Spontaneous Disease Research Articles

Depletion of ApoA5 aggravates spontaneous and diet-induced nonalcoholic fatty liver disease by reducing hepatic NR1D1 in hamsters.

Background: ApoA5 mainly synthesized and secreted by liver is a key modulator of lipoprotein lipase (LPL) activity and triglyceride-rich lipoproteins (TRLs). Although the role of ApoA5 in extrahepatic triglyceride (TG) metabolism in circulation has been well documented, the relationship between ApoA5 and nonalcoholic fatty liver disease (NAFLD) remains incompletely understood and the underlying molecular mechanism still needs to be elucidated. Methods: We used CRISPR/Cas9 gene editing to delete Apoa5 gene from Syrian golden hamster, a small rodent model replicating human metabolic features. Then, the ApoA5-deficient (ApoA5-/-) hamsters were used to investigate NAFLD with or without challenging a high fat diet (HFD). Results: ApoA5-/- hamsters exhibited hypertriglyceridemia (HTG) with markedly elevated TG levels at 2300 mg/dL and hepatic steatosis on a regular chow diet, accompanied with an increase in the expression levels of genes regulating lipolysis and small adipocytes in the adipose tissue. An HFD challenge predisposed ApoA5-/- hamsters to severe HTG (sHTG) and nonalcoholic steatohepatitis (NASH). Mechanistic studies in vitro and in vivo revealed that targeting ApoA5 disrupted NR1D1 mRNA stability in the HepG2 cells and the liver to reduce both mRNA and protein levels of NR1D1, respectively. Overexpression of human NR1D1 by adeno-associated virus 8 (AAV8) in the livers of ApoA5-/- hamsters significantly ameliorated fatty liver without affecting plasma lipid levels. Moreover, restoration of hepatic ApoA5 or activation of UCP1 in brown adipose tissue (BAT) by cold exposure or CL316243 administration could significantly correct sHTG and hepatic steatosis in ApoA5-/- hamsters. Conclusions: Our data demonstrate that HTG caused by ApoA5 deficiency in hamsters is sufficient to elicit hepatic steatosis and HFD aggravates NAFLD by reducing hepatic NR1D1 mRNA and protein levels, which provides a mechanistic link between ApoA5 and NAFLD and suggests the new insights into the potential therapeutic approaches for the treatment of HTG and the related disorders due to ApoA5 deficiency in the clinical trials in future.

The dualistic role of Lyn tyrosine kinase in immune cell signaling: implications for systemic lupus erythematosus.

Systemic lupus erythematosus (SLE, lupus) is a debilitating, multisystem autoimmune disease that can affect any organ in the body. The disease is characterized by circulating autoantibodies that accumulate in organs and tissues, which triggers an inflammatory response that can cause permanent damage leading to significant morbidity and mortality. Lyn, a member of the Src family of non-receptor protein tyrosine kinases, is highly implicated in SLE as remarkably both mice lacking Lyn or expressing a gain-of-function mutation in Lyn develop spontaneous lupus-like disease due to altered signaling in B lymphocytes and myeloid cells, suggesting its expression or activation state plays a critical role in maintaining tolerance. The past 30 years of research has begun to elucidate the role of Lyn in a duplicitous signaling network of activating and inhibitory immunoreceptors and related targets, including interactions with the interferon regulatory factor family in the toll-like receptor pathway. Gain-of-function mutations in Lyn have now been identified in human cases and like mouse models, cause severe systemic autoinflammation. Studies of Lyn in SLE patients have presented mixed findings, which may reflect the heterogeneity of disease processes in SLE, with impairment or enhancement in Lyn function affecting subsets of SLE patients that may be a means of stratification. In this review, we present an overview of the phosphorylation and protein-binding targets of Lyn in B lymphocytes and myeloid cells, highlighting the structural domains of the protein that are involved in its function, and provide an update on studies of Lyn in SLE patients.

Sensory neurons promote immune homeostasis in the lung

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPβ was dependent on JAK1 in the vagus nerve, and CGRPβ suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Single cell transcriptomic heterogeneity between sporadic early and late‐onset of Alzheimer’s disease

AbstractBackgroundRegulatory T‐cells (Tregs) are key modulators of immune responses, and may play a key role in the pathophysiology of various neurodegenerative disorders, including Alzheimer’s disease (AD). However, the impact of Tregs modulation of immune responses during disease progression is poorly understood and still controversial. Thus, the main objective of our ongoing study is to depict a high‐resolution transcriptomic landscape of blood immune cells during spontaneous disease progression, which will facilitate a better understanding of the protective and/or pathogenic immune responses at different stages of the disease1‐3.MethodIn 2019 a Multi‐partner consortium to expand dementia research in Latin America (ReDLat) was created. The Consortium aims to combine genomic, neuroimaging and behavioral (clinical, cognitive, socioeconomic status –SES‐) data to improve dementia characterization and identify novel inroads to treat neurodegeneration in diverse populations. ReDLat initiative supports a basic platform anchored in Argentina, Brazil, Colombia, Peru, Mexico, Chile and USA. allowing us to expand our research and combine genomic, neuroimaging and behavioral (clinical, cognitive, socioeconomic status) data to improve dementia characterization.To investigate changes in individual T cell subsets across progressive clinical conditions we included 4 healthy controls (CTLs), 4 early‐onset sporadic AD (EOAD), 4 late onset AD (LOAD) and 4 Parkinson disease patients (PD) from ReDLat. Peripheral blood mononuclear cells (PBMCs) were isolated and a 5' single‐cell transcriptome and immune repertoire sequencing was performed.ResultsWe identified transcriptionally distinct subpopulations, heterogeneity of the immune system, T cell repertoire diversity and antigen specificity among groups. Interestingly, CD4+CD25+Foxp3+ T‐regs gene expression heterogeneity was observed between sporadic EOAD and LOAD, mainly related to cytotoxicity and inflammation. Our next step is to perform multiple association studies incorporating imaging, sex, cognition and socio‐economics status (SES).ConclusionAlthough full analysis is still in progress, we identified significant changes in individual T‐cell subsets among individuals across the four proposed groups, providing a unique opportunity to identify new risk factors and therapeutics targets for AD.1‐ Fu J (2021) Front. Aging Neurosci.2‐ Cira Dansokho (2016). Brain.3‐Baek H (2016) Oncotarget.

Autoimmune Neutropenia of Infancy Is Almost Exclusively Caused By IgG Antibodies

Background The relevance of IgM antibodies in autoimmune mediated hemocytopenias remains a matter of debate. For autoimmune neutropenia, conflicting results were published. This study was performed as part of the Giessen Neutropenia Registry, which prospectively included children with suspected autoimmune neutropenia. Methods Children with suspected autoimmune neutropenia were prospectively enrolled and data central to their suspected diagnosis were collected over 3 years. Sera were tested for the presence of autoantibodies against neutrophils by granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT). The presence of IgG and IgM antibodies was assessed in a subgroup of patients with positive initial GIFT in whom spontaneous remission of their neutropenia was documented. Results A total of 406 children were included in the study. A remission was reported for 114 patients during the study period. Of those, 96 children (84%) had an initial positive GIFT. Their median age at diagnosis was 11 months (7-17.5), and their mean neutrophil count was 293 (±267)/µL, ranging from 0 to 975. In total 75/96 (78%) of children had at least one documented infection at diagnosis. In 29/96 (30%), a bone marrow aspirate was available. At the documented time point of remission, children were 27 months (18-41) old. In the initial sample send for diagnosis, IgG antibodies against neutrophils were detected in 94/96 children (98%), and IgM antibodies were detected in 2/96 (2%). We did not observe children with autoantibodies of both Ig classes. Conclusion To our knowledge, this is the first study to demonstrate the contribution of IgG or IgM antibodies in a well-defined clinical cohort with a typical course including, spontaneous disease remission. IgG antibodies were present in 98%. Only very rarely, IgM antibodies can be detected. We conclude that in general, screening for IgG autoantibodies is appropriate as a first diagnostic step. In cases with strong clinical suspicion but negative GIFT with anti-IgG, anti-IgM testing may be considered.

Targeted screening of inflammatory mediators in spontaneous degenerative disc disease in dogs reveals an upregulation of the tumor necrosis superfamily.

The regulation of inflammatory mediators in the degenerating intervertebral disc (IVD) and corresponding ligamentum flavum (LF) is a topic of emerging interest. The study aimed to investigate the expression of a broad array of inflammatory mediators in the degenerated LF and IVD using a dog model of spontaneous degenerative disc disease (DDD) to determine potential treatment targets. LF and IVD tissues were collected from 22 normal dogs (Pfirrmann grades I and II) and 18 dogs affected by DDD (Pfirrmann grades III and IV). A qPCR gene array was used to investigate the expression of 80 inflammatory genes for LF and IVD tissues, whereafter targets of interest were investigated in additional tissue samples using qPCR, western blot (WB), and immunohistochemistry. Tumor necrosis factor superfamily (TNFSF) signaling was identified as a regulated pathway in DDD, based on the significant regulation (n-fold ± SD) of various TNFSF members in the degenerated IVD, including nerve growth factor (NGF; -8 ± 10), CD40LG (464 ± 442), CD70 (341 ± 336), TNFSF Ligand 10 (9 ± 8), and RANKL/TNFSF Ligand 11 (85 ± 74). In contrast, TNFSF genes were not significantly affected in the degenerated LF compared to the control LF. Protein expression of NGF (WB) was significantly upregulated in both the degenerated LF (4.4 ± 0.5) and IVD (11.3 ± 5.6) compared to the control group. RANKL immunopositivity was significantly upregulated in advanced stages of degeneration (Thompson grades IV and V) in the nucleus pulposus and annulus fibrosus of the IVD, but not in the LF. DDD involves a significant upregulation of various TNFSF members, with tissue-specific expression profiles in LF and IVD tissues. The differential involvement of TNFSF members within multiple spinal tissues from the same individual provides new insights into the inflammatory processes involved in DDD and may provide a basis to formulate hypotheses for the determination of potential treatment targets.

Microbial exposure during early life regulates development of bile duct inflammation

Objectives The early life microbiome has been linked to inflammatory diseases in adulthood and a role for the microbiome in bile duct inflammation is supported by both human and murine studies. We utilized the NOD.c3c4 mouse model that develops a spontaneous immune-driven biliary disease with a known contribution of the microbiome to evaluate the temporal effects of the early life microbiome. Materials and methods Germ-free (GF) NOD.c3c4 mice were conventionalized into a specific pathogen free environment at birth (conventionally raised, CONV-R) or at weaning (germ-free raised, GF-R) and compared with age and gender-matched GF and conventional (CONV) NOD.c3c4 mice. At 9 weeks of age, liver pathology was assessed by conventional histology and flow cytometry immunophenotyping. Results Neonatal exposure to microbes (CONV-R) increased biliary inflammation to similar levels as regular conventional NOD.c3c4 mice, while delayed exposure to microbes (GF-R) restrained the biliary inflammation. Neutrophil infiltration was increased in all conventionalized mice compared to GF. An immunophenotype in the liver similar to CONV was restored in both CONV-R and GF-R compared to GF mice displaying a proportional increase of B cells and reduction of T cells in the liver. Conclusions Microbial exposure during early life has a temporal impact on biliary tract inflammation in the NOD.c3c4 mouse model suggesting that age-sensitive interaction with commensal microbes have long-lasting effects on biliary immunity that can be of importance for human cholangiopathies.

Regulatory T cells inhibit autoantigen-specific CD4+ T cell responses in lupus-prone NZB/W F1 mice.

Progressive loss of regulatory T cell (Treg)-mediated control over autoreactive effector T cells contributes to the development of systemic lupus erythematosus (SLE). Accordingly, we hypothesized that Treg may also have the capacity to suppress the activation of autoreactive CD4+ T cells that are considered to drive autoimmunity. To investigate whether Treg are involved in the control of autoreactive CD4+ T cells, we depleted CD25+ Treg cells either in vivo or in vitro, or combined both approaches before antigen-specific stimulation with the SLE-associated autoantigen SmD1(83-119) in the NZB/W F1 mouse model either after immunization against SmD1(83-119) or during spontaneous disease development. Frequencies of autoantigen-specific CD4+ T cells were determined by flow cytometry using the activation marker CD154. Both in vitro and in vivo depletion of CD25+ Treg, respectively, increased the frequencies of detectable autoantigen-specific CD4+ T cells by approximately 50%. Notably, the combined in vivo and in vitro depletion of CD25+ Treg led almost to a doubling in their frequencies. Frequencies of autoantigen-specific CD4+ T cells were found to be lower in immunized haploidentical non-autoimmune strains and increased frequencies were detectable in unmanipulated NZB/W F1 mice with active disease. In vitro re-addition of CD25+ Treg after Treg depletion restored suppression of autoantigen-specific CD4+ T cell activation. These results suggest that the activation and expansion of autoantigen-specific CD4+ T cells are partly controlled by Treg in murine lupus. Depletion of Treg therefore can be a useful approach to increase the detectability of autoantigen-specific CD4+ T cells allowing their detailed characterization including lineage determination and epitope mapping and their sufficient ex vivo isolation for cell culture.

Small Molecules Temporarily Induce Neuronal Features in Adult Canine Dermal Fibroblasts.

Several methods have been developed to generate neurons from other cell types for performing regeneration therapy and in vitro studies of central nerve disease. Small molecules (SMs) can efficiently induce neuronal features in human and rodent fibroblasts without transgenes. Although canines have been used as a spontaneous disease model of human central nerve, efficient neuronal reprogramming method of canine cells have not been well established. We aimed to induce neuronal features in adult canine dermal fibroblasts (ACDFs) by SMs and assess the permanency of these changes. ACDFs treated with eight SMs developed a round-shaped cell body with branching processes and expressed neuronal proteins, including βIII-tubulin, microtubule-associated protein 2 (MAP2), and neurofilament-medium. Transcriptome profiling revealed the upregulation of neuron-related genes, such as SNAP25 and GRIA4, and downregulation of fibroblast-related genes, such as COL12A1 and CCN5. Calcium fluorescent imaging demonstrated an increase in intracellular Ca2+ concentration upon stimulation with glutamate and KCl. Although neuronal features were induced similarly in basement membrane extract droplet culture, they diminished after culturing without SMs or in vivo transplantation into an injured spinal cord. In conclusion, SMs temporarily induce neuronal features in ACDFs. However, the analysis of bottlenecks in the neuronal induction is crucial for optimizing the process.

Expression of APP, CDK5, and AKT1 Gene Related to Alzheimer Disease in Brain of Long-tailed Macaques

Amyloid plaques and Neurofibrillary Tangles (NFTs) are known to be key pathological features of Alzheimer disease. To gain a better understanding of this disease, studies were carried out on the Indonesian primates, the long-tailed macaques, using a spontaneous Alzheimer's disease model. Examining and identifying genetic markers involved in plaque formation and NFTs in long-tailed macaques is necessary to reveal their physiological processes. In this study, the expression of genes involved in the development of amyloid plaque (Amyloid Precursor Protein (APP)) and those that control the phosphorylation of tau protein (CDK5 and AKT1) was examined in the long-tailed macaque brain. This study showed that APP, CDK5, and AKT1 may potentially be developed as genetic markers of Alzheimer's disease. Long-tailed macaques exhibited the development of amyloid plaque in the aging brain based on the analysis of the gene expression profile of its biomarker. Furthermore, long-tailed macaques can be optimized for neurodegenerative models.

Gastrointestinal tract pathology of the owl monkey (Aotus spp.).

Owl monkeys are small nocturnal new world primates in the genus Aotus that are most used in biomedical research for malaria. Cardiomyopathy and nephropathy are well-described common diseases contributing to their morbidity and mortality; less is known about lesions affecting the gastrointestinal tract. Records from a 14-year period (2008-2022) at the Keeling Center for Comparative Medicine and Research were queried to identify instances of spontaneous gastrointestinal disease that directly contributed to the cause of death from the 235 adult owl monkeys submitted for necropsy. Of the 235, 10.6% (25/235) had gastrointestinal disease listed as a significant factor that contributed to morbidity and mortality. Diagnoses included candidiasis (3/25), gastric bloat (4/25), and intestinal incarceration and ischemia secondary (11/25), which included intussusception (4/25), mesenteric rent (3/25), strangulating lipoma (2/25), intestinal torsion (1/25), and an inguinal hernia (1/25). Intestinal adenocarcinomas affecting the jejunum (4/25) were the most common neoplasia diagnosis. Oral squamous cell carcinoma (1/25) and intestinal lymphoma (2/25) were also diagnosed. This report provides evidence of spontaneous lesions in the species that contribute to morbidity and mortality.

Correlation of miR-155-5p and BDNF with Prognosis of Patients with Intracerebral Hemorrhage Undergoing CT-guided Minimally Invasive Surgery

Background: Hypertensive intracerebral hemorrhage (HICH) is a spontaneous cerebrovascular disease occuring in the brain parenchyma. Objectives: To characterize the predictive role of miR-155-5p and BDNF in the prognosis of HICH. Methods: All patients with HICH who underwent CT-guided minimally invasive surgery were classified into the good and poor prognosis groups using the modified Rankin Scale (mRS). The level of miR-155-5p was determined by qRT-PCR, and the level of brain-derived neurotrophic factor (BDNF) in serum was determined by ELISA. The relationship between miR-155-5p and BDNF was tested by Pearson correlation and luciferase reporter assay. The logistic regression method was used to determine the risk factors. The ROC curve was drawn to explain the predictive role of miR-155-5p, BDNF, or their combination. Results: A high level of miR-155-5p and a lower level of BDNF were observed in the poor prognosis group. BDNF level was negatively related to the level of miR-155-5p. BDNF is a target of miR-155-5p. BDNF and miR-155-5p were associated with prognosis. BDNF, miR-155-5p or their combination were predictive biomarkers for the prognosis of HICH. Conclusion: BDNF and miR-155-5p were associated with the outcome of HICH patients.

Ets1 and IL17RA cooperate to regulate autoimmune responses and skin immunity to Staphylococcus aureus.

Ets1 is a lymphoid-enriched transcription factor that regulates B- and Tcell functions in development and disease. Mice that lack Ets1 (Ets1 KO) develop spontaneous autoimmune disease with high levels of autoantibodies. Naïve CD4 + T cells isolated from Ets1 KO mice differentiate more readily to Th17 cells that secrete IL-17, a cytokine implicated in autoimmune disease pathogenesis. To determine if increased IL-17 production contributes to the development of autoimmunity in Ets1 KO mice, we crossed Ets1 KO mice to mice lacking the IL-17 receptor A subunit (IL17RA KO) to generate double knockout (DKO) mice. In this study, the status of the immune system of DKO and control mice was assessed utilizing ELISA, ELISpot, immunofluorescent microscopy, and flow cytometric analysis of the spleen, lymph node, skin. The transcriptome of ventral neck skin was analyzed through RNA sequencing. S. aureus clearance kinetics in in exogenously infected mice was conducted using bioluminescent S. aureus and tracked using an IVIS imaging experimental scheme. We found that the absence of IL17RA signaling did not prevent or ameliorate the autoimmune phenotype of Ets1 KO mice but rather that DKO animals exhibited worse symptoms with striking increases in activated B cells and secreted autoantibodies. This was correlated with a prominent increase in the numbers of T follicular helper (Tfh) cells. In addition to the autoimmune phenotype, DKO mice also showed signs of immunodeficiency and developed spontaneous skin lesions colonized by Staphylococcus xylosus. When DKO mice were experimentally infected with Staphylococcus aureus, they were unable to clear the bacteria, suggesting a general immunodeficiency to staphylococcal species. γδ T cells are important for the control of skin staphylococcal infections. We found that mice lacking Ets1 have a complete deficiency of the γδ T-cell subset dendritic epidermal T cells (DETCs), which are involved in skin woundhealing responses, but normal numbers of other skin γδ T cells. To determine if loss of DETC combined with impaired IL-17 signaling might promote susceptibility to staph infection, we depleted DETC from IL17RA KO mice and found that the combined loss of DETC and impaired IL-17 signaling leads to an impaired clearance of the infection. Our studies suggest that loss of IL-17 signaling can result in enhanced autoimmunity in Ets1 deficient autoimmune-prone mice. In addition, defects in wound healing, such as that caused by loss of DETC, can cooperate with impaired IL-17 responses to lead to increased susceptibility to skin staph infections.

Three-dimensional magnetic resonance elastography combining proton-density fat fraction precisely identifies metabolic dysfunction-associated steatohepatitis with significant fibrosis

PurposePatients with metabolic dysfunction-associated steatohepatitis (MASH) and significant fibrosis (fibrosis stage≥2), known as Fibro-MASH, are at increased risk of liver-related outcomes and lower rates of spontaneous disease regression. The aim was to investigate three-dimensional MR elastography (3D-MRE) combining proton-density fat fraction (PDFF) as a means of identifying Fibro-MASH. MethodsForty-eight New Zealand rabbits were fed a high-fat/cholesterol or standard diet to obtain different disease activity and fibrosis stages. Shear stiffness (SS) and Damping Ratio (DR) were derived from 3D-MRE, whereas PDFF was from a volumetric 3D imaging sequence. Steatosis grade, metabolic dysfunction-associated steatotic liver disease activity score (MAS), and fibrosis stage were diagnosed histologically. Serum markers of fibrosis and inflammation were also measured. Correlation and comparison analysis, Receiver operating characteristic curves (ROC), Delong test, logistic regression analysis, and Net reclassification improvement (NRI) were performed. ResultsPDFF correlated with steatosis grade (rho = 0.853). SS increased with developed liver fibrosis (rho = 0.837). DR correlated with MAS grade (rho = 0.678). The areas under the ROC (AUROCs) of SS for fibrosis grading were 0.961 and 0.953 for ≥F2, and ≥ F3, respectively. All the biochemical parameters were considered but excluded from the logistic regression analysis to identify Fibro-MASH. FF, SS, and DR were finally included in the further analysis. The three-parameter model combining PDFF, SS, and DR showed significant improvement in NRI over the model combining SS and PDFF (AUROC 0.973 vs. 0.906, P = 0.081; NRI 0.28, P < 0.05). Conclusion3D-MRE combining PDFF may characterize the state of fat content, disease activity and fibrosis, thus precisely identify Fibro-MASH.

Primary Percutaneous Coronary Intervention in Patients With Spontaneous Coronary Artery Dissection vs Coronary Artery Disease

BackgroundSpontaneous coronary artery dissection (SCAD) is a rare cause of acute myocardial infarction. Revascularization in SCAD remains very challenging and therefore is not recommended as the initial management strategy in stable SCAD without high-risk features. ObjectivesThe aim of this study was to compare in-hospital mortality and 30-day readmission rates between patients with SCAD with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PPCI) and patients with STEMI without SCAD undergoing PPCI. MethodsThis study was conducted using the administrative minimum dataset of the Spanish National Health System (2016-2020). Risk-standardized in-hospital mortality ratios and readmission ratios were calculated, and results were adjusted using propensity score (PS) analyses. ResultsA total of 65,957 episodes of PPCI were identified after exclusions. The crude in-hospital mortality rate was 4.8%. Of these, 315 (0.5%) were SCAD PPCI and 65,642 were non-SCAD PPCI. SCAD PPCI patients were younger and more frequently women than non-SCAD PPCI patients. Crude mortality (5.7% vs 4.8%), risk-standardized in-hospital mortality ratio (5.3% vs 5.3%), and PS-adjusted (315 pairs) mortality (5.7% vs 5.7%) were similar in SCAD PPCI and non-SCAD PPCI patients. In addition, crude (3% vs 3.3%) and PS-adjusted (297 pairs) 30-day readmission rates (3% vs 4%) were also similar in both groups. ConclusionsPPCI, when indicated in patients with STEMI and SCAD, has similar in-hospital mortality and 30-day readmission rates compared with PPCI for atherothrombotic STEMI. These findings support the value of PPCI in selected patients with SCAD.

Spontaneous prion disease in homozygous and heterozygous transgenic mouse models of T188K genetic Creutzfeldt-Jakob disease

Genetic Creutzfeldt-Jakob disease with T188K mutation (T188K gCJD) is the most frequent genetic prion disease in China. To explore the penetration of T188K mutation and the pathogenesis of T188K gCJD, we constructed 2 lines of transgenic mouse models: homozygous Tg188K+/+ mice containing T188K mutation in 2 alleles of human PRNP background and heterozygous Tg188K+/- mice containing 1 allele of T188K human PRNP and 1 allele of the wild-type mouse PRNP. Spontaneous neurological illnesses were identified in all Tg188K mice at their old ages (750–800 days old). About half of the Tg188K mice died prior to the final observation (930 days old). Extensive spongiosis, PrPSc deposit, and reactive gliosis of astrocytes and microglia are neuropathologically identified, showing time-dependent exacerbation. Proteinase K-resistant PrP was detected in the brain, muscle, and intestine tissues, and positive real-time quaking-induced conversion reactions were elicited by the brain and muscle tissues of Tg188K mice. Those data verify that the constructed Tg188K mice highly mimic the clinicopathology of human T188K gCJD, strongly indicating the pathogenicity of T188K mutated PrP.

Endobronchial Valves for Management of Persistent Air Leaks in Patients With Chronic Lung Disease

BackgroundPersistent air leak (PAL) is a challenging problem in patients with spontaneous pneumothorax and chronic lung disease who are poor surgical candidates. Conventional management consists of long-term thoracostomy tube placement; however, in some cases, patients are unable to leave the hospital because of the need for continuous negative pressure. We investigated the application of endobronchial valves (EBVs) in the management of patients with air leak for whom surgical intervention was contraindicated. MethodsEBVs were placed in patients with PALs arising from nonoperative causes. Successful outcomes were defined as the ability to remove the thoracostomy tube from suction, resolution of air leak, and removal of chest tube. ResultsSeventeen patients with PAL requiring negative pressure suction were treated with EBVs. The overall complete successful outcome rate was 82.4%. There was a statistically significant reduction in length of stay after valve placement in the success group compared with the failure group (8.9 days vs 61.7 days; P = .0097). ConclusionsEBVs are a valid option for patients who are not surgical candidates and have failed to respond to conventional management of PAL. This experimental use of EBVs leads to high success rates and shorter length of stay when it is successful.

P-159 Impact of the symbiotic metabolites D-amino acids on preimplantation development

Abstract Study question Are the metabolites derived from the symbiotic microbiota of reproductive tract involved in preimplantation development? Summary answer The metabolites derived from the symbiotic microbiota of reproductive tract, D-amino acid（D-AA）, contributed to preimplantation development. What is known already It has been known that Lactobacillus exist in the vagina as symbiotic bacteria. Though it was thought that Lactobacillus keep the pH environment acidic and prevent infection by pathogenic bacteria, thereby keeping the uterus and fallopian tubes sterile in mammal, we have identified Staphylococcus aureus in oviductal lavage fluid from female mice and confirmed that the antibiotics disrupt the implantation pattern. In human, the vaginal microbiota is reported to be dominated by Lactobacillus, which produces D-amino acids (D-AA). These findings suggest the involvement of bacterial metabolites D-AA in the establishment of human pregnancy. Study design, size, duration 23 women undergoing in vitro fertilization (IVF) treatment at Keio University Hospital from 2019 to 2021 were assessed for eligibility. 3 were excluded due to spontaneous pregnancy or infectious disease. Twenty subjects were allocated to the successful pregnancy group (S-group (8); mean ± standard deviation 35.5 ± 2.8 years) defined as gestational sac detected on ultrasound at 5 weeks' gestation; and to the unsuccessful pregnancy group (U- group (12); 35.5 ± 2.7 years)). Participants/materials, setting, methods Genomic DNA (gDNA) extracted from vaginal and intrauterine samples at each menstrual cycle was used for metagenome analysis at the genus and the species levels by next-generation sequencing, and by quantitative real-time PCR. Amino acids were analyzed using a two-dimensional HPLC. Immunocytochemical staining by a polyclonal antibody to D-AA oxidase (DAO), which catabolizes D-AA, was performed using B6D2F1 mice and mice with a natural point-mutation G181R in DAO gene, which abolishes DAO activity. Main results and the role of chance Both vaginal/uterine bacterial counts were higher in the luteal phase than in the follicular phase. Vaginal bacterial counts in the S-group were lower than those in the U- group. The composition of microbiota of the vaginal samples (N = 23) shifted from the follicular to the luteal phase, with Lactobacillus spp. predominating. However, there was no significant difference in the ratio of Lactobacillus spp. between the S- and U-groups. The intrauterine samples of the S-group contained a high abundance of L. crispatus, L. gasseri, and L. ultunensis, while the U-group contained a high abundance of L. jensenii. Next, we analyzed the Lactobacillus metabolite, D-AA, and detected D-Ser, D-Ala, D-Asn, and D-Pro in the vaginal, uterine, and follicular fluid of the S-group. Follicular fluid that developed into blastocysts contained more D-AA than that of arrested embryos (N = 2). To investigate the significance of D-AA in the preimplantation development, mouse embryos were cultured in amino acid-depleted medium. All of them arrested (0.0%: 0/78), whereas 41% (25/61) of the embryos with D-AA added to the amino acid-depleted medium developed into blastocysts. We confirmed the expression of DAO in oocytes and blastocysts. DAO-null mice had fewer births than wild type. Limitations, reasons for caution The main limitation is the differences of symbiotic microbiota in reproductive tract and embryonic development between mice and humans; it may be difficult to apply the results of this analysis to humans. Wider implications of the findings Our results display a correlation between species-level microbiome profiling and IVF outcomes. In addition, microbiota-derived metabolites D-AA are suggested to be crucial mediators of embryo development. These findings could give rise to the development of new diagnostic techniques and drug. Trial registration number Not applicable

A remnant kidney surgery model in cats induces acute and chronic outcomes that mimic spontaneous chronic kidney disease.

To report acute and chronic outcomes of cats with chronic kidney disease (CKD) induced by a remnant kidney model. 32 purpose-bred cats (n = 15 female, n = 17 male). Cats underwent a 2-stage reduction in renal mass through partial arterial ligation of 1 kidney (day 28) and delayed contralateral nephrectomy (day 0), targeting an 11/12th functional nephrectomy. Acute (days -28 - 29) survival and renal function parameters were compared over time, and the latter were evaluated as predictors for acute mortality. Chronic (days 30 to >1,100) survival, renal function, and morphology were described. Acutely, renal function deteriorated in all cats (mean ± SD baseline and day 28 serum creatinine mean concentration, 1.13 ± 0.23 mg/dL and 3.03 ± 1.20 mg/dL, respectively; P < .001; and GFR, 3.22 mL/min/kg ± 0.12 and 1.21 mL/min/kg ± 0.08, respectively; P < .001). Seven (22%) cats were euthanized after because of clinical signs of uremia after contralateral nephrectomy. Prenephrectomy renal function tests were not significant indicators for survival during this acute phase. Twenty-five cats entered the chronic phase. Ten cats were euthanized at a median of 163 days from nephrectomy because of progressive renal dysfunction. Median survival times were significantly different when stratified by acute kidney injury grade at day 29. Cats in the chronic phase had clinical courses similar to cats with naturally occurring CKD, and most (13/15) were in CKD stage 2. The remnant kidney model is effective at reducing kidney function to an extent that mimics important characteristics of spontaneous CKD in cats.

IMMU-06. SYSTEMIC MRNA VACCINES RESET IMMUNOGENICITY AGAINST DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND DIPG remains a uniformly fatal disease in dire need of new therapies. There are currently no systemic therapies with proven efficacy against these tumors. To overcome these barriers, we developed a systemic mRNA lipid particle (LP) vaccine that localizes to tumors and reticuloendothelial (RE) organs to modulate both innate and adaptive immunity against poorly immunogenic tumors like DIPG. OBJECTIVE We sought to assess whether RNA-LPs encoding for model antigens (e.g. H3K27M) would reprogram innate immunity and simultaneously elicit sustained adaptive immunity against diffuse midline glioma (DMG). RESULTS RNA-LP encoding for model antigens elicit massive recruitment of nearly all monocytes and lymphocytes to RE organs secondary to a danger response mediated by release of orchestrated cytokines (IL-12, TNF-α, IFN-α) and chemokines (CCL2, CCL4, CXCL9-10). This corresponds to increases in absolute numbers of activated DCs and T cells in the RE organs of mice and reprogramming the glioma tumor microenvironment in canines (pet dogs with spontaneous disease). In neonatal mice inoculated midline with established murine K2 gliomas, we observed that RNA-LP vaccines encoding for H3K27M (beginning at ~day 30) elicit significant long-term survivorship, which appears curative in the bulk of animals treated. While H3K27M specific constructs appeared superior, we observed improved survivorship with irrelevant mRNA species (i.e. pp65 and GFP) suggesting these tumors to be particularly sensitive to innate immune modulation. In mice with DMGs, we observed clinical symptomatology of edema/hydrocephalus followed by improvement in many animals suggesting pseudoprogression from intratumoral inflammation and remodeling. CONCLUSION RNA-LPs reprogram the tumor microenvironment of poorly immunogenic tumors in effect making them ‘hot.’ Treatment responses in murine models of advanced DMGs are encouraging. We are advancing a final RNA-LP formulation for FDA-IND submission and early phase clinical trials for DIPG through multi-institutional consortia.