LIVER CHANGES IN HEPATOCEREBRAL DISEASE*

An attempt of pathogenetic analysis of Wilson's disease and Inose's disease, the two most important varieties of hepatocerebral diseases, was made from the facette of the hepatic pathology. The liver changes of a total of 43 human autopsy cases(19 of Wilson's and 24 of Inose's disease) were reevaluated. Concurrently an attempt of experimental production in animals of hepatic lesions compatible with those of Wilson's disease was made. Inose's special type of hepatocerebral disease is divided into two subgroups, a shunt form and a nutritional form. Cerebral damage in the shunt form is attributed to the portal-systemic collaterals, while the clinicopathological survey of the cases with the nutritional form indicates that functional disturbances, related unbalanced diet or endocrine abnormality, seem to cause brain damage. In Wilson's disease, three stages are noted from the analysis of the liver changes; fibrosis or monolobular cirrhosis in the first stage, submassive necrosis or active postnecrotic cirrhosis in the second stage and multilobular cirrhosis or arrested postnecrotic cirrhosis in the third stage. Experimentally, monolobular cirrhosis could be produced in animals by copper administration alone, whereas postnecrotic cirrhosis was first reproduced by a combination of copper administration and low protein diet, anti-kidney serum, or copper nephrosis. It is suggested that for the development of postnecrotic cirrhosis in the second and third stages of Wilson's disease an additional factor other than copper is needed, and that this second factor may be the deficiency of SH- containing aminoacids.     

Formation of quinone methide in the self-cure of quinoid phenol formaldehyde resin

The study of the reaction of some methylenebisphenols with chloranil in ethanol and also of a phenol formaldehyde condensate prepared by acid catalysis, reveals that quinone methide is formed in the self-cure of quinoid phenol formaldehyde resin. The quinone methide reacts with ethanol to give an adduct. The structure of the alcohol adduct is identified by NMR and IR data and by means of pyrolysis.     

A new liver metastatic and peritoneal dissemination model established from the same human pancreatic cancer cell line: analysis using cDNA macroarray

To elucidate the mechanisms of metastasis, we established two sublines HPC-1H5 with a highly liver metastatic cell line and HPC-1P5a with a highly peritoneal disseminating cell line, which were sequentially selected from the parental pancreatic cancer cell line HPC-1. Using these three cell lines, we investigated several biological properties and mRNA levels of differentially-expressed genes involved in cancer metastasis by cDNA macroarray. Microscopic findings for the three cell lines were the same. The tumorigenicity, in vitro growth ability, motile activity, adhesive activity and the production of IL-8 of metastatic sublines were higher than those of parental HPC-1 cells. Particularly, HPC-1H5 cells showed clearly higher levels of IL-8 expression and tumors of HPC-1H5 cells grew faster and bigger than those of HPC-1P5a cells. In cDNA macroarray analysis of HPC-1H5 cells, 22 genes were up-regulated and 44 genes were down-regulated compared with parental HPC-1 cells. In HPC-1P5a cells, 9 genes were up-regulated and 28 genes were down-regulated compared with parental HPC-1 cells. This study provides a demonstration of global gene expression analysis of pancreatic cancer cells with liver metastasis and peritoneal dissemination. Furthermore, our results provide a new insight into the study of liver metastasis and peritoneal dissemination of human pancreatic cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of mutations in the Bruton's tyrosine kinase gene,including a novel genomic rearrangements resulting in large deletion,in Korean X-linked agammaglobulinemia patients

Mutations in the Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). We identified BTK mutations in six patients with presumed XLA from unrelated Korean families. Four out of six mutations were novel: two missense mutations (P565T, C154Y), a point mutation in a splicing donor site (IVS11+1G>A), and a large deletion (a 6.1-kb deletion including BTK exons 11–18). The large deletion, identified by long-distance PCR, revealed Alu-Alu mediated recombination extended from an Alu sequence in intron 10 to another Alu sequence in intron 18, spanning a distance of 6.1 kb. The two known mutations consisted of one missense (G462D) mutation, and a point mutation in a splicing acceptor site (IVS7−9A>G). This study suggests that large genomic rearrangements involving Alu repeats are few but an important component of the spectrum of BTK mutations.     

The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis

PTEN is an important tumor suppressor gene. Hereditary mutation of PTEN causes tumor-susceptibility diseases such as Cowden disease. We used the Cre-loxP system to generate an endothelial cell-specific mutation of Pten (Tie2CrePten) in mice. Tie2CrePten(flox/+) mice displayed enhanced tumorigenesis due to an increase in angiogenesis driven by vascular growth factors. This effect was partially dependent on the PI3K subunits p85alpha and p110gamma. In vitro, Tie2CrePten(flox/+) endothelial cells showed enhanced proliferation/migration. Tie2CrePten(flox/flox) mice died before embryonic day 11.5 (E11.5) due to bleeding and cardiac failure caused by impaired recruitment of pericytes and vascular smooth muscle cells to blood vessels, and of cardiomyocytes to the endocardium. These phenotypes depend strongly on p110gamma rather than on p85alpha and were associated with decreased expression of Ang-1, VCAM-1, connexin 40, and ephrinB2 but increased expression of Ang-2, VEGF-A, VEGFR1, and VEGFR2. Pten is thus indispensable for normal cardiovascular morphogenesis and post-natal angiogenesis, including tumor angiogenesis.     

Neuroexcitatory actions of Tamiflu and its carboxylate metabolite

Oseltamivir (Tamiflu) is now being stockpiled by several governments as a first line treatment for an anticipated outbreak of avian influenza caused by H5N1. However, abnormal behaviors and death associated with the use of Tamiflu have developed into a major issue in Japan where Tamiflu is often prescribed for seasonal influenza. Thus, it is critical to determine neuropsychiatric effects of oseltamivir and to establish methods for safe administration. Using juvenile rats and rat hippocampal slices, we investigated whether oseltamivir has adverse effects on the central nervous system. Systemic injection of oseltamivir (50 mg/kg i.p.) produced no change in behavior within 2 h. However, prior injection of oseltamivir significantly altered the duration of loss of lightning reflex following ethanol injection (3.3 g/kg, i.p.). Ethanol injection in the presence of oseltamivir also resulted in enhanced hypothermia. In the CA1 region of hippocampal slices, oseltamivir (100 μM) induced paired-pulse facilitation in population spikes without changes in excitatory postsynaptic potentials. Similarly, 3 μM oseltamivir carboxylate, the active metabolite of oseltamivir, facilitated neuronal firing, though the facilitation did not involve GABAergic disinhibition. Moreover, oseltamivir carboxylate produced further facilitation following administration of 60 mM ethanol. These findings indicate that oseltamivir has effects on the central nervous system, especially when combined with other agents.     

Spontaneous age-associated amyloidosis in senescence-accelerated mouse (SAM)

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R).Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%) Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm.The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.     

Severe Neutropenia in Japanese Patients with X-Linked Agammaglobulinemia

X-linked agammaglobulinemia (XLA) is clinically characterized by recurrent bacterial infections during early infancy. Although it is not a phagocytic disorder, XLA is sometimes associated with neutropenia. We conducted a nation-wide survey to determine the frequency of neutropenia among Japanese XLA patients. Responses were received from 87 (86%) of 101 patients in which BTK mutations were previously identified, and of these, 16 (18%) had neutropenia. All episodes of neutropenia occurred before initiation of intravenous immunoglobulin (IVIG) replacement therapy. Two XLA patients died of multiple organ failure caused by severe neutropenia and Pseudomonas sepsis before initiation of IVIG replacement therapy. These results suggest that, in some cases, severe bacterial infections in XLA patients might be caused not only by antibody deficiencies but also by neutropenia.     

Molecular cloning and sequence analysis of antigen gene tdpA of Treponema denticola.

We isolated and characterized an immunogenic protein of an oral spirochete, Treponema denticola Johnson. A genomic DNA library constructed with bacteriophage lambda EMBL3 as a vector was immunologically screened with a rabbit antiserum against the whole cells. Using Western immunoblot analysis, we found a particular clone encoding an antigen with a molecular weight of 53,000; we designated the antigen as T. denticola protein A (TdpA). Complete sequence determination revealed an open reading frame of 1,419 bp and a signal peptide sequence that was homologous to that of bacterial lipoprotein. Southern hybridization analysis revealed that the tdpA gene is highly conserved in six tested strains of T. denticola species. Furthermore, we found that sera from some periodontitis patients contained antibody against the TdpA protein, although the reactivities of the antibodies varied among individuals.     

Significance of beta-catenin and pRB pathway components in malignant ovarian germ cell tumours: INK4A promoter CpG island methylation is associated with cell proliferation

To clarify the mechanisms underlying cell cycle promotion in malignant germ cell tumours of the ovary (MGCTOs), beta-catenin and components of the pRB pathway, cyclin D1 and p16, were analysed in relation to cell proliferation. Immunohistochemically, p16 protein was not expressed in a number of MGCTOs (9 of 42 tumours: 21.4%) and was associated with p16 gene (INK4A) promoter 5'-CpG islands methylation. Amplification of the cyclin D1 gene (CCND1) was detected in a small number of MGCTOs (5 of 42 tumours: 13.5%). Reduced expression of p16 due to promoter methylation correlated significantly with increased cell proliferation as evidenced by Ki-67 labelling index (p < 0.001) and mitotic index (p < 0.01). In some tumour types, nuclear localization of beta-catenin has been reported to be associated with beta-catenin gene (CTNNB1) mutation, cyclin D1 overexpression, and increased cell proliferation. Nuclear localization of beta-catenin, which was observed in MGCTOs other than dysgerminoma, was not associated with cyclin D1 expression and increased cell proliferation, but appeared to be related to tumour differentiation. Furthermore, CTNNB1 mutations were not detected in any of the MGCTOs examined. Our results suggest that reduced expression of p16 due to INK4A promoter methylation is one of the principal factors that promote cell proliferation in MGCTOs. Thus, p16 may be a novel target for gene therapies to treat MGCTOs.