Prognostic significance of perirectal lymph node micrometastases in Dukes' B rectal carcinoma: an immunohistochemical study by CAM5.2.

Lymph node metastasis is an important prognostic factor for rectal carcinoma, but only a few attempts at defining the relationship between lymph node micrometastases and prognosis have been made. The purpose of this study was to examine the correlation between the presence of micrometastases and prognosis in patients with rectal carcinoma. Six hundred forty-four lymph nodes were dissected from 42 patients with Dukes' B rectal carcinoma and stained immunohistochemically using a monoclonal antibody, CAM5.2, that binds cytokeratin. Clinicopathological factors, rate of recurrence, and prognosis were compared among patients with and without micrometastases. Micrometastases were detected in 19 lymph nodes (19 of 644 = 2.9%) from 9 patients (9 of 42 = 21.4%). The presence of micrometastases was not related to clinicopathological factors. There were significant differences in recurrence rates (5 of 9 versus 5 of 33, P = 0.02), relapse-free survival rates (P = 0.04), and 10-year survival rates (P = 0.03) between patients with and without micrometastases. Immunohistochemistry successfully identified micrometastatic foci in lymph nodes missed with conventional staining methods. The existence of micrometastases influenced the prognosis in patients with Dukes' B rectal carcinoma.

HBV genome integration and genetic instability in HBsAg-negative and anti-HCV-positive hepatocellular carcinoma in Japan.

The aim of this study is to clarify the existence and the form of HCV RNA and HBV DNA genome integration and genetic instability in liver tissue with HBsAg-negative and anti-HCV-positive HCC. We investigated 16 Japanese patients with HBsAg-negative and anti-HCV-positive HCC. HBV genome integration into host cell genome by Southern hybridization and PCR was examined. Moreover, we analyzed loss of heterozygosity (LOH) and replication errors (RER) of chromosomes 2p, 3p and 17p using the PCR and an autosequencer to determine the three microsatellite regions D2S123, D3S1067, TP53. Eight (50.0%) of 16 were found to have integrated genome of HBV in tumor tissue (T) by PCR. In even the non-tumor regions (NT), seven patients (43.8%) were found to have HBV genome integration. The coincidence between T and NT was found in 4 (25%). Integration of HBV-X gene in T was revealed in three (18.7%), and HBV-integration was confirmed in all NT. No integration of the X gene alone was found in the liver tissue. Five (37.5%) of eight HBV DNA integrated cases simultaneously had HCV RNA minus strand. Concerning the genetic instability, RER were detected in two of 16 (12.5%). RER at 2p; D2S123 was observed in one of 16 (6.2%) and at 3p; D3S1067 was observed in one (6.2%). LOH at the D2S123 locus was observed in one of 12 tumors with heterozygosity (8.3%). There was no genetic instability (LOH or RER) of TP53 which was p53 locus on 17p in T. There was only one case of eight HBV DNA integrated cases (6.2%) with genetic instability of RER of 3p simultaneously in T. In conclusion, the majority of HBsAg-negative and anti-HCV-positive HCC liver tissue was found to have HCV-RNA and HBV DNA integration, and in some samples, HBV DNA integration and genetic instability were concurrently confirmed. It is speculated that multistep carcinogenesis may have been proposed for HCC oncogenetic progression.

Immunohistochemical study of cytochrome P450 2C and 3A in human non-neoplastic and neoplastic tissues.

Organ and cellular distribution and expression constancy of microsomal cytochrome P450 (CYP) 2C and 3A in humans were studied with new polyclonal antibodies to CYP2C (MP-1) and 3A (NF-2) active in formalin-fixed, paraffin-embedded tissues. Antibodies were raised against purified human CYP2C9 and CYP3A4. On western blotting, MP-1 reacted with 2C8, 2C9, 2C18 and 2C19, and NF-2 with 3A4. In both frozen and paraffin sections, hepatocytes showed diffuse immunoreactivity with MP-1 and centrilobular staining with NF-2. In-paraffin sections of 40 kinds of nonneoplastic tissues, epithelium of the small and large intestine, bile duct, nasal mucosa, kidney and adrenal cortex stained positively with both MP-1 and NF-2 antibodies. Epithelium of gastric fundic glands, salivary glands, tracheobronchial glands, Brunner's glands, the prostate, uterine cervix and nasopharynx showed definite reactivity with MP-1. Epithelium of the gastric mucosa with intestinal metaplasia, duodenum, gallbladder and intercalated ducts of the pancreas and chief cells of the parathyroid and the corpus luteum of the ovary reacted with NF-2. Among the neoplastic tissues, MP-1 reacted with pleomorphic adenoma of the salivary gland and carcinomas of six different organs, and NF-2 with those of 7 different organs. These results indicate that CYP2C and CYP3A are distributed widely and organ specifically, as well as being variably expressed in neoplastic and normal states.

Evidence that the elevation of soluble MHC class I antigens in the serum precedes the onset of graft-versus-host disease and is correlated with the severity of the disease in rats.

We examined the changes in the levels of soluble major histocompatibility antigen complex (MHC) class I antigens in the serum under a lethal or nonlethal state of graft-versus-host-disease (GVHD) induced by injecting various doses of PVG rat splenic lymphocytes into (DA x PVG)F1 rats. All rats receiving 4 x 10(8) lymphocytes (lethal dose) died on day 20-36 showing typical features of GVHD, while the injection of 4 x 10(7) cells (nonlethal dose) induced no sign of GVHD. When rats were inoculated with a nonlethal dose of lymphocytes prior to the injection of a lethal dose, all rats survived with or without showing transient GVHD. Preceding the onset of GVHD the levels of soluble class I antigens increased significantly to 1094 +/- 487 ng/ml (mean +/- SD, n = 4) from 3 days after the injection of a lethal dose to the time of death, whilst the levels in the nonlethal dose group remained unchanged. Rats with transient GVHD in the preinoculated group showed the increase of soluble class I antigens to the same extent as rats with lethal GVHD, suggesting that GVHD was systemically ongoing. The levels of soluble class I antigens also correlated with the severity of GVHD as judged by daily observation and histological studies. Rats receiving a lethal dose showed destructive alteration of spleen structure and cellular infiltration in the portal area of the liver before the animals started to show signs of GVHD, whereas rats in the nonlethal dose group exhibited no marked change. These data suggest the possibility of serum soluble class I antigens being not only a diagnostic but also a prognostic marker for GVHD.

Peliosis hepatis and neoplastic/dysplastic lesions in aged male Long-Evans Cinnamon rats: MR imaging with pathologic correlation.

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, abnormally accumulates copper in the liver. There have been a lot of reports on preneoplastic and neoplastic hepatic tumors in LEC rats, but few studies have been focused on other lesions. The aim of this study was to describe the MR findings of the liver of LEC rats with pathologic correlation to characterize the hepatic lesions developed in them. We measured MR images of the liver of six aged (over the age of 70 weeks old) male LEC rats. Measurements of T(1), T(2)-weighted images, and the dynamic and delayed studies after i.v. gadolinium injection were performed. The rats were sacrificed immediately after the measurements, and the diagnosis was histologically made. We identified seven lesions of peliosis hepatis, three neoplastic/dysplastic lesions, three cysts and one cholangiofibrosis. Peliosis hepatis was characterized as showing a significantly long T(2) relaxation time of 57.9 +/- 13.3 ms (mean +/- standard deviation) compared with 41.3 +/- 1.7 ms in normal liver, and prolonged enhancement after a gadolinium injection. Neoplastic/dysplastic lesions tended to show prolonged T(2), and they showed isointensity on T(1)-weighted images. They were best characterized by early enhancement followed by a rapid wash-out after a gadolinium injection. In conclusions, the frequent occurrence of peliosis hepatis observed in the present study suggests this can be a characteristic lesion in aged LEC rats. The characteristic MR findings enable us to distinguish between peliosis hepatis and neoplastic/dysplastic lesions.

MR imaging of non-cancerous hepatic lesions in Long-Evans cinnamon rats.

We measured MR images of the liver of Long-Evans Cinnamon (LEC) rats with pathologic correlation and assessed the effectiveness of MR imaging (MRI) for diagnosis of noncancerous hepatic lesions. T1- and T2-weighted images of their livers were obtained, and the dynamic and delayed studies after intravenous gadolinium injection were also performed. Cholangiofibrosis showed low signal intensity on T1-weighted images and high signal intensity on T2-weighted images. The T2 relaxation time of cholangiofibrosis was significantly prolonged (p < .01), and the signal intensity ratio of this lesion to muscle on T1-weighted images was significantly lower than that of normal liver parenchyma to muscle (p < .01). The lesion was enhanced immediately after gadolinium injection and the enhancement was prolonged. Among three cases of peliosis hepatis identified, one showed heterogeneous intensities on both T1- and T2-weighted images and the other two showed similar intensity pattern to cholangiofibrosis. The characteristic MR appearance of cholangiofibrosis may be useful to distinguish it from hepatocellular carcinoma (HCC).

The role of previous infection of hepatitis B virus in Hbs antigen negative and anti-HCV negative Japanese patients with hepatocellular carcinoma: etiological and molecular biological study.

The aim of this study is to elucidate the important role of the previous infection of HBV, and the relations among HBV genome integration and p53 gene mutation, telomerase activity and genetic instability in liver tissue with HBsAg-negative (NB) and anti-HCV negative (NC) hepatocellular carcinoma (HCC). We examined the backgrounds of 34 NB and NC (NBNC) Japanese patients with chronic liver disease (CLD) patients not associated with HCC and 26 NBNC CLD patients with HCC. HBV genome integration into host cell genome, p53 gene mutation telomerase activity and genetic instability were examined in 6 with NBNC HCC (NBNC-HCC) tumorous tissue (T) and non-tumorous tissues (NT). In the NBNC group, HBV-related antibody positive patients with HCC are significantly more than the patients without HCC. Moreover, concerning the stage of the coexisted liver diseases, in NBNC CLD, LC patients with HCC is 19 of 26 (73.1%) , on the other hand, LC patients without HCC is 16 of 34 (47.1%). LC patients with HCC group is significantly more than that without HCC. Three (50%) of 6 in T and 3 cases (50% ) in NT were found to integrated genome of HBV. p53 gene mutation was observed in 3 (50%) of T. Concerning the telomerase activity, 3 of 6 cases (50%) in T and 1 case in NT was recognized. There was no genetic instability (LOH or RER) of D2S123, D3S1067 and TP 53 in T and NT. Finally in T of NBNC HCC cases, TTVDNA was detected in 3 of 5. Even in the HBsAg-negative and anti-HCV negative HCC cases, CLD coexisting with LC, previous HBV infection and HBVDNA integration were observed. There were a few cases with HBVDNA integration, p53 gene mutation, telomerase activity and genetic instability, simultaneously in HCC tissue, and in some cases, the coexistence with TTVDNA were concurrently confirmed. It is speculated that the important role of the previous infection of HBV may have also been proposed for HCC oncogentic progression in NBNC CLD [corrected].

Screening of milk aspiration in 105 infant death cases by immunostaining with anti-human alpha-lactalbumin antibody.

To determine the frequency and degree of milk aspiration in infant death cases, immunohistochemical examinations were performed on lung sections from 41 sudden death cases and 64 in-hospital death cases using anti-human alpha-lactalbumin antibody. Milk aspiration to some degree was detected in more than half of the sudden death cases and in about one-third of the in-hospital death cases. A semi-quantitative examination of the amount of aspirated milk was subsequently performed in the positive cases. The amount of aspirated milk in the sudden death cases was significantly higher than that in the in-hospital death cases. The frequency distribution of the amount of aspirated milk was similar in shape in both groups. In most cases, a very small amount of aspirated milk was detected. The aspirated milk was assumed to be a result of occasional gastroesophageal reflux or cardiopulmonary resuscitation. However, in five cases, much larger amounts of aspirated milk were found. In these cases, milk aspiration may have been an important part of the cause of death. We concluded that slight milk aspiration is not rare in infant death cases, and that in a few cases, the aspiration is lethal. An immunohistochemical screening test is available to perform a postmortem diagnosis in these cases.

Immunohistochemical detection of human milk components aspirated in lungs of an infant.

We examined an autopsy case of an infant whose cause of death was suspected to be asphyxia due to human breast milk aspiration. In order to demonstrate aspirated milk in the lungs, we conducted immunohistochemical staining using eight antibodies against the components of human milk. Seven of the eight antibodies reacted positively with the substances suspected to be aspirated milk. We concluded that immunohistochemical staining with these antibodies is useful to demonstrate human milk aspiration and provide some keys for detecting the causes of unexpected infant deaths.

Immunohistochemical examination of the lungs in infant death cases using antibodies against milk components.

To examine the use of immunohistochemical staining with antibodies against milk components for detection of aspirated milk on lung sections, eighteen infant death cases were investigated. Immunostaining was performed with anti-human alpha lactalbumin, anti-human IgA, anti-human milk fat globulin 1, and anti-cow whey antibody. Reactivity with each antibody was examined, and semi-quantitative examinations were performed to compare the amount of aspirated milk using anti-human alpha lactalbumin antibody. Materials in the alveoli or bronchioli on lung sections suspected to be aspirated milk showed the most sensitive and clearest reaction with anti-human alpha lactalbumin antibody. Of the eighteen cases, ten cases showed positive reaction with this antibody. The amount of aspirated milk varied widely in each case. In conclusion, immunohistochemical staining with antibodies against human milk components, especially anti-human alpha lactalbumin antibody, can detect small amounts of milk. Using this method, we were able to compare the relative amount of aspirated milk among cases.

Fatal cardiovascular injuries to the unbelted occupant associated with airbag deployment: two case-reports.

We present two forensic autopsy cases of unbelted occupants associated with the airbag deployment in motor vehicle collisions. Both victims suffered from cardiovascular injuries which were the cause of death. Case 1: A 50-year-old man sustained a contusion on the left anterior chest with rib fractures and laceration of the intrapericardial inferior vena cava, the right ventricle, and the right pleuropericardium. Case 2: A 40-year-old man sustained multiple rib fractures, sternal fracture, and the rupture of the right ventricle. Autopsies and vehicle examinations revealed that both victims' chest seemed to strike the steering wheel through the deployed airbag. Therefore, we determined that the source of blunt impact force is the steering wheel through the airbag rather than airbag deployment only. In light of these two cases, we learned that the steering wheel should be considered as the blunt impact force inducing cardiovascular injuries even in cases in which the airbag has been deployed.

[Distribution of pulmonary neuroendocrine cells--an immunohistochemical study of the lung in autopsied infants including sudden infant death syndrome].

The distribution of pulmonary neuroendocrine cells (PNEC) was analyzed immunohistochemically in 14 victims of sudden infant death syndrome (SIDS), and 10 cases of infant death unrelated to SIDS, excluding congenital heart disease. Lung tissue sections were immunostained with antibodies against chromogranin A (CGA), calcitonin (CT) and gastrin-releasing peptide (GRP). CT/GRP immunoreactivity decreased in older infants of each group, while CGA immunoreactivity showed almost no decrease. Serial section analysis showed some PNEC produced CGA, CT and GRP. However, CGA-immunoreactive PNEC sometimes lacked of CT/GRP immunoreactivity. The difference of PNEC distribution between SIDS and the control cases could not be verified. To date, there have been no studies reported of PNEC distribution in infants by using CGA expression. CGA is considered to be the most useful marker for detecting PNEC in infant lung. Our findings suggest that substances produced by PNEC changed with postnatal development both in SIDS and the control group. This result may be one clue to clarifying the development and function of small airways in infants, allowing further progress in SIDS research.

Effects of oseltamivir phosphate (Tamiflu) in human sera on results of microneutralization and hemagglutinin-inhibition tests for H5N2 avian influenza virus.

To determine the influence of oseltamivir phosphate (Tamiflu) on the results of microneutralization and hemagglutinin-inhibition (HI) tests in human sera with H5N2 influenza virus, ten volunteers were administered Tamiflu and blood samples were collected. In the microneutralization test, no consistent effects were observed. However, in the HI test, specimens from all volunteers taken at 4 and 7 h after drug administration showed a higher titer as compared to 0 and 24 h after administration when mammalian cells (horse, guinea pig, and human) were used. These results suggest that the administration of Tamiflu may affect the results of HI tests for H5N2 virus.

Uncomplicated acute diverticulitis of the cecum and ascending colon: sonographic findings in 18 patients.

To determine the sonographic features of uncomplicated acute diverticulitis of the cecum and ascending colon, the sonographic findings in 534 patients who presented with right lower quadrant pain were reviewed. Of these, 18 patients had uncomplicated acute diverticulitis of the cecum and ascending colon. The diagnosis was confirmed by surgery (one patient), clinical course (17 patients), CT (eight patients), or contrast enema (11 patients). On sonography, a round or oval focus of varying echogenicity, which protruded from a segmentally thickened colonic wall and was surrounded by a hyperechoic area, was seen in all 18 patients. These were hypoechoic foci (12 patients), hypoechoic foci with internal strong echoes (three patients), and echogenic shadowing foci with surrounding hypoechoic bands (three patients). Extraluminal gas (one patient) and thickening of lateroconal fascia (six patients) were seen also. Findings of enlarged appendix, frank abscess, and ascites were absent. All patients, including the one who had laparotomy, were successfully treated medically for diverticulitis. Of 515 patients without diverticulitis, in only one patient with acute appendicitis did sonography show a hypoechoic protruding focus. Our experience indicates that the major sonographic finding in patients with uncomplicated acute diverticulitis of the right colon is a hypoechoic round or oval focus protruding from a segmentally thickened colonic wall.

Sudden death due to right ventricular cardiomyopathy.

A 21-year-old man died suddenly at a small party. He had had no clinical signs of cardiac disease except for a slightly abnormal electrocardiogram (occasional premature ventricular contractions) since he was 15 years of age. Autopsy examination revealed cardiomegaly (469 g), with right atrial and ventricular dilatation. The right ventricular myocardium was massively replaced with adipose tissue, and there was one isolated fatty lesion in the right side of the ventricular septum. There were no congenital malformations such as a septal defect or valvular deformity. Histologically, muscular fibers remaining in the right ventricular wall showed neither degenerative nor inflammatory changes. An isolated lesion of the ventricular septum consisted of almost complete replacement of the muscle bundles with adipose tissue. Such a pathologic condition has recently been termed right ventricular cardiomyopathy. Postmortem examination is necessary to make a definite diagnosis of the disease, because in most adult cases of the disease, sudden death occurs before there have been any critical signs.

A small mucinous cystadenocarcinoma of the liver detected by a fluid-fluid level on ultrasonography.

A case of small, borderline malignant biliary mucinous cystic tumor is presented. The patient initially presented to us 18 months earlier to undergo a sigmoid colon resection for sigmoid colon cancer. At that time, a liver cyst measuring 18 x 12 mm was detected. On a follow-up abdominal ultrasonography study for colon cancer, the liver cyst had enlarged to 21 mm in diameter and contained a fluid-fluid level 18 months after surgery. Histological examination of a needle biopsy specimen indicated possible adenocarcinoma. Lateral segmentectomy of the liver was performed. Histopathologically, the tumor was diagnosed as a mucinous cystic tumor, of border line malignancy, which had originated from a bile duct gland. It contained both mucinous and serous components, which were thought to have caused the formation of a fluid-fluid level within the cyst. In this case, the fluid-fluid level demonstrated by ultrasonography was beneficial in the early detection of a cystic tumor of the liver. This case may be the smallest reported cystadenocarcinoma of the liver yet published.

Novel murine autoimmune-mediated liver disease model induced by graft-versus-host reaction and concanavalin A.

BACKGROUND AND AIMS: We have previously reported that cluster of differentiation (CD)4+ T cells induced autoimmune liver diseases in mice with graft-versus-host reaction (GVHR) because of major histocompatibility complex (MHC) class II disparity. To analyze the progression of the autoimmune-related mechanism in the liver, concanavalin A (Con A) was injected in mice undergoing GVHR. The aim of this study is to clarify whether Con A deteriorates murine hepatic lesions induced by GVHR, and to elucidate the participation of the cytokines of liver-infiltrating CD4+ T cells. METHODS: Mice (F1; B6.C-H-2(bm12) x B6) were intravenously injected with B6 T spleen cells. Concanavalin A (15 mg/kg) was administrated 5 days after cell transfer. We examined serum transaminase, antimitochondrial antibodies (AMA), antinuclear antibodies (ANA) and histological changes. Liver-infiltrating CD4+ T cells were sorted and their cytokine mRNA expression was examined by the use of reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Graft-versus-host reaction + Con A mice revealed an elevated serum transaminase, elevated AMA and ANA titers, increased periportal cellular infiltration, piecemeal necrosis and bridging necrosis in the liver. In this group, interferon (IFN)-gamma mRNA expression was more elevated than it was in the GVHR mice. However, there was no difference in the expression of interleukin (IL)-10 mRNA between the two groups. CONCLUSION: The results suggest that Con A deteriorates the GVHR-induced hepatic lesions, and IFN-gamma and IL-10 of CD4+ T cells might be implicated in the progression of autoimmune-related hepatic lesions. This model might offer an aspect for the investigation of progressive mechanisms in T-cell- mediated hepatobiliary injury.

Optimal and effective oral dose of taurine to prolong exercise performance in rat.

The aim of this study was to determine the effective and optimum dose of taurine for exercise performance and to maintain tissue taurine concentration. Rats received a respective daily dose of 0, 20, 100, and 500 mg/kg body weight of taurine (EC and ET-1, -2, -3 groups, respectively) for two weeks, and then, were subjected to treadmill until exhaustion. The running time to exhaustion was significantly prolonged by 25% and 50% in the ET-2 and -3 groups, respectively, compared to that in the EC group accompanied with maintenance of taurine tissue concentrations. Furthermore, the oxidative glutathione per total glutathione ratio in tissues was inhibited in the ET-2 and -3 groups whereas it was higher in the EC group than in both the no exercise and taurine-administered groups. Therefore the effective and optimal doses of oral taurine administration for two weeks on a transient exercise performance were between 100 and 500 mg/kg/day.

Biochemical and histological changes after more than four years of treatment of ursodeoxycholic acid in primary biliary cirrhosis.

We report on three patients with symptomatic, anicteric, and noncirrhotic primary biliary cirrhosis (Ludwig histological stage III at first liver biopsy) who were treated orally with 600 mg/day of ursodeoxycholic acid (UDCA) for more than 4 years. Follow-up liver biopsy was performed twice (at 1-3 and 4-6 years) during treatment. In all cases, during the whole period of up to 4-6 years of UDCA treatment, transaminase, alkaline phosphatase, and gamma-glutamyltranspeptidase levels improved, remaining at subnormal levels compared with pretreatment levels. Moreover, histological stage did not change throughout the UDCA treatment of up to 6 years. The second liver biopsy (at 1-3 years) revealed decreased lymphocytic infiltration in all cases, and bridging fibrosis was decreased in two cases. However, in the third biopsy at 4-6 years, portal inflammation was increased in one case without fibrotic progression; in the other two cases, bridging fibrosis was slightly worsened without portal inflammatory progression. In summary, these three cases show that liver histology was found to be improved, as were blood chemistry and pruritus, during short-term UDCA treatment, but histology results were slightly worse after long-term treatment despite the sustained improvement in biochemistry and pruritus.

Expression of cytochrome P450 3A4 in foveolar epithelium with intestinal metaplasia of the human stomach.

The expression of cytochrome P450 3A4 (CYP3A4) in the foveolar epithelium of the human stomach with intestinal metaplasia was studied using immunohistochemistry, western blotting and reverse transcription polymerase chain reaction (RT-PCR). CYP3A4 was immunohistochemically detected in the foveolar epithelium with intestinal metaplasia, but was not detected in foveolar epithelium without intestinal metaplasia, in the pyloric gland or in the fundic gland of the stomach. Western blotting and RT-PCR demonstrated that CYP3A4 protein and mRNA were expressed in the liver and pyloric gland mucosa with intestinal metaplasia, but not in the fundic gland mucosa without intestinal metaplasia. Possible roles of CYP expression in the gastric mucosa with intestinal metaplasia in human stomach carcinogenesis are briefly discussed.