Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30-mum-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.

The cell biology of atherosclerosis - new developments

In the development of atherosclerotic lesions, three basic processes occur: 1) invasion of the artery wall by leucocytes, particularly monocytes and T-lymphocytes; 2) smooth muscle phenotypic modulation, proliferation, and synthesis of extracellular matrix; and 3) intracellular (macrophage and smooth muscle) lipoprotein uptake and lipid accumulation. Invasion of the vessel wall by leucocytes is mediated through the expression of adhesion molecules on both leucocytes and the endothelium making them 'sticky'. The adhesion molecules are induced by high serum cholesterol levels or complement fragments. Leucocytes which have adhered to the endothelium are chemo-attracted into the vessel wall by cytokines produced by early arriving leucocytes or by low density lipoprotein which has passively passed into the wall, in the process being trapped and oxidised. The oxidised low density lipoprotein is taken up by scavenger receptors (which are not subject to down-regulation) on both macrophages and smooth muscle cells. The overaccumulation of lipid is toxic to the cells and they die contributing to the central necrotic core. The macrophages and T-lymphocytes produce substances which induce smooth muscle cells of the artery wall to change from a 'contractile' (high volume fraction of myofilaments [V(v)myo]) to a 'synthetic' (low V(v)myo) phenotype. In this altered state they respond to growth factors released from macrophages, platelets, regenerating endothelial cells and smooth muscle cells; produce large amounts of matrix; express lipoprotein scavenger receptors; express adhesion molecules for leucocytes; and express HLA-DR following exposure to the T-lymphocyte product, IFN-delta, suggesting that they can become involved in a generalised immune reaction.

Repeated three-dimensional magnetic resonance imaging of atherosclerosis development in innominate arteries of low-density lipoprotein receptor-knockout mice

Background-In vivo methods to evaluate the size and composition of atherosclerotic lesions in animal models of atherosclerosis would assist in the testing of antiatherosclerotic drugs. We have developed an MRI method of detecting atherosclerotic plaque in the major vessels at the base of the heart in low-density lipoprotein (LDL) receptor-knockout (LDLR-/-) mice on a high-fat diet. Methods and Results-Three-dimensional fast spin-echo magnetic resonance images were acquired at 7 T by use of cardiac and respiratory triggering, with approximate to140-mum isotropic resolution, over 30 minutes. Comparison of normal and fat-suppressed images from female LDLR-/- mice I week before and 8 and 12 weeks after the transfer to a high-fat diet allowed visualization and quantification of plaque development in the innominate artery in vivo. Plaque mean cross-sectional area was significantly greater at week 12 in the LDLR-/- mice (0.14+/-0.086 mm(2) [mean+/-SD]) than in wild-type control mice on a normal diet (0.017+/-0.031 mm(2), p

Leukaemia inhibitory factor retards the progression of atherosclerosis

Objective: Our previous studies showed that the pleiotropic cytokine leukaemia inhibitory factor (LIF) inhibits the de novo formation of experimental atherosclerotic lesions. The present study examined whether LIF also inhibits progression of pre-existing atheroma. Methods: Balloon angioplasty was performed on the right carotid arteries of 18 rabbits immediately before placing animals on a cholesterol-enriched diet. After 4 weeks, at which time the intima:media ratio (IN) was 0.99+/-0.12 (n=6), osmotic minipumps containing LIF (n=6) or saline control n=6) were inserted into the peritoneal cavity of each of the remaining rabbits for a further 4 weeks. Arteries were then harvested for analysis. Results: Continuous administration of LIF for the final 4 weeks of an 8-week cholesterol-enriched diet completely inhibited lesion progression in injured carotid arteries (I:M 1.05+/-0.16) compared with the saline-treated group at 8 weeks (1.62+/-0.13; P

A case in variant in cow's milk is atherogenic

Casein is a major protein in cow's milk that occurs in several variant forms, two of which are beta-casein A(1) and beta-casein A(2). The levels of these two proteins vary considerably in milk dependent on the breed of cow, and epidemiology studies suggest that there is a relationship between their consumption and the degree of atherosclerosis. In the present study, the direct effect of consumption of beta-casein A(1) vs beta-casein A(2) on atherosclerosis development was examined in a rabbit model. Sixty rabbits had their right carotid artery balloon de-endothelialised at t = 0, divided randomly into 10 groups (n = 6 per group), then for 6 weeks fed a diet containing 0, 5, 10 or 20% casein isolate, either beta-casein variant A(1) or A(2) made up to 20% milk protein with whey. Some groups had their diets supplemented with 0.5% cholesterol. Blood samples were collected at t = 0, 3 and 6 weeks and rabbits were sacrificed at t = 6 weeks. In the absence of dietary cholesterol, beta-casein A(1) produced significantly higher (P < 0.05) serum cholesterol, LDL, HDL and triglyceride levels than whey diet alone, which in turn produced higher levels than beta-casein A(2). Rabbits fed beta-casein A(1) had a higher percent surface area of aorta covered by fatty streaks than those fed beta-casein A(2) (5.2+/-0.81 vs 1.1+/-0.39, P < 0.05) and the thickness of the fatty streak lesions in the aortic arch was significantly higher (0.04+/-0.010 vs 0.00, P < 0.05). Similarly, the intima to media ratio (I:M) of the balloon injured carotid arteries in A(1) fed animals (0.77+/-0.07) was higher than in those that consumed A(2) (0.57+/-0.04) or whey (0.58+/-0.04), but this did not reach significance. In the presence of 0.5% dietary cholesterol, the thickness of the aortic arch lesions was higher (P < 0.05) in 5, 10 and 20% casein A(1) fed animals compared with their A(2) counterparts, while other parameters were not significantly different. It is concluded that beta-casein A(1)is atherogenic compared with beta-casein A(2). (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

T-cell clonality to Porphyromonas gingivalis and human heat shock protein 60s in patients with atherosclerosis and periodontitis

Individuals with periodontitis have been reported to have a significantly increased risk of developing coronary heart disease. Several studies have demonstrated that the immune response to heat shock protein 60 (HSP60) may be involved in the pathogenesis of both atherosclerosis and chronic periodontitis. To investigate this possible link between these diseases, cellular and humoral immune responses to HSP60 in atherosclerosis patients were compared with those in periodontitis patients and healthy subjects using human and Porphyromonas gingivalis HSP60 (GroEL) as antigens. Antibody levels to both human and P. gingivalis HSP60s were the highest in atherosclerosis patients, followed by periodontitis patients and healthy subjects. Clonal analysis of the T cells clearly demonstrated the presence of not only human HSP60- but also P. gingivalis GroEL-reactive T-cell populations in the peripheral circulation of atherosclerosis patients. Furthermore, these HSP60-reactive T cells seemed to be present in atherosclerotic lesions in some patients. These results suggest that T-cell clones with the same specificity may be involved in the pathogenesis of the different diseases.

Multiple genotypes of Chlamydia pneumoniae identified in human carotid plaque

Chlamydia pneumoniae is an obligate intracellular respiratory pathogen that causes 10% of community-acquired pneumonia and has been associated with cardiovascular disease. Both whole-genome sequencing and specific gene typing suggest that there is relatively little genetic variation in human isolates of C. pneumoniae. To date, there has been little genomic analysis of strains from human cardiovascular sites. The genotypes of C. pneumoniae present in human atherosclerotic carotid plaque were analysed and several polymorphisms in the variable domain 4 (VD4) region of the outer-membrane protein-A (ompA) gene and the intergenic region between the ygeD and uridine kinase (ygeD-urk) genes were found. While one genotype was identified that was the same as one reported previously in humans (respiratory and cardiovascular), another genotype was found that was identical to a genotype from non-human sources (frog/koala).

Hemispheric contributions to lexical ambiguity resolution: Evidence from individuals with complex language impairment following left-hemisphere lesions

Nine individuals with complex language deficits following left-hemisphere cortical lesions and a matched control group (n 5 9) performed speeded lexical decisions on the third word of auditory word triplets containing a lexical ambiguity. The critical conditions were concordant (e.g., coin–bank–money), discordant (e.g., river–bank–money), neutral (e.g., day–bank– money), and unrelated (e.g., river–day–money). Triplets were presented with an interstimulus interval (ISI) of 100 and 1250 ms. Overall, the left-hemisphere-damaged subjects appeared able to exhaustively access meanings for lexical ambiguities rapidly, but were unable to reduce the level of activation for contextually inappropriate meanings at both short and long ISIs, unlike control subjects. These findings are consistent with a disruption of the proposed role of the left hemisphere in selecting and suppressing meanings via contextual integration and a sparing of the right-hemisphere mechanisms responsible for maintaining alternative meanings.

Discourse priming of homophones in individuals with dominant subcortical lesions, cortical lesions, and Parkinson’s disease

An on-line priming experiment was used to investigate discourse-level processing in four matched groups of subjects: individuals with nonthalamic subcortical lesions (NSL) ( n =10), normal control subjects ( n =10), subjects with Parkinsons disease (PD) ( n =10), and subjects with cortical lesions ( n =10). Subjects listened to paragraphs that ended in lexical ambiguities, and then made speeded lexical decisions on visual letter strings that were: nonwords, matched control words, contextually appropriate associates of the lexical ambiguity, contextually inappropriate associates of the ambiguity, and inferences (representing information which could be drawn from the paragraphs but was not explicitly stated). Targets were presented at an interstimulus interval (ISI) of 0 or 1000ms. NSL and PD subjects demonstrated priming for appropriate and inappropriate associates at the short ISI, similar to control subjects and cortical lesion subjects, but were unable to demonstrate selective priming of the appropriate associate and inference words at the long ISI. These results imply intact automatic lexical processing and a breakdown in discourse-based meaning selection and inference development via attentional/strategic mechanisms.

Lesions caused by cardiovascular flukes (Digenea : Spirorchidae) in stranded green turtles (Chelonia mydas)

Evidence of infection with spirorchid flukes (Digenea: Spirorchidae) was sought at necropsy of 96 stranded green turtles, Chelonia mydas, that were examined during the course of a survey of marine turtle mortality in southeastern Queensland, Australia. Three species of spirorchid (Hapalotrema mehrai, H. postorchis, and Neospirorchis schistosomatoides) were identified. Severe disease due to spirorchid fluke infection (spirorchidiasis) was implicated as the principal cause of mortality in 10 turtles (10%), and appeared to be one of multiple severe problems in an additional 29 turtles (30%). Although flukes were observed in only 45% of stranded C. mydas in this study, presumed spirorchid fluke infection was diagnosed in an additional 53% of turtles, based principally on characteristic necropsy lesions and to a lesser extent on the histopathological detection of spirorchid eggs. Characteristic necropsy lesions included miliary spirorchid egg granulomas, which were observed most readily on serosal surfaces, particularly of the small intestine. Cardiovascular lesions included mural endocarditis, arteritis, and thrombosis, frequently accompanied by aneurysm formation. Resolution of thrombi was observed to occur via a combination of granuloma formation about indigestible components (spirorchid fluke egg shells) and exteriorization through the vessel wall, which resulted in granulomatous nodules on the adventitial surface. Septic aortic thrombosis complicated by disseminated bacterial infection, observed in five turtles, was recorded for the first time. Egg granulomas were ubiquitous in turtle tissues throughout this study. Although they generally appeared to be mild or incidental lesions, they were occasionally associated with severe multifocal granulomatous pneumonia or meningitis.

Both CD4(+) and CD8(+) lymphocytes reduce the severity of tissue lesions in murine systemic candidiasis, and CD4(+) cells also demonstrate strain-specific immunopathological effects

The role of T lymphocytes in host responses to sublethal systemic infection with Candida albicans was evaluated by mAb depletion of CD4(+) and CD8(+) cells from BALB/c and CBA/CaH mice, which develop mild and severe tissue damage, respectively. Depletion of CD4(+) lymphocytes from BALB/c mice markedly increased tissue damage, but did not alter the course of infection. In CBA/CaH mice, depletion of CD4+ cells abrogated tissue destruction in both brain and kidney at day 4 after infection, and significantly decreased fungal colonization in the brain. However, the severity of tissue lesions increased relative to controls from day 8 onwards. A small increase in tissue damage was evident in both mouse strains after depletion of CD8(+) cells. There were no major differences between days 4 end 8 after infection in cDNA cytokine profiles of CD4(+) lymphocytes from either BALB/c or CBA/CaH mice. After passive transfer into infected syngeneic recipients, spleen cells from infected CBA/CaH mice markedly increased tissue damage when compared to controls, and also caused a significant increase in fungal colonization in the brain. A similar transfer in BALB/c mice increased the number of inflammatory cells in and around the lesions, but had no effect on the fungal burden in brain and kidney. The data demonstrate that both CD4(+) and CD8(+) lymphocytes contribute to the reduction of tissue damage after systemic infection with C. albicans, and that the development and expression of CD4(+) lymphocyte effector function is influenced by the genetic background of the mouse.

Effects of restricted cochlear lesions in adult cats on the frequency organization of the inferior colliculus

Restricted cochlear lesions in adult animals result in plastic changes in the representation of the lesioned cochlea, and thus in the frequency map, in the contralateral auditory cortex and thalamus. To examine the contribution of subthalamic changes to this reorganization, the effects of unilateral mechanical cochlear lesions on the frequency organization of the central nucleus of the inferior colliculus (ICC) were examined in adult cats. Lesions typically resulted in a broad high-frequency hearing loss extending from a frequency in the range 15-22 kHz. After recovery periods of 2.5-18 months, the frequency organization of ICC contralateral to the lesioned cochlea was determined separately for the onset and late components of multiunit responses to tone-burst stimuli. For the late response component in all but one penetration through the ICC, and for the onset response component in more than half of the penetrations, changes in frequency organization in the lesion projection zone were explicable as the residue of prelesion responses. In half of the penetrations exhibiting nonresidue type changes in onset-response frequency organization, the changes appeared to reflect the unmasking of normally inhibited inputs. In the other half it was unclear whether the changes reflected unmasking or a dynamic process of reorganization. Thus, most of the observed changes were explicable as passive consequences of the lesion, and there was limited evidence for plasticity in the ICC. The implications of the data with respect to the primary locus of the changes and to the manner in which they contribute to thalamocortical reorganization are considered. (C) 2003 Wiley-Liss, Inc.

The assessment of oral cytology utilizing ploidy analysis and virtual microscopy for the early detection of epithelial dysplasia and neoplasia in oral mucosal lesions

Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality which is due, at least in part, to late detection. Precancerous and cancerous oral lesions may mimic any number of benign oral lesions, and as such may be left without investigation and treatment until they are well advanced. Over the past several years there has been renewed interest in oral cytology as an adjuvant clinical tool in the investigation of oral mucosal lesions. The purpose of the present study was to compare the usefulness of ploidy analysis after Feulgen stained cytological thin-prep specimens with traditional incisional biopsy and routine histopathological examination for the assessment of the pre-malignant potential of oral mucosal lesions. An analysis of the cytological specimens was undertaken with virtual microscopy which allowed for rapid and thorough analysis of the complete cytological specimen. 100 healthy individuals between 30 and 70 years of age, who were non-smokers, non-drinkers and not taking any medication, had cytological specimens collected from both the buccal mucosa and lateral margin of tongue to establish normal cytology parameters within a control population. Patients with a presumptive clinical diagnosis of lichen planus, leukoplakia or OSCC had lesional cytological samples taken prior to their diagnostic biopsy. Standardised thin preparations were prepared and each specimen stained by both Feuglen and Papanicolau methods. High speed scanning of the complete slide at 40X magnification was undertaken using the Aperio Scanscope TM and the green channel of the resultant image was analysed after threshold segmentation to isolate only nuclei and the integrated optical density of each nucleus taken as a gross measure of the DNA content (ploidy). Preliminary results reveal that ploidy assessment of oral cytology holds great promise as an adjunctive prognostic factor in the analysis of the malignant potential of oral mucosal lesions.

The usefulness of acetic acid wash and chemiluminescent illumination (Vizilite) in the visualization of oral mucosal white lesions

A diligent and careful examination of the mouth and oral structures has been historically deficient in revealing premalignant and malignant oral lesions. Conventional screening practice for oral neoplastic lesions involves visual scrutiny of the oral tissues with the naked eye under projected incandescent or halogen illumination. Visualization is the principal strategy used to find patients with lesions at risk for malignant transformation; hence, any procedure which highlights neoplastic lesions should aid the clinician. This pilot study examined the usefulness of acetic acid wash and chemiluminescent light (Vizilite) in enhancing visualization of oral mucosal white lesions, and its ability to highlight malignant and potentially malignant lesions. Fifty five patients referred for assessment of a white lesion, were prospectively screened with Vizilite, and an incisional biopsy performed for a definitive diagnosis. The age, sex, and smoking status of all patients were recorded, and all lesions were photographed. The visibility, location, size, border, and presence of satellite lesions, were also recorded. The Vizilite tool enhanced intraoral visualization of 26 white lesions, but it could not distinguish between epithelial hyperplasia, dysplasia, or carcinoma. Indeed, all lesions appeared ‘‘aceto-white’’, regardless of the definitive diagnosis. On one occasion, Vizilite aided in the identification of a satellite lesion that was not observed by routine visual inspection. Vizilite appears to be a useful visualization tool, but it does not aid in the identification of malignant and potentially malignant lesions of the oral mucosa.