Integrative multi-omics profiling in human decedents receiving pig heart xenografts.

In a previous study, heart xenografts from 10-gene-edited pigs transplanted into two human decedents did not show evidence of acute-onset cellular- or antibody-mediated rejection. Here, to better understand the detailed molecular landscape following xenotransplantation, we carried out bulk and single-cell transcriptomics, lipidomics, proteomics and metabolomics on blood samples obtained from the transplanted decedents every 6 h, as well as histological and transcriptomic tissue profiling. We observed substantial early immune responses in peripheral blood mononuclear cells and xenograft tissue obtained from decedent 1 (male), associated with downstream T cell and natural killer cell activity. Longitudinal analyses indicated the presence of ischemia reperfusion injury, exacerbated by inadequate immunosuppression of T cells, consistent with previous findings of perioperative cardiac xenograft dysfunction in pig-to-nonhuman primate studies. Moreover, at 42 h after transplantation, substantial alterations in cellular metabolism and liver-damage pathways occurred, correlating with profound organ-wide physiological dysfunction. By contrast, relatively minor changes in RNA, protein, lipid and metabolism profiles were observed in decedent 2 (female) as compared to decedent 1. Overall, these multi-omics analyses delineate distinct responses to cardiac xenotransplantation in the two human decedents and reveal new insights into early molecular and immune responses after xenotransplantation. These findings may aid in the development of targeted therapeutic approaches to limit ischemia reperfusion injury-related phenotypes and improve outcomes.

Cellular recovery after prolonged warm ischaemia of the whole body.

After cessation of blood flow or similar ischaemic exposures, deleterious molecular cascades commence in mammalian cells, eventually leading to their death1,2. Yet with targeted interventions, these processes can be mitigated or reversed, even minutes or hours post mortem, as also reported in the isolated porcine brain using BrainEx technology3. To date, translating single-organ interventions to intact, whole-body applications remains hampered by circulatory and multisystem physiological challenges. Here we describe OrganEx, an adaptation of the BrainEx extracorporeal pulsatile-perfusion system and cytoprotective perfusate for porcine whole-body settings. After 1 h of warm ischaemia, OrganEx application preserved tissue integrity, decreased cell death and restored selected molecular and cellular processes across multiple vital organs. Commensurately, single-nucleus transcriptomic analysis revealed organ- and cell-type-specific gene expression patterns that are reflective of specific molecular and cellular repair processes. Our analysis comprises a comprehensive resource of cell-type-specific changes during defined ischaemic intervals and perfusion interventions spanning multiple organs, and it reveals an underappreciated potential for cellular recovery after prolonged whole-body warm ischaemia in a large mammal.

Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells.

The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer's disease. To investigate neurogenic potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in humans, macaques, and pigs. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in the adult mouse, pig, and macaque but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific, transcriptomically defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer's disease-related proteins. This resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function.

Regulation of prefrontal patterning and connectivity by retinoic acid.

The prefrontal cortex (PFC) and its connections with the mediodorsal thalamus are crucial for cognitive flexibility and working memory1 and are thought to be altered in disorders such as autism2,3 and schizophrenia4,5. Although developmental mechanisms that govern the regional patterning of the cerebral cortex have been characterized in rodents6-9, the mechanisms that underlie the development of PFC-mediodorsal thalamus connectivity and the lateral expansion of the PFC with a distinct granular layer 4 in primates10,11 remain unknown. Here we report an anterior (frontal) to posterior (temporal), PFC-enriched gradient of retinoic acid, a signalling molecule that regulates neural development and function12-15, and we identify genes that are regulated by retinoic acid in the neocortex of humans and macaques at the early and middle stages of fetal development. We observed several potential sources of retinoic acid, including the expression and cortical expansion of retinoic-acid-synthesizing enzymes specifically in primates as compared to mice. Furthermore, retinoic acid signalling is largely confined to the prospective PFC by CYP26B1, a retinoic-acid-catabolizing enzyme, which is upregulated in the prospective motor cortex. Genetic deletions in mice revealed that retinoic acid signalling through the retinoic acid receptors RXRG and RARB, as well as CYP26B1-dependent catabolism, are involved in proper molecular patterning of prefrontal and motor areas, development of PFC-mediodorsal thalamus connectivity, intra-PFC dendritic spinogenesis and expression of the layer 4 marker RORB. Together, these findings show that retinoic acid signalling has a critical role in the development of the PFC and, potentially, in its evolutionary expansion.

Brain vulnerability and viability after ischaemia.

The susceptibility of the brain to ischaemic injury dramatically limits its viability following interruptions in blood flow. However, data from studies of dissociated cells, tissue specimens, isolated organs and whole bodies have brought into question the temporal limits within which the brain is capable of tolerating prolonged circulatory arrest. This Review assesses cell type-specific mechanisms of global cerebral ischaemia, and examines the circumstances in which the brain exhibits heightened resilience to injury. We suggest strategies for expanding such discoveries to fuel translational research into novel cytoprotective therapies, and describe emerging technologies and experimental concepts. By doing so, we propose a new multimodal framework to investigate brain resuscitation following extended periods of circulatory arrest.

Chemerin Blood Levels are Associated with MRI Measured Volumes of Abdominal Adipose Tissue Compartments and Lifestyle Choices.

Obesity is a low-grade chronic inflammatory state, in which a cytokine chemerin with its immunometabolic modulation has an important role. We aimed to study in a healthy population relationships between serum chemerin levels, lifestyle choices and magnetic resonance imaging (MRI) assessed volumes of abdominal visceral (VAT) and subcutaneous (SAT) adipose tissues, which have different cytokine expression profiles. Overall, 73 healthy participants undertook lifestyle questionnaire and underwent anthropometric measurements along with MRI scanning of abdominal SAT and VAT. Furthermore, complete blood count was determined along with chemerin and fibrinogen serum levels. Regression model for prediction of chemerin serum levels was built after controlling for sex, age and anthropometric measures. Median serum chemerin was 141 (125-195) ng/mL. Serum chemerin had a moderate positive correlation with SAT and VAT volumes. The two most important predictors of chemerin levels were MRI detected SAT and thigh circumference. Independently, chemerin levels were associated with smoking, preference of salty food, and favoring flavor/simplicity of preparation over nutritional value of the food. Serum chemerin seems to be more strongly correlated with the volume of abdominal SAT than VAT, with certain lifestyle choices influencing chemerin levels independently of abdominal fat.

Transcardial gradient of adiponectin, interleukin-6 and tumor necrosis factor-α in overweight coronary artery disease patients.

BACKGROUND: Obesity is associated with coronary artery disease (CAD), where epicardial adipose tissue (EAT) express proatherogenic cytokines (i.e., interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)) and decreases production of beneficial adiponectin. Studies on endocrine role of EAT are mostly based on assessing cytokines' mRNAs, whereas cytokine blood levels might not readily correlate. In order to get better insight into the endocrine role of thickened EAT in CAD, we assessed transcardial gradient of adiponectin, IL-6 and TNF-α. METHODS: We assessed anthropometric and ultrasound measures in cohort of fifty nondiabetic subjects (21 CAD and 29 non-CAD). Blood sampled from aortic root and coronary sinus was assayed for adiponectin, IL-6 and TNF-α, using ELISA. RESULTS: Except thicker EAT in CAD subjects, anthropometric measures were similar (overweight), with higher adiponectin in coronary sinus than in aortic root (p<0.001) in both groups. CAD subjects had lower adiponectin in aortic root (p<0.001) and higher levels of TNF-α in coronary sinus than in aortic root (p=0.05). EAT thickness positively correlated with hip circumference (p=0.038) and negatively correlated with adiponectin levels (for both p<0.05). CONCLUSIONS: Transcardial gradient of adiponectin in non-CAD and CAD overweight subjects was similar, while latter had lower systemic adiponectin level and thicker EAT. EAT with thickening reaches the threshold level of near-maximal down-regulation of adiponectin and its further thickening is not associated with continued decrease of adiponectin production. In CAD patients levels of TNF-α were higher, but IL-6 were not, and these cytokines might be flush out by lymphatic route.