Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

The Influence of Immunization Route on the Adjuvant Effect of alpha-Galactosylceramide

Potent vaccine formulations ideally include adjuvants to activate innate immune responses and enhance antigen-specific adaptive immunity. The synthetic glycolipid alpha-Galactosylceramide (α-GalCer) effectively activates the innate immune mediating NKT cells to produce cytokines and activate downstream immune cells, resulting in development of humoral and cell mediated immune responses to co-administered antigens. While a single intravenous immunization of α-GalCer strongly activates NKT cells, multiple doses by this route are well documented to induce anergy in NKT cells. Anergy is defined as the deficiency in NKT proliferation and cytokine production, including IL-4 and IFNγ. However, our studies have shown that two doses of α-GalCer administered intranasally by the intranasal route leads to reactivation of NKT cells and improved adaptive immune responses after each subsequent dose. I therefore investigated the role of multiple routes of immunization in activation of NKT cells, i.e. anergy versus repeated activation. Specifically, I hypothesized that the differential capacity of NKT cells to produce IFNγ, as a result of route of immunization with α-GalCer, influences the induction of adaptive immune responses to co-administered antigen. Our experimental design utilizes the observation that intranasal immunization primarily induces immune responses in the lungs while intravenous immunization induces responses in the liver. Using intracellular cytokine staining for IFNγ production and Elispot analyses for determining NKT and T cell activation, respectively, it was determined that administering two consecutive intravenous doses resulted in anergy to NKT cells (no IFNγ production) in the liver and lack of adaptive immunity while second immunization by the intranasal route overcame anergy in the lung. The outcome in the other tissues analyzed was mixed and could be the result of tissue microenvironment among others possible reasons. When intranasal dosing preceded systemic, NKT cells were reactivated to produce IFNγ and induced positive adaptive immune responses in the responding lung tissue. These results indicate that the mechanism by which mucosal and systemic immunization routes activate NKT cells may differ in that there is a differential tissue-specific effect induced by each route. Future studies are necessary to determine the reason for these tissue-specific effects and how they relate to NKT cell activation.

Genetics and epidemiology of gallbladder disease in New World native peoples.

Native peoples of the New World, including Amerindians and admixed Latin Americans such as Mexican-Americans, are highly susceptible to diseases of the gallbladder. These include cholesterol cholelithiasis (gallstones) and its complications, as well as cancer of the gallbladder. Although there is clearly some necessary dietary or other environmental risk factor involved, the pattern of disease prevalence is geographically associated with the distribution of genes of aboriginal Amerindian origin, and levels of risk generally correspond to the degree of Amerindian admixture. This pattern differs from that generally associated with Westernization, which suggests a gene-environment interaction, and that within an admixed population there is a subset whose risk is underestimated when admixture is ignored. The risk that an individual of a susceptible New World genotype will undergo a cholecystectomy by age 85 can approach 40% in Mexican-American females, and their risk of gallbladder cancer can reach several percent. These are heretofore unrecognized levels of risk, especially of the latter, because previous studies have not accounted for admixture or for the loss of at-risk individuals due to cholecystectomy. A genetic susceptibility may, thus, be as "carcinogenic" in New World peoples as any known major environmental exposure; yet, while the risk has a genetic basis, its expression as gallbladder cancer is so delayed as to lead only very rarely to multiply-affected families. Estimates in this paper are derived in part from two studies of Mexican-Americans in Starr County and Laredo, Texas.

PLASMACYTOID DENDRITIC CELLS SENSE SKIN INJURY AND PROMOTE WOUND HEALING THROUGH TYPE I INTERFERONS

Plasmacytoid dendritic cells (pDCs) are a rare population of circulating cells, which selectively express intracellular Toll-like receptors (TLR)-7 and TLR-9 and have the capacity to produce large amounts of type I IFNs (IFN-a/b) in response to viruses or host derived nucleic acid containing complexes. pDCs are normally absent in skin but accumulate in the skin of psoriasis patients where their chronic activation to produce IFN-a/b drives the disease formation. Whether pDCs and their activation to produce IFN-a/b play a functional role in healthy skin is unknown. Here we show that pDCs are rapidly and transiently recruited into healthy human and mouse skin upon epidermal injury. Infiltrating pDCs were found to sense nucleic acids in wounded skin via TLRs, leading to the production of IFN-a/b. The production of IFN-a/b was paralleled by a short lived expression of cathelicidins, which form complexes with extracellular nucleic acids and activated pDCs to produce IFN-a/b in vitro. In vivo, cathelicidins were sufficient but not necessary for the induction of IFN-a/b in wounded skin, suggesting redundancy of this pathway. Depletion of pDCs or inhibition of IFN-a/bR signaling significantly impaired the inflammatory response and delayed re-epithelialization of skin wounds. Thus we uncover a novel role of pDCs in sensing skin injury via TLR mediated recognition of nucleic acids and demonstrate their involvement in the early inflammatory process and wound healing response through the production of IFN-a/b.

The dynamic proteome of Lyme disease Borrelia.

The proteome of the spirochete bacterium Borrelia burgdorferi, the tick-borne agent of Lyme disease, has been characterized by two different approaches using mass spectrometry, providing a launching point for future studies on the dramatic changes in protein expression that occur during transmission of the bacterium between ticks and mammals.

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.

Prediction of cardiovascular disease risk factors in Mexican-American children from parental risk factors, and social and demographic characteristics in the HHANES

Objectives. Cardiovascular disease (CVD) including CVD secondary to diabetes type II, a significant health problem among Mexican American populations, originates in early childhood. This study seeks to determine risk factors available to the health practitioner that can identify the child at potential risk of developing CVD, thereby enabling early intervention. ^ Design. This is a secondary analysis of cross-sectional data of matched Mexican American parents and children selected from the HHANES, 1982–1984. ^ Methods. Parents at high risk for CVD were identified based on medical history, and clinical and physical findings. Factor analysis was performed on children's skinfold thicknesses, height, weight, and systolic and diastolic blood pressures, in order to produce a limited number of uncorrelated child CVD risk factors. Multiple regression analyses were then performed to determine other CVD markers associated with these Factors, independently for mothers and fathers. ^ Results. Factor analysis of children's measurements revealed three uncorrelated latent variables summarizing the children's CVD risk: Factor1: ‘Fatness’, Factor2: ‘Size and Maturity’, and Factor3: ‘Blood Pressure’, together accounting for the bulk of variation in children's measurements (86–89%). Univariate analyses showed that children from high CVD risk families did not differ from children of low risk families in occurrence of high blood pressure, overweight, biological maturity, acculturation score, or social and economic indicators. However, multiple regression using the factor scores (from factor analysis) as dependent variables, revealed that higher CVD risk in parents, was significantly associated with increased fatness and increased blood pressure in the children. Father's CVD risk status was associated with higher levels of body fat in his children and higher levels of blood pressure in sons. Mother's CVD risk status was associated with higher blood pressure levels in children, and occurrence of obesity in the mother associated with higher fatness levels in her children. ^ Conclusion. Occurrence of cardiovascular disease and its risk factors in parents of Mexican American children, may be used to identify children at potentially higher risk for developing CV disease in the future. Obesity in mothers appears to be an important marker for the development of higher levels of body fatness in children. ^

A longitudinal comparison of cardiovascular disease risk factors among children and adolescents in the U.S. and Japan: Project HeartBeat!

Blood cholesterol and blood pressure development in childhood and adolescence have important impact on the future adult level of cholesterol and blood pressure, and on increased risk of cardiovascular diseases. The U.S. has higher mortality rates of coronary heart diseases than Japan. A longitudinal comparison in children of risk factor development in the two countries provides more understanding about the causes of cardiovascular disease and its prevention. Such comparisons have not been reported in the past. ^ In Project HeartBeat!, 506 non-Hispanic white, 136 black and 369 Japanese children participated in the study in the U.S. and Japan from 1991 to 1995. A synthetic cohort of ages 8 to 18 years was composed by three cohorts with starting ages at 8, 11, and 14. A multilevel regression model was used for data analysis. ^ The study revealed that the Japanese children had significantly higher slopes of mean total cholesterol (TC) and high density lipoprotein (HDL) cholesterol levels than the U.S. children after adjusting for age and sex. The mean TC level of Japanese children was not significantly different from white and black children. The mean HDL level of Japanese children was significantly higher than white and black children after adjusting for age and sex. The ratio of HDL/TC in Japanese children was significantly higher than in U.S. whites, but not significantly different from the black children. The Japanese group had significantly lower mean diastolic blood pressure phase IV (DBP4) and phase V (DBP5) than the two U.S. groups. The Japanese group also showed significantly higher slopes in systolic blood pressure, DBP5 and DBP4 during the study period than both U.S. groups. The differences were independent from height and body mass index. ^ The study provided the first longitudinal comparison of blood cholesterol and blood pressure between the U.S. and Japanese children and adolescents. It revealed the dynamic process of these factors in the three ethnic groups. ^

Measuring health state preferences for hemophilia: Development of a disease-specific utility instrument

Research has shown that disease-specific health related quality of life (HRQoL) instruments are more responsive than generic instruments to particular disease conditions. However, only a few studies have used disease-specific instruments to measure HRQoL in hemophilia. The goal of this project was to develop a disease-specific utility instrument that measures patient preferences for various hemophilia health states. The visual analog scale (VAS), a ranking method, and the standard gamble (SG), a choice-based method incorporating risk, were used to measure patient preferences. Study participants (n = 128) were recruited from the UT/Gulf States Hemophilia and Thrombophilia Center and stratified by age: 0–18 years and 19+. ^ Test retest reliability was demonstrated for both VAS and SG instruments: overall within-subject correlation coefficients were 0.91 and 0.79, respectively. Results showed statistically significant differences in responses between pediatric and adult participants when using the SG (p = .045). However, no significant differences were shown between these groups when using the VAS (p = .636). When responses to VAS and SG instruments were compared, statistically significant differences in both pediatric (p < .0001) and adult (p < .0001) groups were observed. Data from this study also demonstrated that persons with hemophilia with varying severity of disease, as well as those who were HIV infected, were able to evaluate a range of health states for hemophilia. This has important implications for the study of quality of life in hemophilia and the development of disease-specific HRQoL instruments. ^ The utility measures obtained from this study can be applied in economic evaluations that analyze the cost/utility of alternative hemophilia treatments. Results derived from the SG indicate that age can influence patients' preferences regarding their state of health. This may have implications for considering treatment options based on the mean age of the population under consideration. Although both instruments independently demonstrated reliability and validity, results indicate that the two measures may not be interchangeable. ^

Chronic volatile substance abuse among the adult Kickapoo traditional tribe of Texas: Disease and disability profiles, neuropsychosocial consequences, and social implications for treatment

The purpose of this study, based on secondary data from attendees at a substance abuse clinic for the Kickapoo Healing Grounds in Eagle Pass, Texas, is two fold: (1) to elucidate neuro-behavioral performance of volatile substance abusers in the Kickapoo tribe and (2) to determine factors associated with their treatment completion and rehabilitation as measured by their employment at follow-up. Volatile substance abuse (VSA) is associated with a host of neurological manifestations, and secondary prevention or clinical treatment and rehabilitation remains the mainstay of control efforts. Very little is known about VSA in general, and especially among Native American populations. It is anticipated that the results will help determine and assist other tribes and non-tribal substance abuse centers with treatment planning for volatile substance abusers among Native American populations. ^

Nonlinear tests for genetic studies of complex disease

Linkage and association studies are major analytical tools to search for susceptibility genes for complex diseases. With the availability of large collection of single nucleotide polymorphisms (SNPs) and the rapid progresses for high throughput genotyping technologies, together with the ambitious goals of the International HapMap Project, genetic markers covering the whole genome will be available for genome-wide linkage and association studies. In order not to inflate the type I error rate in performing genome-wide linkage and association studies, multiple adjustment for the significant level for each independent linkage and/or association test is required, and this has led to the suggestion of genome-wide significant cut-off as low as 5 × 10 −7. Almost no linkage and/or association study can meet such a stringent threshold by the standard statistical methods. Developing new statistics with high power is urgently needed to tackle this problem. This dissertation proposes and explores a class of novel test statistics that can be used in both population-based and family-based genetic data by employing a completely new strategy, which uses nonlinear transformation of the sample means to construct test statistics for linkage and association studies. Extensive simulation studies are used to illustrate the properties of the nonlinear test statistics. Power calculations are performed using both analytical and empirical methods. Finally, real data sets are analyzed with the nonlinear test statistics. Results show that the nonlinear test statistics have correct type I error rates, and most of the studied nonlinear test statistics have higher power than the standard chi-square test. This dissertation introduces a new idea to design novel test statistics with high power and might open new ways to mapping susceptibility genes for complex diseases. ^

Racial disparities in CD4 counts at initial HIV-1 diagnosis: Analysis of the Adult Spectrum of HIV disease dataset and public health implications

Introduction. The HIV/AIDS disease burden disproportionately affects minority populations, specifically African Americans. While sexual risk behaviors play a role in the observed HIV burden, other factors including gender, age, socioeconomics, and barriers to healthcare access may also be contributory. The goal of this study was to determine how far down the HIV/AIDS disease process people of different ethnicities first present for healthcare. The study specifically analyzed the differences in CD4 cell counts at the initial HIV-1 diagnosis with respect to ethnicity. The study also analyzed racial differences in HIV/AIDS risk factors. ^ Methods. This is a retrospective study using data from the Adult Spectrum of HIV Disease (ASD), collected by the City of Houston Department of Health. The ASD database contains information on newly reported HIV cases in the Harris County District Hospitals between 1989 and 2000. Each patient had an initial and a follow-up report. The extracted variables of interest from the ASD data set were CD4 counts at the initial HIV diagnosis, race, gender, age at HIV diagnosis and behavioral risk factors. One-way ANOVA was used to examine differences in baseline CD4 counts at HIV diagnosis between racial/ethnic groups. Chi square was used to analyze racial differences in risk factors. ^ Results. The analyzed study sample was 4767. The study population was 47% Black, 37% White and 16% Hispanic [p<0.05]. The mean and median CD4 counts at diagnosis were 254 and 193 cells per ml, respectively. At the initial HIV diagnosis Blacks had the highest average CD4 counts (285), followed by Whites (233) and Hispanics (212) [p<0.001 ]. These statistical differences, however, were only observed with CD4 counts above 350 [p<0.001], even when adjusted for age at diagnosis and gender [p<0.05]. Looking at risk factors, Blacks were mostly affected by intravenous drug use (IVDU) and heterosexuality, whereas Whites and Hispanics were more affected by male homosexuality [ p<0.05]. ^ Conclusion. (1) There were statistical differences in CD4 counts with respect to ethnicity, but these differences only existed for CD4 counts above 350. These differences however do not appear to have clinical significance. Antithetically, Blacks had the highest CD4 counts followed by Whites and Hispanics. (2) 50% of this study group clinically had AIDS at their initial HIV diagnosis (median=193), irrespective of ethnicity. It was not clear from data analysis if these observations were due to failure of early HIV surveillance, HIV testing policies or healthcare access. More studies need to be done to address this question. (3) Homosexuality and bisexuality were the biggest risk factors for Whites and Hispanics, whereas for Blacks were mostly affected by heterosexuality and IVDU, implying a need for different public health intervention strategies for these racial groups. ^

A systematic review and meta-analysis of diabetes disease management programs

Context. Despite the rapid growth of disease management programs, there are still questions about their efficacy and effectiveness for improving patient outcomes and their ability to reduce costs associated with chronic disease. ^ Objective. To determine the effectiveness of disease management programs on improving the results of HbA1c tests, lipid profiles and systolic blood pressure (SBP) readings among diabetics. These three quantitative measures are widely accepted methods of determining the quality of a patient's diabetes management and the potential for future complications. ^ Data Sources. MEDLINE and CINAHL were searched from 1950 to June 2008 using MeSH terms designed to capture all relevant studies. Scopus pearling and hand searching were also done. Only English language articles were selected. ^ Study Selection. Titles and abstracts for the 2347 articles were screened against predetermined inclusion and exclusion criteria, yielding 217 articles for full screening. After full article screening, 29 studies were selected for inclusion in the review. ^ Data Extraction. From the selected studies, data extraction included sample size, mean change over baseline, and standard deviation for each control and experimental arm. ^ Results. The pooled results show a mean HbA1c reduction of 0.64%, 95% CI (-0.83 to -0.44), mean SBP reduction of 7.39 mmHg (95% CI to -11.58 to -3.2), mean total cholesterol reduction of 5.74 mg/dL (95% CI, -10.01 to -1.43), and mean LDL cholesterol reduction of 3.74 mg/dL (95% CI, -8.34 to 0.87). Results for HbA1c, SBP and total cholesterol were statistically significant, while the results for LDL cholesterol were not. ^ Conclusions. The findings suggest that disease management programs utilizing five hallmarks of care can be effective at improving intermediate outcomes among diabetics. However, given the significant heterogeneity present, there may be fundamental differences with respect to study-specific interventions and populations that render them inappropriate for meta-analysis. ^

Genetic epidemiology of gallbladder disease in Mexican Americans and cholesterol 7$\alpha$-hydroxylase gene sequence variation

Among Mexican Americans, the second largest minority group in the United States, the prevalence of gallbladder disease is markedly elevated. Previous data from both genetic admixture and family studies indicate that there is a genetic component to the occurrence of gallbladder disease in Mexican Americans. However, prior to this thesis no formal genetic analysis of gallbladder disease had been carried out nor had any contributing genes been identified.^ The results of complex segregation analysis in a sample of 232 Mexican American pedigrees documented the existence of a major gene having two alleles with age- and gender-specific effects influencing the occurrence of gallbladder disease. The estimated frequency of the allele increasing susceptibility was 0.39. The lifetime probabilities that an individual will be affected by gallbladder disease were 1.0, 0.54, and 0.00 for females of genotypes "AA", "Aa", and "aa", respectively, and 0.68, 0.30, and 0.00 for males, respectively. This analysis provided the first conclusive evidence for the existence of a common single gene having a large effect on the occurrence of gallbladder disease.^ Human cholesterol 7$\alpha$-hydroxylase is the rate-limiting enzyme in bile acid synthesis. The results of an association study in both a random sample and a matched case/control sample showed that there is a significant association between cholesterol 7$\alpha$-hydroxylase gene variation and the occurrence of gallbladder disease in Mexican Americans males but not in females. These data have implicated a specific gene, 7$\alpha$-hydroxylase, in the etiology of gallbladder disease in this population.^ Finally, I asked whether the inferred major gene from complex segregation analysis is genetically linked to the cholesterol 7$\alpha$-hydroxylase gene. Three pedigrees predicted to be informative for linkage analysis by virtue of supporting the major gene hypothesis and having parents with informative genotypes and multiple offspring were selected for this linkage analysis. In each of these pedigrees, the recombination fractions maximized at 0 with a positive, albeit low, LOD score. The results of this linkage analysis provide preliminary and suggestive evidence that the cholesterol 7$\alpha$-hydroxylase gene and the inferred gallbladder disease susceptibility gene are genetically linked. ^