AUTOIMMUNE RESPONSES TO ATHEROSCLEROTIC

Atherosclerosis is a chronic, complex arterial disease characterized by intimal lipid accumulation and inflammation. A unique lipid-binding molecule, namely cluster of differentiation 1d (CD1d), may impact atherosclerosis. Structurally, CD1d acts as a nonpolymorphic cell-surface receptor, resembling the major histocompatibility complex-I (MHC-I). While MHC-I restricts peptide antigen presentation to T cells, CD1d presents lipid antigens to T cells named CD1d-restrictedd T cells. Although increased expression of CD1d has been found in human plaques, the exact nature of CD1d-recognized lipids in atherosclerosis remains to be determined. Three groups of lipids may undergo oxidation in atherosclerosis producing atherogenic lipids: phospholipids, fatty acids, and cholesterol. The central hypothesis is that CD1d recognizes and present oxidative lipids to activate CD1d-restricted T cells, and trigger proinflammatory signal transduction In the first part of this study, oxidative phospholipids were identified and characterized as potential autoantigen for CD1d-restricted T cells. Derived from phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine by oxidization, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) is commonly found in atherosclerotic plaques. Upon stimulation with PGPC, spleen-derived CD1d-restricted T cells produced higher levels of cytokines and proliferated at higher rates than those without PGPC stimulation. CD1d deficiency compromised the PGPC-triggered T cell activation, suggesting that PGPC may function as a potentially novel autoantigen for T cells in atherosclerosis. In the second part of this study, CD1d-mediated proinflammatory signaling was evaluated in murine models. Enhanced CD1 expression occurred in spleens of db/db mice with hyperlipidemia. Tumor necrosis factor-alpha (TNF-α) was increased in db/db spleen, while TNF-α receptor expression augmented in the db/db murine heart, in comparison with those in normal mice. The nuclear factor-κ B (NF-κB) expression was enhanced in the db/db heart, whereas CD1d-null mice showed lower NF-κB, implying the involvement of CD1d in inflammation of the spleen and heart tissues in the mice with hyperlipidemia. The current study has identified PGPC as a novel lipid antigen recognized by CD1d-restricted T cells in atherosclerosis. The animal study has also provided evidence that CD1d regulates NF-κB-mediated proinflammatory signaling. Hence, CD1d-restricted T cell responses to autolipid antigen and mediated inflammatory signal may represent a new molecular pathway that triggers cardiovascular tissue injury in atherosclerosis and hyperlipidemia.

The assumptions and effects of conservative procedures in low -dose risk assessment for setting safety standards

Conservative procedures in low-dose risk assessment are used to set safety standards for known or suspected carcinogens. However, the assumptions upon which the methods are based and the effects of these methods are not well understood.^ To minimize the number of false-negatives and to reduce the cost of bioassays, animals are given very high doses of potential carcinogens. Results must then be extrapolated to much smaller doses to set safety standards for risks such as one per million. There are a number of competing methods that add a conservative safety factor into these calculations.^ A method of quantifying the conservatism of these methods was described and tested on eight procedures used in setting low-dose safety standards. The results using these procedures were compared by computer simulation and by the use of data from a large scale animal study.^ The method consisted of determining a "true safe dose" (tsd) according to an assumed underlying model. If one assumed that Y = the probability of cancer = P(d), a known mathematical function of the dose, then by setting Y to some predetermined acceptable risk, one can solve for d, the model's "true safe dose".^ Simulations were generated, assuming a binomial distribution, for an artificial bioassay. The eight procedures were then used to determine a "virtual safe dose" (vsd) that estimates the tsd, assuming a risk of one per million. A ratio R = ((tsd-vsd)/vsd) was calculated for each "experiment" (simulation). The mean R of 500 simulations and the probability R $<$ 0 was used to measure the over and under conservatism of each procedure.^ The eight procedures included Weil's method, Hoel's method, the Mantel-Byran method, the improved Mantel-Byran, Gross's method, fitting a one-hit model, Crump's procedure, and applying Rai and Van Ryzin's method to a Weibull model.^ None of the procedures performed uniformly well for all types of dose-response curves. When the data were linear, the one-hit model, Hoel's method, or the Gross-Mantel method worked reasonably well. However, when the data were non-linear, these same methods were overly conservative. Crump's procedure and the Weibull model performed better in these situations. ^

A DESCRIPTIVE STUDY OF ANIMAL BITES TO HUMANS IN HOUSTON, TEXAS, JANUARY 1, 1982 - JULY 30, 1982 (RABIES)

This study describes animal bites which occurred within the city of Houston, Texas from January 1, 1982, through July 30, 1982. The Rabies Investigation Branch (RIB) recorded 2,047 bite cases. The study has as its foundation a data base management system that was developed to improve the planning and evaluation of the activities of the Rabies Investigation Branch of the Animal Control Center of the City of Houston Health Department. The study provides information which will allow for the development of a more effective animal bite program to meet the needs of the citizens of Houston. ^

Molecular cross talk between misfolded proteins in animal models of Alzheimer's and prion diseases.

The central event in protein misfolding disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated with different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimer's and prion diseases. For this purpose, we inoculated prions in an Alzheimer's transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion-infected mice compared with their noninoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimer's and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs.

A study of factors regulating the autonomous proliferation of the steroid hormone-induced LJ6195 murine reproductive tract tumor

Neonatal estrogen treatment of BALB/c mice results in the unregulated proliferation of the cervicovaginal epithelium and eventually tumorigenesis. The conversion of the normally estrogen responsive cyclic proliferation of the vaginal epithelium to a continuous estrogen-independent pattern of growth is a complex phenomenon. The aim of this study was to gain an understanding of the mechanism(s) by which steroid hormone administration during a critical period of development alters the cyclic proliferation of vaginal epithelium, ultimately leading to carcinogenesis in the adult animal.^ The LJ6195 murine cervicovaginal tumor was induced by treating newborn female BALB/c mice with 20 $\mu$g 17$\beta$-estradiol plus 100 $\mu$g progesterone for the first 5 days after birth. In contrast to proliferation of the normal vaginal epithelium, proliferation of LJ6195 is not regulated by estradiol. Northern blot analysis of RNA from vaginal tracts of normal mice, neonatal-estrogen treated mice, and LJ6195 indicate that there is an alteration in the expression of several genes such as the estrogen receptor, c-fos, and HER2/neu. In response to neonatal estrogen treatment, the estrogen receptor is down regulated in the murine vaginal tract. Therefore, the estrogen-independent nature of this tissue is established as early as 3 months after treatment. There is strong evidence that the proliferation of LJ6195 is regulated through an autocrine growth pathway. The LJ6195 tumor expresses mRNA for the epidermal growth factor receptor. In addition, conditioned medium from the LJ6195 tumor cell line contains a growth factor(s) with epidermal growth factor-like activity. Conditioned medium from the LJ6195 cell line stimulated the proliferation of the EGF-dependent COMMA D mouse mammary gland cell line in a dose-dependent manner. The addition of an anti-mEGF-antibody to LJ6195 cell cultures significantly decreased growth. These results suggest that the EGF-receptor mediated growth pathway may play a role in regulating the estrogen-independent proliferation of the LJ6195 tumor. ^

A study of the {\it in vivo\/} role of myogenin

Myogenin is a member of the MyoD family of skeletal muscle specific bHLH transcription factors. All of the members of this family have been shown to initiate the muscle differentiation cascade in a variety of nonmuscle cell lines. Many of the properties of the MyoD family have been studied in vitro, but their in vivo roles had not yet been examined. In this thesis, I study the in vivo role of myogenin by creating mice that carry a mutation at the myogenin locus.^ Mice lacking the myogenin protein are born alive, but immobile. Histological examination showed that these mice are severely deficient in skeletal muscle; they show a reduction in the number and density of myofibers. In addition to the reduction in fiber number, these mice express lower levels of a variety of muscle-specific markers. The undifferentiated cells in the muscle forming regions of these mice do express some muscle-specific markers, indicating that these cells are determined but undifferentiated myoblasts. Additional studies show that the major muscle defect arises late in embryogenesis, at a time coincident with secondary myogenesis. Moreover, studies regarding the nature of the remaining myofibers indicate that they are representative of a normal population of myofibers, merely reduced in numbers. In addition, I studied the effects of combining the myogenin mutation with mutations in two other members of the MyoD family, MyoD and myf5. Mice mutant in myogenin + MyoD and myogenin + myf5 show no increase in the severity of the myogenin single mutation, as indicated by histological or molecular examination. These results reveal the unique and essential role of myogenin in mammalian skeletal myogenesis. ^

A study of the relationship between physical activity and cardiovascular fitness and coronary heart disease risk factors in female adolescents

The association of measures of physical activity with coronary heart disease (CHD) risk factors in children, especially those for atherosclerosis, is unknown. The purpose of this study was to determine the association of physical activity and cardiovascular fitness with blood lipids and lipoproteins in pre-adolescent and adolescent girls.^ The study population was comprised of 131 girls aged 9 to 16 years who participated in the Children's Nutrition Research Center's Adolescent Study. The dependent variables, blood lipids and lipoproteins, were measured by standard techniques. The independent variables were physical activity measured as the difference between total energy expenditure (TEE) and basal metabolic rate (BMR), and cardiovascular fitness, VO$\rm\sb{2max}$(ml/min/kg). TEE was measured by the doubly-labeled water (DLW) method, and BMR by whole-room calorimetry. Cardiovascular fitness, VO$\rm\sb{2max}$(ml/min/kg), was measured on a motorized treadmill. The potential confounding variables were sexual maturation (Tanner breast stage), ethnic group, body fat percent, and dietary variables. A systematic strategy for data analysis was used to isolate the effects of physical activity and cardiovascular fitness on blood lipids, beginning with assessment of confounding and interaction. Next, from regression models predicting each blood lipid and controlling for covariables, hypotheses were evaluated by the direction and value of the coefficients for physical activity and cardiovascular fitness.^ The main result was that cardiovascular fitness appeared to be more strongly associated with blood lipids than physical activity. An interaction between cardiovascular fitness and sexual maturation indicated that the effect of cardiovascular fitness on most blood lipids was dependent on the stage of sexual maturation.^ A difference of 760 kcal/d physical activity (which represents the difference between the 25th and 75th percentile of physical activity) was associated with negligible differences in blood lipids. In contrast, a difference in 10 ml/min/kg of VO$\rm\sb{2max}$ or cardiovascular fitness (which represents the difference between the 25th and 75th percentile in cardiovascular fitness) in the early stages of sexual maturation was associated with an average positive difference of 15 mg/100 ml ApoA-1 and 10 mg/100 ml HDL-C. ^

A study of the impact of cigarette advertising on adolescent susceptibility to smoking among 5th-, 8th-, and 12th-grade students in Hempstead and Hitchcock Independent School Districts

This study assessed the impact of cigarette advertising on adolescent susceptibility to smoking in the Hempstead and Hitchcock Independent School Districts. A convenience sample of 217 youths, 10-19 years of age was recruited in the study. Students completed both a paper-and-pencil and a computer-aided questionnaire in April 1996. Adolescents were defined as susceptible to smoking if they could not definitely rule out the possibility of future smoking. For the analysis, an index was devised: a 5-point index of an individual's receptivity to cigarette advertising. The index is determined by the number of positive responses to five survey items (recognizing cigarette brand logos, recognizing cigarette advertisement's pictures, recognizing cigarette brand slogans, evaluating adolescent attitudes toward cigarette advertising, and the degree to which adolescents were exposed to cigarette advertisements). Using logistic regression, we assessed the independent importance of the index in predicting susceptibility to smoking and ever smoking after adjusting for sociodemographic variables, perceived school performance and family composition. Of students surveyed, 54.4% of students appeared to have started the smoking uptake process as measured by susceptibility to smoking. Camel was recognized by the majority of students (88%), followed by Marlboro (41.5%) and Newport (40.1%). The pattern for recognition of the cigarette advertisements was the same as the pattern of market for cigarette. Advertisement featuring the cartoon character Joe Camel was significantly more appealing to adolescents than were advertisements with human models, with animal models, and with text only (p $<$ 0.001). Text only advertisement was significantly less appealing than other types of advertisements. The cigarette advertisement with White models (Marlboro) had significantly higher appeal to White students than to African-American students (p $<$ 0.001). The cigarette advertisement featuring African-American models (Virginia Slims) was significantly more appealing to African-American students than other ethnic groups (p $<$ 0.001). Receptivity to cigarette advertising was to be an important concurrent predictor of past smoking experience and intention to smoke in the future. Adolescents who scored in the fourth quartile of the Index of Receptivity to Cigarette Advertising were 7.54 (95% confidence interval (CI) = 1.92-29.56) times as likely to be susceptible to smoking, and were 4.56 (95% CI = 1.55-13.38) times as likely to have tried smoking, as those who scored in the first quartile of the Index. The findings confirmed the hypothesis that cigarette advertising may be a strong current influence in encouraging adolescents to initiate the smoking uptake process than sociodemographic variables, perceived school performance and family composition. ^

Lab animal allergy surveillance at a private medical school in three dissimilar animal research facilities

Very few studies have described MUP-1 concentrations and measured prevalence of Laboratory Animal Allergy (LAA) at such a diverse institution as the private medical school (MS) that is the focus of this study. Air sampling was performed in three dissimilar animal research facilities at MS and quantitated using a commercially available ELISA. Descriptive data was obtained from an anonymous laboratory animal allergy survey given to both animal facility employees and the researchers who utilize these facilities alike. Logistic regression analysis was then implemented to investigate specific factors that may be predictive of developing LAA as well as factors influencing the reporting of LAA symptoms to the occupational health program. Concentrations of MUP-1 detected ranged from below detectable levels (BDL) to a peak of 22.64 ng/m3 . Overall, 68 employees with symptoms claimed they improved while away from work and only 25 employees reported their symptoms to occupational health. Being Vietnamese, a smoker, not wearing a mask, and working in any facility longer than one year were all significant predictors of having LAA symptoms. This study suggests a LAA monitoring system that relies on self-reporting can be inadequate in estimating LAA problems. In addition, efforts need to be made to target training and educational materials for non-native English speaking employees to overcome language and cultural barriers and address their specific needs. ^

West Nile virus in the Rio Grande Valley: A seroprevalence study of juvenile dogs

This study used canine sentinel surveillance and collected a sample of adult mosquitoes to investigate the potential impact of West Nile virus (WNV) in human populations in the Rio Grande Valley along the Texas-Mexico border. Samples for this study were collected from juvenile dogs two months to one year of age in animal shelters located in the Rio Grande Valley. The sample was comprised of stray dogs in order to include animals with maximum nighttime exposures to Culex mosquitoes. Serum samples were collected following the 2007 WNV transmission season and were tested for IgG antibodies against WNV. Evidence of antibodies to WNV was found in 35.1% of the sample population consisting of 74 dogs. During this same time period, mosquitoes in Brownsville were trapped and morphologically identified to develop greater understanding of the mosquito populations in the region and to further understand other potential mosquito vectors for disease transmission. The vast majority of mosquitoes living in this area were Aedes albopictus (47.6%), Culex quinquefasciatus (23.7%), and Aedes aegypti (20.1%). This study shows that WNV and the vector responsible for WNV transmission are active in the Rio Grande Valley and pose a threat to the human and animal populations. ^

Design of long-term animal bioassay for low-dose extrapolation using a multistage model

Conventional designs of animal bioassays allocate the same number of animals into control and dose groups to explore the spontaneous and induced tumor incidence rates, respectively. The purpose of such bioassays are (a) to determine whether or not the substance exhibits carcinogenic properties, and (b) if so, to estimate the human response at relatively low doses. In this study, it has been found that the optimal allocation to the experimental groups which, in some sense, minimize the error of the estimated response for low dose extrapolation is associated with the dose level and tumor risk. The number of dose levels has been investigated at the affordable experimental cost. The pattern of the administered dose, 1 MTD, 1/2 MTD, 1/4 MTD,....., etc. plus control, gives the most reasonable arrangement for the low dose extrapolation purpose. The arrangement of five dose groups may make the highest dose trivial. A four-dose design can circumvent this problem and has also one degree of freedom for testing the goodness-of-fit of the response model.^ An example using the data on liver tumors induced in mice in a lifetime study of feeding dieldrin (Walker et al., 1973) is implemented with the methodology. The results are compared with conclusions drawn from other studies. ^

A STUDY OF ILLNESS AND RESERVOIRS ASSOCIATED WITH GIARDIA LAMBLIA IN RURAL EGYPT

A longitudinal investigation of the health effects and reservoirs of Giardia lamblia was undertaken in forty households located in a rural Nile Delta region of Egypt. Stool specimens obtained once weekly for six months from two to four year old children were cyst or trophozoite-positive in 42 percent of the 724 examined. The mean duration of excretion in all but one Giardia-negative child was seven and one-half weeks with a range of one to 17 weeks. Clinical symptoms of illness were frequently observed within a month before or after Giardia excretion in stool of children, but a statistical inference of association was not demonstrated.^ Seventeen percent of 697 specimens obtained from their mothers was Giardia-positive for a mean duration of four weeks and a range of one to 18 weeks. Mothers were observed to excrete Giardia in stool less frequently during pregnancy than during lactation.^ Nine hundred sixty-two specimens were collected from 13 species of household livestock. Giardia was detected in a total of 22 specimens from cows, goats, sheep and one duck. Giardia cysts were detected in three of 899 samples of household drinking water.^ An ELISA technique of Giardia detection in human and animal stool was field tested under variable environmental conditions. The overall sensitivity of the assay of human specimens was 74 percent and specificity was 97 percent. These values for assay of animal specimens were 82 percent and 98 percent, respectively.^ Surface antigen studies reported from the NIH Laboratory of Parasitic Diseases show that antigens of three Egyptian human isolates are different from each other and from most other isolates against which they were tested.^ The ubiquity of human and animal fecal contamination combined with estimates of ill days per child per year in this setting are substantial arguments for the introduction of a suggested mass parasite control program to intervene in the cyclical transmission of agents of enteric disease. ^

Anesthetic choices and breast cancer recurrence: A retrospective cohort study of risk factors

Background: The impact of anesthetic techniques for breast cancer surgery traditionally has been centered on the incidence of acute pain syndromes and complications immediately after surgery. Evaluating anesthesia management beyond short-term effects is an emerging science. Several animal studies have concluded that regional anesthesia independently reduces cancer recurrence and metastasis. A small number of retrospective clinical studies indicate that reductions in cancer recurrence are attributable to anesthesia technique; however, individual risk factors need to be taken into consideration. ^ Purpose: The aims were to: 1) investigate differences in patient, disease and treatment factors between women who received surgical treatment for breast cancer with paravertebral regional and general anesthesia compared to women who received general anesthesia alone; 2) explore patient, disease and treatment factors associated with recurrence of breast cancer; and 3) test the association between type of anesthesia and breast cancer recurrence and survival over 22–46 months following surgery. ^ Methods: This retrospective cohort study included 358 patients with stage 0-III disease who received a partial or total mastectomy without axillary node dissection between October 2006 and October 2008 at a large academic cancer center. Follow-up ended in August 2010 with a median follow-up time of 28.8 months. ^ Results: The patient demographics were equally represented across anesthesia groups. Mean BMI (kg/m2) was greater for the patients who received general anesthesia (GA) alone (29±6.8) compared to those that received paravertebral regional block (PVB) with GA (28±5.1), p=0.001. The PVB with GA group had more advanced stages of disease (p=0.01) and longer surgeries (p=0.01) than the GA only group. Breast cancer recurrence was detected in only 1.7% of the study population. The mean age was 51±18 in those who had a recurrence compared to 58±11 in the non-recurrent group (p=0.06). Overall, no association between anesthesia type and recurrence was found (p=0.53), with an unadjusted estimated hazard ratio of 1.84 (95% CI 0.34–10.08). ^ Conclusions: In contrast to previous retrospective studies in cancer patients receiving surgical and anesthesia treatment, this study was unable to detect a difference in relating type of anesthesia with decreased breast cancer recurrence. Nonetheless, a significant association between BMI and type of anesthesia was observed and should be taken into account in future studies. Because the overall rate of recurrence was very small in this population, a larger study would be needed to detect any differences in rates of recurrence attributable to type of anesthesia. ^

A cohort study of body fatness and coronary mortality in middle -aged men

Data from the Chicago Western Electric Study were used to investigate whether central fat distribution, as estimated by the ratio of subscapular-to-triceps skinfold, was associated with 25-year risk of death from coronary heart disease in a cohort of 1,945 middle-aged employed men. Subscapular-triceps skinfold ratio was found positively and significantly associated with risk of coronary death after adjustment for age and body mass index. The age-adjusted proportional hazards regression coefficient was 0.2078 with 95% confidence interval of 0.0087 to 0.4069. A difference of 1.1 in the subscapular-triceps skinfold ratio (the difference between the mean of the fifth quintile and of the first and second quintiles combined) was associated with a relative risk of 1.31 with 95% confidence interval of 1.06 to 1.62. The coefficient was decreased to 0.1961 (95% confidence interval of ($-$0.0028 to 0.3950) after adjustment for diastolic blood pressure, serum cholesterol and cigarette smoking as well as age and body mass index. At least some of the effect of central fat on coronary risk is probably mediated by blood pressure and serum lipids, but whether all of the effect can be accounted for blood pressure and serum lipids is uncertain.^ This study supports the concept that central fat distribution is a risk factor for 25-year risk of coronary death in middle-aged men. ^