Jazon Romilson de Souza Almeida

Autonomic function in patients with functional dyspepsia assessed by 24-hour heart rate variability

The function of the autonomic nervous system was assessed in 23 patients with dysmotility-like functional dyspepsia and 12 healthy volunteers by analysis of 24-hr heart rate variability and a battery of five standardized sympathetic and parasympathetic cardiovascular reflex tests. Measures of heart rate variability were obtained by analysis of ambulatory electrocardiographic recordings using both the time and the frequency domain methods. The values of parameters reflecting vagal activity (HF, rMSSD) were significantly lower in patients with functional dyspepsia. Individual analysis of heart rate variability data identified at least one abnormal parameter of vagal function in seven (30.4%) patients, and in five of these the results of parasympathetic cardiovascular reflex tests were also abnormal. Our results suggest impaired efferent vagal function in a subgroup of patients with functional dyspepsia. This abnormality may play a role in the pathogenesis of the disease in these patients.

Portal Vein Thrombosis and Liver Transplantation: Long Term

Obstruction of the portal vein may be related to constriction by malignant tumors or thrombosis associated with liver disease. We herein have reported our experience with patients undergoing liver transplantation with portal vein thrombosis (PVT) whose diagnosis was made intraoperatively. From September 1991 to May 2009, we studied 27/419 (6.4%) patients with PVT who were evaluated according to the presence of esophagogastric varices, underlying disease, malignancy, and if there was previous surgery, review of medical records on data collected prospectively. We observed 24 (88.9%) patients with PVT grade 1, 2 (7.4%) with grade 2, and 1 (3.7%) with grade 3. The average age of the PVT patients was 47.5 years; the average model for End-Stage Liver Discase score was 18.3, and the predominant diagnosis, hepatitis C cirrhosis. Eighteen underwent a sclerotherapy/ligature. The sensitivity of ultrasound for grade 1 thrombosis was 39.1%; for grade 2, 50%; and for grade 3, 100%. Portal vein thrombectomy was performed in 24 patients. In other patients (grade 2), we performed an anastomosis of the donor portal vein to the recipient gastric vein or to a greater splanchnic collateral vein. In only 1 patient was the graft performed using the donor portal vein-donor iliac vein-recipient superior mesenteric vein. None of the patients displayed PVT in the immediate postoperative period. Actuarial survivals at the years 1, 3, and 5 were 85%, 74%, and 63%, respectively. We concluded that PVT cannot be considered to be a contraindication for liver transplantation.

Evaluation of five DNA extraction methods for detection of H. pylori in formalin-fixed paraffin-embedded (FFPE) liver tissue from patients with hepatocellular carcinoma

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC.

High serum laminin and type IV collagen levels in schistosomiasis mansoni

BACKGROUND Fibrosis is the process of excessive deposition of collagen and other extra cellular matrix components and large amounts of these components have been shown in periovular schistosomal granulomas, especially in the liver. Laminin and type IV collagen have been investigated in various hepatic disorders but their accuracy in fibrosis detection and in the evaluation of its progression in schistosomiasis have not been fully explained. AIM To measure the serum levels of two markers of fibrosis, laminin and type IV collagen in schistosomiasis. PATIENTS AND METHODS Sixty-four patients with different clinical forms of schistosomiasis mansoni: intestinal (group I), hepatointestinal (group II), compensated (group III) and decompensated hepatosplenic (group IV) and 18 healthy volunteers were included. RESULTS Serum type IV collagen and laminin levels were significantly increased in patients compared to controls. At about clinical forms, serum type IV collagen was increased in groups II and IV, compared to controls and was significantly higher in group IV than in group I. Serum laminin was significantly increased in groups II, III and IV and was significantly higher in group IV than in group II. Serum type IV collagen was closely correlated with serum laminin in groups II and IV. CONCLUSIONS Connective tissue marker levels did not correlate with periportal thickness. In schistosomiasis mansoni there is an increase of type IV collagen and laminin levels at the initial stage of the disease, as well as in advanced forms. We also suggest that these markers may be a useful predictor of disease progression.

Bleeding Duodenal Varices Successfully Treated with TIPS

Upper gastrointestinal hemorrhage is a life-threatening complication of liver cirrhosis and portal hypertension. It is well recognized that, despite adequate pharmacologic and endoscopic therapy, there is a 20–35% mortality ratio in the first occurrence of gastrointestinal bleeding [1, 2]. Ruptured esophageal varices, peptic ulcer disease, portal hypertensive gastropathy, and ruptured gastric varices are most commonly associated with bleeding events, and the severity seems to be associated with the degree of liver dysfunction. The most common site of variceal bleeding is the lower esophagus, followed by gastric varices. These can be detected in up to 25% of portal hypertensive, cirrhotic patients [3–5]. Duodenal varices are uncommon among cirrhotic patients [6], and bleeding duodenal varices represent a major challenge for management, as both pharmacological and endoscopic therapy may not fully control hemorrhage. We report a case of a patient diagnosed with liver cirrhosis and portal hypertension on a waiting list for liver transplantation who developed upper gastrointestinal bleeding secondary to duodenal varices, managed with transjugular intrahepatic portosystemic shunt (TIPS). Endoscopic features preand post-TIPS are reviewed.

Orocecal transit time in patients with functional dyspepsia.

Background The importance of intestinal dysmotility in functional dyspepsia is a controversial issue. Goals To investigate the orocecal transit time in patients with functional dyspepsia, as well as a possible association between intestinal transit and the presence of anxiety or Helicobacter pylori infection in these patients. Study The participants in this study were 23 patients with dysmotility-like functional dyspepsia and 24 control subjects. Orocecal transit time was measured by the lactulose hydrogen breath test. The presence of anxiety was assessed by the Hospital Anxiety and Depression (HAD) scale. Results No significant difference in orocecal transit times was found between patients (median, 55 minutes; 95% confidence interval [CI], 40–60 minutes) and control subjects (median, 50 minutes; 95% CI, 40–60 minutes;p = 1). In the assessment, 15 patients (73%) scored for anxiety on the HAD scale, and 15 patients (65%) had positive test results for H. pylori. There was no significant difference in orocecal transit times between the patients with (median, 55 minutes; 95% CI, 40–63 minutes) and those without H. pylori infection (50 minutes; 95% CI, 40–68 minutes;p = 0.85), or between the patients with (median, 45 minutes; 95% CI, 40–68 minutes) and those without (60 minutes; 95% CI, 40–63 minutes;p = 0.77) anxiety. Conclusions Orocecal transit time is within the normal range in patients with functional dyspepsia. Anxiety and H. pylori infection do not seem to influence orocecal transit time in these patients.

Predicting Survival After Liver Transplantation Using Up-to-Seven Criteria in Patients With Hepatocellular Carcinoma

BACKGROUND Liver transplantation is a controversial issue in the initial management of hepatocellular carcinoma (HCC). The aim of this study was to demonstrate that up-to-seven could be useful as a model in evaluating potential candidates for liver transplantation for HCC. METHODS Between January 1997 and December 2010, 84 patients undergoing liver transplantation were analyzed for HCC. Pathologic variables included the number of lesions, maximal tumor diameter, vascular invasion, and tumor grade. The following variables were recorded: Age, gender, recurrence of HCC, survival, donor age, incidental, Milan criteria, Model for End-Stage Liver Disease score, and alpha-fetoprotein (AFP) levels. To evaluate variables we used the Kaplan-Meier method, Cox regression and Kolmogorov-Smirnov test. RESULTS There were 68 (80.9%) patients within up-to-seven criteria and 16 (19.1%) beyond this criteria. AFP levels were 96.4 versus 464.3 ng/dL in patients within versus beyond up-to-seven criteria. The median diameter of the largest nodule was 22.2 versus 32.0 mm in patients within versus beyond up-to-seven criteria, respectively. The median sum of nodules was 1.8 and 5.6 nodules in patients within versus beyond up-to-seven criteria. Vascular invasion was present in 11 (13.0%) patients within versus 7 (8.3%) beyond. Recurrence was observed in 9 (10.7%) patients and only 3 were beyond up-to-seven criteria, 2 had vascular invasion, and another showed poor differentiation. Post liver transplantation survival rates were 87.7%, 74.5%, and 65.3% at 1, 3, and 5 years in patients within up-to-seven versus 80.7%, 51.7%, and 32.1% beyond (P = .03). Similar results were observed when we applied Milan criteria or expanded Milan criteria. The largest nodule was the only predictive factor after proportional hazard Cox regression application (Beta = 0.037; exponent = 1.0377; Wald = 4.542; P = .03). In the present study, up-to-seven criteria could be useful to evaluate potential liver transplant candidates due to HCC.

Improved Detection of Helicobacter pylori DNA in Formalin-fixed Paraffin-embedded (FFPE) Tissue of Patients with Hepatocellular Carcinoma Using Laser Capture Microdissection (LCM)

Dear Editor, Helicobacter DNA has been detected in hepatic tissues from patients with various hepatobiliary diseases, mainly cirrhosis and hepatocellular carcinoma (HCC). Although the role of Helicobacter spp. in pathogenesis of these diseases remains unclear, the available data suggest that Helicobacter infection may play a role in hepatic carcinogenesis [1]. Considering that HCC is one of the most common malignancies with more than 500.000 new tumors diagnosed annually [2], further studies related to H. pylori and development of HCC have fundamental importance on the understanding of its pathogenesis. Formalin-fixed paraffin-embedded (FFPE) tissue represents an extraordinary source for molecular studies as genomic DNA can be extracted from this sample. However, DNA extraction from FFPE tissues is challenging because nucleic acids are commonly fragmented and cross-linked with proteins. Furthermore, methods of DNA extraction from FFPE tissue are generally laborious and time-consuming. Laser capture microdissection (LCM) is a recently developed technique for isolation of pure populations of cells from tissue sections by microscopic visualization. Because of its high precision and accuracy, LCM has been employed in cancer-related studies. In this work, we used LCM technique to improve the detection of H. pylori in FFPE liver from patients with HCC. With this aim, six H. pylori-positive samples detected by polymerase chain reaction (PCR) with H. pylori-specific 16S rRNA primers were selected. The sequence of the sense primer (JW21) was 5′-GCGACCTGCTGGAACATTAC-3′(position 691-710) and the antisense primer (JW22) was 5′-CGTTAGCTCCATTACTGGAGA-3′ (position 829-809) [3]. Tissue samples were cut on 0.17 mm PEN membrane-covered slides (Carl Zeiss, MicroImaging GmbH, G€ ottingen, Germany) and then routine staining with carbol fuchsin was performed [4]. Thereafter, stained bacteria were microdissected using a PALM MicroBeam system (Carl Zeiss, MicroImaging GmbH, G€ ottingen, Germany) and then ejected into the Eppendorf tube cap by a single laser shot (Fig. 1C,D). After microdissection, a digestion buffer was added into Eppendorf for DNA extraction. The crude lysate was directly employed as template for PCR [4]. The samples were further amplified using H. pylori 16S rRNA primers as previously described [3], and amplicons were identified by sequence analysis. Microorganisms resembling H. pylori were observed in hepatic sinus from HCC samples (Fig. 1A,B). The number of cocci was greater than of bacilli as previously described [5]. PCR results showed that all six microdissected samples were positive for 16S rRNA gene and showed 98% similarity to 16S rRNA gene of H. pylori by sequence analysis (GeneBank accession number CP003419.1). Nevertheless, we cannot exclude the possibility of cross-reaction of these primers with other Helicobacter spp. These results demonstrated that LCM can be extensively applied for identification of H. pylori in FFPE liver tissue of HCC patients. Considering that bacteria were mainly found in peritumoral tissue, this technique was highly effective for obtaining a targeted bacterial population within a selected area in the HCC tissue. Beyond that, LCM simplified the H. pylori detection because extracted DNA was used directly as a template for PCR amplification. Further studies will be performed to isolate H. pylori from other tissues using LCM technique.

Prognostic Factors for Hepatocellular Carcinoma Recurrence: Experience With 83 Liver Transplantation Patients

INTRODUCTION Orthotopic liver transplantation (OLT) is a rational therapeutic option for early-stage hepatocellular carcinoma (HCC) providing a potential cure and improving survival. METHODS This retrospective study of a longitudinal cohort used an electronic database collected prospectively from September 1997 to May 2010. The variables were gender, age (years), and alpha-fetoprotein (AFP) level (ng/mL). In explanted livers we observed: microvascular or macrovascular invasion, number of nodules and their largest size, Edmondson-Steiner histological differentiation, incidental tumor transarterial chemoembolization (TACE), Milan criteria, and previous down-staging. RESULTS Five of 83 (6.0%) subjects including 68 (82%) males with a mean time to diagnosis of 9 months experienced tumor relapses. Mean patient age at HCC recurrence was 55.3 years for male and 44.6 years for female subjects. Vascular invasion was detected in 17/83 (20.5%) subjects, namely 2% of macrovascular invasion, and 52.5% with expanded Milan criteria due to an increased number and size of nodules in the explanted livers. An incidental tumor was observed in 29.5% of cases. Preoperative TACE treatment was performed in 13 (15.6%) patients. None of the patients who had a HCC recurrence had undergone TACE. AFP level at the time of recurrence was around 1,900 ng/mL. The predictive factor for mortality was nodule size (P=.04; hazard ratio=0.0269; confidence interval [CI], 95% 0.0094-0.299). CONCLUSION Patients with relapses showed the worst survival and tumor size was a predictive factor for recurrence.

Survival Before and After Model for End-stage Liver Disease Score Introduction on the Brazilian Liver Transplant Waiting List

INTRODUCTION To examine whether the official adoption of Model for End-Stage Liver Disease (MELD) as a criterion for organ allocation was effective, we studied risk factors for patient deaths and the accuracy of the MELD score to predict mortality. METHODS Patients on the waiting list for liver transplantation were divided into two periods depending on whether they were on the waiting list before (period 1) or after (period 2) the MELD introduction in Brazil. The Kaplan-Meier method with log-rank tests were used to study patient survivals. Predictive factors were identified using the Cox regression method. A receiver operating characteristic (ROC) curve was used to analyze Child-Turcotte-Pugh (CTP) and MELD accuracy. RESULTS We analyzed 295 patients in period 1 and 240 in period 2. The survivals after 3, 6, 9, and 12 months in periods 1 and 2, were 95.6%, 90.5%, 84.9%, and 69.6% vs 95.7%, 92.1%, 85.3%, and 83.3%, respectively (P = NS). Multivariate analysis showed CTP, MELD-Na, and albumin levels, besides spontaneous bacterial peritonitis (SBP), to be independent factors related to survival in period 1. In period 2, CTP, creatinine levels, international normalized ratio, besides spontaneous bacterial peritonitis, were the independent factors. The ROC curve for CTP was 0.676 and for MELD, 0.644 (P = .4) in period 1. In period 2, the ROC curve for CTP was 0.680 and for MELD, 0.718 (P = .4). CONCLUSION Patient survival on the waiting list for liver transplantation did not change at 1 year after the introduction of the MELD.