Increased IgD and CD27 Double Negative (DN) B cell population in pediatric onset autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic, inflammatory liver disease of unknown aetiology which requires lifelong immunosuppression. Most therapeutic and outcome studies of AIH have been conducted predominantly in Caucasian (European Ancestry, EA) cohorts, with the exclusion of African American (AA) patients due to inadequate sample size. It is known that AA patients have a severe phenotype of autoimmune diseases and demonstrate a poor response to conventional medical therapy. Understanding cellular and molecular pathways which determine AIH severity and progression in AA patients is likely to lead to the discovery of novel, personalised and better tolerated therapies. The aim of the study is to determine the distinct effector B cell phenotypes which contribute to disease severity and progression of AIH in AA children as compared to their EA cohorts. PBMCs were isolated from blood samples collected from patients visiting Children's Healthcare of Atlanta (CHOA) and were grouped into AA, (n = 12), EA, (n = 11) and controls (n = 12) and were processed for flow cytometry. Markers of B cell development, maturation and activation were assessed namely CD19, CD21, IgD, CD27, CD38, CD11c, CD24, CD138. AA children with AIH demonstrated an expansion of CD19 + ve, Activated Naïve (aN), (CD19+ IgD-/CD27- Double Negative (DN2) ([CD19+/IgD-/CD27++CD38++) cells. Plasmablasts were significantly higher along with Signalling Lymphocytic activation molecule F7 (SLAMF7). Unswitched memory [CD19+] IgD+CD27+ (USM) B cells were significantly contracted in AA patients with AIH. B cell phenotyping reveals a distinct profile in AA AIH patients with a major skewing towards the expansion of effector pathways which have been previously characterised in severe SLE in AA patients. These results suggest that the quantification and therapeutic target of B cell pathway could contribute substantially to the clinical approach to AIH especially in the AA population.

Concordant B and T Cell Heterogeneity Inferred from the Multiomic Landscape of Peripheral Blood Mononuclear Cells in a Crohn's Disease Cohort.

BACKGROUND AND AIMS: Crohn's disease is characterized by inflammation in the gastrointestinal tract due to a combination of genetic, immune, and environmental factors. Transcriptomic and epigenomic profiling of intestinal tissue of Crohn's disease patients have revealed valuable insights into pathology, however have not been conducted jointly on less invasive peripheral blood mononuclear cells (PBMCs). Furthermore, the heterogeneous responses to treatments among individuals with Crohn's disease imply hidden diversity of pathological mechanisms. METHODS: We employed single nucleus multiomic analysis, integrating both snRNA-seq and snATAC-seq of PBMCs with a variety of open source bioinformatics applications. RESULTS: Our findings reveal a diverse range of transcriptional signatures among individuals, highlighting the heterogeneity in PBMC profiles. Nevertheless, striking concordance between three heterogeneous groups was observed across B cells and T cells. Differential gene regulatory mechanisms partially explain these profiles, notably including a signature involving TGFß signaling in two individuals with Crohn's disease. A mutation mapped to a transcription factor binding site within a differentially accessible peak associated with the expression of this pathway, with implications for a personalized approach to understanding disease pathology. CONCLUSIONS: This study highlights how multiomic analysis can reveal common regulatory mechanisms that underlie heterogeneity of PBMC profiles, one of which may be specific to inflammatory disease.

Characterization of Intestinal Mesenchymal Stromal Cells From Patients With Inflammatory Bowel Disease for Autologous Cell Therapy.

Therapy with mesenchymal stromal cells (MSCs) has shown promise in inflammatory bowel disease-leveraging their immunosuppressive and regenerative properties. However, the potential immunogenic complications of allogenic MSCs sourced from different tissues raise concern. Thus, we assessed the fitness and functionality of autologous intestinal MSCs as a potential platform for cellular therapy. Mucosal biopsy-derived MSCs from Crohn's disease (n = 11), ulcerative colitis (n = 12), and controls (n = 14) were analyzed by microscopy and flow cytometry for doubling-time, morphology, differentiation potential, and immunophenotype. Gene expression, cell-subtype composition, along with surface marker and secretome changes after IFN-γ priming were measured by bulk and single-cell RNA sequencing coupled with a 30-plex Luminex panel. MSCs expanded ex vivo demonstrate canonical MSC markers, similar growth kinetics, and tripotency regardless of the patient phenotype. Global transcription patterns were similar at baseline though inflammatory bowel disease (IBD) rectal MSCs showed changes in select immunomodulatory genes. IFN-γ priming resulted in upregulation of shared immunoregulatory genes (particularly in PD-1 signaling) and overrode the transcriptional differences observed at baseline. Furthermore, MSCs secrete key immunomodulatory molecules at baseline and in response to IFN-γ including CXCL10, CXCL9, and MCP-1. Overall, MSCs from IBD patients have normal transcriptional and immunomodulatory properties with therapeutic potential and can be sufficiently expanded.

Assessing Cellular and Transcriptional Diversity of Ileal Mucosa Among Treatment-Naïve and Treated Crohn's Disease.

BACKGROUND: Crohn's disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn's. METHODS: Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn's patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type. RESULTS: We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn's phenotypes, indicating changes in cellular activity. CONCLUSIONS: Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn's disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.

Angiotensin-converting Enzyme-2 (ACE2) Expression in Pediatric Liver Disease.

The membrane protein angiotensin-converting enzyme-2 (ACE2) has gained notoriety as the receptor for severe acute respiratory syndrome coronavirus 2. Prior evidence has shown ACE2 is expressed within the liver but its function has not been fully discerned. Here, we utilized novel methodology to assess ACE2 expression in pediatric immune-mediated liver disease to better understand its presence in liver diseases and its role during infections such as COVID-19. We stained liver tissue with ACE2-specific immunofluorescent antibodies, analyzed via confocal microscopy. Computational deep learning-based segmentation models identified nuclei and cells, allowing the quantification of mean cellular and cytosolic immunofluorescent. Spatial transcriptomics provided high-throughput gene expression analysis in tissue to determine cellular composition for ACE2 expression. ACE2 plasma expression was quantified via enzyme-linked immunosorbent assay. High ACE2 expression was seen at the apical surface of cholangiocytes, with lower expression within hepatocyte cytosol and nonparenchymal cells ( P <0.001). Children with liver disease had higher ACE2 hepatic expression than pediatric control tissue ( P <0.001). Adult control tissue had higher expression than pediatric control ( P <0.001). Plasma ACE2 was not found to be statistically different between samples. Spatial transcriptomics identified cell composition of ACE2-expressing spots containing antibody-secreting cells. Our results show ACE2 expression throughout the liver, with strongest localization to cholangiocyte membranes. Machine learning can be used to rapidly identify hepatic cellular components for histologic analysis. ACE2 expression in the liver may be increased in pediatric liver disease. Future work is needed to better understand the role of ACE2 in chronic disease and acute infections.

Ileal Derived Organoids From Crohn's Disease Patients Show Unique Transcriptomic and Secretomic Signatures.

BACKGROUND & AIMS: We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn's disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. METHODS: RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. RESULTS: We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. CONCLUSIONS: PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes.

Ischemia reperfusion injury induces pyroptosis and mediates injury in steatotic liver thorough Caspase 1 activation.

A steatotic liver is increasingly vulnerable to ischemia reperfusion injury (IRI), and the underlying mechanisms are incompletely defined. Caspases are endo-proteases, which provide critical regulatory connections between cell death and inflammation. Caspase 1 is driven by inflammasomes which are key signaling platforms, that detect sterile stressors (DAMPs), releasing the highly pro-inflammatory cytokine interleukin IL-8 and IL-1ß. To delineate the involvement of Caspase 1 and 11 in hepatocellular injury in steatotic liver undergoing IRI. Male C57BL6/Wild Type and Caspase 1Null, Caspase 11-/- and Caspase 1-/-/11-/- mice were fed a high fat diet (HFD) for 12 weeks. These mice were subjected to 40 min of ischemia followed by 2-24 h of reperfusion. Hepatocellular injury was assessed by histopathologic injury scoring, serum ALT and propidium iodide (PI) uptake, mRNA levels of Caspase 1, IL-1ß by RT PCR, Caspase 1 activity assay and Caspase 1. Specific Caspase 1, inhibitor experiments were carried out. All groups gained similar body weight after a 12-week HFD. Cleaved Caspase 1 protein levels, Caspase 1 mRNA levels were significantly higher in steatotic liver undergoing IRI. Executor of pyroptosis cleaved GSDMD levels were higher in HFD fed mouse compared to lean. In addition, genetic deletion of Caspase 1, Casp1Null mouse expressing Caspase-11 and Caspase 1/11 double knock out demonstrated significant reduction in serum ALT (p < 0.01), Injury Score, (p < 0.0002) but not in Caspase 11 alone. Caspase 1 is the driver of hepatocellular injury in a steatotic liver undergoing IRI, inhibition of which leads to hepatoprotection, thus providing a therapeutic target for clinical use.

African American Pediatric Liver Transplant Recipients Have an Increased Risk of Death After Transferring to Adult Healthcare.

OBJECTIVE: To analyze the long-term outcomes in pediatric liver transplant recipients after they have transferred to an adult provider and assess for racial disparities in health outcomes. STUDY DESIGN: This is a single-center, retrospective review of pediatric patients who underwent liver transplantation between July 1990 and August 2015 at a tertiary healthcare system with a large transplant center. Patient mortality and retransplantation were assessed after transfer to adult care. RESULTS: There were 120 patients who were transferred, of whom 19 did not meet the inclusion criteria. Of the remaining 101 patients, 64 (63%) transferred care to a nearby affiliated tertiary adult facility, 29 (29%) were followed by other healthcare systems, and 8 (8%) were lost to follow-up. Of the patients followed at our affiliated adult center, 18 of the 64 (28%) died. Of those 18 deaths, 4 (22%) occurred within the first 2 years after transfer, and 10 (55%) within 5 years of transfer. Four patients were retransplanted by an adult provider, of whom 2 eventually received a third transplant. African Americans had higher rates of death after transfer than patients of other races (44% mortality vs 16%, representing 67% of all cases of death; P = .032), with nearly 50% mortality at 20 years from time of transplantation. CONCLUSIONS: Death is common in pediatric liver transplant recipients after transfer to adult care, with African Americans having disproportionately higher mortality. This period of transition of care is a vulnerable time, and measures must be taken to ensure the safe transfer of young adults with chronic health care needs.

A two decade long study of disease progression of de novo and recurrent autoimmune hepatitis in the pediatric population.

Recurrent autoimmune hepatitis (rAIH) occurs in patients who undergo liver transplantation (LT) for AIH and de novo AIH (dAIH) is seen in patients who are transplanted for etiologies other than AIH. Whether these are distinct diseases with a similar phenotype remains understudied. The aim of this study was to identify clinical and immunologic factors affecting outcome in patients with dAIH and rAIH. A retrospective review of 387 LT patients from 1997 to 2014 was carried out, and they were followed until 2018. Patients with rAIH or dAIH were identified based on the pre-transplant diagnosis of AIH (or not) and characteristic histology. Liver biopsies were stained with H&E, B-cell marker CD20, and plasma cell marker CD138. Out of 387 patients, 31 were transplanted for AIH, and 8/31 developed rAIH. Of the remaining 356 patients, eight developed dAIH. Compared to the dAIH group, rAIH occurred in older patients, had an earlier onset in the allograft, and had higher IgG and serum ALT levels. It was most commonly seen in African American (AA) patients (87%). rAIH patients had significantly higher CD20 and CD138 positivity in liver biopsies. In addition, they had increased rejection episodes prior to the onset of recurrence, increased graft loss, and mortality. rAIH is a more aggressive disease, and has a preponderance of B cells and plasma cells in the liver tissue as compared to dAIH. The concurrent association with increased graft loss and patient mortality in rAIH warrants further investigations into B cell-targeted therapies.

Soluble PD1 levels are increased with disease activity in paediatric onset autoimmune hepatitis and inflammatory bowel disease.

Introduction: Immune mediated liver diseases entail a broad category which are associated with increased morbidity and mortality amongst the paediatric population. Programmed Death 1 (PD1) is an inhibitory receptor mainly expressed by T cells, and when activated shed into plasma as soluble PD1(sPD1). The AIM of this study was to evaluate sPD1 levels in plasma of paediatric patients with Autoimmune Hepatitis (AIH), Primary Sclerosing Cholangitis (PSC), AIH and PSC overlap, Inflammatory Bowel Disease (IBD) alone, and concurrent PSC/IBD and AIH/IBD in order to identify a biomarker to response or predict relapse verses remission.Methods: Plasma samples were collected from 41 paediatric patients. AIH patients were further categorized into active, incomplete responders and responders, based on response to standard therapy. sPD1 levels were measured and compared between PSC, PSC/AIH, IBD alone, PSC/IBD and AIH/IBD patients and between active AIH, incomplete responders and responders. Flow cytometry was performed to further analyze CD45RA+, CD3CD4, CD8, CCR7, CXCR3, CD38 and PD1.Results: In the AIH group, those with active disease demonstrated a significantly higher sPD1 levels in comparison to responders (*p > .001). However, the incomplete responders didn't show a reduction in sPD1 in comparison to active AIH and patients with IBD alone. Interestingly, patients with PSC showed significantly lower level of sPD1 compared to active AIH (*p < .002), whereas, patients with PSC in conjunction with AIH (*p < .006) or IBD (*p < .02) demonstrated a significant increase in sPD1. In addition, we have observed increased levels of circulating CD4 and CD8 bound PD1 in active AIH but not in PSC or responders suggesting T cells activation. CD4+ PD1 double positive cells demonstrated increased expression of CXCR3. Thus, suggesting the activation of PD1 + T cells is mediating through CXCR3 in Autoimmune hepatitis.Conclusions: Our study demonstrates that sPD1 levels correlate with active disease state of AIH and IBD. sPD1 levels did not correlate with PSC. However, PSC in conjunction with AIH or IBD showed higher levels of sPD1. This suggests that T cell activation plays a critical role in active AIH and IBD but not in PSC. Soluble PDI levels could be used as a clinical biomarker to assess response in patients with AIH and for prospectively monitoring PSC patients for development of IBD or AIH.

Influence of Fat on Differential Receptor Interacting Serine/Threonine Protein Kinase 1 Activity Leading to Apoptotic Cell Death in Murine Liver Ischemia Reperfusion Injury Through Caspase 8.

Current understanding is that receptor interacting serine/threonine protein kinase 1 (RIPK1) can lead to two distinct forms of cell death: RIPK3-mediated necroptosis or caspase 8 (Casp8)-mediated apoptosis. Here, we report that RIPK1 signaling is indispensable for protection from hepatocellular injury in a steatotic liver undergoing ischemia reperfusion injury (IRI) but not in the lean liver. In lean liver IRI, RIPK1-mediated cell death is operational, leading to protection in RIP1 kinase-dead knock-in (RIPK1K45A) mice and necrostatin-1s (Nec1s)-treated lean wild-type (WT) mice. However, when fed a high-fat diet (HFD), RIPK1K45A-treated and Nec1s-treated WT mice undergoing IRI demonstrate exacerbated hepatocellular injury along with decreased RIPK1 ubiquitylation. Furthermore, we demonstrate that HFD-fed RIPK3-/-/Casp8-/- mice show protection from IRI, but HFD-fed RIPK3-/-/Casp8-/+ mice do not. We also show that blockade of RIPK1 leads to increased Casp8 activity and decreases mitochondrial viability. Conclusion: Although more studies are required, we provide important proof of concept for RIPK1 inhibition leading to distinctive outcomes in lean and steatotic liver undergoing IRI. Considering the rising incidence of nonalcoholic fatty liver disease (NAFLD) in the general population, it will be imperative to address this critical difference when treating patients with RIPK1 inhibitors. This study also presents a new target for drug therapy to prevent hepatocellular injury in NAFLD.

Racial disparities in presentation and outcomes of paediatric autoimmune hepatitis.

BACKGROUND & AIMS: Most studies on autoimmune hepatitis (AIH) in children are in predominantly Caucasian cohorts. Paediatric AIH in African Americans (AA) is understudied, with a dearth of clinical predictors of outcome, often leading to serious complications and even mortality. The aim of the study was to define disease presentation, progression, response to therapy and outcomes in paediatric AIH in a well-defined, large, single centre, demographically diverse population. METHODS: We conducted a review of patients with AIH who were followed at this tertiary liver transplant centre. Clinical and laboratory covariates were assessed with regard to disease presentation, progression and outcomes in AA vs Non-AA children. RESULTS: African Americans patients constituted 42% of this cohort. At 1-year follow-up, AA children were receiving significantly higher doses of steroids compared to non-AA. More AA presented with end-stage liver disease (ESLD) with high immunoglobulin G and GGT:platelet ratio. After adjusting for other risk factor variables like gender, age at presentation and ESLD, AA children were at 4.5 times higher risk for significant outcome liver transplant/death within the first 12 months of presentation. Post-transplant, recurrent AIH was seen in 50% of AA vs 8% in non-AA. CONCLUSIONS: African American patients with AIH are more likely to present with ESLD and have an increased early risk for transplantation with high likelihood of disease recurrence post-transplantation. Studies are needed to delineate factors such as inherent biology, genetics and access to care. Early referral and tailored immunosuppressive regimens are required for AA patients with AIH.

Loss of L-selectin-guided CD8+ , but not CD4+ , cells protects against ischemia reperfusion injury in a steatotic liver.

Steatotic liver responds with increased hepatocellular injury when exposed to an ischemic-reperfusion insult. Increasing evidence supports the role of immune cells as key mediators of this injury in a normal (lean) state, but data about their role in a steatotic liver are practically nonexistent. The objective of the current study was to delineate the contribution of specific phenotypes of T cells and adhesion molecules in exacerbated cell death in steatotic liver injury. RNA sequencing was performed on isolated steatotic primary hepatocytes, and T-cell markers were assessed in hepatic lymphocytes after ischemia reperfusion injury (IRI) in high-fat diet (HFD)-fed mice. Cluster of differentiation 8 knockout (CD8-/- ) and CD4-/- mice along with CD8 and L-selectin antibody-treated mice were fed an HFD, and hepatocellular injury was assessed by histology, propidium iodide injection, and alanine aminotransferase after IRI. RNA sequencing demonstrated a strikingly differential gene profile in steatotic hepatocytes versus lean hepatocytes. After injury, the HFD liver showed increased necrosis, infiltrating CD8+ cells, alanine aminotransferase, and proinflammatory cytokines. Hepatic lymphocytes demonstrated increased CD8+ /CD62L+ (L-selectin) cells in HFD-fed mice after IRI. CD8-/- mice and CD8-depleted C57BL/6 mice demonstrated significant protection from injury, which was not seen in CD4-/- mice. L-selectin blockade also demonstrated significant hepatoprotection from IRI. L-selectin ligand MECA-79 was increased in HFD-fed mice undergoing IRI. CONCLUSION: Blockade of CD8 and L-selectin, but not CD4, ameliorated hepatocellular injury, confirming that CD8+ cells are critical drivers of injury in a steatotic liver; this represents a therapeutic target in steatotic liver injury, underlining the importance of development of therapies specific to a steatotic liver. (Hepatology 2017;66:1258-1274).

Contrast-Based Real-Time Assessment of Microcirculatory Changes in a Fatty Liver After Ischemia Reperfusion Injury.

OBJECTIVES: A fatty liver is known to have impairment of microcirculation, which is worsened after ischemia reperfusion injury (IRI). This makes most fatty grafts unsuitable for transplantation, and in the absence of real time assessment of microcirculation this selection has been at best, random. The aim of this study was to demonstrate the utility of a contrast enhanced ultrasound model in quantitative assessment of the microcirculation of a fatty liver. METHODS: We subjected fatty mice to IRI, and blood flow dynamics were assessed before and after the injury. RESULTS: There was a significant increase in the resistive and pulsatility index of the extrahepatic artery and a significant decrease in velocity of the portal vein. There was also a quantifiable decrease in the intrahepatic blood volume, blood flow, time to peak flow, and perfusion index of mice with fatty liver, suggesting that a fatty liver develops hemodynamic abnormalities after IRI, leading to increased hepatocellular injury. CONCLUSIONS: Hemodynamic abnormalities in liver can be reliably quantified using a contrast, enhanced Doppler ultrasound, which is an inexpensive technique with multiple clinical applications. It can be used to assess the quality of the fatty liver donor graft before organ retrieval; for determining live donor candidacy, for making post-IRI recovery prognosis, and for assessing the effectiveness of therapeutic interventions.

Mitigation of autophagy ameliorates hepatocellular damage following ischemia-reperfusion injury in murine steatotic liver.

Ischemia-reperfusion injury (IRI) is a common clinical consequence of hepatic surgery, cardiogenic shock, and liver transplantation. A steatotic liver is particularly vulnerable to IRI, responding with extensive hepatocellular injury. Autophagy, a lysosomal pathway balancing cell survival and cell death, is engaged in IRI, although its role in IRI of a steatotic liver is unclear. The role of autophagy was investigated in high-fat diet (HFD)-fed mice exposed to IRI in vivo and in steatotic hepatocytes exposed to hypoxic IRI (HIRI) in vitro. Two inhibitors of autophagy, 3-methyladenine and bafilomycin A1, protected the steatotic hepatocytes from HIRI. Exendin 4 (Ex4), a glucagon-like peptide 1 analog, also led to suppression of autophagy, as evidenced by decreased autophagy-associated proteins [microtubule-associated protein 1A/1B-light chain 3 (LC3) II, p62, high-mobility group protein B1, beclin-1, and autophagy-related protein 7], reduced hepatocellular damage, and improved mitochondrial structure and function in HFD-fed mice exposed to IRI. Decreased autophagy was further demonstrated by reversal of a punctate pattern of LC3 and decreased autophagic flux after IRI in HFD-fed mice. Under the same conditions, the effects of Ex4 were reversed by the competitive antagonist exendin 9-39. The present study suggests that, in IRI of hepatic steatosis, treatment of hepatocytes with Ex4 mitigates autophagy, ameliorates hepatocellular injury, and preserves mitochondrial integrity. These data suggest that therapies targeting autophagy, by Ex4 treatment in particular, may ameliorate the effects of IRI in highly prevalent steatotic liver.

Radiation fibrosis of the vocal fold: from man to mouse.

OBJECTIVES/HYPOTHESIS: To characterize fundamental late tissue effects in the human vocal fold following radiation therapy. To develop a murine model of radiation fibrosis in order to ultimately develop both treatment and prevention paradigms. DESIGN: Translational study using archived human and fresh murine irradiated vocal fold tissue. METHODS: 1) Irradiated vocal fold tissue from patients undergoing laryngectomy for loss of function from radiation fibrosis was identified from pathology archives. Histomorphometry, immunohistochemistry, and whole-genome microarray, as well as real-time transcriptional analyses, were performed. 2) Focused radiation to the head and neck was delivered to mice in a survival fashion. One month following radiation, vocal fold tissue was analyzed with histomorphometry, immunohistochemistry, and real-time PCR transcriptional analysis for selected markers of fibrosis. RESULTS: Human irradiated vocal folds demonstrated increased collagen transcription, with increased deposition and disorganization of collagen in both the thyroarytenoid muscle and the superficial lamina propria. Fibronectin were increased in the superficial lamina propria. Laminin decreased in the thyroarytenoid muscle. Whole genome microarray analysis demonstrated increased transcription of markers for fibrosis, oxidative stress, inflammation, glycosaminoglycan production, and apoptosis. Irradiated murine vocal folds demonstrated increases in collagen and fibronectin transcription and deposition in the lamina propria. Transforming growth factor (TGF)-ß increased in the lamina propria. CONCLUSION: Human irradiated vocal folds demonstrate molecular changes leading to fibrosis that underlie loss of vocal fold pliability occurring in patients following laryngeal irradiation. The irradiated murine tissue demonstrates similar findings, and this mouse model may have utility in creating prevention and treatment strategies for vocal fold radiation fibrosis.

The glucagon-like peptide-1 receptor agonist Exendin 4 has a protective role in ischemic injury of lean and steatotic liver by inhibiting cell death and stimulating lipolysis.

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase - all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.

A(2B)AR expression in non-immune cells plays an important role in the development of murine colitis.

BACKGROUND: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis. AIMS: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation. METHODS: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. RESULTS: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. CONCLUSIONS: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.

The use of lipid microtubes as a novel slow-release delivery system for laryngeal injection.

OBJECTIVES/HYPOTHESIS: The potential utility of direct injection of bioactive substances in the treatment of vocal fold tissue fibrosis is limited by rapid clearance from the injection site. The objective of this study is to evaluate the potential of a lipid-based microtube delivery system to preserve the biological activity of injected substances and prolong their duration of pharmacological effects in the larynx. STUDY DESIGN: Prospective in vitro and case-control in vivo murine study METHODS: Lipid-based microtubes were loaded with Texas red-dextran (MT-TR) and hepatocyte growth factor (MT-HGF). In vitro and in vivo (using a murine vocal fold injection model) release of MT-TR and MT-HGF were determined to assess duration of microtube-mediated delivery. The biologic effects of MT-HGF on fibroblasts were assessed after treatment in the presence of transforming growth factor (TGF)-ß. RESULTS: In vitro release kinetics demonstrated slow release of MT-TR and MT-HGF, correlating with in vivo results demonstrating persistence of MT-HGF at 4 weeks postinjection. Bioefficacy was maintained, as MT-HGF was shown to inhibit TGF-ß-mediated induction of procollagen mRNA levels in vitro 24 hours after treatment in fibroblast cells. Sustained release of HGF from microtubes at 6 days exacerbated the effects of TGF-ß and increased levels of procollagen mRNA. CONCLUSIONS: Microtubes have significant potential utility as an efficacious means of sustained-release delivery of bioactive agents to the larynx. Atthough the role of HGF as an antifibrotic agent is questioned, its sustained bioefficacy after microtube encapsulation distinguishes microtubes from other delivery vehicles.

Pathologic effects of external-beam irradiation on human vocal folds.

OBJECTIVES: We sought to better characterize pathologic changes that occur in the human vocal fold after radiotherapy for head and neck cancer. METHODS: In a blinded, controlled study of archived tissue, we evaluated postirradiation salvage laryngectomy vocal fold tissue without evidence of malignant disease. Clinical and demographic patient data were collected. In a blinded fashion, irradiated tissue was compared to nonirradiated, benign control tissue. Histomorphometric analysis was used to assess muscle and collagen organization, superficial lamina propria (SLP) and vocal ligament thickness, vocalis muscle fiber area, collagen content, and hyaluronic acid content. Immunohistochemical analysis was used to assess the content of type I collagen, type IV collagen, vimentin, fibronectin, alpha-smooth muscle actin, matrix metalloproteinase 9, and laminin. RESULTS: Twenty irradiated vocal folds were evaluated and compared to control specimens. Collagen and muscle disorganization was noted in the irradiated specimens. The SLP and vocal ligament thicknesses and the mean muscle fiber diameters did not differ significantly. The SLP fibronectin and the vocalis muscle and SLP collagen content were significantly increased in the irradiated vocal folds, and the SLP collagen content increased significantly with time between irradiation and resection. The laminin content of irradiated vocalis muscles was significantly decreased. CONCLUSIONS: Radiotherapy results in significant vocal fold tissue changes. Having more precisely defined these changes, we plan continued investigation seeking targeted preventive and therapeutic interventions for improved vocal quality following radiotherapy.