bluuebunny/crossref_metadata_2025 huggingface dataset:
Oral tolerization to adenoviral proteins permits repeated adenovirus-mediated gene therapy in rats with pre-existing immunity to adenoviruses
Authors: Ilan Yaron , Sauter Bernhard , Chowdhury Namita Roy , Reddy Bhoompally V. , Thummala Narsing R. , Droguett Gustavo , Davidson Anne , Ott Michael , Horwitz Marshall S. , Chowdhury Jayanta Roy
Published: July 2004

Exposure to wild-type adenoviruses is common in humans and results in immune response against adenoviruses. The pre-existing antibodies and a strong secondary humoral and cellular immune response would interfere with gene transfer using recombinant adenoviral vectors. To test whether the secondary immune response can be abrogated by oral tolerization to adenoviral antigens, we immunized bilirubin-UDP-glucuronosyltransferase (BUGT)-deficient jaundiced Gunn rats with a recombinant adenovirus (5 × 109 pfu/rat) expressing the human UDP-glucouronosyltransferase (BUGT1) gene (Ad-hBUGT). Transgene expression was shown by reduction of mean serum bilirubin levels from 7.0 mg/dL to 2.3 mg/dL in 14 days, which then increased gradually to pretreatment levels in 6 weeks. All recipients developed antibodies (1:210) and cytotoxic lymphocytes against the adenovirus. For oral tolerization, we administered to the immunized rats protein extracts of a recombinant adenovirus type 5 (1-1.5 mg/day) via duodenostomy tubes 10 to 40 days after the initial virus injection; control rats received bovine serum albumin. In rats fed adenoviral proteins and the BSA-fed controls, the antibody titers decreased to 1:27 and 1:29, respectively, in 70 days. Lymphocytes from the tolerized rats expressed TGF-β1 upon exposure to antigen-presenting cells primed with adenoviral antigens, whereas IFN-γ expression was undetectable. In contrast, lymphocytes from the BSA-treated control rats expressed IFN-γ but not transforming growth factor β1 (TGF-β1). Seventy days after the first injection in the orally tolerized rats, but not in the controls, a second Ad-hBUGT injection caused human BUGT1 expression again, reducing serum bilirubin levels to those observed after the first injection. In the tolerized rats, serum antibody titers and anti-adenoviral cytotoxic lymphocyte activities continued to decline despite the second injection, whereas the antibody levels were boosted in the non-tolerized group. This results show that by preventing the secondary booster response, oral tolerization permits repeated adenovirus-directed gene transfer despite the presence of a residual antibody titer from a previous adenoviral exposure.
Persistent viremia after recovery from self-limited acute hepatitis B
Authors: Yotsuyanagi Hiroshi , Yasuda Kiyomi , Iino Shiro , Moriya Kyoji , Shintani Yoshizumi , Fujie Hajime , Tsutsumi Takeya , Kimura Satoshi , Koike Kazuhiko
Published: July 2004

To define the duration of viremia in the course of acute hepatitis B, we semiquantitatively determined the levels of hepatitis B virus (HBV) DNA in the sera, using polymerase chain reaction (PCR) coupled with Southern blotting, of non-immunocompromised patients with self-limited acute hepatitis B. In the sera of 10 of 11 patients, HBV DNA, which was presumably coated with viral proteins, was detected for a long period after recovery, even at the final observation times, which ranged from 6 to 19 months after disease onset. To characterize the mode of HBV that was present in serum, we immunoprecipitated immune complexes in sera by the addition of anti-human immunoglobulin G (IgG) and determined the levels of HBV DNA separately in the supernatants and pellets. In the acute phase of hepatitis B, high levels of HBV DNA were detected both in the supernatants and pellets at comparative levels. After the convalescent phase, the amount of HBV DNA in the supernatant decreased with respect to that in the pellets. It is notable that, in most cases, serum HBV persisted as a form of immune complex even after the seroconversion to antibody to hepatitis B surface antigen (anti-HBs). These data suggest that the replication of HBV may persist in some organs, most likely in the liver or peripheral blood cells, for a long period after recovery from acute hepatitis B, and the data indicate the possible transmission of HBV from organ transplantation donors who exhibit serological markers of past infection only.
Characteristics of patients with hepatitis C virus with and without GB virus C/Hepatitis G virus Co-infection and efficacy of interferon alfa
Authors: Enomoto Masaru , Nishiguchi Shuhei , Fukuda Katsuhiko , Kuroki Tetsuo , Tanaka Motoharu , Otani Shuzo , Ogami Masayuki , Monna Takeyuki
Published: July 2004

GB virus C/hepatitis G virus (GBV-C/HGV) infection seems to be common among patients with hepatitis C virus (HCV) infection. We studied retrospectively the proportions of patients with GBV-C/HGV RNA and antibodies to the GBV-C/HGV second envelope protein (anti-E2) among 149 subjects with chronic hepatitis C who had received interferon alfa. The clinical characteristics of patients with GBV-C/HGV RNA or anti-E2 were examined, as was the efficacy of the treatment. Stored serum specimens were tested for GBV-C/HGV RNA by the reverse transcription polymerase chain reaction and for anti-E2 by an enzyme-linked immunosorbent assay. Of the 149 patients before therapy, 8 (5%) had GBV-C/HGV RNA only, 72 (48%) had anti-E2 only, and 4 (3%) had both. The mean age of patients with GBV-C/HGV RNA (some with anti-E2) was significantly less than that of patients with anti-E2 only. Results of laboratory and histological evaluations were not different depending on the presence of GBV-C/HGV RNA or anti-E2. The GBV-C/HGV RNA titer decreased during therapy in all 12 patients with GBV-C/HGV RNA; only 4, with a low titer before therapy and with anti-E2 detected at some time, had sustained clearance of GBV-C/HGV. Our results suggested that half of the patients with chronic hepatitis C had been exposed to GBV-C/HGV, but in almost all, the virus had been cleared; also, even chronic GBV-C/HGV infection did not affect the severity of the disease arising from HCV. Interferon alfa treatment was sometimes effective against GBV-C/HGV, and anti-E2 may be associated with clearance of GBV-C/HGV.
Risk factors for hepatocellular carcinoma and its incidence after interferon treatment in patients with chronic hepatitis C
Authors: Kasahara Akinori , Hayashi Norio , Mochizuki Kiyoshi , Takayanagi Masahide , Yoshioka Kentaro , Kakumu Shinichi , Iijima Akihiro , Urushihara Akihiko , Kiyosawa Kendo , Okuda Michiari , Hino Keisuke , Okita Kiwamu
Published: July 2004

To elucidate the risk factors for liver carcinogenesis and to examine the incidence of hepatocellular carcinoma (HCC) after interferon therapy, 1,022 chronic hepatitis C patients treated with interferon were followed by ultrasonography for 13 to 97 months (median 36 months). Sustained response with prolonged alanine aminotransferase normalization was found in 313 patients, transient response with alanine aminotransferase relapse after therapy in 304, and no response in 405. Forty-six developed HCC, of whom 5 were sustained responders, 9 were transient responders, and 32 were nonresponders. The cumulative incidence of HCC in transient responders was almost equal to that in sustained responders, and it was significantly higher in nonresponders than in sustained and transient responders (P = .0009). The seventh-year cumulative incidence rates of HCC in sustained responders, transient responders, and nonresponders were estimated to be 4.3%, 4.7%, and 26.1%, respectively. However, there was no significant difference in the cumulative incidence of HCC between patients with HCV subtype 1 and 2 (P = .14). Cox regression analysis showed that the risk of HCC development was not elevated in transient responders compared with sustained responders, but that the risk was 7.90-fold higher in nonresponders than in sustained responders (P = .008). Patients ≥55 years of age had a significantly higher risk ratio (4.65) than did those under 55 years of age (P = .006). The risk of HCC development in men was 4.35 times higher than the risk in women (P = .02). However, the degree of fibrosis was not a significant risk factor for the development of HCC (risk ratio, 3.16; P = .052). These results suggest that patients in the high-risk group of HCC after interferon therapy were those who showed no response, those who were older, and those who were male, and that such patients should be carefully followed using ultrasonography.
A randomized study comparing ribavirin and interferon alfa monotherapy for hepatitis C recurrence after liver transplantation
Authors: Gane Edward J. , Lo Su-Kong , Riordan Stephen M. , Portmann Bernard C. , Lau Johnson Y. , Naoumov Nikolai V. , Williams Roger
Published: July 2004

Hepatitis C virus (HCV) infection usually recurs after orthotopic liver transplantation (OLT), and most patients develop graft damage. This study compared the efficacy of interferon alfa (IFN-α) and ribavirin monotherapies in liver transplant recipients with chronic hepatitis C in the graft. Thirty OLT recipients with chronic hepatitis C were randomized to receive either IFN-α (3 MU three times a week) or ribavirin (up to 1.2 g daily) for 24 weeks. Virological, biochemical, and histological responses to treatment were assessed. Twenty-eight patients completed the treatment regimen, two ribavirin-treated patients being withdrawn because of severe hemolysis. Normalization of serum aspartate aminotransferase was achieved in 13 of 14 patients receiving ribavirin (93%) and 6 of 14 patients receiving IFN-α (43%; P = .01). Lobular inflammation was reduced in 9/14 ribavirin-treated (64%) and 3 of 14 IFN-α-treated patients (21%; P = .05), each of whom had a biochemical response. However, the total histological activity index did not improve in either the interferon (P = .43) or the ribavirin (P = .96) group. Posttreatment viremia levels were significantly reduced in IFN-α-treated (P = .05) but not in ribavirin-treated (P = .88) patients. Hemolysis occurred in all ribavirin-treated patients, with serum hemoglobin decreasing to <10 g/dL in 50%. Total leukocyte and lymphocyte counts decreased significantly during ribavirin treatment (P = .02 and P = .004, respectively). We concluded that in patients with chronic hepatitis C after OLT, IFN-α retains an antiviral effect whereas ribavirin is superior in achieving normalization of serum aspartate aminotransferase levels and reducing lobular inflammation, but not the total histological activity index. These findings provide a rationale for combination therapy in the post-OLT setting, although patients must be carefully monitored for hemolysis.
The GDPAL region of the pre-s1 envelope protein is important for morphogenesis of woodchuck hepatitis virus†
Authors: Yu Minshu , Emerson Suzanne U. , Cote Paul , Shapiro Max , Purcell Robert H.
Published: July 2004

The pre-S envelope protein of duck hepatitis B virus (DHBV) contains a region, Asp-Asp-Pro-Leu-Leu (DDPLL), that is specifically required for virus assembly and secretion (Lenhoff and Summers, J Virol 1994;68:4565-4571). We found that amino acids 201 to 205 of the pre-S envelope protein of woodchuck hepatitis virus (WHV) form a conserved amino acid cluster, Gly-Asp-Pro-Ala-Leu (GDPAL), which resembles the DDPLL sequence of DHBV. To determine whether the GDPAL region was functionally equivalent to the DDPLL region, we deleted this region from the pre-S protein of WHV or mutated individual amino acids within the region. The mutant DNA was transfected into human hepatoma cell line Huh7, and the medium was assayed for virion production by immunoprecipitation and Southern blot analysis. We found that an in-frame deletion of this small region inhibited virion formation, suggesting that the GDPAL region of the pre-S envelope protein was required for virus assembly and/or secretion of WHV. Individual replacement of alanine 204, leucine 205, or serine 206 with other amino acid residues did not affect virus production. However, substitution of either aspartic acid 202 with valine or proline 203 with leucine dramatically inhibited WHV production. Furthermore, the GDPAL mutants were individually tested for their abilities to complement a pre-S1 defective genome. The results showed that the GDPAL region functioned as part of the pre-S1 protein but was not required to function as part of the pre-S2 protein.
The impact of traveling to endemic areas on the spread of hepatitis E virus infection: Epidemiological and molecular analyses
Authors: Wu Jaw-Ching , Sheen I-Jane , Chiang Tzen-Yuh , Sheng Wen-Yung , Wang Yuan-Jen , Chan Cho-Yu , Lee Shou-Dong
Published: July 2004

Traveling to endemic areas carries a risk of hepatitis E virus (HEV) infection, but no molecular analysis to document sources of infection is available. Eighteen (38%) of 47 patients with acute non-A, non-B, non-C hepatitis were positive for antibody to HEV (anti-HEV), and 9 (50%) of these were also positive for serum HEV RNA by polymerase chain reaction following reverse transcription. Only 1 (5%) of the 21 patients with acute hepatitis A was positive for HEV RNA. Travel to endemic areas (mostly to China; odds ratio, 22.2; 95% confidence interval, 4.7-105.8) and deeper jaundice (odds ratio, 5.2; 95% confidence interval, 1.01-27.2) were the only factors associated with HEV infection in multivariate analysis. The two HEV isolates from two patients who had traveled to China and the HEV isolate from a patient whose travel history was obscure formed a monophyletic group with the isolates from Guangzhou. The HEV isolates from our patients show a homology of 72% to 78% in nucleotide sequence with the Burma, Beijing, India, Pakistan, and Xiangjiang strains; a homology of 81% to 91% with the Guangzhou strains; and a homology of 76% with the Mexico strain. The close relationship between the Taiwan isolates and the Guangzhou strains was further supported by the short Kimura’s two-parameter distances among them. In summary, HEV infection does occur in this area. Epidemiological and molecular analyses strongly indicate that most cases of HEV infection originated from travel to HEV-endemic areas.
Neurovisual impairment: A frequent complication of alpha-interferon treatment in chronic viral hepatitis
Authors: Manesis Emanuel K. , Moschos Michael , Brouzas Dimitrios , Kotsiras John , Petrou Constantine , Theodosiadis George , Hadziyannis Stephanos
Published: July 2004

Following our earlier observation of clinically evident optic tract neuropathy in patients receiving low-dose interferon (IFN) therapy, we prospectively evaluated 53 consecutive patients treated for chronic hepatitis B or C with a median dose of 3 MU of IFN-a2b thrice weekly. Measurements included routine ophthalmologic evaluation and recordings of visual evoked responses (VER), electro-retinograms (ERG), visual acuity, and visual fields, before, at the end of IFN treatment, and at follow-up visits. Baseline P100 latencies of VERs (base-VER) were abnormally prolonged in 24 patients (32 of 106 eyes, 30.2%); age was the only significant covariate associated with increased risk for an abnormal base-VER by multiple logistic regression (relative risk [RR] 5.3 per each 5-year increase in age). In 45 patients (74 eyes) with normal baseline P100 latencies, the end-of-treatment VERs (end-VER) were significantly prolonged compared with baseline, becoming abnormal in 11 (15 of 74 eyes, 20.3%) (138.8 ± 8.7 vs. 117.7 ± 5.2 ms, P < .001). This subgroup had older age (59.1 ± 11.0 vs. 47.5 ± 15.3, P = .007) and reduced visual sensitivity compared with their own pretreatment measurements (24.5 ± 1.6 vs. 23.0 ± 1.2db, P = .019). Changes of end-VERs by age had a sigmoid distribution with a steep increase of values beyond the 5th decade (R2 = .326, P < .001). In a logistic regression model, significant predictors of abnormal end-VERs were, patients’ age (RR 5.6 per each 5-year increase), presence of hepatitis B virus (HBV) infection (RR 15.1 compared with hepatitis C virus [HCV] infection) and serum cholesterol levels above 240 mg% (RR 7.1 compared with values <240 mg%). Subconjunctival hemorrhages were seen in 2 cases and funduscopic examination revealed cotton wool spots in one other. ERG recordings and the P100 amplitude remained unchanged. After stopping IFN, the treatment-associated neurovisual abnormalities reversed to normal in 7 patients (10 of 15 eyes) and persisted in 5 (5 of 15 eyes, 33.3%) for up to 37 (median 7.3) months observation, all patients remaining clinically asymptomatic. In conclusion, subclinical neurovisual impairment is a frequent, largely unrecognized complication of low-dose IFN therapy, and patients with chronic hepatitis B and older age appear to be most susceptible. This apparently innocuous complication is long lasting, possibly irreversible in some patients, with yet undetermined consequences on visual function.
Relative quantification and mapping of hepatitis C virus by in situ hybridization and digital image analysis†
Authors: Gosálvez Jaime , Rodríguez-Iñigo Elena , Ramiro-Díaz José L. , Bartolomé Javier , Tomás José F. , Oliva Horacio , Carreño Vicente
Published: July 2004

Although several reports concerning the detection of hepatitis C virus (HCV) by in situ hybridization have been published, there are no data concerning the relative viral load in infected hepatocytes or about its relation with serum viremia levels. To address these issues, liver biopsies from 10 patients with chronic hepatitis C were analyzed by in situ hybridization and digital image analysis of hybridization signals. Serum HCV RNA levels were measured using the Amplicor Monitor test. HCV RNA was detected by in situ hybridization in the hepatocytes of the ten liver samples. The hybridization signals were mainly found in the cytoplasm. The relative viral load per infected cell fit the second order polynomial curves in all cases. The minimum and maximum relative viral load per infected hepatocyte differed in the ten cases; however, large differences were not observed in the mean relative viral load among the samples, especially when compared with the increasing values detected for copy number per milliliter in serum. The percentage of infected cells ranged from 4.8% to 87.6% in the ten cases. The percentage of positive cells correlated with the serum viremia levels. Our data suggest that HCV viremia does not depend on the relative viral load per infected cell but on the number of infected hepatocytes.
Determinants of outcome of compensated hepatitis C virus-related cirrhosis
Authors: Serfaty Lawrence , Aumaître Hugues , Chazouillères Olivier , Bonnand Anne-Marie , Rosmorduc Olivier , Poupon Renée E. , Poupon Raoul
Published: July 2004

The aim of this study was to assess the incidence of decompensation (ascites, jaundice, variceal bleeding, and encephalopathy), hepatocellular carcinoma (HCC) and death or liver transplantation in patients with compensated hepatitis C virus (HCV)-related cirrhosis, taking into account the viral genotype and interferon (IFN) therapy. Between 1989 and 1994, 668 patients with no clinical evidence of decompensation were referred to our department for liver biopsy because of positivity for anti-HCV antibodies and elevated aminotransferase activity; 103 of these patients had cirrhosis. The median follow-up was 40 months. Fifty-nine patients were treated with IFN for a mean duration of 11 ± 6 months; 3 (5%) had a prolonged biochemical and virological response. Baseline characteristics of IFN-treated and untreated patients were not significantly different. HCV genotypes (InnoLiPa) were predominantly 1b (48%) and 3a (20%). During follow-up, complications of cirrhosis occurred in 26 patients, HCC in 11 patients, and decompensation not related to HCC in 19 patients. Sixteen patients died, 94% of liver disease. Three patients were transplanted for liver failure. The 4-year risk of HCC was 11.5% (annual incidence 3.3%) and that of decompensation was 20%. Survival probability was 96% and 84% at 2 and 4 years, respectively. In multivariate analysis, the absence of IFN therapy was the only independent factor predictive both for HCC and decompensation. A low albumin level at entry and the absence of IFN therapy were the two independent factors predictive of death or liver transplantation. Probability of survival at 2 and 4 years was significantly different between IFN-treated and untreated patients (respectively 97% and 92% vs 95% and 63%,P< .0001). In conclusion, in patients with compensated HCV-related cirrhosis: 1) complications of cirrhosis are frequent, whatever the viral genotype; and 2) the severity of cirrhosis and the absence of IFN therapy are independently predictive of bad outcome
Diet and gallstones in italy: The cross-sectional MICOL results†
Authors: Attili Adolfo Francesco , Scafato Emanuele , Marchioli Roberto , Marfisi Rosa Maria , Festi Davide
Published: July 2004

Fifteen thousand nine hundred ten men and 13,674 women (age, 30-69 years) were enrolled in an epidemiological survey of the general population, between December 1984 and April 1987. Each participant was submitted to ultrasonography (US) of the gallbladder and completed a food-frequency questionnaire, covering 38 food items. A common portion size was identified and subjects were asked how often each item was consumed. Nutrient intake was computed by multiplying the intake frequency and nutrient content per portion for each item, and then by summing the product over all foods. Each nutrient intake was adjusted for energy intake. Alcohol intake was calculated by summing the consumption of wine, beer, and liquor. Having excluded subjects aware of having gallstones (GS) or previously submitted to cholecystectomy (to avoid prothopatic bias), 787 males and 1,014 females with GS and 14,272 males and 10,836 females without GS were available for analysis. Relative risks (RR) of GS were computed by quintiles of nutrient intake. The overnight fasting period was calculated as the difference between the specified time of dinner and the time of the next meal (breakfast or lunch). A significant negative association was found between RR of GS and total energy intake for males (χ2for trend = 8.37; P = .004), fiber intake for females (χ2 = 5.45; P = .02), and daily alcohol consumption for males (χ2 = 10.86; P = .001). A positive association was observed between RR of GS and carbohydrate (χ2 = 5.95; P = .01 for males; χ2 = 9.39; P = .002 for females) and protein intake only for males (χ2 = 10.92; P = .01). Prevalence of GS was higher among subjects who had an overnight fasting period of over 12 hours than subjects with that of less than 12 hours. (RR: 1.35; 95% CI: 1.01-1.80 for males; RR: 1.28; 95% CI: 1.03-1.60 for females). These data do not confirm that high energy intake is associated with an increased risk of GS. Factors protecting against GS comprise: low carbohydrate (males and females) and protein (males) intakes, high fiber (females) and moderate alcohol intake (males) consumption, and a shorter overnight fasting period for both sexes.
Visual attention in cirrhotic patients: A study on covert visual attention orienting
Authors: Amodio Piero , Marchetti Piergiorgio , Del Piccolo Franco , Campo Giovanni , Rizzo Cristiano , Iemmolo Rosa Maria , Gerunda Giorgio , Caregaro Lorenza , Merkel Carlo , Gatta Angelo
Published: July 2004

Attentional dysfunction, which influences overall cognitive productivity, is not well characterized in cirrhotic patients. The aim of this study was to clarify the features of covert visual attention orienting in cirrhotics without overt hepatic encephalopathy. One hundred consecutive cirrhotic patients and 40 controls were enrolled. Visual covert attention orienting was assessed by the Posner test, which evaluates the effect of a cue on visual reaction times. Patients were characterized by the number connection test (NCT) and electroencephalographic (EEG) spectral analysis. The severity of liver disease was graded using standard laboratory parameters and the Child-Pugh’s classification. Fifty-five psychometric and EEG evaluations were performed in the follow-up of 17 patients to assess the relationship between the variations of psychometric and neurophysiological findings. NCT and quantified-EEG parameters (altered in 19% and 40% of cirrhotic patients, respectively) were linked to each other and to the severity of liver disease. The Posner test showed a delay of visual reaction times in class B-C cirrhotic patients. Reaction times were correlated with ammonia and EEG parameters. The effect of the cue was higher in cirrhotic patients than in controls, particularly in the invalid position. This study suggests that cirrhotic patients have a reduced activity rate and reduced capacity to disengage attention previously focused on a cue. Such alterations are linked to NCT and EEG findings.
Suppression of murine experimental autoimmune hepatitis by T-cell vaccination or immunosuppression
Authors: Lohse Ansgar W. , Dienes Hans-Peter , Büschenfelde Karl-Hermann Meyer zum
Published: July 2004

Patients with autoimmune hepatitis (AIH) usually require immunosuppressive therapy for many years, if not for a lifetime. Experimental immunotherapy such as T-cell vaccination aims at manipulating the immune system in such a way that autoimmunity is specifically regulated to enable long-lasting correction of the disease process. We aimed to test the feasibility of T-cell vaccination as well as conventional immunosuppression in the murine model of experimental autoimmune hepatitis (EAH). EAH was induced in 5- to 7-week-old BALB/c mice by immunization with syngeneic liver homogenate in complete Freund’s adjuvant. For T-cell vaccination, splenocytes were removed from animals 14 days after induction of EAH and from control animals, and activatedin vitroby mitogen stimulation with Concanavalin A (Con A). Activated T cells were irradiated and injected at 5 × 107cells per animal as T-cell vaccine. Immunosuppression in control animals was performed with prednisolone with or without azathioprine. T-cell vaccination with T cells from EAH animals, but not with irrelevant T cells, was able to protect animals from EAH, reducing the average disease severity from 2.2 (±0.3) to 0.5 (±0.3) (P< .01). T-cell vaccination was also able to treat EAH, because application of the vaccine 2 weeks after induction of the disease significantly reduced disease activity at week 4 from 2.4 (±0.4) to 1.1 (±0.2) (P< .05). Both passive transfer of disease and the capacity to protect by T-cell vaccination was mediated by CD4 T cells. Specific cellular recognition of activated disease-inducing T cells could be detected in vaccinated animals. Immunosuppressive drugs could also suppress EAH. Thus, T-cell vaccination in EAH is feasible and effective. Stimulation of a regulatory T-cell network is the likely mechanism of action by which T-cell vaccination can suppress EAH.
An intravital fluorescence microscopic study of hepatic microvascular and cellular derangements in developing cirrhosis in rats
Authors: Vollmar Brigitte , Siegmund Sören , Menger Michael D.
Published: July 2004

Quantitative data defining the relationship between the hepatic microcirculation and the development of liver pathological changes could provide a basis for a better understanding of fibrogenic processes, such as cirrhosis. Therefore, we established the technique of intravital fluorescence microscopy and computer-assisted microcirculation analysis systems in developing cirrhosis in rats with the aim of quantitatively assessing the association of hepatic microvascular morphology with its disordered acinar architecture, and nonparenchymal cell transformation with collagen deposition, parenchymal cell loss, and liver dysfunction. In animals chronically exposed to carbon tetrachloride (CCl4), the most significant microvascular changes progressively observed in vivo were the concomitant appearance of 1) sinusoid-free space around dilated postsinusoidal venules with 2) substituting occurrence of yellow-green autofluorescent collagen deposition, 3) reduction in sinusoidal density, but 4) increase of vascular lumen caused by the formation of shunting vessels bypassing the sinusoids. Present on-line analysis further indicated the local coincidence of changed spatial distribution of Ito cells (accumulation of vitamin A ultraviolet autofluorescence in zone 3) with fibrotic autofluorescent septa, causing significant collapse of parenchymal tissue (hepatocellular bis-benzamide fluorescence) and diminution of hepatocellular excretory function (bile flow). Regression analysis revealed strong correlations between loss of parenchymal tissue and both collagen deposition and sinusoidal rarefication, as well as between sinusoidal rarefication and collagen deposition. Thus, sequential in vivo analysis presented herein provides the new information on the concomitant onset of cellular, fibrotic, and microvascular changes in developing fibrosis/cirrhosis, excluding that distinct cellular or fibrotic alterations are a prerequisite for the manifestation of microcirculatory and vascular derangements or vice versa.
Dietary iron overload as a risk factor for hepatocellular carcinoma in black africans
Authors: Mandishona Eberhard , MacPhail Patrick A. , Gordeuk Victor R. , Kedda Mary-Anne , Paterson Alan C. , Rouault Tracey A. , Kew Michael C.
Published: July 2004

Although the iron-loading disease, hereditary hemochromatosis, has a strong causal association with hepatocellular carcinoma (HCC), the carcinogenic potential of dietary iron overload in Black Africans is not known. We investigated this potential by evaluating iron status, alcohol consumption, markers for hepatitis B (HBV) and C virus (HCV) infections, and exposure to dietary aflatoxin B1 in 24 rural patients with this tumor, 48 race-, sex-, and age-matched hospital-based controls, and 75 related or unrelated close family members of the cancer patients. Iron overload was defined as a raised serum ferritin concentration in combination with a transferrin saturation ≥60%, and was confirmed histologically when possible. Among 24 patients and 48 hospital controls, the risk of developing HCC in the iron-loaded subjects was 10.6 (95% confidence limits of 1.5 and 76.8) relative to individuals with normal iron status, after adjusting for alcohol consumption, chronic HBV and HBC infections, and exposure to aflatoxin B1. The risk of HCC in subjects with HBV infection was 33.2 (7.2, 153.4) (odds ratio [95% confidence limits]), HCV infection 6.4 (0.3, 133.5), and alcohol consumption 2.0 (0.5, 8.2). Aflatoxin B1 exposure did not appear to increase the risk of HCC. The population attributable risk of iron overload in the development of HCC was estimated to be 29%. Among 20 cancer patients and 75 family members, the risk of developing HCC with iron overload was 4.1 (0.5, 32.2). We conclude that dietary iron overload may contribute to the development of HCC in Black Africans.
Prognosis of hepatocellular carcinoma with diabetes mellitus after hepatic resection
Authors: Ikeda Yasuharu , Shimada Mitsuo , Hasegawa Hirofumi , Gion Tomonobu , Kajiyama Kiyoshi , Shirabe Ken , Yanaga Katsuhiko , Takenaka Kenji , Sugimachi Keizo
Published: July 2004

We evaluated the effect of diabetes mellitus on the prognosis of hepatocellular carcinoma after an elective hepatic resection. Of the 342 patients who underwent a hepatic resection between April 1985 and March 1995, 87 (25.4%) were diabetic. Postoperative morbidity was more common among diabetics than among nondiabetics (36.0% vs. 22.5%, P = .0239). The postoperative survival rate and the cancer-free survival rate were also better in patients without diabetes than in those with diabetes (P = .0333, P = .0149). The results of a multivariate analysis show diabetes mellitus to be an independent and prognostic indicator after a hepatic resection with hepatocellular carcinoma. According to the above findings, diabetes mellitus is thus considered to be a risk factor for prognosis after hepatic resection in patients with hepatocellular carcinoma.
Keratinocyte growth factor protects murine hepatocytes from tumor necrosis factor-induced apoptosis in vivo and in vitro
Authors: Senaldi Giorgio , Shaklee Christine L. , Simon Bernadett , Rowan Christopher G. , Lacey David L. , Hartung Thomas
Published: July 2004

Keratinocyte growth factor (KGF) promotes epithelial growth and differentiation and has potent effects on the liver. The coinjection of lipopolysaccharide (LPS) and d -galactosamine (GalN) results in hepatic failure in mice. Mechanistically, LPS-induced tumor necrosis factor (TNF) triggers hepatocyte apoptosis, which is enhanced by GalN-arrested transcription. Similarly, the combination of TNF and actinomycin D (ActD) causes hepatocyte apoptosis in vitro. We studied the effect of KGF on LPS and GalN-induced hepatic failure in vivo and on TNF- and ActD-induced hepatocyte apoptosis in vitro, where it was compared with those of hepatic growth factor (HGF) and epidermal growth factor (EGF). Mice treated with human recombinant KGF (1 mg/kg subcutaneously) 24 hours before intraperitoneal coinjection of LPS and GalN sustained prolonged survival compared with control mice, although overall mortality was not changed. The counts of apoptotic hepatocytes, serum alanine and aspartate transaminases, and DNA fragments in the cytosolic fraction of liver homogenates were higher in control mice than in treated mice 6 hours after LPS and GalN coinjection, before any mortality occurred. In vitro, hepatocytes pretreated with KGF exhibited reduced TNF- and ActD-induced cell damage and DNA fragmentation, similar to hepatocytes pretreated with HGF and EGF. In conclusion, KGF prolongs survival during LPS- and GalN-induced hepatic failure by temporarily protecting hepatocytes against apoptosis. It also protects hepatocytes in vitro against TNF- and ActD-induced apoptosis.
Analysis of the expression pattern of the latent transforming growth factor β binding protein isoforms in normal and diseased human liver reveals a new splice variant missing the proteinase-sensitive hinge region
Authors: Michel Kristin , Roth Sylke , Trautwein Christian , Gong WenRong , Flemming P. , Gressner Axel M.
Published: July 2004

Latent transforming growth factor β binding protein (LTBP), a component of the extracellular matrix (ECM) of various tissues, is important for the secretion of TGF-β and, furthermore, for the storage of TGF-β in ECM. The proteolytic cleavage of LTBP is assumed to be the prerequisite for the activation of TGF-β. We investigated the mRNA expression pattern of the three LTBP isoforms (LTBP-1, -2, -3) and the protein distribution of the components of the large latent TGF-β complex, namely LTBP-1 and -2, latency-associated protein (LAP), and TGF-β, in human liver using reverse-transcription polymerase chain reaction (RT-PCR) and immunhistochemical alkaline phosphatase anti-alkaline phosphatase (APAAP) staining. Parts of explanted livers diagnosed as hepatitis B, hepatitis C, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC) and normal liver tissue were examined. LTBP transcripts were detected in the same manner in all liver specimens. Interestingly, we found a new splice variant of LTBP-1 (LTBP-1D), in which the sequence coding for the proteinase-sensitive hinge region is deleted. The corresponding parts of the human LTBP-2 and LTBP-3 cDNA coding for the hinge region were sequenced and show neither similar proteinase cleavage sites nor deleted cDNA sequences. The proposed proteinase cleavage site of mouse LTBP-3 seems not to be conserved in the human LTBP-3 gene. By immunohistochemistry, LTBP-1, -2, and LAP were detectable in normal and diseased livers and showed a different staining pattern for both LTBP isoforms. By contrast, TGF-β showed a spotted staining pattern in diseased livers only, predominantly in the area of parenchymal cells that are close to fibrotic tissue. This strongly suggests the release of active TGF-β from preexisting latent complexes. The LTBP-1D splice variant, which is probably less sensitive against proteolytic degradation and therefore may protect TGF-β from activation, may have importance for modulating the biological activity of TGF-β in normal and diseased liver.
Expression of co-stimulatory molecules by kupffer cells in chronic hepatitis of hepatitis C virus etiology
Authors: Burgio Vito L. , Ballardini Giorgio , Artini Marco , Caratozzolo Marcello , Bianchi Francesco B. , Levrero Massimo
Published: July 2004

In this paper we show that in viral hepatitis most Kupffer cells (KCs) are activated and express high levels of CD80, CD40, and class-II MHC molecules, thus acquiring the phenotype of professional antigen presenting cells (APCs). Activated KCs display a close contact with CD4+ T lymphocytes and form KCs-T lymphocyte clusters. Clusters are found within the sinusoids, across the sinusoid wall, and within the liver parenchima as well, as a consequence of transendothelial migration (TEM). The positivity of activated KCs for hepatitis C virus (HCV) antigens, which likely reflects phagocytosis of infected hepatocytes, suggests that KCs-T cell clusters represent the morphological expression of the functional interaction between KCs acting as professional APCs and antigen-experienced CD4+ T lymphocytes within the liver. These phenotypic and morphological changes are distinct features of livers in chronic hepatitis patients compared with controls.
Changes in lymph vessels and portal veins in the portal tract of patients with idiopathic portal hypertension: A morphometric study
Authors: Oikawa Hiroki , Masuda Tomoyuki , Sato Shin-Ichiro , Yashima Akiko , Suzuki Kazuyuki , Sato Shunichi , Satodate Ryoichi
Published: July 2004

Little is known about the effects of the pathological process associated with idiopathic portal hypertension (IPH) on hepatic lymph vessels or lymph flow. We used morphometric analysis to examine IPH-associated changes in lymph vessels and branches of the portal vein, with use of immunohistochemical staining for alpha smooth muscle actin. We also quantitated these changes using an image analysis system. The study was conducted with use of liver wedge biopsy material from 10 patients with advanced IPH and 10 control samples from patients with gastric carcinoma without liver disease. The number of lymph vessels, identified by a lack of smooth muscle layer in the wall, and the ratio of the total area of these vessels to that of the portal tract were higher in IPH samples than in the control samples, but the ratio of the area of a single lymph vessel to that of the portal tract in IPH samples was not different from control samples. The number of portal vein branches, characterized by hypertrophy of the smooth muscle layer in IPH samples was not different from control samples. The ratio of the total area of these branches to that of the portal tract, and the ratio of a single portal vein branch to that of the portal tract, were lower in IPH samples than in the control samples. Our results suggest that these morphometric changes in IPH may be associated with a reduction in portal blood flow and increased lymph flow, and that the latter may in turn reduce the high portal vein pressure in idiopathic portal hypertension.
Hepatic stellate cells are not subjected to oxidant stress during iron-induced fibrogenesis in rodents
Authors: Montosi Giuliana , Garuti Cinzia , Martinelli Sabrina , Pietrangelo Antonello
Published: July 2004

Oxidant stress plays a key role in hepatic fibrogenesis. This study was undertaken to assess whether, during iron overload-associated liver fibrosisain vivo, oxidant stress occurs in hepatic stellate cells (HSC) during active fibrogenesis. Gerbils were treated with iron-dextran, and, after hepatic fibrosis developed, livers were subjected to various combination ofin situhybridization and immunocytochemistry analyses. In iron-treated animals, no specific accumulation of ferritin protein was found in collagen mRNA-expressing cells. Moreover, the activity of the iron regulatory protein, the main sensor of cellular iron status, was unchanged in HSC from iron-treated animals. Although a significant amount of malondialdehyde-protein adducts was detected in gerbil liver during fibrogenesis, accumulation of these lipid peroxidation by-products was restricted to iron-laden cells adjacent to activated HSC. In cultured gerbil HSC, iron, aldehydes, and other pro-oxidants were able to enhance the expression of an oxidant stress-responsive gene, heme oxygenase (HO), with no change in collagen mRNA accumulation. In keeping with these findings, we found that,in vivo, activation of HO gene was present in iron-filled nonparenchymal cell aggregates, but absent in HSC. In conclusion, the data indicate that during iron overload-associated fibrogenesis, HSC are not directly subjected to oxidant stress, but are likely to be activated by paracrine signals arising in neighboring cells.
Delayed fas-mediated hepatocyte apoptosis during liver regeneration in mice: hepatoprotective role of TNFα
Authors: Takehara Tetsuo , Hayashi Norio , Mita Eiji , Kanto Tatsuya , Tatsumi Tomohide , Sasaki Yutaka , Kasahara Akinori , Hori Masatsugu
Published: July 2004

Fas-mediated apoptosis is one of the major death processes of hepatocytes in liver diseases. Although compensatory regeneration occurs during liver injury, it has not been determined whether regenerating hepatocytes die by the same apoptotic process as quiescent hepatocytes. To clarify this issue, the hepatocyte apoptotic process, after injection of agonistic anti-mouse Fas, was compared between sham-operated mice and two-thirds partially hepatectomized mice. The onset of hepatocyte apoptosis was retarded in hepatectomized mice, as evidenced by both morphological and biochemical observations, resulting in significantly prolonged animal survival. Flow cytometric analysis revealed similar levels of Fas expression on hepatocytes between hepatectomized mice and sham-operated mice; however, the activation of liver caspase-3-like protease after Fas stimulation was suppressed in hepatectomized mice, whereas pro-caspase-3 expression did not change with or without hepatectomy. Anti-tumor necrosis factor alfa (TNFα), when administered before hepatectomy, partially reversed suppression of caspase-3-like activity after Fas stimulation. Furthermore, the injection of TNFα into untreated mice suppressed caspase-3-like activity and prolonged animal survival after Fas stimulation. These results indicate that Fas-signaling events at the level or upstream of caspase-3-like protease are suppressed during liver regeneration, resulting in delayed hepatocyte apoptosis, and also that TNFα acts as one of the protective factors against Fas-mediated hepatocyte apoptosis.
What strategy should be used for diagnosis of hepatitis C virus infection in clinical laboratories?
Authors: Pawlotsky Jean-Michel , Lonjon Isabelle , Hezode Christophe , Raynard Bruno , Darthuy Francoise , Remire Jocelyne , Soussy Claude-James , Dhumeaux Daniel
Published: July 2004

The aim of this study was to determine a cost-effective strategy for the diagnosis of hepatitis C virus (HCV) infection in clinical laboratories. Anti-HCV antibodies were sought in 3,014 consecutive unselected samples with two different enzyme-linked immunosorbent assays (ELISA). An immunoblot-based confirmatory assay (RIBA3.0) was performed in the samples with at least one ELISA positive or weakly positive. HCV RNA was evaluated using HCV polymerase chain reaction (PCR) in the samples with a weakly positive ELISA, discrepant results of the two ELISAs, or an indeterminate RIBA3.0 pattern. The two ELISAs gave concordant results in 2,957 (98.1%) of the 3,014 samples (negative in 87.9%, positive in 11.8%, and weakly positive in 0.3%), and discrepant results in 57 (1.9%). RIBA3.0 was positive in 338 of the 350 ELISA-positive samples (96.6%) and indeterminate in 12. Six of them were PCR-positive. Among the 8 weakly positive samples, 1 was RIBA3.0-positive, 6 were RIBA3.0-indeterminate, and 1 was RIBA3.0-negative; all were PCR-negative. Among the 57 samples with discrepant ELISA results, 4 were RIBA3.0-positive (none were PCR-positive), 22 were RIBA3.0-indeterminate (1 was PCR-positive), and 31 were RIBA3.0-negative (6 were PCR-positive). In these cases, the clinical context and PCR detection of HCV RNA allowed for definitive classification. In conclusion, one single ELISA determination is necessary for diagnosis of HCV infection in clinical laboratories, and confirmation of positive or weakly positive ELISAs with immunoblot-based confirmatory assays is no longer needed. HCV-RNA detection by PCR helps to resolve weakly positive or negative ELISA results when the clinical context is compatible with hepatitis C.
Broadly reactive antibodies to hypervariable region 1 in hepatitis C virus-infected patient sera: Relation to viral loads and response to interferon
Authors: Hattori Masaki , Yoshioka Kentaro , Aiyama Toshiyuki , Iwata Kazuo , Terazawa Yasuyuki , Ishigami Masatoshi , Yano Motoyoshi , Kakumu Shinichi
Published: July 2004

To clarify the nature of serum anti-hypervariable region 1 (HVR1) antibodies in patients infected with hepatitis C virus (HCV), we assessed the reactivity of 21 patients’ sera with 42 HVR1 proteins by Western blot. HVR1 was expressed as fusion proteins with glutathione S-transferase (GST). The patients’ sera reacted with variable percentages of the HVR1 proteins, and always reacted with HVR1 proteins of the different genotype. In the genotype-1b-infected patients, the percentage of genotype-1b HVR1 proteins reactive with serum correlated significantly with viral loads; the sera reactive with the higher percentages of HVR1 proteins contained the larger viral loads. In addition, it was significantly lower in the responders of interferon (IFN) therapy than in nonresponders. The competition assays indicated that multiple fractions of anti-HVR1 antibodies with different specificity in a serum reacted with different HVR1 proteins, and that, additionally, a single fraction of antibodies often reacted with more than one HVR1 protein through a similar amino acid sequence. In conclusion, serum anti-HVR1 antibodies were broadly reactive by the mechanism of both the cross-reactivity of a single fraction of anti-HVR1 antibodies with more than one HVR1 protein and the presence of multiple fractions of anti-HVR1 antibodies with different specificity in a serum. In genotype-1b-infected patients, the broad reactivity of serum anti-HVR1 antibodies correlated with viral loads and response to IFN. Further studies are necessary to elucidate the correlation among the broad reactivity of sera with multiple HVR1 proteins and clinical features of chronic hepatitis C patients.