement activation and innate immune system and protects the lung from infection by organisms that bind mannose sugar (eg, strep, staph, yeasts, P.Jiveci). MBL deficiency gives an opsonisation defect with reduced phagocytosis by alveolar macrophages. Blood levels are genetically determined, with UK population data showing: 12% severe deficiency 34% moderate deficiency 100–600 pg/ml, 54% normal Serum levels relate to polymorphisms of the MBL2 genes. χ 2 analysis of frequency distribution showed no differences for HC, COPD & IPF>50 years. TB&Sarcoid had higher frequencies of normal MBL levels compared with HC (p=0.001 and 0.024 respectively). IPF The literature shows MBL to consistently have interesting and important central roles in lung defenses via effects on complement, apoptosis and cytokines. Its observed deficiency in young IPF and those with a FH could be a genetic risk factor of relevance, explaining its early occurrence in deficiency and later occurrence in ‘sufficiency’, where it gives protection from insult or injury to the lung.