Postmortem Liver Research Articles

The des-Arg9-bradykinin/B1R axis: Hepatic damage in COVID-19.

Patients infected by the SARS-CoV-2 virus are commonly diagnosed with threatening liver conditions associated with drug-induced therapies and systemic viral action. RNA-Seq data from cells in bronchoalveolar lavage fluid from COVID-19 patients have pointed out dysregulation of kallikrein-kinin and renin-angiotensin systems as a possible mechanism that triggers multi-organ damage away from the leading site of virus infection. Therefore, we measured the plasma concentration of biologically active peptides from the kallikrein-kinin system, bradykinin and des-Arg9-bradykinin, and liver expression of its proinflammatory axis, bradykinin 1 receptor (B1R). We measured the plasma concentration of bradykinin and des-Arg9-bradykinin of 20 virologically confirmed COVID-19 patients using a liquid chromatography-tandem mass spectrometry-based methodology. The expression of B1R was evaluated by immunohistochemistry from post-mortem liver specimens of 27 COVID-19 individuals. We found a significantly higher blood level of des-Arg9-bradykinin and a lower bradykinin concentration in patients with COVID-19 compared to a healthy, uninfected control group. We also observed increased B1R expression levels in hepatic tissues of patients with COVID-19 under all hepatic injuries analyzed (liver congestion, portal vein dilation, steatosis, and ischemic necrosis). Our data indicate that des-Arg9-bradykinin/B1R is associated with the acute hepatic dysfunction induced by the SARS-CoV-2 virus infection in the pathogenesis of COVID-19.

Integration of Liver Glycogen and Triglyceride NMR Isotopomer Analyses Provides a Comprehensive Coverage of Hepatic Glucose and Fructose Metabolism.

Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive measurement of this process starting from their conversion to sugar phosphates, involvement of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products, remains incomplete. Mice were fed a chow diet supplemented with 35 g/100 mL drinking water of a 55/45 fructose/glucose mixture for 18 weeks. On the final night, the sugar mixture was enriched with either [U-13C]glucose or [U-13C]fructose, and deuterated water (2H2O) was also administered. 13C-isotopomers representing newly synthesized hepatic glucose-6-phosphate (glucose-6-P), glycerol-3-phosphate, and lipogenic acetyl-CoA were quantified by 2H and 13C NMR analysis of post-mortem liver glycogen and triglyceride. These data were applied to a metabolic model covering glucose-6-P, PPP, triose-P, and de novo lipogenesis (DNL) fluxes. The glucose supplement was converted to glucose-6-P via the direct pathway, while the fructose supplement was metabolized by the liver to gluconeogenic triose-P via fructokinase-aldolase-triokinase. Glucose-6-P from all carbohydrate sources accounted for 40-60% of lipogenic acetyl-CoA and 10-12% was oxidized by the pentose phosphate pathway (PPP). The yield of NADPH from PPP flux accounted for a minority (~30%) of the total DNL requirement. In conclusion, this approach integrates measurements of glucose-6-P, PPP, and DNL fluxes to provide a holistic and informative assessment of hepatic glucose and fructose metabolism.

Impact of the SARS-CoV-2 Pandemic on Liver Surgery and Liver Transplantations in Germany

The SARS-CoV-2 pandemic has led to restrictions in surgical care worldwide and therefore also posed new challenges to liver surgery. The respective procedures often entail high perioperative risks and resource requirements. However, the indication for liver surgery is frequently without alternatives. To date, there is little knowledge about the impact of the pandemic on liver surgery in Germany. A retrospective data analysis of liver surgery procedures in Germany as well as transplantations was conducted. Evaluations were based on procedure codes recorded between 2010 and 2020 according to diagnosis-related groups (DRG) by the Federal Statistical Office of Germany (Destatis) and data from the German Organ Procurement Organization (Deutsche Stiftung Organtransplantation; DSO). According to DRG procedure codes relating to liver surgery recorded between 2010 and 2020 in Germany, the annual fluctuation for the first year of the pandemic 2020 remained comparable to previous years. Furthermore, the development of post-mortem liver transplantations as well as living liver donations remained stable in Germany in 2020 and 2021. The number of liver surgery procedures in Germany was subject to a dynamic development until 2020, without apparent changes in the first year of the SARS-CoV-2 pandemic. The most frequently performed liver procedures, as well as liver transplantations, remained stable with respect to their annually recorded numbers. Publication of data regarding procedures in liver surgery and transplantation in 2021 need to be awaited and analyzed to evaluate whether the observations presented in this article prove stable any further.

Abstract 785: Multi-omics profiling identifies genetic and epigenetic regulation underlying alcohol induced liver fibrosis in a porcine hepatocellular carcinoma model

Abstract Background: Cirrhosis is a form of end-stage liver disease which manifests with a host of metabolic and hematologic derangements, and is commonly the result of long-term ethanol exposure. Large animal models of cirrhosis, and of one of its common long-term sequelae, hepatocellular carcinoma (HCC), are needed to study novel and emerging therapeutic interventions. Methods: Liver fibrosis was induced in the Oncopig cancer model, a large animal model of HCC, via intrahepatic, intra-arterial ethanol infusion. Post-mortem liver sections from five fibrosis induction and five age-matched controls were harvested for RNA-seq (mRNA and lncRNA), small RNA-seq (miRNA), and reduced representation bisulfite sequencing (RRBS; DNA methylation). Supervised, multi-omic analysis of the 5,000 genes and methylated regions demonstrating the largest inter-sample variance with non-zero counts along with all sequenced miRNA were analyzed using the DIABLO method within the mixOmics 6.8.5 R library. Results: Single component DIABLO latent variable analysis of mRNAs, lncRNAs, miRNAs, and methylated regions was performed to identify the genetic elements (variables) which most reliably discriminated between experimental arms. Analysis of the top 100 discriminatory variables from each ‘omics data set was performed to provide sufficient data for pathway analysis while maintaining separation of the experimental arms within the latent space. IPA pathway analysis of these genes revealed differential regulation of pathways related to hepatic fibrosis, adipogenesis, ketogenesis as well as several other liver-specific metabolic pathways. Identification of the five most discriminatory variables was also performed. Single component, five variable analysis revealed ELOVL2, LIPK, and LAMA3 most reliably differentiated control and fibrosis groups, along with two lncRNA transcripts, XLOC035153 and XLOC036409 which were predicted to target S1PR1 and UHRF1BP1L, respectively. LAMA3, XLOC036409, and its target UHRF1BP1L were overexpressed in Oncopig fibrotic liver samples. UHRF1, a potential binding target for UHRF1BP1L, has previously been implicated as an oncogene for hepatocellular carcinoma. S1PR1 is involved in vasculogenesis and cancer cell motility via the RAC-CDC42 and ERK pathways. Conclusions The differentially regulated genetic elements identified by the multi-omic analyses were largely concordant with known alterations in human alcoholic liver disease. This agreement lends validity to the Oncopig model as a model organism for human alcoholic liver disease. In addition, novel genes, UHRF1BP1L and S1PR1, were associated with porcine liver fibrosis and further work to characterize their precise mechanistic roles in the natural history of this disease is required. Citation Format: Mark Hieromnimon, Lawrence Schook, Ron Gaba, Kyle Schachtschneider. Multi-omics profiling identifies genetic and epigenetic regulation underlying alcohol induced liver fibrosis in a porcine hepatocellular carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 785.

Safety and Feasibility of Contrast-Enhanced Computed Tomography with a Nanoparticle Contrast Agent for Evaluation of Diethylnitrosamine-Induced Liver Tumors in a Rat Model

Safety and feasibility of contrast-enhanced computed tomography (CECT) with a nanoparticulate contrast agent, ExiTron nano 12000, was evaluated in a rat liver tumor model. This study employed eighteen 8-week-old male F344 rats. Six rats given tap water for 8 weeks further divided into two: Control group and Normal Liver with CECT group. Six rats each were given tap water containing diethylnitrosamine (DEN) at 100 ppm for 8 or 14 weeks; Adenoma group and Hepatocellular carcinoma (HCC) group, respectively. Biochemical marker values and adverse events were evaluated after CT imaging. ExiTron nano 12000 was evaluated for the hepatic contrast enhancement, and the detection and measurement of liver nodules by CECT after 8- and 14-weeks administration of DEN. Post-mortem liver specimens were evaluated by hematoxylin-eosin (HE) staining, and the number and size of liver nodules were measured. The HCC group was evaluated for diagnostic concordance between HE-stained and CECT-detected nodules. The contrast agent enhanced liver and was tolerated after CECT in 15 rats. Biochemical parameter values did not differ significantly between the Control and Normal Liver groups. The numbers of CECT-detected nodules in the Adenoma and HCC groups were 14.8 ± 5.1, and 32.4 ± 8.1, respectively. The HCC group had 3.6 ± 2.7 of pathological HCCs, which were identified by CECT. The size of CECT-detected HCCs correlated significantly with that of pathological HCCs (r = 0.966, p < 0.0001). CECT with ExiTron nano 12000 is a safe and feasible method to measure tumors in a rat liver tumor model.

Post-mortem liver and bone marrow iron quantification in haemodialysis patients: A prospective cohort study.

SummaryBackgroundMagnetic resonance liver scans indicate that iron overload is common in haemodialysis (HD) patients. However, histological evidence is scarce.MethodsLiver biopsy and bone marrow aspirate were obtained in the first 24h post mortem from 21 adult HD patients. Biochemical liver iron content (LIC) was quantified by electrothermal atomization atomic absorption spectrophotometry. Tissue iron deposition was graded in the liver and bone marrow using Scheuer and Gale's criteria, respectively.FindingsMedian LIC was 42.5 (22.9-69.7) μmol/g and the majority (n=11; 57%) had mild to moderate liver iron overload (LIC >36 μmol/g). Scheuer grade was 2 (1-3) and 13 (62%) of liver biopsies had increased (> 1) iron deposition. In the bone marrow, median Gale's grade was 3 (3-4) and 9 (45%) patients had increased (>3) iron content.Contrary to old autopsy studies, done in the pre-erythropoiesis-stimulating agents (ESAs) era, both liver and bone marrow were iron replete and showed a positive correlation (r=0.71, p<0.001).Ferritin proved to have a good diagnostic accuracy for liver iron overload (0.87 95% CI 0.71-1.00) with an optimal cut-off value of 422 ng/ml. Haemoglobin was negatively associated with both LIC (r= -0.46, p=0.04) and iron content in the bone marrow (p=0.04). Patients with increased LIC had higher resistance to ESAs (p=0.02), yet no association with previous IV iron therapy.InterpretationIn the majority of HD patients there was iron accumulation in both the liver and bone marrow that associated with anaemia severity and resistance to ESAs, suggesting a blocking mechanism of iron's utilization.FundingNone.

Genetic resistance to Campylobacter coli and Campylobacter jejuni in wild boar (Sus scrofa L.)

We studied the genetic resistance to Campylobacter coli and Campylobacter jejuni in wild boar using both STR analysis and genome-wide association studies (GWAS). A total of 60 wild boars hunted in Tuscany (Italy) during the 2018/2019 hunting season were analyzed and genotyped. During postmortem operations, fecal swabs, liver samples and kidneys were collected. Two groups of animals were considered for the statistical analysis: 28 Campylobacter positive (22 for C. coli and 6 for C. jejuni) and 32 Campylobacter negative. Regarding STR analysis, 15 markers belonging to a marker panel validated by the International Society of Animal Genetics (ISAG) for swine were used: for each marker, alleles and genotype frequencies between the two groups of animals were compared using the Chi-square test and Fisher’s exact tests. To analyze the genetic variability within groups, the following parameters were computed: molecular coancestry coefficients (fij), kinship distance (Dk), inbreeding coefficient (Fi), and genetic similarities (GS). The internal relatedness (IR) was also calculated, and ANOVA was used to verify the relationships between IR and Campylobacter groups. For GWAS, the Geneseek Genomic Profiler Porcine HD (70 k), containing 62,330 SNPs, was used. No differences in the internal relatedness (IR) were observed between the two groups (F = 5.64, P = 0.065) and no significant association between STRs and SNPs and Campylobacter positivity was observed. Although genetic resistance to bacterial diseases is often regulated by multiple genes controlling different processes of the host–pathogen interaction, in our studies no candidate genes that could be directly or indirectly involved in the development of the disease were identified.

Odd chain fatty acid metabolism in mice after a high fat diet.

Epidemiological studies show that higher circulating levels of odd chain saturated fatty acids (FA: C15:0 and C17:0) are associated with lower risk of metabolic disease. These odd chain saturated fatty acids (OCSFA) are produced by α-oxidation in peroxisomes, de novo lipogenesis, from the diet and by gut microbiota. Although present at low concentrations, they are of interest as potential targets to reduce metabolic disease risk. To determine whether OCSFA are affected by obesogenic diets, we have investigated whether high dietary fat intake affects the frequency of OCSFA-producing gut microbiota, liver lipid metabolism genes and circulating OCSFA. FA concentrations were determined in liver and serum from pathogen-free SPF C57BL/6 J mice fed either standard chow or a high fat diet (HFD; 60% calories as fat) for four and twelve weeks. Post-mortem mouse livers were analysed histologically for fat deposition by gas chromatography–mass spectrometry for FA composition and by qPCR for the lipid metabolic genes fatty acid desaturase 2 (FADS2), stearoyl CoA desaturase 1 (SCD1), elongation of long-chain fatty acids family member 6 (ELOVL6) and 2-hydroxyacyl-CoA lyase 1 (HACL). Gut microbiota in faecal pellets from the ileum were analysed by 16S RNA sequencing. A significant depletion of serum and liver C15:0 (>50%; P < 0.05) and liver C17:0 (>35%; P < 0.05) was observed in HFD-fed SPF mice in parallel with hepatic fat accumulation after four weeks. In addition, liver gene expression (HACL1, ELOVL6, SCD1 and FADS2) was lower (>50%; P < 0.05) and the relative abundance of beneficial C3:0-producing gut bacteria such as Akkermansia, Lactobacillus, Bifidobacterium was lower after HFD in SPF mice. In summary, high dietary fat intake reduces serum and liver OCSFA, OCSFA-producing gut microbiota and is associated with impaired liver lipid metabolism. Further studies are required to identify whether there is any beneficial effect of OCSFA and C3:0-producing gut bacteria to counter metabolic disease.

Postmortem Liver Pathology Findings In Patients With Covid-19: A Systematic Review

The coronavirus disease 2019 (COVID-19) pandemic quickly became a major public health challenge with extra-pulmonary manifestations, including liver damage. Postmortem examination is crucial for gaining a better understanding of these manifestations and improving the management of patients. This study summarizes the current knowledge of the postmortem liver pathology of patients with COVID-19. This review was conducted according on studies evaluating the postmortem macroscopic and microscopic findings of liver in patients with COVID-19 by searching four electronic databases (PubMed, Scopus, Google Scholar and Web of Science) until June 2021. From the 317 screened articles, 16 articles examining a total of 332 patients who had died with COVID-19 were selected. The major findings of liver were moderate macro and micro vesicular steatosis with mild sinusoidal dilation, active lobular and portal vein thrombosis, mildly-increased lymphocyte filtration in sinusoidal space and multifocal hepatic necrosis. Also, the most common comorbidities were hypertension and other metabolic diseases. In conclusion, liver damage due to COVID-19 infection occurs with various manifestations in patients who have died with COVID-19. Therefore, monitoring liver function during the course and treatment of this disease is necessary for the better management of the patients and to decrease the mortality rate due to COVID-19.

A case series of drug induced liver injury leading to ACLF after complementary & alternative medicine over-use for COVID-19

Background and Aims: ACLF is a condition in which 2 insults to liver operate simultaneously, 1 being chronic, and other acute. Complementary & alternative medicine (CAM) are important causes for ACLF. India is the birth place of Ayurveda & CAM is considered safe by the common population with around 80% of the population relying on it. CAM consumption has increased in recent years. Due to the pandemic and the focus on improved immunity, the consumption of CAM has gone up. India has reported 4.8 lakh COVID 19 related deaths till December 2021. However, WHO has estimated 4.7 million deaths directly or indirectly related to COVID-19. We documented a case series of CAM related DILI-ACLF, with CAM being consumed for COVID prevention Methods: ACLF established with APASL defining criteria. USG was done to assess for features of CLD. Liver biopsy was done where feasible. Results: Case 1-39-year-old diabetic taking Giloy Kwath for 2 months for COVID prevention. Presenting with jaundice & ascites having MELD score 18 and CTP class B, he had NASH related cirrhosis on biopsy and is still on follow up. Case 2- f/u/c of CTP A alcoholic cirrhosis who consumed a crushed herb for protection against COVID given by a quack for 3 months, with no alcohol intake in 2 years. He presented with jaundice and encephelopathy, had MELD score 38 & CTP C & succumbed to illness. Case 3 49-year-old lady consuming Giloy Kwath for 4 months for COVID prevention. She was diagnosed with AIH type 1 with MELD score 39. She succumbed to illness with post-mortem liver biopsy showed features of AIH cirrhosis Conclusion: CAM is the most common cause of drug induced ACLF. CAM consumption increased during the pandemic and may have lead to increase in indirect COVID related deaths

Postmortem Liver Pathology Findings in Patients With COVID-19: A Systematic Review

Background: The Coronavirus Disease 2019 (COVID-19) pandemic promptly became a significant public health challenge with extra-pulmonary manifestations, including liver damage. Postmortem examination is crucial for gaining a better understanding of these manifestations and improving patient management. This study summarized the current knowledge of the postmortem liver pathology of patients with COVID-19. Methods: This review was conducted on studies evaluating the postmortem macroscopic and microscopic findings of the liver in patients with COVID-19. Accordingly, we searched 4 electronic databases (PubMed, Scopus, Google Scholar, &amp; Web of Science) until June 2021. From the 317 screened articles, 16 articles examining a total of 332 patients who had died due to COVID-19 were selected. Results: The significant findings of the liver were moderate macro and microvesicular steatosis with mild sinusoidal dilation, active lobular and portal vein thrombosis, mildly-increased lymphocyte filtration in sinusoidal space, and multifocal hepatic necrosis. Additionally, the most common comorbidities were hypertension and other metabolic diseases. In conclusion, liver damage due to COVID-19 infection has various manifestations in patients who have expired due to COVID-19. Conclusion: Therefore, monitoring liver function during the course and treatment of this disease is necessary for better patient management and to decrease the COVID-19-induced mortality rate COVID.

Postmortem mitochondrial membrane permeability transition assessment of apoptotic cell death in brain and liver of insulin resistant, ovariectomised rats.

The adverse alterations in mitochondrial functions can affect neuronal function negatively, as they play a crucial role in neuronal plasticity and death. Direct measurements of mitochondrial activity, including membrane potential and ATP production, are not easily achieved in post-mortem brain and liver samples because most organ functions cease to work after death; in fact, with increasing post-mortem intervals (PMI), the brain and liver tissues deteriorate rapidly. Standard procedures of mitochondria isolation, protein determination expressed as BSA equivalent, and spectrophotometric assessment of pore opening at 540 nm were employed. Our results showed that (a) intact mitochondria may be isolated from rat brain and liver of these rats after storage in animal body (in situ) at −20 °C for 7 days (168 h, post-mortem), (b) some population of these mitochondria can still take up exogenous Ca2+ and (c) they can still resist osmotically induced large amplitude swelling in a suitable buffer. The need for mitochondrial purity, structural integrity and abundance for functional studies are common hindrances that can encumber mitochondrial research. Therefore, this study is significant to have shown that PMI up to 7 days did not extensively, diminish MMPT pore status in normal and diabetic, ovariectomised rats. This can be relevant for forensic data mining.

Pathological characteristics of liver sinusoidal thrombosis in COVID-19 patients: A series of 43 cases.

AimCoronavirus disease (COVID‐19) is characterized by pneumonia with secondary damage to multiple organs including the liver. Liver injury (elevated alanine aminotransferase [ALT] and aspartate aminotransferase [AST]) often correlates with disease severity in COVID‐19 patients. The aim of this study is to identify pathological microthrombi in COVID‐19 patient livers by correlating their morphology with liver injury, and examine hyperfibrinogenemia and von Willebrand factor (vWF) as mechanisms of their formation.MethodsForty‐three post‐mortem liver biopsy samples from COVID‐19 patients were obtained from Papa Giovanni XXIII Hospital in Bergamo, Italy. Three morphological features of microthrombosis (sinusoidal erythrocyte aggregation [SEA], platelet microthrombi [PMT], and fibrous thrombi) were evaluated.ResultsWe found liver sinusoidal microthrombosis in 23 COVID‐19 patients (53%) was associated with a higher serum ALT and AST level compared to those without (ALT: 10‐fold, p = 0.04; AST: 11‐fold, p = 0.08). Of 43 livers, PMT and SEA were observed in 14 (33%) and 19 (44%) cases, respectively. Fibrous thrombi were not observed. Platelet microthrombi were associated with increased ALT (p < 0.01), whereas SEA was not (p = 0.73). In COVID‐19 livers, strong vWF staining in liver sinusoidal endothelial cells was associated with significantly increased platelet adhesion (1.7‐fold, p = 0.0016), compared to those with weak sinusoidal vWF (2‐fold, p < 0.0001). Sinusoidal erythrocyte aggregation in 19 (83%) liver samples was mainly seen in zone 2. Livers with SEA had significantly higher fibrinogen (1.6‐fold, p = 0.031) compared to those without SEA in COVID‐19 patients.ConclusionsLiver PMT is a pathologically important thrombosis associated with liver injury in COVID‐19, while SEA is a unique morphological feature of COVID‐19 patient livers. Sinusoidal vWF and hyperfibrinogenemia could contribute to PMT and SEA formation.

Dietary Selenium Requirement for the Prevention of Glucose Intolerance and Insulin Resistance in Middle-Aged Mice.

Although dietary selenium (Se) deficiency or excess induces type 2 diabetes-like symptoms in mice, suboptimal body Se status usually causes no symptoms but may promote age-related decline in overall health. We sought to determine the dietary Se requirement for protection against type 2 diabetes-like symptoms in mice. Thirty mature (aged 4 mo) male C57BL/6J mice were fed a Se-deficient torula yeast AIN-93M diet supplemented with Na2SeO4 in graded concentrations totaling 0.01 (basal), 0.04, 0.07, 0.10, and 0.13 (control) mg Se/kg for 4 mo (n=6) until they were middle-aged (8 mo). Droplets of whole blood were used to determine glucose tolerance and insulin sensitivity in the mice from ages 5 to 8 mo. Postmortem serum, liver, and skeletal muscle were collected to assay for selenoprotein expression and markers of glucose metabolism. Data were analyzed by 1-way ANCOVA with or without random effects for time-repeated measurements using live mice or postmortem samples, respectively. Compared with control, the consumption of basal diet increased (P<0.05) fasting serum insulin (95% CI: 52%, 182%) and leptin (95% CI: 103%, 118%) concentrations in middle-aged mice. Dietary Se insufficiency decreased (P<0.05) 1) glucose tolerance (13-79%) and insulin sensitivity (15-65%) at ≤0.10 mg Se/kg; 2) baseline thymoma viral proto-oncogene phosphorylation on S473 (27-54%) and T308 (22-46%) at ≤0.10 and ≤0.07 mg Se/kg, respectively, in the muscle but not the liver; and 3) serum glutathione peroxidase 3 (51-83%), liver and muscle glutathione peroxidase 1 (32-84%), serum and liver selenoprotein P (28-42%), and liver and muscle selenoprotein H (39-48%) and selenoprotein W (16-73%) protein concentrations at ≤0.04, ≤0.10, ≤0.07, and ≤0.10 mg Se/kg, respectively. Mice fed diets containing ≤0.10 mg Se/kg display impaired glucose tolerance and insulin sensitivity, suggesting increased susceptibility to type 2 diabetes by suboptimal Se status at levels ≤23% of nutritional needs.

Accumulation of alpha-synuclein within the liver, potential role in the clearance of brain pathology associated with Parkinson\u2019s disease

Alpha-synuclein (α-syn) aggregation is the hallmark pathological lesion in brains of patients with Parkinson’s disease (PD) and related neurological disorders characterized as synucleinopathies. Accumulating evidence now indicates that α-syn deposition is also present within the gut and other peripheral organs outside the central nervous system (CNS). In the current study, we demonstrate for the first time that α-syn pathology also accumulates within the liver, the main organ responsible for substance clearance and detoxification. We further demonstrate that cultured human hepatocytes readily internalize oligomeric α-syn assemblies mediated, at least in part, by the gap junction protein connexin-32 (Cx32). Moreover, we identified a time-dependent accumulation of α-syn within the liver of three different transgenic (tg) mouse models expressing human α-syn under CNS-specific promoters, despite the lack of α-syn mRNA expression within the liver. Such a brain-to-liver transmission route could be further corroborated by detection of α-syn pathology within the liver of wild type mice one month after a single striatal α-syn injection. In contrast to the synucleinopathy models, aged mice modeling AD rarely show any amyloid-beta (Aß) deposition within the liver. In human post-mortem liver tissue, we identified cases with neuropathologically confirmed α-syn pathology containing α-syn within hepatocellular structures to a higher degree (75%) than control subjects without α-syn accumulation in the brain (57%). Our results reveal that α-syn accumulates within the liver and may be derived from the brain or other peripheral sources. Collectively, our findings indicate that the liver may play a role in the clearance and detoxification of pathological proteins in PD and related synucleinopathies.

Heroin Fatality in a Feline: A Case Report with Postmortem Liver Concentrations

A case of feline intoxication and fatality with the illicit drug heroin is described. A 5-year-old castrated male domestic shorthair cat was recently diagnosed with an active pneumonitis and left at home for a couple of days under the care of another resident. Upon return, the owner found his cat dead with strong suspicion of foul play. The cat was necropsied by a local veterinary clinic to retrieve the liver for diagnostic toxicology. The postmortem liver sample screened positive for 6-acetylmorphine and 6-acetylcodeine by gas chromatography mass spectrometry. Deconvolution techniques were applied to chromatograms, which revealed the additional presence of morphine and mirtazapine. Subsequent quantitation of mirtazapine, heroin, morphine, 6-acetylmorphine and 6-acetylcodeine was performed by gas chromatography--tandem quadrupole mass spectrometry. Although companion animal fatalities arising from toxicities are a likely consequence of drug abuse in a home, this is the first reported case of a malicious feline fatality resulting from heroin with quantitation of heroin metabolites.

Quantitation of Fentanyl and Metabolites from Liver Tissue Using a Validated QuEChERS Extraction and LC-MS-MS Analysis.

According to the National Institute on Drug Abuse (NIDA), more than one hundred people die every day from opioid overdose. Overdose fatalities have risen as the availability of potent synthetic opioids, such as fentanyl, has increased. A forensic postmortem toxicological specimen is often in various stages of decomposition, experiencing autolysis and putrefaction, which complicates the extraction, creating a difficult challenge for toxicologists. Isolating the target drug, while creating an efficient and simplified analytical scheme, is a goal for most toxicology laboratories. The validation of a quick, easy, cheap, effective, rugged and safe extraction protocol is presented in this study as an alternative analytical method for efficient extraction and detection of fentanyl and its major metabolites: norfentanyl and despropionyl fentanyl (4-ANPP). The liquid Chromatography with tandem mass spectrometry analysis was validated following the American Academy of Forensic Sciences Standards Board (ASB) standard 036 proposed requirements. Evaluated parameters include selectivity, matrix effects (MEs), linearity, processed sample stability, bias, precision and proof of applicability using liver samples from authentic postmortem cases. MEs (represented as percent ionization suppression or enhancement) at low and high concentrations were -10.0% and 1.4% for fentanyl, -2.1% and -0.3% for 4-ANPP and 3.1% and 2.8% for norfentanyl, respectively. Bias for the three analytes ranged from -8.5% to -19.9% for the low concentrations, -3.6% to -14.7% for the medium concentrations and 1.5% to -16.1% for the high concentrations with all being within the ±20% guideline. Precision for the three analytes ranged from 2.2% to 15.1%. The linear range for the fentanyl and norfentanyl was 0.5-100 and 4-ANPP had a linear range of 0.4-80μg/kg. The authentic postmortem liver samples ranged in fentanyl concentrations from 56.6 to 462.3μg/kg with a mean of 149.2μg/kg (n=10). The range of norfentanyl concentrations were 1.9 to 50.0μg/kg with a mean of 14.1μg/kg (n=10). The range of 4-ANPP concentrations were 3.2 to 23.7μg/kg with a mean of 7.5μg/kg (n=7).

Imaging Mass Cytometric Analysis of Postmortem Tissues Reveals Dysregulated Immune Cell and Cytokine Responses in Multiple Organs of COVID-19 Patients.

SARS-coronavirus-2–induced immune dysregulation and inflammatory responses are involved in the pathogenesis of coronavirus disease-2019 (COVID-19). However, very little is known about immune cell and cytokine alterations in specific organs of COVID-19 patients. Here, we evaluated immune cells and cytokines in postmortem tissues, i.e., lungs, intestine, liver, kidneys, and spleen of three patients with COVID-19. Imaging mass cytometry revealed monocyte, macrophage, and dendritic cell (DC) infiltration in the lung, intestine, kidney, and liver tissues. Moreover, in patients with COVID-19, natural killer T cells infiltrated the liver, lungs, and intestine, whereas B cells infiltrated the kidneys, lungs, and intestine. CD11b+ macrophages and CD11c+ DCs also infiltrated the lungs and intestine, a phenomenon that was accompanied by overproduction of the immunosuppressive cytokine interleukin (IL)-10. However, CD11b+ macrophages and CD11c+ DCs in the lungs or intestine of COVID-19 patients did not express human leukocyte antigen DR isotype. In contrast, tumor necrosis factor (TNF)-α expression was higher in the lungs, intestine, liver, and kidneys, but not in the spleen, of all COVID-19 patients (compared to levels in controls). Collectively, these findings suggested that IL-10 and TNF-α as immunosuppressive and pro-inflammatory agents, respectively,—might be prognostic and could serve as therapeutic targets for COVID-19.

Method Validation of Seven Synthetic Cathinones by LC-MS/MS Analysis and the Prevalence of N-Ethylpentylone in Postmortem Casework.

N-ethylpentylone (NEP, ephylone, bk-EBDP) was the most prevalent synthetic cathinone detected by the Miami-Dade Medical Examiner Toxicology Laboratory from 2016-2018. There is limited information regarding the toxicity of NEP, however the few published reports suggest that NEP can cause serious toxic effects and sudden death. The purpose of this publication is to describe a validated LC-MS/MS method for seven synthetic cathinones (methylone, ethylone, butylone, dibutylone, α-PVP, pentylone, and NEP) and to present a detailed summary regarding the presence of NEP in postmortem casework at the Miami-Dade Medical Examiner Department. Post-mortem iliac blood, serum, liver, and brain specimens were prepared by solid-phase extraction with analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS). Analyte linearity was established from 0.01 to 0.5 mg/L on a six-point calibration curve. A total of 101 NEP quantitations were performed using this method. Concentrations in postmortem case samples ranged from 0.01 mg/L to 2.7 mg/L. Iliac blood concentrations averaged 0.312 mg/L with a median of 0.137 mg/L (n=72) across all causes and manners of death. Approximately half of the cases were homicides in which the decedent was the victim of gunshot wounds or stabbing. Two of the three highest concentration cases of NEP (2.7 mg/L and 1.7 mg/L) involved 38-year old white males who were tasered by police prior to death. The psychostimulant effect of NEP may result in an excited delirium and/or hallucinogenic state. The concentration of NEP detected in accidental intoxication and polydrug cases overlapped with those attributed to other causes, including homicides and police involved deaths.

Pathological findings in the postmortem liver of patients with coronavirus disease 2019 (COVID-19)

Although coronavirus disease 2019 (COVID-19) is transmitted via respiratory droplets, there are multiple gastrointestinal and hepatic manifestations of the disease, including abnormal liver-associated enzymes. However, there are not many published articles on the pathological findings in the liver of patients with COVID-19. We collected the clinical data from 17 autopsy cases of patients with COVID-19 including age, sex, Body mass index (BMI), liver function test (alanine aminotransaminase (ALT), aspartate aminotransaminase (AST), alkaline phosphatase (ALP), direct bilirubin, and total bilirubin), D-dimer, and anticoagulation treatment. We examined histopathologic findings in postmortem hepatic tissue, immunohistochemical (IHC) staining with antibody against COVID-19 spike protein, CD68 and CD61, and electron microscopy. We counted the number of megakaryocytes in liver sections from these COVID-19–positive cases. Abnormal liver-associated enzymes were observed in 12 of 17 cases of COVID-19 infection. With the exception of three cases that had not been tested for D-dimer, all 14 patients’ D-dimer levels were increased, including the cases that received varied doses of anticoagulation treatment. Microscopically, the major findings were widespread platelet-fibrin microthrombi, steatosis, histiocytic hyperplasia in the portal tract, mild lobular inflammation, ischemic-type hepatic necrosis, and zone 3 hemorrhage. Rare megakaryocytes were found in sinusoids. COVID-19 IHC demonstrates positive staining of the histiocytes in the portal tract. Under electron microscopy, histiocyte proliferation is present in the portal tract containing lipid droplets, lysosomes, dilated ribosomal endoplasmic reticulum, microvesicular bodies, and coronavirus. The characteristic findings in the liver of patients with COVID-19 include numerous amounts of platelet-fibrin microthrombi, as well as various degrees of steatosis and histiocytic hyperplasia in the portal tract. Possible mechanisms are also discussed.