Expression of S-100 protein in intercalated duct cells of bovine pancreas

Although Mutoh et al. have found intercalated ducts in the pancreatic islets of avians, including the chicken, moorhen (Gallinula chloorpus), and Japanese quail (Coturnix coturnix japonica), and even demonstrated the functions of intercalated duct cells in pancreatic islets, to our knowledge, there have been only a few reports on the relationship between intercalated ducts and islets in mammalia. Accordingly, in this study, we investigated whether intercalated ducts are morphologically related to the islets of cattle, by using S-100 protein as a ductal cell marker for immunocytochemistry and examining the ultrastructure of intercalated ducts and islets electron-microscopically. The results revealed intercalated ducts that reacted positively for S-100 protein within and near islets, with approximately 12% of the islets having intercalated ducts in the vicinity and approximately 1.5% containing intercalated ducts within them. Ultrastructurally, intercalated ducts were also seem to be closely related to the islets. In conclusion, the results of the present study indicate that a close relationship exists between intercalated ducts and islets.     

Mechanisms Involved in the Binding of Thymocytes to Rat Thymic Dendritic Cells

The effects of monoclonal antibodies (mAbs) to cell-surface molecules, divalent cations, and various cell-signaling and metabolic inhibitors on the binding of thymocytes to rat thymic dendritic cells (TDC) were studied using a rosette assay. It was found that TDC/thymocyte adhesion was stronger and faster at 37°C than at 4°C. Flow cytometric analysis demonstrated that bound thymocytes were predominantly CD4⁺CD8⁺ and CD4⁺CD8^-, but in comparison to the phenotype of whole thymocytes, they were enriched in the mature TCRαβ^hi subset. The binding of thymocytes to TDC at 37°C was almost completely dependent on Ca²⁺ and Mg²⁺ and partly on an intact cytoskeleton and calmodulin-dependent protein kinase. The adhesion was independent of new protein synthesis and the activities of protein kinases A and C, tyrosine kinases, as well as phosphotyrosine protein phosphatases. The TDC/thymocyte adhesion at 37°C was partly blocked by anti-LFA-1 (WT.1), anti-CD18 (WT.3), and anti-ICAM-1 (1A29) mAb. MAbs to class II MHC (OX-3 and OX-6), CD4 (W3/25), CD8 (OX-8), and αβTCR (R73) stimulated the adhesion via an LFA-1-dependent pathway, whereas an anti-CD45 mAb (G3C5) stimulated the rosette formation independently of LFA-1. MAbs to CD2 (OX-34), CD11b (ED7), CD11b/c (OX-42), and class I MHC (OX-18) were without significant effects on the adhesion process.     

Identification of Goodpasture antigens in human alveolar basement membrane.

Goodpasture (GP) antigens, protein components reactive with human autoantibodies against glomerular basement membrane (GBM), were identified in human alveolar basement membrane (ABM) using an enzyme-linked immunoassay (ELISA), Western blotting and immunoprecipitation. All six anti-GBM antisera studied, three obtained from patients with glomerulonephritis and pulmonary haemorrhages (i.e. GP syndrome), and three from patients with glomerulonephritis alone, distinctively reacted with collagenase-digested (CD) ABM. Very cationic 22-28 kD and 40-48 kD components were detected by blot analysis combined with two-dimensional gel electrophoresis. These proteins showed some similarities to GP antigens in human GBM with respect to the monomer-dimer composition and charge distribution. Inhibition ELISA revealed that the binding of anti-GBM antisera to CDGBM decreased when they were pre-incubated with CDABM, suggesting that the anti-GBM antisera recognized the same epitope(s) on the GBM and ABM. Heterogeneity of the GP antigens in human ABM was demonstrated by blotting; monomeric antigens were absent or at low levels in the CDABM of three out of 10 normal individuals. In immunoprecipitation, anti-GBM antisera from patients with and without pulmonary haemorrhage showed different reactivities with CDABM. The former antisera precipitated both monomeric and dimeric components, but the latter did not. The observations of variation in monomer-dimer composition of ABM, and the different binding of anti-GBM antisera to it may explain why only some patients with anti-GBM nephritis have lung involvement.     

Ricinus communis agglutinin I blocks the binding of human anti-renal basement membrane antibodies to the kidney.

Whether or not glycosyl moieties of glycoproteins present in human renal basement membranes are related to the sites where anti-basement membrane antibodies bind was examined by blocking experiments using several kinds of lectin. Ricinus communis agglutinin I (RCA I), specific for galactose, blocked the binding of human anti-glomerular basement membrane (GBM) and anti-tubular basement membrane (TBM) antibodies to renal basement membranes. This lectin also diminished the binding of rabbit anti-laminin antibody, but did not inhibit the binding of mouse anti-fibronectin or rabbit anti-human TBM antibodies. These findings suggest that the binding sites of human anti-GBM and anti-TBM antibodies and heteroantibodies to laminin are closely related to the galactose moieties in glycoproteins of human renal basement membranes. Whether the galactose-containing branches are associated with the nephritogenicity of human anti-GBM and anti-TBM antibodies or simply exist adjacently to the antibody binding sites remains to be discerned.     

An invasion-independent pathway of blood-borne metastasis: a new murine mammary tumor model

It is generally believed that active invasion by cancer cells is essential to the metastatic process. In this report, we describe a murine mammary tumor (MCH66) model of metastasis that does not require invasion into the vascular wall of both the primary tumor and the target organ, in this case, the lung. The process involves intravasation of tumor nests surrounded by sinusoidal blood vessels, followed by intravascular tumor growth in the lung, without penetration of the vascular wall during the process. Comparative studies using a nonmetastatic MCH66 clone (MCH66C8) and another highly invasive metastatic cell line (MCH416) suggested that high angiogenic activity and sinusoidal remodeling of tumor blood vessels were prerequisites for MCH66 metastasis. Differential cDNA analysis identified several genes that were overexpressed by MCH66, including genes for the angiogenesis factor pleiotrophin, and extracellular matrix-associated molecules that may modulate the microenvironment toward neovascularization. Our analyses suggest that tumor angiogenesis plays a role in the induction of invasion-independent metastasis. This model should prove useful in screening and development of new therapeutic agents for cancer metastasis.     

Positioning of actin filaments and tension generation in skinned muscle fibres released after stretch beyond overlap of the actin and myosin filaments

Summary Skinned fibres from frog semitendinosus muscle were stretched in relaxing solution from a sarcomere length of 2.5 m to greater sarcomere lengths, and then shortened back to the original length. Fibres could be stretched up to sarcomere lengths of 3.3 m, and reshortened fully. If the original stretch was to a sarcomere length greater than 3.3 m, the extent of recovery was dependent on the magnitude of the stretch and the number of times the stretch/shorten cycle was repeated. When the original stretch was to sarcomere lengths beyond overlap of the thick and thin filaments, the thin filaments did not re-enter the thick filament array but buckled at the A-I junction. If these fibres were subsequently activated and contracted, the thin filaments re-entered the thick filament array, taking up approximately their former positions, and allowing reduced development of isometric tension.Finally, the present observations support the view that calcium-induced interactions of actin and myosin filaments in the presence of ATP play an important role in the organization of myofibrillar structure during differentiation (compare Hayashiet al. 1977; Shimada & Obinata, 1977).     

Selection of higher molecular weight genomic DNA for molecular diagnosis from formalin-fixed material.

The patterns of DNA degradation in frozen, methanol-fixed, and formalin-fixed tissues were investigated by high-performance liquid chromatography (HPLC). The chromatograms all yielded one major peak with or without several extra minor peaks representing molecular weights of preserved genomic DNA. The most characteristic differences were in the retention times of the major peaks, with the earliest major peak occurring in the formalin-fixed tissues, and followed by the methanol-fixed, and frozen tissue samples, in that order. This means that the molecular weight of the DNA from formalin-fixed tissue is much shortened than that recovered from methanol-fixed tissue and frozen tissue. The results also indicated that a small amount of higher molecular weight DNA is still preserved in formalin-fixed tissues. To improve the amplification efficiency of polymerase chain reaction (PCR) analysis of formalin-fixed material, we isolated the higher molecular weight DNA from formalin-fixed, paraffin-embedded tissue from four different organs and compared the amplification efficiencies with those of the crude DNA extract. We used eight sets of oligonucleotide primers producing 262 to 989 base pair (bp) fragments of beta-globin. The results showed that the PCR amplification analyses were more efficient with the isolated higher molecular weight DNA than with the crude DNA extract. Our study demonstrated that not all the DNA in formalin-fixed, paraffin-embedded tissue samples is totally degraded but that a small amount of higher molecular weight DNA persists. The feasibility of molecular diagnosis using formalin-fixed material can be improved by isolating the preserved higher molecular weight DNA by HPLC.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Clonal expansion of multiphenotypic Epstein-Barr virus-infected lymphocytes in chronic active Epstein-Barr virus infection

Chronic active Epstein-Barr virus (EBV) infection has been recognized as clonal non-neoplastic lymphoproliferative diseases. However, some reports of cases with a multiphenotypic expansion of EBV-infected lymphocytes give rise to questions of how EBV infects multiphenotypic lymphocytes and whether chronic active EBV infection is a truly monoclonal lymphoproliferative disease. We report two patients with chronic active EBV infection who showed expansion of multiphenotypic EBV-infected lymphocytes. EBV DNA was detected in CD4+ and CD8+ T cells and in B cells from pleural fluid of one patient and in T and B cells from a cervical lymph node of the other patient by polymerase chain reaction (PCR). Although real-time PCR showed that there were equally high loads of EBV genomes in CD4+ and CD8+ T cells from the pleural fluid, Southern blot hybridization with terminal repeats of the EBV genome showed a single band of the same molecular weight in three tissue samples from the patient. The results indicated biphenotypic expansions of CD4+ and CD8+ T cells infected with the same clone of EBV. Furthermore, bisulfite PCR analysis showed hypermethylated status in the Cp region in the two patients regardless of their cell populations. There has been a discrepancy between clonality and expansion of multiphenotypic EBV-infected lymphocytes. We speculate that lymphoid progenitor cells that have not differentiated into T and B cell progenitors are infected with EBV, resulting in clonal expansion of EBV-infected multiphenotypic cells.