Rajkumar Cheluvappa

Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid

Age-related changes in the hepatic sinusoid, called pseudocapillarization, may contribute to the pathogenesis of dyslipidemia. Caloric restriction (CR) is a powerful model for the study of aging because it extends lifespan. We assessed the effects of CR on the hepatic sinusoid to determine whether pseudocapillarization is preventable and hence a target for the prevention of age-related dyslipidemia. Livers from young (6 months) and old (24 months) CR and ad libitum fed (AL) F344 rats were examined using electron microscopy and immunohistochemistry. In old age, there was increased thickness of the liver sinusoidal endothelium and reduced endothelial fenestration porosity. In old CR rats, endothelial thickness was less and fenestration porosity was greater than in old AL rats. Immunohistochemistry showed that CR prevented age-related decrease in caveolin-1 expression and increase in peri-sinusoidal collagen IV staining, but did not alter the age-related increase of von Willebrands factor. CR reduces age-related pseudocapillarization of the hepatic sinusoid and correlates with changes in caveolin-1 expression.

Pathogenesis of the hyperlipidemia of Gram-negative bacterial sepsis may involve pathomorphological changes in liver sinusoidal endothelial cells.

The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common opportunistic pathogens, especially after liver transplantation. Pathophysiological alterations of liver sinusoidal endothelial cells (LSECs) have far-reaching repercussions on the liver and on metabolism. LSECs are perforated with fenestrations, pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) and the P. aeruginosa toxin, pyocyanin, have marked effects on LSECs. Initial loss of LSEC porosity (defenestration) induced by P. aeruginosa pyocyanin and LPS may confer subsequent immune tolerance to circulating bacterial antigens and toxins. This review collates the known immune responses of the liver to Gram-negative bacterial toxins, with a focus on LSECs. Hyperlipidemia is an important response to Gram-negative bacterial sepsis. The mechanisms proposed for sepsis-associated hyperlipidemia include tissue lipoprotein lipase inhibition and upregulated hepatic triglyceride production. In this review, we propose defenestration of the LSECs by bacterial toxins as an additional mechanism for the hyperlipidemia of sepsis. Given the role of LSECs in hyperlipidemia and liver allograft rejection, LSEC changes induced by P. aeruginosa toxins including LPS and pyocyanin may have significant clinical implications.

Autophagy suppression by appendicitis and appendectomy protects against colitis.

Background:When done at a young age, appendicitis followed by appendectomy (AA) offers protection against ulcerative colitis development in later life. We developed the first ever murine AA model. Using this model, we showed earlier that previous AA ameliorated colitis. We aimed to determine whether autophagy genes contribute to the anti-colitis protection conferred by AA, and if so, to delineate the autophagy-linked genes involved in this. Methods:Mice with 2 laparotomies each served as controls (sham–sham). Distal colons were harvested (4 AA-group colons, 4 sham–sham group colons), and RNA extracted from each. The RNA was taken through microarray analysis or reverse transcription-polymerase chain reaction validation. Gene set enrichment analysis software was used to analyze the microarray data. Results:Out of 28 key autophagy-related genes investigated (VPS15, VPS34, FIP200, ATG03, ATG04A, ATG04B, ATG05, ATG07, ATG10, ATG12, ATG13b, ATG14, ATG16L1, BECN1, GABARAPL1, IRGM1, IRGM2, LAMP2, LC3A, LC3B, RAB7A, UVRAG, NOD2, XBP1, LRRK2, ULK1, ULK2, PTPN2), 7 have genetic associations with inflammatory bowel diseases (ATG16L1, IRGM1, NOD2, XBP1, LRRK2, ULK1, PTPN2). There was slight upregulation of IRGM1, FIP200, and ATG04A (P < 0.05), but no variations with the other 25 genes. In contrast, gene set enrichment analysis revealed that AA downregulated 74 gene sets (associated with 28 autophagy genes) while upregulating only 5 (false discovery rate <5%; P < 0.001) gene sets. Additionally, 22 gene sets associated with the 7 autophagy + inflammatory bowel disease-associated genes were downregulated by AA, whereas only 3 were upregulated. The genes with maximum AA-induced gene set suppression were VPS15, LAMP2, LC3A, XBP1, and ULK1. Conclusions:AA induces profound autophagy suppression in the distal colon. The AA-induced upregulation of individual genes (IRGM1, FIP200, ATG04A) could be a reflection of complex compensatory changes or the initial abnormality that led to the pronounced autophagy suppression. Autophagy suppression by AA may induce lesser antigen processing, leading to lesser cross-reactive immunity between microbes and self-antigens, and subsequent amelioration of colitis.

The effect of Pseudomonas aeruginosa virulence factor, pyocyanin, on the liver sinusoidal endothelial cell

1 Dieulafoy G. Exulceratio simplex. L’intervention chirurgicale dans les hématémèses foudroyantes consécutives à l’exulcération simple de l’estomac. Bull. Acad. Med. 1898; 39: 49–84. 2 Reilly HF, Al-Kawas FH. Dieulafoy’s lesion. Diagnosis and management. Dig. Dis. Sci. 1991; 36: 1702–7. 3 Lee YT, Walmsley RS, Leong RW, Sung JJ. Dieulafoy’s lesion. Gastrointest. Endosc. 2003; 58: 236–43. 4 Juler GL, Labitzke HG, Lamb R, Allen R. The pathogenesis of Dieulafoy’s gastric erosion. Am. J. Gastroenterol. 1984; 79: 195–200. 5 Willemsen PJ, Vanderveken ML, De Caluwe DO, Tielliu IF. Hemobilia: a rare complication of chelecystitis and cholecystolithiasis. Acta Chir. Belg. 1996; 96: 93–4.

T helper type 17 pathway suppression by appendicitis and appendectomy protects against colitis

Appendicitis followed by appendectomy (AA) at a young age protects against inflammatory bowel disease (IBD). We wanted to characterize the role of the T helper type 17 (Th17) system involved in this protective effect. AA was performed on 5‐week‐old male BALB/c mice and distal‐colon samples were harvested. Mice with two laparotomies each served as sham–sham (SS) controls. RNA was extracted from four individual colonic samples per group (AA and SS groups) and each sample microarray‐analysed and reverse transcription–polymerase chain reaction (RT–PCR)‐validated. Gene‐set enrichment analysis (GSEA) showed that the Th17 recruitment factor gene CCL20 was significantly suppressed at both 3 days post‐AA and 28 days post‐AA. Although Th17 cell development differentiation factor genes TGF‐β2 and TGF‐β3 were significantly up‐regulated 3 days post‐AA, GSEA 28 days post‐AA showed that AA down‐regulated 29 gene‐sets associated with TGF‐β1, TGF‐β2 and TGF‐β3 in contrast to none up‐regulated with any of these genes. GSEA showed substantial down‐regulation of gene‐sets associated with Th17 lymphocyte recruitment, differentiation, activation and cytokine expression in the AA group 28 days post‐AA. We conclude that Th17‐system cytokines are kept under control by AA via down‐regulation of proinflammatory CCL20, a rapid down‐regulation of pro‐Th17 cell differentiation genes TGF‐β2 and TGF‐β3, suppression of RORC‐associated gene‐sets, increased protective STAT1 expression and suppression of 81 ‘pro‐Th17’ system gene‐sets. AA suppresses the Th17 pathway leading to colitis amelioration. Further characterization of Th17‐associated genes and biological pathways will assist in the development of better therapeutic approaches in IBD management.

Protective pathways against colitis mediated by appendicitis and appendectomy

Appendicitis followed by appendectomy (AA) at a young age protects against inflammatory bowel disease (IBD). Using a novel murine appendicitis model, we showed that AA protected against subsequent experimental colitis. To delineate genes/pathways involved in this protection, AA was performed and samples harvested from the most distal colon. RNA was extracted from four individual colonic samples per group (AA group and double‐laparotomy control group) and each sample microarray analysed followed by gene‐set enrichment analysis (GSEA). The gene‐expression study was validated by quantitative reverse transcription–polymerase chain reaction (RT–PCR) of 14 selected genes across the immunological spectrum. Distal colonic expression of 266 gene‐sets was up‐regulated significantly in AA group samples (false discovery rates < 1%; P‐value < 0·001). Time–course RT–PCR experiments involving the 14 genes displayed down‐regulation over 28 days. The IBD‐associated genes tnfsf10, SLC22A5, C3, ccr5, irgm, ptger4 and ccl20 were modulated in AA mice 3 days after surgery. Many key immunological and cellular function‐associated gene‐sets involved in the protective effect of AA in experimental colitis were identified. The down‐regulation of 14 selected genes over 28 days after surgery indicates activation, repression or de‐repression of these genes leading to downstream AA‐conferred anti‐colitis protection. Further analysis of these genes, profiles and biological pathways may assist in developing better therapeutic strategies in the management of intractable IBD.

Liver sinusoidal endothelial cells and acute non-oxidative hepatic injury induced by Pseudomonas aeruginosa pyocyanin

The liver sinusoidal endothelial cell (LSEC) is damaged by many toxins, including oxidants and bacterial toxins. Any effect on LSECs of the Pseudomonas aeruginosa virulence factor, pyocyanin, may be relevant for systemic pseudomonal infections and liver transplantation. In this study, the effects of pyocyanin on in vivo rat livers and isolated LSECs were assessed using electron microscopy, immunohistochemistry and biochemistry. In particular, the effect on fenestrations, a crucial morphological aspect of LSECs was assessed. Pyocyanin treatment induced a dose‐dependent reduction in fenestrations in isolated LSECs. In the intact liver, intraportal injection of pyocyanin (11.9 μM in blood) was associated with a reduction in endothelial porosity from 3.4 ± 0.2% (n = 5) to 1.3 ± 0.1% (n = 7) within 30 min. There were decreases in both diameter and frequency of fenestrations in the intact endothelium. There was also a decrease in endothelial thickness from 175.8 ± 5.8 to 156.5 ± 4.0 nm, an endothelial pathology finding previously unreported. Hepatocyte ultrastructure, liver function tests and immunohistochemical markers of oxidative stress (3‐nitrotyrosine and malondialdehyde) were not affected. Pyocyanin induces significant ultrastructural changes in the LSEC in the absence of immunohistochemical evidence of oxidative stress or hepatocyte injury pointing to a novel mechanism for pyocyanin pathogenesis.

A comprehensive evaluation of bladder cancer epidemiology and outcomes in Australia

ObjectiveTo review bladder cancer statistics and management in Australia and identify gaps for future work here.MethodsEvidence was reviewed from GLOBOCAN 2008v2.0, Pubmed, and conference presentations. We also use data from reports from Cancer Council Australia, State Cancer Councils, and Australian Institute of Health and Welfare.ResultsThe incidence and mortality rates of bladder cancer in Australia closely parallel those of other developed countries. Bladder cancer was the 8th most common cause of cancer in men, and the 17th most common cause of cancer in women. Bladder cancer was the 13th most common cause of cancer death in men, and the 17th most common cause of cancer death in women. We briefly review the evidence regarding causality, including nutritional, occupational, and environmental factors. We compare Australian incidence and mortality rates internationally, by state/territory, by socioeconomic strata, and by geographical regions. Importantly, we review evidence on the quality of bladder cancer management in Australia.ConclusionsThe geographical, regional, and socioeconomic differences in Australian bladder cancer statistics may be associated with different patterns of diagnosis and treatment.ImplicationsThe quality of bladder cancer surveillance and cystectomies in Australia requires improvement to conform to global standards and to improve decreasing survival rates.

Pseudomonas aeruginosa and the hyperlipidaemia of sepsis

Pathophysiological alterations of liver sinusoidal endothelial cells (LSECs) impact liver and metabolism. LSEC fenestrations are pores facilitating lipoproteins and macromolecule transfer between blood and hepatocytes. The Gram negative bacterium Pseudomonas aeruginosa is one of the most common opportunistic nosocomial pathogens, especially in post-liver transplant recipients. Gram negative bacterial endotoxin (lipopolysaccharide, LPS) and the P. aeruginosa toxin, pyocyanin, have marked effects on LSECs, including loss of porosity (defenestration). Currently proposed mechanisms for sepsis-hyperlipidaemia, an important response to Gram negative bacterial sepsis, include tissue lipoprotein-lipase inhibition and increased hepatic triglyceride production. Owing to the well-substantiated role of LSECs in liver-allograft rejection and hyperlipidaemia, we propose defenestration of the LSEC is an additional cellular mechanism for sepsis-hyperlipidaemia, including pseudomonal sepsis post-liver transplantation.

Effects of Old Age on Hepatocyte Oxygenation

Abstract:  Hepatic phase I drug metabolism is diminished in old age. It has been suggested that hepatocyte hypoxia and impaired bioenergetics in old age may contribute to this aging change. Therefore, we sought to determine whether old age was associated with in vivo hypoxia in the aged rat liver. Immunohistochemical studies with the nitroimidazole hypoxia marker, pimonidazole, were carried out in livers from young and old rats. Preliminary studies were performed on four young (4‐month‐old) and six old (2‐year‐old) F344 rats to directly visualize the distribution and intensity of pimonidazole staining. There were no significant differences in the distribution or in the intensity of pimonidazole immunohistochemical staining between young and aged rat livers. In conclusion, no major changes in hepatocyte oxygenation were seen in the aged rat liver, and the ATP changes are unlikely to be secondary to hepatocyte hypoxia or impaired oxygen diffusion into the liver. It is thus more likely that age‐related reduction in liver ATP is attributable to mitochondrial dysfunction.