To evaluate the effect of age and other risk factors for atherosclerosis on arterial blood velocity, carotid arteries in 179 healthy individuals ranging from 21 to 102 years old were examined using color Doppler ultrasonography. Velocity in common and internal carotid arteries decreased consecutively from young adults to very elderly people except for peak internal carotid artery velocity. Peak common carotid artery velocity in the elderly (≥ 65 years old) people was inversely associated with age and diastolic blood pressure and directly associated with pulse pressure. Minimum velocity of common carotid artery was inversely correlated with age and diastolic blood pressure in the elderly people. In elderly group, peak internal carotid artery velocity correlated only with serum high-density lipoprotein cholesterol. Minimum internal carotid artery velocity correlated inversely with systolic blood pressure in adults and diastolic blood pressure in elderly people. Blood velocity in the very elderly population approaches the critical level for thrombogenesis.

Purpose To assess the frequency of shedding of herpes simplex virus type 1 (HSV-1) DNA in tears and saliva of asymptomatic individuals. Methods Fifty subjects without signs of ocular herpetic disease participated. Serum samples from all subjects were tested for HSV IgG antibodies by ELISA and for HSV-1 by neutralization assay. HSV-1 DNA copy number and frequency of shedding were determined by real-time PCR analysis of tear and saliva samples collected twice daily for 30 consecutive days. Results Thirty-seven of the 50 subjects (74%) were positive for HSV IgG by ELISA. The percentages of positive eye and mouth swabs were approximately equivalent, 33.5% (941/2806) and 37.5% (1020/2723), respectively. However, the percentage of samples with high HSV-1 genome copy numbers was greater in saliva than in tears, which may have been a result of the sample volume collected. Shedding frequency in tears was nearly the same for males (347/1003; 34.6%) and females (594/1705; 34.8%); in saliva, males had a higher frequency of shedding (457/1009; 45.3% vs. 563/1703; 33.1%, male vs. female, respectively). Overall, 49 of 50 (98%) subjects shed HSV-1 DNA at least once during the course of the 30-day study. Conclusion The percentage of asymptomatic subjects who intermittently shed HSV-1 DNA in tears or saliva was higher than the percentage of subjects with positive ELISA or neutralization antibodies to HSV. Since most HSV transmission occurs during asymptomatic shedding, further knowledge of the prevalence of HSV-1 DNA in tears and saliva is warranted in order to control its spread. Shedding is simple to study, and its suppression might be an efficient way to evaluate new antivirals in humans.

PURPOSE To investigate the corneal virulence of toxin-deficient mutants of Staphylococcus aureus in young and aged mice in a topical inoculation model of keratitis. METHODS Corneas of young and aged A/J mice were scarified and topically inoculated with a log phase S. aureus parent strain (8325-4), an alpha-toxin-deficient mutant (DU1090), or an Agr-defective mutant (ISP546) deficient in production of multiple toxins or with purified alpha-toxin. Slit lamp examination (SLE) and histopathology were performed, and bacterial colony-forming units (CFU) and myeloperoxidase (MPO) activity were determined. RESULTS The infection of young mice with the mutant strains demonstrated significantly lower SLE scores (P < or = 0.0001) and reduced histopathologic changes compared with infections with the parent bacterial strain. Either mutant strain of S. aureus produced SLE scores in aged mice through 9 days after infection (PI) that were significantly lower than those of aged mice similarly infected with the toxin-producing parent strain (P < or = 0.0001). Despite use of identical inocula, the CFU per eye were greater for the parent than the mutant strains from 1 to 5 days PI in the young mice (P < or = 0.0372) and from 1 to 3 days PI in the aged mice (P < or = 0.0018). MPO activities were at the maximum at day 1 PI and were similar overall for all infections. Administration of purified alpha-toxin caused greater gross and histopathologic changes in eyes of aged mice than in those of young mice. CONCLUSIONS Bacterial toxins, and especially alpha-toxin, can mediate corneal disease in mice. Differences in severity of S. aureus keratitis in aged versus young mice correlates with their susceptibility to alpha-toxin.

Objective: To examine whether the atherosclerotic risk from cholesterol is modified by serum glucose level. Methods: Data from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study of 1530 individuals with complete autopsy data for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and hemoglobin A1c (HbA1c) were examined with any atherosclerotic raised lesions (RL) >0% of surface area on any of three arterial specimens as the outcome. A TC/HDL-C ratio was categorized into quintiles and HbA1c was dichotomized as the upper quartile versus lower three quartiles. Odds ratios (ORs) were estimated from logistic regression models adjusting for sex, race, age, body mass index, smoking and hypertension. Results: An interaction product term of TC/HDL × HbA1c was statistically significant (P=0.006) despite adjustment for the main effects and other covariates. In models stratified by HbA1c, ORs (3.0, 3.9, 1.9, 3.5) for four upper quintiles of TC/HDL-C in the upper HbA1c stratum were substantially higher than those in the lower HbA1c stratum (0.9, 1.3, 1.4 and 1.1). Strata differences were even more striking in the subset of those ≥25 years old. Conclusions: These results suggest a synergistic interaction between glucose and cholesterol that magnifies the atherosclerotic risk associated with TC/HDL-C for those with higher HbA1c levels.

Purpose: To define factors that protect the eye from Staphylococcus aureus keratitis and limit tissue damage once keratitis occurs. Methods: Rabbit tears were analyzed for bactericidal and phospholipase A(2) (PLA(2)) activities on S. aureus. Inhibition by spermidine of PLA(2) anti-staphylococcal activity in tears was tested in vitro and in vivo. Rabbits immunized with heat-inactivated alpha-toxin were challenged with intrastromal injection of S aureus. Results: Arachidonic acid was cleaved from S. aureus by purified PLA(2) or rabbit tears. Spermidine inhibited these reactions in vitro and facilitated keratitis in vivo. PLA(2) activity decreased with advanced age and shortly following sleep, but increased with keratitis. Antibody to alpha-toxin significantly reduced corneal damage and epithelial cell sloughing during keratitis. Conclusions: PLA(2) is a major host-defense component of rabbit tears. Alpha-toxin is a major mediator of corneal damage, and antibody to alpha-toxin reduces pathologic changes during keratitis.

PURPOSE To establish, in the scarified mouse eye, a new model of Staphylococcus aureus keratitis suitable for studies of pathogenesis and host defense mechanisms. METHODS Corneas of three strains of mice (BALB/c, A/J, and C57BL/6) were scarified and inoculated with S. aureus strain 8325-4. Mice underwent slit lamp examination (SLE) at 1, 3, 5, 7, and 9 days after infection and were killed. Histopathologic analyses, determination of bacterial colony-forming units (CFU), and myeloperoxidase (MPO) activity assays were performed at each time point. RESULTS S. aureus keratitis developed in both BALB/c and A/J strains of mice, but not in C57BL/6. The BALB/c and A/J strains demonstrated greater susceptibility to infection, as evidenced by significantly higher SLE scores and more viable bacteria per infected eye than in C57BL/6 mice at 5, 7, and 9 days after infection (P <or= 0.0001). Histopathologic analysis and MPO assays of infected A/J mice both revealed an influx of polymorphonuclear leukocytes (PMNs). Histology demonstrated presence of leukocytes in the aqueous humor, migration of PMNs into infected tissue, corneal erosion, and edema in the eyes of infected A/J mice. Whereas infected BALB/c mice demonstrated both PMN migration and corneal edema, eyes of infected C57BL/6 mice failed to show even mild histopathologic changes. CONCLUSIONS These studies demonstrate the establishment of Staphylococcus keratitis in the mouse eye. This model should provide for a large range of future studies that are currently unavailable in the rabbit keratitis model, particularly those requiring a genetically altered host or specific immunologic reagents.

The association between hepatic global DNA methylation measured using pyrosequencing technology and the risk of subclinical atherosclerosis was examined in the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. PDAY is a bi-racial investigation of the natural history of atherosclerosis and its risk factors involving 3013 individuals aged 15–34 years who underwent autopsy after dying of unrelated causes in 1987–1994.Raised atherosclerotic lesions were defined as the sum of the percentages of intimal surface area detected in the right coronary artery and left half of the abdominal and thoracic aorta harboring fibrous plaques, complicated lesions, and calcified lesions during a postmortem pathological examination. To conduct the case–control study, 300 cases selected with the highest raised lesion scores were paired with 300 controls without raised lesions after matching for age, race, and gender.Global DNA methylation was not associated with disease risk in the study population considered as a whole using conditional logistic regression models to analyze matched pairs. Since the estimation of the risk of atherosclerosis associated with inter-individual variation in DNA methylation was similar if unconditional logistic regression was used, subgroup analyses were carried out after adjusting for matching variables. A modest association with methylation levels below the median value was found in white but not in African-American study participants (odds ratio = 1.59, 95% confidence interval = 1.02–2.49, p = 0.04).Hepatic global DNA methylation does not appear to be a definitive determinant of atherosclerosis burden in a postmortem sample of young adults.

Pandit-Bhalla, Meenakshi M.D., M.B.A.*; Diethelm, Lisa M.D.*; Ovella, Ty M.D.*; Sloop, Gregory D. M.D.†; Valentine, Vincent G. M.D.‡ Author Information

The chapter presents a rationale for determining the localization of oxidation-specific epitopes. The chapter also discusses the methods by which these epitopes can be localized with particular attention to immunohistochemistry. Additionally, it describes technical issues associated with immunohistochemistry for oxidation-specific epitopes and provides a protocol for performing immunohistochemistry. The immunolocalization of oxidation-specific epitopes in atherosclerosis are also discussed. The spectrum of illness in which oxidative stress is proposed to play a pathogenic role includes chronic diseases associated with aging, such as atherosclerosis, diabetes mellitus, Alzheimer disease, and aging itself, as well as acute diseases such as ischemia/reperfusion injury and bacterial meningitis. The most research regarding the role of oxidative stress has been in atherosclerosis, the leading cause of mortality in the industrialized world. The distribution of oxidization-specific epitopes can be determined in two ways—biochemically or histologically using immunofluorescence or immunohistochemistry. The chapter focuses on histologic methods of determining the localization of oxidization-specific epitopes. The histologic identification of oxidization-specific epitopes uses antibodies that bind to neoepitopes created by the oxidative modification of a protein.

Atherosclerotic plaque-like lesions are prevalent in synthetic arteriovenous shunts created to provide vascular access for hemodialysis. Similarities to atherosclerotic plaques in native arteries include eccentric location, immunoreactivity for smooth muscle actin, dystrophic calcifications, superimposed thrombi, and foam cells. Fatty streaks were not grossly identified on Sudan IV staining. Because of the similarities to atherosclerosis in native vessels, these findings may have several implications for atherogenesis. The development of raised, fibrous lesions does not require decades. The presence of smooth muscle in atherosclerotic plaque-like lesions does not require a source from tunica media. A precursor fatty streak may not be required for the development of raised, fibrous lesions. Finally, development of atherosclerotic plaque-like lesions does not require putative inflammatory effects from cholesterol or LDL accumulation, or even a native vessel that can respond to injury. The atherosclerotic plaque-like lesions in this study probably developed from organization of mural thrombi.

PURPOSE:To determine the pathogenic role of gamma- and alpha-toxin in a rabbit model of Staphylococcus aureus keratitis.METHODS:S. aureus strains Newman (expressing gamma-toxin), Newman Delta(hlg) (deficient in gamma-toxin), Newman Delta(hlg)/pCU1 hlg(+) (chromosomal gamma-toxin-deficient mutant rescued by a plasmid encoding gamma-toxin), and Newman Delta(hla) (alpha-toxin-deficient) were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE), and bacterial colony-forming units (CFU) per cornea were determined at 15, 20, and 25 hours after infection. Histologic examination of corneas was performed. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain Newman. Western blot analyses of culture supernatants were performed to detect alpha-toxin production.RESULTS:All strains grew equivalently, producing approximately 7 log CFU per cornea at 25 hours after infection. SLE scores at 20 and 25 hours after infection revealed that strains Newman Delta(hlg) and Newman Delta(hla), although virulent, caused significantly less ocular damage and inflammation than their parent or the gamma-toxin genetically rescued strain (P <or= 0.0006). Histologic and SLEs revealed that all strains except Newman Delta(hla) produced corneal erosions. Rabbits immunized actively or passively to alpha-toxin had reduced SLE scores (P <or= 0.0003 and P <or= 0.0033, respectively) and no epithelial erosions when infected with strain Newman. Western blot analysis demonstrated that strains Newman and Newman Delta(hlg), but not Newman Delta(hla), produced alpha-toxin.CONCLUSIONS:These results illustrate that the virulence of strain Newman involves both alpha- and gamma-toxin, with alpha-toxin mediating corneal epithelial erosions. An additional uncharacterized toxin could also be active in damaging the cornea.

PURPOSE To develop a topical inoculation model of Staphylococcus aureus keratitis in which scarification, contact lenses, and spermidine are used to inhibit the host defenses and to investigate the role of alpha-toxin in this infection. METHODS An alpha-toxin-positive parent strain (8325-4), its isogenic alpha-toxin-negative mutant (DU1090), and a genetically rescued form of the mutant (DU1090/pDU1212) were bound to rabbit-specific contact lenses, treated with spermidine (50 mM), and applied to scarified rabbit corneas. Eyes were treated topically with spermidine before and after lens application. Eyes were graded for disease by slit lamp examination (SLE) every 6 hours until 24 hours PI (PI), and erosion diameters were measured. Histopathologic changes and colony forming units (CFUs) of bacteria were determined. RESULTS Spermidine treatment and inoculation of eyes with Staphylococcus on contact lenses resulted in significant increases in both CFUs per cornea (P = 0.0041) and SLE score (P or= 0.1959) multilog increase in CFUs over the inoculum at 24 hours PI. The alpha-toxin-producing strains, 8325-4 and DU1090/pDU1212, caused significantly more disease than the alpha-toxin-deficient mutant DU1090 at 24 hours PI (P

Purpose. To determine the effectiveness of lysostaphin treatment of experimental endophthalmitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Methods. In one experiment, rabbits were injected in the mid-vitreous with 50 or 200 CFU of S. aureus; untreated groups and groups injected intra-vitreally at 8 or 24 hours postinfection with vehicle or lysostaphin (0.1 mg/ml) were compared in terms of CFU/ml vitreous at 24 or 48 hours postinfection. Histopathology of untreated and treated eyes was also compared. To quantify the potency of lysostaphin, additional rabbits were injected with 50 CFU of S. aureus and untreated eyes and eyes treated at 8 hours with 0.001, 0.01 or 0.05 mg/ml were compared in terms of CFU/ml vitreous at 24 hours postinfection. Results. Vitreous of untreated eyes or vehicle-treated eyes injected with 50 or 200 CFU of S. aureus contained 5–10 million CFU/ml at 24 or 48 hours postinfection. All eyes treated with lysostaphin at 8 hours postinfection had less than 1 log CFU/ml in the vitreous (P = 0.0001). Similarly, eyes treated with lysostaphin at 24 hours postinfection had approximately 1 log of CFU/ml at 48 hours postinfection. None of the untreated eyes were sterile and 88% or 50% of the eyes treated at 8 or 24 hours postinfection, respectively, were sterile. Eyes treated with lysostaphin at 8, but not 24, hours postinfection had less pronounced pathologic changes than the untreated eyes (P = 0.002). A significant reduction in the CFU/ml vitreous at 24 hours postinfection was obtained by treating infected eyes at 8 hours postinfection with lysostaphin at concentrations of =0.001 mg/ml (P = 0.0034). Conclusions. Lysostaphin is effective in treating experimental endophthalmitis mediated by MRSA.