Ryoichi Sakamoto

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.

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 No-vaRED 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.

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°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 ofx 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.

Frequency of clinically isolated strains of oral Candida species at Kagoshima University Hospital, Japan, and their susceptibility to antifungal drugs in 2006–2007 and 2012–2013

BackgroundThe isolation frequency and susceptibility to antifungal agents of oral Candida isolates from patients with oral candidiasis (OC) were compared between studies conducted in 2006–2007 and 2012–2013.MethodsA total158 strains was isolated from 112 patients who visited Kagoshima University Hospital for the treatment of OC during the 14-month period from February 2012 and March 2013, and evaluated on the isolation frequency of each Candida strain and the susceptibility against antifungal drugs as compared to those evaluated in 2006–2007.ResultsThere was a higher frequency of xerostomia as a chief complaint and of autoimmune disease in the 2012–2013 study than in the 2006–2007 study. More than 95% of Candida isolates were C. albicans and C. glabrata. In addition, the proportion of the latter increased from 12.3% in the 2006–2007 study to 23.4% in the 2012–2013 study, while the proportion of the former decreased from 86.2% to 72.8%, respectively. C. albicans was isolated in almost all patients, while C. glabrata was only isolated concomitantly with C. albicans. Minimal inhibitory concentrations (MICs) were not significantly different between groups with a few exceptions. Candida isolates, of which MICs surpassed break points, apparently increased for miconazole and itraconazole against C. glabrata in the 2012–2013 study, but this was not statistically significant. As a result, more cases of autoimmune disease, a greater number of C. glabrata isolates, and higher resistance to azoles were seen in the 2012–2013 study than in the 2006–2007 study.ConclusionThese data indicate that with recent increases in C. glabrata infection, a causative fungus of OC, and in C. glabrata resistance to azoles, caution is needed in the selection of antifungal drugs for the treatment of OC.

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.

In vitro antifungal activity against oral candida species using a denture base coated with silver nanoparticles

Although oral Candida easily adheres to denture base materials, many denture detergents are effective only against bacteria but not against Candida. Silver nanoparticles (AgNPs), which are known to have potent antibacterial and antifungal activity, have been used in the prevention of oral candidiasis (OC). We evaluated the adherence of Candida albicans and Candida glabrata on a heat-cured Acron resin piece supported by AgNPs by low-vacuum scanning electron microscopy (SEM) and measuring colony-forming units. C. albicans and C. glabrata increasingly adhered to the resin surface of the control piece over time, but the adhesion AgNP of both Candida species to the AgNP-coated surface was significantly inhibited (P < 0.001). Low-vacuum SEM revealed that C. albicans and C. glabrata on the resin surface of control pieces appeared as oval colonies, with a major axis of 3-4 μm and a smooth cell wall, but those on the AgNP-coated resin surface were less abundant than the control and showed swollen yeast features, with a major axis of more than 5 μm and a corrugated cell wall. Our results suggest a way to prevent denture-associated OC by using denture base materials processed by AgNPs.