Therapeutic effects of vitamin A on experimental cholestatic rats with hepatic fibrosis.

PURPOSE: The aim of this study is to investigate the role of hepatic stellate cells (HSCs) and the effect of vitamin A administration on liver damage induced by bile duct ligation (BDL) and administration of CCl(4). METHODS: Two types of animal model were used; one was BDL as a model of biliary atresia, the other was CCl(4)-induced hepatic fibrosis. Pathological changes of the liver with or without administration of vitamin A were compared by light and electron microscopy with focusing on HSCs in each experimental group. Immunohistochemical examination was performed with anti-keratinocyte growth factor (KGF), anti-alpha-smooth muscle actin (α-SMA), and anti-glial fibrillary acidic protein (GFAP) antibodies, as markers of fibrosis. RESULTS: On light microscopic findings, periportal inflammation with bile ductular proliferation was obvious in BDL group and pericentral necrosis with fatty degeneration was observed in CCl(4) group, both of which were ameliorated by subcutaneous injection of vitamin A. Electron microscopy showed lipid droplets were almost depleted in the HSCs treated with BDL or CCl(4), which improved with vitamin A administration. Immunohistochemistry demonstrated that enhanced expression of all three fibrotic markers in the BDL group was diminished by vitamin A administration. CONCLUSIONS: Although most of our data are qualitative observation, vitamin A may ameliorate hepatic fibrosis in the BDL model by restoring vitamin A in the HSCs.

The 3G5 antigen is expressed in dermal mast cells but not pericytes.

BACKGROUND: It has been shown that the 3G5 antigen recognized by monoclonal antibody 3G5 (mAb 3G5) is a useful marker of pericytes in normal human skin. However, most 3G5 antigen-expressing cells in capillary vessels were stained negatively for alpha-smooth muscle actin (alpha-SMA), a prominent pericyte marker. This study was designed to determine whether the expression of the 3G5 antigen is restricted to specific stages of pericyte development, or if it is expressed in other cells rather than pericytes in capillary vessels. METHODS: 3G5 antigen-expressing cells were detected in normal human skin, granulating tissues from decubitus ulcers and inflammatory psoriatic skin with extensive angiogenesis using double immunofluorescent staining with mAb 3G5 and monoclonal antibodies (mAbs) to various pericyte markers, tryptase and chymase. Furthermore, using immunoelectron microscopy, 3G5 antigen-expressing cells were observed in the granulating tissues. RESULTS: The immunoelectron microscopic findings and double immunofluorescent staining (using mAb 3G5 and either anti-tryptase or anti-chymase mAbs) showed that 3G5 antigen-expressing cells were mast cells in normal skin, granulating tissues and psoriatic skin. CONCLUSIONS: The results indicated that 3G5 antigen is a marker of mast cells, but not of pericytes in normal and diseased skin.

Immunoreactivity of phage library-derived human single-chain antibodies to amyloid beta conformers in vitro.

The pathogenesis of Alzheimer's disease involves conformational changes of A beta. A series of antibodies recognizing a distinct conformation of A beta (snapshot antibody) is useful for both understanding the mechanism of molecular conversion and identifying diagnostic and therapeutic reagents. As A beta with various conformations can be prepared in vitro under varying physicochemical conditions, snapshot antibodies can be isolated by directly binding to target molecules with antibody-displaying phages. We tested the feasibility of this idea. We show a feature of several A beta-reactive antibodies isolated from our human single-chain Fv antibody-phage library and particularly report the characteristics of an scFv clone, B6, selected from the fibrillar A beta 1-42-coated biopanning. B6 bound to fibrillar A beta 1-42 as well as globulomer A beta 1-42 but not to soluble A beta 1-42 or A beta 1-40. B6 inhibited A beta 1-42 fibril formation with 600 nM IC50 in spite of being the monovalent scFv form. Epitope analysis suggested that the binding site might be located at the beta2 sheet of the C-terminus of A beta 1-42. Although it is believed that N-terminus-recognizing antibodies tend to show the capability to inhibit A beta 1-42 fibrillation, B6 is the first human inhibitory antibody recognizing the C-terminus of A beta 1-42.

Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes.

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.

The assessment of cell proliferation during 9,10-dimethyl-1,2-benzanthracene-induced hamster tongue carcinogenesis by means of histone H3 mRNA in situ hybridization.

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 ( P << 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.

Double autoimmunostaining with glycine treatment.

Double autoimmunostaining by a sequential twice-repeated enzyme-labeled polymer method was examined on archival paraffin sections of formalin-fixed human tissue using an autoimmunostaining apparatus to determine optimal conditions for glycine treatment, to select the best combination of dyes for the horseradish peroxidase-hydrogen peroxide reaction, and to investigate mounting methods for preparing permanent specimens. The optimal glycine treatment determined by changing the incubation time in 0.1 M glycine hydrochloride buffer, pH 2.2, was glycine buffer washing three times for 1 min each, with suppression of nonspecific binding of the primary antibody by protein blocking. Combinations of DAB and AEC, SG and AEC with Ultramount, and DAB and VIP or NovaRED and SG with the VectaMount were found usable for the double autoimmunostaining, based on color analysis of the dyes. Pairs of primary antibodies, CD68 and anti-fascin antibodies CD3 and CD79a, and anti-Ki-67 antigen and anti-p53 antibodies were applicable in double autoimmunostaining with appropriate antigen retrieval for each pair of primary antibodies. Consequently, good sequential double autoimmunostaining should include masking the nonspecific binding of primary antibodies, optimal glycine treatment, and selection of adequate dyes and mounting methods.

Cytochemistry of gastric parietal cells with high-pressure freezing followed by freeze-substitution.

Gastric parietal cells were examined for changes in their ultrastructure and distribution of the proton pump during feeding and fasting states in rats. The fundic glands from rats fed ad libitum or fasted with free access to water were cryofixed using high-pressure freezing followed by freeze-substitution in acetone containing osmium or acrolein and then embedded in Epon 812 or Lowicryl K4M resin, respectively. Excellent ultrastructural preservation was achieved. During the feeding state, intracellular canaliculi and numerous microvilli were well developed, while tubulovesicles were poorly developed. In contrast, during the fasting state, the microvilli in the narrowed space of the intracellular canaliculi were tightly packed and the tubulovesicles were enlarged. Ultrathin sections were immunostained with antibodies against the alpha- and beta-subunits of the proton pump, H+ x K(+)-ATPase, using the immunogold method. The labelling was strong and clearly localized in comparison with that obtained using the conventional chemical-fixation method. Each subunit was localized on the membrane of the microvilli, intracellular canaliculi and tubulovesicles. The distribution of subunit proteins varied between the two states. During ad libitum feeding, the immunolabelling was localized strongly on the membranes of the microvilli and intracellular canaliculi. In contrast, the labelling was strong on the tubulovesicle membrane in the fasting state. The results obtained with each anti-subunit antibody by H+ x K(+)-ATPase immunostaining revealed differences in distribution and labelling density between the feeding and fasting states.

Cytochemical investigation of gastric gland component cells with high-pressure freezing followed by freeze-substitution and hydrophilic resin embedding.

Gastric gland component cells were electron-microscopically and immunoelectronmicroscopically examined with high-pressure freezing followed by freeze substitution and a low-temperature embedding resin method and compared to that of the conventional chemical-fixation method. The rat gastric gland was high-pressure frozen, freeze-substituted with acetone-containing osmium or acrolein, and embedded in Epon 812 or Lowicryl K4M, respectively. Using the high-pressure freezing method, the vitreous freezing range reached the depth of 150 microns from the surface. The ultrathin sections from both procedures embedding in Epon 812 and Lowicryl K4M were doubly stained with uranyl acetate and lead acetate, and histochemically or immunohistochemically stained, respectively. In comparison to the conventional chemical fixation method, excellent results were obtained with respect to ultrastructural preservation. The stainings performed in this experiment included periodic acid-thiocarbohydrazide-silver proteinate staining, cationic colloidal cold at pH 2.5 staining, Helix pomatia lectin-staining, anti-alpha or -beta subunit antibodies of H+K(+)-ATPase immunostaining and pepsinogen immunostaining. The staining intensity of those was stronger than that of the conventional immersion-chemical fixation method. In addition to these results, the labels also showed good specific localization. In this paper, we provide a description of the high-pressure freezing followed by freeze substitution and low-temperature embedding resin method compared to the conventional chemical-fixation method. Our results suggest that this method is a suitable tool for ultrastructural and histochemical/immunohistochemical studies at high resolution.

Immunoelectron microscopic analysis of lysosomal deposits in alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum.

Alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency with angiokeratoma corporis diffusum (AKCD) is one of the lysosomal storage diseases. GalNAc(alpha))1-O-Ser/Thr (Tn) is theoretically deposited in lysosomes, but substances with attached galactose and neuraminic (sialic) acid (T and sialosyl Tn, respectively) are excreted in patients' urine. In this study, in two Japanese cases we analyzed the material accumulated in lysosomes using immunoelectron microscopy with mouse antibodies to Tn, sialosyl Tn and T (Thomsen-Friedenreich) antigens in order to find out what substance(s) is really deposited in lysosomes. We found that only GalNAc(alpha)1-O-Ser/Thr (Tn) was actually accumulated in vacuolated lysosomes of vascular endothelial cells, eccrine sweat gland cells, fibroblasts and pericytes. Galactosylation and sialylation of Tn appears to occur in cells other than those in the skin. The results suggest that this disease is caused by a single enzyme deficiency.

Improvement of supersensitive immunohistochemistry with an autostainer: a simplified catalysed signal amplification system.

The ImmunoMax/catalysed signal amplification (CSA) system is a supersensitive method of paraffin immunohistochemistry. It incorporates antigen retrieval, the streptavidin-biotin complex (sABC) method, and the catalysing reporter deposition/catalysing biotinylated tyramide reaction. Strong, non-specific cytoplasmic reaction in the ImmunoMax/CSA is due to endogenous biotin unmasked in the antigen retrieval step. We examined procedures to diminish this non-specific immunoreaction and improved the ImmunoMax/CSA. Antigen retrieval in a hot water bath yielded a smaller endogenous biotin immunoreaction than antigen unmasking in an autoclave. Post-antigen retrieval fixation in buffered 10% formalin solution suppressed the biotin immunoreaction but masked the target antigen, Ki67. Post-reaction washing with 0.1% Tween 20 in Tris-HCl buffer at 35 degrees C did not diminish the endogenous biotin immunoreaction. Animal serum also did not suppress the non-specific immunoreactivity of biotin and antibodies. Because endogenous biotin is detected by duplicated biotin-streptavidin reactions in the ImmunoMax/CSA, we replaced the sABC step with a labelled polymer secondary antibody (the EnVision system)--a simplified CSA system--because the sensitivity of the EnVision system was the same as that of the sABC method. The non-specific immunoreaction induced by the EnVision system was masked competitively by blocking protein. By using an antibody against Ki67 antigen that can react only with the nucleus, we were able to evaluate the non-specific cytoplasmic immunoreaction induced by the detection system. We believe that the simplified CSA system will open up the field of supersensitive paraffin immunohistochemistry.

Histochemical, immunohistochemical, and ultrastructural studies of gingival fibromatosis: a case report.

Gingival fibromatosis is a rare disease characterized by enlargement of the gingiva. The purpose of this study was to analyze a case of idiopathic gingival fibromatosis, using histochemical and immunohistochemical staining and transmission electron microscopy. The patient was a 39-year-old Japanese man, in whom the gingiva was enlarged throughout the entire mandible and maxilla. Specimens of gingival fibromatosis exhibited epithelial hyperplasia and increased amounts of collagen fiber bundles in the connective tissue light-microscopically. Well-developed collagen bundles were strongly stained with Azan and Masson trichrome staining. Immunohistochemically, the gingival connective tissue was specifically stained by type I collagen and vimentin antibodies. Ultrastructurally, the lesion consisted of fibroblasts and mature collagen fibers running in all directions. No myofibroblasts were detected histochemically, immunohistochemically, or ultrastructurally. These findings suggested that this disease may be the result of an increase in collagen synthesis by the fibroblasts and/or that it may be associated with one of the findings of histologic heterogeneity.