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Hyalinized stroma (HS) is a dense, eosinophilic, and amorphous extracellular material in the stroma. HS is observed in several tumors; however, it has not been comprehensively studied in pancreatic intraductal papillary mucinous neoplasm (IPMN) or intraductal oncocytic papillary neoplasm (IOPN). Here, we aimed to evaluate the immunohistochemical and microscopic characteristics of HS in IPMN and IOPN. The prevalence of HS was determined in 168 cases of IPMN, including intestinal type (IPMN-I), gastric type (IPMN-G), and pancreatobiliary type (IPMN-PB), as well as in 11 cases of IOPN. Immunohistochemical staining for laminin and collagen (types I, II, III, IV, and V), as well as Congo red staining were performed in IPMN and IOPN cases containing HS. The prevalence of HS among the IPMN and IOPN specimens was 1.2% (2/168 cases) and 45.5% (5/11 cases), respectively. The prevalence rates of HS in each IPMN subtype were as follows: 2.2% (2/91 cases) in IPMN-G, and 0% in IPMN-PB and IPMN-I. All seven HS cases were positive for collagen I, III, IV, and V but were negative for Congo red staining. Most cases showed negative, focal, or weak expression of laminin and type II collagen. These findings indicate that HS is associated with IOPN and is primarily composed of collagen fibers.

A perivascular epithelioid tumor (PEComa) is a rare tumor probably arising from the perivascular epithelioid cells. Only three cases of pancreatic PEComa have been reported in the English-language literature. The present report describes an extremely rare case of pancreatic PEComa. A 47-year-old Japanese woman complained of lower abdominal pain and a well-demarcated solid tumor was found in the pancreatic head. There was no history of tuberous sclerosis complexes. Pylorus-preserving pancreaticoduodenectomy was thus performed. There was a well-demarcated, solid tumor measuring 17 mm in the pancreatic head. The tumor was composed of a diffuse proliferation of epithelioid tumor cells with many blood vessels but no adipose tissue. The tumor cells expressed HMB45 and alpha-smooth muscle actin. Ultrastructurally, the tumor cells possessed many membrane-bound granules that were positive for HMB45 on immunoelectron microscopy. The results of immunoelectron microscopy show that some PEComas possess not only typical melanosomes or premelanosomes but also aberrant melanosomes.

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer.

Natural killer (NK) cells are specialized lymphocytes which arecharacterized as non-T and non-B cells, as they lack classic T and B cellsurface markers. Recently, NK like immunoreactivity has been identified inendocrine and neuronal tissues as well as in the tumors derived from theneuroectoderm and neuroendocrine system. We examined the expression of NK-1like immunoreactivity in 6 normal pituitary glands and in 55 cases ofneoplastic pituitaries (16 growth hormone (GH) producing adenomas, 14prolactin (PRL) producing adenomas, 4 thyrotropin (TSH) producing adenomas,5 adrenocortocitropin (ACTH) producing adenomas and 16 non-functioningadenomas) immunohistochemically. The expression of the S-100 protein, whichis a marker for folliclo-stellate (FS) cells, which have been reported tosecrete cytokines as immuno-endocrine modulators, were also examined. Innormal pituitary glands, NK-1 was detected in all 6 tissues in the cytoplasmof about 5–10% of the anterior pituitary cells. By serialsectioning and double immunostaining, NK-1 immunopositivity was frequentlyfound to be localized in ACTH cells. The colocalization with other anteriorpituitary hormones such as GH, PRL, the beta-subunit of luteinizing hormone(LHβ), follicle stimulating hormone (FSHβ), TSHβ andα-subunit of glycoprotein (αSU) was not observed. The S-100immunopositive FS cells, which were scattered among hormone producing cells,were closely associated with NK-1 immunoreactive cells in the normalpituitaries. Among the 55 cases of pituitary adenomas, NK-1 was present inall the types of pituitary tumors, and a total of 33 (60.0%)contained NK-1 positive tumor cells. The frequency of NK-1 immunoreactivityin the individual adenoma types was; 14 of 16 GH producing adenomas(87.5%), 7 of 14 PRL producing adenomas (50%), 3 of 4 TSHproducing adenomas (75%), 3 of 5 ACTH producing adenomas(60%), and 5 of 16 nonfuctioning adenomas (31.3%). By doubleimmunostaining, NK-1 was found to be frequently colocalized with ACTH inACTH producing adenomas, and was colocalized with PRL in PRL producingadenomas, or with GH, PRL or the α-subunit in GH producing adenomacells. NK-1 immunoreactive cells were observed in close association withS-100 immunopositive FS cells in the adenomas. Our results may indicate thatNK-1 positive cells may have functions as a paracrine modulators of theirneighboring cells, which includes S-100 positive FS cells.

A total of 145 paraffin-embedded biopsy samples of gastric xanthoma were analyzed for the localization of Helicobacter pylori (HP) antigens. By the indirect immunoperoxidase method using a polyclonal antibody, HP infection was identified on the surface of foveolar cells in 69 (48%) samples. In 38 (55%) of the 69 lesions, the HP antigens were demonstrated in the cytoplasm of xanthoma cells clustered in the actively inflamed lamina propria mucosae. Among the remaining 76 xanthoma lesions negative for HP infection on the epithelial surface, only eight (11%) showed the existence of HP antigens in the foamy histiocytes, and 39 (51%) revealed mild inflammatory change. Monoclonal antibody study using 75 specimens also gave a comparable result. Pre-embedding immuno-electron microscopy using paraffin sections revealed positively labeled rod-shaped bacteria both on the epithelial surface and in the phagosome of the xanthoma cells. These findings strongly suggest that some of the xanthoma lesions are provoked by lamina proprial invasion of surface-infected HP.

Chlamydia trachomatis (CT) and Escherichia coli (EC) antigens were sought in routinely prepared paraffin-embedded sections from 31 cases of acute and chronic epididymitis by indirect immunoperoxidase staining. Chlamydia trachomatis antigens were detected in epithelial cells as cytoplasmic inclusions in samples from six patients (mean age, 43 years) with severe epididymitis, characterized by minimally destructive, periductal, and intraepithelial inflammation with active epithelial proliferation. Squamous metaplasia and formation of lymphoepithelial complexes occasionally were noted. Escherichia coli antigens, common to other pyogenic bacteria, were observed in the cytoplasm of foamy histiocytes in samples from seven patients (mean age, 60 years), characterized by highly destructive epididymitis forming large abscesses and xanthogranulomas. Specimens from 18 patients were negative for either antigen. Pre-embedding immunoelectron microscopy on paraffin-embedded sections demonstrated positive reactions on the cell wall of the chlamydial bodies and rod-shaped bacteria. Escherichia coli-positive cases were accompanied by scrotal pain, pyuria, positive bacterial culture, leukocytosis, accelerated erythrocyte sedimentation rate, and a positive C-reactive protein test. Chlamydia trachomatis-positive cases were clinically indolent and manifested by an epididymal tumor. Chlamydial epididymitis can be distinguished from bacterial epididymitis not only clinically and immunohistochemically but also histologically.

Eosinophilic hyaline inclusions were consistently seen in the perinuclear cytoplasm of suprabasal keratinocytes in lichen amyloidosus. The inclusions, negative with amyloid staining, were immunoreactive for ubiquitin and cytokeratin, and ultrastructurally showed aggregations of fine filaments of two sizes (central thin and peripheral thick). The thin filaments were the main component in the upper epidermal layer. Four monoclonal antibodies (AE1, AE3, KL1 and CAM5.2) and one antiserum (WSS) were used for characterizing cytokeratin expression. The AE1 antibody normally stained the basal cells, but in lichen amyloidosus basal staining mostly disappeared. Instead, groups of suprabasal keratinocytes were labeled, with AE1-reactive inclusions distributed therein. In contrast, the KL1 antibody, showing suprabasal staining, failed to react with the inclusions. The inclusions were weakly reactive with the AE3 and WSS antibodies, which stained all keratinocytes. The CAM5.2 antibody was unreactive. The subepidermal amyloid deposits were negative with all the antibodies. The inclusions were ubiquitinated especially in the granular layer. Immunoelectron microscopy disclosed that ubiquitin was more densely localized in the thin filaments than in the thick ones. This indicated that cytokeratin expression and metabolism are altered in the affected epidermis, and that ubiquitin functions in the process of degradation of abnormal cytokeratin filaments.

Extensive immunohistochemical analyses of the hyperplastic human palatine tonsil disclosed variegated B cell phenotypes on the lymphoid cells among the crypt epithelium. The reticular epithelial network was evident by cytokeratin immunostaining. The reticular epithelium near the crypt lumen was positive for lysozyme. Secretory component was negative, while HLA-DR was frequently expressed. Intramucosal small lymphocytes, densely distributed in the luminal side, consisted mainly of B cells expressing CD19, CD20, CD21, CD22, CD45R, CD74, DBB42, HLA-DR, HLA-DQ, bcl-2 protein and surface IgM. Some B cells revealed mantle zone phenotypes (surface IgD+, CD5+, CD24+, DBA44+, CD10-, DNA7-). Cells of germinocyte phenotype (CD10+, DNA7+) were sparsely seen. A good number of intramucosal lymphoid cells were further labeled for CD11b, a phenotype of so-called B-1 cells. Plasma cells were clustered within the basal half. IgG was their major immunoglobulin class, followed by IgA, IgM and IgD classes. A smaller number of T cells (CD2+, CD3+, CD5+, CD45RO+, TCR alpha beta+) were identified among the epithelium. CD4+ cells predominated over CD8+ cells. TCR gamma delta+ cells were rare. Macrophages (CD68+), dendritic histiocytes (S-100 protein+, CD1+), and natural killer cells (CD16+ or CD57+) were also dispersed. Another unique feature of this lymphoepithelial complex was the existence of HLA-DR- intramucosal intramucosal microvasculature, where lymphocyte recirculation was suggested. Proliferating cell nuclear antigen was detected commonly in the epithelial cells but rarely in the lymphoid cells. Possible lymphoepithelial interactions and morphologic similarities to the thymic medulla are discussed.

パルボウイルスB19 (以下B19) は伝染性紅斑, いわゆるリンゴ病の病原体であり, 成人でも風邪様症状や関節炎を起こし, 特に妊婦が妊娠初期から中期にかけて感染すると胎児水腫などを引き起こし流早死産になることが明らかになってきた. 現在, 産科的に風疹やサイトメガロウイルスにつぐ重要なウイルスとして注目されている.〈症例〉胎齢25週2日の男児死産児. 母親は28歳, 保母. 妊娠10週頃に伝染性紅斑に患, 妊娠22週の超音波検査にて胎児水腫を指摘され当院産科を受診し, B19感染による非免疫性胎児水腫が疑われた. 25週2日子宮内胎児死亡のため人工中絶となった.〈剖検〉全身浮腫と著明な胸腹水の貯留を認めたが外表奇形, 内臓奇形はみられなかった. 剖検時の腹水細胞診では細胞の変性強く核内封入体を有した感染細胞は明らかでなかったが, 酵素抗体間接法 (B19に対するモノクロナール抗体) により感染細胞の細胞質に特異抗原を認めた. また諸臓器 (肝, 脾, 肺, 腎, 骨髄, 胎盤など) に核内封入体を有する感染赤芽球が多数認められ, 酵素抗体法にて陽性が認められた. また胎児胸腹水のPCR法分析および組織電顕にてB19を確認した.〈考察〉本邦の妊娠可能女性の50%から80%はB19抗体陰性といわれており, 感染時に定形的な紅斑を示さないことが多いとされる. またB19IgM陽性妊婦の約10%に胎児水腫が発症したとの報告があり, その致死率も高い. 早期診断のためにも簡便でかつ臨床応用可能な細胞診は有用であると思われた.

In order to elucidate the relationship between secretory pathway and processing for precursor molecule of peptide hormones, we performed immunoelectron microscopic studies to localize POMC-derived peptides in mouse cultured L cells (fibroblasts without secretory granules) and in mouse AtT20 cells (ACTH secreting pituitary tumor cells with secretory granules) which had been transformed with human POMC gene. From the electron microscopic localization patterns, L42 cells were considered to serve as a model of constitutive pathway without processing of POMC, and A53 cells were considered to serve as a model of transgranular (regulated) pathway with processing of POMC. Immunoblotting supported these interpretations.

A total of 17 (10 adrenal and 7 extraadrenal) pheochromocytomas were examined immunohistochemically for the localization of catecholamine-synthesizing enzymes-tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT)-as well as the marker peptides for pheochrornocytomas (i.e., met-enkephalin-arggly-leu [MEAGL]). Normal adult, fetal, and newborn human adrenal medullas were also examined. Six adrenal pheochromocytomas showed immunohistochernically positive staining for PNMT; 5 of these cases demonstrated elevated serum adrenalin levels, This indicated morphofunctional correlation. PNMT-positive cells were mostly positive for TH but did not show co-localization of MEAGL except in 1 case, Absence of co-localization was considered to reflect the physiological condition of the specimen, based on the similar staining in the normal adult adrenal medulla. Primary culture and immunoelectron microscopy suggested the processing and synthesis of adrenalin and MEAGL in the secretory granules.Seven extraadrenal pheochromocytornas were negative for PNMT and suggested the lack of adrenalin synthesis.

Expression changes in subsets of genes occur in the course of altering cell fates, i.e., aging, cell death, and carcinogenesis. These changes simultaneously provide the good candidate as a biomarker for monitoring cancer. We have identified a novel human septin family gene, Bradeion, from adult brain cDNA library by a monoclonal antibody CE5. Northern blot and in situ hybridization analysis showed that Bradeion has two distinct transcripts, approximately 2.2 and 1.7 kb length (alpha and beta, respectively) mainly in brain and slightly in heart, and no expression in any fetal organs. Haplotype analysis placed the gene location at 17q23. The gene contains GTPase motifs highly conserved in the septin family genes that are essential for cytokinesis and cell separation. The transcript of beta form lacks a hydrophobic region, which suggests that this form arises from a single Bradeion gene through unique RNA splicing. Interestingly, this brain-specific Bradeion gene is also expressed in two human cancers, colorectal cancer and malignant melanoma. Ectopic expression of normal Bradeion alpha and beta transcripts were confirmed both in patients' tumor samples and in in vitro cultured human cancer cell lines. Thus the Bradeion provides valuable tools as a tumor-specific and selective marker.

Three specimens of localized amyloidosis of the seminal vesicle surgically removed for prostatic cancer were immuno-histochemically analyzed to clarify the nature of the permanganate-sensitive congophilic subeplthelial deposition. A variety of known amyloidogenic substances and secretory products in the seminal fluid were screened using the indirect immunoperoxldase method. In addition to reactivities with antibodies to amyloid P component and human seminal plasma, the amyloid material was immunoreactive for lactoferrin using a rabbit antiserum and two of three mouse monoclonal antibodies. All the antibodies labeled some of the normal seminal vesicle epithelial cells for this iron-binding, bacteriostatJc glycoprotein. In the prostate without accompanying amyloid deposition, a considerable proportion of the glandular epithelium and secretory material were positive for lactoferrin. Pre-embedding immunoelectron microscopy showed lactoferrin immunoreactivity on the amyloid fibrils. Focal staining of the amyloid for gross cystic disease fluid protein-15 was also observed in two lesions. These findings strongly suggest that lactoferrin is the major constituent in localized senile amyloidosis of the seminal vesicle.

Thirty-one cases of mammary carcinoma were examined immunohistochemically for the expression of transforming growth factor (TGF) beta, fibronectin (FN) and fibronectin receptor (FNR) in order to clarify the reason for the reported relationship between TGF beta expression and a high incidence of lymph node metastasis. It was revealed that TGF beta expression is closely related to the expression of FN, an intercellular matrix protein, and its cellular receptor FNR, one of the integrins. The interaction between FN and FNR in a tumor is considered to form the basis of the invasive nature of carcinoma cells. Thus, it is suggested that TGF beta expression in carcinoma cells induces the interaction between FN and FNR, which may lead to carcinomatous invasion resulting in lymph nodal metastasis.

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.

The major heat shock protein, HSP70, protects cells from a variety of stressful stimuli, while c-myc protein allegedly stimulates the expression of HSP70 by transacting on the HSP70 promotor. The present study was aimed at correlating the expression of HSP70 with that of c-myc protein in benign and malignant breast lesions. Indirect immunoperoxidase and immunoblotting techniques using monoclonal antibodies were employed. Fresh frozen sections were prepared from five fibroadenomas and 59 breast carcinomas. Immunohistochemically, both substances were localized in the nuclei and/or cytoplasm of normal and neoplastic epithelial cells. The stromal cells were positive for c-myc protein but negative for HSP70. The expressions of HSP70 and c-myc protein were comparable to each other in the malignant cells from 37 (63%) carcinomas. In 17 (29%) carcinomas, c-myc protein expression predominated over that of HSP70, whereas in five (8%) carcinomas, HSP70 was predominant. Carcinomas with high-grade nuclear atypia often showed negative HSP70 staining, whereas tumors with nuclear HSP70 localization tended to accompany lower-grade nuclear atypia. A similar relation was also observed between c-myc protein expression and nuclear atypia of the tumor. All five fibroadenomas and most of the non-neoplastic epithelial cells adjacent to cancer showed strong reactivities with both substances. Immunoblot analysis for HSP70 revealed a clear, single band in the extract of tumors with strong HSP70 staining, but no, or only faint, bands were seen in the extract of immunohistochemically HSP70-negative carcinomas. The discrepancy between the expressions of each substance in a certain percentage of breast carcinomas may suggest the presence of HSP70 production mechanisms other than the c-myc protein-triggered promotor pathway.

It is a matter of debate whether the amplification of c-erbB-2 oncogene or production of the oncoprotein in breast cancers correlate with the presence of lymph node metastasis and with a poor prognosis. This study was aimed at elucidating the immunohistochemical localization of oncogene products which are related to cell growth, c-erbB-2 product, epidermal growth factor receptor (EGFR), c-myc protein and estrogen receptor (ER), in benign and malignant lesions of the breast. Fresh frozen sections of 25 breast cancers and 11 fibroadenomas from Japanese women were studied by indirect immunoperoxidase method with proper fixation. C-erbB-2 product and EGFR were localized on the cell membrane whereas c-myc protein and ER were observed in the nuclei. Immunohistochemical expression of oncogene products and ER were not only observed in the mammary carcinomas but also in the fibroadenomas. However immunoreactivities of EGFR and ER were more frequently seen in the fibroadenomas (p less than 0.05). In breast cancers, the incidence of immunoreactivity for c-erbB-2 was higher in the cases with lymph node metastasis than cases without nodal metastasis (p less than 0.05) and there was reciprocal correlation between the expressions of EGFR and ER (p less than 0.05). Regarding the size of the primary tumour, there was no statistically significant correlation with the expressions of c-erbB-2, EGFR, c-myc or ER. Histological grade correlated only with the expression of ER (p less than 0.05).

臨床的に高コレステロール血症3例 (type3) に, 庶糖あるいは澱粉を主とする高糖質食 (250g) を投与し, 血中諸脂質濃度ならびにその脂酸構成を測定した. 庶糖投与時に血中トリグリセリド濃度の著しい増加を知り, 同時に, 同脂質分画, 燐脂質, コレステロールエステルに, パルミチン酸, オレイン酸の増加, リノール酸の減少を認めた.犬を用いた動物実験において, 庶糖より分解される fructose (100g), あるいは, 澱粉より生ずる, glucose (100g) を投与し, 肝切片のトリグリセリド含量ならびにその脂酸構成を測定した. 庶糖投与犬の肝トリグリセリド含量の著しく高いこと, また同分画におけるパルミチン酸オレイン酸の増加と, リノール酸の減少を認めた.以上より, 庶糖は澱粉より脂質合成作用が強いが, それは庶糖より分解される fructose によるものであり, 肝ならびに血中では, 生体内で合成可能なパルミチン酸, オレイン酸の増加を認め, 合成不可能なリノール酸の減少を認めた.さらに, glucose と fructose との脂質合成の差異に関し, その可能性について若干論及した.

SummaryThe uptake and oxidation of FFA by skeletal muscle were studied in anesthetized, control dogs during rest, electrical stimulation, recovery, and DNP administration. Similar studies were also conducted in diabetic animals during rest, stimulation, and recovery. During rest, skeletal muscle removed about 40% of the labeled FFA and oxidized 36% of the removed amount. During skeletal muscle stimulation, the oxidation of FFA increased. During the recovery period, FFA oxidation was very low and accounted for about 12% of the uptake. The DNP also increased FFA oxidation. Both the uptake and oxidation of FFA by skeletal muscle were elevated in diabetic dogs and a significant uptake of β-hydroxybutyrate was also noted. A further increase of FFA oxidation occurred during stimulation. During recovery, only 7% of the removed FFA was oxidized and glucose uptake was high.