Discrete breakpoint mapping and shortest region of overlap of chromosome arm 1q gain and 1p loss in human hepatocellular carcinoma detected by semiquantitative microsatellite analysis

Recurrent chromosomal gain at 1q is one of the most common features of human hepatocellular carcinoma (HCC), but how the gain at 1q contributes to hepatocarcinogenesis is still unclear. To identify the target genes, precise determination of the shortest region of overlap (SRO) and of breakpoints is necessary. Similarly, the role of loss at 1p, which is also a major cytogenetic aberration in HCC, needs to be determined. Fifty HCCs were examined with the aid of 59 microsatellite markers distributed throughout both arms of chromosome 1. To detect allelic gain effectively, the cutoff value of the allelic imbalance index was set at 0.70. Alleles showing imbalance were subjected to multiplex PCR, using a retained allele as an internal control, to determine whether the imbalance was the result of chromosomal gain or loss. The SRO of the gains was defined as D1S2878-D1S2619 (1q23.-q25.3, 16.9 Mb), which involved 36 cases (72%). Gains in the number of copies of certain oncogenes within this region seemed to be critical for the pathogenesis of HCC. In contrast, the centromeric breakpoints of these gains varied, but they tended to occur mainly in the pericentromeric region (26 of 50 cases, 52%). Rearrangement of specific genes associated with the gains is unlikely. On the other hand, the SRO of deletion was defined as D1S2893-D1S450 (1p36.32-p36.22, 5.1 Mb). Four known putative tumor-suppressor genes (TP73, RIZ1, NBL1/DAN, and CDKN2C) were outside the SRO, suggesting the presence of other candidate genes with critical roles in hepatocarcinogenesis.     

Removal of dihydroxy-terminated components from monomethoxy-terminated poly(ethylene glycol)

A preparative method to remove dihydroxy-terminated components in a sample of presumably monomethoxy, monohydroxy-terminated poly(ethylene glycol) (PEG) is presented. Purification of the monomethoxy-terminated component allows one to prepare a diblock copolymer of PEG and poly(lactic acid) (PLLA) free of a PLLA-PEG-PLLA triblock copolymer in various biomedical applications of the copolymer. Efficiency of the purification is compared for high osmotic pressure chromatography (HOPC) and preparative size exclusion chromatography (SEC). In HOPC, various types of porous silica particles, surfaces, solvents, polymer concentrations have been screened for the optimal performance. It was found that HOPC is more efficient than SEC, especially HOPC of 30-40 wt% solutions in water by a column packed with acid-washed controlled pore glass is optimal in producing high-purity fractions.     

Alteration of T cell maturation and proliferation in the mouse thymus induced by serum factors from patients with ulcerative colitis.

Recently it has been reported that patients with ulcerative colitis (UC) often have thymus abnormalities, although the precise mechanisms which induce those abnormalities remain unclear. We have examined the effect of serum fractions from patients with UC and other colonic diseases on mouse thymus to clarify the possible existence of factors which have thymus growth activity. These fractions were separated from sera of patients with UC by gel filtration and anion exchange high performance liquid chromatography. In mice given UC serum fractions; (i) remarkable increases in weight and total cell number of the thymus were observed from day 4 to day 9; (ii) a significant increase in the number of peanut agglutinin (PNA)+ thymus cells was demonstrated using flow cytometry on day 9; (iii) on quantitative analysis of surface antigens the percentage of Lyt-2+ thymus cells decreased and that of L3T4+ thymus cells increased remarkably on day 13; the number of bright Thy-1.2+ cells and of dull Lyt-1+ cells increased. In contrast, the serum fractions from patients with other colonic diseases and from normal persons caused little change in mouse thymus throughout the study. The results suggest that factors fractionated from the serum of patients with UC disturb intra-thymic T cell maturation and enhance the proliferation of thymus cells.     

LIGHT AND ELECTRON MICROSCOPIC STUDY OF NONFUNCTIONING PARATHYROID CARCINOMA

A case of nonfunctioning parathyroid carcinoma in a 69-year-old female has been studied by light and electron microscopy. The tumor, located on the left side of the anterior neck, was well encapsulated by connective tissue but showed invasion to the capsule and to the thyroid. The tumor cells exhibited a trabecular arrangement surrounded by capillary networks but focally showed several ductal structures. They were polygonal in shape, had a large nucleus showing frequent mitosis and poor cytoplasm containing glycogen. Some tumor cells had clear and abundant cytoplasm, and resembled water-clear cells of the parathyroid. Immunohistochemically, no thyroglobulin was demonstrated in the tumor tissue. Electronmicroscopically, the tumor cells with high N/C ratio contained poorly developed cell organelles and abundant glycogen particles. They were poor in secretory granules and had no conglomeration of lipid. Desmosomes and tonoflbrils were observed. The ratio of the reported number of nonfunctioning parathyroid carcinoma to that of functioning one in Japan was compared with that in western countries. No difference of the ratio was found between these two, when identical criteria were employed.     

Insular component as a risk factor of thyroid carcinoma

Forty-four thyroid carcinomas with an Insular component (JC) were reviewed from 2457 tumors diagnosed as papillary (PC) or follicular carcinoma (FC). These tumors were classified as FC with an IC (FCIC; 30 cases) and PC with an IC (PCIC; 14 cases). Both tumors were composed of solid cell nests in some areas and had a tendency toward a characteristic nuclear size: FCIC had a small nucleus and PCIC contained a nucleus of an Intermediate type or a large nucleus similar to that of PC, although there were numerous tumors with an exceptional nuclear size. The mean age and tumor diameter were the highest and largest in FCIC, respectively, followed by PCIC. Among the 44 cases, 17 patients died of the disease, two were alive with the disease and 18 were alive without the disease. From 13 clinicopathological factors, the presence of an IC, age, non-encapsulation, tumor size, vascular invasion and necrosis were found to be independent variables for actual prognosis of FC and PC based on univariate analysis followed by multtvariate analysis. The results of the present study indicate that the presence of an IC is an independent aggressive prognostic factor for patients with PC and FC.     

AN IMMUNO-ELECTRON MICROSCOPIC STUDY ON INTRA and EXTRACELLULAR LOCALIZATION OF ALPHA-FETOPROTEIN IN HUMAN HEPATOCELLULAR CARCINOMA

Intra and extracellular localization of alpha-fetoprotein (AFP) has been studied by an indirect peroxidase labeled antibody method using 12 cases of human hepatocellular carcinoma (HCC). With light microscopic observation, positive immuno-staining for AFP was observed in 6 out of 12 cases and demonstrated as granular or diffuse deposits in the cytoplasm of neoplastic hepatocytes. In electron microscopic studies, 8 cases showed the positive immuno-staining for AFP in the neoplastic hepatocytes. Intracellular antigen was well circumscribed within certain cell organelles with the positive immuno-staining for AFP being observed in perinuclear space, cisternae of rough endoplasmic reticulum (RER), Golgi complexes, and secretory vesicles. In addition, the positive immuno-staining for AFP was observed in bile canaliculus-like space in most cases with increased levels of serum AFP and in some cases which showed normal levels of serum AFP. Furthermore, the positive immuno-staining for AFP was also observed in intercellular, Disse's-like and sinusoid-like spaces, and micropinocytotic and lysosome-like vesicles in the endothelial cells in a few cases which showed excessively high value of serum AFP. ACTA PATHOL. JPN. 37:915–928, 1987.     

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18

Rat Kupffer cell (KC)-mediated cytotoxicity against both thesyngeneic hepatoma cell line AH70 and hepatocytes was evaluatedby changes in mitochondrial function, and the possible roleof ICAM-1/CD18 in the interaction between the cells was studied.Rhodamine 123 fluorescence, a marker of the mitochondrial membranepotential, decreased in AH70 cells after co-culture with KC,while that in hepatocytes was unchanged by co-culture. Thisdecrease was blocked by anti-ICAM-1, anti-CD18 and the Inhibitionof nitric oxide synthesis. Cytometric studies demonstrated thatICAM-1 expression on AH70 cells increased after addition ofIFN-, IL-1ß, tumor necrosis factor (TNF)- or KC, whilein hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatmentinhibited the increase in ICAM-1 expression and the decreasein rhodamine 123 fluorescence on AH70 cells after co-culturewith KC. CD18 on KC was increased only after co-culture withAH70. TNF- but not IFN- was detected in the supernatant of co-culturebetween KC and AH70 cells, and this production was partiallyinhibited by anti-ICAM-1 and anti-CD18. The activity of Induciblenitric oxide synthase in Kupffer cells and the levels of nitritesand nitrates in the co-culture supernatant increased over time,and this increase was attenuated either by addition of NO synthesisinhibitors, anti-ICAM-1 or anti-CD18. These results indicatethat the rat KC causes mitochondrial dysfunction in cancer cellsvia the production of NO and cell-to-cell adhesion via ICAM-1/CD18has an Important role in this cytotoxic process.     

Immune Abnormalities Induced by Human Endogenous Retroviral Peptides: With Reference to the Pathogenesis of Systemic Lupus Erythematosus

P15E is a specific sequence among the envelope gene (env)-encoded transmembrane proteins of exogenous and endogenous retroviruses. A synthetic peptide (CKS-17) that shows homology to this p15E region in several species of retrovirus is known to induce immune abnormalities. In this study, we examined the effect of a synthetic peptide derived from a region of human endogenous retrovirus (HERV) clone 4-1 ( 4-1) similar to sequences of CKS-17 on the induction of systemic lupus erythematosus (SLE)-related immune abnormalities. Our results indicated that this peptide could induce T-cell activation and anergy in normal peripheral blood mononuclear cells, and the peptide could also promote the production of interleukins IL-6 and IL-16. These phenomena are representative immune abnormalities observed in SLE patients. Thus, our findings support the possibility that HERV acts as a pathogen in human SLE.     

Immunohistochemical study on tissue transglutaminase and copper-zinc superoxide dismutase in human myocardium: Its relevance to apoptosis detected by the nick end labelling method

The influence of free radicals on apoptosis was studied in the human heart; 45 autopsy cases were examined by the nick end labelling method (NELM) that detects DNA fragmentation. Immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and tissue transglutaminase (tTG) induced frequently during apoptosis were also studied. Positive immunoreaction for tTG was detected in mucinous degeneration of myocardial cells; these same cells were also positive for CuZn-SOD but negative for NELM. Myocardial cells showing basophilic alterations after haematoxylin and eosin staining were also positive for CuZn-SOD but negative for the other markers examined. Positive nuclear reaction by NELM was only observed in myocardial cells showing contraction band necrosis or irregularly shaped nuclei surrounding recent or long-standing infarcted foci. In these the other two markers were negative. Since mucinous degeneration lacks the distinguishing morphological features of apoptosis, immunoreactive tTG in this lesion may not imply that the cells are undergoing apoptosis. tTG can be induced in non-apoptotic conditions and may not be involved in apoptosis induced by infarction. Histological disassociation between CuZn-SOD expression and apoptosis suggests the possibility of a cytoprotective role played by endogenous CuZn-SOD against free radical generation in the human heart.     

Effects of L-DOPA and L-5-HTP on the visual evoked response

Small doses (10 and 20 mg/kg) of L-DOPA inhibited the amplitude of the visual evoked response (VER), while large doses (40 and 80 mg/kg) enhanced it. Though low doses (12.5 and 25 mg/kg) of L-5-HTP caused a slight increase in amplitude of the VER, the simultaneous administration of 12.5 mg/kg of L-5-HTP and 10 mg/kg of L-DOPA produced a marked enhancement. The peak latency was prolonged after the injection of any doses of L-DOPA, L-5-HTP, or both.