Increased expression of transforming growth factor-alpha in a patient with recurrent hepatocellular carcinoma following partial hepatectomy.

A 45-year-old woman with chronic hepatitis B underwent partial hepatectomy for hepatocellular carcinoma (HCC). However, the HCC recurred 2 months after surgery and rapid progression of the disease resulted in her death. Immunohistochemistry showed that transforming growth factor-alpha (TGFalpha) was barely expressed in the liver specimens obtained at hepatic resection, whereas autopsy specimens were strongly stained with anti-TGFalpha antibody in the cytoplasm of both non-tumourous and tumourous liver cells. A higher level of Ki67 expression, a proliferating marker, was observed in the recurrent HCC, similar to that of TGFalpha. Thus, we speculate that the partial hepatectomy increased the level of TGFalpha leading to recurrence and progression of HCC through an autocrine/paracrine mechanism.

Eosinophilic enteritis observed during alpha-interferon therapy for chronic hepatitis C.

We report a patient with chronic hepatitis C who developed eosinophilic enteritis while being treated with recombinant interferon alpha-2b. He had no history of either allergic disorders or recurring episodes of abdominal cramps, nausea, or diarrhea. He also had had a normal eosinophil count prior to the interferon treatment. After a 12-week course of interferon alpha-2b, he began to complain of severe abdominal pain, diarrhea, and abdominal fullness. His peripheral eosinophil count increased to 45% (absolute count, of 7,610/microl). Abdominal ultrasonography and computed tomography revealed diffuse thickness of the intestinal wall with gross ascites that contained numerous eosinophils. An upper gastrointestinal barium study with small bowel follow-through showed an edematous mucosal layer of the jejunum and ileum. There was a spectacular relief of the patient's subjective symptoms after the administration of prednisolone. Follow-up studies revealed resolution of the ascites and the mucosal layer edema and normalization of the peripheral eosinophil count. Prednisolone was tapered off, but the eosinophilic enteritis did not recur. As there had been no evident exposure to common causative factors for eosinophilic enteritis, we suggest that interferon alpha-2b could thus have played a role in the triggering of the eosinophilic enteritis.

Telling the diagnosis to cancer patients in Japan: attitude and perception of patients, physicians and nurses.

The aim of this study was to clarify patients', physicians', and nurses' perceptions with regard to the communication of diagnosis to cancer patients in Japan. Sixty-three cancer patients, 35 physicians and 21 nurses were enrolled for this study: 54 of the patients wished to be informed of the diagnosis, of whom 34 had actually been told that they had cancer. Physicians did not tell the truth to the remaining 20 patients, of whom seven were not told the diagnosis because family members objected. Twenty-one of the 35 physicians thought that telling the true diagnosis had a positive effect and 27 thought that disclosure of the diagnosis to cancer patients should be promoted. Sixteen of the 21 nurses did not experience any difficulties with patient care after the diagnosis was disclosed. The present study suggests that medical staff and family members should respect the patient's standpoint because patients have the right to know about their own condition. Physicians should first provide the details of the disease to their patients. Thereafter, family members should be informed, but only with the patient's consent.

The natural history and prognostic factors in patients with cirrhosis and gastric fundal varices without prior bleeding.

Objectives and methods: The prognostic factors have not yet been fully evaluated in patients with cirrhosis and gastric fundal varices (FV). We investigated the natural history of 145 patients with cirrhosis and FV with no history of bleeding. Various possible prognostic factors, which include clinical, biochemical, and endoscopical variables, were analyzed using Cox's proportional hazard model. Results: Among the 145 patients with cirrhosis and FV, there were 76 patients in class A, 45 in class B and 24 class C according to Child's classification. Sixty-five patients had concomitant hepatocellular carcinoma at the time of enrollment. Seventy deaths and 34 episodes of the hemorrhage from FV occurred during the mean follow-up period of 26.4 months. The cumulative survival rates at 1, 3, and 5 years were 75, 53 and 34%, respectively. The cause of death was related to gastrointestinal hemorrhage in 18 patients (15 deaths were related to FV hemorrhage), hepatic failure in 22, hepatocellular carcinoma in 22, and other causes in eight patients. In patients with small-, medium-, and large-sized FV, the deaths related to FV hemorrhage were 4, 21 and 54%, respectively. Overall, the death related to FV hemorrhage was 21%. A multiple regression analysis using Cox's model showed hemorrhage from FV, the presence of hepatocellular carcinoma and poor Child's status were all highly significant prognostic factors. Conclusion: The natural history of the patients with cirrhosis and FV was adversely modified by the hemorrhage from FV, concomitant hepatocellular carcinoma and poor hepatic functional reserve. Since the number of deaths related to FV hemorrhage was great in patients with large-sized FV, it is important to identify high-risk large FV and its prophylactic obliteration. Further studies are needed to elucidate the efficacy of prophylactic obliteration of large-sized FV.

Combination of transileocolic vein obliteration and balloon-occluded retrograde transvenous obliteration is effective for ruptured duodenal varices.

Duodenal varices are a rare site of hemorrhage in patients with portal hypertension, but their rupture is a serious and often fatal event. We report a 65-year-old woman who presented with hematemesis and melena. She was admitted to our department because of prolonged shock, despite having received transfusion of a large volume of blood. Upper gastrointestinal endoscopy revealed nodular varices with active bleeding in the second portion of the duodenum. Endoscopic injection sclerotherapy (EIS) was performed using a tissue adhesive agent, alpha-cyanoacrylate monomer, with only temporary benefit. However, anemia continued to progress after the procedure. Therefore, we combined transileocolic vein obliteration (TIO) with balloon-occluded retrograde transvenous obliteration (B-RIO), using 5% ethanolamine oleate with iopamidol to obliterate the varices. Complete hemostasis was achieved without complications. Neither recurrence of varices nor further bleeding has occurred for over 3 years. We conclude that combined TIO and B-RTO, which can obstruct both the feeding and the draining vessels of duodenal varices to retain the sclerosing agent completely in the varices, is a safe and effective hemostatic measure for ruptured duodenal varices, when EIS has failed to accomplish complete hemostasis.

Magnitude of activity in chronic hepatitis C is influenced by apoptosis of T cells responsible for hepatitis C virus.

BACKGROUND: The mechanisms of hepatitis C virus (HCV) persistence are unknown, but down-regulation of immune response in a host is likely to play a major role in it. METHODS: To investigate whether T cell apoptosis contributes to such down-regulation, we compared peripheral T cell apoptosis in patients with chronic hepatitis C (CHC) with the serum titre of HCV-RNA, serum alanine aminotransferase (sALT) levels and its change, or peripheral T cell proliferation to the recombinant core antigen of HCV, JCC-1. RESULTS: The percentage of apoptosis in T cells was 0.30 +/- 0.31% (mean +/- SD) in 44 patients with CHC and 0.10 +/- 0.05% in 10 normal volunteers (P < 0.05). In patients with CHC there was no statistical correlation between apoptosis in T cells and sALT levels, titre of HCV-RNA or T cell proliferation to JCC-1 antigen. But, in patients showing relatively more apoptosis in T cells (more than mean + 2SD of apoptosis in T cells from normal volunteers), sALT levels decreased. CONCLUSIONS: Thus, T cell apoptosis in patients with CHC is considered to cause a reduction in sALT, contributing to HCV persistence in patients with CHC.

Putative hemochromatosis gene mutations and alcoholic liver disease with iron overload in Japan.

It is well known that alcoholic liver disease is associated with iron overload. To study the role of hemochromatosis gene mutations on the pathogenesis of alcoholic liver disease (ALD), we have analyzed C282Y and H63D mutations on the chromosomes obtained from 95 Japanese alcoholics. Patients were divided in two groups [i.e., 64 alcoholic patients with liver damage (group I) and 31 alcoholics without liver damage (group II)]. In group I, biochemical examinations showed that serum levels of iron and ferritin were significantly high, and unsaturated iron binding capacity levels were low, compared with those of group II. An analysis by means of allele-specific polymerase chain reaction demonstrated that C282Y mutation was not observed in both groups I and II. H63D mutation was observed in only two heterozygotes of group I and in one heterozygote of group II. Results could not indicate the relationship between ALD and these mutations. We speculate that other causes of iron overload may exist in ALD with iron overload.

Unresponsiveness of peripheral T cells induced by apoptotic bodies derived from autologous T cells.

Several reports described the dose-dependent effect of Staphylococcus aureus enterotoxin B (SEB) regarding both levels of apoptosis and anergy of T cells. We investigated here whether T-cell apoptosis induced with SEB causes unresponsiveness of naive T cells. Apoptotic bodies were isolated from human T cells stimulated with antigen-presenting cells (APCs) and SEB by the continuous density gradient centrifugation method. When naive T cells were stimulated with APCs and SEB in the presence of apoptotic bodies, their proliferation was dose dependently suppressed and their TCRs were less downregulated than those of T cells stimulated without apoptotic bodies. Furthermore, those T cells were predisposed not to respond to restimulation with fresh APCs and SEB in the absence of apoptotic bodies. These results, taken together with the observation of tight binding of apoptotic bodies to APCs, imply that T cells stimulated in the presence of apoptotic bodies may undergo unresponsiveness due to interruption of contact with APCs.

An analysis of T cell antigen receptor variable beta genes during the clinical course of patients with chronic hepatitis B.

BACKGROUND: The degree of hepatocyte injury in patients with chronic hepatitis B appears consistent with the number of T cells that respond to hepatitis B virus-related antigens. METHODS: By using a polymerase chain reaction (PCR)-based approach, we monitored a ratio of the T cell antigen receptor (TcR) variable (V) beta gene families against a total TcR V beta gene expression in the peripheral T cells obtained from five patients and four healthy controls. RESULTS: In the healthy controls, there was no significant change in the ratios at an interval of four or eight weeks. In contrast, several TcR V beta families showed the significant changes in the ratios of their gene expression during the follow-up period in all patients. No common highly fluctuated TcR V beta, however, was found among the patients. Furthermore, there was no correlation between their changes and serum levels of alanine aminotransferase. CONCLUSIONS: These findings suggest that the skewing of the TcR family with multiclone is the result of T-cell responses to viral antigens in peripheral blood.

Effects of endoscopic variceal ligation on oxygen transport and the arterial lactate levels in patients with cirrhosis.

OBJECTIVE: Despite the increased cardiac output and oxygen delivery, an impaired oxygen uptake has been noted in patients with cirrhosis. We recently observed that endoscopic variceal ligation decreased the cardiac output due to a reduction in the cardiac preload. It is thus possible that a variceal ligation decreases the oxygen delivery and thereby negatively influences tissue oxygenation in patients receiving such treatment. We thus investigated the effects of variceal ligation on oxygen delivery, oxygen uptake, and the arterial lactate levels. METHODS: There were 22 patients with compensated cirrhosis and risky esophageal varices (Child's class A:B=13:9). Twelve patients underwent an endoscopic variceal ligation and 10 patients received gastroscopy as a control. The cardiac function, blood gas status, oxygen delivery, and arterial lactate concentration were also assessed before and after variceal ligation. The oxygen uptake was calculated by the Fick equation. RESULTS: Following variceal ligation, there was an immediate decrease in the cardiac output and oxygen delivery. The reduction in oxygen delivery was associated with a slight but significant increase in the arterial lactate concentration. The decreased oxygen delivery was also associated with a concomitant decrease in the oxygen uptake. In the control subjects, gastroscopy did not alter the systemic hemodynamics, arterial oxygen status, or arterial lactate levels. CONCLUSION: We found a significant decrease in the oxygen delivery in patients undergoing an endoscopic variceal ligation. Such deteriorated tissue oxygenation may be serious especially in patients with a low oxygen transport ability such as in patients with variceal hemorrhage with anemia. However, the clinical significance of these changes remains unclear and further studies are therefore warranted.

In situ detection of insulin-like growth factor II (IGF2) and H19 gene expression in hepatocellular carcinoma.

To assess the relationship between insulin-like growth factor II (IGF2) and H19 gene expression at the cellular level, we have examined the distribution of IGF2 and H19 mRNA by means of an situ hybridization in hepatic malignancies consisting of hepatocellular carcinoma (HCC), cholangiocellular carcinoma (CCC), and metastatic liver cancer (MLC). In HCC, 15 of 27 tumors (56%) and 11 of 27 tumors (41%) demonstrated increased IGF2 and H19 gene expression, respectively. Of 16 HCCs with increased expression of either IGF2 or H19, 10 tumors coexpressed both transcripts at comparable levels. Moreover, the spatiotemporal distribution and the cellular localization of the two gene transcripts were almost identical, suggesting the presence of a reciprocal relation between IGF2 and H19. In addition, 5 HCCs showed increased IGF2 expression without concomitant H19 expression, whereas 1 HCC showed increased H19 expression without IGF2 transcripts. However, 11 HCCs showed no IGF2 or H19 expression. On the other hand, neither IGF2 transcripts nor H19 transcripts were detected in 2 CCCs or 10 MLCs studied. The data suggest that IGF2 and/or H19 gene expression may be characteristic of some HCCs.

Increased expression of proliferating cell nuclear antigen in autoimmune hepatitis in a patient with raised serum concentration of CA19-9.

A 52 year old woman had autoimmune hepatitis and an increased concentration of serum carbohydrate antigen 19-9 (CA19-9). The origin of the raised CA19-9 was studied using immunohistochemistry. Liver biopsy section showed chronic active hepatitis with large numbers of proliferated bile ductules. Immunohistochemical analysis revealed that the proliferated bile ductule cells were positive for proliferating cell nuclear antigen (PCNA) and for CA19-9. It is speculated that the raised serum CA19-9 concentration was derived from proliferated bile ductule cells and these cells, which are positive for PCNA, may be able to produce high concentrations of CA19-9.

Appearance of cross linked proteins in human atheroma and rat pre-fibrotic liver detected by a new monoclonal antibody.

A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

Evaluation of neutrophil functions after experimental abdominal surgical trauma.

OBJECTIVE: In order to determine the effect of surgical trauma on neutrophil functions, we set up an experimental abdominal surgical model using rats and analyzed neutrophil functions. In addition, we measured tumor necrosis factor-alpha (TNF-alpha), cytokine-induced neutrophil chemoattractant/growth-regulated oncogene (CINC/GRO) and nitric oxide (NO) production. MATERIALS AND METHODS: Male Sprague-Dawley rats, 8 weeks old and weighing 250-270 g, underwent laparotomy (4 rats for each experiment). After the operation, neutrophil chemotaxis was assayed using a modified Boyden chamber, and phagocytosis, active oxygen production and adhesion molecule expression were analyzed by flow cytometry. TNF-alpha and CINC/GRO levels were quantified by an immuno-dot-blot assay, and NO levels were measured by the Griess method. At the operation, NO inhibitor, N(G)-monomethyl-L-arginine acetate (L-NMMA, 40 mg) was intraperitoneally administered, and the effect of L-NMMA was studied. RESULTS: After the surgical trauma (24-48 h), blood neutrophil counts significantly increased (p < 0.001), and neutrophil chemotaxis, phagocytosis and active oxygen production were markedly enhanced (p < 0.01). Moreover, up-regulation of Mac-1 and down-regulation of L-selectin on neutrophils were observed (p < 0.05). The levels of TNF-alpha, CINC/GRO and NO increased remarkably in both blood and ascites at 8-48 h after the surgical trauma (p < 0.01): TNF-alpha increased from 194 +/- 9 (the mean +/- SD, n = 4) and 183 +/- 12 pg/ml (preoperation) to 797 +/- 28 and 1045 +/- 137 pg/ml at 24 h in blood and ascites, respectively; CINC/GRO increased from 0.1 +/- 0 and 0.1 +/- 0 ng/ml (pre-operation) to 66.4 +/- 4.5 and 60.3 +/- 17.9 ng/ml at 8 h in blood and ascites, respectively; NO increased from 2.4 +/- 1.0 and 4.2 +/- 1.1 microM (pre-operation) to 11.9 +/- 0.7 and 36.9 +/- 2.1 microM in blood and ascites at 24 h and 48 h in blood and ascites, respectively. Interestingly, L-NMMA treatment significantly reduced the increased levels of TNF-alpha and CINC/GRO and altered the enhanced neutrophil functions (p < 0.05). CONCLUSION: These observations indicate that abdominal surgical trauma induces the production of NO, TNF-alpha and CINC/GRO, and enhances neutrophil functions such as chemotaxis, phagocytosis and active oxygen production. Furthermore, L-NMMA likely modulates the neutrophil functions and the production of TNF-alpha and CINC/GRO after the surgical trauma.

Analysis of human T-cell antigen receptor variable beta gene usage following vaccination with recombinant HBsAg.

We analyzed the TcR Vbeta gene usage before and after vaccination with the hepatitis B vaccine since changes in the TcR Vbeta gene families would be considered to provide preliminary evidence of a mechanism to prevent HBV infection. Six healthy adult volunteers received immunizations. TcR Vbeta usage, T-cell proliferation, and HLA class II alleles were examined in peripheral blood mononuclear cells (PBMC) both before and after vaccination. Furthermore, TcR Vbeta usage in postimmunization PBMC was also compared with PBMC cultured with recombinant HBsAg (rHBsAg). The level of in vitro T-cell proliferation in the presence of rHBsAg increased significantly (P < 0.01) in PBMC isolated after vaccinations. Increases in the different TcR Vbeta genes were also observed in each individual following vaccinations, regardless of the similarity in their HLA alleles. Specific HBV-related antigen-responsive T cells were induced after HB vaccination, without any common restriction for the TcR Vbeta gene families. The mechanism that helps prevent HBV infection was thus found to involve multiclonal alterations in the TcR Vbeta repertoire.

Effects of L-arginine on the systemic, mesenteric, and hepatic circulation in patients with cirrhosis.

Nitric oxide (NO) is known to play an important role in modulating both the hepatic and mesenteric circulation under physiological and pathological conditions. We investigated how L-arginine, a precursor of NO, modifies the hepatic and mesenteric circulation in patients with cirrhosis. The study design was a single-blind controlled study. We measured the systemic and portal hemodynamics before and following intravenous L-arginine and saline infusion using pulsed Doppler ultrasonography in 20 patients with cirrhosis, and then the effects were compared with those found in 20 healthy subjects. In these patients, the effects of L-arginine on hepatic circulation were investigated using hepatic catheterization. L-Arginine infusion induced systemic vasodilation in both the healthy controls and the cirrhotic patients in a similar hemodynamic manner. In these patients, the L-arginine-induced increase in the portal flow was significantly higher than that of cardiac output (CO); however, the relation was the inverse in healthy subjects. Moreover, the L-arginine-induced increase in the portal flow was greater in the cirrhotic patients than that seen in healthy subjects. As a result, L-arginine infusion was thus found to selectively augment the hepatopetal portal blood flow in the cirrhotic liver. In patients, L-arginine infusion induced marked hepatic vasodilation as demonstrated by the reduced hepatic sinusoidal resistance (HSR) and increased estimated hepatic blood flow (EHBF) associated with the ameliorated intrinsic clearance of indocyanine green. Despite the fall in HSR, the hepatic venous pressure gradient (HVPG) increased following L-arginine infusion. The mesenteric and hepatic vascular areas of cirrhosis exhibited an increased susceptibility to the dilator action of L-arginine. These findings suggest that the enhanced NO production in the splanchnic vascular area has an important role in the hepatic circulation in patients with cirrhosis.

Immunohistochemical evidence of insulin-like growth factor II in human small hepatocellular carcinoma with hepatitis C virus infection: relationship to fatty change in carcinoma cells.

It has recently been reported that insulin-like growth factor II (IGF-II) may play a role in the pathogenesis of hepatocellular carcinoma (HCC). We studied the relationship between the expression of IGF-II and fatty change in human small HCC using immunohistochemical staining techniques. Liver biopsy specimens were obtained from 35 patients with HCC (consisting of 15 patients with fatty change and 20 patients without fatty change). All patients had serum markers for the hepatitis C virus (HCV) and histological findings obtained from non-tumourous lesions showed liver cirrhosis or chronic active hepatitis. Immunohistochemical staining was performed using a monoclonal antibody against rat IGF-II. A positive immunoreaction was found in 69% (24/35) of HCC. Insulin-like growth factor II was immunodetected in 80% (12/15) of HCC with fatty change but only in 60% (12/20) of those without fatty change. In most cases, IGF-II was not found in hepatocytes from non-tumourous lesions. We believe this to be the first time that IGF-II has been detected immunohistochemically in small HCC derived from HCV infection. This growth factor was more frequently immunodetected in HCC with fatty change than without. As insulin is an essential factor for the metabolism of fatty acids, IGF-II may play an important role in both fatty degeneration and in the proliferation of HCC cells. Furthermore, immunohistochemical IGF-II staining may contribute to the diagnosis of HCC, particularly in early stages accompanied by fatty change.

Risk factors for hemorrhage from gastric fundal varices.

The incidence and the risk factors of hemorrhage from gastric fundal varices (FV) have not been fully evaluated. We therefore conducted a retrospective and prospective study to define the incidence and risk factors for such episodes. We investigated 132 patients with cirrhosis and gastric FV. Of these 132 patients, 15 patients had hemorrhagic FV at the time of enrollment. The clinical characteristics were compared between these patients and those without a first hemorrhage from FV. In the patients who had never previously bled, the incidence and risk factors were prospectively investigated. The size of FV was greater and red-spot on the FV were more prevalent in patients with hemorrhagic FV. Child's status was also more severe in these patients. In the 117 patients who had never bled, 34 hemorrhages from FV occurred during the follow-up period. The cumulative risk for such hemorrhage at 1, 3, and 5 years was 16%, 36%, and 44%, respectively. A multiple regression analysis (Cox's model) revealed the size of varices, red-spot on the FV, and Child's status to be statistically significant, as well as independent predictors for hemorrhage from FV. The endoscopic criteria (size of the largest varix and presence of red-spot), as well as the hepatic functional reserve, provide the most essential information for predicting a hemorrhage from FV. An estimation of the probability for hemorrhage from FV based on Cox's model may therefore be beneficial in the clinical management of patients with high-risk FV.