Proinflammatory polarization of monocytes by particulate air pollutants is mediated by induction of trained immunity in pediatric asthma.

BACKGROUND: The impact of exposure to air pollutants, such as fine particulate matter (PM), on the immune system and its consequences on pediatric asthma, are not well understood. We investigated whether ambient levels of fine PM with aerodynamic diameter ≤2.5 microns (PM2.5 ) are associated with alterations in circulating monocytes in children with or without asthma. METHODS: Monocyte phenotyping was performed by cytometry time-of-flight (CyTOF). Cytokines were measured using cytometric bead array and Luminex assay. ChIP-Seq was utilized to address histone modifications in monocytes. RESULTS: Increased exposure to ambient PM2.5 was linked to specific monocyte subtypes, particularly in children with asthma. Mechanistically, we hypothesized that innate trained immunity is evoked by a primary exposure to fine PM and accounts for an enhanced inflammatory response after secondary stimulation in vitro. We determined that the trained immunity was induced in circulating monocytes by fine particulate pollutants, and it was characterized by the upregulation of proinflammatory mediators, such as TNF, IL-6, and IL-8, upon stimulation with house dust mite or lipopolysaccharide. This phenotype was epigenetically controlled by enhanced H3K27ac marks in circulating monocytes. CONCLUSION: The specific alterations of monocytes after ambient pollution exposure suggest a possible prognostic immune signature for pediatric asthma, and pollution-induced trained immunity may provide a potential therapeutic target for asthmatic children living in areas with increased air pollution.

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling.

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.

Combining conservation and species-specific differences to determine how human telomerase binds telomeres.

Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human and Tetrahymena thermophila telomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

Structural biology of telomeres and telomerase.

Telomeres are protein-DNA complexes that protect chromosome ends from illicit ligation and resection. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA to counter telomere shortening. Human telomeres are composed of complexes between telomeric DNA and a six-protein complex known as shelterin. The shelterin proteins TRF1 and TRF2 provide the binding affinity and specificity for double-stranded telomeric DNA, while the POT1-TPP1 shelterin subcomplex coats the single-stranded telomeric G-rich overhang that is characteristic of all our chromosome ends. By capping chromosome ends, shelterin protects telomeric DNA from unwanted degradation and end-to-end fusion events. Structures of the human shelterin proteins reveal a network of constitutive and context-specific interactions. The shelterin protein-DNA structures reveal the basis for both the high affinity and DNA sequence specificity of these interactions, and explain how shelterin efficiently protects chromosome ends from genome instability. Several protein-protein interactions, many provided by the shelterin component TIN2, are critical for upholding the end-protection function of shelterin. A survey of these protein-protein interfaces within shelterin reveals a series of "domain-peptide" interactions that allow for efficient binding and adaptability towards new functions. While the modular nature of shelterin has facilitated its part-by-part structural characterization, the interdependence of subunits within telomerase has made its structural solution more challenging. However, the exploitation of several homologs in combination with recent advancements in cryo-EM capabilities has led to an exponential increase in our knowledge of the structural biology underlying telomerase function. Telomerase homologs from a wide range of eukaryotes show a typical retroviral reverse transcriptase-like protein core reinforced with elements that deliver telomerase-specific functions including recruitment to telomeres and high telomere-repeat addition processivity. In addition to providing the template for reverse transcription, the RNA component of telomerase provides a scaffold for the catalytic and accessory protein subunits, defines the limits of the telomeric repeat sequence, and plays a critical role in RNP assembly, stability, and trafficking. While a high-resolution definition of the human telomerase structure is only beginning to emerge, the quick pace of technical progress forecasts imminent breakthroughs in this area. Here, we review the structural biology surrounding telomeres and telomerase to provide a molecular description of mammalian chromosome end protection and end replication.

Structural and functional consequences of a disease mutation in the telomere protein TPP1.

Telomerase replicates chromosome ends to facilitate continued cell division. Mutations that compromise telomerase function result in stem cell failure diseases, such as dyskeratosis congenita (DC). One such mutation (K170Δ), residing in the telomerase-recruitment factor TPP1, provides an excellent opportunity to structurally, biochemically, and genetically dissect the mechanism of such diseases. We show through site-directed mutagenesis and X-ray crystallography that this TPP1 disease mutation deforms the conformation of two critical amino acids of the TEL [TPP1's glutamate (E) and leucine-rich (L)] patch, the surface of TPP1 that binds telomerase. Using CRISPR-Cas9 technology, we demonstrate that introduction of this mutation in a heterozygous manner is sufficient to shorten telomeres in human cells. Our findings rule out dominant-negative effects of the mutation. Instead, these findings implicate reduced TEL patch dosage in causing telomere shortening. Our studies provide mechanistic insight into telomerase-deficiency diseases and encourage the development of gene therapies to counter such diseases.

Opioid and Opiate Immunoregulatory Processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel δ, µ., and κ receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel µ3 opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Associations of Restraint and Seclusion With Race and Ethnicity on an Adolescent Inpatient Psychiatry Service.

There is limited work examining the association of race and ethnicity with restraint and seclusion (R/S) in pediatric inpatient psychiatric units. The present study assessed risk of R/S by race and ethnicity based on a retrospective review of electronic medical records (EMRs) from an adolescent inpatient psychiatric service. Demographic, diagnostic, and R/S data were analyzed for all 1,865 admissions of 1,327 patients from an adolescent unit at a child and adolescent psychiatric hospital from June 2018 to June 2021. R/S occurred in 459 of the admissions. For the purpose of patient privacy and statistical analysis, race was grouped into the following: Black or African American, other (American Indian or Alaskan Native, Asian, multiracial, other), and White. Patients identified as unknown were not included in the analysis. A binary logistic regression with a repeated subject effect regressed R/S onto race and adjusted for age, gender, and length of stay (LOS). There was an overall significant association of R/S and race (χ22 = 16.81, p < .001), but not ethnicity. In a regression model adjusted for age, gender, and LOS, patients identified as Black or African American were at significantly higher risk of R/S compared with patients identified as White (odds ratio = 1.66, p = .036). There was no significant difference in risk of R/S between patients identified as White vs other. Younger age and longer LOS were also significantly associated with R/S. These findings highlight a critical health care disparity related to race on an inpatient adolescent psychiatry service. There is likely a combination of individual and systemic factors leading to discriminatory practices in the use of R/S. Future work will assess potential associations with diagnosis and child welfare involvement and will examine additional characteristics of R/S. Focus groups held with hospital and community stakeholders will guide next steps to address these findings.

Scale invariance in early embryonic development.

The body plan of the fruit fly is determined by the expression of just a handful of genes. We show that the spatial patterns of expression for several of these genes scale precisely with the size of the embryo. Concretely, discrete positional markers such as the peaks in striped patterns have absolute positions along the anterior-posterior axis that are proportional to embryo length, with better than 1% accuracy. Further, the information (in bits) that graded patterns of expression provide about position can be decomposed into information about fractional or scaled position and information about absolute position or embryo length; all of the available information is about scaled position, again with ~ 1% accuracy. These observations suggest that the underlying genetic network exhibits scale invariance in a deeper mathematical sense. Taking this mathematical statement seriously requires that the network dynamics have a zero mode, which connects to many other observations on this system.

Associations of early life stress with leptin and ghrelin in healthy young adults.

BACKGROUND: Childhood adversity is a major risk factor for cardiometabolic health problems. Stress-related changes in diet suggest a role for endocrine factors that influence dietary intake, such as leptin and ghrelin. These hormones influence metabolism and may contribute to the relationship of early adversity, mental, and cardiometabolic health. This study examined levels of leptin and ghrelin in a sample of young adults with and without early life stress (ELS). METHODS: Young adults ages 18-40 (N = 200; 68.5% female) were recruited from the community. Participants with ELS (N = 118) had childhood maltreatment, and a subset, n = 92 (78.0%) also had parental loss, and n = 65 (55.1%) also had a current psychiatric disorder. Control participants (N = 82) had no maltreatment, parental loss, or psychiatric disorders. Standardized interviews and self-reports assessed demographics, adversity, medical/psychiatric history, and health behaviors. Exclusion criteria included medical conditions and current medications other than hormonal contraceptives. Body Mass Index (BMI) and other anthropometrics were measured, and fasting plasma was assayed for total ghrelin and leptin with the Bio-Plex Pro Human Diabetes Panel. RESULTS: While ELS was significantly associated with greater leptin (r = .16, p = .025), a finding which held when adjusted for age and sex (F(3196)= 28.32, p = .011), this relationship was abolished when accounting for BMI (p = .44). Participants with ELS also had significantly lower total ghrelin (r = .21, p = .004), which held adjusting for age and sex (p = .002) and was attenuated (p = .045) when the model included BMI (F=46.82, p < .001). Current psychiatric disorder was also a significant predictor of greater leptin (r = .28, p < .001) and lower ghrelin (r = .29, p = .003). In the model with ELS and covariates, psychiatric disorder remained significant (F=7.26, p = .008) and ELS was no longer significant (p = .87). Associations with severity and recent perceived stress were also examined. CONCLUSION: The relationship of ELS and leptin was no longer significant when accounting for BMI, suggesting potential avenues for intervention. Ghrelin findings persisted after correction for BMI, which may be secondary to physiological differences in the regulation of these hormones (leptin is produced by adipocytes, whereas ghrelin is produced primarily in the GI tract). Lastly, these findings suggest that psychiatric functioning may be a key component contributing to the relationship of lower total ghrelin and childhood adversity.

A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases.

B cells contribute to multiple aspects of autoimmune disorders, and B cell-targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti-human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell-dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell-targeting therapies in treatment of autoimmune conditions without causing B cell depletion.

Structures of an active type III-A CRISPR effector complex.

Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.

Adverse effects of air pollution-derived fine particulate matter on cardiovascular homeostasis and disease.

Air pollution is a rapidly growing major health concern around the world. Atmospheric particulate matter that has a diameter of less than 2.5 µm (PM2.5) refers to an air pollutant composed of particles and chemical compounds that originate from various sources. While epidemiological studies have established the association between PM2.5 exposure and cardiovascular diseases, the precise cellular and molecular mechanisms by which PM2.5 promotes cardiovascular complications are yet to be fully elucidated. In this review, we summarize the various sources of PM2.5, its components, and the concentrations of ambient PM2.5 in various settings. We discuss the experimental findings to date that evaluate the potential adverse effects of PM2.5 on cardiovascular homeostasis and function, and the possible therapeutic options that may alleviate PM2.5-driven cardiovascular damage.

Neuropeptides as signal molecules in common with leukocytes and the hypothalamic-pituitary-adrenal axis.

There exists a bidirectional regulatory circuit between the nervous and immune systems. This regulation has been shown to be mediated in part through neuroendocrine hormones and cytokines. Both systems have receptors for both types of signal molecules. The nervous system has receptors for cytokines and it also synthesizes cytokines. The immune system synthesizes and responds to cytokines. So, it is not too far-fetched to believe that neuroendocrine peptide hormones could bind to leukocytes and modulate immune functions. However, it is not widely known that the immune system also synthesizes functional, neuropeptide hormones. This will be discussed in this paper citing a plethora of evidence. The aim of this paper is to summarize this evidence by using three neuropeptides that are synthesized by leukocytes and modulate immune functions as examples; corticotropin (ACTH), endorphin (END), and corticotropin releasing factor (CRF). The production and action of these three neuropeptides in the immune system will be explained. Finally, the potential physiological role of leukocyte-derived ACTH, END, and CRF in inflammation as a localized hypothalamic-pituitary-like axis is discussed.

Dissecting the telomere-inner nuclear membrane interface formed in meiosis.

Tethering telomeres to the inner nuclear membrane (INM) allows homologous chromosome pairing during meiosis. The meiosis-specific protein TERB1 binds the telomeric protein TRF1 to establish telomere-INM connectivity and is essential for mouse fertility. Here we solve the structure of the human TRF1-TERB1 interface to reveal the structural basis for telomere-INM linkage. Disruption of this interface abrogates binding and compromises telomere-INM attachment in mice. An embedded CDK-phosphorylation site within the TRF1-binding region of TERB1 provides a mechanism for cap exchange, a late-pachytene phenomenon involving the dissociation of the TRF1-TERB1 complex. Indeed, further strengthening this interaction interferes with cap exchange. Finally, our biochemical analysis implicates distinct complexes for telomere-INM tethering and chromosome-end protection during meiosis. Our studies unravel the structure, stoichiometry, and physiological implications underlying telomere-INM tethering, thereby providing unprecedented insights into the unique function of telomeres in meiosis.

Interleukin-6 alters sleep of rats.

Although it is well established that the cytokines tumor necrosis factor (TNF) and interleukin (IL)-1 regulate sleep, there is no direct evidence implicating IL-6 in the regulation/modulation of sleep. We tested the hypotheses that central administration of rat recombinant IL-6 increases non-rapid eye movements (NREM) sleep of rats, and that central administration of anti-IL-6 antibodies reduces NREM sleep. Effective doses of IL-6 (100 and 500 ng) initially enhance NREM sleep, after which NREM sleep may be suppressed. IL-6 induces febrile responses at doses lower (50 ng) than those required to alter sleep. Rapid eye movements (REM) sleep is not altered by the doses of IL-6 tested. Central administration of monoclonal or polyclonal anti-rat IL-6 antibodies does not alter any of the parameters determined in this study. Collectively, these results support the hypothesis that IL-6 possesses sleep modulatory properties. However, this cytokine may not be involved in the regulation of spontaneous sleep in healthy animals because antagonizing the IL-6 system using antibodies does not alter sleep. The interpretation of these data is consistent with those of previous studies demonstrating correlations between increased IL-6 and excessive daytime sleepiness during some pathophysiological conditions.

Evidence for endogenous interleukin-10 during nociception.

Cytokines such as IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha) have been shown to contribute directly to central and peripheral neuropathic pain. Recently, exogenous interleukin-10 (IL-10) was shown to impede development of dynorphin-induced allodynia presumably by inhibiting IL-1beta. We therefore wanted to determine whether endogenous IL-10 had a role in pain perception. By measuring the latency of the paw licking response, we show in IL-10 knockout mice and in normal mice treated with anti-IL-10 that latency times are increased, suggesting that endogenous IL-10 increases nociception. This does not appear to be directly correlated with IL-10's regulation of DREAM, a transcriptional regulator of prodynorphin synthesis.

Comprehensive analysis of genetic polymorphisms in the interleukin-10 promoter: implications for immune regulation in specific ethnic populations.

The association of interleukin-10 (IL-10) promoter single-nucleotide polymorphisms (SNPs) as risk factors for certain inflammatory diseases, viral infections, cancers, and transplant rejection have been the subject of recent studies. The SNPs -1082 G --> A, -819 C --> T, and -592 C --> A, which have been associated with differential IL-10 production, are strongly linked with ethnicity. In this study, we determined the ethnic distribution of IL-10 promoter SNPs and their haplotype rates among Hispanics, African Americans, and Caucasians from Texas and Ashkenazi Jews from New York. Significant differences in prevalence rates of IL-10 SNPs (and their haplotype distribution) were found. African Americans and Hispanics have a lower rate of putative high-producer SNPs and a higher rate of low IL-10 producers when compared to Caucasians or Ashkenazi Jews. No statistically significant differences in allelic frequencies and haplotype rates were observed between Caucasians and Ashkenazi Jews. This study provides critical new information on the ethnic distribution of IL-10 promoter SNPs in a regional U. S. population and is the first to analyze the rate of SNPs in an unstudied ethnic population, Ashkenazi Jews. Knowledge of IL-10 promoter polymorphisms may prove useful in prediction of immunization responses, disease severity, and in the intelligent design of customized immunotherapy.

Orbital penetration associated with tooth extraction.

Three cats and 2 dogs were evaluated for ophthalmologic complications associated with tooth extraction procedures. Orbital penetration leading to ocular and, in one case, brain trauma was secondary to iatrogenic injury from a dental elevator. Outcomes included enucleation of the affected eye in 3 cases, and death from brain abscessation in 1 case. Early treatment or, preferably, referral to a veterinary ophthalmology specialist may prevent such outcomes. Awareness of the anatomical proximity of caudal maxillary tooth roots and the orbit, appropriate interpretation of diagnostic intraoral dental radiographs, and technical proficiency in tooth extraction techniques will minimize these complications in veterinary dental practice.