The aim of this study was to examine retrospective dengue-illness classification using only clinical laboratory data, without relying on X-ray, ultrasound, or percent hemoconcentration. We analyzed data from a study of children who presented with acute febrile illness to two hospitals in Thailand. Multivariable logistic regression models were used to distinguish: (1) dengue hemorrhagic fever (DHF) versus dengue fever (DF), (2) DHF versus DF + other febrile illness (OFI), (3) dengue versus OFI, and (4) severe dengue versus non-severe dengue + OFI. Data from the second hospital served as a validation set. There were 1,227 patients in the analysis. The sensitivity of the models ranged from 89.2% (dengue versus OFI) to 79.6% (DHF versus DF). The models showed high sensitivity in the validation dataset. These models could be used to calculate a probability and classify patients based on readily available clinical laboratory data, and they will need to be validated in other dengue-endemic regions.
Envelope-modified single-cycle simian immunodeficiency virus selectively enhances antibody responses and partially protects against repeated, low-dose vaginal challenge
Immunization of rhesus macaques with strains of simian immunodeficiency virus (SIV) that are limited to a single cycle of infection elicits T-cell responses to multiple viral gene products and antibodies capable of neutralizing lab-adapted SIV, but not neutralization-resistant primary isolates of SIV. In an effort to improve upon the antibody responses, we immunized rhesus macaques with three strains of single-cycle SIV (scSIV) that express envelope glycoproteins modified to lack structural features thought to interfere with the development of neutralizing antibodies. These envelope-modified strains of scSIV lacked either five potential N-linked glycosylation sites in gp120, three potential N-linked glycosylation sites in gp41, or 100 amino acids in the V1V2 region of gp120. Three doses consisting of a mixture of the three envelope-modified strains of scSIV were administered on weeks 0, 6, and 12, followed by two booster inoculations with vesicular stomatitis virus (VSV) G trans-complemented scSIV on weeks 18 and 24. Although this immunization regimen did not elicit antibodies capable of detectably neutralizing SIV(mac)239 or SIV(mac)251(UCD), neutralizing antibody titers to the envelope-modified strains were selectively enhanced. Virus-specific antibodies and T cells were observed in the vaginal mucosa. After 20 weeks of repeated, low-dose vaginal challenge with SIV(mac)251(UCD), six of eight immunized animals versus six of six naive controls became infected. Although immunization did not significantly reduce the likelihood of acquiring immunodeficiency virus infection, statistically significant reductions in peak and set point viral loads were observed in the immunized animals relative to the naive control animals.
We extend the random permutation model to obtain the best linear unbiased estimator of a finite population mean accounting for auxiliary variables under simple random sampling without replacement (SRS) or stratified SRS. The proposed method provides a systematic design-based justification for well-known results involving common estimators derived under minimal assumptions that do not require specification of a functional relationship between the response and the auxiliary variables.
The Women's Health Initiative: The food environment, neighborhood socioeconomic status, BMI, and blood pressure
Using data (n = 60,775 women) from the Women's Health Initiative Clinical Trial (WHI CT)-a national study of postmenopausal women aged 50-79 years-we analyzed cross-sectional associations between the availability of different types of food outlets in the 1.5 miles surrounding a woman's residence, census tract neighborhood socioeconomic status (NSES), BMI, and blood pressure (BP). We simultaneously modeled NSES and food outlets using linear and logistic regression models, adjusting for multiple sociodemographic factors, population density and random effects at the tract and metropolitan statistical area (MSA) level. We found significant associations between NSES, availability of food outlets and individual-level measurements of BMI and BP. As grocery store/supermarket availability increased from the 10th to the 90th percentile of its distribution, controlling for confounders, BMI was lower by 0.30 kg/m(2). Conversely, as fast-food outlet availability increased from the 10th to the 90th percentile, BMI was higher by 0.28 kg/m(2). When NSES increased from the 10th to the 90th percentile of its distribution, BMI was lower by 1.26 kg/m(2). As NSES increased from the 10th to the 90th percentile, systolic and diastolic BP were lower by 1.11 mm Hg and 0.40 mm Hg, respectively. As grocery store/supermarket outlet availability increased from the 10th and 90th percentiles, diastolic BP was lower by 0.31 mm Hg. In this national sample of postmenopausal women, we found important independent associations between the food and socioeconomic environments and BMI and BP. These findings suggest that changes in the neighborhood environment may contribute to efforts to control obesity and hypertension.
Vaccine protection against simian immunodeficiency virus in monkeys using recombinant gamma-2 herpesvirus
Recombinant strains of replication-competent rhesus monkey rhadinovirus (RRV) were constructed in which strong promoter/enhancer elements were used to drive expression of simian immunodeficiency virus (SIV) Env or Gag or a Rev-Tat-Nef fusion protein. Cultured rhesus monkey fibroblasts infected with each recombinant strain were shown to express the expected protein. Three RRV-negative and two RRV-positive rhesus monkeys were inoculated intravenously with a mixture of these three recombinant RRVs. Expression of SIV Gag was readily detected in lymph node biopsy specimens taken at 3 weeks postimmunization. Impressive anti-SIV cellular immune responses were elicited on the basis of major histocompatibility complex (MHC) tetramer staining and gamma interferon enzyme-linked immunospot (ELISPOT) assays. Responses were much greater in magnitude in the monkeys that were initially RRV negative but were still readily detected in the two monkeys that were naturally infected with RRV at the time of immunization. By 3 weeks postimmunization, responses measured by MHC tetramer staining in the two Mamu-A*01(+) RRV-negative monkeys reached 9.3% and 13.1% of all CD8(+) T cells in peripheral blood to the Gag CM9 epitope and 2.3% and 7.3% of all CD8(+) T cells in peripheral blood to the Tat SL8 epitope. Virus-specific CD8(+) T cell responses persisted at high levels up to the time of challenge at 18 weeks postimmunization, and responding cells maintained an effector memory phenotype. Despite the ability of the RRVenv recombinant to express high levels of Env in cultured cells, and despite the appearance of strong anti-RRV antibody responses in immunized monkeys, anti-Env antibody responses were below our ability to detect them. Immunized monkeys, together with three unimmunized controls, were challenged intravenously with 10 monkey infectious doses of SIVmac239. All five immunized monkeys and all three controls became infected with SIV, but peak viral loads were 1.2 to 3.0 log(10) units lower and chronic-phase viral loads were 1.0 to 3.0 log(10) units lower in immunized animals than the geometric mean of unimmunized controls. These differences were statistically significant. Anti-Env antibody responses following challenge indicated an anamnestic response in the vaccinated monkeys. These findings further demonstrate the potential of recombinant herpesviruses as preventive vaccines for AIDS. We hypothesize that this live, replication-competent, persistent herpesvirus vector could match, or come close to matching, live attenuated strains of SIV in the degree of protection if the difficulty with elicitation of anti-Env antibody responses can be overcome.
We address the problem of selecting the best linear unbiased predictor (BLUP) of the latent value (e.g., serum glucose fasting level) of sample subjects with heteroskedastic measurement errors. Using a simple example, we compare the usual mixed model BLUP to a similar predictor based on a mixed model framed in a finite population (FPMM) setup with two sources of variability, the first of which corresponds to simple random sampling and the second, to heteroskedastic measurement errors. Under this last approach, we show that when measurement errors are subject-specific, the BLUP shrinkage constants are based on a pooled measurement error variance as opposed to the individual ones generally considered for the usual mixed model BLUP. In contrast, when the heteroskedastic measurement errors are measurement condition-specific, the FPMM BLUP involves different shrinkage constants. We also show that in this setup, when measurement errors are subject-specific, the usual mixed model predictor is biased but has a smaller mean squared error than the FPMM BLUP which point to some difficulties in the interpretation of such predictors.
The effect of systematic clinical interventions with cigarette smokers on quit status and the rates of smoking-related primary care office visits
BACKGROUND: The United States Public Health Service (USPHS) Guideline for Treating Tobacco Use and Dependence includes ten key recommendations regarding the identification and the treatment of tobacco users seen in all health care settings. To our knowledge, the impact of system-wide brief interventions with cigarette smokers on smoking prevalence and health care utilization has not been examined using patient population-based data. METHODS AND FINDINGS: Data on clinical interventions with cigarette smokers were examined for primary care office visits of 104,639 patients at 17 Harvard Vanguard Medical Associates (HVMA) sites. An operational definition of "systems change" was developed. It included thresholds for intervention frequency and sustainability. Twelve sites met the criteria. Five did not. Decreases in self-reported smoking prevalence were 40% greater at sites that achieved systems change (13.6% vs. 9.7%, p<.01). On average, the likelihood of quitting increased by 2.6% (p<0.05, 95% CI: 0.1%-4.6%) per occurrence of brief intervention. For patients with a recent history of current smoking whose home site experienced systems change, the likelihood of an office visit for smoking-related diagnoses decreased by 4.3% on an annualized basis after systems change occurred (p<0.05, 95% CI: 0.5%-8.1%). There was no change in the likelihood of an office visit for smoking-related diagnoses following systems change among non-smokers. CONCLUSIONS: The clinical practice data from HVMA suggest that a systems approach can lead to significant reductions in smoking prevalence and the rate of office visits for smoking-related diseases. Most comprehensive tobacco intervention strategies focus on the provider or the tobacco user, but these results argue that health systems should be included as an integral component of a comprehensive tobacco intervention strategy. The HVMA results also give us an indication of the potential health impacts when meaningful use core tobacco measures are widely adopted.
INTRODUCTION: Practice-Based Research Networks (PBRNs) and health systems may provide timely, reliable data to guide the development and distribution of public health resources to promote healthy behaviors, such as quitting smoking. The objective of this study was to determine if PBRN data could be used to make neighborhood-level estimates of smoking prevalence. METHODS: We estimated the smoking prevalence in 32 greater Boston neighborhoods (population = 877,943 adults) by using the electronic health record data of adults who in 2009 visited one of 26 Partners Primary Care PBRN practices (n = 77,529). We compared PBRN-derived estimates to population-based estimates derived from 1999-2009 Behavioral Risk Factor Surveillance System (BRFSS) data (n = 20,475). RESULTS: The PBRN estimates of neighborhood smoking status ranged from 5% to 22% and averaged 11%. The 2009 neighborhood-level smoking prevalence estimates derived from the BRFSS ranged from 5% to 26% and averaged 13%. The difference in smoking prevalence between the PBRN and the BRFSS averaged -2 percentage points (standard deviation, 3 percentage points). CONCLUSION: Health behavior data collected during routine clinical care by PBRNs and health systems could supplement or be an alternative to using traditional sources of public health data.
Stiffness control of balance during dual task and prospective falls in older adults: the MOBILIZE Boston Study
Outdoor fallers differ from indoor fallers substantially in demographics, lifestyle, health condition and physical function. Biomechanical predictors of outdoor falls have not been well characterized. Current validated measures of postural deficits, which describe only the overall postural behavior, are predictive of indoor falls but not outdoor falls. We hypothesized that a model-based description of postural muscle tone and reflexes, particularly during dual tasking, would predict outdoor falls. We tested whether postural stiffness and damping from an inverted pendulum model were predictive of future indoor and outdoor falls among older adults from the MOBILIZE Boston Study. The center of pressure data during standing were obtained from 717 participants aged 77.9+/-5.3 years. Participants stood barefoot with eyes open for 30s per trial, in two sets of five standing trials. One set included a serial subtractions task. Postural stiffness and damping values were determined from the postural sway data. After the postural measurements, falls were monitored prospectively using a monthly mail-in calendar over 6-36 months. Associations of postural measures with fall rates were determined using negative binomial regressions. After covariate adjustments, postural stiffness (p=0.02-0.05) and damping (p=0.007-0.1) were associated with lower outdoor falls risk, but not with indoor falls. Results were invariant by direction (anteroposterior versus mediolateral) or by condition (quiet standing versus dual task). Outdoor fall risk may be tied to postural control more so than indoor falls. Dual tasking is likely related to fall risk among older and sicker older adults, but not those relatively healthy.
Long-term effects on cognitive function of postmenopausal hormone therapy prescribed to women aged 50 to 55 years
IMPORTANCE: Postmenopausal hormone therapy with conjugated equine estrogens (CEEs) may adversely affect older women's cognitive function. It is not known whether this extends to younger women. OBJECTIVE: To test whether prescribing CEE-based hormone therapy to postmenopausal women aged 50 to 55 years has longer-term effects on cognitive function. DESIGN: Trained, masked staff assessed participants with an annual telephone-administered cognitive battery that included measures of global and domain-specific cognitive functions. Cognitive testing was conducted an average of 7.2 years after the trials ended, when women had a mean age of 67.2 years, and repeated 1 year later. Enrollment occurred from 1996 through 1999. SETTING: Forty academic research centers. PARTICIPANTS: The study population comprised 1326 postmenopausal women, who had begun treatment in 2 randomized placebo-controlled clinical trials of hormone therapy when aged 50 to 55 years. INTERVENTION: The clinical trials in which the women had participated had compared 0.625 mg CEE with or without 2.5 mg medroxyprogesterone acetate over a mean of 7.0 years. MAIN OUTCOMES AND MEASURES: The primary outcome was global cognitive function. Secondary outcomes were verbal memory, attention, executive function, verbal fluency, and working memory. RESULTS: Global cognitive function scores from women who had been assigned to CEE-based therapies were similar to those from women assigned to placebo: mean (95% CI) intervention effect of 0.02 (-0.08 to 0.12) standard deviation units (P = .66). Similarly, no overall differences were found for any individual cognitive domain (all P > .15). Prespecified subgroup analyses found some evidence that CEE-based therapies may have adversely affected verbal fluency among women who had prior hysterectomy or prior use of hormone therapy: mean treatment effects of -0.17 (-0.33 to -0.02) and -0.25 (-0.42 to -0.08), respectively; however, this may be a chance finding. CONCLUSIONS AND RELEVANCE: CEE-based therapies produced no overall sustained benefit or risk to cognitive function when administered to postmenopausal women aged 50 to 55 years. We are not able to address whether initiating hormone therapy during menopause and maintaining therapy until any symptoms are passed affects cognitive function, either in the short or longer term. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01124773.
Protective efficacy of a global HIV-1 mosaic vaccine against heterologous SHIV challenges in rhesus monkeys
The global diversity of HIV-1 represents a critical challenge facing HIV-1 vaccine development. HIV-1 mosaic antigens are bioinformatically optimized immunogens designed for improved coverage of HIV-1 diversity. However, the protective efficacy of such global HIV-1 vaccine antigens has not previously been evaluated. Here, we demonstrate the capacity of bivalent HIV-1 mosaic antigens to protect rhesus monkeys against acquisition of infection following heterologous challenges with the difficult-to-neutralize simian-human immunodeficiency virus SHIV-SF162P3. Adenovirus/poxvirus and adenovirus/adenovirus vector-based vaccines expressing HIV-1 mosaic Env, Gag, and Pol afforded a significant reduction in the per-exposure acquisition risk following repetitive, intrarectal SHIV-SF162P3 challenges. Protection against acquisition of infection correlated with vaccine-elicited binding, neutralizing, and functional nonneutralizing antibodies, suggesting that the coordinated activity of multiple antibody functions may contribute to protection against difficult-to-neutralize viruses. These data demonstrate the protective efficacy of HIV-1 mosaic antigens and suggest a potential strategy for the development of a global HIV-1 vaccine. PAPERCLIP:
Tailoring community-based wellness initiatives with latent class analysis--Massachusetts Community Transformation Grant projects
INTRODUCTION: Community-based approaches to preventing chronic diseases are attractive because of their broad reach and low costs, and as such, are integral components of health care reform efforts. Implementing community-based initiatives across Massachusetts' municipalities presents both programmatic and evaluation challenges. For effective delivery and evaluation of the interventions, establishing a community typology that groups similar municipalities provides a balanced and cost-effective approach. METHODS: Through a series of key informant interviews and exploratory data analysis, we identified 55 municipal-level indicators of 6 domains for the typology analysis. The domains were health behaviors and health outcomes, housing and land use, transportation, retail environment, socioeconomics, and demographic composition. A latent class analysis was used to identify 10 groups of municipalities based on similar patterns of municipal-level indicators across the domains. RESULTS: Our model with 10 latent classes yielded excellent classification certainty (relative entropy = .995, minimum class probability for any class = .871), and differentiated distinct groups of municipalities based on health-relevant needs and resources. The classes differentiated healthy and racially and ethnically diverse urban areas from cities with similar population densities and diversity but worse health outcomes, affluent communities from lower-income rural communities, and mature suburban areas from rapidly suburbanizing communities with different healthy-living challenges. CONCLUSION: Latent class analysis is a tool that may aid in the planning, communication, and evaluation of community-based wellness initiatives such as Community Transformation Grants projects administrated by the Centers for Disease Control and Prevention.
Multivitamin and mineral use and breast cancer mortality in older women with invasive breast cancer in the women's health initiative
Multivitamin use is common in the United States. It is not known whether multivitamins with minerals supplements (MVM) used by women already diagnosed with invasive breast cancer would affect their breast cancer mortality risk. To determine prospectively the effects of MVM use on breast cancer mortality in postmenopausal women diagnosed with invasive breast cancer, a prospective cohort study was conducted of 7,728 women aged 50-79 at enrollment in the women's health initiative (WHI) in 40 clinical sites across the United States diagnosed with incident invasive breast cancer during WHI and followed for a mean of 7.1 years after breast cancer diagnosis. Use of MVM supplements was assessed at WHI baseline visit and at visit closest to breast cancer diagnosis, obtained from vitamin pill bottles brought to clinic visit. Outcome was breast cancer mortality. Hazard ratios and 95 % confidence intervals (CIs) for breast cancer mortality comparing MVM users to non-users were estimated using Cox proportional hazard regression models. Analyses using propensity to take MVM were done to adjust for potential differences in characteristics of MVM users versus non-users. At baseline, 37.8 % of women reported MVM use. After mean post-diagnosis follow-up of 7.1 +/- 4.1 (SD) years, there were 518 (6.7 %) deaths from breast cancer. In adjusted analyses, breast cancer mortality was 30 % lower in MVM users as compared to non-users (HR = 0.70; 95 % CI 0.55, 0.91). This association was highly robust and persisted after multiple adjustments for potential confounding variables and in propensity score matched analysis (HR = 0.76; 95 % CI 0.60-0.96). Postmenopausal women with invasive breast cancer using MVM had lower breast cancer mortality than non-users. The results suggest a possible role for daily MVM use in attenuating breast cancer mortality in women with invasive breast cancer but the findings require confirmation.
Overcoming inertia: increasing public health departments' access to evidence-based information and promoting usage to inform practice
In 2010, the New England Region-National Network of Libraries of Medicine at University of Massachusetts Medical School received funding to improve information access for public health departments in 6 New England states and Colorado. Public health departments were provided with desktop digital access to licensed e-resources available through special pricing. In January through mid-April 2012, we evaluated the effectiveness of providing access to and training for using e-resources to public health department staff to motivate usage in practice. We found that additional strategies are needed to accomplish this.
BACKGROUND: Of the 22 known measures of nicotine dependence (ND), none capture the overall disease severity of physical dependence alone. Instead, they capture constructs related to dependence, such as perceived risk, psychological addiction, smoker motivations, or smoking related behaviors, but none of the measures include only physical withdrawal symptoms to capture physical dependence on nicotine.
AIM: To develop a range of nicotine dependence measures that capture physical dependence on nicotine.
METHODS: The final measures were developed in a cross-sectional study conducted in three phases: 1) candidate item development through literature review and cognitive interviews, 2) developing and pre-testing the survey, and 3) survey administration and psychometric evaluation to validate three distinct measures. The final survey was conducted at four health clinics and three high schools. Psychometric tests used to select the final measure items included inter-item correlations, sensitivity analyses done by subgroup, item-total correlations, convergent validity tests, and confirmatory factor analysis. The final measures were evaluated using confirmatory factor analysis (CFA), internal reliability, total score distributions, and convergent validity correlations. Relative validity analyses were also conducted using a ratio of F-Statistics to compare the ability of each new measure to differentiate dependent smokers as compared previous measures.
RESULTS: The final sample included 275 smokers ranging from 14 to 76 years old (mean=30.9, SD=16.2), who smoked an average of 11.5 cigarettes per day (range=0-50, SD=9.4). The sample was 86.5% white and 57.5% male. The three new measures developed included: 1) the 4-item Withdrawal-Induced Craving Scale (WICS) used to capture severity of craving, the most common physical withdrawal symptom; 2) the 12- item Nicotine Withdrawal Symptom Checklist (NWSC), which measures both overall disease severity and the severity of a comprehensive list of individual physical withdrawal symptoms including withdrawal-induced craving, anger, anxiety, depression, headache, insomnia, loss of focus, restlessness, and stress; and 3) the 6-item brief NWSC (NWSC-b), a short measure which only captures overall disease severity. All of the new measures exhibited a unidimensional factor structure loading highly on a single factor (thought to be physical dependence). They also correlated highly (over 0.6) and significantly (p<0.001) to a battery of convergent validity indices including four widely used nicotine dependence measures: Hooked on Nicotine Checklist (HONC), the Autonomy Over Tobacco Scale (AUTOS), the Fagerström Test for Nicotine Dependence (FTND), and self-rated addiction.
CONCLUSION: The WICS, NWSC, and NWSC-b provide three distinct validated tools that can be used by researchers, clinicians, and educators to track the progression of physical dependence on nicotine across a range of smoking behaviors and histories.
The worldwide prevalence of obesity and metabolic disease is increasing at an exponential rate and current projections provide no indication of relief. This growing burden of obesity-related metabolic disorders, including type 2 diabetes mellitus (T2DM), highlights the importance of identifying how lifestyle choices, genetics and physiology play a role in metabolic disease and place obese individuals at a greater risk for obesity-related complications including insulin resistance (IR). This increased risk of IR, which is characterized by a decreased response to insulin in peripheral tissues including adipose tissue (AT) and liver, is associated with a chronic, low grade inflammatory state; however, the causative connections between obesity and inflammation remains in question. Experimental evidence suggests that adipocytes and macrophages can profoundly influence obesity-induced IR because adipocyte dysfunction leads to ectopic lipid deposition in peripheral insulin sensitive tissues, and obese AT is characterized by increased local inflammation and macrophage and other immune cell populations. Attempts to delineate the individual roles of macrophage-derived pro-inflammatory cytokines, like tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β), have demonstrated causative roles in impaired systemic insulin sensitivity, adipocyte function and hepatic glucose and lipid metabolism in obese animal models. Thus, the attenuation of macrophage-derived inflammation is an evolving area of interest to provide insight into the underlying mechanism(s) leading to obesity-induced IR.
Thus, in the first chapter of this thesis, I describe experiments to refine the current paradigm of obesity-induced AT inflammation by combining gene expression profiling with computational analysis of two anatomically distinct AT depots, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) to address whether the inflammatory signature of AT is influenced by diet-induced obesity (DIO). Microarray and qRT-PCR analysis data revealed that DIO mouse SAT is resistant to high fat diet (HFD)-induced inflammation and macrophage infiltration, and our data support the current model of obesity-induced visceral adipose tissue macrophage (VATM) enrichment. Our data demonstrated robust increases in VAT pro-inflammatory cytokine expression, which are consistent with the significant increases in macrophage-specific gene expression and consistent with previous reports in which VAT inflammation is enhanced and attributed to classically activated (M1) macrophage infiltration. However, these data are only observed relative to the expression of invariant housekeeping gene expression. When M1-specific genes are expressed relative to macrophage-specific standards like F4/80 expression, these inflammatory makers are unchanged. These data indicate that the changes in the overall inflammatory profile of DIO mouse VAT is because of quantitative changes in adipose tissue macrophage (ATM) number and not qualitative changes in activation state. These observations are consistent with the idea that infiltrating ATMs may have roles other than the previously described role in mediating inflammation in obese adipose tissue.
Hepatic IR occurs partly as a consequence of adipocyte dysfunction because the liver becomes a reservoir for AT-derived fatty acids (FAs), which leads to obesity-related non-alcoholic fatty liver disease (NAFLD). In the second part of my thesis, I used clodronate liposome-mediated macrophage depletion to define the role of macrophages in hepatic lipid metabolism regulation. We discovered that i.p. administration of clodronate liposomes depletes Kupffer cells (KCs) in ob/ob mice without affecting VATM content, whereas clodronate liposomes depletes both KCs and VATMs in DIO mice. To this end, we established that clodronate liposome-mediated KC depletion, regardless of VATM content in obese mice, abrogated hepatic steatosis by reducing hepatic de novo lipogenic gene expression. The observed reductions in hepatic inflammation in macrophage-depleted obese mice led to the hypothesis that IL-1β may be responsible for obesity-induced increased hepatic triglyceride (TG) accumulation. We determined that IL-1β treatment increases fatty acid synthase (Fas) protein expression and TG accumulation in primary mouse hepatocytes. Pharmacological inhibition of interleukin-1 (IL-1) signaling by interleukin-1 receptor antagonist (IL-1Ra) administration recapitulated these results by reducing hepatic TG accumulation and lipogenic gene expression in DIO mice. Thus, these data highlight the importance of the inflammatory cytokine IL-1β in obesity-driven hepatic steatosis and suggests that liver inflammation controls hepatic lipogenesis in obesity.
To this end, the studies described herein provide new insight and appreciation to the multi-functional nature of macrophages and clinical implications for anti-inflammatory therapy in obesity and NAFLD treatment. We demonstrate the complexities of macrophage-mediated functions in insulin sensitive tissues and a role for obesity-induced inflammatory cytokine IL-1β in hepatic lipid metabolism modulation, which is reversed via IL-1Ra intervention. The use of anti-inflammatory therapy to ameliorate obesity-associated NAFLD was perhaps the most important contribution to this body of work and is full of promise for future clinical application. It is likely that the future of therapeutics will be multi-faceted and combine therapeutic approaches to enhance glucose tolerance and overall health in obese, IR and T2DM patients.
Investigating Tumor Suppressors in the DNA Damage Response: Caretakers of the Genome and Biomarkers to Predict Therapeutic Response: A Dissertation
Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA).
My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome.
The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.
How do embryos develop with such poise from a single zygote to multiple cells with different identities, and yet survive? At the four-cell stage of the C. elegans embryo, only the blastomere EMS adopts the endo-mesoderm identity. This fate requires SKN-1, the master regulator of endoderm and mesoderm differentiation. However, in the absence of the RNA binding protein POS-1, EMS fails to fulfill its fate despite the presence of SKN-1. pos-1(-) embryos die gutless. Conversely, the RNA binding protein MEX-5 prevents ectoderm blastomeres from adopting the endo-mesoderm identity by repressing SKN-1. mex-5(-) embryos die with excess muscle at the expense of skin and neurons.
Through forward and reverse genetics, I found that genes gld-3/Bicaudal C, cytoplasmic adenylase gld-2, cye-1/Cyclin E, glp-1/Notch and the novel gene neg-1 are suppressors that restore gut development despite the absence of pos-1. Both POS-1 and MEX-5 bind the 3’UTR of neg-1 mRNA and its poly(A) tail requires GLD-3/2 for elongation. Moreover, neg-1 requires MEX-5 for its expression in anterior ectoderm blastomeres and is repressed in EMS by POS-1. Most neg-1(-) embryos die with defects in anterior ectoderm development where the mesoderm transcription factor pha-4 becomes ectopically expressed. This lethality is reduced by the concomitant loss of med- 1, a key mesoderm-promoting transcription factor.
Thus the endo-mesoderm identity of EMS is determined by the presence of SKN- 1 and the POS-1 repression of neg-1, whose expression is promoted by MEX-5. Together they promote the anterior ectoderm identity by repressing mesoderm differentiation. Such checks and balances ensure the vital plurality of cellular identity without the lethal tyranny of a single fate.
Approximately half of all emergency department (ED) visits are primary-care sensitive (PCS) – meaning that they could potentially be avoided with timely, effective primary care. Reducing undesirable types of healthcare utilization (including PCS ED use) requires the ability to define, measure, and predict such use in a population.
In this retrospective, observational study, we quantified ED use in 2 privately insured populations and developed ED risk prediction models. One dataset, obtained from a Massachusetts managed-care network (MCN), included data from 2009-11. The second was the MarketScan database, with data from 2007-08. The MCN study included 64,623 individuals enrolled for at least 1 base-year month and 1 prediction-year month in Massachusetts whose primary care provider (PCP) participated in the MCN. The MarketScan study included 15,136,261 individuals enrolled for at least 1 base-year month and 1 prediction-year month in the 50 US states plus DC, Puerto Rico, and the US Virgin Islands.
We used medical claims to identify principal diagnosis codes for ED visits, and scored each according to the New York University Emergency Department algorithm. We defined primary-care sensitive (PCS) ED visits as those in 3 subcategories: nonemergent, emergent but primary-care treatable, and emergent but preventable/avoidable.
We then: 1) defined and described the distributions of 3 ED outcomes: any ED use; number of ED visits; and a new outcome, based on the NYU algorithm, that we call PCS ED use; 2) built and validated predictive models for these outcomes using administrative claims data; 3) compared the performance of models predicting any ED use, number of ED visits, and PCS ED use; 4) enhanced these models by adding enrollee characteristics from electronic medical records, neighborhood characteristics, and payor/provider characteristics, and explored differences in performance between the original and enhanced models.
In the MarketScan sample, 10.6% of enrollees had at least 1 ED visit, with about half of utilization scored as PCS. For the top risk group (those in the 99.5th percentile), the model’s sensitivity was 3.1%, specificity was 99.7%, and positive predictive value (PPV) was 49.7%. The model predicting PCS visits yielded sensitivity of 3.8%, specificity of 99.7%, and PPV of 40.5% for the top risk group.
In the MCN sample, 14.6% (±0.1%) had at least 1 ED visit during the prediction period, with an overall rate of 18.8 (±0.2) visits per 100 persons and 7.6 (±0.1) PCS ED visits per 100 persons. Measuring PCS ED use with a threshold-based approach resulted in many fewer visits counted as PCS, discarding information unnecessarily. Out of 45 practices, 5 to 11 (11-24%) had observed values that were statistically significantly different from their expected values. Models predicting ED utilization using age, sex, race, morbidity, and prior use only (claims-based models) had lower R2 (ranging from 2.9% to 3.7%) and poorer predictive ability than the enhanced models that also included payor, PCP type and quality, problem list conditions, and covariates from the EMR, Census tract, and MCN provider data (enhanced model R2 ranged from 4.17% to 5.14%). In adjusted analyses, age, claims-based morbidity score, any ED visit in the base year, asthma, congestive heart failure, depression, tobacco use, and neighborhood poverty were strongly associated with increased risk for all 3 measures (all P<.001).
CD8+ T Cell and NK Responses to a Novel Dengue Epitope: A Possible Role for KIR3DL1 in Dengue Pathogenesis: A Dissertation
Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections contributing to pathology following DENV infection. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein, NS126-34. We predicted higher frequencies of NS126-34-specific CD8+ T cells in PBMC from individuals undergoing secondary, rather than primary, DENV infection due to the expansion of memory CD8+T cells. We generated a tetramer against this epitope (B57-NS126-34TET) and used it to assess the frequencies and phenotype of antigen-specific T cells in samples from a clinical cohort of children with acute DENV infection established in Bangkok, Thailand. High tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS126-34-specific, other DENV epitope-specific CD8+ T cells, as well as total CD8+ T cells, expressed an activated phenotype (CD69+ and/or CD38+) during acute infection. In contrast, expression of CD71 was largely limited to DENV-specific CD8+ T cells. In vitro stimulation of CD8+ T cell lines, generated against three different DENV epitopes, indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides with substantial upregulation of CD71 detected to peptides which also elicited strong functional responses. CD71 may therefore represent a useful marker of antigenspecific T cell activation.
During the course of our analysis we found substantial binding of B57-NS126-34 TET to CD8- cells. We demonstrated that the B57-NS126-34 TET bound KIR3DL1, an inhibitory receptor on natural killer (NK) cells. NK sensitive target cells presenting the NS126-34 peptide in the context of HLA-B57 were able to dampen functional responses of only KIR3DL1+ NK cells. Analysis of the activation of an NK enriched population in our Thai cohort revealed peak activation during the critical time phase in patients with severe dengue illness, dengue hemorrhagic fever, compared to people with mild illness.
Our data identified CD71 as biologically useful marker to study DENV-specific CD8+ T cell responses and highlighted the role of viral peptides in modulating NK cell activation through KIR-MHC class I interactions during DENV infection.