Immunohistochemical Determination of Hepatic Cytochrome P-4502E1 in Formalin-Fixed, Paraffin-Embedded Sections

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. lmmunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 ± 0.3 vs. 0.8 ± 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 ± 2.1 vs. 7.9 ± 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (r_s= 0.67, p < 0.005) or intensity ± zonal staining scores (r_s= 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embedded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.     

Fracture risk in men with prostate cancer: A population‐based study

Fractures are increased among men with prostate cancer, especially those on androgen‐deprivation therapy (ADT), but few data are available on men with localized prostate cancer. The purpose of this investigation was to estimate fracture risk among unselected community men with prostate cancer and systematically assess associations with ADT and other risk factors for fracture. In a population‐based retrospective cohort study, 742 Olmsted County, MN, men with prostate cancer first diagnosed in 1990–1999 (mean age 68.2 ± 8.9 years) were followed for 6821 person‐years. We estimated cumulative fracture incidence, assessed relative risk by standardized incidence ratios, and evaluated risk factors in time‐to‐fracture regression models. All together, 482 fractures were observed in 258 men (71 per 1000 person‐years). Overall fracture risk was elevated 1.9‐fold, with an absolute increase in risk of 9%. Relative to rates among community men generally, fracture risk was increased even among men not on ADT but was elevated a further 1.7‐fold among ADT‐treated compared with untreated men with prostate cancer. The increased risk following various forms of ADT was accounted for mainly by associations with pathologic fractures (14% of all fractures). Among men not on ADT (62% of the cohort), more traditional osteoporosis risk factors were implicated. In both groups, underlying clinical characteristics prompting different treatments (indication bias) may have been partially responsible for the associations seen with specific therapies. To the extent that advanced‐stage disease and pathologic fractures account for the excess risk, the effectiveness of fracture prevention among men with prostate cancer may be limited. © 2011 American Society for Bone and Mineral Research     

Novel applications of external anal sphincter muscle sarcomere length to enhance the anal canal function

Background Our recent studies show that the external anal sphincter muscle (EAS) operates at a sarcomere length range which is below optimal. In this study, we tested the hypothesis that by surgically increasing sarcomere length and bringing it close to the optimal length, EAS muscle function and anal canal pressure can be enhanced. Methods Rabbits (n = 25) were anesthetized and subjected to either a sham or an EAS plication of different length by placing sutures at two locations, at a distance of 13%, 20%, 28%, or 35% of the circumferential length of the anal canal. Anal canal pressures were recorded before and after the plication. Anal canal was harvested and the EAS muscle sarcomere length was measured using laser diffraction. Key Results Electrical stimulation of the EAS muscle resulted in a stimulus‐dependent increase in the anal canal pressure (mmHg) and EAS muscle stress (mN mm^?2). A significant increase in maximal pressure (212 ± 13 after compared with 139 ± 22 before plication) as well as stress (166 ± 10 after as compared with 88 ± 14 before plication) was recorded at 20% plication length. Passive anal canal stress at 20% plication was not significantly different compared with the sham group. The mean sarcomere lengths with sham and 20% plication were 2.11 and 2.60 μm, respectively. Conclusions & Inferences EAS plication resulted in a length‐dependent increase in EAS muscle sarcomere length with an optimal sarcomere length at 20% plication. These considerations may help guide repair of anal sphincter muscle defects in the humans.     

Inflammation and Repair in Viral Hepatitis C

Hepatitis C viral infection (HCV) results in liver damage leading to inflammation and fibrosis of the liver and increasing rates of hepatic decompensation and hepatocellular carcinoma (HCC). However, the host’s immune response and viral determinants of liver disease progression are poorly understood. This review will address the determinants of liver injury in chronic HCV infection and the risk factors leading to rapid disease progression. We aim to better understand the factors that distinguish a relatively benign course of HCV from one with progression to cirrhosis. We will accomplish this task by discussion of three topics: (1) the role of cytokines in the adaptive immune response against the HCV infection; (2) the progression of fibrosis; and (3) the risk factors of co-morbidity with alcohol and human immunodeficiency virus (HIV) in HCV-infected individuals. Despite recent improvements in treating HCV infection using pegylated interferon alpha (PEGIFN-α) and ribavirin, about half of individuals infected with some genotypes, for example genotypes 1 and 4, will not respond to treatment or cannot be treated because of contraindications. This review will also aim to describe the importance of IFN-α-based therapies in HCV infection, ways of monitoring them, and associated complications.     

Beneficial effects versus toxicity of medium-chain triacylglycerols in rats with NASH

BACKGROUND/AIMS: Replacing long-chain triacylglycerols (LCT) with medium-chain triacylglycerols (MCT) reduces alcohol-induced liver injury. Because of the similarity of the pathogenesis of alcohol-induced liver damage and non-alcoholic steatohepatitis (NASH), our aim was to assess whether MCT is also beneficial in NASH. METHODS: We used a rat NASH model in which corn oil (35% of total calories) was isocalorically replaced with MCT. RESULTS: Partial replacement of LCT did not ameliorate hepatic fat accumulation, 4-hydroxynonenal, collagen type I and its mRNA but it increased TNF-alpha and its mRNA (p<0.001). However, in rats given the high-fat diet restricted to 2/3 of the amount they were consuming, these adverse effects decreased, with and without MCT including less liver steatosis and lower triacylglycerols, but without beneficial effects of MCT. When 70% of the fat calories were replaced with MCT with no LCT remaining in the diet, no steatosis developed and hepatic TNF-alpha was low. When all MCT were given with carbohydrates (instead of LCT) hepatic TNF-alpha also decreased (p<0.001). CONCLUSIONS: MCT are not hepatotoxic, provided the diet contains no significant amount of LCT. Total replacement of dietary LCT with MCT fed ad libitum is beneficial whereas partial replacement becomes hepatotoxic, unless the dietary intake is restricted.     

Frequency-plane analysis of normal and pathological ECG signals for disease identification

In this paper a frequency plane analysis of both normal and diseased ECG signals is performed specifically for disease identification. Image processing techniques are used to develop an automated data acquisition package of 12 lead ECG signals from paper records. A regeneration domain is also developed to check the captured pattern with the original wave shape. A QRS complex detector with an accuracy level ～98.4% in up to 30% signal to noise level is developed. Discrete Fourier transform (DFT) is performed to obtain the frequency spectrum of every ECG signal. Some interesting amplitude and phase response properties of chest lead V2, V3, V4, V6 and limb lead I, II, III, AVL, AVF are seen. Both amplitude and phase properties are different for normal and diseased subjects and can serve an important role in disease identification. A statistical analysis of amplitude property is carried out to show that this property is significantly different for normal and diseased subjects.     

Presumed circle area ratio of the prostate in a community‐based group of men

OBJECTIVE

To determine the normal values for the presumed circle area ratio (PCAR) in a group of community‐based men, and to determine whether PCAR is associated with specific urological outcomes.

PATIENTS AND METHODS

The study was a cross‐sectional analysis among 328 Caucasian men (94% participation) residing in Olmsted County, Minnesota, USA. The PCAR was measured during prostatic ultrasonography. Lower urinary tract symptoms (LUTS) were measured using the American Urologic Association Symptom Index. The peak urinary flow rate was measured by a uroflowmeter, and the postvoid residual volume (PVR) was assessed using the BladderScan^TM BVM 6500 (Verathon, Bothell, WA, USA). Correlations between PCAR and presence of LUTS, peak urinary flow rate, and PVR were determined using Spearman correlation coefficients. Unadjusted and adjusted odds ratios (ORs) were calculated using logistic regression to determine the associations between PCAR thresholds and categorical urological outcomes.

RESULTS

The median (interquartile range) PCAR was 0.85 (0.81–0.88). After adjusting for age and total prostate volume, men who had PCARs of >0.90 were more likely to have elevated overall and obstructive symptom scores (OR 2.95, 95% confidence interval 1.39–6.25, and 3.47, 1.63–7.39, respectively).

CONCLUSION

PCAR might add further information beyond total prostate volume when predicting the development of obstructive LUTS.     

Immunohistochemical localization of ethanol-inducible P450IIE1 in the rat alimentary tract

To determine whether P450IIE1, a microsomal P450 enzyme inducible by ethanol in the liver, is also present and inducible in the alimentary tract, corresponding frozen tissue sections were prepared from rats pair-fed liquid diets containing 36% of total calories as either ethanol or carbohydrate (control) for 3 weeks. Immunohistochemical staining was performed using the peroxidase-antiperoxidase method after tissue sections were reacted with antibody against human P450IIE1. In control animals, immunoreactive P450IIE1 was detected only in duodenal and jejunal villous cells. After ethanol treatment, the content of P450IIE1 increased in duodenal and jejunal villi, and the enzyme was now also found in squamous epithelial cells of the cheek mucosa, tongue, esophagus, and forestomach, and in surface epithelium of the proximal colon. P450IIE1 was neither expressed nor induced by alcohol in the epithelium of stomach fundic and antral mucosa, ileum, distal colon, and rectum. When considered together with the xenobiotic activation properties of P450IIE1, these results may partly explain why alcohol abuse is a risk factor for cellular damage or cancer or both in those alimentary tract tissues in which P450IIE1 is inducible by chronic ethanol intake.