Autopsy Tissue Research Articles

Glutathione level variations in brain and blood in healthy aging and Alzheimer’s disease continuum: A systematic review

AbstractBackgroundOxidative stress (OS) plays an important role in healthy and pathological aging, particularly Alzheimer’s disease (AD). To protect against the harmful effects of OS, the body uses antioxidants, the most prevalent being glutathione (reduced form, GSH) (Dwivedi et al., 2020). The consistency of GSH perturbations with age and in the AD continuum has not been systematically assessed. We address this knowledge gap by conducting a systematic review of GSH variations in brain and blood in healthy aging and the AD continuum.MethodPubMed searches were conducted up to January 2023. 15 keyword combinations were used to identify studies investigating GSH levels in (i) brain (in‐vivo using magnetic resonance spectroscopy (MRS) and in autopsy tissue using biochemical assays) and/or (ii) blood (plasma, serum) using biochemical assays (Fig1A,B). We considered individuals between 18‐39 years (yrs) as young‐adults, 40‐59 yrs as middle‐aged, and ≥60 yrs as old‐adults.Result42 studies met inclusion criteria (Fig1A,B; Fig2). BRAIN : Good to excellent reproducibility was reported by 12 MRS studies (3T, N = 6; 4T, N = 1; 7T, N = 5). For studies investigating age‐specific changes in cognitively unimpaired adults (CU, N = 6), GSH levels were significantly reduced in the majority (50%; temporal, occipital, limbic) of ROIs investigated in old‐ relative to young‐ or middle‐aged adults. Relative to CU, while brain GSH levels were reported to be lower by the majority (5/7) of AD dementia studies, MCI studies lacked consensus, with decreases (N = 3), increases (N = 2) and no significant change (N = 2) reported. BLOOD : Blood GSH levels were lower with increasing age in CU (5/7), and further decreased in patients (5/7).In both brain and blood, the majority (4/7) of AD continuum studies assessing cognition reported poorer cognitive functions with lower GSH levels.ConclusionOur systematic review demonstrates that (a) in‐vivo MRS measurements of GSH are reliable; (b) GSH levels in CU generally decrease with increasing age; and (c) GSH levels are reduced in AD relative to age‐matched CU, and reduced GSH is associated with cognitive impairment. Interventions to increase GSH levels may therefore have beneficial effects in AD.

ZCCHC17 Modulates Neuronal RNA Splicing and Supports Cognitive Resilience in Alzheimer's Disease.

ZCCHC17 is a putative master regulator of synaptic gene dysfunction in Alzheimer's disease (AD), and ZCCHC17 protein declines early in AD brain tissue, before significant gliosis or neuronal loss. Here, we investigate the function of ZCCHC17 and its role in AD pathogenesis using data from human autopsy tissue (consisting of males and females) and female human cell lines. Co-immunoprecipitation (co-IP) of ZCCHC17 followed by mass spectrometry analysis in human iPSC-derived neurons reveals that ZCCHC17's binding partners are enriched for RNA-splicing proteins. ZCCHC17 knockdown results in widespread RNA-splicing changes that significantly overlap with splicing changes found in AD brain tissue, with synaptic genes commonly affected. ZCCHC17 expression correlates with cognitive resilience in AD patients, and we uncover an APOE4-dependent negative correlation of ZCCHC17 expression with tangle burden. Furthermore, a majority of ZCCHC17 interactors also co-IP with known tau interactors, and we find a significant overlap between alternatively spliced genes in ZCCHC17 knockdown and tau overexpression neurons. These results demonstrate ZCCHC17's role in neuronal RNA processing and its interaction with pathology and cognitive resilience in AD, and suggest that the maintenance of ZCCHC17 function may be a therapeutic strategy for preserving cognitive function in the setting of AD pathology.

NIMG-35. RADIO-PATHOMIC MAPS OF TUMOR PROBABILITY IDENTIFY GLIOBLASTOMA INVASION BEYOND 5-ALA GUIDED RESECTION MARGINS THAT EVENTUALLY EXHIBIT TUMOR RECURRENCE

Abstract Current standard of care for glioblastoma includes surgery followed by chemotherapy and radiation, with extent of resection strongly correlated with survival. There is a need to improve the detection of tumor invasion beyond traditional imaging methods, as the full extent of tumor is known to be underestimated. Fluorescence-guided surgery with 5-Aminolevulinic acid (5-ALA) allows surgeons to visualize tumor cells beyond what is seen within traditional MR imaging margins. In this study, we compared tumor probability maps (TPMs) that detect tumor outside of contrast enhancement (CE) to the 5-ALA-guided resection cavity in glioblastoma patients. 3 pathologically confirmed glioblastoma patients that followed standard of care and underwent 5-ALA surgery were included. Patient 1 was a 56-year-old male with radiographic recurrence at 26 days, patient 2 was a 61-year-old male with radiographic recurrence at 130 days, and patient 3 was a 72-year-old male with cystic recurrence at 84 days, all post 5-ALA surgery. A previously published algorithm, using T1, T1C, FLAIR, and ADC as input and autopsy tissue as ground truth, was used to generate TPMs. Pre-surgical TPMs were qualitatively compared to pre-surgical CE, post-5-ALA surgical resection cavities, and radiographic recurrence seen as CE. In patients 1 and 2, TPMs identified areas suspicious of tumor that was not resected and later exhibited radiographic recurrence. Patient 3 had cystic recurrence only identified on post-resection T1C. In all patients, the extent of 5-ALA resection was greater than CE volume, but smaller than TPM identified tumor. These findings suggest that there was residual tumor that was unidentified via 5-ALA and traditional imaging but was identified by the TPMs. Future studies could utilize these maps in conjunction with 5-ALA to maximize surgical resections and prolong overall survival.

NIMG-14. AUTOPSY-BASED RADIO-PATHOMIC MAPS REVEAL DEMOGRAPHIC FACTORS IMPACT THE PRESENCE OF TUMOR OUTSIDE CONTRAST ENHANCEMENT IN GLIOMA PATIENTS

Abstract Gliomas are vastly heterogenous tumors with wide-ranging pathological signatures; as such, there is a pressing need to understand how individualized factors affect tumor growth patterns to develop treatment plans and improve prognosis. This study used autopsy-based radio-pathomic maps of tumor probability to test the hypothesis that differences in patient sex and age impact the presence of non-enhancing tumor. This study used pre-surgical imaging and demographic data from 501 glioma patients from the UCSF-PDGM dataset. Specifically, T1, T1C, FLAIR, and ADC images were used to generate maps of tumor probability using a previously validated model. This model used autopsy tissue aligned to imaging as ground truth to develop models sensitive to identifying areas of non-enhancing tumor outside of traditional imaging signatures. Thresholded maps were used to delineate areas of tumor presence, and the non-enhancing tumor volume was computed for each patient as the predicted high probability tumor volume within non-enhancing FLAIR hyperintensity. A general linear model was then fit to assess the effect of age and sex on non-enhancing tumor volumes, using tumor grade and contrast-enhancing volume as covariates. Male patients trended towards greater non-enhancing tumor volumes compared to female patients (B=5676.6, p=0.053). Older patients were also found to have less non-enhancing tumor volume compared to younger patients (B=-261.1, p=0.015). These results suggest that demographic characteristics influence the progression of tumor beyond the angiogenic tumor mass and may therefore influence treatment efficacy and prognoses. Future research is essential to understanding how these sex differences relate to the worse prognosis historically observed for males with glioma, and how the reduced presence of non-enhancing tumor in older patients relates to a reduction in plasticity-induced tumor propagation.

NIMG-22. COMPARISON OF NON-INVASIVE, ML-GENERATED WHOLE-BRAIN SPATIAL RADIOPATHOMIC MAPS DERIVED FROM ANATOMICAL AND DIFFUSION-WEIGHTED MRI ON PATIENTS WITH NEWLY DIAGNOSED-GLIOMA

Abstract INTRODUCTION Spatial heterogeneity in the glioma microenvironment is difficult to capture through singular biopsy samples. Identification of malignant tumor regions can help guide diagnosis and treatment planning. This study compares radiopathomic maps from 2 strategies derived from anatomical and diffusion-weighted MRI: 1) a support vector machine model trained on an MRI dataset with tissue samples of known spatial coordinates to generate spatial maps of probability of KI-67 and cellularity, and 2) a bagged random-forest model derived from 40+ person glioma brain bank of autopsy tissue coregistered to end-of-life MRI to generate spatial maps of cellularity and tumor probability (TPM). We hypothesize that TPMs from autopsy samples will correlate with KI-67 predictions, while cellularity values among methods will correlate. METHODS Spatial maps of tumor pathology from autopsy- and tissue sample-based models were generated on 55 patients newly-diagnosed with glioblastoma that were scanned with anatomical, diffusion-weighted, and metabolic imaging. Mean values were extracted from each map within the T2-hyperintense, non-enhancing lesion (NEL), the contrast-enhancing lesion (CEL), normal-appearing white matter (NAWM), and 5mm diameter spheres centered at the location where tissues were taken. Spearman correlation analysis was used to elucidate the strength of the linear relationship within the selected regions between the two maps. RESULTS Correlations between KI-67 and TPM values were highest and most significant at the tissue sample locations and CEL (ρ=0.306, p=0.002 and ρ=0.312, p=0.044 respectively). Cellularity maps generated from the autopsy-based model showed no significant correlation with newly diagnosed cellularity values at tissue sample locations, potentially highlighting the influence of treatment. CONCLUSION This study examines the current state of ML-generated spatial mapping, highlighting similarities within CEL, but need to account for the effects of subsequent treatment and progression when comparing tumor beyond the CEL between models derived between different time points.

TMIC-11. SPATIAL PROFILING OF TUMOR VASCULATURE AT AUTOPSY IN BRAINSTEM GLIOMAS

Abstract BACKGROUND Diffuse midline glioma (DMG) is a lethal pediatric tumor, and new treatment strategies are urgently needed. To design therapies that can cross the blood-tumor barrier, we designed a pilot study to investigate protein expression on DMG tumor vasculature using spatial profiling, with attention to targetable surface proteins. METHODS We identified 5 patients and 4 controls consented to institutional tissue banking protocols with adequate autopsy tissue from multiple anatomical regions within the brainstem. Portions of formalin-fixed, paraffin-embedded tissue were arranged into tissue microarrays profiled using the nanoString GeoMX platform. Briefly, a multiplexed cocktail of 51 primary antibodies tagged with UV-photocleavable oligonucleotide barcodes and fluorescent morphology markers were applied to tissues and 10-14 regions of interest (ROIs) were segmented by vessel area for each patient. ROIs were processed using UV light and barcodes were quantified using ex situ digital counting. RESULTS Five patients were included in the pilot study with average age 9.8 years (range 8-13), and overall survival was 9.5 months (range 3-18 months). Three tumors were classified as DMG, H3K27-mutant, WHO grade 4; one as high grade glioma without identified histone alteration but with recurrent DMG drivers (ACVR1 and PIK3CA mutations) and one as infiltrating atypical glioma with high proliferative rate and no DMG driver mutations. Controls ranged from 4-15 years and did not have pathological changes in the brain at death. Protein abundance was compared between cases and control and within cases where tumor-adjacent normal tissue was identified. Several proteins were significantly more abundant on tumor vasculature compared to non-tumor adjacent or control brainstem vessels. Validation studies in primary tissue are ongoing. CONCLUSIONS Digital spatial profiling is a promising technology for investigating tumor vasculature and is feasible in autopsy specimens. Work is ongoing to validate candidate biomarkers and expand the cohort to develop a vascular atlas for DMG.

Local Inflammation But Not Kidney Cell Infection Associated with High APOL1 Expression in COVID-Associated Nephropathy.

Key Points In coronavirus disease-19 biopsies, detection of severe acute respiratory syndrome coronavirus 2 was rare with no evidence of viral replication, whereas autopsy tissue failed quality control.In patients with FSGS, apolipoprotein L1 expression differed by degree of immune cell infiltrates, with some podocytes exhibiting up to 18-fold higher expression.In COVAN, the predicted high induction of apolipoprotein L1 expression occurs in a pattern consistent with the stochastic nature of FSGS pathology.

Aberrant colon metabolome and the sudden infant death syndrome.

The Sudden Infant Death Syndrome (SIDS) has been associated with increased peripheral serotonin and an abnormal colonic microbiome, suggesting the colonic metabolome may also be abnormal. This study addresses this potential correlation by comparing colonic autopsy tissue from SIDS to age-matched non-SIDS controls. Untargeted metabolomic analysis by mass spectrometry is used to assess human colonic metabolomic differences including serotonin. Expression of genes associated with colonic serotonin synthesis and transport (TPH1, TPH2, DDC, SCL6A4) is measured by qRT-PCR. Microbiome analysis is performed to compare the SIDS and non-SIDS colonic microbiome. Unsupervised hierarchical cluster and principal component analyses of metabolomic data shows increased variability in the SIDS cohort and separation of SIDS cases from the non-SIDS controls. There is a trend toward increased serotonin in the SIDS cohort but there is no significant difference in expression of the serotonin synthesis and transport genes between SIDS and non-SIDS control cohorts. Microbiome analysis shows no significant difference between the SIDS and non-SIDS control cohorts. This study demonstrates increased variability in the colonic metabolome and a trend towards increased colonic serotonin in SIDS. The underlying cause of colon metabolomic variability, and its potential role in SIDS pathogenesis, warrants further investigation. The key message of this article is that SIDS is associated with an aberrant colonic metabolome. This is a novel observation suggesting another component in the pathophysiology underlying SIDS. Investigation of why the colonic metabolome is aberrant may offer new insights to SIDS pathogenesis and new strategies to reduce risk.

Prevalence and distribution of Lewy pathology in a homeless population.

The homeless population experience significant inequalities in health, and there is an increasing appreciation of the potential of lifestyle factors in the development of neurodegenerative diseases, including Parkinson's disease. We performed a study on the prevalence and distribution of pathological alpha-synuclein deposition throughout the central and peripheral nervous systems in a homeless population. Forty-four homeless individuals consecutively available for autopsy were recruited. Immunohistochemistry was performed using 5G4 antibody recognizing disease-associated forms of alpha-synuclein, complemented by phospho-synuclein antibody on autopsy tissues collected from 18 regions of the brain and spinal cord, as well as the right and left olfactory bulb, the cauda equina, the extramedullary portion of the vagus nerve, and 27 sites of peripheral organs. The study cohort consisted of 38 males and 6 females, median age 58 years (range 32-67). Lewy-related pathology was present in the brains of three male cases. One showed Braak stage 2 (60 years old), and two stage 4 (56 and 59 years old). One of the Braak stage 4 cases had Lewy-related pathology in the spinal cord, the cauda equina, and the extramedullary portion of the vagus nerve. Examination of 27 sites of peripheral organs found that all three cases with Lewy-related pathology present in the brain were devoid of peripheral organ alpha-synuclein pathology. Multiple system-type alpha-synuclein pathology was not found. Our study, representing a snapshot of the homeless population that came to autopsy, suggests that alpha-synuclein pathology is prevalent in the homeless supporting further study of this vulnerable population.

Altered sphingolipid pathway in SARS-CoV-2 infected human lung tissue.

The SARS-CoV-2 mediated COVID-19 pandemic has impacted millions worldwide. Hyper-inflammatory processes, including cytokine storm, contribute to long-standing tissue injury and damage in COVID-19. The metabolism of sphingolipids as regulators of cell survival, differentiation, and proliferation has been implicated in inflammatory signaling and cytokine responses. Sphingosine-kinase-1 (SK1) and ceramide-synthase-2 (CERS2) generate metabolites that regulate the anti- and pro-apoptotic processes, respectively. Alterations in SK1 and CERS2 expression may contribute to the inflammation and tissue damage during COVID-19. The central objective of this study is to evaluate structural changes in the lung post-SARS-CoV-2 infection and to investigate whether the sphingolipid rheostat is altered in response to SARS-CoV-2 infection. Central and peripheral lung tissues from COVID-19+ or control autopsies and resected lung tissue from COVID-19 convalescents were subjected to histologic evaluation of airspace and collagen deposisiton, and immunohistochemical evaluation of SK1 and CERS2. Here, we report significant reduction in air space and increase in collagen deposition in lung autopsy tissues from patients who died from COVID-19 (COVID-19+) and COVID-19 convalescent individuals. SK1 expression increased in the lungs of COVID-19+ autopsies and COVID-19 convalescent lung tissue compared to controls and was mostly associated with Type II pneumocytes and alveolar macrophages. No significant difference in CERS2 expression was noted. SARS-CoV-2 infection upregulates SK1 and increases the ratio of SK1 to CERS2 expression in lung tissues of COVID-19 autopsies and COVID-19 convalescents. These data suggest an alteration in the sphingolipid rheostat in lung tissue during COVID-19, suggesting a potential contribution to the inflammation and tissue damage associated with viral infection.

Immuno-reactivity evaluation of Mce-truncated subunit candidate vaccine against Mycobacterium avium subspecies paratuberculosis challenge in the goat models

BackgroundDetection of an appropriate antigen with high immunogenicity can be a big step in the production of an effective vaccine for control of Johne’s disease (JD). The aim of this study was to evaluate the efficacy of Mce-truncated protein as a subunit vaccine candidate for the control of JD in experimentally challenged goats.Materials and methodsSix healthy goat kids were immunized with Mce-truncated protein, and two goats were kept as controls. All kids were twice challenged orally with live Mycobacterium avium subspecies paratuberculosis(MAP) strain and half the goats from both the categories were sacrificed at 7 and 10 months after start of challenge study. Culture of MAP was performed from all the necropsied tissues to determine the true JD infection status.ResultsMce-truncated protein only reacted with pooled vaccinated goat sera in western-blot. A significant increase in humoral immune response against Mce protein was also observed in vaccinated goats. Compared to the control group, vaccinated goats gained higher body weights and none of them shed MAP or showed histopatological lesions or colonization of MAP in their necropsy tissues.ConclusionsThe new Mce protein based vaccine provided significant immunity in goats as they could meet the challenge with live MAP bacilli. Although the vaccine used in this study showed the high potential as a new effective vaccine for the control of JD, further validation study is still required to successfully implement the vaccine for JD control program.

Integrated Analysis of Immune Infiltration and Hub Pyroptosis-Related Genes for Multiple Sclerosis.

Studies on overall immune infiltration and pyroptosis in patients with multiple sclerosis (MS) are limited. This study explored immune cell infiltration and pyroptosis in MS using bioinformatics and experimental validation. The GSE131282 and GSE135511 microarray datasets including brain autopsy tissues from controls and MS patients were downloaded for bioinformatic analysis. The gene expression-based deconvolution method, CIBERSORT, was used to determine immune infiltration. Differentially expressed genes (DEGs) and functional enrichments were analyzed. We then extracted pyroptosis-related genes (PRGs) from the DEGs by using machine learning strategies. Their diagnostic ability for MS was evaluated in both the training set (GSE131282 dataset) and validation set (GSE135511 dataset). In addition, messenger RNA (mRNA) expression of PRGs was validated using quantitative real-time polymerase chain reaction (qRT-PCR) in cortical tissue from an experimental autoimmune encephalomyelitis (EAE) model of MS. Moreover, the functional enrichment pathways of each hub PRG were estimated. Finally, co-expressed competitive endogenous RNA (ceRNA) networks of PRGs in MS were constructed. Among the infiltrating cells, naive CD4+ T cells (P=0.006), resting NK cells (P=0.002), activated mast cells (P=0.022), and neutrophils (P=0.002) were significantly higher in patients with MS than in controls. The DEGs of MS were screened. Analysis of enrichment pathways showed that the pathways of transcriptional regulatory mechanisms and ion channels associating with pyroptosis. Four PRGs genes CASP4, PLCG1, CASP9 and NLRC4 were identified. They were validated in both the GSE135511 dataset and the EAE model by using qRT-PCR. CASP4 and NLRC4 were ultimately identified as stable hub PRGs for MS. Single-gene Gene Set Enrichment Analysis showed that they mainly participated in biosynthesis, metabolism, and organism resistance. ceRNA networks containing CASP4 and NLRC4 were constructed. MS was associated with immune infiltration. CASP4 and NLRC4 were key biomarkers of pyroptosis in MS.

Pathogenesis of an experimental coinfection of porcine parainfluenza virus 1 and influenza A virus in commercial nursery swine

Porcine parainfluenza virus 1 (PPIV-1) is a recently characterized swine respirovirus. Previous experimental studies reported PPIV-1 replicates in the porcine respiratory tract causing minimal clinical disease or lesions. However, it is unknown if PPIV-1 co-infections with viral respiratory pathogens would cause respiratory disease consistent with natural infections reported in the field. The objective of this study was to evaluate if PPIV-1 increases the severity of influenza A virus respiratory disease in swine. Fifty conventional, five-week-old pigs were assigned to one of three challenge groups (n = 15) or a negative control group (n = 5). Pigs were challenged with a γ-cluster H1N2 influenza A virus in swine (IAV-S; A/Swine/North Carolina/00169/2006), PPIV-1 (USA/MN25890NS/2016), inoculum that contained equivalent titers of IAV-S and PPIV-1 (CO-IN), or negative control. Clinical scores representing respiratory disease and nasal swabs were collected daily and all pigs were necropsied five days post inoculation (DPI). The CO-IN group demonstrated a significantly lower percentage of pigs showing respiratory clinical signs relative to the IAV-S challenge group from 2 to 4 DPI. The IAV-S and CO-IN groups had significantly lower microscopic composite lesion scores in the upper respiratory tract compared to the PPIV-1 group although the IAV-S and CO-IN groups had significantly higher microscopic composite lung lesion scores. Collectively, PPIV-1 did not appear to influence severity of clinical disease, macroscopic lesions, or alter viral loads detected in nasal swabs or necropsy tissues when administered as a coinfection with IAV-S. Studies evaluating PPIV-1 coinfections with different strains of IAV-S, different respiratory pathogens or sequential exposure of PPIV-1 and IAV-S are warranted.

Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins bind to host mitochondrial proteins, likely inhibiting oxidative phosphorylation (OXPHOS) and stimulating glycolysis. We analyzed mitochondrial gene expression in nasopharyngeal and autopsy tissues from patients with coronavirus disease 2019 (COVID-19). In nasopharyngeal samples with declining viral titers, the virus blocked the transcription of a subset of nuclear DNA (nDNA)-encoded mitochondrial OXPHOS genes, induced the expression of microRNA 2392, activated HIF-1α to induce glycolysis, and activated host immune defenses including the integrated stress response. In autopsy tissues from patients with COVID-19, SARS-CoV-2 was no longer present, and mitochondrial gene transcription had recovered in the lungs. However, nDNA mitochondrial gene expression remained suppressed in autopsy tissue from the heart and, to a lesser extent, kidney, and liver, whereas mitochondrial DNA transcription was induced and host-immune defense pathways were activated. During early SARS-CoV-2 infection of hamsters with peak lung viral load, mitochondrial gene expression in the lung was minimally perturbed but was down-regulated in the cerebellum and up-regulated in the striatum even though no SARS-CoV-2 was detected in the brain. During the mid-phase SARS-CoV-2 infection of mice, mitochondrial gene expression was starting to recover in mouse lungs. These data suggest that when the viral titer first peaks, there is a systemic host response followed by viral suppression of mitochondrial gene transcription and induction of glycolysis leading to the deployment of antiviral immune defenses. Even when the virus was cleared and lung mitochondrial function had recovered, mitochondrial function in the heart, kidney, liver, and lymph nodes remained impaired, potentially leading to severe COVID-19 pathology.

Meta-analysis of Transcriptomic Data from Lung Autopsy and Cellular Models of SARS-CoV-2 Infection.

Severe COVID-19 is a systemic disorder involving excessive inflammatory response, metabolic dysfunction, multi-organ damage, and several clinical features. Here, we performed a transcriptome meta-analysis investigating genes and molecular mechanisms related to COVID-19 severity and outcomes. First, transcriptomic data of cellular models of SARS-CoV-2 infection were compiled to understand the first response to the infection. Then, transcriptomic data from lung autopsies of patients deceased due to COVID-19 were compiled to analyze altered genes of damaged lung tissue. These analyses were followed by functional enrichment analyses and gene-phenotype association. A biological network was constructed using the disturbed genes in the lung autopsy meta-analysis. Central genes were defined considering closeness and betweenness centrality degrees. A sub-network phenotype-gene interaction analysis was performed. The meta-analysis of cellular models found genes mainly associated with cytokine signaling and other pathogen response pathways. The meta-analysis of lung autopsy tissue found genes associated with coagulopathy, lung fibrosis, multi-organ damage, and long COVID-19. Only genes DNAH9 and FAM216B were found perturbed in both meta-analyses. BLNK, FABP4, GRIA1, ATF3, TREM2, TPPP, TPPP3, FOS, ALB, JUNB, LMNA, ADRB2, PPARG, TNNC1, and EGR1 were identified as central elements among perturbed genes in lung autopsy and were found associated with several clinical features of severe COVID-19. Central elements were suggested as interesting targets to investigate the relation with features of COVID-19 severity, such as coagulopathy, lung fibrosis, and organ damage.

Evaluation of a Set of miRNAs in 26 Cases of Fatal Traumatic Brain Injuries.

In forensic medicine, identifying novel biomarkers for use as diagnostic tools to ascertain causes of death is challenging because of sample degradation. To that aim, a cohort (n = 26) of fatal traumatic brain injuries (TBIs) were tested for three candidate miRNAs (namely, miR-124-3p, miR-138-5p, and miR144-3p). For each case, three FFPE specimens (coup area (CA), contrecoup area (CCA), and the corpus callosum (CC)) were investigated, whereas the FFPE brain tissues of 45 subjects (deceased due to acute cardiovascular events) were used as controls. Relative quantification via the ∆∆Ct method returned significantly higher expression levels of the three candidate miRNAs (p < 0.01) in the TBI cases. No difference was detected in the expression levels of any miRNA investigated in the study among the CA, CCA, and CC. Furthermore, the analyzed miRNAs were unrelated to the TBI samples' post-mortem intervals (PMIs). On the contrary, has-miR-124-3p ahashsa-miR-144-3p were significantly correlated (p < 0.01) with the agonal time in TBI deaths. Since the RNA was highly degraded in autoptic FFPE tissues, it was impossible to analyze the mRNA targets of the miRNAs investigated in the present study, highlighting the necessity of standardizing pre-analytical processes even for autopsy tissues.

Smooth muscle differentiation of coronary intima in autopsy tissues after sirolimus-eluting stent implantation

BackgroundIn coronary atherosclerotic disease, the proliferation of intimal smooth muscle cells (SMCs) is regarded as beneficial with respect to stable and unstable plaques, but is thought detrimental in discussions on coronary stent restenosis. To resolve this discrepancy, we focused on the quality, not quantity, of intimal SMCs in coronary atherosclerotic disease. MethodsAutopsied coronary artery specimens from seven patients implanted with bare metal stents (BMS), three with paclitaxel-eluting stents (PES), and 10 with sirolimus (rapamycin)-eluting stents (SES) were immunostained for SMC markers. Cultured human coronary artery SMCs were also treated with sirolimus and paclitaxel. ResultsIntimal SMC differentiation, estimated by the ratio of h-caldesmon+ cells to α-smooth muscle actin+ (α-SMA+) cells, was significantly increased whereas dedifferentiation, estimated from the ratio of fibroblast activation protein alpha (FAPα)+ cells to α-SMA+ cells, was significantly decreased, in tissues of SES compared with BMS cases. No difference in the degree of differentiation was found between PES and BMS cases or between the three groups in nonstented arteries used as controls. Correlation analyses for each field of view revealed a significant positive correlation between h-caldesmon and calponin staining but significant negative correlations with FAPα staining in α-SMA+ cells. Cultured SMCs were shorter (dedifferentiated) and showed an increased FAPα/α-SMA protein when treated with paclitaxel, whereas they became elongated (differentiated) and showed increased calponin/α-SMA proteins with sirolimus. ConclusionsThe SMCs of the coronary intima may differentiate after SES implantation. SMC differentiation may explain both the plaque stabilization and reduced risk of reintervention associated with SES.

Microglia mediate neurocognitive deficits by eliminating C1q-tagged synapses in sepsis-associated encephalopathy.

Sepsis-associated encephalopathy (SAE) is a severe and frequent complication of sepsis causing delirium, coma, and long-term cognitive dysfunction. We identified microglia and C1q complement activation in hippocampal autopsy tissue of patients with sepsis and increased C1q-mediated synaptic pruning in a murine polymicrobial sepsis model. Unbiased transcriptomics of hippocampal tissue and isolated microglia derived from septic mice revealed an involvement of the innate immune system, complement activation, and up-regulation of lysosomal pathways during SAE in parallel to neuronal and synaptic damage. Microglial engulfment of C1q-tagged synapses could be prevented by stereotactic intrahippocampal injection of a specific C1q-blocking antibody. Pharmacologically targeting microglia by PLX5622, a CSF1-R inhibitor, reduced C1q levels and the number of C1q-tagged synapses, protected from neuronal damage and synapse loss, and improved neurocognitive outcome. Thus, we identified complement-dependent synaptic pruning by microglia as a crucial pathomechanism for the development of neuronal defects during SAE.

Viable SARS-CoV-2 Omicron sub-variants isolated from autopsy tissues.

Pulmonary and extrapulmonary manifestations have been described after infection with SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). The virus is known to persist in multiple organs due to its tropism for several tissues. However, previous reports were unable to provide definitive information about whether the virus is viable and transmissible. It has been hypothesized that the persisting reservoirs of SARS-CoV-2 in tissues could be one of the multiple potentially overlapping causes of long COVID. In the present study, we investigated autopsy materials obtained from 21 cadaveric donors with documented first infection or reinfection at the time of death. The cases studied included recipients of different formulations of COVID-19 vaccines. The aim was to find the presence of SARS-CoV-2 in the lungs, heart, liver, kidneys, and intestines. We used two technical approaches: the detection and quantification of viral genomic RNA using RT-qPCR, and virus infectivity using permissive in vitro Vero E6 culture. All tissues analyzed showed the presence of SARS-CoV-2 genomic RNA but at dissimilar levels ranging from 1.01 × 102 copies/mL to 1.14 × 108 copies/mL, even among those cases who had been COVID-19 vaccinated. Importantly, different amounts of replication-competent virus were detected in the culture media from the studied tissues. The highest viral load were measured in the lung (≈1.4 × 106 copies/mL) and heart (≈1.9 × 106 copies/mL) samples. Additionally, based on partial Spike gene sequences, SARS-CoV-2 characterization revealed the presence of multiple Omicron sub-variants exhibiting a high level of nucleotide and amino acid identity among them. These findings highlight that SARS-CoV-2 can spread to multiple tissue locations such as the lungs, heart, liver, kidneys, and intestines, both after primary infection and after reinfections with the Omicron variant, contributing to extending knowledge about the pathogenesis of acute infection and understanding the sequelae of clinical manifestations that are observed during post-acute COVID-19.

Axonal Transport in Coats’ Disease and Congenital Oculodentodigital Syndrome

Introduction: Axoplasmic transport blockade has been demonstrated in acute experimental glaucoma in animals but has not been documented in childhood glaucoma. The purpose of this study was to detail findings and axoplasmic transport status in one eye of a two-year old male with Coat’s disease using the two eyes from a 10-month-old female with Oculodentodigital Syndrome (ODDS) as controls for background axoplasmic transport. We document for the first-time axonal transport block in the lamina cribrosa (LC) in a blind and painful right eye associated with Coats’ disease, (Case 1). Enucleation was done for suspected retinoblastoma without imaging. Histopathology demonstrated myriad macrophages migrated from the vitreous infiltrate into the anterior chamber and meshwork explaining the glaucoma. Two eyes from Case 2, diagnosed at autopsy as having congenital Oculodentodigital Syndrome (ODDS), with a history of surgically normalized intraocular pressures were included as controls for the Coats’ case immunohistochemistry (IHC) and to report additional findings in this rare disorder. Methods: We reviewed available history and performed histopathology on one enucleated Coats’ globe and two additional autopsy eyes from an ODDS case with routine staining of paraffin-processed tissues and amyloid precursor protein (APP-A4) IHC, the only available IHC marker for orthograde axonal transport in human surgical or autopsy tissues. The two eyes from the 10-month-old infant with ODDS had normal IOPs following trabeculotomies at five months of age. Results: In addition to typical pathology for Coats’ the lamina cribrosa portion of the optic nerve from Case 1 demonstrated marked orthograde axonal transport block. Both eyes from Case 2 with ODDS had a history of congenital glaucoma treated medically before bilateral trabeculotomies at five months-age with normalization of IOPs. Descemet scrolls found OD were judged residual to goniotomy. Both globes from Case 2 demonstrated multiple anomalies previously reported in ODDS including microphthalmos, optic disc dysplasia OD, and features of persistent hyperplastic primary vitreous (PHPV) OU. The right eye also demonstrated spontaneous retinal detachment prompting an exam under general anaesthesia at 10-months age when, during induction, she expired with malignant tachycardia. Retinal ganglion cells (RCGs) labeled with APP-A4 OU, but optic nerve LC axons demonstrated only background accumulations of the IHC marker. Conclusions: Orthograde axonal transport was interrupted in the LC of the Coats’ nerve due to secondary inflammatory glaucoma. The angle remained open histologically in-spite-of-early neovascularization and pupillary margin ectropion. Block of orthograde axonal transport in the LC has been well established as the site of initial optic nerve damage in glaucoma in experimental studies and this report reinforces its importance to the pathophysiology of glaucoma optic nerve damage. The primary importance of the two ODDS globes for our report was as comparison controls for background APP-A4 staining in the Coats’ case. Our additional findings in the ODDS eyes suggest delayed spontaneous retinal detachment, and lens epithelial abnormalities are additional anomalies to add to the list of known malformations in this rare disorder. Case 2 also serves as a caution in ODDS regarding vulnerability to combinations of medications when general anaesthesia is contemplated.