Characterization of "Off-Target" Immune Modulation Induced by Live Attenuated Yellow Fever Vaccine.

BACKGROUND: Live attenuated vaccines alter immune functions and are associated with beneficial outcomes. We previously demonstrated that live attenuated yellow fever virus (YFV) vaccine (LA-YF-Vax) dampens T-cell receptor (TCR) signaling in vitro via an RNA-based mechanism. We examined study participants before and after LA-YF-Vax to assess TCR-mediated functions in vivo. METHODS: Serum samples and peripheral blood mononuclear cells (PBMCs) were obtained before and after LA-YF-Vax (with or without additional vaccines) or quadrivalent influenza vaccine. TCR-mediated activation was determined by interleukin 2 release or phosphorylation of the lymphocyte-specific Src kinase. TCR-regulating phosphatase (protein tyrosine phosphatase receptor type E [PTPRE]) expression was also measured. RESULTS: Compared with prevaccination findings, LA-YF-Vax recipient PBMCs demonstrated transient reduction in interleukin 2 release after TCR stimulation and PTPRE levels, unlike in control participants who received quadrivalent influenza vaccine. YFV was detected in 8 of 14 participants after LA-YF-Vax. After incubation of healthy donor PBMCs in serum-derived extracellular vesicles prepared from LA-YF-Vax recipients, TCR signaling and PTPRE levels were reduced after vaccination, even in participants without detectable YFV RNA. CONCLUSIONS: LA-YF-Vax reduces TCR functions and PTPRE levels after vaccination. Extracellular vesicles from serum recapitulated this effect in healthy cells. This likely contributes to the reduced immunogenicity for heterologous vaccines after LA-YF-Vax administration. Identification of specific immune mechanisms related to vaccines should contribute to understanding of the "off-target," beneficial effects of live vaccines.

Trends in Bullying Victimization and Social Unsafety for Sexually and Gender Diverse Students.

Research has documented trends in bullying victimization for sexually diverse adolescents in the US, but trends regarding school social unsafety are understudied and there is a dearth of research examining these trends for gender diverse adolescents. This study aimed to identify disparities in bullying victimization and feelings of social unsafety in schools for sexually and gender diverse adolescents. Data stem from the 2014 (N = 15,800; M age = 14.17, SD = 1.50), 2016 (N = 22,310; M age = 14.17, SD = 1.49), and 2018 (N = 10,493; M age = 14.02, SD = 1.52) survey cycles of the Social Safety Monitor, a Dutch cross-sectional school-based study. Findings indicate that sexual orientation disparities remained relatively small, but stable over time, while gender diverse adolescents remained more likely to be victimized and feel unsafe in school, with larger disparities overall. Monitoring these trends is highly relevant, especially considering recent negative developments regarding societal acceptance of sexual and gender diversity.

Gender Sexuality Alliances and School Safety: Who Benefits Most, and Do Additive School-Led Practices Strengthen the Link?

While Gender and Sexuality Alliances (GSAs) are associated with higher acceptance of sexual diversity and lower bullying-victimization, it is unclear which individual and school-level attributes strengthen these associations. Nationally representative data (N = 1,567 students; Mage = 15.4, SD = 0.16; 34% boys, 66% girls, 51% heterosexual, 49% sexually-diverse after propensity score matching) in 139 Dutch secondary schools were used. Multilevel regression analyses revealed that GSA presence was linked to more inclusive attitudes about sexual diversity and a safer disclosure climate among sexually-diverse students, and lower general bullying-victimization when the school had a GSA combined with school practices to tackle bullying. School professionals and researchers are recommended to recognize the significance of individual and school-level factors that affect GSA correlates.

Heterogenous associations between Gender-Sexuality Alliances and LGBTQ adolescents' maladjustment across individual victimization level.

Gender-Sexuality Alliances (GSAs), which are student-initiated school clubs for LGBTQ youth and allies, can reduce victimization for lesbian, gay, bisexual, transgender, and queer (LGBTQ) youth. This preregistered study identified heterogeneous correlates of GSAs, based on data from an anonymous survey of LGBTQ adolescents aged 13-17 years living in the United States (N = 10,588). In line with the healthy context paradox (Pan et al. [Child Development, 92, 2021, and 1836]), the presence of a GSA exacerbated associations between LGBTQ-based victimization and depressive symptoms, lower self-esteem, and lower academic grades-particularly in transgender youth. Inclusive settings, such as GSAs, might prevent increasing disparities by including tailored strategies to monitor and support more vulnerable, victimized LGBTQ youth.

Characterization of cardiac beta-adrenergic receptors in the guinea pig heart: application to study of beta-adrenergic receptors in shock models.

The myocardial response to catecholamines is significantly diminished in many types of shock or heart failure. The guinea pig heart is an ideal model for the study of shock, as it is relatively inexpensive, and the cardiovascular system of the guinea pig most closely resembles that of the human. Using this model, we have developed techniques to characterize and quantitate changes in beta-adrenergic receptors (beta AR) in the guinea pig heart after burn injury. Preliminary experiments were performed to determine the optimum binding conditions, e.g., incubation time and conditions, protein concentrations, rinsing, etc. Additional experiments were conducted using agonists and antagonists to characterize the rank order of potency and stereospecificity of the beta AR. Crude membrane preparations (50 micrograms/250 microliters) from sham-burned and burned hearts were incubated with 8-10 concentrations of 125I-cyanopindolol (10-450 pM) at 37 degrees C for 1 hr. Under these conditions, binding assays were linear with respect to protein concentration and time. Alprenolol (10 microM) was used to determine nonspecific binding. The membrane preparations used in this study bound both agonists and antagonists with a rank order of potency and stereospecificity characteristic of a beta-adrenergic receptor. Finally, agonist competition curves were performed with isoproterenol in the presence and absence of Gpp(NH)p to determine receptor regulation by the Gs protein. Analysis using computer-assisted techniques suggests that the fraction of high-affinity beta-receptors is significantly reduced after burn injury (41.2 +/- 4.7%) compared to sham-burned controls (54 +/- 2%, P < or = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS)

Burn-induced alterations in cardiac beta-adrenergic receptors.

Previous studies in our laboratory have demonstrated that burn injury (45% total body surface area, 3rd-degree scald burn) diminishes contractile and relaxation function in the isolated perfused guinea pig heart. The mechanisms responsible for the burn-mediated dysfunction are not well understood. Therefore the purpose of this study was to examine the inotropic response to isoproterenol, a beta-adrenergic agonist, and burn-induced alterations in beta-adrenergic receptors (beta-AR) in adult guinea pig hearts. Isoproterenol dose-response curves were generated in isolated perfused hearts from sham-burned and burned guinea pigs. In addition, binding studies were performed using [125I]iodocyanopindolol on hearts from sham-burned and burned guinea pigs. Both the functional response and sensitivity to isoproterenol were significantly diminished 24 h after burn injury. beta-AR density (binding capacity, Bmax) and affinity were determined by Scatchard analysis. Agonist competition curves were performed in the presence or absence of 0.1 mM 5'-guanylyl imidodiphosphate. There was no difference in Bmax in membranes from sham-burned and burned hearts; however, the affinity of beta-AR was significantly decreased after burn injury compared with sham burn [dissociation constant = 32.5 +/- 1.9 (mean +/- SE), n = 10, vs. 26.7 +/- 1.7 pM, n = 10, P = 0.039]. Furthermore, the fraction of receptors in a high-affinity state (those functionally coupled to Gs protein) was significantly decreased after burn injury compared with sham burn (41.2 +/- 4.7%, n = 9, vs. 54 +/- 2%, n = 9, P = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of the terminal mannosyl units of lipoarabinomannan from a virulent strain of Mycobacterium tuberculosis to human macrophages.

Recent studies from this laboratory have demonstrated that macrophage phagocytosis of virulent strains (Erdman and H37Rv), but not the attenuated H37Ra strain of Mycobacterium tuberculosis, is mediated by phagocyte mannose receptors (MR) in addition to complement receptors (CR1 and the leukocyte integrins CR3 and CR4). Lipoarabinomannan (LAM) is a major surface lipoglycan of M. tuberculosis. LAM from the Erdman strain (Man-LAM) contains mannose oligosaccharides at the terminal portions of the molecule. This study investigated the ability of ManLAM to serve as a microbial ligand in adherence to human monocyte-derived macrophages (MDM). Polystyrene microspheres were coated with known amounts of purified ManLAM, LAM without the terminal mannosyl units from an avirulent mycobacterium (AraLAM), lipomannan (LM), or buffer and incubated with MDM monolayers in the absence of serum. The presence of LAM on microspheres was confirmed by indirect immunofluorescence studies. Microspheres coated with ManLAM demonstrated a more than threefold increase in adherence to MDM when compared with microspheres coated with AraLAM, LM, or buffer and the low levels of adherence of microspheres in the latter three groups were comparable. Compared with control monolayers, selective down-modulation of MDM MR on a mannan substrate abrogated the enhanced adherence of microspheres mediated by ManLAM. Adherence of microspheres coated with AraLAM, LM, or buffer was not influenced by MR modulation. To confirm the importance of the terminal mannosyl units of ManLAM in the enhanced adherence of ManLAM microspheres to MDM, these units were selectively removed by exomannosidase treatment. The structure of LAM products before and after enzyme treatment was confirmed by high performance anion exchange chromatography with pulsed amperometric detection. Removal of the terminal mannosyl units abolished the capacity of ManLAM to mediate enhanced adherence of microspheres to MDM. Finally, preincubation of Erdman M. tuberculosis with CS-40, a mAb directed against LAM, resulted in a consistent inhibition of adherence of the bacteria to MDM (up to 49% inhibition), confirming a role for ManLAM on intact bacteria in adherence to MDM. Thus, we provide evidence for a novel receptor-ligand pathway in phagocytosis of M. tuberculosis that consists of MR on macrophages and mannosyl units at the terminal end of ManLAM, a major microbial surface lipoglycan.

Cardiac contractile and calcium transport function after burn injury in adult and aged guinea pigs.

Cardiac dysfunction occurs after a major burn injury regardless of age; whether burn trauma causes greater myocardial contractile depression in the older subject due to reduced cardiac reserves that normally occur with adult aging is not known. The cellular basis for burn-induced cardiac dysfunction is not known, but several studies have suggested that alterations occur in the rate of Ca2+ delivery to the contractile proteins as well as in the rate of Ca2+ removal from the sarcoplasm by the sarcoplasmic reticulum (SR). To determine if age-related differences in the cardiac contractile response to burn injury are associated with differences in SR Ca2+ transport, an isolated heart preparation was used to examine mechanical function, and myocardial homogenate preparations were used to assess SR Ca2+ transport. Guinea pigs from both age groups (adult, 6-8 months, and senescent, 34-36 months of age) were divided into two subgroups--control and 45% cutaneous scald burn. Cardiac dysfunction associated with adult aging alone was indicated by lower systolic pressure and lower rates of left ventricular (LV) pressure rise and fall, as well as decreased responses to isoproterenol, exogenous Ca2+, increased coronary flow rate, and electrical pacing. Myocardial depression in senescent control hearts was accompanied by a decreased maximal Ca2+ uptake in myocardial homogenates, suggesting that altered SR calcium transport may contribute to the diminished contractile function associated with aging. Burn injury impaired cardiac function in all animals regardless of age as evidenced by a leftward shift of LV function curves and altered responses to receptor- and nonreceptor-mediated inotropic interventions. However, the percentage change in cardiac function after burn injury was similar in both age groups compared to those of their respective controls. Significant alterations in SR Ca2+ transport were observed in myocardial homogenates isolated from both adult and senescent hearts after burn injury. Our data confirm that burn injury induced cardiac contractile dysfunction as well as alterations in SR Ca2+ transport function regardless of age, and we conclude that altered SR Ca2+ transport function contributes, in part, to the diminished cardiac function after burn injury.

Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages.

Phagocytosis of the virulent Erdman and H37Rv strains of Mycobacterium tuberculosis, but not that of the attenuated H37Ra strain, by human macrophages is mediated by the mannose receptor (MR) in addition to complement receptors. We have recently determined that a major capsular lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serves as a ligand for the MR during phagocytosis of bacteria. In this study we directly compare uptake of Erdman, H37Rv, and H37Ra LAM by human macrophages and assess the relative contribution of the MR in this process. Microspheres coated with LAM served as model phagocytic particles for studies of LAM as a capsular ligand. Uptake (37 degrees C) of LAM microspheres by monocyte-derived macrophages was greatest for Erdman LAM and intermediate for H37Rv and H37Ra LAM compared with that of buffer microspheres or microspheres coated with LAM from a nontuberculosis strain of mycobacterium (AraLAM). Inhibition of microsphere uptake in the presence of mannan or mannose-BSA was highest for Erdman LAM (75 +/- 8 and 50 +/- 7%, respectively) and H37Rv LAM (57 +/- 13 and 21 +/- 5%, respectively) relative to H37Ra LAM (36 +/- 16 and 22 +/- 11 %, respectively). Inhibition of microsphere uptake in the presence of anti-MR Ab followed a similar pattern: Erdman LAM (80 +/- 9%) > H37Rv LAM (53 +/- 1%) > H37Ra LAM (26 +/- 12%). Attachment (4 degrees C) of microspheres coated with Erdman LAM, H37Rv LAM, and H37Ra LAM was enhanced 12-, 5-, and 4-fold, respectively, compared with that of microspheres coated with AraLAM, and mannose-BSA inhibited attachment of these microspheres by 82 +/- 7, 69 +/- 8, and 12 +/- 17%. Galactose-BSA did not inhibit attachment of any LAM microsphere groups. Chromatographic analyses of mild acid hydrolysates of LAM from Erdman, H37Rv, and H37Ra all revealed the major terminal dimannosyl units. These studies demonstrate differences in the ability of LAM from different M. tuberculosis strains to mediate adherence to macrophages and to serve as ligands for the macrophage MR despite the presence of terminal dimannosyl units. Thus, these studies point toward other subtle structural alterations in LAM among strains that influence initial interactions with human phagocytes.

Age-related changes in myocardial relaxation and sarcoplasmic reticulum function.

Rapid regulation of relaxation is essential to allow the heart to alter stroke volume in response to stress. Inasmuch as Ca2+ transport by the sarcoplasmic reticulum (SR) is an important determinant of relaxation, the purpose of this study was to examine developmental differences in the ability of isoproterenol to alter relaxation time and to determine if these differences were associated with age-related changes in Ca2+ transport by the SR in isolated, perfused adult and newborn guinea pig hearts. Control values of the time constant of isovolumic relaxation (tau) were 37.8 +/- 5.9 ms in adult hearts (n = 8) and 31.6 +/- 5.3 ms in newborn hearts (n = 6). With maximum isoproterenol stimulation, the decrease in tau was significantly greater in adult (51.1 +/- 8.8%, mean +/- SD) compared with that in newborn (26.3 +/- 3.1%, P less than or equal to 0.0001) hearts. Ca2+ uptake, Ca2(+)-dependent adenosinetriphosphatase activity, and Ca2+ pump density were all significantly greater in SR vesicles isolated from adult hearts compared with values measured in SR vesicles from newborn hearts. We conclude that developmental differences in the capacity of the SR to sequester Ca2+ may contribute to age-related differences in the functional response of the heart to isoproterenol.