Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity.

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo.

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.

Precursor Frequency and Affinity Determine B Cell Competitive Fitness in Germinal Centers, Tested with Germline-Targeting HIV Vaccine Immunogens.

How precursor frequencies and antigen affinities impact interclonal B cell competition is a particularly relevant issue for candidate germline-targeting HIV vaccine designs because of the in vivo rarity of naive B cells that recognize broadly neutralizing epitopes. Knowing the frequencies and affinities of HIV-specific VRC01-class naive human B cells, we transferred B cells with germline VRC01 B cell receptors into congenic recipients to elucidate the roles of precursor frequency, antigen affinity, and avidity on B cell responses following immunization. All three factors were interdependently limiting for competitive success of VRC01-class B cells. In physiological high-affinity conditions using a multivalent immunogen, rare VRC01-class B cells successfully competed in germinal centers (GC), underwent extensive somatic hypermutation, and differentiated into memory B cells. The data reveal dominant influences of precursor frequency, affinity, and avidity for interclonal GC competition and indicate that germline-targeting immunogens can overcome these challenges with high-affinity multimeric designs.

Factors in B cell competition and immunodominance.

The majority of all vaccines work by inducing protective antibody responses. The mechanisms by which the B cells responsible for producing protective antibodies are elicited to respond are not well understood. Interclonal B cell competition to complex antigens, particularly in germinal centers, has emerged as an important hurdle in designing effective vaccines. This review will focus on recent advances in understanding the roles of B cell precursor frequency, B cell receptor affinity for antigen, antigen avidity, and other factors that can substantially alter the outcomes of B cell responses to complex antigens. Understanding the interdependence of these fundamental factors that affect B cell responses can inform current vaccine design efforts for pathogens with complex proteins as candidate immunogens such as HIV, influenza, and coronaviruses.

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models.

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.

Increased Peripheral Blood Neutrophil Activation Phenotypes and Neutrophil Extracellular Trap Formation in Critically Ill Coronavirus Disease 2019 (COVID-19) Patients: A Case Series and Review of the Literature.

BACKGROUND: Increased inflammation has been well defined in coronavirus disease 2019 (COVID-19), while definitive pathways driving severe forms of this disease remain uncertain. Neutrophils are known to contribute to immunopathology in infections, inflammatory diseases, and acute respiratory distress syndrome, a primary cause of morbidity and mortality in COVID-19. Changes in neutrophil function in COVID-19 may give insight into disease pathogenesis and identify therapeutic targets. METHODS: Blood was obtained serially from critically ill COVID-19 patients for 11 days. Neutrophil extracellular trap formation (NETosis), oxidative burst, phagocytosis, and cytokine levels were assessed. Lung tissue was obtained immediately postmortem for immunostaining. PubMed searches for neutrophils, lung, and COVID-19 yielded 10 peer-reviewed research articles in English. RESULTS: Elevations in neutrophil-associated cytokines interleukin 8 (IL-8) and interleukin 6, and general inflammatory cytokines IFN-inducible protien-19, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 1ß, interleukin 10, and tumor necrosis factor, were identified both at first measurement and across hospitalization (P < .0001). COVID-19 neutrophils had exaggerated oxidative burst (P < .0001), NETosis (P < .0001), and phagocytosis (P < .0001) relative to controls. Increased NETosis correlated with leukocytosis and neutrophilia, and neutrophils and NETs were identified within airways and alveoli in lung parenchyma of 40% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected lungs available for examination (2 of 5). While elevations in IL-8 and absolute neutrophil count correlated with disease severity, plasma IL-8 levels alone correlated with death. CONCLUSIONS: Literature to date demonstrates compelling evidence of increased neutrophils in the circulation and lungs of COVID-19 patients. Importantly, neutrophil quantity and activation correlates with severity of disease. Similarly, our data show that circulating neutrophils in COVID-19 exhibit an activated phenotype with enhanced NETosis and oxidative burst.

Sleep Duration, Sleep Apnea, and Gray Matter Volume.

This study aimed to evaluate the effect of sleep duration on brain structures in the presence versus absence of sleep apnea in middle-aged and older individuals. The study investigated a population-based sample of 2,560 individuals, aged 49-80 years. The presence of sleep apnea and self-reported sleep duration were examined in relation to gray matter volume (GMV) in total and lobar brain regions. We identified ranges of sleep duration associated with maximal GMV using quadratic regression and bootstrap sampling. A significant quadratic association between sleep duration and GMV was observed in total and lobar brain regions of men with sleep apnea. In the fully adjusted model, optimal sleep durations associated with peak GMV between brain regions ranged from 6.7 to 7.0 hours. Shorter and longer sleep durations were associated with lower GMV in total and 4 sub-regions of the brain in men with sleep apnea.

Association of pericardial adipose tissue with left ventricular structure and function: a region-specific effect?

BACKGROUND: The independent role of pericardial adipose tissue (PAT) as an ectopic fat associated with cardiovascular disease (CVD) remains controversial. This study aimed to determine whether PAT is associated with left ventricular (LV) structure and function independent of other markers of general obesity. METHODS: We studied 2471 participants (50.9 % women) without known CVD from the Korean Genome Epidemiology Study, who underwent 2D-echocardiography with tissue Doppler imaging (TDI) and computed tomography measurement for PAT. RESULTS: Study participants with more PAT were more likely to be men and had higher cardiometabolic indices, including blood pressure, glucose, and cholesterol levels (all P < 0.001). Greater pericardial fat levels across quartiles of PAT were associated with increased LV mass index and left atrial volume index (all P < 0.001) and decreased systolic (P = 0.015) and early diastolic (P < 0.001) TDI velocities, except for LV ejection fraction. These associations remained after a multivariable-adjusted model for traditional CV risk factors and persisted even after additional adjustment for general adiposity measures, such as waist circumference and body mass index. PAT was also the only obesity index independently associated with systolic TDI velocity (P < 0.001). CONCLUSIONS: PAT was associated with subclinical LV structural and functional deterioration, and these associations were independent of and stronger than with general and abdominal obesity measures.

Relationship of obstructive sleep apnoea severity and subclinical systemic atherosclerosis.

Obstructive sleep apnoea (OSA) is a common form of sleep disordered breathing. Untreated OSA might accelerate atherosclerosis, potentially increasing the cardiovascular disease burden in patients. The present study aimed to evaluate the association between objectively measured OSA severity and the presence of subclinical systemic atherosclerosis using noninvasive measurements, including tomographic quantification of the calcium burden.A total of 2157 participants of the Korean Genome and Epidemiology Study, who were free of structural heart disease and underwent both in-home polysomnography and chest computed tomography, were cross-sectionally analysed. Participants were divided into three groups based on the severity of OSA: no OSA (apnoea-hypopnoea index (AHI) <5 events·h-1, n=1096), mild OSA (AHI 5- <15 events·h-1, n=700) and moderate-to-severe OSA (AHI ≥15 events·h-1, n=361). Calcium deposits in the thoracic aorta and coronary arteries were measured by the Agatston score.Participants with moderate-to-severe OSA were 1.6 times (95% CI 1.18-2.15 times; p=0.002) more likely to have ascending thoracic aorta calcification (≥100 units) than those without OSA, after adjustment for cardiovascular risk factors. In addition, the association between moderate-to-severe OSA and ascending thoracic aorta calcification of subjects with higher epicardial fat volume was slightly stronger than that in patients without OSA and in the lowest epicardial fat volume tertile (OR 2.11, 95% CI 1.30-3.43).Severity of OSA in the general population was independently associated with subclinical systemic atherosclerosis. These findings highlight the potential importance of severe OSA, especially in subjects with higher epicardial fat, as a possible predictive factor for systemic atherosclerosis and cardiovascular disease.

Neck Circumference and Cerebral Gray Matter Volume.

BACKGROUND: While obesity is linked with brain atrophy and dementia incidence, associations with regional adiposity are uncertain. Our goal was to determine whether cerebral gray matter volume is related to neck circumference (NC), a measure of regional adiposity having unique relationships with metabolic disorders and cardiovascular disease. METHODS: Magnetic resonance imaging and NC were cross-sectionally assessed from 2011 to 2014 in a population-based sample of 2916 men and women in the Korean Genome and Epidemiology Study. RESULTS: For men, NC was inversely associated with total and regional gray matter in the frontal, temporal, and occipital lobes after adjusting for age and intracranial brain volume. Associations were especially strong in the presence of diabetes. With further adjustment for indices of body composition and other characteristics, total and frontal gray matter in diabetic men were lowered by 6.1 mL (95% confidence interval: 2.5-9.7, P=0.004) and 2.9 mL (95% confidence interval: 1.0-4.9, P=0.017), respectively, per SD increase in NC (2.3 cm). For men without diabetes, and in all women, associations were less apparent. CONCLUSIONS: In men with diabetes, NC was inversely associated with cerebral gray matter volume. The link between neck anthropometry and brain aging in diabetic men warrants further consideration.

Association of brain heptachlor epoxide and other organochlorine compounds with lewy pathology.

BACKGROUND: Organochlorine pesticides are associated with an increased risk of Parkinson's disease. A preliminary analysis from the Honolulu-Asia Aging Study suggested that heptachlor epoxide, a metabolite from an organochlorine pesticide extensively used in Hawaii, may be especially important. This was a cross sectional analysis to evaluate the association of heptachlor epoxide and other organochlorine compounds with Lewy pathology in an expanded survey of brain organochlorine residues from the longitudinal Honolulu-Asia Aging Study. METHODS: Organochlorines were measured in frozen occipital or temporal lobes in 705 brains using gas chromatography with mass spectrometry. Lewy pathology was identified using hematoxylin and eosin- and α-synuclein immunochemistry-stained sections from multiple brain regions. RESULTS: The prevalence of Lewy pathology was nearly doubled in the presence versus the absence of heptachlor epoxide (30.1% versus 16.3%, P < 0.001). Although associations with other compounds were weaker, hexachlorobenzene (P = 0.003) and α-chlordane (P = 0.007) were also related to Lewy pathology. Most of the latter associations, however, were a result of confounding from heptachlor epoxide. Neither compound was significantly related to Lewy pathology after adjustment for heptachlor epoxide. In contrast, the association of heptachlor epoxide with Lewy pathology remained significant after adjustments for hexachlorobenzene (P = 0.013) or α-chlordane (P = 0.005). Findings were unchanged after removal of cases of PD and adjustment for age and other characteristics. CONCLUSIONS: Organochlorine pesticides are associated with the presence of Lewy pathology in the brain, even after exclusion of PD cases. Although most of the association is through heptachlor epoxide, the role of other organochlorine compounds is in need of clarification. © 2018 International Parkinson and Movement Disorder Society.

Effects of Omitting Non-confounding Predictors From General Relative-Risk Models for Binary Outcomes.

BACKGROUND: The effects, in terms of bias and precision, of omitting non-confounding predictive covariates from generalized linear models have been well studied, and it is known that such omission results in attenuation bias but increased precision with logistic regression. However, many epidemiologic risk analyses utilize alternative models that are not based on a linear predictor, and the effect of omitting non-confounding predictive covariates from such models has not been characterized. METHODS: We employed simulation to study the effects on risk estimation of omitting non-confounding predictive covariates from an excess relative risk (ERR) model and a general additive-multiplicative relative-risk mixture model for binary outcome data in a case-control setting. We also compared the results to the effects with ordinary logistic regression. RESULTS: For these commonly employed alternative relative-risk models, the bias was similar to that with logistic regression when the risk was small. More generally, the bias and standard error of the risk-parameter estimates demonstrated patterns that are similar to those with logistic regression, but with greater magnitude depending on the true value of the risk. The magnitude of bias and standard error had little relation to study size or underlying disease prevalence. CONCLUSIONS: Prior conclusions regarding omitted covariates in logistic regression models can be qualitatively applied to the ERR and the general additive-multiplicative relative-risk mixture model without substantial change. Quantitatively, however, these alternative models may have slightly greater omitted-covariate bias, depending on the magnitude of the true risk being estimated.

Effects of Social Development Intervention in Childhood on Adult Life at Ages 30 to 39.

Elementary schools can be effective sites for universal preventive interventions. Less is known about how long effects of intervention in elementary grades last. Can they improve outcomes in adulthood? To test effects of a social developmental intervention in the elementary grades on adult life through the 30s, the Seattle Social Development Project, a nonrandomized controlled trial, followed all consenting 5th-grade students (N = 808) from 18 Seattle public elementary schools from age 10 (in 1985) to age 39 (in 2014), with 88% retention. The sample was gender balanced and ethnically and economically diverse. The full intervention, called Raising Healthy Children, continued from Grades 1 through 6 and consisted of teacher in-service training in classroom management and instructional methods; cognitive, social, and refusal skills training for children; and parent workshops in child behavior monitoring and management, academic support, and anticipatory guidance. Using structural equation modeling, we examined intervention effects from age 30 to age 39 across 9 constructs indicating 3 domains of adult life: health behavior, positive functioning, and adult health and success. An omnibus test across all 9 constructs indicated a significant positive overall intervention-control difference. Examined individually, significant intervention effects included better health maintenance behavior, mental health, and overall adult health and success. Significant effects were not found on substance use disorder symptoms, sex-risk behaviors, or healthy close relationships in the 30s. Results indicate that sustained, theory-based, multicomponent intervention in the elementary grades can produce lasting changes in health maintenance, mental health, and adult functioning through the 30s.

The GS Protein-coupled A2a Adenosine Receptor Controls T Cell Help in the Germinal Center.

T follicular helper (TFH) cells have been shown to be critically required for the germinal center (GC) reaction where B cells undergo class switch recombination and clonal selection to generate high affinity neutralizing antibodies. However, detailed knowledge of the physiological cues within the GC microenvironment that regulate T cell help is limited. The cAMP-elevating, Gs protein-coupled A2a adenosine receptor (A2aR) is an evolutionarily conserved receptor that limits and redirects cellular immunity. However, the role of A2aR in humoral immunity and B cell differentiation is unknown. We hypothesized that the hypoxic microenvironment within the GC facilitates an extracellular adenosine-rich milieu, which serves to limit TFH frequency and function, and also promotes immunosuppressive T follicular regulatory cells (TFR). In support of this hypothesis, we found that following immunization, mice lacking A2aR (A2aRKO) exhibited a significant expansion of T follicular cells, as well as increases in TFH to TFR ratio, GC T cell frequency, GC B cell frequency, and class switching of GC B cells to IgG1. Transfer of CD4 T cells from A2aRKO or wild type donors into T cell-deficient hosts revealed that these increases were largely T cell-intrinsic. Finally, injection of A2aR agonist, CGS21680, following immunization suppressed T follicular differentiation, GC B cell frequency, and class switching of GC B cells to IgG1. Taken together, these observations point to a previously unappreciated role of GS protein-coupled A2aR in regulating humoral immunity, which may be pharmacologically targeted during vaccination or pathological states in which GC-derived autoantibodies contribute to the pathology.

Germinal Center Hypoxia Potentiates Immunoglobulin Class Switch Recombination.

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high-affinity, class-switched Abs. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper cells, and plasmacytes, as well as lower expression of ICOS on T follicular helper cells. Importantly, this reversal of GC hypoxia decreased Ag-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the Ag-specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation.

Emotional and Behavioral Correlates of Persisting Specific Learning Disabilities in Written Language during Middle Childhood and Early Adolescence.

Parents completed the Behavior Assessment System for Children-Second Edition Parent Report Scale (BASC2-PRS) while their children (94 boys, 61 girls; M=11 years-11 months) were given tests. Evidence-based profiles of multiple test scores and history (emergence and persistence) were used to assign to groups without specific learning disabilities in written language (SLDs-WL) (n= 42 control) or with SLDs-WL: (n=29 dysgraphia, n=65 dyslexia, or n=19 oral and written language learning disability [OWL LD]). Parent ratings fell in the clinical or at risk ranges for some individuals in all groups, but mean BASC2-PRS ratings showed nine significant main effects for group (n=4): Behavioral Symptoms Index, Internalizing Problems Composite, Adaptive Skills Composite, two Clinical Scales (Atypicality and Attention Problems), and four Adaptive Scales (Adaptability, Activities of Daily Living, Leadership, and Functional Communication). Each SLDs-WL group differed significantly from the control group on these nine ratings, except dysgraphia on Atypicality and dyslexia on Adaptive Composite, Adaptability, and Leadership; and each correlated with one or more hallmark impairments associated with a specific SLD-WL. In an fMRI study (without OWL LD), the dysgraphia and dyslexia groups, but not control group, showed connectivity with amygdala; BASC2 PRS Internalizing Problems Composite (internal stress) correlated with amygdala connectivity from two cortical regions involved in written word processing and production for all groups (N=40). Applications to assessing emotional and behavioral correlates of SLDs-WL for educational services and future research are discussed.

Effective Instruction for Persisting Dyslexia in Upper Grades: Adding Hope Stories and Computer Coding to Explicit Literacy Instruction.

Children in grades 4 to 6 (N=14) who despite early intervention had persisting dyslexia (impaired word reading and spelling) were assessed before and after computerized reading and writing instruction aimed at subword, word, and syntax skills shown in four prior studies to be effective for treating dyslexia. During the 12 two-hour sessions once a week after school they first completed HAWK Letters in Motion© for manuscript and cursive handwriting, HAWK Words in Motion© for phonological, orthographic, and morphological coding for word reading and spelling, and HAWK Minds in Motion© for sentence reading comprehension and written sentence composing. A reading comprehension activity in which sentences were presented one word at a time or one added word at a time was introduced. Next, to instill hope they could overcome their struggles with reading and spelling, they read and discussed stories about struggles of Buckminister Fuller who overcame early disabilities to make important contributions to society. Finally, they engaged in the new Kokopelli's World (KW)©, blocks-based online lessons, to learn computer coding in introductory programming by creating stories in sentence blocks (Tanimoto and Thompson 2016). Participants improved significantly in hallmark word decoding and spelling deficits of dyslexia, three syntax skills (oral construction, listening comprehension, and written composing), reading comprehension (with decoding as covariate), handwriting, orthographic and morphological coding, orthographic loop, and inhibition (focused attention). They answered more reading comprehension questions correctly when they had read sentences presented one word at a time (eliminating both regressions out and regressions in during saccades) than when presented one added word at a time (eliminating only regressions out during saccades). Indicators of improved self-efficacy that they could learn to read and write were observed. Reminders to pay attention and stay on task needed before adding computer coding were not needed after computer coding was added.

The Relationship Between Marijuana and Conventional Cigarette Smoking Behavior from Early Adolescence to Adulthood.

Longitudinal analyses investigated (a) the co-occurrence of marijuana use and conventional cigarette smoking within time and (b) bidirectional associations between marijuana and conventional cigarette use in three developmental periods: adolescence, young adulthood, and adulthood. A cross-lag model was used to examine the bidirectional model of marijuana and conventional cigarette smoking frequency from ages 13 to 33 years. The bidirectional model accounted for gender, school-age economic disadvantage, childhood attention problems, and race. Marijuana use and conventional cigarette smoking were associated within time in decreasing magnitude and increased cigarette smoking predicted increased marijuana use during adolescence. A reciprocal relationship was found in the transition from young adulthood to adulthood, such that increased conventional cigarette smoking at age 24 years uniquely predicted increased marijuana use at age 27 years, and increased marijuana use at age 24 years uniquely predicted more frequent conventional cigarette smoking at age 27 years, even after accounting for other factors. The association between marijuana and cigarette smoking was found to developmentally vary in the current study. Results suggest that conventional cigarette smoking prevention efforts in adolescence and young adulthood could potentially lower the public health impact of both conventional cigarette smoking and marijuana use. Findings point to the importance of universal conventional cigarette smoking prevention efforts among adolescents as a way to decrease later marijuana use and suggest that a prevention effort focused on young adults as they transition to adulthood would lower the use of both cigarette and marijuana use.

Evaluation of Community-Level Effects of Communities That Care on Adolescent Drug Use and Delinquency Using a Repeated Cross-Sectional Design.

The Communities That Care (CTC) prevention system has shown effects on reducing incidence and prevalence of problem behaviors among a panel of youth followed from 5th through 12th grade. The present report examines whether similar intervention effects could be observed using a repeated cross-sectional design in the same study. Data were from a community-randomized trial of 24 US towns. Cross-sectional samples of sixth, eighth, and tenth graders were surveyed at four waves. Two-stage ANCOVA analyses estimated differences between CTC and control communities in community-level prevalence of problem behaviors for each grade, adjusting for baseline prevalence. No statistically significant reductions in prevalence of problem behaviors were observed at any grade in CTC compared to control communities. Secondary analyses examined intervention effects within a "pseudo cohort" where cross-sectional data were used from sixth graders at baseline and tenth graders 4 years later. When examining effects within the pseudo cohort, CTC compared to control communities showed a significantly slower increase from sixth to tenth grade in lifetime smokeless tobacco use but not for other outcomes. Exploratory analyses showed significantly slower increases in lifetime problem behaviors within the pseudo cohort for CTC communities with high, but not low, prevention program saturation compared to control communities. Although CTC demonstrated effects in a longitudinal panel from the same community-randomized trial, we did not find similar effects on problem behaviors using a repeated cross-sectional design. These differences may be due to a reduced ability to detect effects because of potential cohort effects, accretion of those who were not exposed, and attrition of those who were exposed to CTC programming in the repeated cross-sectional sample.