Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis.

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development.

Exploiting ensemble learning to improve prediction of phospholipidosis inducing potential.

Phospholipidosis is characterized by the presence of excessive accumulation of phospholipids in different tissue types (lungs, liver, eyes, kidneys etc.) caused by cationic amphiphilic drugs. Electron microscopy analysis has revealed the presence of lamellar inclusion bodies as the hallmark of phospholipidosis. Some phospholipidosis causing compounds can cause tissue specific inflammatory/retrogressive changes. Reliable and accurate in silico methods could facilitate early screening of phospholipidosis inducing compounds which can subsequently speed up the pharmaceutical drug discovery pipelines. In the present work, stacking ensembles are implemented for combining a number of different base learners to develop predictive models (a total of 256 trained machine learning models were tested) for phospholipidosis inducing compounds using a wide range of molecular descriptors (ChemMine, JOELib, Open babel and RDK descriptors) and structural alerts as input features. The best model consisting of stacked ensemble of machine learning algorithms with random forest as the second level learner outperformed other base and ensemble learners. JOELib descriptors along with structural alerts performed better than the other types of descriptor sets. The best ensemble model achieved an overall accuracy of 88.23%, sensitivity of 86.27%, specificity of 90.20%, mcc of 0.765, auc of 0.896 with 88.21 g-means. To assess the robustness and stability of the best ensemble model, it is further evaluated using stratified 10×10 fold cross validation and holdout testing sets (repeated 10 times) achieving 84.83% mean accuracy with 0.708 mean mcc and 88.46% mean accuracy with 0.771 mean mcc respectively. A comparison of different meta classifiers (Generalized linear regression, Gradient boosting machines, Random forest and Deep learning neural networks) in stacking ensemble revealed that random forest is the better choice for combining multiple classification models.

Amino acid pattern in the liver and blood of fattening turkeys suffering from hepatic lipidosis.

Hepatic lipidosis (HL) is a well-known disease in fattening and in parent turkey flocks. Among others, dietary effects like (a lack of) essential amino acids (AA) as lipotrophic factors (e.g., methionine) have been considered as potentially predispositing for HL. Several studies have reported abnormal AA profiles in hepatic diseases of humans and other livestock. The ratio of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) in plasma is used to predict hepatic cirrhosis. In this study, the state of supply of AA was investigated by comparing non-affected (NA) animals and those affected by HL. The AA pattern in the liver and blood can provide potential indications of pathogenesis of HL. In cooperation with German poultry veterinarians, 3 cases of HL on 3 different fattening turkey farms were visited (13/14 wk old, "B.U.T. Big 6" and "TP7"). Overall, 73 birds were examined, of which 42 birds suffered from HL and 31 were not affected. Feeding samples of the respective actual feed were taken and analyzed. The selection of animals was carried out (NA randomly) by clinical signs such as apathy and dyspnea and the diagnosis was made at necropsy, which could be confirmed by crude fat content in liver tissue (HL: 309, NA: 155). In liver tissue, the CP and AA contents were lower among animals with HL than among NA (P < 0.05). In blood samples, the sum of AA, ammonia, and urea was more than 3 times higher among animals with HL (431 mg/dL serum) than among NA (114 mg/dL serum; P < 0.01). The ratio of BCAA to AAA was also significantly different between the groups (HL: 0.85, NA: 1.42; P < 0.05). In the case of HL, entire herds were not affected and the "non-affected" ones were comparable with healthy slaughtered animals. There seems to be a clear change in protein and AA metabolism of HL animals, which could lead to an optimization in feeding practice in repeated cases of HL.

PEPCK-C reexpression in the liver counters neonatal hypoglycemia in Pck1 del/del mice, unmasking role in non-gluconeogenic tissues.

Whole body cytosolic phosphoenolpyruvate carboxykinase knockout (PEPCK-C KO) mice die early after birth with profound hypoglycemia therefore masking the role of PEPCK-C in adult, non-gluconeogenic tissues where it is expressed. To investigate whether PEPCK-C deletion in the liver was critically responsible for the hypoglycemic phenotype, we reexpress this enzyme in the liver of PEPCK-C KO pups by early postnatal administration of PEPCK-C-expressing adenovirus. This maneuver was sufficient to partially rescue hypoglycemia and allow the pups to survive and identifies the liver as a critical organ, and hypoglycemia as the critical pathomechanism, leading to early postnatal death in the whole-body PEPCK-C knockout mice. Pathology assessment of survivors also suggest a possible role for PEPCK-C in lung maturation and muscle metabolism.

Hepatic Lipidosis in a Research Colony of Big Brown Bats (Eptesicus fuscus).

During a nearby construction project, a sudden decrease in food intake and guano production occurred in an outdoor colony of big brown bats (Eptesicus fuscus), and one animal was found dead. Investigation revealed that the project was generating a large amount of noise and vibration, which disturbed the bats' feeding. Consequently the bats were moved into an indoor enclosure away from the construction noises, and the colony resumed eating. Over the next 3 wk, additional animals presented with clinical signs of lethargy, weight loss, ecchymoses, and icterus and were necropsied. Gross necropsy of the affected bats revealed large, pale yellow to tan, friable livers with rounded edges that floated when placed in 10% neutral-buffered formalin. Some bats had ecchymoses on the webbing and skin and gross perirenal hemorrhage. Histologic examination showed hepatic and renal tubular lipidosis. The clinical and pathologic signs of hemorrhage and icterus were suggestive of hepatic failure. Hepatic lipidosis was attributed to stress and inappetence associated with environmental perturbations. Once the environmental stressor was removed, the colony morbidity and mortality decreased. However, 2 y later, a series of new environmental stressors triggered additional deaths associated with hepatic lipidosis. Over a 9-y period, 21 cases of hepatic lipidosis were diagnosed in this bat colony.

Apolipoprotein E deficiency and high-fat diet cooperate to trigger lipidosis and inflammation in the lung via the toll-like receptor 4 pathway.

Apolipoprotein E deficiency (ApoE(-/-)) combined with a high-fat Western-type diet (WD) is known to activate the toll-like receptor (TLR4) pathway and promote atherosclerosis. However, to date, the pathogenic effects of these conditions on the lung have not been extensively studied. Therefore, the present study examined the effects of ApoE(-/-) and a WD on lung injury and investigated the underlying mechanisms. ApoE(-/-) and wild-type mice were fed a WD or normal chow diet for 4, 12 and 24 weeks. Lung inflammation, lung cholesterol content and cytokines profiles in broncho-alveolar lavage fluid (BALF) were determined. TLR4 and its main downstream molecules were analyzed with western blot analysis. In addition, the role of the TLR4 pathway was further validated using TLR4-targeted gene silencing. The results showed that ApoE(-/-) mice developed lung lipidosis following 12 weeks of receiving a WD, as evidenced by an increased lung cholesterol content. Moreover, dependent on the time period of receiving the diet, those mice exhibited pulmonary inflammation, which was manifested by initial leukocyte recruitment (at 4 weeks), by increased alveolar septal thickness and mean linear intercept as well as elevated production of inflammation mediators (at 12 weeks), and by granuloma formation (at 24 weeks). The expression levels of TLR4, myeloid differentiation primary response 88 (MyD88) and nuclear factor kappa B were markedly upregulated in ApoE(-/-) WD mice at week 12. However, these effects were ameliorated by shRNA-mediated knockdown of TLR4. By contrast, ApoE(-/-) ND or wild-type WD mice exhibited low-grade or no inflammation and mild lipidosis. The levels of TLR4 and MyD88 in those mice showed only minor changes. In conclusion, ApoE deficiency acts synergistically with a WD to trigger lung lipidosis and inflammation at least in part via TLR4 signaling.

Exudación lipídica masiva y desprendimiento de retina tras braquiterapia y termoterapia transpupilar combinada en melanoma de coroides / Massive lipid exudation and retinal detachment after combined brachytherapy and transpupillary thermotherapy in choroidal melanoma

Caso clínico: Varón de 59 años con melanoma coroideo en ojo izquierdo. Se realiza tratamiento con braquiterapia mediante placa (iodo-125), apareciendo exudación masiva, desprendimiento de retina y grandes placas de depósitos lipídicos una semana después. Se monitoriza la evolución mediante funduscopia y ecografía mensualmente y una vez reabsorbido el fluido subretiniano se realiza termoterapia transpupilar (TTT) de la masa tumoral irradiada, 9 meses tras la intervención. Tres meses después se producen roturas retinianas con siembra vítrea que hacen necesaria la enucleación. Discusión: La terapia combinada con placas de braquiterapia y TTT puede asociar complicaciones severas que requieran la enucleación (AU)

Case report: A 59 year-old male with choroidal melanoma in the left eye who underwent plaque brachytherapy (iodine 125). One week after surgery, massive exudation with retinal detachment and lipid exudation was observed. Evolution was assessed with funduscopy and ultrasound every month. Nine months after surgery transpupillary thermotherapy (TTT) was performed over the fluid-free irradiated residual tumour. Three months after this procedure, new retinal breaks appeared in the treated area with vitreous seeding that required enucleation. Discussion: Combined treatment with plaque brachytherapy and TTT may associate severe complications that may require enucleation of the involved eye (AU)

Pathological changes associated with white striping in broiler breast muscles.

White striping is a condition in broiler chickens characterized grossly by the occurrence of white striations, seen parallel to the direction of muscle fibers, on broiler breast fillets and thighs. Based on visual evaluation of the intensity of white striping, breast fillets can be categorized into normal (NORM), moderate (MOD), and severe (SEV) categories. This study was undertaken to evaluate the details of changes in histology as well as proximate composition occurring in the fillets with respect to the 3 degrees of white striping. In experiment 1, representative breast fillets for each degree of white striping (n = 20) were collected from 45-d-old broilers, approximately 2 h postmortem. From each fillet, 2 skeletal muscle samples were obtained and fixed in 10% neutral buffered formalin. To identify and differentiate the histological changes, slides were prepared and stained using hematoxylin and eosin, Masson's Trichrome, and Oil Red O stains. In experiment 2, samples with 3 degrees of white striping were collected from 57-d-old birds for conducting proximate analysis. Major histopathological changes observed in the MOD and SEV samples consisted of loss of cross striations, variability in fiber size, floccular/vacuolar degeneration and lysis of fibers, mild mineralization, occasional regeneration (nuclear rowing and multinucleated cells), mononuclear cell infiltration, lipidosis, and interstitial inflammation and fibrosis. Microscopic lesions were visually scored for degeneration and necrosis, fibrosis, and lipidosis. The scale used to score the samples ranged from 0 (normal) to 3 (severe). There was an increase (P < 0.05) in mean scores for degenerative or necrotic lesions, fibrosis, and lipidosis as the degree of white striping increased from NORM to SEV. The results from the histopathological study were supported by the findings from proximate analysis confirming that the fat and protein contents of muscle increased (P < 0.05) and decreased (P < 0.05), respectively, as the degree of white striping increased. In conclusion, the histopathological changes occurring in white striping indicate a degenerative myopathy that could be associated with increased growth rate in birds.

[Pathogenesis of lipid storage diseases]. / Patogeneza lipidowych chorób spichrzeniowych.

Lipidoses are rare genetic disorders characterized by defects of the digestive system that impair the way the body uses dietary fat. When the body is unable to properly digest fats, lipids such as cholesterol, sphingolipids or glycolipids may accumulate in body tissues in abnormal amounts. It has been also suggested that molecular mechanisms leading to development of human diseases, including obesity, diabetes type II and atherosclerosis, consist of impaired transport and storage of lipids, as well as disturbed structure and function of lipid membrane microdomains. In this review we discuss probable mechanisms, including role of lipid membrane microdomains, which may participate in pathogenesis of lipid storage diseases such as Niemann-Pick type A/B and type C, as well as Gaucher type I diseases.

Glomerular basement membrane lipidosis in Alagille syndrome.

Alagille syndrome is characterized by a paucity of interlobular bile ducts with chronic cholestasis, cardiac, skeletal, and eye abnormalities and is associated predominantly with JAG1 mutations. Various renal abnormalities have been sporadically described. The classic renal histopathology described in Alagille syndrome is mesangiolipidosis, with lipid deposits predominately confined to the mesangium and minimal deposition within the glomerular basement membrane (GBM). We report a 5-year-old girl with Alagille syndrome who presented with persistent subnephrotic proteinuria and renal tubular acidosis. A renal biopsy showed GBM irregularities (mimicking membranous glomerulonephritis), mesangial sclerosis, and focal segmental glomerulosclerosis (FSGS) on light microscopy. Electron microscopy revealed few lipid inclusions within the mesangium but extensive inclusions along the GBM. These findings are mostly consistent with those reported previously in Alagille syndrome. However, the histologic distribution of lipid vacuoles is seemingly reversed in this patient and is uniquely accompanied by FSGS, emphasizing the spectrum of renal histopathology seen in Alagille syndrome. The proteinuria observed in this patient is likely attributed to significant GBM lipid deposition, which over time may contribute to the development of FSGS.

Screening for the drug-phospholipid interaction: correlation to phospholipidosis.

Phospholipid bilayers represent a complex, anisotropic environment fundamentally different from bulk oil or octanol, for instance. Even "simple" drug association to phospholipid bilayers can only be fully understood if the slab-of-hydrocarbon approach is abandoned and the complex, anisotropic properties of lipid bilayers reflecting the chemical structures and organization of the constituent phospholipids are considered. The interactions of drugs with phospholipids are important in various processes, such as drug absorption, tissue distribution, and subcellular distribution. In addition, drug-lipid interactions may lead to changes in lipid-dependent protein activities, and further, to functional and morphological changes in cells, a prominent example being the phospholipidosis (PLD) induced by cationic amphiphilic drugs. Herein we briefly review drug-lipid interactions in general and the significance of these interactions in PLD in particular. We also focus on a potential causal connection between drug-induced PLD and steatohepatitis, which is induced by some cationic amphiphilic drugs.

Hepatic lipidosis in a black-headed python (Aspidites melanocephalus).

The number of people keeping reptiles in captivity has markedly increased, and widespread inappropriate husbandry will result in an increasing incidence of the diagnosis of hepatic lipidosis(HL). It may become apparent that some species of snake have predisposition to develop HL. In the specific instance of the genus Aspidites, alterations to the usual husbandry that limit feeding and thereby body weight should markedly lessen the chance of the animal's developing HL. It is important for clinicians to develop a more aggressive approach to these cases and to build a bank of data with which to promote understanding of this common malady.

Drug-induced phospholipidosis: issues and future directions.

Numerous drugs containing a cationic amphiphilic structure are capable of inducing phospholipidosis in cells under conditions of in vivo administration or ex vivo incubation. The principal characteristics of this condition include the reversible accumulation of polar phospholipids in association with the development of unicentric or multicentric lamellated bodies within cells. There is an abundance of data providing an understanding of potential mechanisms for the induction of phospholipidosis; however, the process is likely to be complex and may differ from one drug to another. The functional consequences of the presence of this condition on cellular or tissue function are not well understood. The general consensus is that the condition is an adaptive response rather than a toxicological manifestation; however, additional studies to examine this question are needed. Until this issue is resolved, concerns about phospholipidosis will continue to exist at regulatory agencies. Procedures for the screening of potential phospholipogenic candidate compounds are available. In contrast, a clear need exists for the identification of valid biomarkers to assess the development of phospholipidosis in preclinical and clinical studies.

Rare infiltrative lung diseases: a challenge for clinicians.

Rare diffuse infiltrative lung diseases are a challenge for clinicians, radiologists, and pathologists for at least three reasons: (a) their low incidence and prevalence hamper the acquisition of expertise and frequently the diagnosis is delayed; (b) therapeutic actions are mainly empirical and based on steroid use, and (c) pathogenetic events are difficult to explain and only recently new therapeutic measures taking advantage of innovative genetic and/or immunopathogenetic studies have been suggested. In this review rare diffuse lung disorders are briefly discussed (pulmonary alveolar proteinosis, inherited lipidoses, acute eosinophilic pneumonia, amyloidosis, pulmonary ossification, pulmonary alveolar microlithiasis). The list is obviously not exhaustive and arbitrarily chosen. The intent is, however, to emphasize that in this difficult field multidisciplinary expertise and the knowledge of the most recent pathogenetic mechanisms have the main role in diagnosis and treatment.