Periodontal Inflamed Surface Area (PISA) associates with composites of salivary cytokines.

BACKGROUND: Periodontal disease (PerioD) is a chronic, complex inflammatory condition resulting from the interaction between subgingival dysbiotic bacteria and the host immune response leading to local inflammation. Since periodontal inflammation is characterized by multiple cytokines effects we investigated whether Periodontal Inflamed Surface Area (PISA), a continuous measure of clinical periodontal inflammation is a predictor of composite indexes of salivary cytokines. METHODS AND FINDINGS: In a cross-sectional study of 67 healthy, well-educated individuals, we evaluated PISA and several cytokines expressed in whole stimulated saliva. Two salivary cytokine indexes were constructed using weighted and unweighted approaches based on a Principal Component Analysis [named Cytokine Component Index (CCI)] or averaging the (standardized) level of all cytokines [named Composite Inflammatory Index (CII)]. In regression analysis we found that PISA scores were significantly associated with both salivary cytokine constructs, (CCI: part R = 0.51, p<0.001; CII: part R = 0.40, p = 0.001) independent of age, gender and BMI showing that single scores summarizing salivary cytokines correlated with severity of clinical periodontal inflammation. CONCLUSIONS: Clinical periodontal inflammation may be reflected by a single score encompassing several salivary cytokines. These results are consistent with the complexity of interactions characterizing periodontal disease. In addition, Type I error is likely to be avoided.

Electronic Cigarette Aerosol Modulates the Oral Microbiome and Increases Risk of Infection.

The trend of e-cigarette use among teens is ever increasing. Here we show the dysbiotic oral microbial ecology in e-cigarette users influencing the local host immune environment compared with non-smoker controls and cigarette smokers. Using 16S rRNA high-throughput sequencing, we evaluated 119 human participants, 40 in each of the three cohorts, and found significantly altered beta-diversity in e-cigarette users (p = 0.006) when compared with never smokers or tobacco cigarette smokers. The abundance of Porphyromonas and Veillonella (p = 0.008) was higher among vapers. Interleukin (IL)-6 and IL-1ß were highly elevated in e-cigarette users when compared with non-users. Epithelial cell-exposed e-cigarette aerosols were more susceptible for infection. In vitro infection model of premalignant Leuk-1 and malignant cell lines exposed to e-cigarette aerosol and challenged by Porphyromonas gingivalis and Fusobacterium nucleatum resulted in elevated inflammatory response. Our findings for the first time demonstrate that e-cigarette users are more prone to infection.

Rapid diagnosis of Zika virus through saliva and urine by Loop-mediated isothermal amplification (LAMP).

Background: Zika virus (ZIKV) is a single-stranded RNA virus and member of the Flaviviridae family. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood greatly simplifying the assay. Loop-mediated Isothermal Amplification (LAMP) is a rapid assay that detects nucleic acids, including ZIKV RNA. Aim: The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. Method: A total of 131 samples (68 saliva and 63 urine samples) from 69 subjects in the acute phase of ZIKV infection, and confirmed positive for ZIKV by blood analysis through real time-PCR, were collected and analyzed by Reverse Transcriptase Loop-mediated Isothermal Amplification (RT-LAMP). Results: From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 min, and from the 63 urine samples, 25 (39.7%) were positive with the average Tp of 15.8 min. Saliva detected more samples (p = 0.0042) and had faster Tp (p = 0.0176) as compared with urine. Conclusion: Saliva proved to be a feasible alternative to diagnose ZIKV infection during the acute phase by LAMP.

Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

Zika Virus Specific Diagnostic Epitope Discovery.

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for drug discovery, therapeutic target identification, and developing of diagnostics. Here, we present a protocol to discover specific Zika virus (ZIKV) diagnostic peptides using a high-density peptide microarray. A human serum sample validated for ZIKV infection was incubated with a high-density peptide microarray containing the entire ZIKV protein translated into 3,423 unique 15 linear amino acid (aa) residues with a 14-aa residue overlap printed in duplicate. Staining with different secondary antibodies within the same array, we detected peptides that bind to Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies present in serum. These peptides were selected for further validation experiments. In this protocol, we describe the strategy followed to design, process, and analyze a high-density peptide microarray.

A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti-HIV Antibodies and Viral RNA.

OBJECTIVE: We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. METHODS: We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention. RESULTS: The finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample. CONCLUSION: The ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for the "Test and Treat" and "Treatment as Prevention" approaches for controlling the HIV epidemic.

Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification.

Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject's blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200-2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

Design aspects of a case-control clinical investigation of the effect of HIV on oral and gastrointestinal soluble innate factors and microbes.

INTRODUCTION: The impaired host defense system in HIV infection impacts the oral and gastrointestinal microbiota and associated opportunistic infections. Antiretroviral treatment is predicted to partially restore host defenses and decrease the oral manifestation of HIV/AIDS. Well-designed longitudinal studies are needed to better understand the interactions of soluble host defense proteins with bacteria and virus in HIV/AIDS. "Crosstalk" was designed as a longitudinal study of host responses along the gastrointestinal (GI) tract and interactions between defense molecules and bacteria in HIV infection and subsequent therapy. PURPOSE: The clinical core formed the infrastructure for the study of the interactions between the proteome, microbiome and innate immune system. The core recruited and retained study subjects, scheduled visits, obtained demographic and medical data, assessed oral health status, collected samples, and guided analysis of the hypotheses. This manuscript presents a well-designed clinical core that may serve as a model for studies that combine clinical and laboratory data. METHODS: Crosstalk was a case-control longitudinal clinical study an initial planned enrollment of 170 subjects. HIV+ antiretroviral naïve subjects were followed for 9 visits over 96 weeks and HIV uninfected subjects for 3 visits over 24 weeks. Clinical prevalence of oral mucosal lesions, dental caries and periodontal disease were assessed. RESULTS: During the study, 116 subjects (47 HIV+, 69 HIV-) were enrolled. Cohorts of HIV+ and HIV- were demographically similar except for a larger proportion of women in the HIV- group. The most prevalent oral mucosal lesions were oral candidiasis and hairy leukoplakia in the HIV+ group. DISCUSSION: The clinical core was essential to enable the links between clinical and laboratory data. The study aims to determine specific differences between oral and GI tissues that account for unique patterns of opportunistic infections and to delineate the differences in their susceptibility to infection by HIV and their responses post-HAART.

An electrochemiluminescence assay for gp340 (DMBT1).

Gp340 is a member of the scavenger receptor cysteine-rich family of innate immune molecules and also functions as a tumor suppressor. This study describes a picogram-level assay using electrochemiluminescence technology on the MesoScale Discovery platform. Antibodies were evaluated and the best pair was used to assay whole-mouth stimulated saliva and cervical/vaginal lavage. The assay was tested using specimens obtained from healthy volunteers to determine if gp340 concentration in saliva correlates with levels in vaginal lavage fluid. Interestingly, no correlation was determined between gp340 content in these two fluids.

Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis.

BACKGROUND: A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. METHODS: A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. RESULTS: This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. CONCLUSIONS: The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples.

Patient and provider acceptance of oral HIV screening in a dental school setting.

In 2006, the Centers for Disease Control and Prevention (CDC) recommended routine HIV screening in health care settings regardless of the patient's level of risk. This pilot study was developed in response to the suggestion by some health care professionals that dental settings would be appropriate for expansion of HIV testing. This project consisted of two parts: oral fluid HIV testing of patients in the clinic of a dental school and a survey of the clinical dental faculty members' attitudes about acceptability of routine HIV testing in the dental clinic. When patients' agreement to participate in oral fluid HIV testing was examined, 8.2 percent of the patients contacted by the clinic administration staff completed testing. When approached by a faculty member or student during the dental visit admission and tested during the dental visit, however, 88.2 percent completed testing. Of the faculty members who took the survey, 27.4 percent were neutral, 26.4 percent were somewhat in agreement, and 32.1 percent were willing to incorporate HIV testing into routine dental care. In this pilot study, HIV testing of dental patients was most successful when a dental care provider approached patients about testing. If consent was given, the testing was performed during the visit. For the faculty members, the major barrier to testing was a lack of protocol familiarity.

Finger-actuated, self-contained immunoassay cassettes.

The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity.

Stroke rates: 1980-2000: the Minnesota Stroke Survey.

In this paper, the authors report trends in hospitalized stroke rates among Minneapolis-St. Paul, Minnesota (population 2.6 million) metropolitan area residents aged 30-74 years from 1980 to 2000. Cases were identified from lists of discharge diagnoses provided by hospitals serving the target population. Age-adjusted, sex-specific stroke attack rates were computed for each survey year by using 5 different diagnostic definitions: 2 based purely on International Classification of Diseases, Ninth Revision (ICD-9) codes and 3 including clinical and neuroimaging criteria. Stroke rates, as measured by a highly specific clinical definition, remained stable from 1980 to 2000 for women. For men, these rates declined modestly from 1980 to 1990 and leveled off during 1990-2000. In contrast, use of stroke-related ICD-9 discharge codes declined significantly from 1980 to 2000: 35% among men and 16% among women. Neuroimaging use increased significantly from 75% of cases in 1980 to 98% in 2000. Short-term (28-day) stroke survival improved significantly, by 16% for women and 12% for men, from 1980 to 2000. The decline in stroke ICD-9 code usage reflects the influence of increased neuroimaging on discharge coding. The improved short-term survival in the face of stable, clinically defined stroke rates may imply treatment advances or ascertainment of less severe strokes, possibly masking a true decline in stroke rates.

Trends in cigarette smoking: the Minnesota Heart Survey, 1980-1982 through 2000-2002.

This paper reports population-based secular trends in smoking prevalence and tobacco exposure among smokers. The Minnesota Heart Survey (MHS) assessed smoking in probability samples in the seven-county Minneapolis-St. Paul metropolitan area. Five surveys were conducted in 1980-1982, 1985-1987, 1990-1992, 1995-1997, and 2000-2002 using similar sampling strategies and consistent protocols. Participants were metropolitan area residents of Minnesota, aged 25-74 years, with the addition of 75-84-year-olds in the last three surveys. In men, age-adjusted self-reported prevalence of current smoking steadily declined from 32.9% in 1980-1982, to 23.0% in 1995-1997, and to 20.6% in 2000-2002. In women, self-reported smoking was 31.8% in 1980-1982, 18.5% in 1995-1997, and 19.5% in the latest survey. Age-adjusted self-reported quantity of cigarettes consumed among smokers declined over the same period. Changes from 1995-1997 to 2000-2002 were not significant. Compared with Whites, Black participants had higher levels of smoking and later onset of the decline in smoking prevalence. A decline in smoking prevalence seems to have leveled off or reversed between the two most recent survey periods (1995-1997 through 2000-2002). Focus on smoking cessation should continue, especially in the subpopulation that smokes more than the majority.

Relation of length of hospital stay in acute myocardial infarction to postdischarge mortality.

Hospital length of stay (LOS) after acute myocardial infarction (AMI) has steadily decreased because of both improved treatments and cost considerations. Early discharge may adversely affect some patients who might benefit from extended monitoring. The Minnesota Heart Survey was a population-based study of patients with AMI in acute-care hospitals in the Minneapolis-St. Paul, Minnesota, metropolitan area. Medical records were abstracted for a random sample of patients hospitalized with AMI in 1985, 1990, 1995, and 2001. Case fatality rates, adjusted for age and gender, were identified using mortality data from the index hospitalization and Minnesota death certificates. A total of 4,940 patients with a validated AMI were identified from the combined 1985 (n = 1,306), 1990 (n = 1,550), 1995 (n = 1,087), and 2001 (n = 515) surveys. Median LOSs were 9, 8, 6, and 4 days, respectively. Patients hospitalized

Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in patients with congestive heart failure and chronic kidney disease.

BACKGROUND: Patients with coexistent heart failure and chronic kidney disease (CKD) have a poor prognosis, possibly related to the underuse of standard medical therapies--angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARB). METHODS: We performed a retrospective analysis of the Minnesota Heart Survey, identifying patients hospitalized in 2000 in the Minneapolis-St Paul metropolitan area with heart failure. The main outcome measure was the association of ACE-I and ARB use on 30-day and 1-year mortality, stratified by glomerular filtration rate (GFR). RESULTS: Compared to patients with heart failure with preserved renal function (GFR > or = 90 mL/min), patients with severely impaired renal function (GFR <15 mL/min) were far less likely to receive ACE-I or ARB during hospitalization (52.0% vs 69.5%, P < .0001) or at discharge (50.5% vs 65.1%, P < .0001). Worsening renal function was associated with increased mortality, both at 30 days and at 1 year. The inhospital use of either an ACE-I or ARB was associated with significantly reduced 30-day mortality (OR 0.45, 95% CI 0.28-0.59) after adjusting for multiple risk factors. Similarly, the discharge prescription of either an ACE-I or ARB was associated with a significant reduction in adjusted 1-year mortality (OR 0.72, 95% CI 0.58-0.91). However, among patients on dialysis, there was no benefit of ACE-I or ARB on either 30-day or 1-year mortality. CONCLUSIONS: Angiotensin-converting enzyme inhibitors and ARB are underused in patients with heart failure with chronic kidney disease. Given the reduction in 30-day and 1-year mortality, these medications should be considered in most patients with heart failure, independent of underlying renal function. Among patients on hemodialysis, further investigation is warranted.

Development of a microfluidic device for detection of pathogens in oral samples using upconverting phosphor technology (UPT).

Confirmatory detection of diseases, such as HIV and HIV-associated pathogens in a rapid point-of-care (POC) diagnostic remains a goal for disease control, prevention, and therapy. If a sample could be analyzed onsite with a verified result, the individual could be counseled immediately and appropriate therapy initiated. Our group is focused on developing a microfluidic "lab-on-a-chip" that will simultaneously identify antigens, antibodies, RNA, and DNA using a single oral sample. The approach has been to design individual modules for each assay that uses similar components (e.g., valves, heaters, metering chambers, mixers) installed on a polycarbonate base with a common reporter system. Assay miniaturization reduces the overall analysis time, increases accuracy by simultaneously identifying multiple targets, and enhances detector sensitivity by upconverting phosphor technology (UPT). Our microfluidic approach employs four interrelated components: (1) sample acquisition-OraSure UPlink collectors that pick-up and release bacteria, soluble analytes, and viruses from an oral sample; (2) microfluidic processing-movement of microliter volumes of analyte, target analyte extraction and amplification; (3) detection of analytes using UPT particles in a lateral flow system; and (4) software for processing the results. Ultimately, the oral-based microscale diagnostic system will detect viruses and bacteria, associated pathogen antigens and nucleic acids, and antibodies to these pathogens.

Dietary intake of heterocyclic amines and benzo(a)pyrene: associations with pancreatic cancer.

OBJECTIVE: Heterocyclic amines (HCA) and polycyclic aromatic hydrocarbons, formed in temperature- and time-dependent manners during the cooking of meat, are mutagens and carcinogens. We sought to assess the association between dietary intake of HCA and benzo(a)pyrene [B(a)P] and exocrine pancreatic cancer in a population-based case-control study. METHODS: Subjects (193 cases and 674 controls) provided information on their usual meat intake and preparation method, e.g., stewed, fried, or grilled/barbecued, etc. Meat doneness preferences were measured using photographs that showed internal doneness and external brownness. We used a meat-derived HCA, B(a)P, and mutagen database with a questionnaire to estimate intake of PhIP, DiMeIQx, MeIQx, B(a)P, and mutagenic activity (revertants/g of daily meat intake). Data were analyzed with unconditional logistic regression. RESULTS: In analyses adjusted for age, sex, smoking, education, race, and diabetes, the odds ratio and 95% confidence interval for the highest compared with the lowest quintile were as follows: PhIP, 1.8 (1.0-3.1); DiMeIQx, 2.0 (1.2-3.5); MeIQx, 1.5 (0.9-2.7); B(a)P, 2.2 (1.2-4.0); and mutagenic activity, 2.4 (1.3-4.3). CONCLUSIONS: HCAs and B(a)P from well-done barbecued and pan-fried meats may be associated with increased risk for pancreatic cancer.

Hospitalized heart failure: rates and long-term mortality.

BACKGROUND: Heart failure has been called the "new epidemic of cardiovascular disease," but few studies have described key epidemiologic measures of the syndrome in geographically defined US populations. METHODS AND RESULTS: We obtained lists of discharge diagnosis codes in 1995 from 22 Minneapolis-St. Paul metropolitan area hospitals; identified patients 35 to 84 years old with a heart failure discharge code; and sampled and abstracted 50% of the hospital records. To identify heart failure-related hospitalizations, we applied 6 published definitions of the syndrome to the sample and selected cases that met at least 4 of the 6 definitions (n = 2887). The patient cohort was followed for 5 to 6 years to ascertain deaths. The rate of hospitalized heart failure ranged from a few dozen hospitalized patients per 100,000 residents ages 35 to 44 years to more than 2000 per 100,000 residents ages 75 to 84, and was consistently higher among men than among women (age-adjusted rate ratio 1.46; 95% CI 1.39-1.54). Within 1-year of the index admission, 37% of male patients and 30% of female patients have died-10 times the annual mortality of the source population. By the end of the follow-up, cumulative mortality reached 72% in men and 66% in women. In multivariable regression of the hazard of death on age, sex, and left ventricular ejection fraction (LVEF), age was a strong determinant of mortality and male patients had modestly higher hazard of death than female patients (adjusted hazard ratio, 1.29; 95% CI 1.18-1.41). LVEF was not a strong predictor of death. CONCLUSION: A heart failure-related hospitalization is a marker of grave prognosis: only one quarter to one third of the patients survives 5 years after admission. Both the risk of hospitalization for heart failure and the risk of subsequent death are moderately higher in men than in women. LVEF, when measured in the context of heart failure-related hospitalization, is not a strong predictor of death.

Meat intake and cooking techniques: associations with pancreatic cancer.

Heterocyclic amines (HCAs), and polycyclic aromatic hydrocarbons (PAHs), formed in temperature and time-dependent manners during cooking of meat, may increase the risk of certain cancers. As these compounds could be carcinogenic for the pancreas, we assessed meat intake, preparation methods, and doneness preferences as risk factors for exocrine pancreatic cancer. In a case-control study (cases=193, controls=674), subjects provided information on their usual meat intake and how it was cooked, e.g. fried, grilled or barbecued (BBQ), etc. Meat doneness preferences were measured using photographs that showed internal doneness and external brownness with a numerical scale. Data were analyzed with unconditional logistic regression. Odds ratios (ORs) increased with increased intake of grilled/BBQ red meat in an analysis adjusted for age, sex, smoking, education, race, and diabetes. Based on amount of BBQ meat consumed, the OR and 95% confidence interval (CI) for the fifth quintile relative to the reference group (quintiles 1 and 2) was 2.19 (1.4, 3.4). Findings were not substantively changed by further adjustment for calories, total fat, fruit and vegetables, or alcohol consumption (from a food frequency questionnaire (FFQ)). Other meat variables did not show statistically significant associations with risk nor did they substantively alter the findings for BBQ. These included total meat, processed meat, total red meat, total white meat, total broiled meat, total fried meat, or total meat cooked by means other than grilling. We conclude that grilled red meat intake is a risk factor for pancreatic cancer and that method of meat preparation in addition to total intake is important in assessing the effects of meat consumption in epidemiologic studies.