Reactive metabolite of trovafloxacin activates inflammasomes: Implications for trovafloxacin-induced liver injury.

Trovafloxacin is a quinolone antibiotic drug with broad-spectrum activity, which was withdrawn from a global market relatively soon after approval because of serious liver injury. The characteristics of trovafloxacin-induced liver injury are consistent with an idiosyncratic reaction; however, the details of the mechanism have not been elucidated. We examined whether trovafloxacin induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested ciprofloxacin, levofloxacin, gatifloxacin, and grepafloxacin for their ability to activate inflammasomes. Drug bioactivation was performed with human hepatocarcinoma functional liver cell-4 (FLC-4) cells, and THP-1 cells (human monocyte cell line) were used for the detection of inflammasome activation. The supernatant from the incubation of trovafloxacin with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells that had been incubated with trovafloxacin, heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and inflammasome activation in THP-1 cells by the FLC-4 supernatant. These results suggest that reactive metabolites of trovafloxacin can cause the release of DAMPs from hepatocytes that can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by trovafloxacin, which, in some patients, can cause immune-related liver injury.

Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis.

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO2 concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.

Streamlining robotic-assisted abdominoperineal resection.

BACKGROUND: Robot-assisted surgery has proven to be a safe and feasible approach for the management of rectal cancer, including abdominoperineal resection (APR). However, it often incurs longer operative times and higher costs. This study aimed to overcome these limitations by adopting a synchronous approach utilizing an optimized team composition. METHODS: Data on patients who underwent robot-assisted APR at our facility between June 2022 and June 2023 were analyzed. The key points of the optimized approach included the following: At the start of the surgery, the surgeon performed an anococcygeal ligament resection from the perineal side while the bedside assistants set up the ports. Then, through console manipulation, the presacral fascia, elevated by previously placed gauze, was easily and safely incised, providing access to the perineal region. RESULTS: A total of nine patients were included in this study. The median operation time was 231 min, and the intraoperative blood loss was 170 ml. The operation time was reduced to 167.5 min, and the blood loss was 80.5 ml in cases without a trainee. Surgical site infections, classified as Clavien-Dindo grade II complications, were observed in two cases, but no obvious urinary or erectile dysfunction was observed. CONCLUSION: The study results indicate that the challenges associated with APR can be efficiently addressed without requiring additional personnel by streamlining team composition and the synchronous approach. This optimization strategy minimizes the need for a larger surgical team, while maximizing the utilization of surgical time and resources.

Decreased plasma acetaminophen glucuronide/acetaminophen concentration ratio warns the onset of acetaminophen-induced liver injury.

Acetaminophen (APAP)-induced liver injury (AILI) is the most common cause of acute liver failure. Although the mechanisms that trigger AILI are well known, it is less understood how to halt AILI progression and facilitate liver recovery. Therefore, it is necessary to understand the pathophysiology of APAP hepatotoxicity in patients and to examine predictive/preventive markers. In a clinical study, we had a case in which aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels increased in a patient with a low ratio of APAP glucuronide concentration (AP-G)/APAP plasma concentration. Then a reverse translational study was conducted for clarifying this clinical question. The relationship between plasma AP-G/APAP concentration ratio and the levels of AST and ALT was examined by in vivo and in vitro experiments. In in vivo experiments, 10-week-old rats showed lower UGT activity, lower AP-G/APAP concentration ratios, and higher AST and ALT levels than 5-week-old rats. This suggests an inverse correlation between the AP-G/APAP concentration ratio and the AST, ALT levels in APAP-treated rats. Furthermore, as a result of the in vitro experiment, it was confirmed that the cell viability decreased when the AP-G/APAP concentration ratio in the culture medium decreased. Since the decrease in the plasma AP-G/APAP concentration ratio appears earlier than the increase of AST and ALT levels, the ratio might be a presymptomatic marker of AILI. When APAP is used for a long time, it is recommended to perform therapeutic drug monitoring of the AP-G/APAP concentration ratio, which is a predictive/preventive marker of AILI.

Amiodarone, Unlike Dronedarone, Activates Inflammasomes via Its Reactive Metabolites: Implications for Amiodarone Adverse Reactions.

Amiodarone is a benzofuran derivative used to treat arrhythmias, but its use is limited by adverse reactions. There is evidence that some of the severe adverse reactions such as liver injury and interstitial lung disease are immune-mediated; however, details of the mechanism have not been elucidated. We tested the ability of amiodarone to induce the release of danger-associated molecular patterns (DAMPs) that activate inflammasomes. Human hepatocarcinoma functional liver cell-4 (FLC-4) cells were used for drug bioactivation, and the detection of inflammasome activation was performed with the human macrophage cell line, THP-1 cells. Amiodarone is known to be oxidized to reactive quinone metabolites. The supernatant from the incubation of amiodarone with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells with amiodarone, the heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and activation of THP-1 inflammasomes by the FLC-4 supernatant. These results suggested that the reactive quinone metabolites of amiodarone can cause the release of DAMPs from hepatocytes which can activate inflammasomes. Dronedarone, a safer analog of amiodarone, did not activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by amiodarone, which in some patients, can cause immune-related adverse events. In addition, our data suggest that drugs that block the effects or the formation of IL-1ß would provide better treatment of amiodarone-induced immune-related adverse reactions.

Rivaroxaban Attenuates Right Ventricular Remodeling in Rats with Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition that frequently results in right ventricular (RV) remodeling. The objectives of this study are to investigate effects of rivaroxaban on RV remodeling in a rat model of PAH, created with Sugen5416 and chronic hypoxia, and the in vitro effects of rivaroxaban on human cardiac microvascular endothelial cells (HCMECs). To create the PAH model, male Sprague-Dawley rats were subcutaneously injected with Sugen5416 (20 mg/kg) and exposed to 2 weeks of hypoxia (10% O2), followed by 2 weeks of exposure to normoxia. The animals were then divided into 2 groups with or without administration of rivaroxaban (12 mg/kg/d) for a further 4 weeks. HCMECs were cultured under hypoxic conditions (37 °C, 1% O2, 5% CO2) with Sugen5416 and with or without rivaroxaban. In the model rats, RV systolic pressure and Fulton index increased by hypoxia with Sugen5416 were significantly decreased when treated with rivaroxaban. In HCMECs, hypoxia with Sugen5416 increased the expression of protease-activated receptor-2 (PAR-2) and the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor-kappa B (NF-κB), while treatment with rivaroxaban significantly suppressed the expression of these proteins. Rivaroxaban attenuated RV remodeling in a rat model of PAH by reducing ERK, JNK and NF-κB activation. Rivaroxaban has the possibility of providing additive effects on RV remodeling in patients with PAH.

Prognostic significance of ß2-adrenergic receptor expression in patients with surgically resected colorectal cancer.

PURPOSE: The ß2-adrenergic receptor (ß2AR) is highly expressed in various human cancers. The prognostic significance of its expression in patients with colorectal cancer (CRC) remains unclear. The aim of this study was to assess the prognostic role of ß2AR expression in patients with surgically resected CRC. METHODS: One hundred and forty-seven patients with surgically resected CRC were examined using immunohistochemistry. The expression of ß2AR was assessed in the specimens of resected primary tumors. RESULTS: ß2AR was expressed in 52.3% of the patients' tumors. ß2AR expression was significantly associated with T factor, N factor, and tumor cell proliferation (Ki-67 labeling index). Univariate analysis demonstrated that T factor, N factor, tumor stage, lymphatic permeation, vascular invasion, perineural invasion, ß2AR expression, and Ki-67 labeling index were significant prognostic factors for worse disease-free survival (DFS); all but T factor were also significant predictors for worse overall survival (OS). Multivariate analysis confirmed that expression of ß2AR was a significant prognostic marker for predicting worse DFS and OS. CONCLUSION: ß2AR expression was identified as a significant independent prognostic factor in patients with surgically resected CRC.

The 2-Hydroxyiminostilbene Metabolite of Carbamazepine or the Supernatant from Incubation of Hepatocytes with Carbamazepine Activates Inflammasomes: Implications for Carbamazepine-Induced Hypersensitivity Reactions.

Although the pathophysiology of carbamazepine-induced idiosyncratic or hypersensitivity reactions is unclear, they are presumed to be immune mediated, involving a complex interaction between drug metabolism and activation of the immune system. Cell stress can be caused by reactive metabolites, and this has the potential to release damage-associated molecular patterns (DAMPs), which are responsible for activation of the immune system. Idiosyncratic drug reactions occur mainly in the liver because of its role in drug metabolism and reactive metabolite formation. DAMPs can activate inflammasomes, which may be a common mechanism by which DAMPs lead to an immune response. In the present study, we investigated whether carbamazepine induces the release of DAMPs by using human hepatocarcinoma functional liver cell-4 (FLC-4) cells for bioactivation of carbamazepine. THP-1 cells, a human macrophage cell line, were used for detecting inflammasome activation. We found that increased caspase-1 activity and production of interleukin-1ß by THP-1 cells were caused by the supernatant from the incubation of carbamazepine with FLC-4 cells. In the supernatant, heat shock protein 60 was significantly increased. In addition, 2-hydroxyiminostilbene, which is a metabolite of carbamazepine, activated inflammasomes. These results suggest that the reactive iminoquinone metabolite can directly activate inflammasomes or that stressed hepatocytes cause the release of DAMPs, which are responsible for inflammasome activation. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients. SIGNIFICANCE STATEMENT: A metabolite of carbamazepine, 2-hydroxyiminostilbene itself, and the damage-associated molecular patterns released from hepatocytes incubated with carbamazepine activated inflammasomes. The activation of inflammasomes may be an important step in the immune system activation by carbamazepine, which can lead to hypersensitivity reactions in some patients.

Interaction of platinum agents, cisplatin, carboplatin and oxaliplatin against albumin in vivo rats and in vitro study using inductively coupled plasma-mass spectrometory.

The protein binding rates (PBR) of platinum-containing agents cisplatin (CDDP), carboplatin (CBDCA) and oxaliplatin (L-OHP) have been reported as 98%, 25-50% and 98%, respectively. To investigate the protein-binding properties of albumin with cisplatin, carboplatin and oxaliplatin, inductively coupled plasma mass spectrometry (ICP-MS) was used to measure their plasma concentration in rats over time. The study also examined the effects of cisplatin, carboplatin and oxaliplatin-binding on albumin in vitro, using CD spectrometry and native-polyacrylamide gel electrophoresis (native PAGE). The ratios of PBR to irreversible PBR, of cisplatin and oxaliplatin were 98%:98% and 90%:87%, respectively, indicating a higher affinity for irreversible binding with albumin. That of carboplatin was 25%:10%, indicating 60-70% reversible binding with albumin. The plasma protein binding rate concentrations of cisplatin, carboplatin and oxaliplatin after in vivo administration were 96%, 15% and 80%, respectively. The CD spectrometry of albumin was unaffected by cisplatin, carboplatin and oxaliplatin binding. Though similar protein binding rates were observed with oxaliplatin and cisplatin, oxaliplatin had a higher mobility rate during PAGE. It was confirmed that the binding of cisplatin and oxaliplatin with albumin affected its electric charge but not the structure. In conclusion, cisplatin and oxaliplatin bind irreversibly with albumin in plasma and may irreversibly interact with tissue protein and/or DNA. The difficulties involved with predicting the tissue concentrations of cisplatin and oxaliplatin from their plasma concentration inhibits their therapeutic drug monitoring. On the contrary, carboplatin, like some generic drugs, reversibly binds to plasma proteins. It is, therefore, possible to conduct therapeutic drug monitoring for carboplatin.

The 1st step initiation essential for allergen-specific IgE antibody production upon the 2nd step: Induction of non-specific IgE+ small B cells containing secondly-sensitized allergen-specific ones in mice firstly-sensitized with an allergen.

There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.

Factor Xa inhibition by rivaroxaban attenuates cardiac remodeling due to intermittent hypoxia.

Patients with obstructive sleep apnea (OSA) have a high prevalence of atrial fibrillation (AF). Rivaroxaban, a coagulation factor Xa inhibitor, has recently been reported to show pleiotropic effects. This study investigated the influence of rivaroxaban on cardiac remodeling caused by intermittent hypoxia (IH). Male C57BL/6J mice were exposed to IH (repeated cycles of 5% oxygen for 1.5 min followed by 21% oxygen for 5 min) for 28 days with/without rivaroxaban (12 mg/kg/day) or FSLLRY, a protease-activated receptor (PAR)-2 antagonist (10 µg/kg/day). IH caused endothelial cell degeneration in the small arteries of the right atrial myocardium and increased the level of %fibrosis and 4-hydroxy-2-nonenal protein adducts in the left ventricular myocardium. IH also increased the expression of PAR-2 as well as the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and nuclear factor-kappa B (NF-κB) were increased in human cardiac microvascular endothelial cells. However, rivaroxaban and FSLLRY significantly suppressed these changes. These findings demonstrate that rivaroxaban attenuates both atrial and ventricular remodeling induced by IH through the prevention of oxidative stress and fibrosis by suppressing the activation of ERK and NF-κB pathways via PAR-2. Treatment with rivaroxaban could potentially become a novel therapeutic strategy for cardiac remodeling in patients with OSA and AF.

Contributions of caspase-8 and -9 to liver injury from CYP2E1-produced metabolites of halogenated hydrocarbons.

1. Drug-induced liver injury is difficult to predict at the pre-clinical stage. This study aimed to clarify the roles of caspase-8 and -9 in CYP2E1 metabolite-induced liver injury in both rats and cell cultures in vitro treated with carbon tetrachloride (CCl4), halothane or sevoflurane. The human hepatocarcinoma functional liver cell line was maintained in 3-dimensional culture alone or in co-culture with human acute monocytic leukemia cells. 2. In vivo, laboratory indices of liver dysfunction and histology were normal after administration of sevoflurane. CCl4 treatment increased blood AST/ALT levels, liver caspase-3 and -9 activities and liver malondialdehyde, accompanied by centrilobular hepatocyte necrosis. Halothane increased AST/ALT levels, caspase-3 and -8 activities (but not malondialdehyde) concomitant with widespread hepatotoxicity. In vitro, CCl4 treatment increased caspase-9 activity and decreased both mitochondrial membrane potential (MMP) and cell viability. In co-culture, halothane increased caspase-8 activity and decreased MMP and cellular viability. There were no toxic responses in CYP2E1 knockdown in monoculture and co-culture. 3. CYP2E1-inducing compounds play a pivotal role in halogenated hydrocarbon toxicity. 4. Changes in hepatocyte caspase-8 and -9 activities could be novel biomarkers of metabolites causing DILI, and in pre-clinical development of new pharmaceuticals can predict nascent DILI in the clinical stage.

Supernatant from Hepatocyte Cultures with Drugs That Cause Idiosyncratic Liver Injury Activates Macrophage Inflammasomes.

There is increasing evidence that most idiosyncratic drug-induced liver injury (IDILI) is immune mediated, and in most cases, reactive metabolites appear to be responsible for the induction of this immune response. Reactive metabolites can cause cell damage with the release of damage-associated molecular patterns (DAMPs), which is thought to be involved in immune activation. Presumably, the reason that the liver is a common target of idiosyncratic drug reactions is because it is the major site of drug metabolism and reactive metabolite formation. Inflammasomes can be activated by DAMPs, and this may be a common mechanism by which DAMPs initiate an immune response. In this study, we tested the ability of drugs to induce the release of DAMPs that activate inflammasomes. The drugs tested were amodiaquine and nevirapine; both are associated with significant incidences of severe IDILI. The hepatocytes were a human hepatocarcinoma functional liver cell-4 (FLC-4) cell line. For the detection of inflammasome activation, we used the human macrophage cell line, THP-1 cells. We found that the supernatant from the incubation of both drugs with FLC-4 cells for 7 days led to increased caspase-1 activity and production of IL-1ß by THP-1 cells. However, amodiaquine alone also directly activated THP-1 cells. This is presumably because the myeloperoxidase in THP-1 cells can bioactivate amodiaquine to a reactive metabolite. In contrast, nevirapine requires cytochromes P450 for reactive metabolite formation and therefore required incubation with hepatocytes. These results support the hypothesis that reactive metabolites of drugs can cause the release of DAMPs, which in turn can activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by drugs, which in some patients can lead to IDILI. Our in vitro model is simple and convenient for evaluating inflammasome activation, and this may be a method to screen drugs for IDILI risk.

A Novel Analytical Method of Cisplatin Using the HPLC with a Naphthylethyl Group Bonded with Silica Gel (πNAP) Column.

Cisplatin is the most widely used anticancer drug in the world. Mono-chloro and none-chloro complexes of cisplatin may be believed to be the activated compounds. The separation of these compounds using octa decyl silyl column or aminopropylsilyl silica gel column is difficult because of high-reactivity and structural similarity. In this study, cisplatin, hydroxo complexes, and OH-dimer were determined by HPLC using a naphthylethyl group bonded with silica gel (πNAP) column. The analytical conditions of HPLC were as follows: analytical column, πNAP column; wave length, 225 nm; column temperature, 40°C; mobile phase, 0.1 M sodium perchlorate, acetonitrile, and perchloric acid (290 : 10 : 3), flow rate, 1.0 mL/min. Sample (20 µL) was injected onto the HPLC system. Retention time of cisplatin, mono-chloride, OH-dimer, and none-chloride was 3.2, 3.4, 3.6, and, 4.3-6.6 min, respectively. Measurable ranges with this method were 1×10-5 to 4×10-3 M for cisplatin. Correlation coefficient of the calibration curves of cisplatin was 0.999 (p<0.01). The within- and between-day variations of coefficient of variation (CV) were 5% or lower. In this study, injectable formulations in physiological saline solution, water for injection, 5% glucose solution, and 7% sodium bicarbonate precisely were measured the stability and compositional changes upon mixing by πNAP column rather than C18 column. We successfully determined cisplatin, hydroxo complexes, and OH-dimer by HPLC using a πNAP column. Thus the measurement of cisplatin (cis-diamminedichloro-platinum(II), cis-[PtCl2(NH3)2]) (CDDP) should be done using a πNAP column rather than a C18 column or aminopropylsilyl silica gel column.

In vivo heat-stimulus-triggered osteogenesis.

Several studies have reported that heat stress stimulates the activity of osteoblastic cells in vitro. However, few have addressed the effects of heat stress on osteogenesis in vivo, nor have the optimal temperatures for bone formation been determined. The aim of the present study was to investigate the effects of hyperthermia treatment on osteogenesis in a rat tibial defect model. Forty-four Sprague Dawley rats were divided into two groups with or without hyperthermia treatment. A 3-mm circular defect in the proximal tibia filled with magnetite cationic liposomes embedded in alginate beads was subjected to hyperthermia treatment (43-46 °C). Radiological assessment at 2 weeks after the treatment showed that significantly stimulated osteogenesis was observed in the hyperthermia group as compared to the control group (p = 0.003). Histomorphometrical analysis at 2 weeks revealed a significant increase of newly formed bone in the hyperthermia group, compared with the control group (p < 0.001). Area of newly formed bone in each hyperthermia group was significantly increased as compared with the control group (43 °C; p = 0.005, 44 °C; p = 0.019, 45 °C; p = 0.003, and 46 °C; p = 0.003, respectively). Alkaline phosphatase was overexpressed at the surfaces of newly formed bone adjacent to magnetite cationic liposome implantation. Our results demonstrate for the first time that heat stimulus accelerates osteogenesis in vivo, and may thus be of interest as a novel and promising tool to induce osteogenesis clinically as well.

Hydrogen gas attenuates embryonic gene expression and prevents left ventricular remodeling induced by intermittent hypoxia in cardiomyopathic hamsters.

The prevalence of sleep apnea is very high in patients with heart failure (HF). The aims of this study were to investigate the influence of intermittent hypoxia (IH) on the failing heart and to evaluate the antioxidant effect of hydrogen gas. Normal male Syrian hamsters (n = 22) and cardiomyopathic (CM) hamsters (n = 33) were exposed to IH (repeated cycles of 1.5 min of 5% oxygen and 5 min of 21% oxygen for 8 h during the daytime) or normoxia for 14 days. Hydrogen gas (3.05 vol/100 vol) was inhaled by some CM hamsters during hypoxia. IH increased the ratio of early diastolic mitral inflow velocity to mitral annulus velocity (E/e', 21.8 vs. 16.9) but did not affect the LV ejection fraction (EF) in normal Syrian hamsters. However, IH increased E/e' (29.4 vs. 21.5) and significantly decreased the EF (37.2 vs. 47.2%) in CM hamsters. IH also increased the cardiomyocyte cross-sectional area (672 vs. 443 µm(2)) and interstitial fibrosis (29.9 vs. 9.6%), along with elevation of oxidative stress and superoxide production in the left ventricular (LV) myocardium. Furthermore, IH significantly increased the expression of brain natriuretic peptide, ß-myosin heavy chain, c-fos, and c-jun mRNA in CM hamsters. Hydrogen gas inhalation significantly decreased both oxidative stress and embryonic gene expression, thus preserving cardiac function in CM hamsters. In conclusion, IH accelerated LV remodeling in CM hamsters, at least partly by increasing oxidative stress in the failing heart. These findings might explain the poor prognosis of patients with HF and sleep apnea.

Characterization of time-course morphological features for efficient prediction of osteogenic potential in human mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hBMSCs) represents one of the most frequently applied cell sources for clinical bone regeneration. To achieve the greatest therapeutic effect, it is crucial to evaluate the osteogenic differentiation potential of the stem cells during their culture before the implantation. However, the practical evaluation of stem cell osteogenicity has been limited to invasive biological marker analysis that only enables assaying a single end-point. To innovate around invasive quality assessments in clinical cell therapy, we previously explored and demonstrated the positive predictive value of using time-course images taken during differentiation culture for hBMSC bone differentiation potential. This initial method establishes proof of concept for a morphology-based cell evaluation approach, but reveals a practical limitation when considering the need to handle large amounts of image data. In this report, we aimed to scale-down our proposed method into a more practical, efficient modeling scheme that can be more broadly implemented by physicians on the frontiers of clinical cell therapy. We investigated which morphological features are critical during the osteogenic differentiation period to assure the performance of prediction models with reduced burden on image acquisition. To our knowledge, this is the first detailed characterization that describes both the critical observation period and the critical number of time-points needed for morphological features to adequately model osteogenic potential. Our results revealed three important observations: (i) the morphological features from the first 3 days of differentiation are sufficiently informative to predict bone differentiation potential, both activities of alkaline phosphatase and calcium deposition, after 3 weeks of continuous culture; (ii) intervals of 48 h are sufficient for measuring critical morphological features; and (iii) morphological features are most accurately predictive when early morphological features from the first 3 days of differentiation are combined with later features (after 10 days of differentiation).

Human hepatocarcinoma functional liver cell-4 cell line exhibits high expression of drug-metabolizing enzymes in three-dimensional culture.

The expression levels of CYP and uridine diphosphate-glucuronosyl transferase (UGT) are lower in hepatocellular carcinoma cell lines than in human primary hepatocytes. However, a functional liver cell (FLC)-4 cell line that has a greater capacity to secrete liver-specific proteins than other hepatocellular carcinoma cells has recently been established. A three-dimensional culture using Engelbreth-Holm-Swan (EHS) gel induces the secretion of liver-specific proteins via the induction of hepatocyte nuclear factor-4α (HNF-4α). The aim of this study was to evaluate the mRNA expression of the enzymes CYP and UGT in FLC-4 and HepG2 cells in monolayer and three-dimensional cultures using EHS gel. The mRNA levels of HNF-4α, albumin, pregnane X receptor (PXR), constitutive androstane receptor (CAR), CYPs (1A2, 2E1, 2C8, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) were determined using real-time reverse transcription (RT) PCR. In a monolayer culture, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C9, 2C19, 2D6, and 3A4) and UGTs (1A1, 1A6, and 1A9) were higher in FLC-4 cells than in HepG2 cells. In FLC-4 cells, the mRNA expression levels of HNF-4α, albumin, PXR, CAR, CYPs (2E1, 2C8, 2C19, and 3A4) and UGTs (1A1, 1A6, 1A9, and 2B7) significantly increased in three-dimensional culture. FLC-4 cells cultured in EHS gel showed significantly increased expression levels of CYPs and UGTs. The results of this study suggest that human hepatocellular carcinoma FLC-4 cells cultured in EHS gel show potential for use in studying in vitro drug metabolism.

The structurally diverse phytocannabinoids cannabichromene, cannabigerol and cannabinol significantly inhibit amyloid ß-evoked neurotoxicity and changes in cell morphology in PC12 cells.

BACKGROUND: Phytocannabinoids (pCBs) have been shown to inhibit the aggregation and neurotoxicity of the neurotoxic Alzheimer's disease protein beta amyloid (Aß). We characterized the capacity of six pCBs-cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), cannabidivarin (CBDV), cannabidiol (CBD) and Δ9 -tetrahydrocannabinol (Δ9 -THC)-to disrupt Aß aggregation and protect against Aß-evoked neurotoxicity in PC12 cells. METHODS: Neuroprotection against lipid peroxidation and Aß-induced cytotoxicity was assessed using the MTT assay. Transmission electron microscopy was used to visualize pCB effects on Aß aggregation and fluorescence microscopy, with morphometrics and principal component analysis to assess PC12 cell morphology. RESULTS: CBD inhibited lipid peroxidation with no significant effect on Aß toxicity, whilst CBN, CBDV and CBG provided neuroprotection. CBC, CBG and CBN inhibited Aß1-42 -induced neurotoxicity in PC12 cells, as did Δ9 -THC, CBD and CBDV. CBC, CBN and CBDV inhibited Aß aggregation, whilst Δ9 -THC reduced aggregate density. Aß1-42 induced morphological changes in PC12 cells, including a reduction in neuritic projections and rounded cell morphology. CBC and CBG inhibited this effect, whilst Δ9 -THC, CBD and CBDV did not alter Aß1-42 effects on cell morphology. CONCLUSIONS: These findings highlight the neuroprotective activity of CBC, CBG and CBN as novel pCBs associated with variable effects on Aß-evoked neurite damage and inhibition of amyloid ß aggregation.

Prediction of antibody production performance change in Chinese hamster ovary cells using morphological profiling.

Monoclonal antibodies (mAbs) represent a significant segment of biopharmaceuticals, with the market for mAb therapeutics expected to reach $200 billion in 2021. Chinese Hamster Ovary (CHO) cells are the industry standard for large-scale mAb production owing to their adaptability and genetic engineering capabilities. However, maintaining consistent product quality is challenging, primarily because of the inherent genetic instability of CHO cells. In this study, we address the need for advanced technologies for quality monitoring of host cells in biopharmaceuticals. We highlight the limitations of traditional cell assessment techniques such as flow cytometry and propose a noninvasive, label-free image-based analysis method. By utilizing advanced image processing and machine learning, this technique aims to non-invasively and quantitatively evaluate subtle quality changes in suspension cells. The research aims to investigate the use of morphological analysis for identifying subtle alterations in mAb productivity of CHO cells, employing cells stimulated by compounds as a model for this study. Our results show that the mAb productivity of CHO cells (day 8) can be predicted only from their early morphological profile (day 3). Our study also discusses the importance of strategic methods for forecasting host cell mAb productivity using morphological profiles, as inferred from our machine learning models specialized in predictive score prediction and anomaly prediction.