To find a practical method to establish hepatopulmonary syndrome (HPS) in rats for use as an experimental model system.Forty male Sprague-Dawley rats were equally divided into a normal group (injected subcutaneously with 3 mL/kg of olive oil for 12 weeks), abdominal compartment syndrome (ACS) group (injected subcutaneously with 3 mL/kg olive oil for 12 weeks, followed by an intraperitoneal injection of 4% succinylated gelatin and maintanence of 20 mmHg abdominal pressure for 3 h), cirrhosis group (injected subcutaneously with 40% carbon tetrachloride (CCl4) in olive oil twice weeklyfor 12 weeks, with first dose doubled), and an ACS+ cirrhosis (HPS model) group (CCl4-induced, followed by the intraperitoneal injection with succinylated gelatin and 3 h of 20 mmHg abdominal pressure). The mice were sacrificed to perform blood gas analysis and to assess lung pathology. Comparisons between two groups were carried out by non-parametric analysis, and multiple comparisons were carried out by the Kruskal-Wallis H test.Blood gas analyses showed significant differences in the values of pH for the normal group (7.41+/-0.04), the ACS group (7.22+/-0.06), the cirrhosis group (7.53+/-0.04), and the HPS model group (7.47+/-0.02) (P less than 0.05). The ACS group and the HPS model group showed significantly different values of partial pressure of oxygen (PaO2; 58.57+/-5.41 and 58.20+/-3.19 mm Hg) and of alveolar-arterial oxygen difference (AaDO2; 83.86+/-28.49 and 84.80+/-11.82 mm Hg) than the normal group and the cirrhosis group (PaO2: 86.67+/-1.37 and 85.00+/-2.53 mm Hg; AaDO2: 38.17+/-9.20 and 37.00+/-6.23 mm Hg) (P less than 0.05). Pathological analysis of the lungs from the ACS group revealed widened alveolar septa, different-sized alveolar spaces, reduced lung capacity, edema and hemorrhage in some of the alveolar cavities, and telangiectasia in the alveolar walls. The lungs from the cirrhosis group also showed widened alveolar septa, different-sized alveolar spaces, and reduced lung capacity, but were distinct in the features of inflammatory cell infiltration, and hyperemia in the pulmonary vessels. The lungs from the HPS model group showed all of the features of both the lungs from the ACS and cirrhosis groups, but also showed macrophage accumulation and microthrombi in the pulmonary vessels.Inducing ACS in the setting of CCL4-induced cirrhosis in a rat generates pathological features that adequately mirror those of HPS and may represent a useful experimental model for in vivo studies of HPS.

To determine the efficacy of the plant-derived bioflavonoid, quercetin, for treating nonalcoholic fatty liver disease (NAFLD) by using a rat model, and to investigate the molecular mechanism underlying its therapeutic effects.One-hundred Sprague-Dawley rats were randomly assigned into the normal control group (normal group), untreated NAFLD model control group (model group), 75 mg/kg/day quercetin treatment group (low-dose group), and 300 mg/kg/day quercetin treatment group (high-dose group). The NAFLD rat model was established by providing four weeks of a high-fat diet; the normal group received normal rat chow diet. The quercetin treatments were administered for eight weeks after model establishment and control groups received simultaneous gavages of isotonic saline, with continuation of the respective diets. At the end of the eight weeks (experimental week 12), the rats were sacrificed for liver and serum collection. Intergroup differences in liver index, fasting blood glucose (FBG), triglycerides (TG), interleukin (IL)-18, IL-10, malondialdehyde (MDA), and histopathological features were assessed by independent samples t-test (normal vs. model), one-way ANOVA (model vs. treatments), and least significant difference t-test (pairwise comparisons); correlations were assessed by Pearson's correlation coefficient.Compared with the normal group, the model group showed significantly higher liver index (t=-2.327), FBG (t=-3.482), TG (t=-0.302), and serum IL-18 (t=-2.704) (all P less than 0.05), but significantly lower IL-10 (t=2.622, P less than 0.05); the MDA level was also higher in the model group, but the difference was not significant (t=-1.083, P less than 0.05). Livers from the model group showed obvious histological features of inflammation (lymphocyte and neutrophil infiltration) and steatosis (cytoplasmic lipid droplets). Inflammation was positively correlated with IL-18 (P less than 0.05), but negatively correlated with IL-10 (P less than 0.05), while steatosis was negatively correlated with IL-10 (P less than 0.05). Compared to the model group, quercetin treatment (both low- and high-dose) led to significant decreases in the liver index, FBG and IL-18 (all, P less than 0.01), and significant increase in IL-10 (P less than 0.05); however, the changes in liver index, FBG and IL-10 were not significantly different between the low- and high-dose treatment groups, but the high-dose of quercetin did induce a significantly greater decrease in IL-18 than the low-dose (P less than 0.05).NAFLD rats have higher serum levels of IL-18 but lower levels of IL-10 than their healthy counterparts, and these differential cytokine expressions may be related to liver inflammation and steatosis. Quercetin treatment may help to delay the progression of NAFLD, possibly by adjusting the balance of inflammatory cytokines.

To explore the role and mechanism of the Fas/Fas ligand (FasL) system and its downstream signaling pathway related to the progression of alcoholic steatohepatitis and liver fibrosis.Eighteen C57BL/6J mice were randomly divided into three groups: controls; alcoholic steatohepatitis model, given four-weeks of a 4% ethanol-containing Lieber-DeCarli liquid diet; alcoholic steatohepatitis and liver fibrosis model, given the four-week alcohol diet followed by twice weekly intraperitoneal injections of carbon tetrachloride (5% olive oil solution; 2 mL/kg dose) during the fifth to eighth weeks. Mice in the model groups were sacrificed at the end of week 4 and 8, respectively, along with control mice for comparative analyses. Liver tissue sections were evaluated for hepatocellular apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. The mRNA expression of Fas, FasL, cysteine aspartate-specific proteases 3 (caspase 3), and cytochrome P450 2E1 (CYP 2E1) in liver tissues was detected by reverse transcription (RT)-PCR, visualized by ethidium bromide staining, and normalized to the gray-value of GAPDH expression. The protein expression of Fas and caspase 3 were detected by western blotting (b-actin normalized), and of FasL and CYP 2E1 by immunohistochemistry staining. Intergroup differences and statistical significance were evaluated by single factor analysis of variance and the least squares difference-t test or the Kruskal-Wallis H test and the Mann-Whitney U test.The number of apoptotic cells in the liver sections was significantly higher in both model groups with alcoholic steatohepatitis (vs. controls) and the amount in the alcoholic steatohepatitis plus liver fibrosis model was significantly higher than that in the model with only alcoholic steatohepatitis. In addition, activation of Fas, FasL and its downstream signaling pathway showed an increasing trend with extent of liver injury. The hepatic mRNA (by RT-PCR) and protein (by western blotting) normalized expression levels in the controls, alcoholic steatohepatitis models, and alcoholic steatohepatitis plus liver fibrosis models were, respectively: Fas mRNA: 0.50+/-0.05, 0.61+/-0.10, 0.76+/-0.03 (H=12.137, P less than 0.05), protein: 0.52+/-0.14, 0.86+/-0.10, 0.99+/-0.09 (F=12.758, P less than 0.01); FasL mRNA: 0.31+/-0.03, 0.53+/-0.02, 1.02+/-0.04 (F=153.260, P less than 0.01); caspase 3 mRNA: 0.86+/-0.11, 0.85+/-0.05, 1.33+/-0.16 (F=8.740, P less than 0.01), protein: 0.40+/-0.03, 0.69+/-0.06, 1.02+/-0.10 (F=90.785, P less than 0.01); CYP 2E1 mRNA: 0.72+/-0.14, 1.00+/-0.15, 1.30+/-0.20 (H=4.713, P less than 0.01). The changes in hepatic FasL and CYP 2E1 expression detected by immunohistochemistry were consistent with the mRNA expression.Activation of Fas/FasL and its downstream signaling pathway, which induces hepatocellular apoptosis, contributes to the development of alcoholic steatohepatitis and liver fibrosis.

To characterize the clinical, laboratory, imaging and pathological features of primary sclerosing cholangitis (PSC) and investigate the impact of ursodeoxycholic acid (UDCA) therapy on patient prognosis.The medical records of 22 patients diagnosed with PSC between 2002 and 2011 were retrospectively reviewed. The PSC diagnosis had been made in patients with suspect biochemical abnormalities following evaluation by magnetic resonance cholangiopancreatography (MRCP) and/or endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous transhepatic cholangiography (PTC). Fibrosis and inflammaton were assessed by immunohistochemical analyses of tissue biopsies. Outcome of patients treated with UDCA (13-15 mg/kg/day, oral) were compared to that of patients without UDCA treatment by the X2 or corrected X2 tests.Among the 22 PSC patients, the majority was male (n=15) and presented with fatigue, dark urine, and body weight loss (n=15). Four cases had ulcerative colitis. At admission, all 22 cases showed elevated levels of alkaline phosphatase[ALP: (348+/-184) U/L], 19 cases showed elevated alanine aminotransferase [ALT: (94.0+/-67.0) U/L] and aspartate aminotransferase [AST: (98.0+/-67.0) U/L], and 15 cases showed elevated levels of total bilirubin (99.0+/-115.0) mumol/L and direct bilirubin (74.4+/-92.4 mumol/L. ERCP examination showed segmental intrahepatic bile duct stenosis with expansion, and stiff and enlarged gallbladder bile ducts, but unclear findings for the common bile ducts and pancreatic ducts. MRCP showed beading of the intrahepatic bile duct, stiffness of the bile duct wall, and dilation of the common bile duct. Fibrosis and inflammation were observed in the bile ducts, along with hyperplasia and the typical features of "onion skin" fibrosis and fibrous obliterative cholangitis. Five of the 10 patients treated with UDCA improved, and seven of the 12 patients in the non-UDCA treatment group improved. There was no statistically significant difference in outcome between the groups (paired X2=0.333, corrected X2=0.083, P more than 0.05).PSC patients were predominantly male and the common clinical manifestations were fatigue, dark urine, and body weight loss. At admission, serum biochemical indicators of cholangitis were increased significantly and subsequent imaging studies confirmed the suspected diagnosis by showing obvious characteristic changes. UDCA treatment did not significantly improve patient prognosis.

To determine the expression of nerve growth factor (NGF) in hepatic tissues and serum of patients with primary liver cancer (PLC), and to investigate the relationship of serum NGF levels with clinicopathological features of PLC.Hepatocellular carcinoma (HCC) samples and patient-matched tumor-adjacent liver samples were collected from 26 PLC patients to assess the mRNA and protein expressions of NGF by reverse transcription-PCR, western blotting (b-actin normalized), and immunohistochemistry. In addition, serum samples were collected from 40 PLC patients, 40 liver cirrhosis patients, 40 chronic hepatitis patients (including hepatitis B or C virus infections), and 30 healthy (normal) controls. The serum levels of NGF were measured by enzyme-linked immunosorbent assay. Intergroup differences were assessed by the t-test, and correlation with sex, age, presence of cirrhosis, tumor size, and TNM classification were assessed by the Kruskal-Wallis H test followed the Mann-Whitney U test.HCC tissues showed higher mRNA and protein expressions of NGF than the corresponding tumor-adjacent non-HCC tissues. Hepatic NGF expression was mainly localized to the tumor cell cytoplasm. Serum NGF expression was significantly higher in PLC patients (33.86+/-16.11 pg/ml) and cirrhosis patients (20.57+/-9.73 pg/ml) than in normal controls (11.13+/-6.12 pg/ml) and chronic hepatitis patients (13.20+/-6.23 pg/ml) (P less than 0.01). Furthermore, when the PLC patients were stratified according to tumor size and TNM stage, the serum NGF level was found to be significantly higher in patients with tumors more than 5 cm (vs. less than 5 cm; U=83.000, P=0.002) or of TNM stage III/IV (vs. stage I/II; U=103.500, P=0.009).Elevated expression of NGF in liver cancer tissues and serum of PLC patients is related with tumor size and TNM staging. These findings suggest that NGF may play a role in HCC tumorigenesis and/or that serum NGF may represent a prognostic marker of PLC.

To investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and liver fibrosis using a C57BL/6J mouse model of chronic inflammation.Twelve male C57BL/6J mice were given a high-fat diet (15.0% fat, 1.25% cholesterol, 0.5% cholic acid) and randomly assigned to the normal control group (n=6; subcutaneously injected with 0.5 mL of isotonic saline, every other day for 14 weeks) or the chronic inflammation model group (n=6; subcutaneously injected with of 0.5 mL of 10% casein, every other day for 14 weeks). At the end of week 14, the animals were sacrificed and blood was collected from the left ventricle for serological analysis of inflammatory markers and lipid profile, including serum amyloid A (SAA), interleukin-6 (IL-6), total cholesterol (TC) and free cholesterol (FC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL)). Extracted liver tissues were divided for use in histological analysis (lipid accumulation and fibrosis evaluated by Oil Red O, Sirius red and Masson's trichrome staining) and quantitative fluorescence real-time PCR (to measure b-actin normalized expression of TNF-a MCP1, SREBP-2, LDLr, HMGCoA-r, ATF-6, GRP78, BMP-7, TGF-b, and collagens type I and type IV). Comparisons between groups were made by the two-samples t-test or Satterthwaite t-approximation test, collagen type I and type IV.Compared to the normal control group, the inflammation model group showed elevated serum IL-6 (12.55+/-4.75 vs. 32.41+/-7.42 pg/mL, P less than 0.01), reduced serum TC (14.82+/-1.56 vs. 10.62+/-0.48 mmol/L, P less than 0.01), up-regulated hepatic TNF-a mRNA expression (1.05+/-0.35 vs. 2.12+/-0.72, P less than 0.01), and elevated hepatic TC (12.10+/-2.57 vs. 23.21+/-8.75 mmol/L, P less than 0.05). In addition, the inflammation group showed abnormal lipid deposition, and increased and thickened reticular fibers. The livers of the inflammation group also showed up-regulated mRNA expression of SREBP-2 (normal control: 1.01+/-0.19 vs. 2.63+/-0.13, P less than 0.05), GRP78 (1.07+/-0.47 vs. 2.21+/-0.99, P less than 0.05), TGF-b (1.01+/-0.14 vs. 1.38+/-0.28, P less than 0.05), and collagen type I (1.02+/-0.27 vs. 1.71+/-0.51, P less than 0.05) and down-regulation of BMP-7 (1.01+/-0.15 vs. 0.55+/-0.25, P less than 0.01).Activation of the inflammatory system exacerbates hepatic cholesterol accumulation and hepatic fibrosis in C57BL/6J mice.

To observe the effects of Wnt3a on proliferation and, activation of hepatic stellate cells (HSCs) and their the expression of the transforming growth factor beta (TGFb) and /Smad signaling factors of rat hepatic stellate cells line in vitro using a rat HSC line.Sychronized HSC-T6 cells were stimulated with various concentrations of recombinant Wnt3a (50, 100, 200, 250 and 300 ng/mL). Unstimulated cells served as controls. Edu Effects on proliferation were determined by EdU (5-ethynyl-2'-deoxyuridine) incorporation assay and fluorescence microscopy.analysis was used to observe the proliferation of the hepatic stellate cells stimulated by different concentration of recombinant Wnt3a, and theEffects on the protein expression of TGFb/Smad signaling factors was assessed by western blot detection (gray-value analysis) of alpha-smooth muscle actin (a-SMA), a-SMA, TGFb1, Smad3, and and Smad7; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected as the normalization control in the hepatic stellate cells was observed by Western blot analysis .The correlation was also observed. The significance of inter-group differences was assessed by one-way ANOVA, and correlations were determined using bivariate statistical modeling.In general, HSCThe proliferation of hepatic stellate cells increased after the addition ofin response to Wnt3a stimulation for 24 h, reaching its peak atThe maximum proliferation rate was observed with the 200 ng/mL Wnt3a concentration (63.00+/-2.30%), and it increased dramatically compared with those inwhich was significantly higher than the proliferation rates of the unstimulated control cells, and the cells stimulated with 50, 100 and 150 ng/mLl group (P less than 0.05), but the increase was not significantly different from that in the compared cells stimulated with 250 and 300 ng/mLl group,it had no obvious increase(P more than 0.05).; The Wnt3a stimulation also led to time-dependent increases in the protein expressions of a-SMA, TGFb1, and Smad3 increased with the addition of Wnt3a and the extension of time . For all three, The maximal amount of increased protein expressiony all reached to thewas maximal produced by stimulation when hepatic stellate cells were treated bywith 300 ng/mLl Wnt3a for 48 h hours,and the rations of(normalized gray- values:s of a-SMA, 1.0860+/-0.0101; TGFb1, 1.0346+/-0.0118; Smad3, to GAPDH were 1.0860+/-0.0101, 1.0346+/-0.0118, 1.0306+/-0.0122)respectively. HoweverIn contrast, the Wnt3a stimulation led to concentration- and time-depenent decreases in Smad7 expressionvaried inversely, with to them with the minimal ration of it to GAPDHthe maximal decrease occurring with 300 ng/mL Wnt3a for 48 h (0.7736+/-0.0139) after being treated by 300 ng/ml Wnt3a for 48h. The comparison was remarkably discrepant, (P less than 0.05).There were positive correlations between a-SMA expression and was found to be positively correlated to TGFb1, Smad3 (r=0.968, P less than 0.05) and; Smad3 (r=0.997, P less than 0.01), but a-SMA and Smad7 had negatively correlated to Smad7 ion(r=0.960, P less than 0.05).Wnt3a can increase thestimulates proliferation as well asand activation of ratthe hepatic stellate cellsHSCs , and upregulate modifies the expression of TGFb/Smad signaling factors, of the hepatic stellate cells, andwhich may promote the hepatic fibrosis.

To explore the factors influencing failure of an immunization to interrupt perinatal (mother-to-child) transmission of hepatitis B virus (HBV).Between June 2006 and March 2010, a total of 1355 pregnant women testing positive for the hepatitis B surface antigen (HBsAg), at gestational weeks 20 to 42, and without use of antiviral or immunomodulatory drugs during the pregnancy were prospectively recruited to the study. The mothers were given a choice of receiving hepatitis B immunoglobulin (HBIG; three 200 IU intramuscular injections give at four-week intervals starting from gestation week 28) or not. All neonates (1360, including five sets of twins) received hepatitis B vaccine (10 mug) plus HBIG (200 IU) combined immunization within 24 h of birth, as early as possible. Peripheral venous blood samples were collected from the neonates within 24 h of birth and at 7 and 12 months of age for detection of HBV markers, including hepatitis B e antigen (HBeAg) and HBV DNA. The infants were classified according to HBV perinatal transmission status (infection group and non-infection group) and various factors (maternal-related: age, gravidity, parity; pregnancy/birth-related: threatened premature labor, complications; neonate-related: sex, birth weight, apgar score) were compared between the two groups by using non-conditional logistic regression analysis to determine their potential influence on failure of immunization to inhibit transmission.After 12 months of follow-up, 1.54% (21/1360) of the neonates had presented with HBV infection. Analysis of the HBV-infected neonates revealed differences in infection rates between neonates born to mothers with HBIG injection (2.22% vs. without HBIG injection: 1.11%, P less than 0.05) and caesarean section (1.35% vs. vaginal delivery: 1.73%) but neither reached statistical significance (P less than 0.05); only the practice of breastfeeding showed a significant difference for infection rate, with neonates fed artificial formula having higher infection rate (3.13%) than the breastfed neonates (0.27%, P less than 0.05). The neonate HBV infection rate was also significantly higher for neonates born to HBeAg-positive mothers (4.44% vs. HBeAg-negative mothers: 0%, P less than 0.05) and HBV DNA-positive mothers (3.13% vs. HBV DNA-negative mothers: 0%, P less than 0.05). When the mothers were stratified by serum level of HBV DNA, there was a significant difference in HBV-infected neonates born to mothers with more than or equal to 1*10(7) IU/ml(6.01% vs. 10(3)-10(6) IU/ml: 0.56% and less than 1*10(3) IU/ml: 0%, both P less than 0.05). Logistic regression analysis indicated that the independent risk factors for HBV perinatal transmission despite immunization were maternal serum HBeAg-positive status (relative risk (RR)=31.74, 95% confidence interval (CI): 3.88-259.38) and maternal HBV DNA of more than or equal to107 copies/mL (RR=22.58, 95% CI: 4.75-107.40).Failure of vaccine plus HBIG to interrupt mother-to-child transmission of HBV is influenced by maternal serum HBeAg-positive status and maternal HBV DNA of more than or equal to107 copies/mL.

To investigate the relation of thyroid function with hashimoto thyroiditis (HT, an autoimmune disease of unknown etiology also known as chronic lymphocytic thyroiditis) in patients with chronic hepatitis C (CHC) receiving treatment with pegylated-interferon-alpha (Peg-IFNa) based on the observation that HT is common among individuals undergoing IFN-based therapy.One-hundred-and-seven patients with chronic hepatitis C were enrolled for study between January 2008 and December 2010. Thyroid function was assessed by electrochemiluminescence assays to detect serum levels of anti-thyroid peroxidase (A-TPO) antibodies, thyroid stimulating hormore (TSH), and free thyroxine (FT4) prior to initiation of the IFN-based therapy. The treatment strategies (drugs, doses, schedules) were designed according to HT status (CHC with HT, or CHC without HT). Patients were monitored during the 24 weeks of treatment (including measuring serum alanine aminotransferae (ALT), TSH, and FT4 every two to four weeks, and HCV RNA every four weeks) so that the IFNa dose could be adjusted and thryoid medications (levothyroxine sodium or methimazole) added as necessary. The response rate at end of treatment (week 24) was assessed.Twenty-one of the CHC patients were diagnosed with HT, and the incidence of thyroid dysfunction among the CHC patients with HT was 71.4% (15/21); among the CHC patients with no HT, the incidence of thyroid dysfunction was significantly lower (30.2% (26/86), X2 = 12.1995, P less than 0.01). In the CHC patients with HT, 90.5% (19/21) had serum levels of A-TPO antibodies that were more than or equal to 2-times higher than the normal value at the end of treatment. Of the 15 CHC patients with HT and thyroid dysfunction, 73.3% (11/15) continued to show thyroid dysfunction at the end of treatment. Hypothyroidism was the most common form of thyroid dysfunction observed (4/11), and all of those patients responded to levothyroxine sodium treatment. The virological response rates of the two groups (CHC with HT and CHC without HT) were not significantly different at any time point examined (treatment week 4, 12, and 24, P more than 0.05).The incidence of thyroid dysfunction is significantly higher among CHC patients with HT than among CHC patients without HT. If suspected, these patients should be carefully monitored because the clinical symptoms of thyroid dysfunction are not obvious and the drug therapy should be carefully adjusted to minimize the thyroid dysfunction while maximizing the antiviral effect.

To investigate the effects of quercetin on serum levels of resistin and interleukin (IL)-18 and incidence of insulin resistance (IR) in nonalcoholic fatty liver disease (NAFLD) using a rat model.NAFLD was induced in Sprague-Dawley rats by administering a high-fat diet for four weeks. The model rats were then treated with quercetin (oral gavage administration; low dose group: 75 mg/kg/day, high dose group: 300 mg/kg/day) for eight weeks. Untreated model rats served as controls. Serum levels of resistin, triglyceride (TG), IL-18, fasting plasma glucose (FPG), fasting insulin (FINS), and malondialdehyde (MDA) were measured by standard biochemical assays before and after the quercetin administration. In addition, the insulin resistance index (HOMA-IR) was calculated and pathological changes in liver were observed by histological analysis.Compared to the untreated model rats, the quercetin treated model rats showed significantly lower serum resistin (5.98 vs. 2.70), serum IL-18 (10.93 vs. 8.21), FPG (7.45 vs. 4.99), FINS (12.69 vs. 8.59), and HOMA-IR (4.22 vs. 1.87) (all P less than 0.01). Compared to the untreated model group, the high dose group showed significantly lower TG (t = 4.70) and MDA (t = 5.14) (both P less than 0.01). Serum levels of resistin and IL-18, and levels of TG, FPG and FINS were found to be positively correlated with HOMA-IR and the degree of liver disease (r more than 0, all P less than 0.05). The degree of degeneration was decreased in accordance with the dosages of quercetin, as compared to the untreated model group (U = 4.41 and 2.19, both P less than 0.05), and the pathological degree was less extensive in the high dose group than in the low dose group (U = 2.44, P less than 0.01).Quercetin treatment reduces levels of inflammatory cytokines and improves lipid peroxidation and IR in NAFLD rats, and its beneficial effects appear to increase with higher dosage.