Histomorphological, Histochemical and Ultrastructural Studies on the Healthy Liver of Yemen Veiled Chameleon (Chamaeleo calyptratus) in Southern Saudi Arabia / Estudios Histomorfológicos, Histoquímicos y Ultraestructurales en el Hígado Sano del Camaleón Velado de Yemen (Chamaeleo calyptratus) en el Sur de Arabia Saudita

SUMMARY: The livers of reptiles are being studied as a model for the link between the environment and hepatic tissue. There have been few investigations on the histology of reptile livers, and very few or no studies have examined the histology of liver of veiled chameleon (Chamaeleo calyptratus). This paper describes the histomorphological, histochemical and ultrastructural characterization of the liver of veiled chameleons in southern Saudi Arabia. Seven Chamaeleo calyptratus were captured in the summer season in Abha City, Aseer region, southern Saudi Arabia. Chamaeleon liver samples were processed for histomorphology, histochemistry and ultrastructure analyses. Morphologically liver of Chamaeleo calyptratus was observed as a large dark brown organ with lighter speckles, which represent melanin deposits. It located at the ventral part of abdominal cavity forward of the stomach. Its dimensions approximately were 3.7 x 2 cm. The liver was a bilobed organ divided into two lobes, right and left lobes. The right one was bigger than the others. The gallbladder was well developed and had an elongated shape, situated between the two lobes and contained the bile for the digestion. Microscopically, the liver was found to be covered by a thick layer of connective tissue, which formed the hepatic capsule. Hepatic parenchyma probably appeared in cross sections as hepatic glandular-like alveoli "acini" or follicular structures with various diameters, each acinus contains approximately four to six hepatocytes, surrounded by sinusoidal capillaries filled with abundant melanomacrophages, which are absent in birds and mammals. Melanomacrophages are common in the hepatic parenchyma's perisinusoidal areas, particularly near portal spaces. Hepatocytes are polyhedral or pyramidal with and mostly contained large, rounded nuclei mostly peripherally located, with prominent dark oval nucleoli. Some of nuclei are eccentric or central position. The cytoplasm appeared spongy or vacuolated and more eosinophilic when stained by hematoxylin-eosin and strongly reactive to PAS staining technique, indicating abundant glycogen content. The reticular fibers that surround hepatocytes, blood arteries, and sinusoids supported the hepatic parenchyma. The blood sinusoids are seen interspersed among hepatocytes of varying sizes. The sinusoidal lumen was bordered by flattened endothelial cells and includes elliptical nucleated erythrocytes and liver macrophages as phagocytes, which are also known as Kupffer cells. Branches of the portal vein, hepatic artery, small bile duct, and lymph vessels were detected in the hepatic portal area "tract" or triad which made up of connective. Hematopoietic tissue was observed in subcapsular region and portal triads. Ultrastructurally, the hepatocyte appeared polyhedric containing a single large rounded basal or eccentric vesicular nucleus with prominent nucleolus. Extensive network of rough endoplasmic reticulum (RER) often arranged in an array parallel to the nuclear membrane with many mitochondria, and Golgi apparatus were described. The cytoplasm contained glycogen granules, vesicles or vacuoles scattered throughout the cytoplasm especially at the apical region were reported. The bile canaliculi and the hepatic "Kupffer" cells were also discussed. This is the first study on the histological characterization of the healthy liver of Yemen veiled chameleon in southern Saudi Arabia. The findings reported here should be used as a reference to compare with the pathological abnormalities of the liver in this animal.

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Machine perfusion preservation with hemoglobin based oxygen vesicles alleviate ultrastructural damages in porcine liver donated after cardiac death.

Midthermic machine perfusion (MMP) of post-circulatory arrest donor liver grafts has the advantage of preserving the functional ultrastructure of hepatocytes in donor grafts. It was reported that oxygenation during MMP reduces portal venous resistance and increases bile production. The MMP with hemoglobin-based oxygen vesicles (HbV) keeps the lower aspartate aminotransferase level (an indicator of liver injury) and maintains the functional ultrastructure of mitochondria in the hepatocytes. To evaluated differences of ultrastructural damages in donor livers between the MMP with and without HbV, porcine liver grafts after 60 min of warm ischemia were perfused at 22°C for 4 h with or without HbV, and a part of liver grafts were analyzed by transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The remaining grafts were perfused with autologous blood at 38°C for 2 h in an isolated liver reperfusion model (IRM) that mimics the inside of the body after transplantation, and then analyzed by TEM and OM-SEM. Hepatocytes after MMP had small round mitochondria with rod-shaped cristae and reticulovesicular rough endoplasmic reticulum (rER) in both HbV(+) and HbV(-) livers. After IRM of HbV(+) livers, the well-developed lamellar rER was often found in hepatocytes. Liver sinusoidal endothelial cells (LSECs) after MMP contained some large vacuolar structures containing amorphous garbage in the cytoplasm, and their size along with appearance frequency were smaller and lower, respectively, in HbV(+) livers than HbV(-). Oxygenation during the MMP by using HbV suppressed the ultrastructural damages in donor livers, in particular for the LSECs. RESEARCH HIGHLIGHTS: Liver sinusoidal endothelial cells after midthermic machine perfusion had large vacuolar organelles with amorphous garbage. Oxygenation during the perfusion made them less and smaller, ultrastructurally supporting its utility.

Liver in guinea pig: Scanning and transmission electron microscopic studies.

The liver performs many functions, which support metabolism, immunity, digestion, detoxification, vitamin storage, and so on. The liver is the storage organ for fat-soluble vitamins, iron and copper. Six adult healthy guinea pigs of 16-32 weeks of age (Irrespective of sex) were procured from the Department of Laboratory Animal Medicine, TANUVAS as per ethical committee approval. Animals were dissected as per CPCSEA norms and liver pieces were utilized for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) study. The study was performed to document the ultrastructural details of liver of guinea pigs by SEM and TEM. By SEM, the liver parenchyma appeared as lobular with anastomosed one cell thick hepatic cell plates, which appeared as continuous sheets with small to large holes representing sinusoids. The sinusoidal lumen was lined by two types of cells namely endothelial cells and Kupffer cells. Endothelial cells had numerous large and small cytoplasmic fenestrations. Presence of Kupffer cells along with Ito cells were observed in the sinusoids. By TEM, hepatocytes had angular shape with sinusoidal face and bile canalicular face. Hepatocytes had oval nucleus with peripheral heterochromatin and central electron lucent granules and a prominent nucleolus. Bile canaliculi were formed by two adjacent hepatocytes. Fenestrated endothelial cells with flat and elongated shape were found to be in the formation of hepatic sinusoidal wall. A narrow space of Disse was found between the sinusoidal endothelium and hepatocytes. Kupffer cells and pit cells were found within the sinusoidal lumen. Ito cells were found in the space of Disse. RESEARCH HIGHLIGHTS: By scanning electron microscopy, the liver parenchyma appeared as lobular with anastomosed one cell thick hepatic cell plates, which appeared as continuous sheets with small to large holes representing sinusoids. By transmission electron microscopy, hepatocytes had angular shape with sinusoidal face and bile canalicular face. Hepatocytes had oval nucleus with peripheral heterochromatin and central electron lucent granules and a prominent nucleolus.

Histological and ultrastructural changes observed in testicles, epididymides, seminal vesicles and liver of rat after intraperitoneal administration of aluminum and indium.

BACKGROUND: Aluminum (Al) and indium (In) have been largely used in medicine, pharmacy, dentistry, manufacturing, engineering, clothing as well as food processing and packaging. Our previous study showed that In was accumulated as electron-dense materials in lysosomes of Sertoli and Leydig testicular cells and the liver ones, when administered to male rats as soluble form. For this reason, we have undertaken to confirm whether Al have the same behavior as In and to enlarge this behavior to other organs of the male reproductive system: epididymis and seminal vesicle. METHODS: Experiments were performed on 24 adult male Wistar rat weighing approximately 250 g. Animals were divided to 3 groups, received Al, In or saline solution as 7 chronic intraperitoneal injections over a period of two weeks and were sacrificed 24 h after the last injection. For ultrastructure study we used The Transmission Electron Microscopy (TEM). RESULTS: The TEM showed the presence of electron-dense granules in lysosomes of testicular cells (Sertoli and Leydig cells), and in the principal epididymal and seminal vesicle cells of Al and In treated rats. Impairments were observed in the endoplasmic reticulum and mitochondria and many vacuoles were identified in the cells cytoplasm. Our results concluded that lysosomes of Leydig and Sertoli cells, principal epididymis, and seminal vesicle cells as well as liver cells, played a central role in the extraction and concentration of Al and In under insoluble form after their introduction into the body as a soluble route. This mechanism intended to protect the organism against exogenous toxic and non-recognized mineral elements after their intrusion into the body. CONCLUSION: It looks important to proceed with the study of Al and In impact on the endocrine and exocrine functions of the male rat reproductive system.

Ultrastructural changes of donor livers in liver transplantation indicate hepatocytes injury.

The use of electron microscopy (EM) can provide details about cells and tissue down to the nanometer level. We aim to observe ultrastructural changes in the donor liver by EM and analyze the relationship with prognosis. Data from 89 liver transplant recipients were collected and analyzed for recovery of graft function. The results revealed significantly higher organelle injury scores in the primary liver graft nonfunction (PNF) group. High-score group had higher peak alanine aminotransferases, peak aspartate aminotransferases, and peak international normalized ratio (p = .041, .006 and .036, respectively). Warm ischemia time, score of rough endoplasmic reticulum and nucleus was larger in low-score group (p = .007, .006, and .025, respectively). Patients in high-score group had a significantly short survival time (60.0% vs. 92.9%, p = .0039). No significant difference was found in the analysis of 3-year survival rate (60% vs. 84.5%, p = .07). EM is one of feasible and effective strategy for evaluating the quality of donor liver and the patient's prognosis. Ultrastructural changes under EM indicate hepatocytes injury and a high score indicates a worse outcome in early period but does not affect long-term survival. RESEARCH HIGHLIGHTS: We showed that ultrastructural changes in the donor liver by electron microscopy indicate hepatocytes injury and could be a reference for prognosis. A high score indicates a worse outcome in early period but does not affect long-term survival. It is rare in the current researches.

Attenuation of hepatic ischemia­reperfusion injury by adipose stem cell­derived exosome treatment via ERK1/2 and GSK­3ß signaling pathways.

Exosomes are an emerging therapeutic tool for the treatment of tissue injuries. In the present study, the protective effect of isolated exosomes from adipose­derived stem cells (ADSCs­exo) against hepatic ischemia­reperfusion (I/R) injury was explored. Hepatic I/R injury was achieved by inducing ischemia for 60 min followed by reperfusion for 2 and 6 h. Pre­treatment with ADSCs­exo revealed a significant reduction in necrosis and apoptosis in liver tissue induced by I/R injury. Hypoxic oxidative stress was managed by exosome­mediated reduced reactive oxygen species and increased superoxide dismutase that in turn protected mitochondrial damage and apoptosis. Reduction in inflammatory mediators such as IL­1ß and TNF­α was also observed and protection of hepatocytes from I/R injury was evidenced by a significant decrease in biochemical markers of liver damage (alanine transaminase, aspartate transaminase and lactate dehydrogenase). Exosomal prostaglandin E2 (PGE2)­mediated ERK1/2 and GSK­3ß phosphorylation were revealed to increase Bcl­2 and decrease Bax expression with mitochondrial permeability transition pore­inhibition which may be considered a prime mechanism of exosome­mediated hepatoprotection. In conclusion, our results indicated that ADSCs­exo pre­treatment is effective in protecting liver I/R injury.

Loss of Hepatic Transcription Factor EB Attenuates Alcohol-Associated Liver Carcinogenesis.

Alcohol is a well-known risk factor for hepatocellular carcinoma. Autophagy plays a dual role in liver cancer, as it suppresses tumor initiation and promotes tumor progression. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and autophagy, which is impaired in alcohol-related liver disease. However, the role of TFEB in alcohol-associated liver carcinogenesis is unknown. Liver-specific Tfeb knockout (KO) mice and their matched wild-type (WT) littermates were injected with the carcinogen diethylnitrosamine (DEN), followed by chronic ethanol feeding. The numbers of both total and larger tumors increased significantly in DEN-treated mice fed ethanol diet than in mice fed control diet. Although the number of tumors was not different between WT and L-Tfeb KO mice fed either control or ethanol diet, the number of larger tumors was less in L-Tfeb KO mice than in WT mice. No differences were observed in liver injury, steatosis, inflammation, ductular reaction, fibrosis, and tumor cell proliferation in DEN-treated mice fed ethanol. However, the levels of glypican 3, a marker of malignant hepatocellular carcinoma, markedly decreased in DEN-treated L-Tfeb KO mice fed ethanol in comparison to the WT mice. These findings indicate that chronic ethanol feeding promotes DEN-initiated liver tumor development, which is attenuated by genetic deletion of hepatic TFEB.

Patterns of energetic substrate modifications in response to feeding in boas, Boa constrictor (Serpentes, Boidae).

Ambush-foraging snakes that ingest large meals might undergo several months without eating when they use the internal reserves to support the energetic costs of living. Then, morphological and physiological processes might be orchestrated during the transition from fasting to the postprandial period to rapidly use the energetic stores while the metabolic rate is elevated in response to food intake. To understand the patterns of substrates deposition after feeding, we accessed the morphological and biochemical response in Boa constrictor snakes after two months of fasting and six days after feeding. We followed the plasma levels of glucose, total proteins, and total lipids, and we performed the stereological ultrastructural analysis of the liver and the proximal region of the intestine to quantify glycogen granules and lipid droplets. In the same tissues and stomach, we measured the activity of the enzyme fructose-1,6-biphosphatase (FBPase1) involved in the gluconeogenic pathway, and we measured pyruvate kinase (PK) and lactate dehydrogenase (LDH) enzymatic activities involved in the anaerobic pathway in the liver. Briefly, our results indicated an increase in boas' plasma glucose one day after meal intake compared to unfed snakes. The hepatic glycogen reserves were continuously restored within days after feeding. Also, the enzymes involved in the energetic pathways increased activity six days after feeding in the liver. These findings suggest a quick restoring pattern of energetic stores during the postprandial period.

Inflammation and Liver Cell Death in Patients with Hepatitis C Viral Infection.

Hepatitis C virus (HCV)-induced liver disease contributes to chronic hepatitis. The immune factors identified in HCV include changes in the innate and adaptive immune system. The inflammatory mediators, known as "inflammasome", are a consequence of the metabolic products of cells and commensal or pathogenic bacteria and viruses. The only effective strategy to prevent disease progression is eradication of the viral infection. Immune cells play a pivotal role during liver inflammation, triggering fibrogenesis. The present paper discusses the potential role of markers in cell death and the inflammatory cascade leading to the severity of liver damage. We aim to present the clinical parameters and laboratory data in a cohort of 88 HCV-infected non-cirrhotic and 25 HCV cirrhotic patients, to determine the characteristic light microscopic (LM) and transmission electron microscopic (TEM) changes in their liver biopsies and to present the link between the severity of liver damage and the serum levels of cytokines and caspases. A matched HCV non-infected cohort was used for the comparison of serum inflammatory markers. We compared the inflammation in HCV individuals with a control group of 280 healthy individuals. We correlated the changes in inflammatory markers in different stages of the disease and the histology. We concluded that the serum levels of cytokine, chemokine, and cleaved caspase markers reveal the inflammatory status in HCV. Based upon the information provided by the changes in biomarkers the clinician can monitor the severity of HCV-induced liver damage. New oral well-tolerated treatment regimens for chronic hepatitis C patients can achieve cure rates of over 90%. Therefore, using the noninvasive biomarkers to monitor the evolution of the liver damage is an effective personalized medicine procedure to establish the severity of liver injury and its repair.

Spermidine Supplementation Protects the Liver Endothelium from Liver Damage in Mice.

Chronic liver diseases are multifactorial and the need to develop effective therapies is high. Recent studies have shown the potential of ameliorating liver disease progression through protection of the liver endothelium. Polyamine spermidine (SPD) is a caloric restriction mimetic with autophagy-enhancing properties capable of prolonging lifespan and with a proven beneficial effect in cardiovascular disease in mice and humans. We evaluated the use of dietary supplementation with SPD in two models of liver disease (CCl4 and CDAAH diet). We analyzed the effect of SPD on endothelial dysfunction in vitro and in vivo. C57BL/6J mice were supplemented with SPD in the drinking water prior and concomitantly with CCl4 and CDAAH treatments. Endothelial autophagy deficient (Atg7endo) mice were also evaluated. Liver tissue was used to evaluate the impact of SPD prophylaxis on liver damage, endothelial dysfunction, oxidative stress, mitochondrial status, inflammation and liver fibrosis. SPD improved the endothelial response to oxidative injury in vitro and improved the liver endothelial phenotype and protected against liver injury in vivo. SPD reduced the overall liver oxidative stress and improved mitochondrial fitness. The absence of benefits in the Atg7endo mice suggests an autophagy-dependent effect of SPD. This study suggests SPD diet supplementation in early phases of disease protects the liver endothelium from oxidative stress and may be an attractive approach to modify the chronic liver disease course and halt fibrosis progression.

Normothermic machine perfusion attenuates hepatic ischaemia-reperfusion injury by inhibiting CIRP-mediated oxidative stress and mitochondrial fission.

Extracellular cold-inducible RNA-binding protein (CIRP) is a proinflammatory mediator that aggravates ischaemia-reperfusion injury (IRI). Normothermic machine perfusion (NMP) could effectively alleviate the IRI of the liver, but the underlying mechanism remains to be explored. We show that human DCD livers secreted a large amount of CIRP during static cold storage (CS), which is released into the circulation after reperfusion. The expression of CIRP was related to postoperative IL-6 levels and liver function. In a rat model, the CIRP expression was upregulated during warm ischaemia and cold storage. Then, rat DCD livers were preserved using CS, hypothermic oxygenated machine perfusion (HOPE) and NMP. C23, a CIRP inhibitor, was administrated in the HOPE group. Compared with CS, NMP significantly inhibited CIRP expression and decreased oxidative stress by downregulating NADPH oxidase and upregulating UCP2. NMP markedly inhibited the mitochondrial fission-related proteins Drp-1 and Fis-1. Further, NMP increased the mitochondrial biogenesis-related protein, TFAM. NMP significantly reduced inflammatory reactions and apoptosis after reperfusion, and NMP-preserved liver tissue had higher bile secretion and ICG metabolism compared to the CS group. Moreover, C23 administration attenuated IRI in the HOPE group. Additionally, HL-7702 cells were stimulated with rhCIRP and C23. High rhCIRP levels increased oxidative stress and apoptosis. In summary, NMP attenuates the IRI of DCD liver by inhibiting CIRP-mediated oxidative stress and mitochondrial fission.

ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials.

Alterations in lipid metabolism might contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, no pharmacological agents are currently approved in the United States or the European Union for the treatment of NAFLD. Two parallel phase 2a studies investigated the effects of liver-directed ACC1/2 inhibition in adults with NAFLD. The first study ( NCT03248882 ) examined the effects of monotherapy with a novel ACC1/2 inhibitor, PF-05221304 (2, 10, 25 and 50 mg once daily (QD)), versus placebo at 16 weeks of treatment; the second study ( NCT03776175 ) investigated the effects of PF-05221304 (15 mg twice daily (BID)) co-administered with a DGAT2 inhibitor, PF-06865571 (300 mg BID), versus placebo after 6 weeks of treatment. The primary endpoint in both studies was percent change from baseline in liver fat assessed by magnetic resonance imaging-proton density fat fraction. Dose-dependent reductions in liver fat reached 50-65% with PF-05221304 monotherapy doses ≥10 mg QD; least squares mean (LSM) 80% confidence interval (CI) was -7.2 (-13.9, 0.0), -17.1 (-22.7, -11.1), -49.9 (-53.3, -46.2), -55.9 (-59.0, -52.4) and -64.8 (-67.5, -62.0) with 16 weeks placebo and PF-05221304 2, 10, 25 and 50 mg QD, respectively. The overall incidence of adverse events (AEs) did not increase with increasing PF-05221304 dose, except for a dose-dependent elevation in serum triglycerides (a known consequence of hepatic acetyl-coenzyme A carboxylase (ACC) inhibition) in 23/305 (8%) patients, leading to withdrawal in 13/305 (4%), and a dose-dependent elevation in other serum lipids. Co-administration of PF-05221304 and PF-06865571 lowered liver fat compared to placebo (placebo-adjusted LSM (90% CI) -44.6% (-54.8, -32.2)). Placebo-adjusted LSM (90% CI) reduction in liver fat was -44.5% (-55.0, -31.7) and -35.4% (-47.4, -20.7) after 6 weeks with PF-05221304 or PF-06865571 alone. AEs were reported for 10/28 (36%) patients after co-administered PF-05221304 and PF-06865571, with no discontinuations due to AEs, and the ACC inhibitor-mediated effect on serum triglycerides was mitigated, suggesting that PF-05221304 and PF-06865571 co-administration has the potential to address some of the limitations of ACC inhibition alone.

Preservation of collagen in the soft tissues of frozen mammoths.

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

Curcumin activation of nuclear factor E2-related factor 2 gene (Nrf2): Prophylactic and therapeutic effect in nonalcoholic steatohepatitis (NASH).

BACKGROUNDS: Modern dietary habits have been associated with Nonalcoholic Steatohepatitis (NASH). Curcumin is a natural herbal found to suppress cellular oxidative states and could be beneficial in NASH. This study investigates the effect of curcumin in an animal model of NASH. MATERIALS AND METHODS: Fifty rats were allocated into five groups. Control, High Fat Diet (HFD), curcumin prophylactic (CP) and therapeutic (CT) groups. HFD regimen was given for 16 weeks. Curcumin was given along with HFD (prophylactic) or after establishment of the model for two weeks (therapeutic). Livers and blood samples were harvested for histological, biochemical, and molecular studies. KEY FINDINGS: Livers from HFD groups showed vascular, inflammatory, cellular degenerative and fibrotic changes. The hepatic damage was reflected by the increased serum liver enzymes. HFD groups showed excessive fibrotic change. Interestingly, curcumin administration as prophylactic or therapeutic significantly preserved and/or restored liver structure. This was evidenced by the normalization of the liver enzymes, preservation and/or reversibility of cellular changes and the decrease of the stage of fibrosis. Nuclear factor E2-related factor 2 gene (Nrf2) expression showed no changes in the HFD groups, however it showed upregulation in curcumin treated groups. Thus, the protective and therapeutic effect of curcumin could be induced through upregulation of the Nrf2 gene. Curcumin has a beneficial prophylactic and therapeutic effect that could hinder the development and/or treat NASH in susceptible livers. SIGNIFICANCE: Curcumin has a beneficial prophylactic and therapeutic effect that could hinder the development and/or treat NASH in susceptible livers.

Role of mitochondria-endoplasmic reticulum coupling in lycopene preventing DEHP-induced hepatotoxicity.

Di (2-ethylhexyl) phthalate (DEHP) is a hazardous compound used as a plasticizer in plastic products. As a natural carotenoid, lycopene (LYC) is considered an effective protective agent against various types of organ damage. The present study aimed to investigate the role of mitochondria-endoplasmic reticulum (ER) coupling in LYC preventing DEHP-induced hepatotoxicity. The mice were treated with LYC (5 mg kg-1) and/or DEHP (500 or 1000 mg kg-1). In the present study, LYC prevented DEHP-induced histopathological changes including fibrosis and glycogen storage in the liver. Additionally, LYC alleviated DEHP-induced ultrastructural injury of mitochondria and ER. LYC had the underlying preventability against DEHP-induced mitochondrial dynamics imbalance including an increase in fission and a decrease in fusion. Furthermore, DEHP induced mitochondria-associated endoplasmic reticulum membrane (MAM) disorder-induced ER stress through the ER unfolded protein response (UPRER), but LYC alleviated these alterations. Therefore, LYC prevented DEHP-induced hepatic mitochondrial dynamics and MAM disorder, leading to ER stress. The present study provides novel evidence of mitochondria-ER coupling as a target for LYC that prevents DEHP-induced hepatotoxicity.

Ultrastructural Profile Combined with Immunohistochemistry of a Hepatic Progenitor Cell Line in Pediatric Autoimmune Hepatitis: New Insights into the Morphological Pattern of the Disease.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I-the most undifferentiated progenitor cells; III-intermediate hepatocyte-like cells; II-intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare-those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Depdc5 deficiency exacerbates alcohol-induced hepatic steatosis via suppression of PPARα pathway.

Alcohol-related liver disease (ALD), a condition caused by alcohol overconsumption, occurs in three stages of liver injury including steatosis, hepatitis, and cirrhosis. DEP domain-containing protein 5 (DEPDC5), a component of GAP activities towards Rags 1 (GATOR1) complex, is a repressor of amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In the current study, we found that aberrant activation of mTORC1 was likely attributed to the reduction of DEPDC5 in the livers of ethanol-fed mice or ALD patients. To further define the in vivo role of DEPDC5 in ALD development, we generated Depdc5 hepatocyte-specific knockout mouse model (Depdc5-LKO) in which mTORC1 pathway was constitutively activated through loss of the inhibitory effect of GATOR1. Hepatic Depdc5 ablation leads to mild hepatomegaly and liver injury and protects against diet-induced liver steatosis. In contrast, ethanol-fed Depdc5-LKO mice developed severe hepatic steatosis and inflammation. Pharmacological intervention with Torin 1 suppressed mTORC1 activity and remarkably ameliorated ethanol-induced hepatic steatosis and inflammation in both control and Depdc5-LKO mice. The pathological effect of sustained mTORC1 activity in ALD may be attributed to the suppression of peroxisome proliferator activated receptor α (PPARα), the master regulator of fatty acid oxidation in hepatocytes, because fenofibrate (PPARα agonist) treatment reverses ethanol-induced liver steatosis and inflammation in Depdc5-LKO mice. These findings provide novel insights into the in vivo role of hepatic DEPDC5 in the development of ALD.

The Recruitment-Secretory Block ("R-SB") Phenomenon and Endoplasmic Reticulum Storage Diseases.

In this article, we review the biological and clinical implication of the Recruitment-Secretory Block ("R-SB") phenomenon. The phenomenon refers to the reaction of the liver with regard to protein secretion in conditions of clinical stimulation. Our basic knowledge of the process is due to the experimental work in animal models. Under basal conditions, the protein synthesis is mainly carried out by periportal (zone 1) hepatocytes that are considered the "professional" synthesizing protein cells. Under stimulation, midlobular and centrolobular (zones 2 and 3) hepatocytes, are progressively recruited according to lobular gradients and contribute to the increase of synthesis and secretion. The block of secretion, operated by exogenous agents, causes intracellular retention of all secretory proteins. The Pi MZ phenotype of Alpha-1-antitrypsin deficiency (AATD) has turned out to be the key for in vivo studies of the reaction of the liver, as synthesis and block of secretion are concomitant. Indeed, the M fraction of AAT is stimulated for synthesis and regularly exported while the Z fraction is mostly retained within the cell. For that reason, the phenomenon has been designated "Recruitment-Secretory Block" ("R-SB"). The "R-SB" phenomenon explains why: (a) the MZ individuals can correct the serum deficiency; (b) the resulting immonohistochemical and electron microscopic (EM) patterns are very peculiar and specific for the diagnosis of the Z mutation in tissue sections in the absence of genotyping; (c) the term carrier is no longer applicable for the heterozygous condition as all Pi MZ individuals undergo storage and the storage predisposes to liver damage. The storage represents the true elementary lesion and consequently reflects the phenotype-genotype correlation; (d) the site and function of the extrahepatic AAT and the relationship between intra and extracellular AAT; (e) last but not least, the concept of Endoplasmic Reticulum Storage Disease (ERSD) and of a new disease, hereditary hypofibrinogenemia with hepatic storage (HHHS). In the light of the emerging phenomenon, described in vitro, namely that M and Z AAT can form heteropolymers within hepatocytes as well as in circulation, we have reviewed the whole clinical and experimental material collected during forty years, in order to evaluate to what extent the polymerization phenomenon occurs in vivo. The paper summarizes similarities and differences between AAT and Fibrinogen as well as between the related diseases, AATD and HHHS. Indeed, fibrinogen gamma chain mutations undergo an aggregation process within the RER of hepatocytes similar to AATD. In addition, this work has clarified the intriguing phenomenon underlying a new syndrome, hereditary hypofibrinogenemia and hypo-APO-B-lipoproteinemia with hepatic storage of fibrinogen and APO-B lipoproteins. It is hoped that these studies could contribute to future research and select strategies aimed to simultaneously correct the hepatocytic storage, thus preventing the liver damage and the plasma deficiency of the two proteins.

Characterization of perfusion decellularized whole animal body, isolated organs, and multi-organ systems for tissue engineering applications.

To expand the application of perfusion decellularization beyond isolated single organs, we used the native vasculature of adult and neonatal rats to systemically decellularize the organs of a whole animal in situ. Acellular scaffolds were generated from kidney, liver, lower limb, heart-lung system, and a whole animal body, demonstrating that perfusion decellularization technology is applicable to any perfusable tissue, independent of age. Biochemical and histological analyses demonstrated that organs and organ systems (heart-lung pair and lower limb) were successfully decellularized, retaining their extracellular matrix (ECM) structure and organ-specific composition, as evidenced by differences in organ-specific scaffold stiffness. Altogether, we demonstrated that organs, organ systems and whole animal bodies can be perfusion decellularized while retaining ECM components and biomechanics.

Characterization of an Acidic Polysaccharides from Carrot and Its Hepatoprotective Effect on Alcoholic Liver Injury in Mice.

The characteristics of acidic polysaccharides extracted from Daucus carota L. var. sativa Hoffm were investigated and its hepatoprotective effects on alcoholic liver injury were determined in the mice model. A carrot polysaccharide (CPS-I: Carrot polysaccharide-I) with the molecular weight of 3.40×104  kDa was isolated from Daucus carota L. and purified by diethylaminoethyl-52 and Sephadex G-150 column chromatography. The components were analyzed by HPLC, which revealed that CPS-I consisted of galacturonic acid, rhamnose, xylose, arabinose, fructose, and galactose at a relative ratio of 1 : 3.16 : 1.13 : 5.53 : 3.45 : 7.76. Structural characterization analysis suggested that CPS-I was mainly composed of →6)-ß-D-Galp-(1→ and →5)-α-L-Araf-(1→. The hepatoprotective effect of CPS-I was evaluated by alcoholic liver injury mice model. The results showed that the administration of CPS-I (300 mg/kg/day) alleviated the alcoholic liver injury in mice by increasing the levels of ADH and ALDH and reducing oxidative stress. CPS-I ameliorated the pathological changes of liver characterized by lipid accumulation, and reduced the number of lipid droplets.