Potential therapeutic role of melatonin in hepatobiliary diseases: current evidence and clinical observations.

Melatonin (MEL) is produced and secreted by the pineal gland as well as the small intestine, liver, retina, lymphocytes, and melanocytes in the skin in both experimental animals as well as in humans. While pineal and retinas MEL is closely related to the light/dark cycle, the production of MEL by other so called extrapineal tissues is independent of such circadian rhythm. Among the primary mechanisms of action of MEL in humans, the most important are interaction of MEL with specific receptors (M1, M2, M3) and the MEL 'scavenging' activity against the formation of free oxygen metabolites as a result of MEL's ability to transfer free electrons and stimulation of the expression of redox reaction enzymes. In addition, MEL binds to intracellular proteins such as calmodulin, thereby affecting the course of cell cycle, and it has been shown to activate of nuclear receptors belonging to the retinoid orphan receptors/retinoid Z receptors (ROR/RZR) subfamily. MEL exerts regulatory effects on the master clock regulating diurnal rhythms. This updated review presents current view on the synthesis and metabolism of MEL and the growing body of experimental evidence transferable to the practical medicine supporting a pleiotropic molecule beneficial effects on the health including protection against various organ abnormalities, including internal organs such as the liver. Although the beneficial effects of MEL in various types of liver damage have been well documented in experimental studies, there are relatively few studies on liver dysfunction in humans. Considering the worldwide obesity pandemic often associated with the occurrence of steatohepatitis and cirrhosis, the beneficial effects of MEL in liver pathology should be proven in randomized trials involving patients presenting with hepatic disorders.

The emering role of helminths in treatment of the inflammatory bowel disorders.

The problem of immune-mediated diseases, such as inflammatory bowel disorders (IBDs), still remains a significant clinical and therapeutic problem. Therefore, the tendency to search for safer and more effective methods of reducing their incidence and increasing the efficiency of therapy of this group of diseases is understandable. Recently, attention has been drawn to the potential therapeutic influence of intestinal helminths on the inflammatory process induced by the immune response, as well as the observed significant potential of these organisms for modulating the host immune response, which is beneficial both for the dwelling parasite and the host with an IBD. It has been proven that the effects of certain intestinal helminths on the host immune system are complex and omni-directional. They involve the modulation of TLRs expression, causing proliferation and activation of TH2 lymphocytes, leading to proliferation of regulatory T cells (TREG), and production of immunomodulatory proteins, such as cystatins and glycoprotein ES-62. In the developing countries of Africa, South America and Asia, where the level of personal and environmental hygiene is relatively low, the incidence of autoimmune diseases is also significantly lower. Limited exposure to common bacterial and parasitic pathogens in populations of very highly developed countries has probably contributed to depletion of immunological memory and the development of hypersensitivity mechanisms. Thus, reasonable suggestions have been made that the host-parasite biocenotic relationship between humans and nematodes of the gastrointestinal tract can be considered as a mutualism, rather than a typical parasitism, and may in the future be used as an alternative therapeutic model for IBD patients.

The effects of long-term melatonin treatment on plasma liver enzymes levels and plasma concentrations of lipids and melatonin in patients with nonalcoholic steatohepatitis: a pilot study.

The present study represents the follow-up of our initial observations designed to investigate whether in patients with nonalcoholic steatohepatitis (NASH) the beneficial effect of 12-week course of melatonin (MT) on liver enzymes could be maintained with prolonged period of treatment and to analyze whether biochemical treatment responses could be sustainable after melatonin discontinuation. Forty two patients with histologically proven NASH (30 treated with melatonin 2x5 mg daily, 12 controls receiving placebo) enrolled to our previous 3-month study agreed to take part of subsequent 12 weeks treatment followed by 12-week follow-up period. Enrolled patients had biochemical determinations every six weeks during the melatonin treatment period and again after 12 weeks of follow-up. Significant reduction in median alanine aminotransferase (ALT) levels between baseline and week 18, week 24 and follow-up was observed in both MT-treated and control group: 43% and 31%, 42% and 33%, 32% and 31%. Aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (GGT) levels decrease significantly only in MT-treated group. In MT-treated group mean percentage change in AST level below baseline at week 18, at week 24 and at follow-up was 45%, 33% (p<0.05) and 8% (ns), respectively. The evolution of GGT levels was as follows: the mean percentage reduction in GGT below baseline level at week 18, 24 and follow-up was: 48%, 52% and 38% (p<0.05), respectively. In both MT-treated and control group plasma cholesterol, triglicerydes and glucose concentrations as well as plasma alkaline phosphatase persisted within normal values during the prolonged study period. Plasma concentration of melatonin (pg/ml) in MT-treated group averaged 7.5±3.5 at baseline and increased to 52.5±17.5 at 24th week. The results of our study demonstrating beneficial effect of melatonin on liver enzymes in patients with NASH would seem to encourage further controlled trials of melatonin given over a longer period of time with liver histology as end point.

The pilot study of 3-month course of melatonin treatment of patients with nonalcoholic steatohepatitis: effect on plasma levels of liver enzymes, lipids and melatonin.

The mechanism by which nonalcoholic fatty liver disease (NAFLD) progresses into nonalcoholic steatohepatitis (NASH) is unknown, however, the major process is oxidative stress with increased production of reactive oxygen species and excessive inflammatory cytokine generation. To date, there are no effective treatments for NASH and the published data with treatment using antioxidants are not satisfactory. Melatonin (MT), the potent endogenous antioxidant secreted in circadian rhythm by pinealocytes and in large amounts in the digestive system, was reported to improve oxidative status and to exert beneficial effects in NASH pathology in experimental animals, but no study attempted to determine the possible effectiveness of MT in humans with NASH. In this study, 42 patients (12 placebo controls and 30 MT-treated) with histological evidence (liver biopsy) of NASH and no history of alcohol abuse, were included. The treatment group took melatonin (2x5 mg/daily orally), while controls were treated with placebo. At baseline no significant differences between the groups were found for age, body mass index (BMI) as well as for plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and concentrations of cholesterol, triglycerides (TG), glucose and MT. During the study period plasma ALT level and cholesterol concentration decreased significantly in both MT-treated and control groups, however AST and GGT levels decreased significantly only in MT-treated groups. Median value of AST level at baseline was 76.5 (64.2-114.2) IU/L and its percentage decrease at 4, 8 and 12 week was 20, 36 and 38%, resp. Baseline GGT median level was 113 (75.7-210.7) IU/L and its mean percentage decrease at week 4, 8 and 12 was 46, 48 and 47%, resp. Plasma ALP levels did not change significantly during MT treatment. Median value of plasma concentrations of MT (pg/mL) in MT-treated group rose from 7.5 (5.0-14.25) at baseline to 35.5(18.8-110.0), 43.5(17.0-102.5) and 49.5(18.0-99.5) at the end of 4, 8 and 12 week of treatment, respectively. Plasma levels of TG and glucose as well as BMI in controls and MT-treated patients were not significantly different from baseline. This study demonstrates for the first time in humans that three months treatment with MT significantly improves plasma liver enzymes in patients with NASH without causing any side-effect. Plasma MT levels during the whole period of MT treatment persisted above that at baseline. Our findings show that treatment with MT significantly improves plasma liver enzymes in NASH patients, but larger cohort trials and longer treatment with MT are required before this indole could be included into the spectrum of the NASH treatment.

Altered basal and postprandial plasma melatonin, gastrin, ghrelin, leptin and insulin in patients with liver cirrhosis and portal hypertension without and with oral administration of melatonin or tryptophan.

This investigation was designed to assess the effects of oral administration of melatonin (10 mg) and tryptophan (Trp) (500 mg) on fasting and postprandial plasma levels of melatonin, gastrin, ghrelin, leptin and insulin in 10 healthy controls and in age-matched patients with liver cirrhosis (LC) and portal hypertension. Fasting plasma melatonin levels in LC patients were about five times higher (102 +/- 15 pg/mL) than in healthy controls (22 +/- 3 pg/mL). These levels significantly increased postprandially in LC patients, but significantly less so in controls. Treatment with melatonin or L-Trp resulted in a further significant rise in plasma melatonin, both under fasting and postprandial conditions, particularly in LC patients. Moreover, plasma gastrin, ghrelin, leptin and insulin levels under fasting and postprandial conditions were significantly higher in LC subjects than in healthy controls and they further rose significantly after oral application of melatonin or Trp. This study shows that: (a) patients with LC and portal hypertension exhibit significantly higher fasting and postprandial plasma melatonin levels than healthy subjects; (b) plasma ghrelin, both in LC and healthy controls reach the highest values under fasting conditions, but decline postprandially, especially after oral application of melatonin or Trp; and (c) plasma melatonin, gastrin, ghrelin and insulin levels are altered significantly in LC patients with portal hypertension compared with that in healthy controls possibly due to their portal systemic shunting and decreased liver degradation.

Only live Helicobacter pylori is capable of caspase-3 dependent apoptosis induction in gastric mucosa epithelial cells.

Physiological process of cell death, apoptosis, plays a beneficial role in organism survival, but in some pathologies, like gastric Helicobacter pylori (Hp) infection, this process may turn against the host organism causing tissue damage. Knowledge of the mechanisms controlling apoptosis may have potential significance in treatment of these pathologic states. Therefore, we sought to determine whether apoptosis induced in the gastric epithelial cells exposed to live Hp involves the alteration in heat shock protein 70 (HSP70) expression and activation of caspase-3 in peroxisome proliferator-activated receptors (PPARgamma dependent manner). Experiments were performed with KATO III, gastric epithelial cells, exposed to CagA and Vac A positive live Hp, water Hp extracts or Hp culture supernatant over different time periods. Total cellular RNA and proteins were isolated for PCR, western-blot and EMSA studies. Genomic DNA was isolated to analyze apoptosis status. We propose new model of Hp induced HSP70 dependent, caspase-3 executed apoptosis in human gastric epithelium. KATO III cells exposed to Hp, showed an increase in caspase-3 activity accompanied and preceeded by activation of nuclear translocation of PPARg peaking at 48 h of culture. Moreover, heat shock factor 1 (HSF-1) bound up with phosphorylated STAT-3 was unable to activate HSP70 protein synthesis in KATO III exposed to Hp. Lack of protective effect of HSP70, activation of caspase-3--dependent apoptosis pathway caused by Hp and alteration of the bax/bcl-2 cellular equilibrium led to gastric epithelial cell death. The observed phenomenon might be helpful in understanding of the mechanism of Hp related gastrointestinal tract diseasess, especially gastric cancer.

Molecular mechanism of protection against chemically and gamma-radiation induced apoptosis in human colon cancer cells.

The involvement of peroxisome proliferator-activated receptors (PPARs) in the cancer cell elimination through apoptosis is a generally accepted fact. However, some reports indicate that the activation of PPARgamma is directly responsible for carcinogenesis. Caco-2 cells, a human adenocarcinoma cells, were used as a model of colon cancer. Cell cultures (5 x 10(6) cell per dish) were pretreated for 24 h with PPAR gamma agonists ciglitazone (CI, 1 x 10(-6)M) and retinoic acid (RA, 1 x 10(-6)M) and part of the cultures were subsequently subjected to gamma-radiation (photons) with therapeutic dose of 2,5 Gy. Total cellular RNA and proteins (cytoplasmic and nuclear) were isolated 24h after cultures irradiation or 48 h after stimulation in the non irradiated part of experiment to preserve the equal growth time for all samples. gamma-Irradiation of the cells abolished nuclear translocation of PPARgamma under its agonists treatment and preserved PPARgamma in the cytoplasmic pool. But it did not affect the HSP 70 expression in response to ciglitazone and retinoic acid. Moreover, combined gamma-irradiation and CI/RA treatment of the cells changed the equilibrium between Bax and Bcl-2 mRNA to anti apoptotic state with increased expression of Bcl-2 and almost abolished expression of Bax. In conclusion, this paper provides an evidence for the anti-apoptotic action of PPARgamma agonists used along with the gamma-radiation. Moreover, it shows that the up-regulated HSP70, in response to PPARgamma agonists in gamma-irradiated cultures promotes cell survival.

Helicobacter pylori in liver diseases.

At the present stage of knowledge, the participation of the Helicobacter bacteria in the pathology of liver and the bile tract in humans has not been univocally documented. However, apparent are the premises so as to go on performing the examinations under discussion since the said participation cannot be excluded. If the more direct evidence of the etiologic role of the Helicobacter in the pathology of liver were available, it would create the chances for the more effective treatment of patients than the case has been so far. Cancer commonly derives from the chronic inflammation and infection and in case of hepatocellular carcinoma (HCC), may arise either from local liver derived progenitor cells (LPCs) or bone marrow originated stem cells (BMSCs) and future studies should disclose the role of either type of cells and of inflammatory factors such as generated by Helicobacter infection in the liver pathophysiology.

Sympathovagal balance fluctuates during colonoscopy.

BACKGROUND AND STUDY AIM: Colonoscopy is a common gastroenterological procedure for investigation of the bowel. The main side effects of colonoscopy are pain during investigation, cardiovascular complications and very rarely even death. The aim of this study was to compare the continuous fluctuation of heart rate variability (HRV) components during colonoscopy under normal conditions, analgesia/sedation, and total intravenous anesthesia. PATIENTS AND METHODS: 37 consecutive patients (aged 35 - 65), were randomly allocated to three groups: no sedation (control group 1); analgesia/sedation (group 2); and total intravenous anesthesia (group 3). Holter electrocardiography and subsequent frequency domain analysis were undertaken. The low-frequency (LF, 0.04 - 0.15 Hz) and the high-frequency (HF, 0.15 - 0.40 Hz) components were estimated using spectral analysis in the usual way. Normalized units (nu) were calculated from the following equations: LFnu = LF/(LF + HF), and HFnu = HF/(LF + HF). RESULTS: Groups 2 and 3 were found to have a significantly lower HFnu and higher LFnu than group 1 essentially throughout the procedure. A one-way analysis of variance and t-test confirmed that the differences were significant when the colonoscope reached the splenic flexure as were the LF/HF balances at the splenic and hepatic flexures and the cecum. The percentage change in LF/HF was also analyzed, and it was found that in group 3 the mean change was over 136 % when the colonoscope reached the sigmoid flexure, which was significantly higher than in the other two groups. CONCLUSION: Most changes in HRV components occurred during colonoscopy of the left side of the bowel. Analgesia/sedation and total intravenous anesthesia increased HRV by increasing the LF component.

Progastrin and its products from patients with chronic viral hepatitis and liver cirrhosis.

BACKGROUND: Gastrin and its precursor, progastrin, are synthesized in the stomach, particularly when infected with Helicobacter pylori, and they are metabolized, at least in part, in the liver. However, little is known about their levels in various hepatic diseases. METHODS: This study was carried out on 147 patients including chronic hepatitis B (n = 35), hepatitis C (n = 52) and liver cirrhosis (n = 60) of class A (n = 38), class B (n = 15) and class C (n = 7) (Child-Pugh classification) and age- and sex-matched healthy controls (n = 65). The diagnosis of chronic hepatitis was confirmed by liver biopsy in all patients, whereas the diagnosis of liver cirrhosis was based on clinical and laboratory findings. Liver biopsy was done in 38 out of 60 patients. Blood samples were collected under basal conditions and separated plasma samples were kept frozen at -70 degrees C until radioimmunoassay of progastrin and its products, including bioactive amidated gastrins. RESULTS: Median (range) plasma concentrations of total progastrin product and amidated gastrin in control subjects were 147.5 (73-345) pM and 33 (15-65), respectively. These concentrations in hepatitis B and C were not significantly different from those in controls. In cirrhosis (classes A, B and C), the concentrations of the progastrin and of gastrin were significantly (P < 0.05) higher than in controls reaching, respectively, 253.5 (135-683 pM) and 47.5 (17-385) pM. Both progastrin and gastrin levels were significantly higher in H. pylori-positive than in negative cirrhotic patients. Antibodies against H. pylori were present in about 50% of controls, 68% of hepatitis B, 57% of hepatitis C and in 83% in cirrhosis patients. The difference in H. pylori prevalence between cirrhosis and controls was statistically significant. CONCLUSIONS: Plasma levels of progastrin and gastrin are significantly increased in cirrhotic patients and this could be attributed to reduced metabolism of these peptides in liver cirrhosis and to their increased release due to H. pylori infection rate in this disease.

Short-term changes of serum IL-2 and IL-6 induced by interferon alpha-2b in patients with chronic hepatitis C.

BACKGROUND: The standard therapy of chronic hepatitis C with interferon alpha (IFN alpha) and ribavirin has established but limited efficacy. The prognostic factors of treatment are still under investigation. IL-2 and IL-6 are key cytokines involved in activation of B and T lymphocytes and thus in humoral and cellular responses; they are also deeply involved in generation and maintenance of inflammatory processes. The aim of the study was to evaluate the short-term influence of INF alpha-2b on serum IL-2 and IL-6 levels in sustained responders (SR) and non-responders (NR). MATERIAL AND METHODS: Altogether 12 patients (7 males and 5 females) chronically infected with HCV (anti-HCV positive, HCV-RNA positive by PCR) were enrolled to the study. Patients were treated with IFN 3 MU tiw for 6 months and then they were followed for another 6 months. Five patients responded to the treatment (sustained responders-SR)-Group I, seven patients did not respond (non-responders-NR)-Group II. Serum concentrations of IL-2 and IL-6 were assessed by ELISA before ['0'] and at 1st ['1'], 2nd ['2'], 3rd ['3'], 6th ['4'] and 12th ['5'] hour after the first IFN injection. CONCLUSIONS: Interferon alpha-2b induced short-term increase of serum IL-2 concentrations in SR but not in NR. Serum IL-6 level increased both in SR and NR but this effect was more pronounced and persisted longer in sustained responders.

Cardiac sympathovagal balance during endoscopic retrograde cholangiopancreatography.

BACKGROUND AND STUDY AIMS: Cardiopulmonary complications have been reported during upper gastrointestinal endoscopy and endoscopic retrograde cholangiopancreatography (ERCP). The aim of this study was to evaluate the sympathovagal response to the stretching of the common bile duct caused by contrast medium injection during ERCP. PATIENTS AND METHODS: The 16 patients included in the study were assigned to two groups according to the size of common bile duct. Group A consisted of patients with normal-sized bile ducts, while Group B patients had dilated common bile ducts. The heart rate variability (HRV) technique was used to assess the sympathovagal balance. The HRV data were gathered using the Holter technique, and frequency domain analysis revealed two main spectral components of HRV: low frequency (LF) and high frequency (HF). RESULTS: In patients with normal-sized bile ducts, contrast injection initiated a rapid increase in the power spectra (ms2) of both the HF and LF components. In patients with dilated common bile ducts, this phenomenon was not observed. CONCLUSIONS: During ERCP, contrast injection into a dilated common bile duct does not stimulate the autonomic nervous system in the same way as it does when the common bile duct is of normal size.

Activation of plasma contact and coagulation systems and neutrophils in the active phase of ulcerative colitis.

We have shown that the contact (kallikrein-kinin) system is involved in the pathogenesis of experimental enterocolitis. We now investigate activation of the contact and coagulation pathways, platelets, and neutrophils in active and inactive ulcerative colitis patients as compared to normal controls. In active ulcerative colitis patients, a significant decrease of plasma prekallikrein, high molecular weight kininogen, and C1 inhibitor levels was observed as compared with controls, as well as prekallikrein activation on western blots. Significant elevation of prothrombin fragment (F1 + 2), which indicates thrombin generation, and elastase-alpha 1-antitrypsin complexes, reflecting neutrophil activation, were found in patients with active disease. Plasma beta-thromboglobulin, a marker of platelet activation, was elevated in both active and inactive disease and appears to be a feature of ulcerative colitis. Activation of contact and coagulation pathways, as well as neutrophils, may mediate inflammation in the active phase of ulcerative colitis.

A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1.

The function of the conserved Phe 100 residue of RNase T1 (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kcat reduced 75-fold and a small increase in Km for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T1 complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.