Breed, smaller weight, and multiple injections are associated with increased adverse event reports within three days following canine vaccine administration.

OBJECTIVE: To evaluate patient and vaccine factors associated with adverse events (AEs) recorded within 3 days of vaccine administration in a large cohort of dogs. ANIMALS: 4,654,187 dogs vaccinated in 16,087,455 office visits in a 5-year period at 1,119 hospitals of a corporate practice. METHODS: Electronic medical records of dogs vaccinated between January 1, 2016, and December 31, 2020, were searched for diagnoses of possible AEs recorded within 3 days of administration of vaccines without concurrent injectable heartworm preventative. Patient risk factors (age, sex, breed, and weight) and number and type of vaccine were extracted from records. ORs (and 95% CIs) for risk factors were estimated via multivariable logistic regression mixed models with patient as a random effect. RESULTS: AEs were recorded following 31,197 vaccination visits (0.19%, or 19.4/10,000 visits). Reported AE rates increased from 1 to 4 vaccines administered and among individual vaccines were greatest for rabies vaccine. AE rate was generally inversely related to body weight, with largest rates in dogs ≤ 5 kg. The largest AE rates were noted in French Bulldogs and Dachshunds (ORs > 4 compared to mixed-breed dogs). CLINICAL RELEVANCE: Risk factor information can be used to update vaccination protocols and client communication. Breed differences may indicate genetics as the primary risk factor for adverse vaccine reactions following vaccinations.

Younger dogs of specific breeds are more likely to experience a rare adverse event after administration of extended-release injectable moxidectin heartworm preventive.

OBJECTIVE: To estimate the incidence of and identify patient risk factors for an acute adverse event in dogs after administration of a sustained-release injectable heartworm preventive product. ANIMALS: Canine patients that received the injectable heartworm preventive product during routine preventive care visits. METHODS: Retrospective analysis of electronic medical records of canine visits within a large network of primary care veterinary clinics in which the product was administered from January 1, 2016, through December 31, 2020. Visits during which vaccination(s) were also administered were excluded from analysis. Identification of acute adverse events was based on diagnostic entries and other clinical presentations suggestive of an adverse event within 3 days of product administration. Data were analyzed using mixed-effects logistic regression. RESULTS: In the 5-year study period, 1,399,289 visits with 694,030 dogs led to an incidence estimate of approximately 14.3 events/10,000 doses. Regression analysis found younger dogs and 7 breeds (relative to mixed-breed dogs) to have statistically significant greater odds of an event. CLINICAL RELEVANCE: Understanding of incidence and patient risk factors provides veterinary professionals and dog owners more information when deciding on heartworm preventive options for their dog when considering risk for adverse event in dogs of certain ages or breeds.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Stratification of Companion Animal Life Stages from Electronic Medical Record Diagnosis Data.

Aging is a complex, multifactorial process, where different life stages reflect changes in metabolic processes, immune capacities, and genetic/epigenetic repertoires. With accumulating exposure to environmental stresses and deterioration of physiological functions, body systems become more prone to low-grade chronic inflammation and an increasing range of pathologies. We hypothesized that differential susceptibility to diseases across life span reflects phased changes in an organism's physiological capacity that may highlight when interventions may be appropriately used. Furthermore, the number of life stages may vary between species and be impacted by signalment such as breed. We tested this hypothesis using disease diagnoses data from veterinary electronic medical records containing almost 2 million cats and over 4 million dogs. Bi-clustering (on rates of disease diagnoses) and adaptive branch pruning were used to identify age clusters that could be used to define adult life stages. Clustering among diagnoses were then interpreted within the context of each defined life stage. The analyses identified 5 age clusters in cats and 4 age clusters within each of the 4 canine breed size categories used. This study, using population scale data for two species, one with differential size and life expectancies, is the first to our knowledge to use disease diagnosis data to define adult life stages. The life stages presented here are a result of a data-driven approach to age and disease stratification and are intended to support conversations between clinicians and clients about appropriate health care recommendations.

Mitf is required for T cell maturation by regulating dendritic cell homing to the thymus.

Thymic dendritic cells (DCs) promote immune tolerance by regulating negative selection of autoreactive T cells in the thymus. How DC homing to the thymus is transcriptionally regulated is still unclear. Microphthalmia-associated transcription factor (Mitf) is broadly expressed and plays essential roles in the hematopoietic system. Here, we used Mitf-mutated mice (Mitfvit/vit) and found enlargement of the thymus and expansion of CD4/CD8 double-positive T cells. Mitf was highly expressed in a subset of thymic DCs among the hematopoietic system. Genetic mutation or pharmacological inhibition of Mitf in DCs decreased the expression levels of Itga4, which are critical molecules for the homing of DCs to the thymus. Further, inhibition of Mitf decreased thymic DC number. These results suggest a pivotal role of Mitf in the maintenance of T cell differentiation by regulating the homing of DC subsets within the thymus.

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction.

Canonically, hormones are produced in the endocrine organs and delivered to target tissues. However, for steroids, the concept of tissue intracrinology, whereby hormones are produced in the tissues where they exert their effect without release into circulation, has been proposed, but its role in physiology/disease remains unclear. The meibomian glands in the eyelids produce oil to prevent tear evaporation, which reduces with aging. Here, we demonstrate that (re)activation of local intracrine activity through nicotinamide adenine dinucleotide (NAD+)-dependent circadian 3ß-hydroxyl-steroid dehydrogenase (3ß-HSD) activity ameliorates age-associated meibomian gland dysfunction and accompanying evaporative dry eye disease. Genetic ablation of 3ß-HSD nullified local steroidogenesis and led to atrophy of the meibomian gland. Conversely, reactivation of 3ß-HSD activity by boosting its coenzyme NAD+ availability improved glandular cell proliferation and alleviated the dry eye disease phenotype. Both women and men express 3ß-HSD in the meibomian gland. Enhancing local steroidogenesis may help combat age-associated meibomian gland dysfunction.

Dynamic stem cell selection safeguards the genomic integrity of the epidermis.

Maintaining genomic integrity and stability is crucial for life; yet, no tissue-driven mechanism that robustly safeguards the epithelial genome has been discovered. Epidermal stem cells (EpiSCs) continuously replenish the stratified layers of keratinocytes that protect organisms against various environmental stresses. To study the dynamics of DNA-damaged cells in tissues, we devised an in vivo fate tracing system for EpiSCs with DNA double-strand breaks (DSBs) and demonstrated that those cells exit from their niches. The clearance of EpiSCs with DSBs is caused by selective differentiation and delamination through the DNA damage response (DDR)-p53-Notch/p21 axis, with the downregulation of ITGB1. Moreover, concomitant enhancement of symmetric cell divisions of surrounding stem cells indicates that the selective elimination of cells with DSBs is coupled with the augmented clonal expansion of intact stem cells. These data collectively demonstrate that tissue autonomy through the dynamic coupling of cell-autonomous and non-cell-autonomous mechanisms coordinately maintains the genomic quality of the epidermis.

EGFR-mediated epidermal stem cell motility drives skin regeneration through COL17A1 proteolysis.

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis-mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration.

Stem cell spreading dynamics intrinsically differentiate acral melanomas from nevi.

Early differential diagnosis between malignant and benign tumors and their underlying intrinsic differences are the most critical issues for life-threatening cancers. To study whether human acral melanomas, deadly cancers that occur on non-hair-bearing skin, have distinct origins that underlie their invasive capability, we develop fate-tracing technologies of melanocyte stem cells in sweat glands (glandular McSCs) and in melanoma models in mice and compare the cellular dynamics with human melanoma. Herein, we report that glandular McSCs self-renew to expand their migratory progeny in response to genotoxic stress and trauma to generate invasive melanomas in mice that mimic human acral melanomas. The analysis of melanocytic lesions in human volar skin reveals that genetically unstable McSCs expand in sweat glands and in the surrounding epidermis in melanomas but not in nevi. The detection of such cell spreading dynamics provides an innovative method for an early differential diagnosis of acral melanomas from nevi.

Obesity accelerates hair thinning by stem cell-centric converging mechanisms.

Obesity is a worldwide epidemic that predisposes individuals to many age-associated diseases, but its exact effects on organ dysfunction are largely unknown1. Hair follicles-mini-epithelial organs that grow hair-are miniaturized by ageing to cause hair loss through the depletion of hair follicle stem cells (HFSCs)2. Here we report that obesity-induced stress, such as that induced by a high-fat diet (HFD), targets HFSCs to accelerate hair thinning. Chronological gene expression analysis revealed that HFD feeding for four consecutive days in young mice directed activated HFSCs towards epidermal keratinization by generating excess reactive oxygen species, but did not reduce the pool of HFSCs. Integrative analysis using stem cell fate tracing, epigenetics and reverse genetics showed that further feeding with an HFD subsequently induced lipid droplets and NF-κB activation within HFSCs via autocrine and/or paracrine IL-1R signalling. These integrated factors converge on the marked inhibition of Sonic hedgehog (SHH) signal transduction in HFSCs, thereby further depleting lipid-laden HFSCs through their aberrant differentiation and inducing hair follicle miniaturization and eventual hair loss. Conversely, transgenic or pharmacological activation of SHH rescued HFD-induced hair loss. These data collectively demonstrate that stem cell inflammatory signals induced by obesity robustly represses organ regeneration signals to accelerate the miniaturization of mini-organs, and suggests the importance of daily prevention of organ dysfunction.

Mutant ASXL1 induces age-related expansion of phenotypic hematopoietic stem cells through activation of Akt/mTOR pathway.

Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.

Label-free quality control and identification of human keratinocyte stem cells by deep learning-based automated cell tracking.

Stem cell-based products have clinical and industrial applications. Thus, there is a need to develop quality control methods to standardize stem cell manufacturing. Here, we report a deep learning-based automated cell tracking (DeepACT) technology for noninvasive quality control and identification of cultured human stem cells. The combination of deep learning-based cascading cell detection and Kalman filter algorithm-based tracking successfully tracked the individual cells within the densely packed human epidermal keratinocyte colonies in the phase-contrast images of the culture. DeepACT rapidly analyzed the motion of individual keratinocytes, which enabled the quantitative evaluation of keratinocyte dynamics in response to changes in culture conditions. Furthermore, DeepACT can distinguish keratinocyte stem cell colonies from non-stem cell-derived colonies by analyzing the spatial and velocity information of cells. This system can be widely applied to stem cell cultures used in regenerative medicine and provides a platform for developing reliable and noninvasive quality control technology.

Distinct types of stem cell divisions determine organ regeneration and aging in hair follicles.

Hair follicles, mammalian mini-organs that grow hair, miniaturize during aging, leading to hair thinning and loss. Here we report that hair follicle stem cells (HFSCs) lose their regenerative capabilities during aging owing to the adoption of an atypical cell division program. Cell fate tracing and cell division axis analyses revealed that while HFSCs in young mice undergo typical symmetric and asymmetric cell divisions to regenerate hair follicles, upon aging or stress, they adopt an atypical 'stress-responsive' type of asymmetric cell division. This type of division is accompanied by the destabilization of hemidesmosomal protein COL17A1 and cell polarity protein aPKCλ and generates terminally differentiating epidermal cells instead of regenerating the hair follicle niche. With the repetition of these atypical divisions, HFSCs detach from the basal membrane causing their exhaustion, elimination and organ aging. The experimentally induced stabilization of COL17A1 rescued organ homeostasis through aPKCλ stabilization. These results demonstrate that distinct stem cell division programs may govern tissue and organ aging.

Evaluation of the proliferative potential of skin keratinocytes and fibroblasts isolated from critical limb ischemia patients.

Impaired wound healing in critical limb ischemia (CLI) results from multiple factors that affect many cell types and their behavior. Epidermal keratinocytes and dermal fibroblasts play crucial roles in wound healing. However, it remains unclear whether these cell types irreversibly convert into a non-proliferative phenotype and are involved in impaired wound healing in CLI. Here, we demonstrate that skin keratinocytes and fibroblasts isolated from CLI patients maintain their proliferative potentials. Epidermal keratinocytes and dermal fibroblasts were isolated from the surrounding skin of foot wounds in CLI patients with diabetic nephropathy on hemodialysis, and their growth potentials were evaluated. It was found that keratinocytes from lower limbs and trunk of patients can give rise to proliferative growing colonies and can be serially passaged. Fibroblasts can also form colonies with a proliferative phenotype. These results indicate that skin keratinocytes and fibroblasts maintain their proliferative capacity even in diabetic and ischemic microenvironments and can be reactivated under appropriate conditions. This study provides strong evidence that the improvement of the cellular microenvironments is a promising therapeutic approach for CLI and these cells can also be used for potential sources of skin reconstruction.

NUAK2 localization in normal skin and its expression in a variety of skin tumors with YAP.

BACKGROUND: NUAK2 is a critical gene that participates in the carcinogenesis of various types of cancers including melanomas. However, the expression patterns of NUAK2 in normal skin and in various types of skin tumors have not been fully elucidated to date. OBJECTIVES: To elucidate the distribution and localization of NUAK2 expression in normal skin, and characterize the expression patterns of NUAK2 and YAP in various types of skin tumors. METHODS: In this study, we characterized the expression of NUAK2 in tissues by developing a novel NUAK2-specific monoclonal antibody and using that to determine NUAK2 expression patterns in normal skin and in 155 cases of various types of skin tumors, including extramammary Paget's disease (EMPD), squamous cell carcinoma (SCC), Bowen's disease (BD), actinic keratosis (AK), basal cell carcinoma (BCC) and angiosarcoma (AS). Further, we analyzed the expression patterns of YAP and p-Akt in those tumors. RESULTS: Our analyses revealed that NUAK2 is expressed at high frequencies in EMPD, SCC, BD, AK, BCC and AS. The expression of p-Akt was positively correlated with tumor size in EMPD (P = 0.001). Importantly, the expression of NUAK2 was significantly correlated with YAP in SCC (P = 0.012) and in BD (P = 0.009). CONCLUSIONS: Our results suggest that the YAP-NUAK2 axis has critical importance in the tumorigenesis of SCC and BD, and that therapeutic modalities targeting the YAP-NUAK2 axis may be an effective approach against skin tumors including SCC and BD.

Automated collective motion analysis validates human keratinocyte stem cell cultures.

Identification and quality assurance of stem cells cultured in heterogeneous cell populations are indispensable for successful stem cell therapy. Here we present an image-processing pipeline for automated identification and quality assessment of human keratinocyte stem cells. When cultivated under appropriate conditions, human epidermal keratinocyte stem cells give rise to colonies and exhibit higher locomotive capacity as well as significant proliferative potential. Image processing and kernel density estimation were used to automatically extract the area of keratinocyte colonies from phase-contrast images of cultures containing feeder cells. The DeepFlow algorithm was then used to calculate locomotion speed of the colony area by analyzing serial images. This image-processing pipeline successfully identified keratinocyte stem cell colonies by measuring cell locomotion speed, and also assessed the effect of oligotrophic culture conditions and chemical inhibitors on keratinocyte behavior. Therefore, this study provides automated procedures for image-based quality control of stem cell cultures and high-throughput screening of small molecules targeting stem cells.

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

IGF-1R deficiency in human keratinocytes disrupts epidermal homeostasis and stem cell maintenance.

BACKGROUND: Epidermal stem cells (ESCs) are keratinocytes that reside in the basal layer of the epidermis and mediate epidermal homeostasis. Insulin-like growth factor 1 (IGF-1) signaling through its receptor (IGF-1R) has been identified as an important regulator in rodent skin development and differentiation. However, the role of IGF-1/IGF-1R signaling in human keratinocytes is not yet well understood. OBJECTIVE: This study aimed to clarify the role of IGF-1/IGF-1R signaling in human epidermal homeostasis. METHODS: IGF-1R specific knockout (KO) HaCaT keratinocytes were generated by CRISPR-Caspase-9-mediated non-homologous end joining frame-shift mutations. Further, the behavior of these keratinocytes in epidermal homeostasis was investigated using reconstructed epidermis and human skin equivalents. RESULTS: IGF-1R KO HaCaT keratinocytes were successfully established and produced thin epidermis in three-dimensional culture models. Keratin10-positive cells were frequently found in the basal layer of the reconstructed epidermis. CONCLUSIONS: IGF-1/IGF-1R signaling was demonstrated to play a key role in maintaining human epidermal homeostasis. This method provides a new framework to investigate gene function in human epidermal homeostasis.

Stem cell competition orchestrates skin homeostasis and ageing.

Stem cells underlie tissue homeostasis, but their dynamics during ageing-and the relevance of these dynamics to organ ageing-remain unknown. Here we report that the expression of the hemidesmosome component collagen XVII (COL17A1) by epidermal stem cells fluctuates physiologically through genomic/oxidative stress-induced proteolysis, and that the resulting differential expression of COL17A1 in individual stem cells generates a driving force for cell competition. In vivo clonal analysis in mice and in vitro 3D modelling show that clones that express high levels of COL17A1, which divide symmetrically, outcompete and eliminate adjacent stressed clones that express low levels of COL17A1, which divide asymmetrically. Stem cells with higher potential or quality are thus selected for homeostasis, but their eventual loss of COL17A1 limits their competition, thereby causing ageing. The resultant hemidesmosome fragility and stem cell delamination deplete adjacent melanocytes and fibroblasts to promote skin ageing. Conversely, the forced maintenance of COL17A1 rescues skin organ ageing, thereby indicating potential angles for anti-ageing therapeutic intervention.