Matrix metalloproteinase-9 expression is associated with the presence of Chlamydia pneumoniae in human coronary atherosclerotic plaques.

OBJECTIVE: To investigate the association between Chlamydia pneumoniae and matrix metalloproteinase-9 (MMP-9) in atherosclerotic plaques. DESIGN: 31 coronary atherosclerotic plaque specimens were studied by immunohistochemistry, polymerase chain reaction (PCR), and reverse transcription PCR for the presence of C pneumoniae antigen and genomic DNA, and of MMP-9 protein and transcripts. RESULTS: Immunohistochemical analysis identified a strong association between the presence of C pneumoniae antigen and production of MMP-9 in coronary atherosclerotic plaques (p = 0.001). Furthermore, analysis of the intralesional amount of C pneumoniae and MMP-9 indicated an increased number of cells positive for MMP-9 in arterial sections that had increased C pneumoniae positivity (p < 0.05). CONCLUSIONS: This study provides evidence of an association between expression of MMP-9 and the intravascular presence of C pneumoniae and may suggest a potential pathological mechanism whereby C pneumoniae may contribute to the progression of coronary atherosclerosis.

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.

Hypertrophic cardiomyopathy: histopathological features of sudden death in cardiac troponin T disease.

BACKGROUND: Patients with hypertrophic cardiomyopathy (HCM) are at increased risk of premature death; this is particularly apparent for patients with mutations of the troponin T gene. Myocyte disarray and interstitial fibrosis, pathological features of HCM, may be determinants in these deaths. The relation between genotype, pathological phenotype, and mode of death has not been explored. METHODS AND RESULTS: Seventy-five hearts with HCM were examined. DNA was available in 50 for screening of the troponin T gene. The macroscopic findings, percentage of disarray, percentage of fibrosis, and percentage of small-vessel disease were correlated with the genotype. A troponin T mutation was identified in 9 of the 50 patients, 8 of whom died suddenly. Patients with a troponin T mutation were younger (mean age, 21.0 years [range, 6 to 37] versus 39.1 years [range, 14 to 72]; P<0.0001), had more sudden death (P=0.02), and had lower heart weights, less fibrosis, and greater disarray than other HCM patients (mean heart weight, 380.3+/-105.4 versus 585.0+/-245.7 g, P=0.002; mean fibrosis, 0.7+/-0.4% versus 2.6+/-2.8%, P=0.001; mean disarray, 46.2+/-7.2% versus 24.1+/-15.9%, P<0.0001; and mean small-vessel disease, 11.7+/-14.6 versus 14.1+/-8.7, P=0.6, respectively). Similarly, patients with troponin T mutations who died suddenly had lower heart weights and greater disarray than patients who died suddenly with unknown genotype (ie, troponin T mutation excluded) (mean heart weight, 429.8+/-75.4 versus 559.6+/-204.43 g, P=0.04, and mean disarray, 40.1+/-9.4% versus 20.2+/-12.6%, P=0.002, respectively). CONCLUSIONS: Patients with troponin T mutations had severe disarray, with only mild hypertrophy and fibrosis. These patients died suddenly and at an especially early age. We propose that extensive myocyte disarray in the absence of marked hypertrophy is the pathological substrate for sudden death 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.

Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease).

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an autosomal dominant heart muscle disorder that causes arrhythmia, heart failure, and sudden death. Previously we mapped the genetic locus for the triad of autosomal recessive ARVC, palmoplantar keratoderma, and woolly hair (Naxos disease) to chromosome 17q21, in which the gene for plakoglobin is encoded. This protein is a key component of desmosomes and adherens junctions, and is important for the tight adhesion of many cell types, including those in the heart and skin. METHODS: We studied 19 individuals with Naxos disease, as well as unaffected family members and unrelated individuals from the neighbouring Greek islands of Naxos and Milos. Gene sequence was determined by reverse transcriptase PCR from RNA isolated from the skin of an affected individual and mutations in other cases were confirmed by restriction-enzyme analysis. FINDINGS: A homozygous 2 base pair deletion in the plakoglobin gene was identified only in the 19 affected individuals. This deletion caused a frameshift and premature termination of the protein, which was shown by western blot analysis. 29 clinically unaffected family members were heterozygous for the mutation; 20 unrelated individuals from Naxos and 43 autosomal dominant ARVC probands were homozygous for the normal allele. INTERPRETATION: The finding of a deletion in plakoglobin in ARVC suggests that the proteins involved in cell-cell adhesion play an important part in maintaining myocyte integrity, and when junctions are disrupted, cell death, and fibrofatty replacement occur. Therefore, the discovery of a mutation in a protein with functions in maintaining cell junction integrity has important implications for other dominant forms of ARVC, related cardiomyopathies, and other cutaneous diseases.

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.

A new mutation of the cardiac troponin T gene causing familial hypertrophic cardiomyopathy without left ventricular hypertrophy.

AIM: To screen for a mutation of the cardiac troponin T gene in two families where there had been sudden deaths without an increase in left ventricular mass but with myocardial disarray suggesting hypertrophic cardiomyopathy. METHODS: DNA from affected individuals from both families was used to screen the cardiac troponin T gene on an exon by exon basis. Mutation screening was achieved by polymerase chain reaction and direct sequencing. Where appropriate, a mutation was confirmed by restriction digest. RESULTS: A novel missense mutation of exon 9 was found in the affected individuals of one of the families. This mutation at amino acid 94 resulted in the substitution of arginine for leucine and was not found in 100 normal control samples. A mutation of the cardiac troponin T gene was excluded in the second family. CONCLUSIONS: A mutation of the gene for the sarcomeric protein cardiac troponin T can cause familial hypertrophic cardiomyopathy with marked myocyte disarray and frequent premature sudden death in the absence of myocardial hypertrophy at clinical or macroscopic level.

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.

Meta-analysis of the association of enteroviruses with human heart disease.

The role of viruses in the genesis of both dilated cardiomyopathy (DCM) and acute myocarditis remains uncertain. Modern molecular techniques such as polymerase chain reaction (PCR) and in situ hybridisation are sensitive means of detecting viral genomic material in human myocardial tissue and may help to resolve the quest. Meta-analysis of the papers in the literature records studies of both acute myocarditis and DCM where molecular techniques were used to demonstrate enteroviruses. This review studies information from the published literature as well as statistical analysis of the cumulative molecular data relating enteroviruses to DCM, and to compare these findings with the information available on the role of enteroviruses in acute myocarditis. Twelve papers reported studies in acute myocarditis, of which 11 found higher percentages of enteroviral RNA positivity in the diseased population, giving an overall odds ratio of 4.4. Seventeen papers reported studies in DCM, with 11 recording higher positivity rates in these patients. Cumulative analysis of these data suggests an overall odds ratio of 3.8. The causative role of enteroviruses in acute myocarditis, particularly in children, is supported by meta-analysis of the available literature. The data on DCM is suggestive of an association but a proportion of the studies are negative.

Antiviral activity of human immunodeficiency virus type 1 protease inhibitors in a single cycle of infection: evidence for a role of protease in the early phase.

The antiviral activities of two substrate-based inhibitors of human immunodeficiency virus type 1 (HIV-1) protease, UK-88,947 and Ro 31-8959, were studied in acute infections. H9 and HeLaCD4-LTR/beta-gal cells were infected either with HIV-1IIIB or a replication-defective virus, HIV-gpt(HXB-2). Both inhibitors were capable of blocking early steps of HIV-1 replication if added to cells prior to infection. Partial inhibition was also obtained by addition of inhibitor at the time of or as late as 15 min after infection. The inhibitors were ineffective if added 30 min postinfection. The inhibitory effects were studied by cDNA analysis with PCR followed by Southern blot hybridization and by infectivity assays allowing quantitation of HIV-1 in a single cycle of replication. When UK-88,947-treated H9 cells were coinfected with HIV-1 and human T-cell leukemia virus type I only the replication of HIV-1 was inhibited, demonstrating viral specificity. Pretreating the infectious virus stocks with the inhibitors also prevented replication, indicating that the inhibitors block the action of the viral protease and not a cellular protease. A panel of primer sets was used to analyze cDNA from cell lysates by PCR amplification at 4 and 18 h postinfection. Four hours after infection, viral specific cDNA was detected with all of the four primer pairs used: R/U5, nef/U3, 5' gag, and long terminal repeat (LTR)/gag. However, after 18 h, only the R/U5 and nef/U3 primer pairs and not the 5' gag or LTR/gag primer pair were able to allow amplification of cDNA. The results suggest a crucial role of HIV-1 protease in the early phase of viral replication. Although it is not clear what early steps are affected by the protease, it is likely that the target is the NC protein, as referred from our previous reports of the in situ cleavage of the nucleocapsid (NC) protein by the viral protease inside lentiviral capsids. The results suggest that it is not the inhibition of initiation and progression of reverse transcription but the stability of full-size unintegrated cDNA which is affected in the presence of protease inhibitors. Alternatively, the cleavage of the NC protein may be required for the proper formation of preintegration complex and/or for its transport to the nucleus.

Growth characteristics of human herpesvirus-6: comparison of antigen production in two cell lines.

In order to optimize viral antigen production, the growth characteristics of human herpesvirus-6 (HHV-6) (strain AJ) were studied in two cell lines: HSB-2 and JJHAN. The cells were infected with different multiplicities of infection (moi) and viral growth was monitored by appearance of cytopathic effect (CPE), total and viable cell count, immunofluorescence test, immunoblotting, and electron microscopy. Although > or = 70% of JJHAN cells showed CPE when infected at high moi, only 5% of the cells contained viral antigens when tested with immunofluorescence. In contrast the percentage of cells showing fluorescence in HSB-2 cells reached > or = 30% when infected at > or = 1:50. More than 10 polypeptides of molecular weight ranging between 31-140 kD appeared in the HSB-2 cultures by immunoblotting while only 3 polypeptides were detected in the JJHAN cultures at high moi. Different stages of virus maturation were seen in the HSB-2 cells by electron microscopy but the replication of the virus in JJHAN cells appeared to be restricted. For the purpose of antigen production the optimal conditions for the AJ strain of HHV-6 were found to be culturing in HSB-2 cells at a concentration of 1:25-1:50 infected to uninfected cells and harvesting after 7 days.

Cross reaction of antibodies to a glycine/alanine repeat sequence of Epstein-Barr virus nuclear antigen-1 with collagen, cytokeratin, and actin.

P62 is a synthetic peptide which corresponds to the glycine/alanine repeat sequence of Epstein-Barr virus nuclear antigen-1. It is the main epitope recognised by anti-rheumatoid arthritis nuclear antigen antibodies. It was shown previously that anti-P62 antibodies were raised fourfold in patients with rheumatoid arthritis compared with controls. To examine the possibility that this increase was due to cross reactive autoantibodies binding to P62, anti-P62 antibodies from serum samples taken from 10 patients with rheumatoid arthritis and five healthy controls were purified by affinity chromatography. Immunoglobulin G anti-P62 antibodies purified from four of 10 serum samples from patients with rheumatoid arthritis also reacted with human epidermal keratin, denatured collagen type II and actin, but not with influenza antigens, as determined by enzyme linked immunosorbent assay (ELISA). Anti-P62 antibodies in serum samples from healthy controls and patients with rheumatoid arthritis reacted with epidermal keratin by immunoblotting. It is suggested that antibodies to the glycine/alanine repeat sequence of Epstein-Barr nuclear antigen-1 recognise homologous epitopes on keratin, actin, and collagen. It is also possible that molecular mimicry between a major epitope on the Epstein-Barr virus and several autoantigens might contribute to the breakdown of tolerance and autoimmunity in patients with rheumatoid arthritis.

HIV-1 proteinase is required for synthesis of pro-viral DNA.

HIV-1 proteinase activity is thought to occur primarily post-integration by cleaving the viral Gag and Gag-Pol polyproteins. Its role in the pre-integration stages of viral replication, however, has not been studied in detail. Here we report that a synthetic peptide analogue, UK-88,947, which is a specific inhibitor of purified HIV-1 proteinase, inhibits the processing of the viral polyproteins in cultures of HIV-1 infected cells and prevents the formation of mature, infectious virions. Analysis of DNA from HIV-1 infected cells treated with UK-88,947 showed that viral DNA synthesis was inhibited when the compound was added to cultures one hour before infection. Similar results were obtained when AZT was used. Neither HIV-1 reverse transcriptase or the replication of FIV are inhibited by UK-88,947.

Congenital and maternal cytomegalovirus infections in a London population.

OBJECTIVE: To determine if women at risk of having babies infected with cytomegalovirus (CMV) can be identified antenatally. DESIGN: Prospective serological and demographic study of pregnant women and virological study of their newborn infants. SETTING: Teaching hospital in London. SUBJECTS: 3315 pregnant women and 2737 of their babies. MAIN OUTCOME MEASURES: Quantitative detection of CMV IgG antibodies; qualitative detection of CMV IgM antibodies; demographic characteristics of mothers; qualitative and quantitative titration of CMV viruria in newborn. RESULTS: Congenital CMV infection was found in nine newborn babies (0.33%) two of whom had symptoms. Serological testing of the nine mothers showed four primary and five recurrent infections; both of the symptomatic children were born in the latter group. Testing for CMV specific IgM antibodies or quantitation of IgG antibodies in early pregnancy sera could not differentiate those women at risk of giving birth to babies infected or damaged by CMV from the rest of the population. Quantitation of viruria confirmed that those babies most at risk of CMV disease have the highest titres of CMV. CONCLUSIONS: (i) Since laboratory tests in pregnant women cannot reliably identify fetuses at risk of disease, screening for asymptomatic maternal infection coupled with termination of pregnancy cannot be recommended. (ii) Since 'immune' women can still give birth to babies affected by CMV, we propose that future CMV vaccines should be used to immunize children with the aim of eradicating CMV infection in preference to selective immunization of sero-susceptible females.