Expression of unfolded protein response genes in post-transplantation liver biopsies.

BACKGROUND: Cholestatic liver diseases are a major source of morbidity and mortality that can progress to end-stage liver disease and hyperbilirubinemia is a hallmark of cholestasis. There are few effective medical therapies for primary biliary cholangitis, primary sclerosing cholangitis and other cholestatic liver diseases, in part, due to our incomplete understanding of the pathogenesis of cholestatic liver injury. The hepatic unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum stress that is important in the pathogenesis of many liver diseases and recent animal studies have demonstrated the importance of the UPR in the pathogenesis of cholestatic liver injury. However, the role of the UPR in human cholestatic liver diseases is largely unknown. METHODS: RNA was extracted from liver biopsies from patients after liver transplantation. RNA-seq was performed to determine the transcriptional profile and hepatic UPR gene expression that is associated with liver injury and cholestasis. RESULTS: Transcriptome analysis revealed that patients with hyperbilirubinemia had enhanced expression of hepatic UPR pathways. Alternatively, liver biopsy samples from patients with acute rejection had enhanced gene expression of LAG3 and CDK1. Pearson correlation analysis of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels demonstrated significant correlations with the hepatic expression of several UPR genes, as well as genes involved in hepatic bile acid metabolism and inflammation. In contrast, serum alkaline phosphatase levels were correlated with the level of hepatic bile acid metabolism gene expression but not liver UPR gene expression. CONCLUSIONS: Overall, these data indicate that hepatic UPR pathways are increased in cholestatic human liver biopsy samples and supports an important role of the UPR in the mechanism of human cholestatic liver injury.

Profiling the liver graft.

PURPOSE OF REVIEW: Achieving operational tolerance remains a priority in liver transplantation. Although several biomarkers of tolerance and rejection have been identified, few have been reproducible and validated across centers, and therefore have yet to reach clinical practice. Here we summarize findings from prior seminal studies and review current developments in profiling the liver allograft. RECENT FINDINGS: Substantial efforts and progress have been made in the recent years towards the discovery of reliable biomarkers that can predict and guide successful immunosuppression withdrawal. Recent studies have also investigated the transcriptomic signatures underlying not only acute rejection but also subclinical inflammation and chronic allograft injury. SUMMARY: As new genomic and sequencing technologies continue to develop, clinical trials are underway to validate biomarkers of tolerance, as well as better understand the mechanisms of both acute and subclinical rejection, with the goal of maximizing allograft survival. Altogether, this will hopefully enable the implementation of immunosuppression withdrawal protocols into clinical practice and make operational tolerance reliably attainable in the near future.

Liver-related mortality is similar among men and women with cirrhosis.

BACKGROUND & AIMS: Sex-based differences are known to significantly contribute to outcomes in patients with chronic liver diseases; however, the role of patient sex in cirrhosis is unclear. We aimed to study the relationship between patient sex and cirrhosis. METHODS: We analyzed a cohort of 20,045 patients with cirrhosis using a Chicago-wide electronic health record database that was linked with the United Network for Organ Sharing and cause of death data from the state death registry. Adjusted Cox survival analyses and competing risk analyses were performed to obtain subdistribution hazard ratios (HRs) for liver-related cause of death. RESULTS: Female and male patients had similar age, racial distribution, insurance status, and comorbidity status by Elixhauser score. Females had higher rates of cholestatic liver disease (17.1% vs. 6.2%, p <0.001) and non-alcoholic steatohepatitis (29.8% vs. 21.2%, p <0.001) than males. They were less likely to have portal hypertensive complications and had lower peak MELD-Na scores during follow-up. Female sex was associated with a decreased hazard of all-cause mortality (adjusted HR 0.85; 95% CI 0.80-0.90). This effect was attenuated when liver-related mortality was examined (subdistribution HR 0.93; 95% CI 0.87-1.00). No significant difference was noted for women who were 'ever-listed' in competing risk analyses for either all-cause mortality (subdistribution HR 1.09; 95% CI 0.88-1.35) or liver-related death (subdistribution HR 1.12; 95% CI 0.87-1.43), despite lower rates of listing (7.5% vs. 9.8%; p <0.001) and transplant (3.5% vs. 5.2%; p <0.001). CONCLUSIONS: In this longitudinal study of patients with cirrhosis, female sex was associated with a survival advantage likely driven by lower rates of non-liver-related death. Women were not at an increased risk of liver-related death despite lower rates of listing and transplantation. LAY SUMMARY: Patient sex is an important contributor in many chronic diseases, including cirrhosis. Prior studies have suggested that female sex is associated with worse outcomes. We analyzed a cohort of 20,045 patients with cirrhosis using a Chicago-wide electronic health record database. Using multivariate competing risk analyses, we found that female sex in cirrhosis is actually associated with a lower risk of all-cause mortality and has no association with liver-related mortality. Our findings are novel because we show that women with cirrhosis have a similar risk of liver-related death as their male counterparts, despite lower rates of listing and transplantation.

Cardioembolic Sources in Patients With Small Single Subcortical Infarcts.

Transthoracic echocardiogram (TTE) is widely used as part of the work-up for ischemic stroke. However, the added utility of TTE in the management of small single subcortical infarcts (SSSI) has not been extensively evaluated. Therefore, we aimed to determine the frequency of high-risk and medium-risk cardioembolic sources diagnosed by TTE in SSSI patients, and whether the findings altered clinical management. We performed a retrospective analysis of a prospective cohort of patients with confirmed acute ischemic stroke enrolled in a single-center observational registry between August 2012 and July 2014. We assessed infarct topography on brain magnetic resonance imaging, blinded to final stroke subtype and treatment, to identify patients with SSSI. We defined SSSI as lesions in the corona radiata, basal ganglia, thalamus, or brainstem measuring <1.5 cm in maximal diameter. We excluded patients with atrial fibrillation and large artery stenosis >50%, and assessed the frequency of cardioembolic sources found by TTE in the remaining patients. Among 908 patients, 75 (8.3%) had SSSI, of which 4 (5.3%) had atrial fibrillation and 6 (8%) had symptomatic large artery stenosis. TTE revealed cardiac abnormalities in 46% of the remaining patients (mean age: 65.1 y, 52% female, 58% white). The most common findings were dilated left atrium (29%), patent foramen ovale (14%), and atrial septal aneurysm (7.7%). Anticoagulants were prescribed in 3 (4.6%) patients on the basis of TTE results. TTE identified potential cardioembolic sources in almost half of the patients, but altered antithrombotic management in only 5% of patients with SSSI.

Analysis of population inquiry on practices for ultraviolet radiation protection.

UV radiation exposure is one of the key modifiable risk factors for skin cancer. Hence, patient education regarding skin protection and sunscreen use is of tremendous importance to public health. To better understand patient practices regarding skin protection in a population level, we looked into the Internet search behavior of the US-based population. We investigated patient inquires on the United States Food and Drug Administration (FDA) announcements regarding sunscreen use by quantifying search terms such as "broad spectrum sunscreen", "sunscreen" and "sunblock" with Google Trends, a novel methodology for understanding internet search practices. Our findings show that "broad spectrum sunscreen" searches were significantly increased post 2011 FDA announcements, which suggest increased public awareness regarding the importance of broad spectrum protection. It is encouraging these preliminary results indicate that skin protection practices are being increasingly investigated by the general public and may serve as a novel approach for identifying areas of improvement regarding patient education on the reduction of the risk for skin cancer.

The microbiota regulates susceptibility to Fas-mediated acute hepatic injury.

Whereas a significant role for intestinal microbiota in affecting the pathogenesis and progression of chronic hepatic diseases is well documented, the contribution of the intestinal flora to acute liver injury has not been extensively addressed. Elucidating the influence of the intestinal microbiota on acute liver inflammation would be important for better understanding the transition from acute injury to chronic liver disease. Using the Concanavalin A (ConA)-induced liver injury model in laboratory mice, we show that the severity of acute hepatic damage varies greatly among genetically identical mice raised in different environments and harboring distinct microbiota. Through reconstitution of germ-free (GF) mice, and the co-housing of conventional mice, we provide direct evidence that manipulation of the intestinal flora alters susceptibility to ConA-induced liver injury. Through deep sequencing of the fecal microbiome, we observe that the relative abundance of Ruminococcaceae, a Gram(+) family within the class Clostridia, but distinct from segmented filamentous bacteria, is positively associated with the degree of liver damage. Searching for the underlying mechanism(s) that regulate susceptibility to ConA, we provide evidence that the extent of liver injury following triggering of the death receptor Fas varies greatly as a function of the microbiota. We demonstrate that the extent of Fas-induced liver injury increases in GF mice after microbiota reconstitution, and decreases in conventionally raised mice following reduction in intestinal bacterial load, by antibiotic treatment. We also show that the regulation of sensitivity to Fas-induced liver injury is dependent upon the toll-like receptor signaling molecule MyD88. In conclusion, the status and composition of the intestinal microbiota determine the susceptibility to ConA-induced acute liver injury. The microbiota acts as a rheostat, actively modulating the extent of liver damage in response to Fas triggering.

Liver inflammation in a mouse model of Th1 hepatitis despite the absence of invariant NKT cells or the Th1 chemokine receptors CXCR3 and CCR5.

The specific mechanisms that mediate CD4(+) T-cell-mediated liver injury have not been fully elucidated. CD4(+) invariant natural killer T (iNKT) cells are required for liver damage in some mouse models of hepatitis, while the chemokine receptors CXCR3 and CCR5 are considered dominant Th1 chemokine receptors involved in Th1 trafficking in inflammatory conditions. BALB/c-Tgfb1(-/-) mice spontaneously develop Th1 hepatitis. Here, we directly test the hypotheses that iNKT cells or the Th1-cell chemokine receptors CXCR3 and CCR5 are required for development of liver disease in Tgfb1(-/-) mice. Tgfb1(-/-) mouse livers exhibited significant increases in iNKT cells and in ligands for CXCR3 or CCR5. Tgfb1(-/-) mice were rendered deficient in iNKT cells, CXCR3, CCR5, or both CXCR3 and CCR5, by cross-breeding with appropriate knockout mice. Tgfb1(-/-) mice developed severe liver injury, even in the absence of functional CD1d/iNKT cells, CXCR3, CCR5, or both CXCR3 and CCR5. Liver CD4(+) T cells accumulated to high numbers, and spleen CD4(+) T-cell numbers declined, regardless of the functionality of the CXCR3/CCR5 response pathways. Similarly, dendritic cells and macrophages accumulated in Tgfb1(-/-) livers even when CXCR3 and CCR5 were knocked out. Th1-associated cytokines (IFN-γ, TNF-α, IL-2) and chemokines (CXCL9, CXCL10) were strongly overexpressed in Tgfb1(-/-) mice despite knockouts in CD1d, CXCR3, or CCR5. These studies indicate that the cellular and biochemical basis for CD4(+) T-cell-mediated injury in liver can be complex, with myriad pathways potentially involved.

Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants.

Human Immunodeficiency Virus-type 1 (HIV-1) binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA) transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV-1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA) sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α), a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.