Disorganization of the splenic microanatomy in ageing mice.

The precise mechanisms responsible for immunosenescence still remain to be determined, however, considering the evidence that disruption of the organization of primary and secondary lymphoid organs results in immunodeficiency, we propose that this could be involved in the decline of immune responses with age. Therefore, we investigated the integrity of the splenic microarchitecture in mice of increasing age and its reorganization following immune challenge in young and old mice. Several differences in the anatomy of the spleen with age in both the immune and stromal cells were observed. There is an age-related increase in the overall size of the white pulp, which occurs primarily within the T-cell zone and is mirrored by the enlargement of the T-cell stromal area, concurrent to the distinct boundary between T cells and B cells becoming less defined in older mice. In conjunction, there appears to be a loss of marginal zone macrophages, which is accompanied by an accumulation of fibroblasts in the spleens from older animals. Furthermore, whereas the reorganization of the white pulp is resolved after several days following antigenic challenge in young animals, it remains perturbed in older subjects. All these age-related changes within the spleen could potentially contribute to the age-dependent deficiencies in functional immunity.

Reduced regulatory T cell diversity in NOD mice is linked to early events in the thymus.

The thymic natural regulatory T cell (Treg) compartment of NOD mice is unusual in having reduced TCR diversity despite normal cellularity. In this study, we show that this phenotype is attributable to perturbations in early and late stages of thymocyte development and is controlled, at least in part, by the NOD Idd9 region on chromosome 4. Progression from double negative 1 to double negative 2 stage thymocytes in NOD mice is inefficient; however, this defect is compensated by increased proliferation of natural Tregs (nTregs) within the single positive CD4 thymocyte compartment, accounting for recovery of cellularity accompanied by loss of TCR diversity. This region also underlies the known attenuation of ERK-MAPK signaling, which may preferentially disadvantage nTreg selection. Interestingly, the same genetic region also regulates the rate of thymic involution that is accelerated in NOD mice. These findings highlight further complexity in the control of nTreg repertoire diversity.

Time-series transcriptional profiling yields new perspectives on susceptibility to murine osteoarthritis.

OBJECTIVE: Chronological age is a powerful epidemiologic risk factor for osteoarthritis (OA), a multifactorial disease that is characterized by articular cartilage (AC) degradation. It is unclear from a molecular perspective how aging interacts with OA to produce this risk to AC integrity. To address this key question, we used in vivo time-course analysis of OA development and murine interstrain variability in natural susceptibility to OA to examine changes in non-OA-prone CBA mice versus OA-prone STR/Ort mice, which develop disease that bears significant histologic resemblance to human OA. Through global transcriptome profiling, we attempted to discover the molecular signature linked with both OA vulnerability and progression. METHODS: Affymetrix Mouse Gene 1.0 ST Array profiles were generated from AC samples derived from CBA and STR/Ort mice at 3 different ages, corresponding to the stages prior to, at, and late after the natural onset of OA in the STR/Ort mice. RESULTS: We found that the OA in STR/Ort mice exhibited a molecular phenotype resembling human OA, and we pinpointed a central role of NF-κB signaling and the emergence of an immune-related signature in OA cartilage over time. We discovered that, strikingly, young healthy AC has a highly expressed skeletal muscle gene expression program, which is switched off during maturation, but is intriguingly retained in AC during OA development in STR/Ort mice. CONCLUSION: This study is the first to show that AC chondrocytes share a high-abundance gene-expression program with skeletal muscle. We show that failure to switch this program off, as well as the restoration of this program, is associated with inappropriate expression of NF-κB signaling pathways, skeletal muscle-related genes, and induction and/or progression of OA.

An evolutionary perspective on the mechanisms of immunosenescence.

There is an accumulating body of evidence that a decline in immune function with age is common to most if not all vertebrates. For instance, age-associated thymic involution seems to occur in all species that possess a thymus, indicating that this process is evolutionary ancient and conserved. The precise mechanisms regulating immunosenescence remain to be resolved, but much of what we do know is consistent with modern evolutionary theory. In this review, we assess our current knowledge from an evolutionary perspective on the occurrence of immunosenescence, we show that life history trade-offs play a key role and we highlight the possible advantages of the age-related decline in thymic function.

It's not all equal: a multiphasic theory of thymic involution.

Regression of the thymus is a key feature of immunosenescence, which coincides with a decrease in T cell output and contributes to the restriction of the T cell repertoire in the elderly, leading to increased susceptibility to illness and disease. However, the mechanisms involved in thymic involution are still not fully known. Although, it is often believed that thymic involution occurs during the onset of puberty, increasing data suggests alterations to the thymus happen much earlier in life. Therefore, the changes in the thymus and subsequent thymic function may not just be an ageing phenomenon. In this article, we propose that there are several, non-linear, phases to thymic atrophy, which are regulated by different mechanisms, including the familiar age-dependent thymic involution and a much earlier growth-dependent thymic involution.

BTN1A1, the mammary gland butyrophilin, and BTN2A2 are both inhibitors of T cell activation.

Butyrophilin (BTN) genes encode a set of related proteins. Studies in mice have shown that one of these, BTN1A1, is required for milk lipid secretion in lactation, whereas butyrophilin-like 2 is a coinhibitor of T cell activation. To understand these disparate roles of BTNs, we first compared the expression and functions of mouse Btn1a1 and Btn2a2. Btn1a1 transcripts were not restricted to lactating mammary tissue but were also found in virgin mammary tissue and, interestingly, spleen and thymus. In confirmation of this, BTN1A1 protein was detected in thymic epithelial cells. By contrast, Btn2a2 transcripts and protein were broadly expressed. Cell surface BTN2A2 protein, such as the B7 family molecule programmed death ligand 1, was upregulated upon activation of T cells. We next examined the potential of both BTN1A1 and BTN2A2 to interact with T cells. Recombinant Fc fusion proteins of murine BTN2A2 and, surprisingly BTN1A1, bound to activated T cells, suggesting the presence of one or more receptors on these cells. Immobilized BTN-Fc fusion proteins, but not MOG-Fc protein, inhibited the proliferation of CD4 and CD8 T cells activated by anti-CD3. BTN1A1 and BTN2A2 also inhibited T cell metabolism, IL-2, and IFN-gamma secretion. Inhibition of proliferation was not abrogated by exogenous IL-2 but could be overcome following costimulation with high levels of anti-CD28 Ab. These data are consistent with a coinhibitory role for mouse BTNs, including BTN1A1, the BTN expressed in the lactating mammary gland and on milk lipid droplets.

Evidence of conserved neuroendocrine interactions in the thymus: intrathymic expression of neuropeptides in mammalian and non-mammalian vertebrates.

The function of lymphoid organs and immune cells is often modulated by hormones, steroids and neuropeptides produced by the neuroendocrine and immune systems. The thymus intrinsically produces these factors and a comparative analysis of the expression of neuropeptides in the thymus of different species would highlight the evolutionary importance of neuroendocrine interaction in T cell development. In this review, we highlight the evidence which describes the intrathymic expression and function of various neuropeptides and their receptors, in particular somatostatin, substance P, vasointestinal polypeptide, calcitonin gene-related peptide and neuropeptide Y, in mammals (human, rodent) and non-mammals (avian, amphibian and teleost), and conclude that neuropeptides play a conserved role in vertebrate thymocyte development.

Preventing and responding to child sexual abuse: Organizational efforts.

BACKGROUND: Child sexual abuse (CSA) remains an ongoing threat to the wellbeing of children who might be victimized, to the liberty of individuals who might engage in abusive behavior, and to the reputations and livelihood of organizations in which abuse might occur. In the U.S., millions of children participate in youth-serving organizations (YSOs), and it is known that a proportion of CSA occurs in these settings. Despite the severity of these threats, there is little knowledge of the steps that organizations take to prevent and respond to CSA. OBJECTIVE: Our study aimed to index current measures to prevent and respond to CSA in organizational settings, using four of the largest U.S.-based YSOs as exemplars. METHODS: In partnership with our partner YSOs, we completed a qualitative analysis of 74 organizational documents including written policies, codes of conduct, trainings, and other documents that formalize these four organizations' current CSA prevention and intervention efforts. RESULTS: These organizations collectively implement hundreds of distinct measures aimed at preventing, detecting, and responding to CSA. These measures were categorized under eight overarching themes, including: overall commitment to child safety, code of conduct, training and education, assessment, implementation and monitoring, screening and hiring, reporting and responding to child sexual abuse, youth problem sexual behavior, and boundaries for teen leaders and young adult staff. CONCLUSION: Findings from the current study, outlining key prevention and policy areas undertaken by participating YSOs, offer a starting point for discussion about core elements needed to keep children safe from sexual abuse in YSO settings.

The developmentally regulated expression of Twisted gastrulation reveals a role for bone morphogenetic proteins in the control of T cell development.

The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of decapentaplegic (dpp) and its orthologs, the bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. We have uncovered a role for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4(-)CD8(-) double-negative (DN) thymocytes and their differentiation to the CD4(+)CD8(+) double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4(+) or CD8(+) single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals.

Early-life nutrition influences thymic growth in male mice that may be related to the regulation of longevity.

Nutrition and growth rate during early life can influence later health and lifespan. We have demonstrated previously that low birthweight, resulting from maternal protein restriction during pregnancy followed by catch-up growth in rodents, was associated with shortened lifespan, whereas protein restriction and slow growth during lactation increased lifespan. The underlying mechanisms by which these differences arise are unknown. In the present study, we report that maternal protein restriction in mice influences thymic growth in early adult life. Offspring of dams fed a low-protein diet during lactation (PLP offspring) had significant thymic growth from 21 days to 12 weeks of age, whereas this was not observed in control mice or offspring of dams fed a low-protein diet during pregnancy (recuperated offspring). PCNA (proliferating-cell nuclear antigen) and SIRT1 (silent information regulator 1) protein levels at 21 days of age were significantly higher in the thymus from both PLP mice (P<0.001 and P<0.05 respectively) and recuperated mice (P<0.001 and P<0.01 respectively) compared with controls. At 12 weeks, PLP mice maintained a higher SIRT1 level, whereas PCNA was decreased in the thymus from recuperated offspring. This suggests that mitotic activity was initially enhanced in the thymus from both PLP and recuperated offspring, but remained sustained into adulthood only in PLP mice. The differential mitotic activity in the thymus from PLP and recuperated mice appeared to be influenced by changes in sex hormone concentrations and the expression of p53, p16, the androgen receptor, IL-7 (interleukin-7) and the IL-7 receptor. In conclusion, differential thymic growth may contribute to the regulation of longevity by maternal diet.

Functional analysis of neuropeptides in avian thymocyte development.

The function of lymphoid organs and immune cells is often modulated by peptides and hormones produced by the neuroendocrine and immune systems. We have previously reported the intrathymic expression of neuropeptides in the thymus of different species and that neuropeptides can influence murine thymocyte development in vitro. To further explore the evolutionary nature of neuroendocrine interactions in the thymus, we identified the expression of calcitonin-gene-related peptide, neuropeptide Y, somatostatin (SOM), substance P and vasointestinal polypeptide, as well as their receptors on chicken thymic epithelial cells (TEC) and thymocytes by immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). All the studied neuropeptides and their receptors were found to be expressed in both TEC and thymocytes, suggesting that intrathymic neuroendocrine interactions may take place within the avian thymus. In order to elucidate whether such interactions play a role in avian thymocyte development, neuropeptides and their antagonists were added to embryonic thymus organ cultures and found to influence chicken thymopoiesis. In particular, an antagonist of SOM increased the proportion of double-positive thymocytes, while SOM itself appeared to inhibit the early stages of thymocyte development. Taken together, these data provide further evidence to suggest that neuropeptides play a conserved role in vertebrate thymocyte development.

Nutritional Programming Effects on the Immune System.

The relationship between patterns of early growth and age-associated diseases such as type 2 diabetes and cardiovascular disease is well established. There is also strong evidence from both human and animal studies that early environmental factors such as maternal nutrition may influence lifespan. Interestingly, more recent studies have demonstrated that nutritional programming in early life effects immunity, such that altered lifespan can also lead to programmed changes in immune function. Here we describe the use of immunohistology and flow cytometry techniques to study two key immune lymphoid organs: one that is involved in developing immune cells (thymus) and another which is the site of immune activation (spleen).

Polymorphisms in the canine IL7R 3'UTR are associated with thymic output in Labrador retriever dogs and influence post-transcriptional regulation by microRNA 185.

Interleukin-7 (IL-7) and its receptor (IL-7R) are essential for T cell development in the thymus, and changes in the IL-7/IL-7R pathway have been implicated in age-associated thymic involution which results in a reduction of naïve T cell output. The aim of this study was to investigate the relationship between IL7 and IL7R genetic variation and thymic output in dogs. No single nucleotide polymorphisms (SNPs) were identified in the canine IL7 gene, but a number were present in the canine IL7R gene. Polymorphisms in the IL7R exon 8 and 3'UTR were found to be associated with signal joint T cell receptor excision circle (sj-TREC) values (a biomarker of thymic output) in young and geriatric Labrador retrievers. Additionally, one of the SNPs in the IL7R 3'UTR (SNP 14 c.1371 + 446 A > C) was found to cause a change in the seed-binding site for microRNA 185 which, a luciferase reporter assay demonstrated, caused changes in post-transcriptional regulation, and therefore might be capable of influencing IL-7R expression. The research findings suggest a genetic link between IL7R genotype and thymic output in dogs, which might impact on immune function as these animals age and provide further evidence of the involvement of IL-7/IL-7R pathway in age-associated thymic involution.

Perturbation of the T cell receptor repertoire occurs with increasing age in dogs.

Immunosenescence is the gradual deterioration in immune system function associated with ageing. This decline is partly due to involution of the thymus, which leads to a reduction in the output of naive T cells into the circulating lymphocyte pool. Expansion of existing naive and memory T cell populations, to compensate for the reduction in thymic output, can lead to reduced diversity in the T cell repertoire with increasing age, resulting in impairment of immune responses to novel antigenic challenges, such as during infection and vaccination. Since associations between T cell repertoire and age have only been examined in a limited number of species, to gain further insights into this relationship, we have investigated age-related changes in the canine T cell receptor (TCR) repertoire. Blood samples were obtained from Labrador retriever dogs of varying ages and variation in the complementary determining region 3 (CDR3) of the T cell receptor beta (TCRB) chain was investigated. CDR3 size spectratyping was employed to evaluate clonal expansion/deletion in the T cell repertoire, allowing identification of profiles within individual variable (V) region families that skewed away from a Gaussian distribution. Older dogs (10-13 years) were found to have an increased number of TCRB V gene spectratypes that demonstrated a skewed distribution, compared with young dogs (≤3 years). Additionally, there was a reduction in the number of clonal peaks present in the spectratypes of old dogs, compared with those of young dogs. The study findings suggest that there is an age-associated disturbance in the diversity of the T cell receptor repertoire in dogs.

Spatial correlation coefficient images for ultrasonic detection.

In ultrasonics, image formation and detection are generally based on signal amplitude. In this paper, we introduce correlation coefficient images as a signal-amplitude independent approach for image formation. The correlation coefficients are calculated between A-scans digitized at adjacent measurement positions. In these images, defects are revealed as regions of high or low correlation relative to the background correlations associated with noise. Correlation coefficient and C-scan images are shown to demonstrate flat-bottom-hole detection in a stainless steel annular ring and crack detection in an aluminum aircraft structure.

Toward a more comprehensive analysis of the role of organizational culture in child sexual abuse in institutional contexts.

This article draws on a report prepared for the Australian Royal Commission into Institutional Responses to Child Sexual Abuse (Palmer et al., 2016) to develop a more comprehensive analysis of the role that organizational culture plays in child sexual abuse in institutional contexts, where institutional contexts are taken to be formal organizations that include children among their members (referred to here as "youth-serving organizations"). We begin by integrating five strains of theory and research on organizational culture from organizational sociology and management theory into a unified framework for analysis. We then elaborate the main paths through which organizational culture can influence child sexual abuse in youth-serving organizations. We then use our unified analytic framework and our understanding of the main paths through which organizational culture can influence child sexual abuse in youth-serving organizations to analyze the role that organizational culture plays in the perpetration, detection, and response to child sexual abuse in youth-serving organizations. We selectively illustrate our analysis with case materials compiled by the Royal Commission into Institutional Responses to Child Sexual Abuse and reports of child sexual abuse published in a variety of other sources. We conclude with a brief discussion of the policy implications of our analysis.

A fully human antitumor immunoRNase selective for ErbB-2-positive carcinomas.

We report the preparation and characterization of a novel, fully human antitumor immunoRNase (IR). The IR, a human RNase and fusion protein made up of a human single chain variable fragment (scFv), is directed to the ErbB-2 receptor and overexpressed in many carcinomas. The anti-ErbB-2 IR, named hERB-hRNase, retains the enzymatic activity of the wild-type enzyme (human pancreatic RNase) and specifically binds to ErbB-2-positive cells with the high affinity (K(d) = 4.5 nm) of the parental scFv. hERB-hRNase behaves as an immunoprotoxin and on internalization by target cells becomes selectively cytotoxic in a dose-dependent manner at nanomolar concentrations. Administered in five doses of 1.5 mg/kg to mice bearing an ErbB-2-positive tumor, hERB-hRNase induced a dramatic reduction in tumor volume. hERB-hRNase is the first fully human antitumor IR produced thus far, with a high potential as a poorly immunogenic human drug devoid of nonspecific toxicity, directed against ErbB-2-positive malignancies.

An Age-Associated Decline in Thymic Output Differs in Dog Breeds According to Their Longevity.

The age associated decline in immune function is preceded in mammals by a reduction in thymic output. Furthermore, there is increasing evidence of a link between immune competence and lifespan. One approach to determining thymic output is to quantify signal joint T cell receptor excision circles (sj-TRECs), a method which has been developed and used in several mammalian species. Life expectancy and the rate of aging vary in dogs depending upon their breed. In this study, we quantified sj-TRECs in blood samples from dogs of selected breeds to determine whether there was a relationship between longevity and thymic output. In Labrador retrievers, a breed with a median expected lifespan of 11 years, there was an age-associated decline in sj-TREC values, with the greatest decline occurring before 5 years of age, but with sj-TREC still detectable in some geriatric animals, over 13 years of age. In large short-lived breeds (Burnese mountain dogs, Great Danes and Dogue de Bordeaux), the decline in sj-TREC values began earlier in life, compared with small long-lived breeds (Jack Russell terriers and Yorkshire terriers), and the presence of animals with undetectable sj-TRECs occurred at a younger age in the short-lived breeds. The study findings suggest that age-associated changes in canine sj-TRECs are related to breed differences in longevity, and this research highlights the use of dogs as a potential model of immunosenescence.

The effects of aging and maternal protein restriction during lactation on thymic involution and peripheral immunosenescence in adult mice.

Environmental factors such as nutrition during early life can influence long-term health, a concept termed developmental programming. Initial research was focused towards the effects on metabolic health but more recent studies have demonstrated effects on parameters such as lifespan and immunity. In this study we report that maternal protein restriction during lactation in mice, that is known to prolong lifespan, slows aging of the central and peripheral immune systems. Offspring of dams fed a postnatal low-protein (PLP) diet during lactation had a significant increase in thymic cellularity and T cell numbers across their lifespan compared to controls, and a less marked age-associated decrease in thymocyte cluster of differentiation (CD) 3 expression. PLP animals also demonstrated increased relative splenic cellularity, increased naïve: memory CD4+ and CD8+ T cell ratios, increased staining and density of germinal centres, and decreased gene expression of p16 in the spleen, a robust biomarker of aging. A slower rate of splenic aging in PLP animals would be expected to result in decreased susceptibility to infection and neoplasia. In conclusion nutritionally-induced slow postnatal growth leads to delayed aging of the adaptive immune system, which may contribute towards the extended lifespan observed in these animals.

A new human antitumor immunoreagent specific for ErbB2.

PURPOSE: Our aim was to isolate a novel human mini-antibody(scFv) that specifically targets ErbB2-positive cancer cells. ErbB2, a tyrosinekinase receptor, is overexpressed in clinically significant tumors, such as breast, ovary, and lung carcinomas. In normal tissues, it is expressed only in certain epithelial cell types. EXPERIMENTAL DESIGN: A large phagemid library (Griffin.1 library) of human scFv was used for the isolation of the ErbB2-specific scFv. A very effective strategy was developed for the isolation, consisting in a double subtractive selection, the use of two different combinations of "positive," i.e., ErbB2-bearing, and "negative" cell lines. RESULTS: Here we report the isolation of the first human anti-ErbB2 mini-antibody endowed with antitumor action. Both in its soluble and phage format, it binds specifically to ErbB2, inhibits its autophosphorylation, is internalized by target cells, and exerts a strong and specific antiproliferative action on ErbB2-positive target cells. A correlation was found between the extent of this antiproliferative effect and the expression levels of ErbB2 on target cells, with a strong cytotoxicity for hyper-expressing cells, such as SKBR3, in which apoptosis was evidenced. CONCLUSIONS: This scFv is a potentially effective immunoreagent for diagnostics and therapeutics of certain cancers, both as a readily diffused molecule in solid tumors and as an essential asset for the construction of fully human anticancer drugs.