Discovery of serum biomarkers of alcoholic fatty liver in a rodent model: C-reactive protein.

BACKGROUND: Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model. METHODS: Serum samples derived from animals with AFL, NAFL, or liver fibrosis were characterized and compared using two-dimensional differential gel electrophoresis. A matrix-assisted laser desorption ionization-time of flight tandem mass spectrometer in conjunction with mascot software was used for protein identification. Subsequently, Western blotting and flexible multi-analyte profiling were used to measure the expressions of the putative biomarkers present in the serum of animals and clinical patients. RESULTS: Eight differential putative biomarkers were identified, and the two most differentiated proteins, including upregulated C-reactive protein (CRP) and downregulated haptoglobin (Hp), were further investigated. Western blotting validated that CRP was dramatically higher in the serum of AFL compared to healthy controls and other animals with liver disease of NAFL or liver fibrosis (p < 0.05). Moreover, we found that CRP and Hp were both lower in liver fibrosis of TAA-induced rats and clinical hepatitis C virus-infected patients. CONCLUSION: The results suggest that increased levels of CRP are an early sign of AFL in rats. The abnormally elevated CRP induced by ethanol can be used as a biomarker to distinguish AFL from normal or otherwise diseased livers.

Novel biomarkers predict liver fibrosis in hepatitis C patients: alpha 2 macroglobulin, vitamin D binding protein and apolipoprotein AI.

BACKGROUND: The gold standard of assessing liver fibrosis is liver biopsy, which is invasive and not without risk. Therefore, searching for noninvasive serologic biomarkers for liver fibrosis is an importantly clinical issue. METHODS: A total of 16 healthy volunteers and 45 patients with chronic hepatitis C virus (HCV) were enrolled (F0: n = 16, F1: n = 7, F2: n = 17, F3: n = 8 and F4: n = 13, according to the METAVIR classification). Three serum samples of each fibrotic stage were analyzed by two-dimension difference gel electrophoresis (2D-DIGE). The differential proteins were identified by the cooperation of MALDI-TOF/TOF and MASCOT; then western blotting and Bio-Plex Suspension Array were used to quantify the protein levels. RESULTS: Three prominent candidate biomarkers were identified: alpha 2 macroglobulin (A2M) is up regulated; vitamin D binding protein (VDBP) and apolipoprotein AI (ApoAI) are down regulated. The serum concentration of A2M was significantly different among normal, mild (F1/F2) and advanced fibrosis (F3/F4) (p < 0.01). The protein levels of VDBP and ApoAI were significantly higher in normal/mild fibrosis, when compared to those in advanced fibrosis (both p < 0.01). CONCLUSIONS: This study not only reveals three putative biomarkers of liver fibrosis (A2M, VDBP and ApoAI) but also proves the differential expressions of those markers in different stages of fibrosis. We expect that combination of these novel biomarkers could be applied clinically to predict the stage of liver fibrosis without the need of liver biopsy.

Etiology and treatment of childhood peptic ulcer disease in Taiwan: a single center 9-year experience.

BACKGROUND/PURPOSE: Peptic ulcer disease (PUD) in children is relatively rare as compared with adults. This study aimed to assess the etiology, clinical and histological characteristics, and treatment of PUD in children. METHODS: All children aged < 18 years with an endoscopic diagnosis of PUD were enrolled in a tertiary referral center. The demographic data, clinical, endoscopic, and histological findings were compared between patients with different causes of PUD. RESULTS: From 1234 endoscopic examinations, 67 (5.4%) children (median age, 11.4 years) with gastric ulcer (GU; n=27) or duodenal ulcer (DU; n=40) were included. Thirty-two (47.7%) of them had Helicobacter pylori infection and 11 (16.5%) had previous use of non-steroidal anti-inflammatory drugs (NSAIDs). Non-H. pylori, non-NSAID PUD was found in 24 (35.8%) patients. Children with H. pylori-related PUD had a significantly higher mean age, antral chronic inflammatory score, rate of familial PUD, and presence of DU and nodular gastritis than those with NSAID-related and non-H. pylori, non-NSAID PUD (p < 0.01). In contrast, children with NSAID-related PUD had a higher rate of upper gastrointestinal bleeding, associated with acute febrile disease, than those with H. pylori-related and non-H. pylori, non-NSAID PUD (p < 0.05). All but two patients with non-H. pylori, non-NSAID PUD were disease free after H. pylori eradication and proton pump inhibitor treatment for 1-2 months. CONCLUSION: In children, H. pylori-related PUD is associated with familial peptic ulcer and the presence of DU. However, short-term NSAID use is correlated highly with GU. The outcome of childhood PUD is good.

Gender difference of circulating ghrelin and leptin concentrations in chronic Helicobacter pylori infection.

BACKGROUND: Both ghrelin and leptin are important appetite hormones secreted from the stomach. We examined whether demographic background, Helicobacter pylori infection, or its related gastritis severity could be associated with circulating ghrelin and leptin levels. METHODS: This study prospectively enrolled 341 dyspeptic patients (196 females, 145 males), who had received endoscopy to provide the gastric specimens over both antrum and corpus for histology reviewed by the updated Sydney's system. The fasting blood sample of each patient was obtained for total ghrelin and leptin analysis. RESULTS: Without H. pylori infection, there were similar ghrelin levels between female and male patients. In the H. pylori-infected patients, the males had lower plasma ghrelin levels than females (1053 vs. 1419 pg/mL, p < .001). Only in males, not in females, the H. pylori infection and its related acute and chronic inflammation scores were significantly associated with a lower ghrelin level (p < or = .04). The multivariate regression disclosed that only the chronic inflammation score independently related to a lower ghrelin level. Only in males, the ghrelin levels ranked in a downward trend for the gastritis feature as with limited-gastritis, with antrum-predominant gastritis, and with corpus-gastritis (1236, 1101, and 977 pg/mL). Leptin level was not related to H. pylori-related gastritis, but positively related to body mass index. CONCLUSION: There should be a gender difference to circulating total ghrelin levels, but not leptin levels, in response to H. pylori infection and its related chronic gastritis.

Aflatoxin exposure and hepatitis C virus in advanced liver disease in a hepatitis C virus endemic area in Taiwan.

This community-based study evaluated the role of aflatoxin exposure in advanced liver disease in hepatitis C virus (HCV)-endemic townships. Preventive health examination was performed on 314 adults > or = 40 years of age recruited from HCV-endemic townships in Tainan, Taiwan. Aflatoxin-albumin in serum was quantified by a new enzyme-linked immunosorbent assay method. After adjusting serum albumin levels and platelet counts, aflatoxin-Bi albumin adducts was still an independent risk factor for advanced liver disease among all 314 residents (> 8 versus < or = 8 (AFBi)-albumin/albumin; OR = 2.29, 95% CI = 1.23-4.27, P = 0.009) and particularly in anti-HCV-positive subjects (OR = 2.09, 95% CI = 1.09-4.0, P = 0.026). Levels of AFB1-albumin/albumin were significantly related to ultrasonographic parenchyma scores (P < 0.001, one-way ANOVA) in all and anti-HCV-positive subjects. The findings indicated aflatoxin exposure may be associated with advanced liver disease in chronic hepatitis C patients in HCV-endemic regions in Taiwan.

Childhood functional abdominal pain and Helicobacter pylori infection.

BACKGROUND/AIMS: Like functional gastrointestinal disorders in adults, the exact etiology of childhood functional abdominal pain (FAP) is unclear. Among those proposed etiologies, chronic Helicobacter pylori (HP) infection plays an important role. Therefore, we conducted this study to investigate the relationship of childhood FAP and chronic HP infection. METHODOLOGY: From 1998 to 2000, pediatric patients referred to our outpatient department under the impression of recurrent abdominal pain were carefully evaluated, and only subjects with FAP were recruited for study. Endoscopic examinations for rapid urease test and histology were performed in each patient, and [13C]-urea breath test was also performed in each patient. Twelve months later, all the procedures were repeated again for follow-up study. Status of FAP and HP infection of each subject was re-evaluated again for further comparisons. RESULTS: In total, 135 patients participated in the primary survey, and 114 of them were successfully followed 12 months later. The results of primary endoscopic examinations were as follows: 43.7% normal results, 19.3% esophagitis, 15.6% gastroduodenitis, 13.3% peptic ulcer diseases (PUD), and 7.4% nodular gastritis. The overall prevalence of HP infection was 23.7% (32/135) at the primary survey. At the follow-up study, 77 (67.5%) patients still suffered from FAP. Persistence of FAP was noted among 59 of 84 (70.2%) non-HP infected patients, and in 13 (86.7%) of the 15 HP-infected, non-eradicated patients in the follow-up study, which did not show statistical significance (P=0.13). CONCLUSIONS: Aside from subjects with PUD, 72.7% of FAP children suffered from similar symptoms during the 12-month follow-up, and HP infection status did not significantly influence the disease courses.

Capsule 13C-urea breath test for the diagnosis of Helicobacter pylori infection.

AIM: To compare the accuracy of capsule 13C-urea breath test (UBT) with conventional invasive methods for the diagnosis of Helicobacter pylori infection. METHODS: One hundred patients received CLO test, histological examination, culture and 100- or 50-mg capsule UBT for the diagnosis of H pylori infection. H pylori infection was defined as those with positive culture or positive results from both histology and CLO test. RESULTS: Both the sensitivity and specificity of the 100-mg capsule UBT (n = 50) were 100%. The sensitivity and specificity of the 50-mg capsule UBT (n = 50) were 96.4 and 100%, respectively. Taken together, the accuracy of capsule UBT (n = 100) was higher than that of CLO test, histology and culture (100% vs 92%, 91% and 89%, respectively; P = 0.035, 0.018 and 0.005, respectively). Our data showed that the optimal timing of sampling for 100- and 50-mg capsule UBT was 15-30 and 6-15 min, respectively. CONCLUSION: Capsule UBT has a higher accuracy compared with biopsy-based tests. It is an ideal method for the diagnosis of H pylori infection.

Accuracy of three diagnostic tests used alone and in combination for detecting Helicobacter pylori infection in patients with bleeding gastric ulcers.

OBJECTIVE: Accuracy of diagnostic methods for detecting Helicobacter pylori (H. pylori) infection among patients with bleeding peptic ulcers has not been thoroughly investigated. The aim of this study was to compare the diagnostic tests and their combined usage in detection of H. pylori infection in patients with bleeding gastric ulcers and without the use of nonsteroidal anti-inflammatory drugs. METHODS: A total of 57 patients who presented with bleeding gastric ulcers by endoscopy were enrolled. The status of H. pylori was identified by performing the rapid urease test (RUT), histology and (13)C-labeled urea breath test (UBT). The criteria for having H. pylori infection was a minimum of two positive tests. RESULTS: The prevalence of H. pylori infection in our patient group was 80.7%. Among the three tests used: RUT, histology, and UBT, sensitivities were 56.5%, 97.8% and 100%, while specificities were 100%, 45.5% and 81.8%, respectively. The overall accuracies of the tests were 78.3%, 71.6% and 90.9%, respectively. Although UBT obtained significantly higher accuracy than histology (P = 0.02) as opposed to RUT (P = 0.11), UBT had significantly higher sensitivity than RUT (P < 0.001). In terms of combining any two of the three tests, more accuracy (98.9%) was achieved when both UBT and histology were used to confirm the diagnosis of the other. Conversely, failure to use combined tests generated the potential of missing a proper H. pylori diagnosis. CONCLUSIONS: UBT is superior to the other two tests in bleeding gastric ulcers. RUT lacks sensitivity for detection of H. pylori infection. However, the concomitant use of UBT and histology seems to be more accurate when gastric ulcers present with bleeding.

Comparison of rabeprazole-based four- and seven-day triple therapy and omeprazole-based seven-day triple therapy for Helicobacter pylori infection in patients with peptic ulcer.

BACKGROUND AND PURPOSE: Rabeprazole is a new proton pump inhibitor producing rapid inhibition of gastric acid secretion. This may potentiate the inhibitory effect of antibiotics against Helicobacter pylori. This study compared the efficacy, safety, and tolerability of 4- and 7-day rabeprazole-based triple therapies versus 7-day omeprazole-based triple therapy. METHODS: A total of 70 H. pylori-infected peptic ulcer patients were randomly assigned to 1 of 3 groups: RAC4 (rabeprazole 20 mg, amoxicillin 1000 mg, and clarithromycin 500 mg twice daily for 4 days), RAC7 (rabeprazole 20 mg, amoxicillin 1000 mg, and clarithromycin 500 mg twice daily for 7 days), and OAC7 (omeprazole 20mg, amoxicillin 1000 mg, and clarithromycin 500 mg twice daily for 7 days). Endoscopy, Campylobacter-like organism (CLO) test, H. pylori culture, and 13C-urea breath test were performed before randomization and 8 weeks after the start of triple therapy. RESULTS: Intention-to-treat (ITT) eradication rates for the RAC4, RAC7, and OAC7 groups were 87% (20/23), 83%(19/23), and 88% (21/24), respectively, and per-protocol (PP) eradication rates were 91% (20/22), 95% (19/20), and 100% (21/21), respectively. There was no significant difference among the ITT or PP eradication rates of the 3 groups. All 3 regimens were well tolerated and compliance was excellent. CONCLUSIONS: One-week RAC and 1-week OAC are equally effective for H. pylori eradication in peptic ulcer patients. The duration of RAC triple therapy can be shortened to 4 days without compromising its efficacy.

Discovery of tumor markers for gastric cancer by proteomics.

Gastric cancer (GC) has a high rate of morbidity and mortality among various cancers worldwide. The development of noninvasive diagnostic methods or technologies for tracking the occurrence of GC is urgent, and searching reliable biomarkers is considered.This study intended to directly discover differential biomarkers from GC tissues by two-dimension-differential gel electrophoresis (2D-DIGE), and further validate protein expression by western blotting (WB) and immunohistochemistry (IHC).Pairs of GC tissues (gastric cancer tissues and the adjacent normal tissues) obtained from surgery was investigated for 2D-DIEG.Five proteins wereconfirmed by WB and IHC, including glucose-regulated protein 78 (GRP78), glutathione s-transferase pi (GSTpi), apolipoprotein AI (ApoAI), alpha-1 antitrypsin (A1AT) and gastrokine-1 (GKN-1). Among the results, GRP78, GSTpi and A1ATwere significantlyup-regulated and down-regulated respectively in gastric cancer patients. Moreover, GRP78 and ApoAI were correlated with A1AT for protein expressions.This study presumes these proteins could be candidates of reliable biomarkers for gastric cancer.

Human neutrophil peptides 1-3 as gastric cancer tissue markers measured by MALDI-imaging mass spectrometry: implications for infiltrated neutrophils as a tumor target.

OBJECTIVE: Human neutrophil peptides (HNPs) -1, -2 and -3 are significantly upregulated and were reported as biomarkers in gastric cancer (GC). However, the tissue location and function of HNPs 1-3 are still unclear in GC, and the spatial distribution of the triad needs to be disclosed. The aims of this study were to investigate the distribution and relationships among HNPs-1, -2 and -3, and assess whether infiltrated neutrophils accumulate in gastric tumor. METHODS: In this study, paired samples (n=33) of the GC tissues and adjacent normal tissues from the same patients were obtained from surgery. Expression of HNPs 1-3 were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The distributions of the HNPs 1-3 in GC tissues were investigated. After verification of HNPs-1 by immunohistochemistry, infiltrated neutrophils were also detected. Then, an in vitro assay was used to observe the binding capacity and measure the cytotoxic effect of HNPs-1 against AGS cells. RESULTS: Comparing to neighboring normal tissue, expressional level of HNPs 1-3 were significantly higher and their distributions overlapped in cancerous tissues of GC patients with high abundance in the lamina propria, whereas HNPs-1 was identified as the highest major peak. Moreover, HNPs-1, -2 and -3 correlated with each other. Besides, we also observed that increased infiltrated neutrophils accumulating in GC tissues, indicating that a strong positive correlation between HNPs 1-3 and infiltrated neutrophils. In addition, the further investigated demonstrated that the major peptide, HNPs-1, was statistically increased with the advance of tumor development from the early to advanced stage of GC (p< 0.05). Moreover, we also noticed that HNPs-1 with a great binding capacity to GC AGS cells in vitro can inhibit tumor cell growth. CONCLUSIONS: Our results suggest that neutrophil secreted peptides, HNPs 1-3, increased in the GC tissues and could be used as potential biomarkers detected using MALDI-TOF MS, implying that elevated neutrophils may be used as a tumor target for tumor treatment. The binding capacity of HNPs-1 with GC cells implies that tracking molecules conjugated with HNPs-1 could be applied as a specific probe for GC diagnoses.

More economic 25 mg 13C-urea breath test can be effective in detecting primary Helicobacter pylori infection in children.

BACKGROUND AND AIM: The high cost of the 13C-urea breath test (UBT) limits its wide application for both epidemiological and clinical studies for diagnosing Helicobacter pylori infection. This study examined if a lower-dose UBT, applying 1 mg/kg of bodyweight (maximum 25 mg, UBT(25)), could introduce cost savings while preserving high diagnostic yields for primary H. pylori infection. METHODS: Children aged less than 16 years were recruited after obtaining consent. Those children with administration of antibiotics or proton pump inhibitors within 1 month of the tests were excluded. Positive tests for both the UBT with 50 mg urea (UBT(50)) and the H. pylori stool antigen (HpSA) were qualifying criteria for H. pylori infection. Negative results for both indicated non-infection. The UBT(25) was conducted 1 week after the UBT(50). The cut-off points for the UBT(25) ranging from 2delta to 5delta were examined for their sensitivity, specificity and accuracy rates. RESULTS: A total of 153 children were recruited (55% male; mean age 9.1 +/- 3.5 years). Both the UBT(50) and HpSA test were positive in 18 (13.1%) and negative in 119 children, respectively. The sensitivity and specificity of the UBT(25) were optimally achieved at 88.9% (95% confidence interval [CI]: 71.4-100) and 95.0% (95% CI: 91.1-99.9), judged with a cut-off point at 3.5delta. The diagnostic accuracy was significantly higher for children older than 7 years than for those younger than 7 years (98%vs 85%, P = 0.009). CONCLUSION: Lower-dose UBT titration by bodyweight can cut costs while maintaining a highly reliable method to screen primary H. pylori infection in children older than 7 years, which is generally beyond school age.

Short-term recurrent abdominal pain related to Helicobacter pylori infection in children.

BACKGROUND AND AIM: The causal relationship between Helicobacter pylori infection and recurrent abdominal pain in children is still under debate. This study assessed the relationship between H. pylori infection and recurrent abdominal pain (RAP) in preschool and school children. METHODS: A total of 1271 preschool and school children completed a questionnaire to define the RAP or short-term RAP (SRAP) with pain duration from 2 weeks to 3 months. The serum samples of 118 children with RAP, 60 with SRAP and 212 control children without abdominal pain were all tested for anti-H. pylori IgG. Children with abdominal pain and anti-H. pylori seropositivity were followed for 1 year to assess the relationship of H. pylori infection and recurrent abdominal pain. RESULTS: The prevalence rates of RAP and SRAP in children were 9.8% (124/1271) and 5.5% (70/1271), respectively. Children with SRAP had a higher anti-H. pylori seropositive rate than those with RAP (25%vs 5%, P < 0.001) and control (25%vs 9%, P = 0.001). Among children with SRAP, the epigastric pain was related to H. pylori infection (P = 0.002). One year later, 71% (15/21) of the follow-up children (15 with SRAP, six with RAP) became symptom free regardless of the persistence of H. pylori. CONCLUSION: H. pylori infection is more commonly found in children with short-term RAP, and presentation of epigastric pain in these cases can be considered as a warning alarm to screen for H. pylori infection.

Children of Helicobacter pylori-infected dyspeptic mothers are predisposed to H. pylori acquisition with subsequent iron deficiency and growth retardation.

BACKGROUND: We tested whether Helicobacter pylori-infected dyspeptic mothers had a higher rate of H. pylori infection in their children, and whether such H. pylori-infected children were predisposed to iron deficiency or growth retardation. MATERIALS AND METHODS: A total of 163 children from 106 dyspeptic mothers (58 with and 48 without H. pylori infection) were enrolled to evaluate body weight, height, hemoglobin, serum ferritin, and H. pylori infection using the 13C-urea breath test. A questionnaire was used to evaluate demographic factors of each child. RESULTS: The rate of H. pylori infection in children with H. pylori-infected dyspeptic mothers was higher than that of children with noninfected mothers (20.5% vs. 5.3%; p<.01, OR: 4.6, 95% CI: 1.5-14.2). The rate of H. pylori infection in children elevated as the number of their H. pylori-infected siblings increased (p<.01). For children below 10 years of age, H. pylori infection was closely related to low serum ferritin and body weight growth (p<.05). CONCLUSION: The children of H. pylori-infected dyspeptic mothers had an increased risk for such infection. The risk further increased once their siblings were infected. H. pylori infection in pre-adolescent children may determine iron deficiency and growth retardation.