Yasunori Fujioka

Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats

Abstract This report deals with the mechanism involved in the cerebellar degeneration following experimental methylmercury poisoning of male Wistar rats. The cerebellar granule cells of animals that exhibited typical hind leg paresis were shrunken and displayed marked nuclear pyknosis. At the ultrastructural level, the nuclei of these cells were condensed and fragmented, features which are characteristic of apoptosis. In situ staining for DNA strand breaks revealed that the pyknotic nuclei were positively labeled. DNA fragmentation was confirmed by agarose gel electrophoresis; a ladder pattern of multiples of approximately 200-base pair fragments, typical of apoptosis, was observed with the cerebellar DNA of the methylmercury-treated animals. These observations suggest that the degeneration of cerebellar granule cells by alkyl mercury compounds involves an apoptotic process.

Expression of vascular endothelial growth factor in human myocardial infarction.

SummaryTo define mechanisms that may influence collateral circulation and angiogenesis, we investigated vascular endothelial growth factor (VEGF) mRNA expression in human hearts. In non-ischemic human hearts, VEGF mRNA was not detected in vessels, but was found in cardiomyocytes. In hearts with myocardial infarction, the intensity of the VEGF signal was much higher in smooth muscle cells of arterioles adjacent to necrosis and in infiltrating macrophages than in myocytes around the site of the necrosis. This study suggests that levels of VEGF expression are high in smooth muscle cells and macrophages around infarcted areas after myocardial infarction and that VEGF may play a role in promoting collateral circulation and angiogenesis in human ischemic hearts.

Pulmonary tumor thrombotic microangiopathy in patients with gastric carcinoma: an analysis of 6 autopsy cases and review of the literature.

Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare clinicopathological entity causing severe pulmonary hypertension (PH). Its histological features include widespread tumor emboli of the small arteries and arterioles of the lung, associated with thrombus formation and fibrocellular and fibromuscular intimal proliferation. Although PTTM has drawn increased attention as a fatal complication of gastric carcinoma (GC), comprehensive studies are still lacking. In order to clarify clinical and pathological features of GC-induced PTTM, recent autopsy cases were analyzed with a review of the literature. Of 36 autopsy cases with GC, 6 (16.7%) were affected by PTTM. Four were male and 2 female, with a mean age of 72.7 years. Three patients presented with PTTM-related clinical manifestations and died of PTTM. They showed clear morphological evidence of PH. The other 3 patients had PTTM as an incidental finding irrespective of clinical manifestations or PH. No patient was diagnosed antemortem as PTTM. All PTTM cases were associated with advanced GC, with a histology of adenocarcinoma of poorly differentiated type (n=4) or signet-ring cell type (n=2). Expression of tissue factor and vascular endothelial growth factor was confirmed immunohistochemically in tumor cells in all cases. The results were all in line with previous studies. In addition, the current study revealed vascular lesions characteristic of PTTM morphology to be present exclusively in the lung. In conclusion, our study shows a 16.7% incidence of PTTM in GC patients, with half of them developing PH and dying of PTTM, confirming a clinical significance as a non-negligible lethal complication of GC. In addition to many known clinicopathological characteristics of PTTM, the current study pointed to some PTTM issues requiring clarification, including the pathogenesis of the exclusive pulmonary distribution of vascular lesions of PTTM. Since details remain to be elucidated, interdisciplinary research is a high priority with a close collaboration between pathologists and clinicians in order to overcome this lethal condition.

Experimental infection of germ-free mice with hyper-toxigenic enterohaemorrhagic Escherichia coli O157:H7, strain 6.

A mouse enterohaemorrhagic Escherichia coli (EHEC) infection model was developed with a combination of germ-free (GF) mice and hyper-toxigenic EHEC (HT-EHEC) O157:H7 strain 6. The HT-EHEC strain 6 produced both Shiga-like toxin (SLT)-1 1.0 microg/mI and SLT-2 8.2 microg/ml in vitro. Eight-week-old germ-free mice were inoculated intragastrically with 5.0 x 10(7) cfu of HT-EHEC strain 6. All GF mice challenged with the HT-EHEC developed ruffled fur and convulsion of limbs or hindleg weakness within 3 days after the challenge, culminating in death within 5 days. The HT-EHEC colonised well at a level of 10(8) - 10(9) cfu/g of faeces 5 days after the challenge. Both SLT-1 and SLT-2 were demonstrated in the faeces of the mice for 5 days after challenge. Histological examination of the colons of the infected mice showed congestion of the lamina propria mucosa, mild inflammatory cell infiltration and goblet cell depletion. In proximal tubules of the renal cortex, epithelial swelling with scattered necrotic cells was recognised. Endothelial swelling and mononuclear cell infiltration were also observed in the glomeruli. The brain showed acute neuronal necrosis in the cerebrum and slight loss of Purkinje cells in the cerebellum. Passive immunisation with anti-SLT antisera prolonged the life of the mice without anyneural symptoms. Microscopically, all tissue specimens from the passively immunised mice were not remarkable. These results indicate that the infection of GF mice with HT-EHEC is a useful animal model to study the pathogenicity of SLT-producing E. coliand the toxins.

Distribution of the high-affinity choline transporter in the human and macaque monkey spinal cord

The distribution of the high-affinity choline transporter (CHT) was determined in the human and macaque monkey spinal cord using in situ hybridization histochemistry and immunohistochemistry. Signals for CHT mRNA were observed in somatic motor neurons, sympathetic preganglionic neurons, and neurons in the medial part of lamina VII. The mRNA for CHT was co-localized in single neurons with the mRNAs for vesicular acetylcholine transporter and cholineacetyltransferase. These same cholinergic neuronal groups were labeled by immunohistochemistry for human CHT. Of somatic motor neurons, smaller cell bodies of gamma-motor neurons were labeled very intensely, whereas larger cell bodies of alpha-motor neurons showed various degrees of labeling from weak to moderately intense. Human CHT is thus a novel cholinergic marker, which not only labels cholinergic neurons, but also reveals their heterogeneity.

Immunohistological study of globoid cell leukodystrophy

We examined three autopsy cases of globoid cell leukodystrophy (GLD) with different survival, using immunohistochemistry and in situ nick end labeling (ISEL). The white matter lesion was pronounced in the corona radiata, corpus callosum and cerebellar peduncles in three cases, where a spongy state developed, in addition to the neuronal loss in the thalamus, cerebellum and inferior olivary nucleus. Ramified microglia, being immunoreactive for ferritin and HLA-DR alpha, were scattered in the white matter, and some of them also had immunoreactivity for TNF-alpha. Both the small-sized and large-sized globoid cells showed immunoreactivity for ferritin KP-1 and NCAM, while some of the small-sized globoid cells were also immunoreactive for HLA-DR alpha and TNF-alpha. As the survival became longer, the occurrence of the globoid cells decreased, however, they were commonly observed in the corpus callosum and cerebellar peduncle in three cases. T lymphocytes immunoreactive for LCA, UCHL-1 and CD3 were increased around the vessels in the white matter. ISEL stained nuclei of mononuclear cells in the white matter in two cases with short survival, although the cell origin was not verified. ISEL also visualized a few nuclei of the small-sized globoid cells in one case. On the other hand, immunostainings against cell death proteins such as bcl-2 family members and p53 failed to identify any significant changes. These data suggest that the immunological step and to a lesser extent the apoptotic process may partly be involved in the myelin breakdown and glial pathology in GLD, as reported in the twitcher mouse, a murine model of GLD.

Dural band pathology in syringomyelia with Chiari type I malformation

Surgical material taken from the ‘outer layer’ of thickened dura mater (dural band) at the craniovertebral junction of eight cases of syringomyelia with Chiari type I malformation was histologically examined in comparison with four autopsy cases as controls. The dural band was thickened and there were increased numbers of collagen fibers which showed fiber splitting, hyalinous nodule, calcification and/or ossification. These changes were not observed in the four control cases. Thus, it is suggested that the thickening of the dura mater may be a causative factor of syringomyelia with Chiari type I malformation. In addition, the histology of the thickened dura mater suggests the condition may be a consequence of birth injury in these patients.

Diagnostic significance of high mobility group I(Y) protein expression in intraductal papillary mucinous tumors of the pancreas

Introduction Overexpression of the high mobility group I(Y), [HMGI(Y)], gene/proteins has been demonstrated in many types of human malignancies, suggesting that HMGI(Y) may play a vital role in the oncogenic transformation of cells. Aims To analyze HMGI(Y) expression in intraductal papillary mucinous tumor (IPMT) of the pancreas to verify whether determination of the HMGI(Y) expression level could provide any diagnostic advantages in the pathological diagnosis of IPMT. Methodology Thirty-three different lesions from 25 patients with IPMT, including 20 with mild dysplasia, 7 with moderate dysplasia, and 6 with carcinoma, were analyzed immunohistochemically with use of an HMGI(Y)-specific antibody. Results Immunohistochemical analysis revealed that, although no significant immunoreactivity was found in cases of normal pancreatic duct or mild dysplasia, 28.6% (2/7) of moderate dysplasia showed multifocal immunoreactivity with moderate intensity. In contrast, in 50% (3/6) of the cases of carcinoma, intense multifocal or diffuse immunoreactivity occurred, almost equivalent to that observed in cases of duct cell carcinoma, whereas in the remaining 3 cases of carcinoma only a faint focal immunoreactivity occurred. Histologic examination revealed that these HMGI(Y)-positive carcinomas had an invasive growth pattern, whereas the HMGI(Y)-negative carcinomas were either carcinomas in situ or tumors with minimal invasion. Thus, an increased expression level of HMGI(Y) proteins was closely associated with the malignant phenotype in IPMT. Conclusion On the basis of these findings, we propose that HMGI(Y) proteins could play an important role(s) in a multistage process of carcinogenesis of IPMT and that the HMGI(Y) protein level could serve as a potential diagnostic marker, which may enable the identification of tumor cells with potential to be biologically malignant.

HHV8-negative primary effusion lymphoma of the peritoneal cavity presenting with a distinct immunohistochemical phenotype

Primary effusion lymphoma (PEL) has been recognized as a body‐cavity‐based lymphoma that was originally reported to be associated with human herpes virus 8 (HHV8) infection, and was frequently found in human immunodeficiency virus‐positive (HIV) patients. Here we describe an autopsy case of PEL of the peritoneal cavity in an immunocompetent patient. Cytological analysis of tumor cells within ascites revealed immunocytochemical features of keratin positivity and CD45 negativity. At autopsy, the presence of a massive volume of ascites as well as diffuse tumor cell infiltrates within the serosa of the intestine and mesenterium were observed. Tumor cells were morphologically similar to anaplastic large‐cell lymphoma, but were immunohistochemically positive for keratin and epithelial membrane antigen (EMA). They also showed no reactivity to representative lymphocyte surface markers including CD45, in addition to being negative for CD30 and p80NPM/ALK. Molecular analysis of the tumor cells revealed monoclonality of the immunoglobulin heavy‐chain gene rearrangement which demonstrated a lymphoma of the B‐cell lineage. Furthermore, HHV8 was not detected by immunohistochemical analysis, PCR or nested PCR technique. Based on these results, we consider the present case to be an HHV8‐negative PEL with keratin and EMA positivity.

Bcl-2 Expression in Non-Small Cell Lung Cancers: Higher Frequency of Expression in Squamous Cell Carcinomas with Earlier pT Status

Abnormal expression of the bcl-2 gene product (Bcl-2) has been found in a wide variety of tumors, including lung cancer. In the present study, a total of 116 tumor specimens from surgically resected non-small cell lung cancer (NSCLC) patients, that were previously studied for p53 protein expression, were analyzed with immunohistochemistry for Bcl-2 expression. Forty (34%) of 116 tumor specimens showed Bcl-2 expression, which was found to occur more frequently in males than females (p = 0.049) and to be associated with smoking (p = 0.047). Bcl-2 expression was more frequently observed in squamous cell carcinomas (27 of 51, 53%) than in adenocarcinomas (12 of 55, 22%; p = 0.002), and in pT1 tumors (11 of 13, 85%) than in pT2 and pT3 tumors (16 of 38, 42%) in squamous cell carcinomas (p = 0.01). Bcl-2 expression did not correlate either with p53 protein status. We compared Bcl-2 expression in primary tumors and metastatic tumors of regional lymph nodes. Of 11 cases with Bcl-2-negative primary tumors, 10 were Bcl-2-negative in metastatic tumors except 1 case. In contrast, of 10 cases with Bcl-2-positive primary tumors, 6 lost Bcl-2 expression in metastatic tumors, while the remaining 4 cases still showed Bcl-2 expression in metastatic tumors. In the 89 potentially curatively treated patients, those with Bcl-2-positive and Bcl-2-negative tumors did not show a significant difference in survival (5-year survival rates, 56 and 42%, respectively, p = 0.2 by the generalized Wilcoxon test). These data indicate that Bcl-2 expression is frequently observed in squamous cell carcinomas with early pT status, and that it does not predict prognosis of patients with NSCLC.