Hepatocyte-Derived Exosomes Promote Liver Immune Tolerance: Possible Implications for Idiosyncratic Drug-Induced Liver Injury.

Most idiosyncratic drug-induced liver injury appears to result from an adaptive immune attack on the liver. Recent evidence suggests that the T-cell response may be facilitated by the loss of immune tolerance. In this study, we explored the hypothesis that constitutively released hepatocyte-derived exosomes (HDE) are important for maintaining normal liver immune tolerance. Exosomes were isolated from the conditioned medium of primary human hepatocytes via polymer precipitation. Mock controls were prepared by processing fresh medium that was not hepatocyte exposed with precipitation reagent. THP-1 monocytes were then treated with HDE or an equivalent volume of mock control for 24 h, followed by a 6-h stimulation with LPS. HDE exposure resulted in a significant decrease in the LPS-induced media levels of interleukin-1ß and interleukin-8. Gene expression profiling performed in THP-1 cells just prior to LPS-induced stimulation identified a significant decrease among genes associated with innate immune response. MicroRNA (miRNA) profiling was performed on the HDE to identify exosome contents that may drive immune suppression. Many of the predicted mRNA target genes for the most abundant microRNAs in HDE were among the differentially expressed genes in THP-1 cells. Taken together, our data suggest that HDE play a role in maintaining normal liver immune tolerance. Future experiments will explore the possibility that drugs causing idiosyncratic liver injury promote the loss of homeostatic HDE signaling.

Parental Adjustment following Pediatric Burn Injury: The Role of Guilt, Shame, and Self-Compassion.

Objective: The purpose of this study was to examine the association between factors: guilt, shame, self-compassion, and parents' psychological adjustment to their child's burn injury. Methods: Participants were 91 parents and primary caregivers (63 mothers, 25 fathers, 3 other) of 71 children recruited on the ward or at outpatient clinics during the first 8 weeks following their child's burn injury. In 20 cases, both parents participated, while for 51 children only one parent participated. Participants completed questionnaires which assessed adjustment (symptoms of depression, anxiety, and posttraumatic stress syndrome [PTSS]) as well as guilt, shame, and self-compassion. Results: Multilevel analysis indicated that feelings of guilt and shame were associated with poorer adjustment in parents, while parents who rated high in self-compassion reported fewer symptoms of depression and PTSS. Guilt and shame showed a differing pattern of effects with shame explaining more variance for anxiety and depression. Length of hospitalization predicted PTSS; however, the remaining injury factors (size of burn, requiring a skin graft) were unrelated to parental adjustment. Conclusions: Health-care professionals should pay close attention to families' subjective injury experiences. Screening for psychological distress should be offered to all families regardless of the size and severity of the burn injury.

miR-122 Release in Exosomes Precedes Overt Tolvaptan-Induced Necrosis in a Primary Human Hepatocyte Micropatterned Coculture Model.

Idiosyncratic drug-induced liver injury (IDILI) is thought to often result from an adaptive immune attack on the liver. However, it has been proposed that the cascade of events culminating in an adaptive immune response begins with drug-induced hepatocyte stress, release of exosomal danger signals, and innate immune activation, all of which may occur in the absence of significant hepatocelluar death. A micropatterned coculture model (HepatoPac) was used to explore the possibility that changes in exosome content precede overt necrosis in response to the IDILI drug tolvaptan. Hepatocytes from 3 human donors were exposed to a range of tolvaptan concentrations bracketing plasma Cmax or DMSO control continuously for 4, 24, or 72 h. Although alanine aminotransferase release was not significantly affected at any concentration, tolvaptan exposures at approximately 30-fold median plasma Cmax resulted in increased release of exosomal microRNA-122 (miR-122) into the medium. Cellular imaging and microarray analysis revealed that the most significant increases in exosomal miR-122 were associated with programmed cell death and small increases in membrane permeability. However, early increases in exosome miR-122 were more associated with mitochondrial-induced apoptosis and oxidative stress. Taken together, these data suggest that tolvaptan treatment induces cellular stress and exosome release of miR-122 in primary human hepatocytes in the absence of overt necrosis, providing direct demonstration of this with a drug capable of causing IDILI. In susceptible individuals, these early events may occur at pharmacologic concentrations of tolvaptan and may promote an adaptive immune attack that ultimately results in clinically significant liver injury.

Dressing changes in a burns unit for children under the age of five: A qualitative study of mothers' experiences.

This study aimed to investigate the experiences of mothers who had attended their child's burn dressing changes. Participants were recruited from a burns unit based within a children's hospital. Face-to-face interviews were conducted with five mothers of children under the age of five who had undergone a series of dressing changes taking place on the burns unit. The interview guide explored parents' experience of initial and subsequent dressing changes. Participants were prompted to explore their expectations, thoughts, feelings and behaviours associated with these experiences. The interviews were recorded and transcribed verbatim. Transcripts were analysed using interpretative phenomenological analysis. The analysis identified four themes: 'needing to fulfil the responsibilities associated with being a mother'; 'emotional synchrony between mother and child'; 'being informed and knowing what to expect'; and 'the importance of establishing rapport with nurses performing dressing changes'. Findings from this research can inform services to help optimise mothers' experiences of dressing changes in this stage of pediatric burn care.

Subtoxic Alterations in Hepatocyte-Derived Exosomes: An Early Step in Drug-Induced Liver Injury?

Drug-induced liver injury (DILI) is a significant clinical and economic problem in the United States, yet the mechanisms that underlie DILI remain poorly understood. Recent evidence suggests that signaling molecules released by stressed hepatocytes can trigger immune responses that may be common across DILI mechanisms. Extracellular vesicles released by hepatocytes, principally hepatocyte-derived exosomes (HDEs), may constitute one such signal. To examine HDE alterations as a function of drug-induced stress, this work utilized prototypical hepatotoxicant acetaminophen (APAP) in male Sprague-Dawley (SD) rats, SD rat hepatocytes, and primary human hepatocytes. HDE were isolated using ExoQuick precipitation reagent and analyzed by quantification of the liver-specific RNAs albumin and microRNA-122 (miR-122). In vivo, significant elevations in circulating exosomal albumin mRNA were observed at subtoxic APAP exposures. Significant increases in exosomal albumin mRNA were also observed in primary rat hepatocytes at subtoxic APAP concentrations. In primary human hepatocytes, APAP elicited increases in both exosomal albumin mRNA and exosomal miR-122 without overt cytotoxicity. However, the number of HDE produced in vitro in response to APAP did not increase with exosomal RNA quantity. We conclude that significant drug-induced alterations in the liver-specific RNA content of HDE occur at subtoxic APAP exposures in vivo and in vitro, and that these changes appear to reflect selective packaging rather than changes in exosome number. The current findings demonstrate that translationally relevant HDE alterations occur in the absence of overt hepatocellular toxicity, and support the hypothesis that HDE released by stressed hepatocytes may mediate early immune responses in DILI.

Co-culture of Hepatocytes and Kupffer Cells as an In Vitro Model of Inflammation and Drug-Induced Hepatotoxicity.

Immune-mediated drug-induced hepatotoxicity is often unrecognized as a potential mode of action due to the lack of appropriate in vitro models. We have established an in vitro rat donor-matched hepatocyte and Kupffer cell co-culture (HKCC) model to study immune-related responses to drug exposure. Optimal cell culture conditions were identified for the maintenance of co-cultures based on cell longevity, monolayer integrity, and cytokine response after lipopolysaccharide (LPS) exposure. Hepatocyte monocultures and HKCCs were then used to test a subset of compounds associated with hepatotoxic effects with or without LPS. Cytokine levels and metabolic activity (cytochrome P450 3A [Cyp3A]) were measured after a 48-h exposure to monitor endotoxin-induced changes in acute phase and functional end points. LPS-activated HKCCs, but not hepatocyte monocultures, treated with trovafloxacin or acetaminophen, compounds associated with immune-mediated hepatotoxicity, showed LPS-dependent decreases in interleukin-6 production with concomitant increases in Cyp3A activity. Differential endotoxin- and model-dependent alterations were observed in cytokine profiles and Cyp3A activity levels that corresponded to specific compounds. These results indicate the utility of the HKCC model system to discern compound-specific effects that may lead to enhanced or mitigate hepatocellular injury due to innate or adaptive immune responses.

In vitro photodynamic inactivation of Candida species and mouse fibroblasts with phenothiazinium photosensitisers and red light.

In the present study, the in vitro susceptibilities of five Candida spp. to photodynamic antimicrobial chemotherapy (PACT) with four phenothiazinium derivatives, methylene blue (MB), new methylene blue N (NMBN), toluidine blue O (TBO) and the novel pentacyclic phenothiazinium photosensitiser S137, in combination with red light were investigated. The efficacy of each PS was determined, initially, based on its minimal inhibitory concentration (MIC). Additionally, we evaluated the effect of the photodynamic treatment with NMBN and S137 on Candida survival and on the mouse fibroblast cell line L929. MICs varied both among PS and species and decreased with light dose increase. For most treatments (species and fluences) NMBN and S137 showed the lowest MICs. MICs for NMBN and S137 were <2.5 µM for all the Candida species when a fluence of 25 J cm⁻² was used. PACT with NMBN (fluence of 15 J cm⁻²) resulted in reductions in survival from 0.3 log (Candida krusei) to 3 logs (C. parapsilosis). PACT with S137 was more effective than with NMBN. Fluence of 15 J cm⁻² resulted in reductions in survival from 1 log (C. krusei) to 3 logs (C. parapsilosis) and fluence of 25 J cm⁻² resulted in a reduction of approximately 2 logs (C. krusei) and between 3 and 4 logs in survival of the other 4 species of Candida. In vitro relative toxicities of the phenothiazinium PS to mammalian cells exhibited a similar trend to the antifungal data, i.e. greater toxicity and phototoxicity with NMBN and S137 compared to the established PS.

Structural and functional analysis of the human nuclear xenobiotic receptor PXR in complex with RXRα.

The human nuclear xenobiotic receptor PXR recognizes a range of potentially harmful drugs and endobiotic chemicals but must complex with the nuclear receptor RXRα to control the expression of numerous drug metabolism genes. To date, the structural basis and functional consequences of this interaction have remained unclear. Here we present 2.8-Å-resolution crystal structures of the heterodimeric complex formed between the ligand-binding domains of human PXR and RXRα. These structures establish that PXR and RXRα form a heterotetramer unprecedented in the nuclear receptor family of ligand-regulated transcription factors. We further show that both PXR and RXRα bind to the transcriptional coregulator SRC-1 with higher affinity when they are part of the PXR/RXRα heterotetramer complex than they do when each ligand-binding domain is examined alone. Furthermore, we purify the full-length forms of each receptor from recombinant bacterial expression systems and characterize their interactions with a range of direct and everted repeat DNA elements. Taken together, these data advance our understanding of PXR, the master regulator of drug metabolism gene expression in humans, in its functional partnership with RXRα.

Adult attachment style and childhood interpersonal trauma in non-epileptic attack disorder.

Non-epileptic attack disorder (NEAD) poses a significant clinical problem but is poorly understood. Attachment theory provides a framework for understanding the development and maintenance of NEAD and the contribution of childhood abuse and neglect to these processes. A cross-sectional design was used to study attachment style and early traumatic experiences in individuals with NEAD (N=17) compared to those with epilepsy (N=26). A significant difference in predominant attachment style between the two groups was found, with fearful attachment occurring more frequently in the NEAD group. Abuse and neglect were also significantly more common in the NEAD patients. Both early traumatic experiences and fearful attachment added significantly to the predictive power of a logistic regression equation after controlling for anxiety and dysthymia. The findings suggest a link between disturbed attachment and NEAD and have clinical implications for therapeutic intervention with this group.