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SUMMARY: Phytase is used in nearly all broiler diets at varying levels and new commercial sources have been developed in recent years. A series of 5 experiments at 4 different universities were conducted to investigate broiler performance metrics and tibia mineralization from 2 mixer-added, Escherichia coli-derived, 6-phytases, Optiphos, a coated phytase, and Optiphos Plus, a recently developed phytase product selected to be intrinsically heat stable, independent of a coating. Treatments for each experiment included a negative control (NC) and the NC + varying levels of Optiphos and Optiphos Plus up to 1,500 FTU/kg. Experimental diets were fed to male broiler chicks for 21 d. On d 21, broilers were euthanized and tibiae were excised to determine tibia mineralization. Experiments 1–4 utilized pelleted diets while Experiment 5 utilized a mash diet. For each experiment, treatments were arranged by a formulated phytase level and source factorial, using a randomized complete block design. In addition, slope-ratio models were created using data from all experiments to evaluate responses to analyzed phytase levels. Positive live performance and tibia mineralization responses to phytase activity were observed (P < 0.05) in all 5 experiments. Performance and bone mineral metrics continued to improve up to approximately 1,500 FTU/kg in a curvilinear fashion, demonstrating a value of super-dosing, the degree employed being dependent on the product used and the cost-benefit economics of implementation. When comparing nonlinear response slopes across all analyzed phytase levels and experiments, the data showed that efficacy of Optiphos Plus was approximately 20% higher than that of Optiphos at similar analyzed levels (P < 0.05).

Germline epigenetic programming, including genomic imprinting, substantially influences offspring development. Polycomb Repressive Complex 2 (PRC2) plays an important role in Histone 3 Lysine 27 trimethylation (H3K27me3)-dependent imprinting, loss of which leads to growth and developmental changes in mouse offspring. In this study, we show that offspring from mouse oocytes lacking the PRC2 protein Embryonic Ectoderm Development (EED) were initially developmentally delayed, characterised by low blastocyst cell counts and substantial growth delay in mid-gestation embryos. This initial developmental delay was resolved as offspring underwent accelerated fetal development and growth in late gestation resulting in offspring that were similar stage and weight to controls at birth. The accelerated development and growth in offspring from Eed-null oocytes was associated with remodelling of the placenta, which involved an increase in fetal and maternal tissue size, conspicuous expansion of the glycogen-enriched cell population, and delayed parturition. Despite placental remodelling and accelerated offspring fetal growth and development, placental efficiency, and fetal blood glucose levels were low, and the fetal blood metabolome was unchanged. Moreover, while expression of the H3K27me3-imprinted gene and amino acid transporter Slc38a4 was increased, fetal blood levels of individual amino acids were similar to controls, indicating that placental amino acid transport was not enhanced. Genome-wide analyses identified extensive transcriptional dysregulation and DNA methylation changes in affected placentas, including a range of imprinted and non-imprinted genes. Together, while deletion of Eed in growing oocytes resulted in fetal growth and developmental delay and placental hyperplasia, our data indicate a remarkable capacity for offspring fetal growth to be normalised despite inefficient placental function and the loss of H3K27me3-dependent genomic imprinting.

EB1 is a key cellular protein that delivers regulatory molecules throughout the cell via the tip-tracking of growing microtubule plus-ends. Thus, it is important to understand the mechanism for how EB1 efficiently tracks growing microtubule plus-ends. It is widely accepted that EB1 binds with higher affinity to GTP-tubulin subunits at the growing microtubule tip, relative to GDP-tubulin along the microtubule length. However, it is unclear whether this difference in affinity alone is sufficient to explain the tip-tracking of EB1 at growing microtubule tips. Previously, we found that EB1 binds to exposed microtubule protofilament-edge sites at a ~70 fold faster rate than to closed-lattice sites, due to diffusional steric hindrance to binding. Thus, we asked whether rapid protofilament-edge binding could contribute to efficient EB1 tip tracking. A computational simulation with differential EB1 on-rates based on closed-lattice or protofilament-edge binding, and with EB1 off-rates that were dependent on the tubulin hydrolysis state, robustly recapitulated experimental EB1 tip tracking. To test this model, we used cell-free biophysical assays, as well as live-cell imaging, in combination with a Designed Ankyrin Repeat Protein (DARPin) that binds exclusively to protofilament-edge sites, and whose binding site partially overlaps with the EB1 binding site. We found that DARPin blocked EB1 protofilament-edge binding, which led to a decrease in EB1 tip tracking on dynamic microtubules. We conclude that rapid EB1 binding to microtubule protofilament-edge sites contributes to robust EB1 tip tracking at the growing microtubule plus-end.

The transmission of DNA through extracellular vesicles (EVs) represents a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. We aimed to investigate the potential for vertical DNA transmission within maternal endometrial EVs to the pre-implantation embryo and describe any effect on embryo bioenergetics. We discovered that the human endometrium secretes all three general subtypes of EV - apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs) - into the human endometrial fluid (EF) within the uterine cavity. EVs become uniformly secreted into the EF during the menstrual cycle, with the proportion of different EV populations remaining constant; however, MVs contain significantly higher levels of mitochondrial (mt)DNA than ABs or EXOs. During the window of implantation, MVs contain an eleven-fold higher level of mtDNA when compared to cells-of-origin within the receptive endometrium, which possesses a lower mtDNA content and displays the upregulated expression of mitophagy-related genes. Furthermore, we demonstrate the internalization of EV-derived nuclear-encoded (n)DNA/mtDNA by trophoblast cells of murine embryos, which associates with a reduction in mitochondrial respiration and ATP production. These findings suggest that the maternal endometrium suffers a reduction in mtDNA content during the preconceptional period, that nDNA/mtDNA become packaged into secreted EVs that the embryo uptakes, and that the transfer of DNA to the embryo within EVs occurs alongside the modulation of bioenergetics during implantation.

Abstract Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 individual retrievable cell sample ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Background: Men diagnosed with Klinefelter syndrome (KS) commonly exhibit non-obstructive azoospermia or rarely having sperm in their ejaculate, rendering them traditionally considered sterile prior to the introduction of intracytoplasmic sperm injection (ICSI). The presence of mosaic KS may mask the classical phenotype, resulting in underdiagnosis throughout their lifetime. Surgical sperm retrieval through Microdissection Testicular Sperm Extraction (Micro-TESE) combined with ICSI has become the gold standard approach, maximizing reproductive outcomes in these individuals. However, it is noteworthy that approximately 7% of men with KS may exhibit sperm in their ejaculate, providing an opportunity for them to achieve biological parenthood through ICSI.Case Presentation: In this report, we present an exceptional case of a 45-year-old man with Mosaic KS and severe oligozoospermia who successfully achieved pregnancy utilizing ICSI with freshly ejaculated sperm. Remarkably, this case represents the oldest recorded instance of a man with Klinefelter syndrome fathering his own biological child using sperm derived from fresh ejaculate.Conclusion: Although this case is exceedingly rare, it underscores the critical importance of exhausting all possibilities to facilitate biological parenthood in men with KS before considering alternative options such as sperm donation or adoption. By recognizing the potential for successful conception using ejaculated sperm in this population, we can provide individuals with mosaic KS the opportunity to fulfill their desire for biological offspring.

Motion: For The Outdated Significance of Morphological Assessment in Embryo Selection and the Rise of Advanced Technologies in Reproductive Medicine This symposium lecture presents a compelling debate, shedding light on the diminishing relevance of traditional static morphological assessment as a primary criterion for embryo. The speaker aims to challenge the long-standing reliance on morphological evaluation and advocate for the integration of cutting-edge technologies as a more reliable and objective means of embryo quality assessment. During the debate, the speaker will examine the limitations of morphological assessment, acknowledging its subjective nature, potential bias, and lack of comprehensive insight into embryonic development. The traditional morphological assessment based solely on visual appearance may not adequately predict an embryo’s true potential for successful implantation and healthy pregnancy. A variety of available new technologies will be highlighted during the lecture, showcasing alternatives to morphological embryo assessment. Attendees will gain insight into how these innovative methods can offer more objective and precise measures of embryo quality, enhancing the success rates of assisted reproductive procedures. The symposium will foster an engaging environment, encouraging active participation and thoughtful discussions among attendees from the reproductive medicine community. Participants will have the opportunity to challenge prevailing notions and explore the implications of adopting new technologies in clinical practice. In summary, this debate aims to catalyze a paradigm shift in embryo evaluation, emphasizing the need to move beyond traditional morphological assessment towards embracing advanced technologies, to optimize embryo selection and lead to improved outcomes in IVF. Motion: Against Morphological Assessment of Embryos is Not Outdated In the era of new technologies and artificial intelligence, looking good has never been more important! On-time progression through specific events is requisite for human preimplantation development and provides important visual insights into the health and physiology of the embryo. The formation of 2 distinct pronuclei, the times and patterns of cleavage, and how the cells compact are all key markers of development. Following compaction, the formation of the blastocyst is not only a temporally regulated event, but also an energetically demanding process, one that reflects the metabolic activity (a biomarker of viability) of the embryo. Further, at the blastocyst stage it is possible to visualise and quantitate the formation of both the inner cell mass (ICM) and trophectoderm, with all parameters of blastocyst development linked to transfer outcomes. The development of time-lapse incubation, which can be described as embryo morphology over time, has led to the incorporation of several key morphological features to support embryo selection, further reflecting the significance of embryo morphology. Interestingly, AI algorithms used for embryo selection reflect predominantly those morphological parameters that are known to affect transfer outcomes, such as blastocyst quality. While only specific molecular tests can accurately determine the precise chromosomal composition of an embryo, key morphological features and events can also be used to tag those embryos at greatest risk of being genetically abnormal. In conclusion, key morphological developmental events indirectly inform us of the physiology of the embryo (degree of blastocyst expansion reflects metabolic activity for example), and hence this is why morphological grading systems (such as the Gardner Grade for blastocysts) have been shown to be effective in embryo selection for over the past 25 years, and further have proven to be as useful as AI or biomarker analysis for the majority of patients.

The issues surrounding the culture of the preimplantation human embryo are distinct from those associated with the propagation of somatic cells, given that the starting point of culture, the pronucleate oocyte, and the final stage of development, the blastocyst, are as distinct as any two cell types can be with regards to their nutrient requirements and metabolism. Furthermore, the cleavage stage period of embryo development is exquisitely sensitive to its environment and has limited capacity to regulate against disruptions in its surroundings, especially changes in pH. Historically the media used in human IVF, such as Human Tubal Fluid medium, were based on simple salt solutions but lacked key regulators of blastomere function, specifically amino acids. A resurgence of research in embryo metabolism and physiology in the 1980s paved the way for the development of Sequential media, designed not only to accommodate the changing requirements of the embryo itself (both in terms of carbohydrates and amino acids), but which were also based on available data on the dynamic/differing composition of the Fallopian tube and uterus. Sequential media, especially when used in combination with isolettes, were shown to be highly effective in supporting blastocyst development and significantly increased implantation rates, thereby facilitating the worldwide movement to single embryo transfer. However, while this approach partially satisfied the changing nutrient requirements of the embryo, and further facilitated the removal of toxins built up in the culture drop over 48h, such as ammonium, it necessitated moving embryos. Unless undertaken using warmed gassed isolettes, moving embryos inevitably resulted in shifts in temperature and pH. With the advent of time-lapse, there began a growing movement towards the use of one-step media in order to keep the embryo within an imaging incubator. To facilitate this, hybrid type media were formulated, and while they do not accommodate the dynamic requirements of the preimplantation period, their use meant embryos were not exposed to changes in either temperature or pH. To date, clinical data have reported similar outcomes using both media approaches, plausibly due to a trade-off of the inherent limitations of either approach. An alternative approach proposed decades ago, is to provide the embryo with a gradient of nutrients within a perfusion system, thereby facilitating a dynamic culture environment without the need to move embryos. Unfortunately, until recently, the technology to support this concept has not been available. However, the recent development of 3D printing using 2-photo lithography, has made it possible to create embryo-specific perfusion systems. Hence, we are now able to evaluate the potential of such dynamic systems and could find ourselves on the verge of a new era in embryo culture.

The authors add to published empirical findings that an appropriate range of validity evidence for achievement tests, including science and mathematics, has either not been gathered, not reported, or is not accessible for independent review. The current study focuses on a sample of published, peer reviewed science intervention studies from a single journal using science achievement measures, over an 11-years period, with some discussion of validity, and finds results similar to previous studies that were conducted in other STEM areas and broader contexts. The consensus of the educational measurement profession is that validity is assessed by the extent to which evidence and theory support the proposed interpretation of test scores for proposed uses. What is troublesome is that a shortfall in validity evidence raises concerns about faulty, or insubstantial, test score interpretation to inform student short-term and long-term education and career trajectories, and to inform curricula intervention improvements. Discussion cautions readers that though the study findings report shortfalls in some kinds of validity evidence, this simply raises a flag for the test user to consider what kinds of validity evidence apply to her test use. It also raises a flag for the profession to explore the reasons for shortfalls.

Abstract Background While involvement in the legal system offers an opportunity to educate, screen, and treat high-risk youth, research shows that staff attitudes toward these practices can serve as barriers to implementation. The current study investigates the degree to which JJ staff endorse HIV prevention, testing, and treatment linkage practices with youth under community supervision and examines differences between individuals who supervise youth (e.g., juvenile probation officer) and those working in non-supervisory roles (e.g., case manager, assessment specialist). Methods Juvenile justice staff consenting to participation in JJ-TRIALS completed an initial staff survey (N = 501). Survey items measured perceived importance of HIV/STI prevention (4 items); perceived importance of HIV/STI testing (7 items); and perceived importance of HIV/STI treatment linkage (8 items). Results Confirmatory Factor Analysis (CFA) was computed (SAS CALIS procedure) for each of the three domains. Findings suggest that while staff recognize that youth are at risk for HIV/STIs and require provision of HIV/STI prevention and treatment linkage, attitudes concerning the importance of procuring or providing testing services for youth is substantially lower. Furthermore, analytic models comparing staff with and without supervision responsibilities (computed using SAS PROC MIXED) indicated that attitudes differed by site and staff responsible for supervision rated HIV treatment linkage practices as less important compared to non-supervising staff. Conclusions Establishing partnerships with health agencies equipped with resources and skillsets to provide HIV/STI testing and related services may be an effective model to promote greater awareness and use of best practices among JJ staff and more effectively address the unmet needs of this high-risk population of youth.

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm’s potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

Abstract Background Sepsis survivors often develop chronic critical illness (CCI) and demonstrate the persistent inflammation, immunosuppression, and catabolism syndrome predisposing them to long-term functional limitations and higher mortality. There is a need to identify biomarkers that can predict long-term worsening of physical function to be able to act early and prevent mobility loss. N-terminal pro-brain natriuretic peptide (NT-proBNP) is a well-accepted biomarker of cardiac overload, but it has also been shown to be associated with long-term physical function decline. We explored whether NT-proBNP blood levels in the acute phase of sepsis are associated with physical function and muscle strength impairment at 6 and 12 months after sepsis onset. Methods This is a retrospective analysis conducted in 196 sepsis patients (aged 18–86 years old) as part of the University of Florida (UF) Sepsis and Critical Illness Research Center (SCIRC) who consented to participate in the 12-month follow-up study. NT-proBNP was measured at 24 h after sepsis onset. Patients were followed to determine physical function by short physical performance battery (SPPB) test score (scale 0 to12—higher score corresponds with better physical function) and upper limb muscle strength by hand grip strength test (kilograms) at 6 and 12 months. We used a multivariate linear regression model to test an association between NT-proBNP levels, SPPB, and hand grip strength scores. Missing follow-up data or absence due to death was accounted for by using inverse probability weighting based on concurrent health performance status scores. Statistical significance was set at p ≤ 0.05. Results After adjusting for covariates (age, gender, race, Charlson comorbidity index, APACHE II score, and presence of CCI condition), higher levels of NT-proBNP at 24 h after sepsis onset were associated with lower SPPB scores at 12 months (p

Abstract Background Defining the mechanisms that establish and regulate the transmission of epigenetic information from parent to offspring is critical for understanding disease heredity. Currently, the molecular pathways that regulate epigenetic information in the germline and its transmission to offspring are poorly understood. Results Here we provide evidence that Polycomb Repressive Complex 2 (PRC2) regulates paternal inheritance. Reduced PRC2 function in mice resulted in male sub-fertility and altered epigenetic and transcriptional control of retrotransposed elements in foetal male germ cells. Males with reduced PRC2 function produced offspring that over-expressed retrotransposed pseudogenes and had altered preimplantation embryo cleavage rates and cell cycle control. Conclusion This study reveals a novel role for the histone-modifying complex, PRC2, in paternal intergenerational transmission of epigenetic effects on offspring, with important implications for understanding disease inheritance.

Deep-seated educational discourses have blamed low-income communities for their youth’s lack of high school completion. These deficit discourses reflect top–down knowledge hierarchies and a lack of knowledge democracy in education (de Sousa Santos 2007; Hall & Tandon 2017; Visvanathan 2009), and they are in need of critical and diverse knowledge reckoning by low-income communities themselves. This article relays how a community-university participatory action research (PAR) partnership became a dynamic site of knowledge democracy from which to counter and transform deficit-based knowledge systems imposed on economically disadvantaged communities. Steeped in the generative enactments of PAR, storytelling, ecological metaphor, strength-based approaches and the arts, this article explores a low-income/social housing community’s knowledge practices that are energising and growing its community power to support the success of their youth in school. These seven knowledge practices are narrated through the ecological metaphor of trees, specifically via a co-constructed PAR team narrative called the Tree of Community Knowledge and Engagement. In the telling and retelling of this counternarrative-in-the-making, this article embodies knowledge democracy. Here, community members’ energising knowledge practices are recognised as invaluable forms of everyday educational knowing and leadership for their youth. This article further explores three broad ways of knowing that reside within and across community members’ seven knowledge practices: lived knowing, interconnected knowing and participatory/power-in-relation knowing. The three community ways of knowing illustrate how the community is growing its power to support youth’s success via a transformative educational worldview, from which other schools and universities could learn and, indeed, thrive.

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

Reprogramming to pluripotency involves drastic restructuring of both metabolism and the epigenome. However, induced pluripotent stem cells (iPSC) retain transcriptional memory, epigenetic memory, and metabolic memory from their somatic cells of origin and acquire aberrant characteristics distinct from either other pluripotent cells or parental cells, reflecting incomplete reprogramming. As a critical link between the microenvironment and regulation of the epigenome, nutrient availability likely plays a significant role in the retention of somatic cell memory by iPSC. Significantly, relative nutrient availability impacts iPSC reprogramming efficiency, epigenetic regulation and cell fate, and differentially alters their ability to respond to physiological stimuli. The significance of metabolites during the reprogramming process is central to further elucidating how iPSC retain somatic cell characteristics and optimising culture conditions to generate iPSC with physiological phenotypes to ensure their reliable use in basic research and clinical applications. This review serves to integrate studies on iPSC reprogramming, memory retention and metabolism, and identifies areas in which current knowledge is limited.

Metabolism has been shown to alter cell fate in human pluripotent stem cells (hPSC). However, current understanding is almost exclusively based on work performed at 20% oxygen (air), with very few studies reporting on hPSC at physiological oxygen (5%). In this study, we integrated metabolic, transcriptomic, and epigenetic data to elucidate the impact of oxygen on hPSC. Using 13C-glucose labeling, we show that 5% oxygen increased the intracellular levels of glycolytic intermediates, glycogen, and the antioxidant response in hPSC. In contrast, 20% oxygen increased metabolite flux through the TCA cycle, activity of mitochondria, and ATP production. Acetylation of H3K9 and H3K27 was elevated at 5% oxygen while H3K27 trimethylation was decreased, conforming to a more open chromatin structure. RNA-seq analysis of 5% oxygen hPSC also indicated increases in glycolysis, lysine demethylases, and glucose-derived carbon metabolism, while increased methyltransferase and cell cycle activity was indicated at 20% oxygen. Our findings show that oxygen drives metabolite flux and specifies carbon fate in hPSC and, although the mechanism remains to be elucidated, oxygen was shown to alter methyltransferase and demethylase activity and the global epigenetic landscape.

Human induced pluripotent stem cells (iPSCs) can be differentiated in vitro into bona fide cardiomyocytes for disease modelling and personalized medicine. Mitochondrial morphology and metabolism change dramatically as iPSCs differentiate into mesodermal cardiac lineages. Inhibiting mitochondrial fission has been shown to promote cardiac differentiation of iPSCs. However, the effect of hydrazone M1, a small molecule that promotes mitochondrial fusion, on cardiac mesodermal commitment of human iPSCs is unknown. Here, we demonstrate that treatment with M1 promoted mitochondrial fusion in human iPSCs. Treatment of iPSCs with M1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. The pro-fusion and pro-cardiogenic effects of M1 were not associated with changes in expression of the α and β subunits of adenosine triphosphate (ATP) synthase. Our findings demonstrate for the first time that hydrazone M1 is capable of promoting cardiac differentiation of human iPSCs, highlighting the important role of mitochondrial dynamics in cardiac mesoderm lineage specification and cardiac development. M1 and other mitochondrial fusion promoters emerge as promising molecular targets to generate lineages of the heart from human iPSCs for patient-specific regenerative medicine.

Aging is associated with an increased incidence of cancer. One contributing factor could be modulation of immune cells responsible for anti-tumor responses, such as dendritic cells (DCs) and T cells. These immunological changes may also impact the efficacy of cancer immunotherapies in the elderly. The effects of healthy aging on DCs and T cells, and their impact on anti-mesothelioma immune responses, had not been reported. This study examined DCs and T cells in young (2–5 months; equivalent to 16–26 human years) and elderly (20–24 months; equivalent to 60–70 human years) healthy and mesothelioma-bearing C57BL/6J mice. During healthy aging, elderly lymph nodes adopted a regulatory profile, characterized by: (i) increased plasmacytoid DCs, (ii) increased expression of the adenosine-producing enzyme CD73 on CD11c+ cells, and (iii) increased expression of multiple regulatory markers (including CD73, the adenosine A2B receptor, CTLA-4, PD-1, ICOS, LAG-3, and IL-10) on CD8+ and CD4+ T cells, compared to lymph nodes from young mice. Although mesotheliomas grew faster in elderly mice, the increased regulatory status observed in healthy elderly lymph node DCs and T cells was not further exacerbated. However, elderly tumor-bearing mice demonstrated reduced MHC-I, MHC-II and CD80 on CD11c+ cells, and decreased IFN-γ by CD8+ and CD4+ T cells within tumors, compared to young counterparts, implying loss of function. An agonist CD40 antibody based immunotherapy was less efficient at promoting tumor regression in elderly mice, which may be due to: (i) failure of elderly CD8+ T cells to up-regulate perforin, and (ii) increased expression of multiple regulatory markers on CD11c+ cells and T cells in elderly tumor-draining lymph nodes (including CD73, PD-1, ICOS, LAG-3, and TGF-β). Our findings suggest that checkpoint blockade may improve responses to immunotherapy in elderly hosts with mesothelioma, and warrants further investigation.

Most cancers emerge in the elderly, including lung cancer and mesothelioma, yet the elderly remain an underrepresented population in pre-clinical cancer studies and clinical trials. The immune system plays a critical role in the effectiveness of many anti-cancer therapies in young hosts via tumor-specific T cells. However, immunosuppressive macrophages can constitute up to 50% of the tumor burden and impair anti-tumor T cell activity. Altered macrophage phenotype and function during aging may further impact anti-tumor T cell responses. Yet, the impact of macrophages on anti-tumor T cell responses and immunotherapy in the elderly is unknown. Therefore, we examined macrophages and their interaction with T cells in young (3 months) and elderly (20–24 months) AE17 mesothelioma-bearing female C57BL/6J mice during tumor growth. Mesothelioma tumors grew faster in elderly compared with young mice, and this corresponded with an increase in tumor-associated macrophages. During healthy aging, macrophages increase in bone marrow and spleens suggesting that these sites have an increased potential to supply cancer-promoting macrophages. Interestingly, in tumor-bearing mice, bone marrow macrophages increased proliferation whilst splenic macrophages had reduced proliferation in elderly compared with young mice, and macrophage depletion using the F4/80 antibody slowed tumor growth in young and elderly mice. We also examined responses to treatment with intra-tumoral IL-2/anti-CD40 antibody immunotherapy and found it was less effective in elderly (38% tumor regression) compared to young mice (90% regression). Tumor-bearing elderly mice decreased in vivo anti-tumor cytotoxic T cell activity in tumor draining lymph nodes and spleens. Depletion of macrophages using F4/80 antibody in elderly, but not young mice, improved IL-2/anti-CD40 immunotherapy up to 78% tumor regression. Macrophage depletion also increased in vivo anti-tumor T cell activity in elderly, but not young mice. All the tumor-bearing elderly (but not young) mice had decreased body weight (i.e., exhibited cachexia), which was greatly exacerbated by immunotherapy; whereas macrophage depletion prevented this immunotherapy-induced cachexia. These studies strongly indicate that age-related changes in macrophages play a key role in driving cancer cachexia in the elderly, particularly during immunotherapy, and sabotage elderly anti-tumor immune responses.