C. Panella

Nutritional Supplementation With Branched-Chain Amino Acids in Advanced Cirrhosis: A Double-Blind, Randomized Trial

BACKGROUND & AIMS The role of oral supplementation with branched-chain amino acids (BCAA) in advanced cirrhosis is far from settled. A nutritional approach might prevent progressive liver failure and improve nutritional parameters and quality of life. METHODS A multicenter, randomized study comparing 1-year nutritional supplementation with BCAA against lactoalbumin or maltodextrins was performed in 174 patients with advanced cirrhosis. Primary outcomes were the prevention of a combined end point (death and deterioration to exclusion criteria), the need for hospital admission, and the duration of hospital stay. Secondary outcomes were nutritional parameters, laboratory data and Child-Pugh score, anorexia, health-related quality of life, and need for therapy. RESULTS Treatment with BCAA significantly reduced the combined event rates compared with lactoalbumin (odds ratio, 0.43; 95% confidence interval, 0.19-0.96; P = 0.039) and nonsignificantly compared with maltodextrins (odds ratio, 0.51; 95% confidence interval, 0.23-1.17; P = 0.108). The average hospital admission rate was lower in the BCAA arm compared with control treatments (P = 0.006 and P = 0.003, respectively). In patients who remained in the study, nutritional parameters and liver function tests were, on average, stable or improved during treatment with BCAA and the Child-Pugh score decreased (P = 0.013). Also, anorexia and health-related quality of life (SF-36 questionnaire) improved. Long-term compliance with BCAA was poor. CONCLUSIONS In advanced cirrhosis, long-term nutritional supplementation with oral BCAA is useful to prevent progressive hepatic failure and to improve surrogate markers and perceived health status. New formulas are needed to increase compliance.

Clarithromycin-Resistant Genotypes and Eradication of Helicobacter pylori

Context Point mutations in the peptidyltransferase region of the 23S ribosomal RNA gene may be responsible for Helicobacter pylori clarithromycin resistance. Contribution This study related mutations to eradication rates in 156 adults treated with clarithromycin regimens for H. pylori infection. Eradication was successful in 14 of 15 patients with either A2142G or A2142C point mutations but in only 11 of 23 patients with the A2143G point mutation. Cautions This was a post hoc subgroup study of selected participants in a multicenter randomized trial. Implications The A2143G point mutation may be associated with a low eradication rate of H. pylori infection. The Editors Helicobacter pylori infection plays a major role in peptic ulcer disease, low-grade mucosa-associated lymphoid tissue lymphoma, and gastric cancer (1), and its eradication dramatically affects the natural history of both peptic ulcer and gastric lymphoma (2). European and U.S. guidelines advised the use of triple therapies (proton-pump inhibitor, clarithromycin plus amoxicillin, or metronidazole) for 7 to 14 days to cure this infection (3, 4). However, H. pylori resistance against clarithromycin is increasing worldwide, reducing the success rate of standard triple therapies to mean values as low as 18% to 44% (5-7). Novel culture-free polymerase chain reaction (PCR)based assays have allowed the detection of the genetic mutations that are involved in the mechanisms of clarithromycin resistance (8, 9). In detail, A2143G and A2142G transitions are the most prevalent point mutations in Europe and the United States (10, 11), while the A2144G mutation is more frequent in Asia (12, 13). Although such genetic mutations have been associated with different degrees of bacterial resistance in vitro, data are still conflicting (7, 14). Moreover, no study has assessed the role of these different mutations on H. pylori treatment outcome. In a recent multicenter study, a novel sequential regimen, consisting of a simple dual therapy given for the first 5 days followed by a triple therapy for the remaining 5 days, achieved a very high cure rate as compared with standard triple therapy (92% vs. 74%) (15). Whether such a high cure rate may depend on increased efficacy of the sequential regimen against the clarithromycin-resistant strains is unknown. We wanted to evaluate the role of different point mutations in the success of eradication therapy and to compare the efficacy of standard triple therapy and the sequential regimen for these mutations. Methods Study Design To assess the role of primary clarithromycin resistance in therapeutic outcome, we designed a post hoc subgroup analysis of a previous study involving 8 Italian centers (15). In detail, we selected patients from those who were previously enrolled by our 2 centers to participate in a multicenter study between January and December 2001 (Figure). Demographic and clinical characteristics of patients enrolled in our substudy were similar to those of patients enrolled in the original multicenter study. Briefly, in the original study, Zullo and colleagues (15) allocated patients who were never treated for H. pylori infection, according to a computer-generated randomization list drawn in each center, to receive standard 7-day treatment (20 mg of rabeprazole, 500 mg of clarithromycin, and 1 g of amoxicillin twice daily) or 10-day sequential therapy (20 mg of rabeprazole plus 1 g of amoxicillin twice daily for the first 5 days followed by 20 mg of rabeprazole, 500 mg of clarithromycin, and 500 mg of tinidazole twice daily for the remaining 5 days). To assess H. pylori status, the investigators performed upper endoscopy with several gastric biopsies for histology (Giemsa staining), a rapid urease test, and a standard 13C-urea breath test at entry and at 4 to 6 weeks after therapy. Investigators considered infections to be eradicated when all 3 test results were negative and considered treatment to have failed when at least 1 test result was positive. The local ethics committee approved the protocol, and all participants gave written informed consent. Figure. Flow chart showing data of patients recruited in this substudy from the previous multicenter study. For our current study, we selected 75 of 192 patients who were treated with standard triple therapy and 81 of 185 patients who were treated with the sequential regimen in the 2 participating centers. We recruited patients consecutively from the randomization lists of the previous study, independent of the eradication status. The final study sample included 58 of 140 patients whose infections were eradicated and 17 of 52 patients whose infections were not eradicated after standard triple therapy and 76 of 177 patients whose infections were eradicated and 5 of 8 whose infections were not eradicated after the sequential regimen. Clarithromycin Resistance Assessment We assessed the 3 point mutations (A2142C, A2142G, and A2143G) of clarithromycin resistance by using a validated real-time PCR, as reported elsewhere (16). Briefly, we extracted the DNA by using NucleoSpin Tissue (Macherey-Nagel GmbH & Co., Dren, Germany), according to the manufacturers instructions, applied on paraffin-embedded sections. We applied the same procedure to homogeneous bacterial cultures of H. pylori (positive and negative controls), for which clarithromycin resistance had been previously assessed with Etest (AB BIODISK, Solna, Sweden). We estimated final DNA concentrations by ultraviolet absorbance at 260 nm. Preparation of the Probes and Primers We designed TaqMan minor groove binder (MGB) probes and primers to hybridize with wild-type and mutant DNA by using the Primer Express program and Custom TaqMan SNP Genotyping Assay service (Applied Biosystems, Foster City, California) that synthesized the primers and probes for each mutation. Genotyping Assay The assay reagents for the genotyping single nucleotide mutation from the Assays-by-Design service (Applied Biosystems) consisted of a 40X mix of unlabeled PCR primers and TaqMan MGB probes (FAM and VIC fluorochrome dyelabeled). These assays were designed for the genotyping of specific mutations. Each assay enables scoring of both genotypes in a single well. Since a recent study showed that the conjugation of MGB to oligonucleotides stabilizes nucleic acid duplexes, causing a dramatic increase in oligonucleotide melting temperature (17, 18), we used an attachment of the MGB, which enables the use of shorter fluorogenic probes, thus resulting in improved mismatch discrimination. Our probes were distinguished by being labeled with different fluorescent reporter dyes (FAM dye and VIC dye). A substantial increase in FAM or VIC dye fluorescence indicated homozygosity for the FAM- or VIC-specific allele, while an increase in both signals indicated heterozygosity (19). Real-Time PCR Assay and Allelic Discrimination We performed the real-time PCR procedure according to the method of Wada and colleagues (20). We enclosed positive and negative controls in each assay. We analyzed fluorescence of hybridized probes by multicomponent graphics, where we examined dye-labeled (FAM and VIC), background, and passive control (ROX fluorochrome dye-labeled) fluorescence and expressed them as normalized reporter signal (Rn). We clustered all samples by using the maximum likelihood algorithm based on the ratio of normalized reporter dye signal. The result of the analysis yields 3 major clusters corresponding to the 3 genotypic constituents: wild-type homozygous, mutated-type homozygous, and heterozygous. Characterization of Positive and Negative Controls by Amplification and Sequencing of the Hp23S Fragment We obtained the Hp23S fragment by PCR amplification of H. pylori extracted DNA from homogeneous bacterial cultures (strains with and without clarithromycin resistance, previously assessed by Etest) by using primer Hp23-F (5-CCACAGCGAT GTG GTCTCAG-3) and Hp23-R (5-CTCCATAAGAGCCAAAGCCC-3) according to conventional PCR assay (21). Before sequencing, we purified the PCR products by using the Wizard PCR preps (Promega, Madison, Wisconsin). We performed the sequencing reaction with the same primers for PCR, as described by Sanger and colleagues (22), by using the Dye Terminator 3.1 Ready Reaction Kit (Applied Biosystems) as indicated by the manufacturer. We performed sequencing on the 2 strands of each PCR product with the automated ABI Prism 377 DNA Sequencer (Applied Biosystems) and aligned the resulting nucleotide sequence by using the Sequence Navigator software package (Applied Biosystems). Statistical Analysis We determined sample size before the start of the study on the basis of the available data in the literature. In detail, an eradication rate ranging from 18% to 44% was reported after standard triple therapy in patients with primary clarithromycin-resistant strains (5-7), whereas the sequential regimen eradicated the infection in 79% of such patients (15). Assuming a high eradication rate for the triple therapy (45%) and a relatively poor success rate for the sequential regimen (70%) in patients with primary clarithromycin-resistant strains, we calculated that at least 68 patients per group were needed to detect a statistically significant difference with 0.8 power and an level of 0.05 (2-sided). After the study was completed, we realized that our sample size estimate provided the necessary number of clarithromycin-resistant patients and should have been inflated, on the basis of a presumed overall rate of clarithromycin resistance, to provide an estimate of total sample size. We compared eradication rates by H. pylori clarithromycin-resistant strain mutation (A2142C, A2142G, and A2143G) by using the Fisher exact test or chi-square test, as appropriate. We determined point mutation groupings after reviewing eradication rates by individual mutation. We compared clinical characteristics among the different groups by using the Student t-test for unpaired da

Sequential treatment for Helicobacter pylori does not share the risk factors of triple therapy failure.

Background : Predicting factors for the outcome of conventional Helicobacter pylori triple therapy have been identified. Of these, the presence of the CagA gene is a strong predictor of successful treatment. Our preliminary data show that this factor becomes irrelevant when sequential therapy is used.

Sex hormone-related functions in regenerating male rat liver

Sex hormone receptors were quantitated in normal male rat liver and in regenerating liver at several different times after partial (70%) hepatectomy. Both estrogen and androgen receptor content were altered dramatically by partial hepatectomy. Total hepatic content and nuclear retention of estrogen receptors increased, with the zenith evident 2 days after partial hepatectomy, corresponding to the zenith of mitotic index. Serum estradiol increased after 1 day, and reached a maximum at 3 days after surgery. In contrast, total and nuclear androgen receptor content demonstrated a massive decline at 1, 2, and 3 days after resection. Serum testosterone displayed a parallel decline. In addition, hepatic content of two androgen-responsive proteins was reduced to 15% and 13% of normal values during this period. The activity of these various proteins during regeneration of male rat liver is comparable to that observed in the liver of normal female rats. Taken together, these results indicate that partial hepatectomy induces a feminization of certain sexually dimorphic aspects of liver function in male rats. Furthermore, these data provide evidence that estrogens, but not androgens, may have an important role in the process of liver regeneration.

Oral tacrolimus long-term therapy in patients with Crohn's disease and steroid resistance.

To report the results of a prospective, open‐label, uncontrolled study in 13 patients affected by Crohn’s disease with resistance to steroids.

Phenotypic and genotypic Helicobacter pylori clarithromycin resistance and therapeutic outcome: benefits and limits

INTRODUCTION Primary clarithromycin resistance is increasing worldwide, and it has been regarded as the main factor reducing the efficacy of Helicobacter pylori therapy. However, the clinical consequence of either phenotypic or genotypic resistance still remains unclear. This study aimed to evaluate: (i) the concordance between phenotypic (culture) and genotypic (real-time PCR) tests in assessing primary clarithromycin resistance; and (ii) the role of both in therapeutic outcome. METHODS A post hoc subgroup study was selected from a double-blind, placebo-controlled trial, enrolling 146 patients with dyspepsia or peptic ulcers never previously treated. Real-time PCR and Etest on bacterial culture for assessing clarithromycin resistance were performed. [(13)C]urea breath test (UBT), histology and rapid urease tests at entry and UBT after 4-8 weeks were used to assess infection and eradication. All patients received a 10 day therapy. RESULTS Prevalence of clarithromycin phenotypic resistance was significantly lower as compared with genotypic resistance (18.4% versus 37.6%, P < 0.001). A concordance between the two methods was present in 71.2% of cases. A significant difference in the eradication rate was seen between clarithromycin-susceptible and -resistant strains, when assessed with either Etest (92.4% versus 55.5%, P < 0.001) or a PCR-based method (94.5% versus 70.9%; P < 0.001). Of note, the eradication rate showed the lowest value (30.7%) when phenotypic bacterial resistance was genetically linked to the A2143G point mutation. CONCLUSIONS This study showed that: (i) there is a relevant discordance between the two methods; and (ii) phenotypic clarithromycin resistance markedly reduces H. pylori eradication when it is linked to a specific point mutation.

Two new treatment regimens for Helicobacter pylori eradication: a randomised study

BACKGROUND Several studies have found a fairly low Helicobacter pylori eradication rate using a standard 7-day triple therapy in Italy. Recently, two new therapeutic schedules have been proposed with an eradication rate higher than 90%. This study compared the efficacy of these two treatment regimens. PATIENTS AND METHODS A total of 131 patients with Helicobacter pylori infection and either non-ulcer dyspepsia (73 patients] or peptic ulcer (58 patients) were enrolled. Helicobacter pylori infection was assessed by rapid urease test and histology on gastric biopsies. Patients were randomised to receive either a 5-day course of ranitidine bismuth citrate 400 mg bid, clarithromycin 500 bid, and tinidazole 500 bid, or a 10-day course of omeprazole 20 mg bid plus amoxycillin 1 g bid for the first 5 days, and omeprazole 20 mg bid, clarithromycin 500 mg bid and tinidazole 500 mg bid for the remaining 5 days. Eradication was assessed by endoscopy 4-6 weeks after therapy. RESULTS Overall, 4 patients (2 for each treatment group) were lost to follow-up. Helicobacter pylori eradication rates were 67.2% (95% confidence interval: 55.7-78.7) and 65.2% (95% confidence interval: 53.7-76.6) at per protocol and intention-to-treat analyses, respectively, after the 5-day regimen, and 96.8% (95% confidence interval: 92.5-100) and 93.8% (95% confidence interval: 88-99.7) after the 10-day regimen (p<0.05). Both treatments were well tolerated, and no major side-effects were reported. CONCLUSIONS The 5-day regimen gave disappointing results, while the eradication rate after the 10-day regimen was very high.

Primary clarithromycin resistance in Italy assessed on Helicobacter pylori DNA sequences by TaqMan real-time polymerase chain reaction

Helicobacter pylori clarithromycin resistance is increasing worldwide and different mutations are involved in its mechanisms. Recently, molecular methods have been proposed to assess these mutations.

Lamivudine and alpha-interferon in combination long term for precore mutant chronic hepatitis B

BACKGROUND/AIMS Alpha-interferon (alpha-IFN) and lamivudine are the two licensed drugs for patients with chronic hepatitis B, however, their efficacy in precore mutant chronic hepatitis B is limited. The aim of this study was to investigate the efficacy of 1 year alpha-IFN-lamivudine combination therapy for anti-HBe/hepatitis B virus- (HBV)-DNA positive patients. METHODS Between 1997 and 1999, 29 consecutive anti-HBe/HBV-DNA positive patients entered this prospective pilot study. Patients received 100mg lamivudine orally daily and alpha-IFN 6 million units (MU) three times weekly for 52 weeks. All patients were followed-up for 12 months after stopping therapy. Primary end points were loss of serum HBV-DNA and alanine transaminase normalization at week 52. RESULTS Overall, the end-treatment biochemical and virological response was 93% while the sustained response at week 104 was 14%. HBV-DNA negative patients did not experience a viral breakthrough during treatment; no tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase (YMDD) variant emerged. At week 52, 46% of patients with paired liver biopsies slides available, showed an histological improvement (histological activity index > or =2). CONCLUSIONS Combination of lamivudine and interferon for 1 year is followed by high end-treatment virological and biochemical response rates, by improvement of liver histology and by the prevention of the emergence of YMDD mutation; however, the sustained response rate remains low.

Sequential treatment for Helicobacter pylori eradication in duodenal ulcer patients: improving the cost of pharmacotherapy

Background : Several studies have shown that Helicobacter pylori eradication rates with standard 7‐day triple therapy are unsatisfactory. A novel 10‐day sequential treatment regimen recently achieved a significantly higher eradication rate. To improve the pharmacotherapeutic cost, we evaluated whether an acceptable eradication rate could be achieved in peptic ulcer patients by halving the dose of clarithromycin.