Pentamidine blocks hepatotoxic injury in mice.

Toxin-induced liver diseases lack effective therapies despite increased understanding of the role factors such as an overactive innate immune response play in the pathogenesis of this form of hepatic injury. Pentamidine is an effective antimicrobial agent against several human pathogens, but studies have also suggested that this drug inhibits inflammation. This potential anti-inflammatory mechanism of action, together with the development of a new oral form of pentamidine isethionate VLX103, led to investigations of the effectiveness of this drug in the prevention and treatment of hepatotoxic liver injury. Pretreatment with a single injection of VLX103 in the d-galactosamine (GalN) and lipopolysaccharide (LPS) model of acute, fulminant liver injury dramatically decreased serum alanine aminotransferase levels, histological injury, the number of terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells and mortality compared with vehicle-injected controls. VLX103 decreased GalN/LPS induction of tumor necrosis factor (TNF) but had no effect on other proinflammatory cytokines. VLX103 prevented the proinflammatory activation of cultured hepatic macrophages and partially blocked liver injury from GalN/TNF. In GalN/LPS-treated mice, VLX103 decreased activation of both the mitochondrial death pathway and downstream effector caspases 3 and 7, which resulted from reduced c-Jun N-terminal kinase activation and initiator caspase 8 cleavage. Delaying VLX103 treatment for up to 3 hours after GalN/LPS administration was still remarkably effective in blocking liver injury in this model. Oral administration of VLX103 also decreased hepatotoxic injury in a second more chronic model of alcohol-induced liver injury, as demonstrated by decreased serum alanine and aspartate aminotransferase levels and numbers of TUNEL-positive cells. CONCLUSION: VLX103 effectively decreases toxin-induced liver injury in mice and may be an effective therapy for this and other forms of human liver disease. (Hepatology 2017;66:922-935).

Macrophage autophagy limits acute toxic liver injury in mice through down regulation of interleukin-1ß.

BACKGROUND & AIMS: Overactivation of the innate immune response underlies many forms of liver injury including that caused by hepatotoxins. Recent studies have demonstrated that macrophage autophagy regulates innate immunity and resultant tissue inflammation. Although hepatocyte autophagy has been shown to modulate hepatic injury, little is known about the role of autophagy in hepatic macrophages during the inflammatory response to acute toxic liver injury. Our aim therefore was to determine whether macrophage autophagy functions to down regulate hepatic inflammation. METHODS: Mice with a LysM-CRE-mediated macrophage knockout of the autophagy gene ATG5 were examined for their response to toxin-induced liver injury from D-galactosamine/lipopolysaccharide (GalN/LPS). RESULTS: Knockout mice had increased liver injury from GalN/LPS as determined by significant increases in serum alanine aminotransferase, histological evidence of liver injury, positive terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, caspase activation and mortality as compared to littermate controls. Levels of proinflammatory tumor necrosis factor and interleukin (IL)-6 hepatic mRNA and serum protein were unchanged, but serum IL-1ß was significantly increased in knockout mice. The increase in serum IL-1ß was secondary to elevated hepatic caspase 1 activation and inflammasome-mediated cleavage of pro-IL-1ß to its active form. Cultured hepatic macrophages from GalN/LPS-treated knockout mice had similarly increased IL-1ß production. Dysregulation of IL-1ß was the mechanism of increased liver injury as an IL-1 receptor antagonist prevented injury in knockout mice in concert with decreased neutrophil activation. CONCLUSIONS: Macrophage autophagy functions to limit acute toxin-induced liver injury and death by inhibiting the generation of inflammasome-dependent IL-1ß.

Autophagy confers resistance to lipopolysaccharide-induced mouse hepatocyte injury.

During sepsis, bacterial products, particularly LPS, trigger injury in organs such as the liver. This common condition remains largely untreatable, in part due to a lack of understanding of how high concentrations of LPS cause cellular injury. In the liver, the lysosomal degradative pathway of autophagy performs essential hepatoprotective functions and is induced by LPS. We, therefore, examined whether hepatocyte autophagy protects against liver injury from septic levels of LPS. Mice with an inducible hepatocyte-specific knockout of the critical autophagy gene Atg7 were examined for their sensitivity to high-dose LPS. Increased liver injury occurred in knockout mice, as determined by significantly increased serum alanine aminotransferase levels, histological evidence of liver injury, terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, and effector caspase-3 and -7 activation. Hepatic inflammation and proinflammatory cytokine induction were unaffected by the decrease in hepatocyte autophagy. Although knockout mice had normal NF-κB signaling, hepatic levels of Akt1 and Akt2 phosphorylation in response to LPS were decreased. Cultured hepatocytes from knockout mice displayed a generalized defect in Akt signaling in response to multiple stimuli, including LPS, TNF, and IL-1ß. Akt activation mediates hepatocyte resistance to TNF cytotoxicity, and anti-TNF antibodies significantly decreased LPS-induced liver injury in knockout mice, indicating that the loss of autophagy sensitized to TNF-dependent liver damage. Hepatocyte autophagy, therefore, protects against LPS-induced liver injury. Conditions such as aging and steatosis that impair hepatic autophagy may predispose to poor outcomes from sepsis through this mechanism.

Orthovoltage X-Rays Exhibit Increased Efficacy Compared with γ-Rays in Preclinical Irradiation.

Radionuclide irradiators (137Cs and 60Co) are commonly used in preclinical studies ranging from cancer therapy to stem cell biology. Amidst concerns of radiological terrorism, there are institutional initiatives to replace radionuclide sources with lower energy X-ray sources. As researchers transition, questions remain regarding whether the biological effects of γ-rays may be recapitulated with orthovoltage X-rays because different energies may induce divergent biological effects. We therefore sought to compare the effects of orthovoltage X-rays with 1-mm Cu or Thoraeus filtration and 137Cs γ-rays using mouse models of acute radiation syndrome. Following whole-body irradiation, 30-day overall survival was assessed, and the lethal dose to provoke 50% mortality within 30-days (LD50) was calculated by logistic regression. LD50 doses were 6.7 Gy, 7.4 Gy, and 8.1 Gy with 1-mm Cu-filtered X-rays, Thoraeus-filtered X-rays, and 137Cs γ-rays, respectively. Comparison of bone marrow, spleen, and intestinal tissue from mice irradiated with equivalent doses indicated that injury was most severe with 1-mm Cu-filtered X-rays, which resulted in the greatest reduction in bone marrow cellularity, hematopoietic stem and progenitor populations, intestinal crypts, and OLFM4+ intestinal stem cells. Thoraeus-filtered X-rays provoked an intermediate phenotype, with 137Cs showing the least damage. This study reveals a dichotomy between physical dose and biological effect as researchers transition to orthovoltage X-rays. With decreasing energy, there is increasing hematopoietic and intestinal injury, necessitating dose reduction to achieve comparable biological effects. SIGNIFICANCE: Understanding the significance of physical dose delivered using energetically different methods of radiation treatment will aid the transition from radionuclide γ-irradiators to orthovoltage X-irradiators.

Differential effects of JNK1 and JNK2 inhibition on murine steatohepatitis and insulin resistance.

UNLABELLED: Activation of c-Jun N-terminal kinase (JNK) has been implicated as a mechanism in the development of steatohepatitis. This finding, together with the reported role of JNK signaling in the development of obesity and insulin resistance, two components of the metabolic syndrome and predisposing factors for fatty liver disease, suggests that JNK may be a central mediator of the metabolic syndrome and an important therapeutic target in steatohepatitis. To define the isoform-specific functions of JNK in steatohepatitis associated with obesity and insulin resistance, the effects of JNK1 or JNK2 ablation were determined in developing and established steatohepatitis induced by a high-fat diet (HFD). HFD-fed jnk1 null mice failed to develop excessive weight gain, insulin resistance, or steatohepatitis. In contrast, jnk2(-/-) mice fed a HFD were obese and insulin-resistant, similar to wild-type mice, and had increased liver injury. In mice with established steatohepatitis, an antisense oligonucleotide knockdown of jnk1 decreased the amount of steatohepatitis in concert with a normalization of insulin sensitivity. Knockdown of jnk2 improved insulin sensitivity but had no effect on hepatic steatosis and markedly increased liver injury. A jnk2 knockdown increased hepatic expression of the proapoptotic Bcl-2 family members Bim and Bax and the increase in liver injury resulted in part from a Bim-dependent activation of the mitochondrial death pathway. CONCLUSION: JNK1 and JNK2 both mediate insulin resistance in HFD-fed mice, but the JNK isoforms have distinct effects on steatohepatitis, with JNK1 promoting steatosis and hepatitis and JNK2 inhibiting hepatocyte cell death by blocking the mitochondrial death pathway.

Systematic Selective Sampling of Cholecystectomy Specimens Is Adequate to Detect Incidental Gallbladder Adenocarcinoma.

Many gallbladder adenocarcinomas (ACs) are detected incidentally in routine cholecystectomy specimens, yet sampling practices vary when intestinal metaplasia (IM) or dysplasia are found via routine sampling. Our practice has been to submit 5 additional sections when IM is found, but cases with dysplasia are entirely submitted. We sought to determine an appropriate sampling protocol when encountering these findings. We retrospectively identified cholecystectomy specimens with these features over a 26-month period, yielding 48 of 4059 (1%) cases. Four pathologists independently classified the (2 longitudinal and 1 cystic duct margin) original sections into 1 of 3 categories (IM, low-grade dysplasia [LGD] or high-grade dysplasia [HGD]); initial findings were correlated with final diagnoses. Sixteen (33%) cases had additional findings upon further sampling, including LGD (n=10) or HGD (n=4) and AC (n=2). HGD always accompanied malignancy. We prospectively analyzed 39 of 3133 (1%) additional cholecystectomy specimens, initially submitting the same routine sections. We submitted 5 random sections from cases with IM. Cases with LGD were first examined with 1 additional section per centimeter. All remaining tissue was submitted in all of these cases and separately reviewed. Cases with HGD were entirely submitted as both test cases with HGD in initial sections ultimately showed carcinoma. This protocol detected all cases of HGD and AC. Patients with clear cystic duct margins did not experience neoplastic progression, even if dysplasia was present elsewhere. We conclude gallbladders with HGD should be entirely submitted, LGD may be representatively sampled, and routine sampling is adequate for IM.

Impaired macrophage autophagy increases the immune response in obese mice by promoting proinflammatory macrophage polarization.

Recent evidence that excessive lipid accumulation can decrease cellular levels of autophagy and that autophagy regulates immune responsiveness suggested that impaired macrophage autophagy may promote the increased innate immune activation that underlies obesity. Primary bone marrow-derived macrophages (BMDM) and peritoneal macrophages from high-fat diet (HFD)-fed mice had decreased levels of autophagic flux indicating a generalized impairment of macrophage autophagy in obese mice. To assess the effects of decreased macrophage autophagy on inflammation, mice with a Lyz2-Cre-mediated knockout of Atg5 in macrophages were fed a HFD and treated with low-dose lipopolysaccharide (LPS). Knockout mice developed systemic and hepatic inflammation with HFD feeding and LPS. This effect was liver specific as knockout mice did not have increased adipose tissue inflammation. The mechanism by which the loss of autophagy promoted inflammation was through the regulation of macrophage polarization. BMDM and Kupffer cells from knockout mice exhibited abnormalities in polarization with both increased proinflammatory M1 and decreased anti-inflammatory M2 polarization as determined by measures of genes and proteins. The heightened hepatic inflammatory response in HFD-fed, LPS-treated knockout mice led to liver injury without affecting steatosis. These findings demonstrate that autophagy has a critical regulatory function in macrophage polarization that downregulates inflammation. Defects in macrophage autophagy may underlie inflammatory disease states such as the decrease in macrophage autophagy with obesity that leads to hepatic inflammation and the progression to liver injury.

Valvulitis requiring triple valve surgery as an initial presentation of systemic lupus erythematosus.

Cardiac involvement is common in systemic lupus erythematosus. Classically, the term "verrucous endocarditis" was used to describe the noninfective vegetations seen on pathological inspection of heart valves. The wide use of echocardiography has led to increased frequency of detection of valve abnormalities, most commonly leaflet thickening. The vast majority of patients with systemic lupus erythematosus and valvular involvement are asymptomatic, with only a small minority progressing to hemodynamically significant pathology, generally after long disease duration. We report a patient with systemic lupus erythematosus and associated antiphospholipid syndrome, whose first presentation of disease consisted of severe, symptomatic valvular regurgitation of the mitral, aortic, and tricuspid valves requiring triple valve surgery.

Fulminant liver failure secondary to haemorrhagic dengue in an international traveller.

Dengue infections are caused by a single-stranded RNA virus, which has four serotypes (DEN 1-4); mosquitoes of the genus Aedes serve as vectors of transmission. Risk factors for dengue infection are related to both the host and virus. Age, gender, immune status, and genetic background of the host all contribute to the severity of dengue infection. Recently, international travel to endemic areas has also been identified as a major risk factor for both primary and secondary dengue infection. Dengue remains a diagnostic challenge, given its protean nature, ranging from mild febrile illness to profound shock. The most severe manifestation of dengue infection is dengue shock syndrome, which has an estimated mortality rate close to 50%. Dengue shock syndrome typically presents with increased anion gap metabolic acidosis, disseminated intravascular coagulation, severe hypotension, and jaundice. Liver involvement appears to occur more frequently when infections involve DEN-3 and DEN-4 serotypes. While hepatocellular damage has been reported previously in dengue infection, acute liver failure is an extremely rare occurrence in adults. We report a patient with dengue shock syndrome who presented with acute liver failure and hepatic encephalopathy after recent travel to an endemic area.

Surviving severe acute respiratory distress syndrome: utility of open-lung biopsy and large doses of dexamethasone.

The etiology of acute respiratory distress syndrome is wide and mortality is extremely high. We describe a patient dying from severe acute respiratory distress syndrome who had a tremendous recovery after receiving dexamethasone (1 g daily). This patient required positive end-expiratory pressure (up to 18 mm/Hg) and fractional inspiratory oxygen (up to 100%). Thirty-six hours after the large dose of corticosteroids, the respiratory mechanics and oxygenation were acceptable for extubation. Acute respiratory distress syndrome was proven and other etiologies of respiratory failure were ruled out by a bedside open-lung biopsy. The biopsy proven acute respiratory distress syndrome dramatically resolved with this salvage therapy. High-dose usage of corticosteroids for acute respiratory distress syndrome has tremendous potential.