The T1799A point mutation is present in posterior uveal melanoma.

An activating mutation in exon 15 of the BRAF gene is present in a high proportion of cutaneous pigmented lesions. Until recently this mutation had however only been identified in one case of posterior uveal melanoma. Despite this apparent lack of the BRAF mutation, inappropriate downstream activation of the Ras/Raf/MAPK pathway has been described in posterior uveal melanoma. Based on the already recognised morphological and cytogenetic heterogeneity in uveal melanoma, we hypothesised that the BRAF mutation may be present in uveal melanoma but only in some of the tumour cells. In this study, we analysed 20 ciliary body and 30 choroidal melanomas using a nested PCR-based technique resulting in the amplification of a nested product only if the mutation was present. This sensitive technique can identify mutated DNA in the presence of wild-type DNA. The mutation was identified in 4 of 20 (20%) ciliary body and 11 of 30 (40%) choroidal melanomas. Further analysis of separate areas within the same choroidal melanoma demonstrated that the mutation was not present in the entire tumour. In conclusion, the T1799A BRAF mutation is present in a proportion of posterior uveal melanomas but within these tumours the distribution of the mutation is heterogeneous.

Alphagan allergy may increase the propensity for multiple eye-drop allergy.

AIMS: Since its introduction in 1996, brimonidine tartrate 0.2% ophthalmic solution (Alphagan, Allergan) twice daily has become established as an effective intra ocular pressure-lowering treatment. While the efficacy of Alphagan cannot be questioned, we gained the clinical impression that the drug has an unacceptably high rate of allergy. Of greater concern, we suspected that patients suffering from local Alphagan allergy had a higher rate of allergy to subsequently used topical preparations. We analysed data from a large scale study of glaucoma patients to establish whether our suspicions were correct. SUBJECTS AND METHODS: We have created a database of the entire glaucoma treatment histories for consecutive patients attending a single consultant's clinics (DMIM) at Glasgow Royal Infirmary between May 1999 and September 2001. All have undergone medical treatment for primary open angle glaucoma, ocular hypertension, or normal tension glaucoma. Patients with any other form of glaucoma, and patients in whom a full record of treatment was not available were excluded from the study. RESULTS: Alphagan was discontinued due to allergy on 73 per 100,000 patient treatment days. This was a far higher frequency than for other preparations. In patients allergic to both Alphagan and another preparation (Timoptol, Trusopt and Xalatan), the mean interval between the first and second allergy was shorter when Alphagan allergy occurred first. This was statistically significant in Timoptol and Trusopt cross-reactivity. CONCLUSIONS: Alphagan has high allergenicity, and may increase the likelihood of allergy to subsequently used preparations.

Bilateral uveal melanoma: a series of four cases.

AIM: To describe the occurrence of bilateral primary choroidal melanoma in four patients. METHODS: All patients attending the Liverpool Ocular Oncology Centre with uveal melanoma between January 1993 and February 2002 were identified, and those with bilateral primary choroidal melanoma were reviewed. Their presentation and management are described. RESULTS: Four patients, all female, were identified. Patient 1 presented with a right juxtapapillary melanoma at the age of 64, which was treated with krypton laser and endoresection, and then when aged 73 required proton beam radiotherapy for a melanoma in her left eye. Patient 2 presented at the age of 82 with bilateral choroidal melanomas and underwent simultaneous bilateral plaque radiotherapy. Patient 3 presented with bilateral choroidal melanomas at the age of 75 and was treated initially with bilateral proton beam radiotherapy. Patient 4 was treated at the age of 54 with right plaque radiotherapy for a choroidal melanoma, and 3 years later needed plaque radiotherapy for a melanoma in her other eye. CONCLUSION: Bilateral choroidal melanoma is possible and should be a consideration in the continuing management of patients with choroidal melanoma.

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle.

AIMS: To produce a method of distinguishing between type 1 and 2 skeletal muscle fibres that would be more economical and reproducible than the standard ATPase method and be applicable to both fixed and frozen tissue. Because the ATPase method has been accepted as the basis for fibre identification for the past 50 years, the new method should not give significantly different results. METHODS: Isoforms of myosin correlate with isoforms of myofibrillar ATPase and an immunohistochemical (IHC) double labelling protocol was devised using monoclonal antibodies to fast and slow myosin. This required one tissue section rather than four. The results of the two methods were compared by means of morphometric analysis of skeletal muscle biopsies from 20 normal healthy volunteers. RESULTS: There were no significant differences (p = 0.57) in the percentages of type 1 (46% using the IHC method v 48% using ATPase) or type 2 fibres (54% v 52%, respectively). The 2a and 2b subtypes were distinguished easily. Analysis of variance revealed that cross sectional area (mu m(2)), diameter (mu m), form factor, and density of fibre staining (a measure of substrate-enzyme or protein) were all similar. The method worked equally well on fixed material. CONCLUSION: An IHC method based on the fast and slow isoforms of myosin shows no significant differences in fibre type analysis from the standard ATPase method although it provides important advantages because it is applicable to fixed (including archival) material, is economical and reproducible, and yields a permanent preparation.

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection.

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

Effects of angiotensin II on remodelling of the airway and the vasculature in the rat.

Airway remodelling occurs in chronic asthma. Angiotensin II promotes growth in cardiovascular remodelling. Since the renin-angiotensin system is activated in acute severe asthma, we hypothesized that angiotensin II has a role in airway remodelling. A total of 14 young male Wistar rats were randomly divided into two groups. All received 2-week infusions of bromodeoxyuridine, and the experimental group also received angiotensin II. Blood pressure rose in the angiotensin II-infused group [mean levels: pre-infusion, 134.9 (S.D. 14.7) mmHg; post-infusion, 197.1 (22.5) mmHg], and expression of renin mRNA in the renal juxtaglomerular cells was suppressed in these animals. The proportion of bromodeoxyuridine-positive cell nuclei was no different in the airways of control and angiotensin II-infused animals for smooth muscle [mean bromodeoxyuridine index: control, 8. 6% (S.E.M. 1.1%); angiotensin II, 9.3% (1.1%)], epithelium [control, 16.7% (2.3%); angiotensin II, 16.0% (2.2%)] and adventitia [control, 26.4% (2.2%); angiotensin II, 26.6% (2.4%)]. In the arteries, bromodeoxyuridine indices were higher in the angiotensin II-infused rats [18.4% (2.3%)] than in the control animals [9.4% (2.8%)], but no difference was found in the veins [12% (2.9%) and 11.4% (2.6%) respectively]. Morphometry of the airway wall and mesenteric vasculature was no different in the two groups. Therefore a 2-week infusion of angiotensin II increases blood pressure and DNA synthesis in the mesenteric arteries, but does not cause airway remodelling, in the rat.

Association between antibodies to heat shock protein 65 and coronary atherosclerosis. Possible mechanism of action of Helicobacter pylori and other bacterial infections in increasing cardiovascular risk.

INTRODUCTION: There is growing evidence that the immune response is involved in atherosclerosis. Antibodies to heat shock protein 60/65 have been shown to be a risk factor for carotid atherosclerosis and been proposed as a diagnostic marker of atherosclerosis. In addition, it has been suggested that the immune response to heat shock protein 60/65 may be a link between exposure to microorganisms and increased cardiovascular risk. AIMS: (1) To investigate the association between anti-shock protein 65 titre and coronary atherosclerosis. (2) To assess whether anti-mhsp65 titre is a useful diagnostic marker of atherosclerosis; (3) To examine the influence of Helicobacter pylori infection on anti-heat shock protein 65 titre. METHODS AND RESULTS: In the first study we measured anti-heat shock protein 65 titres in 136 consecutive male subjects admitted for routine coronary angiography. Anti-heat shock protein 65 titres correlated with both the severity and extent of coronary atherosclerosis and the relationship remains statistically significant for the presence of atherosclerosis (P = 0.012) after adjustment for possible confounding influences. However the association had insufficient sensitivity to be a useful clinical test. In the second study we recruited 100 patients with confirmed active H. pylori infection and double blindly randomized them to eradication therapy or placebo. Successful eradication of H. pylori led to a significant fall in anti-heat shock protein 65 titres (from a mean of 256.4 AU.ml-1 to 137.5 AU. ml-1. P = 0.033). CONCLUSION: These results raise the possibility that exposure to H. pylori and other micro-organisms lead to an increased risk of clinically manifest coronary artery disease by an autoimmune process.

Vascular remodelling in intramyocardial resistance vessels in hypertensive human cardiac transplant recipients.

OBJECTIVE: Cardiac transplant recipients often develop hypertension as a side effect of immunosuppressive treatment. The aim of this study was to use the serial endomyocardial biopsies taken to monitor rejection to study the early and sequential arterial changes in human myocardial resistance arteries as hypertension develops. METHODS: At least 14 biopsies were studied from each of 23 patients, divided into a normotensive group (12 patients with a diastolic pressure never greater than 90 mm Hg) and a hypertensive group (11 patients with more than 10% of diastolic pressure measurements above 100 mm Hg). Morphometric analysis of between 30 and 50 arteries and arterioles in two widely separated histological levels from each biopsy was undertaken using an Optomax image analyser. RESULTS: There was a correlation between blood pressure, particularly diastolic pressure, and rate of medial thickening of intramyocardial coronary resistance arteries and arterioles (P = 0.0025). There was also a correlation between serum cyclosporin A concentrations and mean artery wall thickness (P = 0.003). CONCLUSIONS: Hypertension and cyclosporin A treatment are associated with significant wall thickening of intramyocardial resistance vessels in cardiac allograft recipients. These changes may be functionally and clinically important.

An assessment of the distortion of arteries due to sectioning in endomyocardial biopsies.

Arteries are usually studied morphometrically after pressurized fixation and resin embedding. These procedures are impracticable when dealing with diagnostic biopsies. The accuracy of arterial morphometry is determined partly by the degree of tissue distortion during section preparation. The axial ratios of 7340 arteries were measured in 353 endomyocardial biopsies from 23 patients and then compared with those expected from mathematical modelling. An excess of elliptical arteries was found. The distribution of orientation of the long axes of these best fitted a simulated 10 per cent linear distortion in the direction of microtomy. In conclusion, these results suggest that although there is some tissue distortion during sectioning, useful data may be obtained from morphometry of arteries in routinely processed endomyocardial biopsies.

Investigations on the renin-angiotensin system in acute severe asthma.

The renin-angiotensin system is activated in acute severe asthma. The precise mechanism of activation is at present unknown, but may involve, beta2-agonists, catecholamines or proteases released in airway inflammation. This study aims to identify potential factors involved in the activation of the renin-angiotensin system in acute asthma. Forty asthmatics with severe exacerbations of asthma, assessed by measurement of peak expiratory flow rate (mean (SD) 35 (18)% predicted), oxygen saturation (94 (4)%) and pulse rate (108 (16) beats x min(-1)) were recruited. Nineteen (48%) asthmatics had elevated plasma angiotensin II levels (median (interquartile range) 10.9 (4.3-23.5) pg x mL(-1) (normal range 3-12 pg x mL(-1))) and 10 (25%) had elevated plasma renin concentration (22.0 (10.0-50.0) microU x mL(-1) (normal range 9-50 microU x mL(-1))). Plasma renin and angiotensin II correlated strongly, implying renin-dependent angiotensin II formation. No correlation was found between plasma salbutamol, adrenaline, nor-adrenaline, endothelin-1, histamine, eosinophilic cationic protein, serum angio-tensin-converting enzyme (ACE) activity, total immunoglobulin E (IgE), urea and electrolytes, indicators of the severity of the attack, atopic status, blood pressure and renin or angiotensin II levels. We conclude that although a subpopulation of asthmatics appear to have raised renin and angiotensin II during attacks of acute, severe asthma, the mechanism of activation of the renin-angiotensin system remains unclear.

Hepatitis virus infection and liver disease in injecting drug users who died suddenly.

AIM: To determine the extent of liver damage resulting from infection with hepatitis B, C and D viruses (HBV, HCV and HDV) in intravenous drug users (IDUs). METHODS: Liver sections taken at necropsy performed to investigate the cause of sudden death in 48 IDUs were scored for necroinflammatory activity and fibrosis. Evidence of infection was by detection of viral antibodies in serum, hepatitis B surface antigen (HBsAg) and HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Evidence of HCV infection was present in 43 (90%) of 48 serum samples. Six (12%) HBsAg positive serum samples had markers indicative of chronic HBsAg carriage, including three with antibody directed against HDV. Evidence of past HBV infection was found in 27 (69%) of 39 HBsAg negative serum samples. HIV was detected in one (2%) of 48 samples. In five (10%) of 48 samples there was no evidence of current or past infection with HCV, HBV or HIV. All 43 liver sections from HCV positive IDUs scored > or = 1 for necroinflammatory activity, whereas three IDUs without HCV scored 0. Scores for stage of fibrosis were > or = 1 in 15 (35%) of 43 and zero of five IDUs, respectively. Fibrosis scores of > or = 3 were seen only in three IDUs positive for HBV, HDV and HCV. CONCLUSION: Inflammatory activity in the liver is present in a high proportion of IDUs in Glasgow and is strongly associated with HCV infection. Severe chronic liver damage was limited to HBsAg carriers superinfected with HDV and HCV.

Hepatocyte proliferation and serum hepatocyte growth factor levels in patients with alcoholic hepatitis.

BACKGROUND/METHODS: Hepatocyte growth factor is thought to be important in stimulating growth of the liver following injury. In this study we have measured serum levels of hepatocyte growth factor together with hepatocyte proliferation in liver biopsies, by detection of the Ki-67 antigen, in 23 patients with alcoholic hepatitis. RESULTS: Serum hepatocyte growth factor was elevated in all patients (median 0.9 ng/ml; range 0.6-7.7 ng/ml; normal < 0.5 ng/ml) and there was a positive correlation between hepatocyte growth factor levels and hepatocyte proliferation in the biopsies. CONCLUSIONS: These results demonstrate that in acute alcoholic hepatitis the liver proliferates in response to injury and suggest that hepatocyte growth factor may be one of the growth factors responsible for this proliferative activity.

Hepatocyte growth factor/scatter factor expression and c-met in primary breast cancer.

Hepatocyte growth factor/scatter factor (HGF/SF) is a fibroblast-derived cytokine whose receptor is encoded by c-met. Activation of c-met promotes tumour cell proliferation, dissociation, invasiveness and angiogenesis. Aberrant expression of HGF/SF or c-met may play a role in tumour progression. HGF/SF and c-met were determined in 73 breast cancers (median follow up: 61 months) and 10 samples of tumour-free breast tissue. HGF/SF was detected at significantly higher concentrations in breast cancers (median 350, range 58-1604 ng per 100 mg total protein) when compared with normal breast tissue (median 108, range 66-213 ng per 100 mg total protein) (P < 0.001). C-met was detected in all 10 samples of tumour-free breast tissue and in 26 breast cancers. HGF/SF concentrations correlated with disease relapse (P < 0.001) and reduced overall survival (P < 0.001). Tumours with detectable c-met correlated significantly with disease-relapse (P = 0.012). Multivariate analysis demonstrated a significant interaction between HGF/SF and c-met in relation to disease-relapse (P = 0.014). These results suggest a biological interaction involving HGF/SF and c-met in promoting tumour progression in breast cancer.

Antimycobacterial hsp65 and rheumatoid factor titres in a population of normal twins: evidence of genetic control of rheumatoid factor.

Rheumatoid arthritis (RA) is an autoimmune disease and rheumatoid factor (RF), anti-IgG, has been implicated in the pathogenesis, but the exact etiology remains unclear. There are data to suggest and infectious trigger to the autoimmune process, and mycobacteria are considered a candidate. Immunization of various animals with mycobacterial heat shock protein 65 (mhsp65) protects against subsequent autoimmune arthritis in a number of experimental models. Elevated anti-mhsp65 titres have been demonstrated in RA patients, together with specific T cells isolated from inflamed synovium. Mycobacterial hsp65 has also been implicated in other autoimmune disease and in atherosclerosis. The anti-mhsp65 and RF (IgG, IgM and IgA isotypes) titres were assayed by ELISA in 123 pairs of normal twins (61 monozygotic and 62 dizygotic, age 14-79 years), to examine the population distribution and inter-relationship of these antibodies. In addition, we studied the effects of age, sex, genetics and environment on antibody titres. IgG-RF and IgM-RF were detectable in all subjects and IgA-RF in 41 subjects. None of the RF isotypes showed any significant dependence on age or sex. There was a statistically significant correlation between twins for the IgG-RF and IgM-RF, and a positive but not significant correlation for the IgA-RF. All three correlations were stronger for monozygotic than dizygotic twins, reaching statistical significance for IgM-RF (P < 0.001), and this indicates that there is a genetic influence on RF titres. Anti-mhsp65 titres were detectable in 90.5% of the study group with a range of 0.15-19.7 AU/ml. There were weak correlations between twins, stronger for dizygotic than monozygotic twins. This suggests that familial influences on anti-mhsp65 titres are very small, with no evidence of any genetic influence at all. There was no significant relationship of anti-mhsp65 titre with age, sex or RF titres.

The IgE and IgG antibody responses to aerosols of Nephrops norvegicus (prawn) antigens: the association with clinical hypersensitivity and with cigarette smoking.

Raised levels of serum IgE antibodies to prawn antigens were found in 15 of 26 seafood factory process workers with respiratory symptoms and in one of 26 case-matched asymptomatic controls (P < 0.001). Raised IgG antibody titres against the same antigens were found in 18 subjects in each symptom grouping, and the median titres of this antibody did not differ significantly between the groups. The prawn-specific IgE antibody response was significantly associated with atopy (IgE antibody response to common allergens) and with a history of cigarette smoking, confirmed by level of serum cotinine, a major nicotine metabolite. Non-atopic non-smokers were unlikely to become sensitized. The titre of the prawn-specific IgE antibody correlated with the duration of exposure and with the duration of symptoms. Discriminant analysis of the serological profile (anti-prawn IgE, total IgE and cotinine) was sufficient to assign individuals correctly into symptomatic or asymptomatic categories in 77% of subjects. The titres of the IgE and IgG antibody responses to prawn antigens did not correlate, and the main factor which seemed to determine the antibody isotype response to these inhaled antigens was cigarette smoking. IgE antibody was produced mainly by smokers, whereas IgG antibody was the predominant isotype produced by non-smokers.

Autoantibody associations with MHC class II antigens in scleroderma and autoimmune vasculitis.

HLA-DR haplotypes in patients with scleroderma and vasculitis were compared with those in healthy controls from the Scottish population to investigate whether any associations exist between MHC antigens and development of specific autoantibodies. In patients with systemic vasculitis the presence of any antibodies against neutrophil cytoplasmic antigens (ANCA) was associated with an increased frequency of DR8 [p < 0.004], and no patients expressed the DR5 antigen. However, no significant differences were observed when these patients were subdivided into those with anti-myeloperoxidase (MPO) antibodies or anti-proteinase-3 (PR3) antibodies. Scleroderma patients as a whole showed a lower frequency of DR7 than controls [5.1% cf 28% in control population, p < 0.002]. Following subdivision by autoantibody profile, patients with circulating anti-centromere antibody (ACA) showed an increased frequency of DR1 compared to the control population [p < 0.001]. No scleroderma patient without ACA expressed this haplotype. Associations between MHC and some autoantibodies suggest that antigen presentation could lead to their production.

Detection of lymphocyte Fc gamma receptor-blocking factors by the EA rosette inhibition assay. Refinement of the conventional method and development of a novel flow-cytometric assay.

Serum factors which interact with human peripheral blood lymphocyte Fc gamma receptors (Fc gamma Rs) may be detected in vitro by the EA rosette inhibition assay (EARIA). This assay has been used to detect circulating immune complexes and certain alloantibodies directed against cell surface antigens situated in close proximity to Fc gamma Rs. Three main types of FcR-blocking factor have been demonstrated by the EARIA in human serum following exposure to alloantigens. A strong correlation was observed between the presence of one of these FcR-blocking factors (FcBF1) and human renal allograft survival. This factor was previously shown to bind preferentially to CD32+ B cells and to inhibit antibody synthesis. In this study we have shown that detection of FcBF1 by the EARIA depends on the type of erythrocyte and on the amount of antibody used to sensitise the erythrocytes. Furthermore, we have developed a flow-cytometric version of the EARIA which is rapid, reproducible and, most importantly, objective. Inter-laboratory comparisons using this standardised EARIA should now be possible.

Differential binding of acapsulate and encapsulated strains of Cryptococcus neoformans to human neutrophils.

Investigations into mechanisms of binding of encapsulated and acapsulate strains of Cryptococcus neoformans by human neutrophils were performed, using a monolayer assay. The two strains bound to neutrophils by different mechanisms although both had an absolute requirement for opsonization with complement components in normal human serum for binding to occur. Neutrophil binding of encapsulated yeasts required conformational changes in actin yet did not appear to lead to phagocytosis of the organism. A maximum of 12 acapsulate cells bound per neutrophil compared with only four encapsulated yeasts. Cytochalasin D treatment reduced the maximum numbers able to bind per neutrophil by 50%. The encapsulated cryptococci appeared to compete with each other for binding to neutrophils whereas the acapsulate yeast cells bound to neutrophils in an approximately Poisson distribution, suggesting independent binding. Binding of acapsulate cryptococci did not require actin filaments and appeared to trigger phagocytosis. Thus, the capsule of C. neoformans appeared to inhibit binding and internalization by neutrophils.

The relative roles of C4A and C4B in prevention of immune precipitation, solubilisation and immune adherence.

C4A and C4B levels were measured in serum from 246 normal individuals. Complement-mediated solubilisation, assayed using alkaline phosphatase anti-alkaline phosphatase immune complexes (IC), correlated with both C4A and C4B levels. However, C4A and C4B levels showed no correlation with solubilisation of bovine serum albumin (BSA) ICs, or with the prevention of immune precipitation of BSA or alkaline phosphatase ICs, nor with immune adherence assayed using thyroglobulin and BSA ICs.

Induction of hypoferremia and modulation of macrophage iron metabolism by tumor necrosis factor.

The effects of recombinant tumor necrosis factor (TNF), tumor necrosis serum (TNS), recombinant interleukin 1 (IL-1), and prostaglandin E2 on serum iron parameters and iron handling by macrophages in mice have been investigated. Recombinant TNF caused a significant decrease in serum iron levels after 6 hours, but none of the mediators caused significant changes in total iron binding capacity at this time, although TNS caused a significant increase in total iron binding capacity after 24 hours. Peritoneal macrophages taken from mice 6 hours after inoculation of the mediators were pulsed with 59Fe, 125I-transferrin-antitransferrin immune complexes, and subsequent degradation of the complexes and release of iron were investigated. Both recombinant TNF and TNS caused significant increases in uptake and degradation of the complexes, but with recombinant TNF this was not accompanied by a corresponding increase in iron release. IL-1 and prostaglandin E2 also caused increased degradation of the immune complexes, but uptake of the complexes and iron release were unaffected. When peritoneal macrophages from normal mice were treated with the mediators in vitro and then pulsed with labeled immune complexes, recombinant TNF caused a significant decrease in iron release, but none of the other mediators had any effect. None of the mediators affected uptake or degradation of the immune complexes. These results suggest that TNF, rather than IL-1, mediates the hypoferremia of inflammatory disease and that alterations in the ability of macrophages to handle iron may be responsible.