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 microM 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.

The effect of old age on liver oxygenation and the hepatic expression of VEGF and VEGFR2.

In old age, the liver contains less ATP and hypoxia-responsive genes are upregulated. Age-related changes in hepatic perfusion and the liver sinusoidal endothelial cell (LSEC) could contribute to this altered hepatic oxygen-dependent metabolism by causing intrahepatocytic hypoxia. Furthermore, age-related changes in the LSEC ('pseudocapillarization') have been partially induced by ATP depletion. To investigate whether there is intracellular hypoxia in the old rat liver, pimonidazole immunohistochemistry in intact livers and ATP levels in isolated LSECs were studied from young and old rats. There were no age-related changes. To determine whether defenestration of the LSEC could impair oxygen diffusion, pimonidazole immunohistochemistry was performed in rats treated with poloxamer 407. Despite defenestration, there was no change in pimonidazole staining. Immunohistochemistry was then performed to determine whether there are age-related changes in VEGF and VEGFR2. VEGF staining was not associated with age. However, there was an increase in perisinusoidal VEGFR2 expression with increasing age. In conclusion, liver hypoxia does not occur in old age and LSEC pseudocapillarization does not constitute an oxygen-diffusion barrier. There are no age-related changes in VEGF expression but an increase in perisinusoidal VEGFR2 expression, which has implications for the effects of aging on the hepatic sinusoid.

Effects of old age on hepatocyte oxygenation.

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.

Low microsatellite instability is associated with poor prognosis in stage C colon cancer.

PURPOSE: The significance of low microsatellite instability (MSI-L) in colorectal cancer is poorly understood. No clear biologic distinction has been found between MSI-L and microsatellite stable (MSS) colorectal cancer, and these two phenotypes are usually combined when analyzed against the well-defined high MSI (MSI-H) phenotype. Evidence is emerging that an O(6)-methylguanine DNA methyltransferase (MGMT) gene defect is associated with MSI-L. Therefore, to further define this phenotype, we undertook a detailed analysis of the prognostic significance of MSI-L and loss of MGMT expression in colon cancer. PATIENTS AND METHODS: The study cohort was 183 patients with clinicopathologic stage C colon cancer who had not received adjuvant therapy. We analyzed MSI status, MGMT, and mismatch repair protein expression, as well as MGMT and p16 promoter hypermethylation. RESULTS: We showed that MSI-L defines a group of patients with poorer survival (P = .026) than MSS patients, and that MSI-L was an independent prognostic indicator (P = .005) in stage C colon cancer. Loss of MGMT protein expression was associated with the MSI-L phenotype but was not a prognostic factor for overall survival in colon cancer. p16 methylation was significantly less frequent in MSI-L than in MSI-H and MSS tumors and was not associated with survival. CONCLUSION: MSI-L characterizes a distinct subgroup of stage C colon cancer patients, including the MSI-L subset of proximal colon cancer, who have a poorer outcome. Neither the MGMT defect nor p16 methylation are likely to contribute to the worse prognosis of the MSI-L phenotype.

Epithelial and stromal cell urokinase plasminogen activator receptor expression differentially correlates with survival in rectal cancer stages B and C patients.

Urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for colorectal cancer (CRC) patient survival. However, CRC uPAR expression remains controversial, especially regarding cell types where uPAR is overexpressed (e.g., epithelium (uPARE) or stroma-associated cells (uPARS)) and associated prognostic relevance. In this study, two epitope-specific anti-uPAR monoclonal antibodies (MAbs) could discriminate expression of uPARE from uPARS and were used to examine this association with survival of stages B and C rectal cancer (RC) patients. Using immunohistochemistry, MAbs #3937 and R4 were used to discriminate uPARE from uPARS respectively in the central and invasive frontal regions of 170 stage B and 179 stage C RC specimens. Kaplan-Meier and Cox regression analyses were used to determine association with survival. uPAR expression occurred in both epithelial and stromal compartments with differential expression observed in many cases, indicating uPARE and uPARS have different cellular roles. In the central and invasive frontal regions, uPARE was adversely associated with overall stage B survival (HR = 1.9; p = 0.014 and HR = 1.5; p = 0.031, respectively) reproducing results from previous studies. uPARS at the invasive front was associated with longer stage C survival (HR = 0.6; p = 0.007), reflecting studies demonstrating that macrophage peritumoural accumulation is associated with longer survival. This study demonstrates that different uPAR epitopes should be considered as being expressed on different cell types during tumour progression and at different stages in RC. Understanding how uPARE and uPARS expression affects survival is anticipated to be a useful clinical prognostic marker of stages B and C RC.

Correlations between integrin ανß6 expression and clinico-pathological features in stage B and stage C rectal cancer.

Integrin ανß6 is highly expressed in a range of human cancers and frequently correlates with patient survival. This study examines correlations between ανß6 expression and patient clinico-pathological features in Stage B and Stage C rectal cancer, including overall survival. Expression of ανß6 was measured in 362 Stage B or C rectal cancer tissue samples at the tumour central region, invasive tumour front and adjacent non-neoplastic mucosa using immunohistochemistry. Distribution of ανß6 was found to be significantly higher at the invasive front compared to central regions of the tumour (p<0.001) or adjacent non-neoplastic mucosa (p<0.001) suggesting ανß6 plays a role in tumour cell invasion. However, integrin ανß6 expression was not associated with clinico-pathological features or overall survival indicating it is not an independent prognostic marker differentiating Stage B or C rectal cancer. Previous ανß6 studies have suggested the expression of ανß6 is involved in the earlier stages (i.e. Stages A/B) of tumour progression rather than the later stages (i.e. Stages C/D). However, our study has revealed that in rectal cancer ανß6 expression does not increase between Stages B and C, but may occur earlier, namely before or during Stage B cancer.

A case of severe 1,25-dihydroxyvitamin D-mediated hypercalcemia due to a granulomatous disorder.

BACKGROUND: Profound hypercalcemia is usually due to underlying malignancy. CASE: We describe a case of granulomatous myositis presenting with extreme hypercalcemia of 20.1 mg/dl and generalized weakness that did not resolve despite rapid correction of serum calcium. The disease process was unmasked by cholecalciferol supplementation. Initial search for a malignant process yielded no diagnosis, but an elevated 1,25-dihydroxyvitamin D level, in the setting of a suppressed PTH and undetectable PTHrP, pointed to the presence of excessive 1α-hydroxylase activity. METHODS AND RESULTS: Biopsy of the vastus lateralis muscle showed extensive granulomatous myositis. Immunohistochemical staining for 1α-hydroxylase was localized to the multinucleated giant cells and histiocytes. Musculoskeletal magnetic resonance imaging showed involvement of proximal muscle groups of both thighs and upper limbs. CONCLUSION: Measurement of vitamin D metabolites is pivotal in diagnosing 1,25-dihydroxyvitamin D-mediated hypercalcemia. Granulomatous disease can occasionally cause profound hypercalcemia and needs to be considered in the differential diagnosis. 1,25-Dihydroxyvitamin D-mediated hypercalcemia is responsive to glucocorticoid therapy.

Identification of distinctive protein expression patterns in colorectal adenoma.

PURPOSE: As a pre-malignant precursor, adenoma provides an ideal tissue for proteome profiling to investigate early colorectal cancer development and provide possible targets for preventive interventions. The aim of this study was to identify patterns of differential protein expression that distinguish colorectal adenoma from normal tissue. EXPERIMENTAL DESIGN: Twenty paired samples of adenoma and normal mucosa were analysed by 2-DE and MALDI-TOF/TOF MS to detect proteins with ≥2-fold differential expression. RESULTS: Four proteins were up-regulated in adenoma (Annexin A3, S100A11, S100P and eIF5A-1) and three were down-regulated (Galectin-1, S100A9 and FABPL). S100P, galectin-1, S100A9 and FABPL expression was localised by immunohistochemistry. CONCLUSIONS AND CLINICAL RELEVANCE: Distinctive patterns of in vivo protein expression in colorectal adenoma were identified for the first time. These proteins have important functions in cell differentiation, proliferation and metabolism, and may play a crucial role in early colorectal carcinogenesis. The ability to recognise premalignant lesions may have important applications in cancer prevention.