Detection of Chlamydia pneumoniae in atherosclerotic tissue: a comparative study of PCR and immunocytochemistry.

The reported prevalence of Chlamydia pneumoniae in atherosclerotic tissue appears to depend on the detection system used. This introduces problems in determining the role of C. pneumoniae in atherosclerosis. This study analyses the sensitivity and performance of molecular diagnostic methods for the detection of C. pneumoniae and polymerase chain reaction (PCR) inhibitors in atheromatous tissue. Atherosclerotic tissue taken from 30 coronary endarterectomies, nine coronary arteries from explanted hearts, 16 carotid and two femoral endarterectomies are studied. Nested PCR (nPCR) assays targeting the PstI restriction fragment, the OmpA gene and the CRP operon of the chlamydial genome and immunocytochemistry (ICC) are used. Internal controls (IC) are constructed to co-amplify with the specific amplicons and identify the presence of inhibitor. The OmpA, PstI and CRP operon PCR assays had similar analytical sensitivities. However, the OmpA PCR was most affected by PCR inhibitors. Despite this, eight samples (14%) tested positive in the OmpA nPCR and no positives were found using the PstI or CRP operon nPCRs. Primary isolates of C. pneumoniae obtained from 12 patients with acute respiratory infection were positive in all three assays. Of the 48 specimens available for ICC, 33 (69%) were positive for chlamydial antigens. These included samples found positive by PCR. Dilution of samples to eliminate PCR inhibitors may have contributed to the discordant ICC and PCR results. The OmpA PCR, when used with an IC to identify samples with PCR inhibitors, is a reliable tool. However, the sensitivity of the ICC methods justifies their continued use.

Absence of viral nucleic acids in early and late dilated cardiomyopathy.

OBJECTIVE: To investigate whether viral infection acts as a trigger factor for the development of dilated cardiomyopathy in genetically predisposed individuals with a family history of disease. SETTING: Patients attending the cardiomyopathy unit in a cardiac tertiary referral centre. DESIGN: Nested polymerase chain reaction (nPCR) was used to determine whether enteroviral, adenoviral, or cytomegaloviral nucleic acids were detectable in the myocardium of 19 asymptomatic relatives of patients with dilated cardiomyopathy; all these relatives had echocardiographic abnormalities thought to represent early disease. Explanted hearts from patients with end stage dilated cardiomyopathy were also studied and were compared with 25 controls (ischaemic heart disease (21), valvar heart disease (2), hypertrophic cardiomyopathy (1), restrictive cardiomyopathy (1)). Myocardial tissue from two fatal cases of culture positive coxsackie myocarditis was used as a positive control. RESULTS: No viral nucleic acid was detected in any group other than in those with myocarditis. Spiking of random wells with purified recombinant viral nucleic acids confirmed the sensitivity and reproducibility of the assays. CONCLUSIONS: Myocardial viral infection is not detectable in relatives of patients with dilated cardiomyopathy who are suspected of having early disease. There is no evidence that viruses act as a trigger factor for initiating the dilated cardiomyopathy in these patients.

Haemochromatosis gene mutations in idiopathic dilated cardiomyopathy.

BACKGROUND: Two common mutations of the haemochromatosis associated gene (HFE) (cys282tyr (C282Y) and his63asp (H63D)) have been implicated in haemochromatosis and as modulators in cardiovascular disease. OBJECTIVE: To investigate the role of these mutations in the pathogenesis of idiopathic dilated cardiomyopathy. DESIGN AND SETTING: Case-control and prospective cohort study of patients attending a cardiomyopathy unit in a tertiary referral cardiac centre. METHODS: 207 unrelated white patients with dilated cardiomyopathy, followed up for 259 patient years, and 200 controls were tested for HFE C282Y and H63D mutations by polymerase chain reaction and restriction digestion. RESULTS: 31/207 patients (15%) v 24/200 controls (12%) carried C282Y (adjusted odds ratio (OR) 1.2 (95% confidence interval 0.7 to 2.2)), 74/207 (36%) v 53/200 (27%) carried H63D (OR 1.6 (1.1 to 2.5)), and 10/207 (4.8%) v 4/200 (2%) were compound heterozygotes (OR 2.6 (0.8 to 8.5)). Four patients and six controls were H63D homozygous and one was C282Y homozygous. There was a progressive increase in mean serum iron ([Fe]) and transferrin saturations from patients with no mutation ([Fe] = 16.3 micromol/l, transferrin saturation = 23.7%) through H63D heterozygotes (17.5 micromol/l, 25.8%), C282Y heterozygotes (17.1 micromol/l, 26.6%), H63D homozygotes (20.0 micromol/l, 33.5%), compound heterozygotes (26.8 micromol/l, 41.7%), and C282Y homozygotes (34 micromol/l, 71%). At follow up (median 90 months) the rate of death or cardiac transplantation was 52/207 (25%). C282Y heterozygotes had less ventricular dilatation (mean (SD): 59.9 (1.7) mm v 64.9 (0.9) mm, p < 0.05), better fractional shortening (24 (1. 7)% v 18.8 (1.4)%, p < 0.01), and a trend towards improved survival without transplantation. [Fe] and transferrin saturation did not correlate with disease severity and were not associated with reduced survival. CONCLUSIONS: The frequency of the H63D mutation is significantly increased in patients with idiopathic dilated cardiomyopathy. As H63D has a relatively minor effect on iron status, the mechanism of this association may be unrelated to iron metabolism.

Immunological analysis of the tegument phosphoprotein ppUL83 of human cytomegalovirus.

Immunological properties of the tegument phosphoprotein, ppUL83, of human cytomegalovirus (HCMV), expressed using a replication deficient recombinant adenovirus vector (RAd83) are described. The initial characterisation of this protein was carried out by immunofluorescence (IF), immunoprecipitation (RIP) and immunoblotting using nine mouse monoclonal antibodies (Mabs) directed against five linear and four conformational epitopes of ppUL83. The reactivity of the recombinant protein with the Mabs was similar to that observed with native ppUL83, although, the kinetics of its expression was in agreement with expression derived from the HCMV major immediate early promoter (MIEP). The recombinant antigen was used successfully in an Enzyme Immunoassay (EIA) for the detection of IgG class antibodies in 171 sequential sera taken from 21 heart transplant recipients. Comparison of HCMV-infected and RAd83-infected cell extracts in this experiment showed that recombinant antigen could substitute whole virus extracts as a single well-characterised protein in EIA. Serum IgG avidity measurements, using the recombinant ppUL83, differentiated between primary and past HCMV infections in the population studied.

Identification of three HLA-A*0201-restricted cytotoxic T cell epitopes in the cytomegalovirus protein pp65 that are conserved between eight strains of the virus.

The Ag specificity of the CTL response against CMV is directed almost entirely to a single CMV tegument protein, the phosphoprotein pp65. We report the identification of three peptides derived from the protein pp65 that displayed a high or intermediate binding to HLA-A*0201 molecules, which were also able to induce an in vitro CTL response in peripheral blood lymphocytes from CMV seropositive individuals. The peptide-specific CTLs generated were capable of recognizing the naturally processed pp65 either presented by CMV-infected cells or by cells infected with an adenovirus construct expressing pp65 in an HLA-A*0201-restricted manner. Thus, we were able to demonstrate responses to subdominant CTL epitopes in CMV-pp65 that were not detected in polyclonal cultures obtained by conventional stimulations. We also found that the amino acid sequences of the three peptides identified as HLA-A*0201-restricted CTL epitopes were conserved among different wild-type strains of CMV obtained from renal transplant patients, an AIDS patient, and a congenitally infected infant, as well as three laboratory strains of the virus (AD169, Towne and Davis). These observations suggest that these pp65 CTL peptide epitopes could potentially be used as synthetic peptide vaccines or for other therapeutic strategies aimed at HLA-A*0201-positive individuals, who represent approximately 40% of the European Caucasoid population. However, strain variation must be taken in consideration when the search for CTL epitopes is extended to other HLA class I alleles, because these mutations may span potential CTL epitopes for other HLA molecules, as it is described in this study.

Epitope mapping of mouse monoclonal antibodies to the ppUL83 lower matrix phosphoprotein of human cytomegalovirus.

Of nine mouse monoclonal antibodies (MAbs) directed against the lower matrix protein (pp65; ppUL83) of human cytomegalovirus (HCMV), all immunoprecipitated the 65-kDa protein. Only five were reactive by Western blotting, however, and four of these mapped to linear antigenic epitopes located between amino acids 184-195 (MAb C6), 343-357 (MAb C11), 448-462 (MAb C5), and 448-459 (MAb C13). The epitope specificity of the fifth antibody (MAb C3) and the four that recognised nonlinear sites could not be determined. Competition binding studies using HCMV antigen extracted from productively infected human embryonic lung fibroblasts (HELF), in an enzyme immunoassay (EIA), showed that three of the antibodies reactive with linear epitopes and two of those reactive with conformational epitopes (MAbs C3, C6, C11, C14, and C18), were unique in their binding specificities. MAb C4 competed with MAb C8 and MAb C5 competed with MAb C13 for binding to ppUL83. One of the linear epitopes identified, corresponding to amino acids SAFVFPTKDVAL (MAb C6), was an epitope described previously for CD8+ cytotoxic T lymphocytes.