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Introduction: Cytokine growth factors have been ineffective in infected wounds (>105 bacteria/g of tissue) for several reasons. Bacteria degrade the polypeptides themselves, and this effect is accentuated in the presence of tissue cells when excessive matrix metalloproteinases are produced. Also, the growth factor receptors for several cytokines have been shown to have decreased expression in chronic wounds.Materials and Methods: The effects of genomics-based KGF-2 (repifermin) on healing of infected wounds were examined in 3 experiments using a rodent model of a chronically infected (>105 bacteria/g of tissue) granulating wound. Serial wound area measurements were compared among animals treated with vehicle, 2 doses of KGF-2, and KGF-2 in combination with GM-CSF. Binding of biotinylated KGF-2 was compared with binding of a biotinylated irrelevant protein control in sections of both noninfected and infected granulation tissue. Serial biopsies of the wounds for quantitative bacteriology confirmed the wounds were infected throughout the experiments.Results: In all 3 experiments, infected wounds healed faster when topically treated with KGF-2 (P <0.05). The effect was seen most dramatically toward the end of the wound closure curve when the wounds were 75% to 90% closed. Using GM-CSF in combination or in sequence with KGF-2 did not enhance the activity of KGF-2 alone. There was no clear decrease in KGF-2 binding in infected versus noninfected wounds. In contrast, sections incubated with the control-irrelevant protein were uniformly negative. Bacterial counts confirmed the wounds remained at a bacterial tissue level of >105 organisms/g.Conclusion: Repifermin (genomics-based KGF-2) appears more robust than other reported growth factors in the presence of high levels of bacteria. This appears to be the result of its lesser degree of degradation in the presence of bacteria and tissue fibroblasts and the ability of its receptors to remain functional in infected tissue. [ABSTRACT FROM AUTHOR]

Objective: Chronic ear disease presents a unique challenge to otolaryngologists in both rural and urban settings. Cholesteatoma remains a difficult disease to treat in rural populations due to limited healthcare access and high risk of recurrence. The purpose of this study was to determine if there are differences in surgical outcomes among patients with acquired cholesteatoma residing in rural versus urban settings., Study Design: Single-surgeon retrospective case series with chart review., Setting: Tertiary care private otolaryngology practice., Patients: One hundred twenty-two patients presenting to the Kentuckiana ENT otology and neurotology practice from January 2011 to May 2017., Main Outcome Measures: Surgical outcomes including recurrence, air-bone gap improvement, ossicular integrity, and complications were reviewed and compared between the rural and urban cohorts., Results: Presence of postoperative residual cholesteatoma (OR = 8.667, 95% CI = 2.022-37.141, p = 0.008) and number of surgeries per patient (OR = 5.185, 95% CI = 1.086-24.763, p = 0.024) were significantly increased among patients in rural nonmetropolitan areas. No significant differences were found when comparing risk of recurrence, size of cholesteatoma, presence of complications, air-bone gap improvement, and ossicular chain integrity. There were significantly more second-look surgeries performed in privately insured patients (OR = 8.582, 95% CI = 1.937-38.017, p = 0.001)., Conclusions: Patients in rural communities have an increased number of surgeries and postoperative risk for residual cholesteatoma compared to patients residing in urban settings. This study provides the basis for larger, multicenter, prospective examinations of outcomes among urban versus rural patients, which would enable a better understanding of difference in surgical outcomes between rural and urban cohorts.Level of Evidence: IV., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2020, Otology & Neurotology, Inc.)

Posttraumatic stress disorder (PTSD) and substance use disorders (SUD) are highly comorbid among the veteran population. Impulsivity, particularly negative and positive urgency, are prevalent within this dual-diagnosis population and associated with negative outcomes. One possible correlate of negative/positive urgency is intolerance of uncertainty (IU). IU is associated with exacerbated PTSD symptom severity and increased risk for substance use. However, few studies have examined the link between IU and negative/positive urgency in dual-diagnosis populations. This study aimed to examine whether there was a significant association between trait IU and baseline negative and positive urgency in veterans seeking treatment for both PTSD and SUD. In a sample of 114 veterans from a 6-week residential treatment program, IU was significantly associated with higher negative and positive urgency. Further research is warranted to extend these findings and examine whether IU plays an important role in negative/positive urgency for dual-diagnosis populations.

Posttraumatic stress disorder (PTSD) is characterized by strong negative emotions, often in response to trauma cues or reminders. Subsequent emotion regulation strategies impact the maintenance of PTSD symptoms and other trauma-related outcomes (depression, substance use). This study aimed to examine a range of trauma-cued emotions to enhance our understanding of changes following treatment and their potential role in improving relevant outcomes. Participants included 67 veterans diagnosed with PTSD and a substance use disorder who completed a dual diagnosis residential program that used cognitive processing therapy. At pre- and posttreatment, we measured 8 negative emotions following a trauma recall and PTSD symptoms, depressive symptoms, and negative urgency (impulsivity following negative emotions) as treatment outcomes. We used t -tests to assess changes at posttreatment and a within-subjects mediational analysis to test whether changes in trauma-cued emotions mediated treatment outcomes. Participants reported moderate, significant decreases for 5 emotions at posttreatment: anger at self, disgust at self, fear, guilt, and sadness ( d ≥ 0.50), whereas nonsignificant changes were found for anger at others, disgust at others, and shame. Mediation analyses indicated greater reductions in trauma-cued sadness had a significant indirect effect on improvement in PTSD symptoms, depressive symptoms, and negative urgency. Reductions in disgust at self and fear also demonstrated a significant indirect effect on depressive symptom improvement. In this dual diagnosis program, veterans reported a significant reduction in some, but not all, trauma-cued emotions, and improvements in only select emotions accounted for a significant portion of improvement in relevant treatment outcomes.

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes 1 . Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.

The objective was to validate a scientific method for characterizing equine metacarpophalangeal joint (MCPJ) motion in the nonfatigued and fatigued states using a single horse at trot, slow canter, and fast canter. One healthy Thoroughbred gelding exercised on a treadmill to exhaustion (fatigued state) (heart rate >190 BPM and blood lactate >10 mmol/L) while bilateral MCPJ angular data were acquired using electrogoniometry. Blood lactate and heart rate reflected transition from nonfatigued to fatigued states with increasing exercise duration and treadmill speed. Electrogoniometry consistently demonstrated: increase in mean MCPJ maximum extension angle with onset of fatigue; altered extension and flexion angular velocities with onset of fatigue; and increasing stride duration and decreasing stride frequency with onset of fatigue. The method allowed a preliminary but comprehensive characterization of the dynamic relationship between MCPJ kinematics and fatigue, prompting the need for multisubject studies that may enhance our ability to moderate exercise-related distal limb injury in equine athletes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Dysfunctional trauma-related cognitions are important in the emergence and maintenance of posttraumatic stress disorder (PTSD) and the modification of such cognitions is a proposed mechanism of trauma treatment. However, the authors are not aware of any research examining trauma-related cognitions as a treatment mechanism in a sample of individuals with comorbid PTSD and substance use disorder (SUD). Accordingly, the present study sought to address this gap in the literature and examined the relationship between trauma-related cognitions and treatment outcomes within a sample of seventy-two veterans diagnosed with PTSD and SUD. Veterans completed a 6-week day CPT-based treatment program that included cognitive processing therapy as a central component. Measures of trauma-related cognitions, PTSD symptoms, depressive symptoms, and trauma-cued substance craving were completed at pre- and post-treatment. As expected, trauma-related cognitions were associated with several PTSD-related variables prior to treatment. Furthermore, results of a within-subjects mediational analysis indicated that maladaptive trauma-related cognitions decreased during the treatment program and accounted for a significant portion of the variance in the reduction of PTSD and depressive symptoms at post-treatment. This study provides support for the position that attempts to modify dysfunctional trauma-related cognitions among veterans with co-occurring PTSD and SUD can lead to desirable treatment outcomes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Resilience has been associated with less severe psychiatric symptomatology and better treatment outcomes among individuals with posttraumatic stress disorder (PTSD) and substance use disorders. However, it remains unknown whether resilience increases during psychotherapy within the comorbid PTSD and substance use disorder population with unique features of dual diagnosis, including trauma cue-related cravings. We tested whether veterans seeking psychotherapy for comorbid PTSD and substance use disorder reported increased resilience from pre- to posttreatment. We also tested whether increased resilience was associated with greater decreases in posttreatment PTSD and substance use disorder symptoms., Methods: Participants were 29 male veterans (M age = 49.07 years, SD = 11.24 years) receiving six-week residential day treatment including cognitive processing therapy for PTSD and cognitive behavioral therapy for substance use disorder. Resilience, PTSD symptoms, and trauma cue-related cravings were assessed at pre- and posttreatment., Results: Veterans reported a large, significant increase in resilience posttreatment (M diff = 14.24, t = -4.22, p < .001, d = 0.74). Greater increases in resilience were significantly associated with fewer PTSD symptoms (β = -0.37, p = .049, sr = -.36) and trauma-cued cravings (β = -0.39, p = .006, sr = -.38) posttreatment when controlling for pretreatment scores and baseline depressive symptoms., Conclusions: Results suggest that evidence-based psychotherapy for comorbid PTSD and substance use disorder may facilitate strength-based psychological growth, which may further promote sustained recovery.

Objective: Individuals with co-occurring posttraumatic stress disorder (PTSD) and substance use disorder report heightened levels of numerous risky and health-compromising behaviors, including aggressive behaviors. Given evidence that aggressive behavior is associated with negative substance use disorder treatment outcomes, research is needed to identify the factors that may account for the association between PTSD and aggressive behavior among patients with substance use disorder. Thus, the goal of this study was to examine the role of impulsivity dimensions (i.e., negative urgency, lack of premeditation, lack of perseverance, and sensation seeking) in the relations between probable PTSD status and both verbal and physical aggression., Methods: Participants were 92 patients in residential substance use disorder treatment (75% male; 59% African American; M age = 40.25) who completed self-report questionnaires., Results: Patients with co-occurring PTSD-substance use disorder (vs. substance use disorder alone) reported significantly greater verbal and physical aggression as well as higher levels of negative urgency and lack of premeditation. Lack of premeditation and lack of perseverance were significantly positively associated with verbal aggression, whereas negative urgency, lack of premeditation, and lack of perseverance were significantly positively associated with physical aggression. The indirect relation of probable PTSD status to physical aggression through negative urgency was significant., Conclusions: Results highlight the potential utility of incorporating skills focused on controlling impulsive behaviors in the context of negative emotional arousal in interventions for physical aggression among patients with co-occurring PTSD-substance use disorder.

Given that rates of PTSD, particularly among military populations, are increasing, it is critical to gain a better understanding of factors associated with treatment response. Low distress tolerance (DT), conceptualized as the perceived or actual inability to tolerate negative emotional states, may impacts veterans' responses to PTSD treatment. Low DT has been associated with more severe PTSD symptoms in clinical and non-clinical samples; however, its impact on PTSD symptomatology across treatment has yet to be assessed. We examined the impact of changes in DT, from intake to discharge, on post-treatment PTSD symptom severity within two samples of veterans recruited from Veterans Affairs residential PTSD treatment facilities in the northwestern and southern United States (Total N=86; 87% male; 46% White, 39% Black, 9% Latino, 6% Other). Veterans completed the Distress Tolerance Scale and PTSD Checklist (PCL) at intake and discharge from residential PTSD treatment. Regression analyses revealed that, within each veteran sample, those with the greatest improvements in DT had the lowest PCL total and subscale scores at discharge after controlling for respective intake PCL scores. This suggests increases in DT across treatment help explain the degree of benefits experienced by veterans following PTSD treatment., (Published by Elsevier Ltd.)

Objectives: With the recent federal mandate that all U.S. health care settings transition to ICD-10 billing codes, empirical evidence is necessary to determine if the DSM-5 designations map to their respective ICD-10 diagnostic categories/billing codes. The present study examined the concordance between DSM-5 and ICD-10 cannabis use disorder diagnoses., Method: Data were derived from routine clinical assessments of 6871 male and 801 female inmates recently admitted to a state prison system from 2000 to 2003. DSM-5 and ICD-10 diagnostic determinations were made from algorithms corresponding to the respective diagnostic formulations., Results: Past 12-month prevalence rates of cannabis use disorders were comparable across classification systems. The vast majority of inmates with no DSM-5 diagnosis continued to have no diagnosis per the ICD-10, and a similar proportion with a DSM-5 severe diagnosis received an ICD-10 dependence diagnosis. Most of the variation in diagnostic classifications was accounted for by those with a DSM-5 moderate diagnosis in that approximately half of these cases received an ICD-10 dependence diagnosis while the remaining cases received a harmful use diagnosis., Conclusions: Although there appears to be a generally high level of agreement between diagnostic classification systems for those with no diagnosis or those evincing symptoms of a more severe condition, concordance between DSM-5 moderate and ICD-10 dependence diagnoses was poor. Additional research is warranted to determine the appropriateness and implications of the current DSM-5 coding guidelines regarding the assignment of an ICD-10 dependence code for those with a DSM-5 moderate diagnosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The risk of developing a substance use disorder (SUD) is significantly higher among veterans with posttraumatic stress disorder (PTSD). Veterans with this co-occurrence have poorer outcomes than singly diagnosed veterans, which may be related to two risk factors: intolerance uncertainty (IU) and low tolerance of emotional distress (TED). We hypothesized low TED and high IU would independently and interactively relate to heightened PTSD symptomatology and trauma-cue elicited SUD cravings. A sample of 70 veterans (M age=50; 95% men; 65% Black) with co-occurring PTSD-SUD was recruited. The Posttraumatic Stress Disorder Checklist (PCL), Craving Questionnaire, Distress Tolerance Scale, and Intolerance of Uncertainty Scale were administered. In general, low TED and high IU were significantly correlated with the PCL total and subscale scores. When examined within regression models, low TED was associated with elevated PCL scores and trauma-cue elicited SUD cravings; IU was not. However, there was a significant interaction between IU and TED; veterans with elevated IU and low TED had higher PCL Total, Hyperarousal, and Intrusions scores. This highlights the importance of assessing TED and IU among veterans with co-occurring PTSD-SUD, as these risk factors may not only be prognostic indicators of outcomes, but also treatment targets., (Published by Elsevier Ltd.)

Despite emerging evidence for the comorbidity of posttraumatic stress disorder (PTSD) and migraine, few studies have examined the relation of PTSD and migraine, particularly among clinical populations at-risk for both conditions (e.g., substance-dependent patients). This study examined the role of PTSD symptoms in migraine and headache-related disability within a sample of 153 substance-dependent inpatients (37.25% female, Mean age 36.46). PTSD symptoms predicted both migraine and headache-related disability above and beyond gender, depression and anxiety symptoms, the experience of a Criterion A traumatic event, and current alcohol use disorder. Findings highlight the strong association between migraine and PTSD symptoms in a unique population at risk for both conditions.

Background: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus., Methods: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination., Results: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy., Conclusions: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).

Substance use disorder (SUD) is highly prevalent among military populations and associated with a wide range of negative outcomes. The goal of the present study was to explicate the relations among negative affect, emotion dysregulation, and urges to engage in risky behaviors among military veterans in residential SUD treatment. Emotion dysregulation (overall and three dimensions: access to emotion regulation strategies, impulse control, and emotional awareness) mediated the relation between negative affect and urges to engage in risky behaviors. Findings highlight the potential utility of treatments targeting emotion dysregulation in reducing risky behaviors among military veterans with SUD.

Objective: To determine the proportion of dog breeders who fed diets meeting the Association of American Feed Control Officials regulations for nutritional adequacy for reproduction and growth and to investigate factors that influenced feeding practices of breeders., Design: Web-based cross-sectional survey., Sample: 2,067 dog breeders from the United States and Canada., Procedures: A self-administered, anonymous, Web-based questionnaire was used to collect information on breeder demographics and feeding practices during 3 life stages of dogs: adult maintenance for nonpregnant dogs, gestation-lactation, and puppy growth. Appropriateness of commercial diets for each life stage was determined by respondent-reported nutritional adequacy statements on product labels. Data were also collected regarding breeder criteria for diet selection and sources of nutrition information., Results: A substantial number of breeders reported feeding commercial diets not intended for that life stage during gestation-lactation (126/746 [16.9%]) and puppy growth (57/652 [8.7%]). Additionally, approximately one-seventh of breeders reported feeding home-prepared diets for ≥ 1 life stage. Unsubstantiated health and marketing information influenced diet selection of many breeders. Veterinarians, although generally viewed as a trusted source of nutrition information, were consulted by only 823 of 1,669 (49.3%) breeders and were viewed less favorably by breeders feeding home-prepared diets, compared with the opinion of breeders feeding commercial diets., Conclusions and Clinical Relevance: Veterinarians should consider taking a more proactive role in directing dog breeders and other pet owners toward scientifically substantiated sources of diet information and in explaining the importance of current nutritional standards for reproduction and early development of dogs.

The present study examines anxiety and disgust responding during exposure to trauma cues as a function of gender and posttraumatic stress disorder (PTSD). Trauma exposed adults without PTSD were compared to adults with PTSD during a script-driven imagery procedure that exposed each participant to individualized traumatic event cues. Anxiety responding during exposure to an individualized traumatic event script was not associated with gender, PTSD, or interaction of gender and PTSD in the present study. However, gender did moderate the relation between disgust responding and PTSD, such that females with PTSD reported more disgust during the script in comparison to females without PTSD and males with and without PTSD. Heart rate during the individualized trauma script was significantly higher among males with PTSD compared to males without PTSD and females with PTSD. Implications of these findings for conceptualizing how gender differences in emotional and physiological responding contribute to development and course of PTSD are discussed.

The current study compared the psychometric properties and clinical/research utility of four distinct factor/subscale models of alcohol craving (three factor-derived models, and one rationally derived model) as measured by the Alcohol Craving Questionnaire-Now in social (n=52) and alcohol dependent (n=71) drinkers. All participants completed a self-report measure of alcohol abuse in addition to engaging in a structured interview and cue reactivity protocol. Participants provided self-reported craving, as well as desire to approach or avoid drinking, during a cue exposure task using separate analog scales. Factor/subscale models were compared in terms of internal consistency, convergent and divergent validity, and ability to predict cue-elicited approach and craving in addition to diagnostic status. All models demonstrated high levels of internal consistency, convergent and divergent validity, and the ability to predict both cue-elicited craving and alcohol dependence status. Specific strengths and weaknesses of each model are examined and the theoretical, clinical, and research utility of the current findings are discussed.

The current study represents the first examination of covariation biases in contamination fear. Using an undergraduate sample we examined covariation bias for specific emotion outcomes (fear specific and disgust specific) associated with contamination stimuli in high contamination fear (HCF; n=32) and low contamination fear (LCF; n=30) individuals. Following random stimulus-outcome presentation participants provided estimations on the proportion of each presented stimulus-expression pairing. Analyses revealed a specific bias for the over-estimation of fear and disgust contingencies among the HCF group, but not the LCF group. The current study also revealed a specific covariation bias among HCF, not LCF, participants to over-estimate the contingency between contamination stimuli and fear outcomes, not disgust outcomes. Further, results indicate that HCF individuals significantly under-estimate the covariation among contamination stimuli and safety outcomes compared to LCF participants. These findings are discussed in terms of theoretical implications for information processing biases in anxiety disorders.

This study examined the specificity of disgust in predicting avoidance in blood/injury (BI) phobia. Participants high (n=38) and low (n=46) in BI fear completed measures of disgust across multiple domains and severity of BI-related fear. They then completed three randomly presented behavioral avoidance tasks (BATs) that consisted of exposure to a 15'' severed deer leg (BI task), a live spider (spider task), and a 'contaminated' cookie (cookie task). Fainting symptoms associated with each BAT were recorded as well. When controlling for gender and BI fear group membership, mutilation disgust contributed unique variance to avoidance on the BI task and animal disgust contributed unique variance to avoidance on the spider task. None of the disgust domains contributed unique variance to avoidance on the cookie task. For the high BI fear group, self-reported fainting symptoms were more pronounced during the BI and spider BAT than during the cookie BAT. Although mutilation disgust was significantly associated with self-reported fainting symptoms on the BI task among the high BI fear group, this relationship became nonsignificant when controlling for BI-related fear severity. Implications of the domain specificity of disgust and its relevance for understanding fainting responses in BI phobia are discussed.

Research has begun to implicate the role of disgust in the etiology of specific phobias and obsessive-compulsive disorder (OCD). However, it remains unclear if the association between disgust and specific anxiety disorder symptoms is an artifact of trait anxiety or a potential mechanism through which trait anxiety effects specific anxiety disorder symptoms. The present study employed structural equation modeling to differentiate disgust from trait anxiety in the prediction of four types of specific anxiety disorder symptoms in a non-clinical sample (N=352). Results indicate that disgust and trait anxiety latent factors were independently related to spider fears, blood-injection-injury (BII) fears, general OCD symptoms, and OCD washing concerns. However, when both variables were simultaneously modeled as predictors, latent disgust remained significantly associated with the anxiety disorder symptoms, whereas the association between latent trait anxiety and the anxiety disorder symptoms became non-significant or was substantially reduced. Statistical tests of intervening variable effects converged in support of disgust as a significant intervening variable between trait anxiety and spider fears, BII fears, and OCD symptoms (particularly washing concerns). The relevance of these findings for future research investigating the role of disgust in specific anxiety disorders is discussed.

The current study examined scrupulosity in 352 unselected college students as measured by the 19-item Penn Inventory of Scrupulosity (PIOS). Confirmatory factor analysis yielded support for a two-factor model of the 19-item PIOS. However, item-level analyses provided preliminary support for the validity of a 15-item PIOS (PIOS-R) secondary to the removal of items 2, 6, 15, and 10. The two domains of scrupulosity identified on the PIOS-R consisted of the Fear of Sin and the Fear of God. Both domains and total scrupulosity scores were strongly related to obsessive-compulsive symptoms. Scrupulosity also showed significant, but more modest correlations with a broad range of other measures of psychopathology symptoms (i.e., state anxiety, trait anxiety, negative affect, disgust sensitivity, specific fears). However, only obsessive-compulsive symptoms and trait anxiety contributed unique variance to the prediction of scrupulosity. Examination of specific obsessive-compulsive symptom dimensions revealed that only obsessions contributed unique positive variance to the prediction of Fear of God. However, OCD obsessions, washing, and hoarding symptoms contributed unique positive variance to the prediction of Fear of Sin. These findings are interpreted in the context of future research elucidating the relationship between scrupulosity and obsessive-compulsive symptom dimensions.

Complex regional pain syndromes (CRPS, type I and type II) are devastating conditions that can occur following soft tissue (CRPS type I) or nerve (CRPS type II) injury. CRPS type I, also known as reflex sympathetic dystrophy, presents in patients lacking a well-defined nerve lesion, and has been questioned as to whether or not it is a true neuropathic condition with an organic basis. As described here, glabrous and hairy skin samples from the amputated upper and lower extremity from two CRPS type I diagnosed patients were processed for double-label immunofluorescence using a battery of antibodies directed against neural-related proteins and mediators of nociceptive sensory function. In CRPS affected skin, several neuropathologic alterations were detected, including: (1) the presence of numerous abnormal thin caliber NF-positive/MBP-negative axons innervating hair follicles; (2) a decrease in epidermal, sweat gland, and vascular innervation; (3) a loss of CGRP expression on remaining innervation to vasculature and sweat glands; (4) an inappropriate expression of NPY on innervation to superficial arterioles and sweat glands; and (5) a loss of vascular endothelial integrity and extraordinary vascular hypertrophy. The results are evidence of widespread cutaneous neuropathologic changes. Importantly, in these CRPS type I patients, the myriad of clinical symptoms observed had detectable neuropathologic correlates.

Anxiety sensitivity (AS) refers to the fear of anxiety-related symptoms based upon the belief that the sensations have harmful consequences. Although the most popular existing measure is the Anxiety Sensitivity Index (ASI), the Anxiety Sensitivity Profile (ASP) was developed as an alternative and theoretically improved assessment of the multifaceted nature of the AS construct. Nevertheless, there has been a paucity of research on this measure. We evaluated the psychometric properties and factor structure of the ASP in two large, geographically diverse undergraduate samples who completed the ASP and measures of anxiety and depression. Exploratory factor analysis revealed four lower order ASP factors in both samples: (1) fear of arousal-related symptoms, (2) fear of cognitive dyscontrol and dissociation, (3) fear of gastrointestinal symptoms, and (4) fear of cardiac symptoms. The fear of cardiac symptoms factor was relatively unstable in both studies. Correlations between the ASP factors and related variables were consistent with AS theory. The strengths and limitations of the ASP are offered as well as the implications of our findings for the nature and assessment of AS.

Objective: The aims of the current study were to determine if impulsivity serves as a risk factor for eating disorder behavior and to examine whether different risk outcomes are obtained depending on the assessment strategy used to measure impulsivity., Method: Three independent studies are reported, each of which examined the relationship of impulsivity and eating disorder behavior in a prospective longitudinal design with adolescent subjects recruited from both public and private schools. Individuals displaying eating disorder behavior at initial assessments were not included in the analyses, to ensure that we were testing the role of impulsivity in the onset of eating disorder behavior., Results: Trait impulsivity, measured with traditional personality scales, failed to predict the onset of eating disorder behavior in all three studies. However, when behavioral constructs associated with impulsivity, such as delinquency or substance abuse, were examined, they significantly predicted the onset of eating disorder behavior in most of the analyses conducted., Discussion: These results provide moderate support for the idea that impulsivity serves as a risk factor for the onset of eating disorder behavior. However, this is only true when more objective behavioral measures were utilized.

Surface proteins in Gram-positive bacteria are anchored to the cell wall by the action of sortase enzymes. The Staphylococcus aureus sortase A (SrtA) protein anchors proteins by recognizing a cell wall sorting signal containing the amino acid sequence LPXTG. To understand how SrtA binds this sequence, we carried out NMR studies of new peptidyl-cyanoalkene and peptidyl-sulfhydryl inhibitors that contain the sorting signal sequence LPAT. These studies combined with amino acid mutagenesis identified a catalytically important and conserved binding surface formed by residues A118, T180, and I182. Compatible with its recently proposed role as a general base, R197 is also shown to be required for catalysis.

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.

Tn916 and related conjugative transposons are clinically significant vectors for the transfer of antibiotic resistance among human pathogens, and they excise from their donor organisms using the transposon-encoded integrase ((Tn916)Int) and excisionase ((Tn916)Xis) proteins. In this study, we have investigated the role of the (Tn916)Xis protein in stimulating excisive recombination. The functional relevance of (Tn916)Xis binding sites on the arms of the transposon has been assessed in vivo using a transposon excision assay. Our results indicate that in Escherichia coli the stimulatory effect of the (Tn916)Xis protein is mediated by sequence-specific binding to either of its two binding sites on the left arm of the transposon. These sites lie in between the core and arm sites recognized by (Tn916)Int, suggesting that the (Tn916)Xis protein enhances excision in a manner similar to the excisionase protein of bacteriophage lambda, serving an architectural role in the stabilization of protein-nucleic acid structures required for strand synapsis. However, our finding that excision in E. coli is significantly enhanced by the host factor HU, but does not depend on the integration host factor or the factor for inversion stimulation, defines clear mechanistic differences between Tn916 and bacteriophage lambda recombination.

Intubation in the child presenting with severe viral tracheobronchitis or prior subglottic injury can be detrimental to the child and the subglottis. Intubation may lead to further mucosal ischemia, scar, subglottic stenosis, or failed extubation requiring a tracheotomy. Heliox is a combination of helium and oxygen that produces less-dense gas exchange. Its use leads to a decrease in turbulent airflow, which may obviate the need for intubation. Here we report our experience using heliox as initial therapy in 14 consecutive children presenting with severe airway distress and the need for intubation. (Five had viral tracheobronchitis, 5 had inflammatory exacerbation of subglottic stenosis, and 4 had acute iatrogenic subglottic injury.) In 10 of the 14 children, intubation, which can lead to mucosal injury and scarring, was avoided by the use of heliox therapy. Of the 4 children in whom heliox therapy failed, 3 had a prior history of subglottic stenosis. Heliox is a relatively safe and reliable alternative to intubation of children with severe subglottic edema or injury. Heliox should be considered before intubation for selected children with subglottic inflammation and severe airway distress.

Background: Laryngeal electromyography (LEMG) is a valuable diagnostic/prognostic test for patients with suspected laryngeal neuromuscular disorders., Objective: To report our experience with diagnostic LEMG at the Center for Voice Disorders of Wake Forest University and to evaluate the impact of LEMG on clinical management., Methods: Retrospective chart review of 415 patients who underwent diagnostic LEMG over a 5-year period (1995-1999)., Results: Of 415 studies, 83% (346 of 415) were abnormal, indicating a neuropathic process. LEMG results altered the diagnostic evaluation (eg, the type of radiographic imaging) in 11% (46 of 415) of the patients. Unexpected LEMG findings (eg, contralateral neuropathy) were found in 26% (107 of 415) of the patients, and LEMG results differentiated vocal fold paralysis from fixation in 12% (49 of 415). Finally, LEMG results altered the clinical management (eg, changed the timing and/or type of surgical procedure) in 40% (166 of 415) of the patients., Conclusions: LEMG is a valuable diagnostic test that aids the clinician in the diagnosis and management of laryngeal neuromuscular disorders.

The surgical management of children with tracheal stenosis and obstruction is complicated by the perioperative needs of pressure ventilation and indwelling endotracheal tubes. These factors predispose to surgical failure and anastomotic breakdown, restenosis. and pneumomediastinum. The use of extracorporeal membrane oxygenation (ECMO) to manage ventilation during tracheal repair allows better visualization at the surgical site and obviates the need for indwelling endotracheal tubes and high-pressure ventilation. Six children were treated with elective ECMO at a tertiary care hospital. All 6 underwent successful surgical repair, and 4 of the 6 were ultimately extubated. There were no significant complications at the surgical site. There was 1 death from postoperative complications, and 2 patients required tracheotomy. One tracheotomy was performed for upper airway obstruction secondary to retrognathia, and this patient was subsequently decannulated. Medical complications were confined to 2 patients and included sepsis, hyperbilirubinemia, seizure disorder, renal failure, intracranial hemorrhage, and hydrocephalus. Elective ECMO provides a reliable perioperative technique for airway management of children with tracheal stenosis or obstruction. This technique offers the advantage of improved visibility at the operative site and eliminates the need for high-pressure ventilation, thereby likely reducing the risk of perioperative morbidity.

We present the results of a rational mutagenesis and binding-affinity study of the three-stranded beta-sheet-DNA interface in the complex formed by the amino-terminal DNA-binding domain of the Tn916 integrase protein and its cognate binding site. The relative importance of interfacial contacts present in its NMR-derived solution structure have been tested through mutagenesis, fluorescence anisotropy, and intrinsic quenching DNA-binding assays. We find that seven protein-DNA hydrogen bonds (two base-specific and five to phosphate groups) significantly contribute to the level of affinity. These interactions span the entire DNA-binding surface on the protein, but primarily originate from residues in only two strands of the sheet and loop L2. Interestingly, we show that highly populated, precisely defined intermolecular hydrogen bonds in the ensemble of conformers are invariably important for DNA-binding, implying that NMR-derived solution structures provide direct insight into the energetics of recognition. Unusual three-stranded beta-sheet-DNA interfaces have recently been discovered in three unrelated protein-DNA complexes. A comparative analysis of these structures reveals similar sheet positioning, the presence of two invariant interfacial contacts to the phosphodiester backbone, and two semi-conserved base-specific hydrogen bonds. Two of these conserved contacts significantly contribute to the affinity of the integrase-DNA complex, suggesting that the three-stranded beta-sheet DNA-binding motif exhibits conserved principles of recognition., (Copyright 2000 Academic Press.)

The repressor protein of bacteriophage Mu establishes and maintains lysogeny by shutting down transposition functions needed for phage DNA replication. It interacts with several repeated DNA sequences within the early operator, preventing transcription from two divergent promoters. It also directly represses transposition by competing with the MuA transposase for an internal activation sequence (IAS) that is coincident with the operator and required for efficient transposition. The transposase and repressor proteins compete for the operator/IAS region using homologous DNA-binding domains located at their amino termini. Here we present the solution structure of the amino-terminal DNA-binding domain from the repressor protein determined by heteronuclear multidimensional nuclear magnetic resonance spectroscopy. The structure of the repressor DNA-binding domain provides insights into the molecular basis of several temperature sensitive mutations and, in combination with complementary experiments using flourescence anisotropy, surface plasmon resonance, and circular dichroism, defines the structural and biochemical differences between the transposase and repressor DNA-binding modules. We find that the repressor and enhancer domains possess similar three-dimensional structures, thermostabilities, and intrinsic affinities for DNA. This latter result suggests that the higher affinity of the full-length repressor relative to that of the MuA transposase protein originates from cooperative interactions between repressor protomers and not from intrinsic differences in their DNA-binding domains. In addition, we present the results of nucleotide and amino acid mutagenesis which delimits the minimal repressor DNA-binding module and coarsely defines the nucleotide dependence of repressor binding.

The integrase protein catalyzes the excision and integration of the Tn916 conjugative transposon, a promiscuous genetic element that spreads antibiotic resistance in pathogenic bacteria. The solution structure of the N-terminal domain of the Tn916 integrase protein bound to its DNA-binding site within the transposon arm has been determined. The structure reveals an interesting mode of DNA recognition, in which the face of a three-stranded antiparallel beta-sheet is positioned within the major groove. A comparison to the structure of the homing endonuclease I-Ppol-DNA complex suggests that the three-stranded sheet may represent a new DNA-binding motif whose residue composition and position within the major groove are varied to alter specificity. The structure also provides insights into the mechanism of conjugative transposition. The DNA in the complex is bent approximately 35 degrees and may, together with potential interactions between bound integrase proteins at directly repeated sites, significantly bend the arms of the transposon.

The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.

Using tumor necrosis factor (TNF) inhibition in dog blood as a measure of efficacy, and canine emesis as a measure of toxicity, we were able to assign a therapeutic index to rolipram, a prototypic anti-inflammatory compound. Because both assays were performed in the same species, the ambiguities associated with comparing the physiologic effects of drugs on various species was avoided. Rolipram, a standard phosphodiesterase type IV inhibitor, was a prototypic test compound characterized by a number of cardiovascular and central nervous system side effects, as well as its in vitro and in vivo inhibition of TNF. Initial experiments with canine whole blood incubated with lipopolysaccharide resulted in nanogram-per-milliliter concentrations of TNF that could be significantly reduced by in vitro addition of a 0.03 microM concentration of rolipram. Because rolipram inhibited canine TNF production in vitro, a protocol was devised in which TNF inhibitory activity was measured in a series of blood samples from dogs infused with increasingly high doses of rolipram. This yielded the efficacy half of the therapeutic index, whereas the emetogenic dose represented the side effect portion of the index. Rolipram was infused stepwise into conscious dogs at gradually increasing doses. The infusion was stopped when vomiting occurred, and the cumulative dose was reported as the emetic dose. Rolipram caused emesis in dogs at a cumulative dose of 0.1 mg/kg. At each dose of rolipram, blood was collected. The whole blood was incubated in vitro with lipopolysaccharide to induce TNF production, which in turn was quantified by the L929 bio-assay. Theoretically, if the rolipram infusion raised blood values high enough, the rolipram in whole blood would inhibit TNF production and be reflected by a lack of TNF activity in the L929 assay. In this assay system, rolipram's 50% effective dose in the TNF assay was always at least 33-fold lower than its emetic dose of 0.1 mg/kg. This gave rolipram a therapeutic index of at least 33:1 (0.003 versus 0.1 mg/kg) on the basis of its activity in a canine efficacy model (TNF inhibition) and a toxicity model (emesis induction). Experimental compounds were tested for their emetic dose as well as TNF 50% effective dose, with the goal of obtaining a therapeutic index better than that of rolipram. Thus the coupling of cytokine activity with overt toxicity was used to arrive at the therapeutic index of a compound. The therapeutic index was used to rank compounds as to their efficacy/toxicity profile. This ranking was used to eliminate several anti-inflammatory compounds that had a therapeutic index less than that of rolipram.

Inhibitors of cyclic nucleotide phosphodiesterases are known to suppress lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production in vitro in human monocytes. The most potent of these have selectivity for type IV PDEs, suggesting that this class of PDE is the major type involved in the regulation of human TNF-alpha production. Using compounds of two distinct chemical structural classes, a quinazolinedione (CP-77059) and a 4 arylpyrrolidinone (rolipram), we show here that PDE-IV-specific inhibitors are also potent in suppressing LPS-induced TNF-alpha production in vitro in sodium periodate-elicited murine macrophages (IC50s of 1 and 33, respectively). We then report the in vivo anti-inflammatory effect of PDE-IV inhibition in five murine models of inflammation: (i) elevation of serum TNF-alpha induced by a sublethal LPS injection; (ii) LPS-induced endotoxic shock; (iii) LPS/galactosamine-induced endotoxic shock; (iv) carrageenan-induced paw oedema; and (v) adjuvant arthritis. Following a sublethal (5 micrograms/mouse) injection of LPS, serum TNF-alpha levels in mice peaked sharply, reaching concentrations of 3-12 ng/ml 90 min after injection. In this sublethal LPS assay, CP-77059 was about 30 times more potent than rolipram, with a minimum effective dose of 0.1 mg/kg versus 3 mg/kg for rolipram. This rank order is in keeping with the relative in vitro IC50s for CP-77059 and rolipram, as well as their relative Ki against the human PDE-IV enzyme (46 nM and 220 nM, respectively). In LPS-induced endotoxic shock, rolipram and CP-77059 at relatively high doses of 30 and 10 mg/kg, respectively, significantly reduced serum TNF-alpha levels, and also inhibited mortality 66%. In the LPS/galactosamine shock model, in which mice are rendered exquisitely sensitive to LPS by co-injection with galactosamine, only 0.1 microgram of LPS/mouse is necessary for serum TNF-alpha elevation and death. Both rolipram and the CP-77059 caused dose-dependent reduction of serum TNF-alpha and lethality. In the carrageenan-induced paw oedema model, in which there is a pronounced local TNF-alpha response (without a serum TNF-alpha elevation), rolipram significantly inhibited paw swelling as well as localized TNF-alpha levels in the paw. In the adjuvant arthritis model, a chronic model of inflammation also possessing localized TNF-alpha elevation in the inflamed paw, rolipram and CP-77059 suppressed ankle swelling and radiological evidence of joint damage. These data are consistent with a major role for PDE-IV in regulation of TNF-alpha production and inflammatory responses in murine systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.

Elevated tumor necrosis factor (TNF) levels have been reported in various models of acute and chronic inflammation and used by many investigators to determine the stage of disease and effectiveness of treatment. Because of the documented involvement of TNF in the mechanism of septic shock, experiments were done to determine whether serum TNF levels paralleled the pathology in endotoxic shock and other models of inflammation. When mice received an intraperitoneal injection of lipopolysaccharide, serum TNF levels increased dramatically, peaking 90 minutes after injection. In a dose-response experiment with lipopolysaccharide alone, we found no correlation between serum levels of TNF and survival rate of mice. All three lipopolysaccharide concentrations resulted in comparable elevations of serum TNF, yet only in the high-dose group did the animals die. In a second model of endotoxic shock, TNF-alpha levels in serum were again compared with the survival rate of mice receiving lipopolysaccharide plus galactosamine. As in the first model, we found no relationship between the level of TNF in mouse serum and mouse survival rate. The two lowest concentrations of lipopolysaccharide/galactosamine induced identically low levels of serum TNF, yet in one group all of the animals survived and in the other all died. Discrepancies between serum TNF level and mortality rate were also seen in drug treatment experiments. GI 147404X, a standard phosphodiesterase type IV inhibitor, inhibited lipopolysaccharide/galactosamine-induced elevation of serum TNF by 90% at doses of 1 and 10 mg/kg. However, the high dose resulted in 66% protection while the low dose afforded no protection.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

The primary purpose of this study was to determine whether local levels of tumor necrosis factor (TNF) were elevated in chronically inflamed joints in rats with adjuvant-induced arthritis (AA). We also wished to develop methodology for the quantitative measurement of joint TNF, and to examine the effects of known anti-inflammatory agents on joint TNF levels. TNF levels were measured in joints from AA rats taken during the systemic phase (day 20) of arthritic disease. Using the L929 bioassay, joint extracts from AA rats had significantly greater TNF levels (1054 +/- 147 pg/g tissue) than joint extracts from normal rats (110 +/- 42 pg/g tissue). Administration of ibuprofen failed to significantly inhibit TNF levels in the joint at a time point when paw swelling was significantly reduced. The immunomodulating agents, methotrexate, cyclosporin A (CSA) and HWA486 profoundly inhibited both joint TNF levels and paw swelling. The specificity of this assay for TNF was supported by studies with a polyclonal rabbit anti-mouse TNF antibody which neutralized 78-87% of the TNF activity in the joint extracts. Our studies demonstrate a quantitative increase in local TNF expression in adjuvant arthritis and support a role for TNF in chronic inflammation.

Proliferating cell nuclear antigen (PCNA) was evaluated as a marker of cell proliferation in formalin-fixed rat liver tissue through a comparative study with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). The comparison was conducted through the introduction of a dual immunohistochemical procedure that allows the simultaneous detection of the two antigens. The results of this study suggest that although statistically similar indexes for each can be achieved, what has been reported to be the "S-phase fraction" of PCNA-labeled nuclei is significantly different from the population of cells marked by BrdU. The data also suggest that the reason for this difference is that the "S-phase fraction" of PCNA-labeled nuclei is the population of cells in late G1- and early S-phases. BrdU, by comparison, is incorporated into cells only during DNA synthesis. Therefore, although BrdU and PCNA labeling techniques may both be effective for evaluating cell proliferation rates, it must be recognized that labeling indices derived from each are not entirely synonymous. The method presented here for the simultaneous labeling of PCNA and BrdU antigens may have utility in studies of cell cycle perturbations.