Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells.

The adaptive immune response is reliant on a T cell's ability to migrate through blood, lymph, and tissue in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time under highly specific environmental conditions. These conditions may help elucidate mechanisms that regulate migration, improving our understanding of T cell kinetics and providing strong mechanistic evidence that is difficult to attain through animal experiments. A deeper understanding of the molecular interactions that impact cell migration is important to develop improved therapeutics.

Clinical validation of a cell-free DNA fragmentome assay for augmentation of lung cancer early detection.

Lung cancer screening via annual low-dose computed tomography (LDCT) has poor adoption. We conducted a prospective case-control study among 958 individuals eligible for lung cancer screening to develop a blood-based lung cancer detection test that when positive is followed by an LDCT. Changes in genome-wide cell-free DNA (cfDNA) fragmentation profiles (fragmentomes) in peripheral blood reflected genomic and chromatin characteristics of lung cancer. We applied machine learning to fragmentome features to identify individuals who were more or less likely to have lung cancer. We trained the classifier using 576 cases and controls from study samples, and then validated it in a held-out group of 382 cases and controls. The validation demonstrated high sensitivity for lung cancer, and consistency across demographic groups and comorbid conditions. Applying test performance to the screening eligible population in a five-year model with modest utilization assumptions suggested the potential to prevent thousands of lung cancer deaths.

Immune Cell Migration to Cancer.

Immune cell migration is required for the development of an effective and robust immune response. This elegant process is regulated by both cellular and environmental factors, with variables such as immune cell state, anatomical location, and disease state that govern differences in migration patterns. In all cases, a major factor is the expression of cell surface receptors and their cognate ligands. Rapid adaptation to environmental conditions partly depends on intrinsic cellular immune factors that affect a cell's ability to adjust to new environment. In this review, we discuss both myeloid and lymphoid cells and outline key determinants that govern immune cell migration, including molecules required for immune cell adhesion, modes of migration, chemotaxis, and specific chemokine signaling. Furthermore, we summarize tumor-specific elements that contribute to immune cell trafficking to cancer, while also exploring microenvironment factors that can alter these cellular dynamics within the tumor in both a pro and antitumor fashion. Specifically, we highlight the importance of the secretome in these later aspects. This review considers a myriad of factors that impact immune cell trajectory in cancer. We aim to highlight the immunotherapeutic targets that can be harnessed to achieve controlled immune trafficking to and within tumors.

PD-1Hi CAR-T cells provide superior protection against solid tumors.

Chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising treatment option for several hematologic cancers. However, efforts to achieve the same level of therapeutic success in solid tumors have largely failed mainly due to CAR-T cell exhaustion and poor persistence at the tumor site. Although immunosuppression mediated by augmented programmed cell death protein-1 (PD-1) expression has been proposed to cause CAR-T cell hypofunction and limited clinical efficacy, little is known about the underlying mechanisms and immunological consequences of PD-1 expression on CAR-T cells. With flow cytometry analyses and in vitro and in vivo anti-cancer T cell function assays, we found that both manufactured murine and human CAR-T cell products displayed phenotypic signs of T cell exhaustion and heterogeneous expression levels of PD-1. Unexpectedly, PD-1high CAR-T cells outperformed PD-1low CAR-T cells in multiple T cell functions both in vitro and in vivo. Despite the achievement of superior persistence at the tumor site in vivo, adoptive transfer of PD-1high CAR-T cells alone failed to control tumor growth. Instead, a PD-1 blockade combination therapy significantly delayed tumor progression in mice infused with PD-1high CAR-T cells. Therefore, our data demonstrate that robust T cell activation during the ex vivo CAR-T cell manufacturing process generates a PD-1high CAR-T cell subset with improved persistence and enhanced anti-cancer functions. However, these cells may be vulnerable to the immunosuppressive microenvironment and require combination with PD-1 inhibition to maximize therapeutic functions in solid tumors.

Belonging in diverse contexts: Sociability among same-ethnic and cross-ethnic peers.

Ethnically and racially diverse schools provide students opportunities to socially interact with both same- and cross-ethnic peers that can shape their sense of belonging within a school. This study investigates the extent to which same- or cross-ethnic friends influence feelings of school belonging in two large, diverse U.S. high schools (total N = 4,461; 9th-12th grade; 49.6% girls). Employing a longitudinal social network analytic approach, using stochastic actor-oriented modeling, this study found that students become more similar or stay similar to their same-ethnic friends, but not cross-ethnic friends, with no clear indication that students select friends based on similar levels of belonging. These novel findings highlight how feelings of school belonging are fostered through sociability in same-ethnic friend groups. Implications for interventions and other approaches to enhance school belonging are discussed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

The role of school friendship stability, instability, and network size in early adolescents' social adjustment.

This study examined longitudinal associations between early adolescents' school friendship stability, instability, and network size and their perceived social adjustment. The final sample consisted of 430 early adolescents residing in seven Midwestern schools (52% female, 47% Black, 42% White, 5% Hispanic or Latinx, and 6% Other). School friendship stability, instability, and network size were assessed via students' peer nominations of their same-grade friendships collected midway through their 7th and 8th grade years. Students also self-reported on several measures of social adjustment (their social satisfaction, social confidence, and school belonging). Results indicated having new friendships and having a larger friendship network at school were each more predictive of early adolescents' perceived social adjustment than was having maintained friendships or a consistent friend group within this context. However, school friendship stability consistently predicted greater perceived school belonging, whereas having a primarily new friend group and/or having lost more or most of a prior friend group predicted lower levels of this perception. Having lost school friendships was not predictive of early adolescents' social adjustment except when losses composed a significant proportion of their school friendship network (i.e., they had lost more or most of their school friendships from the year before). These findings provide valuable insights about peer friendship dynamics and social adjustment during a life stage critical to social development. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Changes in behavioral correlates of social status during early adolescence: Does school context matter?

This study investigated the role of school context in changes in the behaviors associated with having high social status during early adolescence. Three waves of surveys were collected from students (N = 542, 53% girls; 44% Black, 44% White, 5% Hispanic/Latinx, and 7% other; 60% free/reduced-fee lunch) in the middle of their sixth, seventh, and eighth grade school years. Peer nominations were used to assess two types of social status (peer acceptance and popularity) and three behavioral reputations (academic, prosocial, and physical aggression). Approximately half of the students made a transition from an elementary school to a larger middle school after sixth grade and the other half attended the same school from kindergarten through eighth grade (K-8). Across time and school configurations, students who were well-liked were perceived to be academically oriented, prosocial, and not physically aggressive. In contrast, the reputations of popular students differed in the two school configurations. In the K-8 context, popularity became increasingly associated with academic and prosocial behavior, and less characterized by physical aggression over time. In the transition context, popularity was associated with academic and prosocial behavior in elementary school, but these associations were null upon the transition to middle school in seventh grade. When students moved into eighth grade, popular students were increasingly academically oriented. In the transition group, popularity was also characterized by physical aggression at all three grades. Findings highlighted the significance of school context for the development of popularity. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

School Attachment and Violent Attitudes Preventing Future Violent Behavior Among Youth.

Evidence derived from social information theories support the existence of different underlying cognitive mechanisms guiding violent behavior through life. However, a few studies have examined the contribution of school variables to those cognitive mechanisms, which may help explain violent behavior later in life. The present study examines the relationship between school attachment, violent attitudes, and violent behavior over time in a sample of urban adolescents from the U.S. Midwest. We evaluated the influence of school attachment on violent attitudes and subsequent violent behavior. We used structural equation modeling to test our hypothesis in a sample of 579 participants (54.9% female, 81.3% African American). After controlling for gender and race, our results indicated that the relationship between school attachment and violent behavior over time is mediated by violent attitudes. The instrumentalization of the school context as a learning environment aiming to prevent future violent behavior is also discussed.

Coping with Discrimination from Peers and Adults: Implications for Adolescents' School Belonging.

School belonging is a key indicator of students' academic well-being that is threatened by adults' and peers' transgressions of discrimination. Moreover, the hierarchical power structure at school enables adults and peers to enact ethnic-racial discrimination differently, which is also more or less salient among Black, Asian American, and Latinx youth. Therefore, this study aimed to disentangle the links between adult and peer-perpetrated racial discrimination at school, five distinct coping strategies, and school belonging across ethnic-racial groups. Participants were 1686 students in grades 9-12. These results indicated that adolescents who reported peer discrimination also reported greater proactive and aggressive coping. Black youth who reported more adult discrimination also reported more proactive coping, whereas Asian and Latinx youth who reported more peer discrimination reported more proactive coping. Peer discrimination was indirectly associated with greater school belonging via proactive coping, whereas adult discrimination was directly and negatively related to belonging. These findings suggest that adolescents may be selecting to proactively cope when faced with the discrimination source they most often navigate.

Antipsychotic Behavioral Phenotypes in the Mouse Collaborative Cross Recombinant Inbred Inter-Crosses (RIX).

Schizophrenia is an idiopathic disorder that affects approximately 1% of the human population, and presents with persistent delusions, hallucinations, and disorganized behaviors. Antipsychotics are the standard pharmacological treatment for schizophrenia, but are frequently discontinued by patients due to inefficacy and/or side effects. Chronic treatment with the typical antipsychotic haloperidol causes tardive dyskinesia (TD), which manifests as involuntary and often irreversible orofacial movements in around 30% of patients. Mice treated with haloperidol develop many of the features of TD, including jaw tremors, tongue protrusions, and vacuous chewing movements (VCMs). In this study, we used genetically diverse Collaborative Cross (CC) recombinant inbred inter-cross (RIX) mice to elucidate the genetic basis of antipsychotic-induced adverse drug reactions (ADRs). We performed a battery of behavioral tests in 840 mice from 73 RIX lines (derived from 62 CC strains) treated with haloperidol or placebo in order to monitor the development of ADRs. We used linear mixed models to test for strain and treatment effects. We observed highly significant strain effects for almost all behavioral measurements investigated (P < 0.001). Further, we observed strong strain-by-treatment interactions for most phenotypes, particularly for changes in distance traveled, vertical activity, and extrapyramidal symptoms (EPS). Estimates of overall heritability ranged from 0.21 (change in body weight) to 0.4 (VCMs and change in distance traveled) while the portion attributable to the interactions of treatment and strain ranged from 0.01 (for change in body weight) to 0.15 (for change in EPS). Interestingly, close to 30% of RIX mice exhibited VCMs, a sensitivity to haloperidol exposure, approximately similar to the rate of TD in humans chronically exposed to haloperidol. Understanding the genetic basis for the susceptibility to antipsychotic ADRs may be possible in mouse, and extrapolation to humans could lead to safer therapeutic approaches for schizophrenia.

Non-infectious thoracic discitis: A diagnostic and management dilemma. A report of two cases with review of the literature.

Discitis/ Osteomyelitis is an inflammatory process involving an intervertebral disc and the adjacent vertebral bodies. Infection is the most common cause of discitis, which is often spontaneous and hematogenous in origin. However, many noninfectious processes affecting the spine such as pseudarthrosis in ankylosing spondylitis, amyloidosis, destructive spondyloarthropathy of hemodialysis, Modic changes type 1, neuropathic arthropathy, calcium pyrophosphate dehydrate (CPPD) spondyloarthropathy and gout can mimic infectious discitis/ osteomyelitis. To determine whether a particular patient's spinal process is due to an infectious versus non-infectious cause can be challenging. Although clinical findings and laboratory studies including erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) can be helpful in the diagnosis of bacterial discitis/osteomyelitis due to their high sensitivity; however, their specificity is low. Moreover, both the infectious and non-infectious discitis can appear quite similar on the imaging studies. We present two cases of thoracic discitis with adjacent vertebral osteomyelitis of probable non-infectious etiology. Both were managed with instrumented fusion for stabilization. We also discuss a range of noninfectious causes of discitis/spondylitis and their radiological features which can help differentiate from infectious processes.

Validation of a Single-Nucleotide Polymorphism-Based Non-Invasive Prenatal Test in Twin Gestations: Determination of Zygosity, Individual Fetal Sex, and Fetal Aneuploidy.

We analyzed maternal plasma cell-free DNA samples from twin pregnancies in a prospective blinded study to validate a single-nucleotide polymorphism (SNP)-based non-invasive prenatal test (NIPT) for zygosity, fetal sex, and aneuploidy. Zygosity was evaluated by looking for either one or two fetal genome complements, fetal sex was evaluated by evaluating Y-chromosome loci, and aneuploidy was assessed through SNP ratios. Zygosity was correctly predicted in 100% of cases (93/93; 95% confidence interval (CI) 96.1%-100%). Individual fetal sex for both twins was also called with 100% accuracy (102/102; 95% weighted CI 95.2%-100%). All cases with copy number truth were also correctly identified. The dizygotic aneuploidy sensitivity was 100% (10/10; 95% CI 69.2%-100%), and overall specificity was 100% (96/96; 95% weighted CI, 94.8%-100%). The mean fetal fraction (FF) of monozygotic twins (n = 43) was 13.0% (standard deviation (SD), 4.5%); for dizygotic twins (n = 79), the mean lower FF was 6.5% (SD, 3.1%) and the mean higher FF was 8.1% (SD, 3.5%). We conclude SNP-based NIPT for zygosity is of value when chorionicity is uncertain or anomalies are identified. Zygosity, fetal sex, and aneuploidy are complementary evaluations that can be carried out on the same specimen as early as 9 weeks' gestation.

Analytical Validation of a Single-nucleotide Polymorphism-based Donor-derived Cell-free DNA Assay for Detecting Rejection in Kidney Transplant Patients.

BACKGROUND: Early detection of rejection in kidney transplant recipients holds the promise to improve clinical outcomes. Development and implementation of more accurate, noninvasive methods to detect allograft rejection remain an ongoing challenge. The limitations of existing allograft surveillance methods present an opportunity for donor-derived cell-free DNA (dd-cfDNA), which can accurately and rapidly differentiate patients with allograft rejection from patients with stable organ function. METHODS: This study evaluated the analytical performance of a massively multiplexed polymerase chain reaction assay that targets 13 962 single-nucleotide polymorphisms, characterized and validated using 66 unique samples with 1064 replicates, including cell line-derived reference samples, plasma-derived mixtures, and transplant patient samples. The dd-cfDNA fraction was quantified in both related and unrelated donor-recipient pairs. RESULTS: The dd-cfDNA assay showed a limit of blank of 0.11%, a limit of detection and limit of quantitation of 0.15% for unrelated donors, and limit of blank of 0.23%, a limit of detection and limit of quantitation of 0.29% for related donors. All other metrics (linearity, accuracy, and precision) were observed to be equivalent between unrelated and related donors. The measurement precision of coefficient of variation was 1.8% (repeatability, 0.6% dd-cfDNA) and was <5% for all the different reproducibility measures. CONCLUSIONS: This study validates the performance of a single-nucleotide polymorphism-based massively multiplexed polymerase chain reaction assay to detect the dd-cfDNA fraction with improved precision over currently available tests, regardless of donor-recipient relationships.

Adolescents' Friendships, Academic Achievement, and Risk Behaviors: Same-Behavior and Cross-Behavior Selection and Influence Processes.

This study examined to what extent adolescents' and their friends' risk behaviors (i.e., delinquency and alcohol use) hinder or promote their academic achievement (grade point average [GPA]), and vice versa. Longitudinal data were used (N = 1,219 seventh- to ninth-grade adolescents; Mage  = 13.69). Results showed that risk behaviors negatively affected adolescents' GPA, whereas GPA protected against engaging in risk behaviors. Moreover, adolescents tended to select friends who have similar behaviors and friends' behaviors became more similar over time (same-behavior selection and influence). Furthermore, although same-behavior effects seemed to dominate, evidence was found for some cross-behavior selection effects and a tendency in seventh grade for cross-behavior influence effects. Concluding, it is important to investigate the interplay between different behaviors with longitudinal social network analysis.

Social Status and Classroom Behavior in Math and Science during Early Adolescence.

Social status is a salient feature of the classroom peer ecology in early adolescence, yet research has not examined how it plays out within the domains of math and science. The current study investigated the behavioral profiles of cool and admired youth (n = 739, 51% female) in 5th and 6th grade math and science classes. "Cool" youth were perceived by peers as academically oriented, prosocial, and not disruptive. When grade level differences were found, they tended to favor 5th graders, such that cool youth had even more positive profiles in 5th grade compared to 6th grade. Admiration was associated with a more adaptive pattern of behaviors than coolness, and grade level differences were less pronounced. Cross-lagged models revealed some reciprocal relationships between behavior and social status. Implications for teachers are discussed, namely that attention to social status dynamics is important for creating a positive social climate that supports early adolescent engagement in math and science classes.

It's Lonely at the Top: Adolescent Students' Peer-perceived Popularity and Self-perceived Social Contentment.

Popularity is highly desired among youth, often more so than academic achievement or friendship. Recent evidence suggests being known as "popular" among peers (perceived popularity) may be more detrimental during adolescence than being widely well-liked (sociometric popularity). Thus, this study sought to better understand how two dimensions of popularity (perceived and sociometric) may contribute to adolescents' own perceptions of satisfaction and happiness regarding their social life at school, and hypothesized that "being popular" would have a more complex (and curvilinear) association with adolescents' social contentment than previously considered by linear models. Adolescents' peer popularity and self-perceived social contentment were examined as both linear and curvilinear associations along each status continuum in a series of hierarchical regressions. Participants were 767 7th-grade students from two middle schools in the Midwest (52% female, 46% White, 45% African American). Perceived and sociometric popularity were assessed via peer nominations ("most popular" and "liked the most", respectively). Self-reported social satisfaction, best friendship quality, social self-concept, and school belonging were assessed as aspects of social contentment. The results indicated that both high and low levels of perceived popularity, as well as high and low levels of sociometric popularity, predicted lower perceptions of social satisfaction, poorer best friendship quality, and lower social self-concept than youth with moderate levels of either status. Implications to promote adolescents' psychosocial well-being by targeting popularity's disproportionate desirability among youth are discussed.

Optimizing Detection of Kidney Transplant Injury by Assessment of Donor-Derived Cell-Free DNA via Massively Multiplex PCR.

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3%) versus BL (0.6%), OI (0.7%), and STA (0.4%) (p < 0.0001 all comparisons). The SNP-based dd-cfDNA assay discriminated active from non-rejection status with an area under the curve (AUC) of 0.87, 88.7% sensitivity (95% CI, 77.7⁻99.8%) and 72.6% specificity (95% CI, 65.4⁻79.8%) at a prespecified cutoff (>1% dd-cfDNA). Of 13 patients with AR findings at a routine protocol biopsy six-months post transplantation, 12 (92%) were detected positive by dd-cfDNA. This SNP-based dd-cfDNA assay detected allograft rejection with superior performance compared with the current standard of care. These data support the feasibility of using this assay to detect disease prior to renal failure and optimize patient management in the case of allograft injury.

Characteristics of likability, perceived popularity, and admiration in the early adolescent peer system in the United States and China.

High social status youth are often influential in the peer system. Thus, they may serve as agents of cultural socialization if they exhibit characteristics that reflect cultural values (e.g., interdependence). This research examined the behavior that contributes to high social status in the United States and China. At each of 3 waves, 934 early adolescents (M age = 12.7 years at Wave 1) made behavioral (i.e., prosocial behavior and academic engagement) and social status (i.e., likability, perceived popularity, and admiration) nominations of their peers. Positive behavior was predictive of higher social status in both the United States and China, but this was stronger in China. In the United States, there was a tendency for positive behavior to be less predictive of perceived popularity than other forms of social status (e.g., likability); however, this tendency was not evident in China. (PsycINFO Database Record