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The patient was an 80-year-old woman with combined pulmonary fibrosis and emphysema. She was diagnosed with pulmonary pleomorphic carcinoma in the right upper lobe, which relapsed 18 months after the operation. Computed tomography showed a mass in contact with the posterior wall of the lower part of the stomach. The patient was treated with two cycles of pembrolizumab, but the disease progressed. She was treated with S-1 as second-line therapy, resulting in tumor-shrinking after two cycles. Progression was not observed over the next twelve months. We report a rare case involving S-1 after immune checkpoint inhibitor treatment.

An 84-year-old man, who had received artificial pneumothorax for pulmonary tuberculosis 67 years previously, complained of severe chest pain. Chest CT revealed chronic pyothorax with multiple heterogeneously enhanced cavity lesions in the wall of the right intrathoracic space. 18FDG-PET revealed that the lesions showed an abnormal uptake. CT-guided biopsy was performed and he was diagnosed with pyothorax-associated lymphoma (PAL); the histological diagnosis was diffuse large B cell lymphoma (DLBCL). Furthermore, immunohistochemical staining revealed that the tumor cells were positive for EBNA-2 and LMP-1, suggesting that the latent gene products of Epstein-Barr virus were associated with the development of PAL. The patient was treated with chemotherapy, including rituximab; however, the treatment was discontinued due to the development of severe delirium after chemotherapy. We should keep in mind that elderly patients with a long history of chronic pyothorax are at risk of developing malignant lymphoma. We report the present case with a brief review of the literature.

Aims: To examine whether or not the tight junction-associated transmembrane protein occludin is expressed in rosette or gland-like structures in human rectal carcinoid tumours. The tight junction is crucial for the formation and maintenance of organized tubular structures in glandular epithelia. Previous studies have reported the presence of glandular structures in carcinoid tumours, though they are not believed to arise from glandular epithelium. Methods and results: The expression profiles of occludin in 40 carcinoid tumours were examined immunohistochemically, using an anti-occludin monoclonal antibody. In eight (20%) samples of typical carcinoid tumours, a small number of rosette-like tubular structures outlined by occludin were detected. Conclusions: Tight junction-associated molecules, including occludin, are thought to be one of the most characteristic structural markers of polarized glandular structures. The results of the present study provide supportive evidence that carcinoid tumour cells are capable of glandular differentiation.

The tight junctions of the glandular epithelium are crucial for the maintenance of cell polarity, separating the plasma membrane into apical and basolateral domains. Thus abnormalities of the tight junctions may result in the structural disturbances of glandular epithelial neoplasia. In this study we introduced an anti-occludin monoclonal antibody for semiquantitative assay of the occludin expression in tissue sections of human normal and neoplastic endometrial epithelia using the Adobe Photoshop and NIH Image programs. Normal endometrial glands and samples of endometrial hyperplasia and endometrioid carcinoma grade 1 fully expressed occludin at the apical cell border. In endometrioid carcinomas grades 2 and 3, however, occludin disappeared in solid areas of the carcinomatous tissues. Occludin was also found at the apical borders of the cancer cells that formed glandular structures. Occludin expression decreased progressively in parallel with the increase in carcinoma grade, and the decreased occludin expression correlated with myometrial invasion and lymph node metastasis. These results suggest that the loss of tight junctions has a close relationship with structural atypia in the progression of human endometrial carcinomas and their malignant potential.

Tight junctions are intercellular junctions adjacent to the apical end of the lateral membrane surface. They have two functions, the barrier (or gate) function and the fence function. The barrier function of tight junctions regulates the passage of ions, water, and various macromolecules, even of cancer cells, through paracellular spaces. The barrier function is thus relevant to edema, jaundice, diarrhea, and blood-borne metastasis. On the other hand, the fence function maintains cell polarity. In other words, tight junctions work as a fence to prevent intermixing of molecules in the apical membrane with those in the lateral membrane. This function is deeply involved in cancer cell biology, in terms of loss of cell polarity. Of the proteins comprising tight junctions, integral membrane proteins occludin, claudins, and JAMs have been recently discovered. Of these molecules, claudins are exclusively responsible for the formation of tight-junction strands and are connected with the actin cytoskeleton mediated by ZO-1. Thus, both functions of tight junctions are dependent on the integrity of the actin cytoskeleton as well as ATP. Mutations in the claudin14 and the claudin16 genes result in hereditary deafness and hereditary hypomagnesemia, respectively. Some pathogenic bacteria and viruses target and affect the tight-junction function, leading to diseases. In this review, the relationship between tight junctions and human diseases is summarized.

This study we presents a novel anti-occludin monoclonal antibody that can be used for formalin-fixed, paraffin-embedded tissue sections. The relationships between aberrant localization of carcinoembryonic antigen (CEA) and abnormalities of tight junctions were studied in human colorectal cancers by this antibody. Abnormalities in the cell surface expression of CEA have been shown to be characteristic of human colorectal cancer cells. Cancer cells that participated in the formation of glandular structures expressed occludin at the apical cell border and CEA was expressed more apically than occludin. Where cancer cells showed solid nests without glandular structures, occludin was completely lost and CEA was demonstrated in a diffuse pattern throughout the cells. These findings suggest that the polarized apical expression of CEA in neoplastic glandular structures depends on the expression of occludin and the fence function of tight junctions. During tumour progression, loss of occludin may lead to the loss of membrane polarity and the non-polarized expression of CEA. The antibody described provides a powerful tool for the study of tight junctions in surgically resected human tissue.

The retina is protected from somatic circulation by the blood-retinal barrrier (BRB) composed of tight junctions between retinal vascular endothelial cells (the inner BRB) and those between retinal pigment epithelial cells (the outer BRB). Our recent studies showed that glial cell line-derived neurotrophic factor (GDNF) secreted from astrocytes regulates the permeability of the BBB. In the present study, we immunohistochemically examined the expression of GDNF, neurturin (NTN) and their receptors, GFRalpha1 for GDNF and GFRalpha2 for NTN, because the capillaries of the inner BRB show specialization very similar to the blood-brain barrier (BBB). GDNF and NTN were detected in glial fibrillary acidic protein (GFAP)-positive cells, including Müller cells. GFRalpha1 and GFRalpha2 were localized in von Willebrand factor-positive cells. GDNF and NTN enhanced the barrier function of endothelial cells derived from porcine brain cortex. These results strongly suggest that the barrier function of the BRB is regulated by GDNF and NTN secreted from glial cells, like the BBB.

Sixteen gastrointestinal stromal tumors (GISTs) were studied by immunohistochemical analysis and an ultrastructural procedure. The tumor locations were as follows: esophagus (2), stomach (7), small intestine (3), and large intestine (4). Four of the lesions were classified as malignant, 2 as borderline, and 10 as benign. On the basis of the immunohistochemical analysis, the tumors were classified as follows: 1 as myogenic type, 2 as Schwann cell type, 8 as Cajal cell type (including 2 gastrointestinal autonomic nerve tumors, GANTs), and 5 as mixed-cell type. In each subtype the phenotype was compared to the ultrastructural findings. Myogenic and Schwann cell type revealed ultrastructurally smooth muscle differentiation and schwannian tumor. All 8 tumors of the Cajal cell type revealed interdigitating cytoplasmic processes with occasional clusters of filopodia. Two tumors were subdivided as GANT. Five tumors of mixed-cell type were composed of a mixture of cells with variable myogenic features or variable neural differentiation. We confirmed in this study that immunohistochemical analysis reflected electron microscopic findings.

Epithelioid disorganization is a hallmark of gastrointestinal cancers and is believed to be associated with malignant phenotypes such as invasiveness and the potentiality for metastasis. Although tight junctions (TJs) are known to be crucial for the maintenance of polarized organization of the gastrointestinal epithelium, changes in the TJ proteins in human cancers have not yet been fully elucidated. In this report, we investigated the expression and localization of three TJ proteins-barmotin (7H6 antigen), occludin, and ZO-1-in three phenotypically different human colon cancer cell lines exhibiting differnt grades of epithelioid organization. All three proteins were localized at the most apical part of the cell border corresponding to the site of TJs in T84 cells, in which epithelioid organization was well preserved. In contrast, in COLO320DM cells, which showed no epithelioid phenotypes, occludin was not detectable at either the protein or mRNA level, although barmotin and ZO-1 were present in the cytoplasm. In the third cell line, DLD-1, which showed an epithelioid phenotype intermediate between T84 and COLO320DM, aberrant expression of occludin was found in the basolateral cell membrane. On the other hand, barmotin was present in the cytoplasm, whereas ZO-1 was localized at the cell border. These observations showed that changes in the expression of TJ proteins occur in close correlation with epithelioid disorganization in human colon cancers.

The intestinal epithelial barrier restricts the passage of potentially toxic substances into the systemic circulation and is considered to be mostly mediated by tight junctions, though the mechanisms involved in the regulation of intestinal tight junctions are not yet fully understood. In the present study, we examined whether bacterial lipopolysaccharide (LPS) altered the barrier function of tight junction and localization of tight junctional proteins, ZO-1 and 7H6 antigen, in IEC-6 intestinal cells. Administration of LPS to the basolateral surface of IEC-6 cells disrupted the barrier function and caused the disappearance of 7H6 antigen from the cell border, whereas LPS administered to the apical surface altered neither the barrier function nor the localization of 7H6 antigen in IEC-6 cells. On the other hand, the localization of ZO-1 was not influenced by these treatments of LPS. These results suggest that the interaction of LPS with the basolateral surface of intestinal epithelial cells disrupts the barrier function and 7H6 antigen take part in the maintenance of the barrier function in IEC-6 cells. J. Cell. Physiol. 171:284–290, 1997. © 1997 Wiley-Liss, Inc.

Human glioma cell line KG-1C contains GM3 ganglioside as its sole glycolipid. The degree of M2590 antibody binding to GM3 was found to be regulated by the cell density; the percentage of positive cells in FACS analysis decreased from approximately 20% to close to none as the cells increased their density from sparse to confluent. The contents of GM3 with different cell densities were consistent, being more than 0.4 μmol/g of the cellular weight, which was high enough to be recognized by the antibody. Trypsin treatment of the cells did not increase antibody reactivity. The extracted GM3 retained its antigenicity, being intensely stained with M2590 on a TLC plate; there was no change in chromatographic mobility either, indicating no modification of its chemical structure. The fluorescent microscope disclosed scattered dot-like staining of GM3, particularly at the periphery of the cells. We were able to expose cryptic GM3 fully within 12 h by dispersion of the cells to a sparse density. Surface labeling of GM3 with the use of limited sodium periodate oxidation of sialylated residue equally labeled GM3 either from the confluent cells or the sparse cells. Disassembly of actin filaments with cytochalasin B (10 μM) partially exposed cryptic GM3 of confluent cells, indicating reversibility of the crypticity. All together, the results indicate that cryptic GM3 actually exists on the cell surface, hidden from the surface not by other molecules but by other mechanisms associated with the cellular architecture. We are beginning to explore the possibility of selective localization of GM3 in small caves or folds of the cell membrane produced upon cell-to-cell contact.

The capillary endothelium of the central nervous system functions as a highly selective permeability barrier, corresponding to the blood-brain barrier (BBB). In the present study, we assessed the roles of the tight junction-associated proteins ZO-1 and 7H6 in the development of brain endothelial barrier function. Capillary endothelial cells (ECs) of porcine brain were cultured and treated with a combination of astrocyte conditioned medium (CM), 8-(4-chlorophenylthio) cyclic AMP (cAMP), and phosphodiesterase inhibitor. Barrier function induced within 10h was more than 10 fold higher in terms of transendothelial electrical resistance (TER) and permeability to inulin and mannitol. Concomitantly, 7H6 antigen was significantly induced at the cell border, but the expression of ZO-1 did not change significantly between the control and treated cells. These results showed the importance of astrocyte CM and cAMP for the induction of the BBB and suggested that astrocyte CM and cAMP may function differently in the induction of 7H6 antigen in brain capillary ECs.

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21(waf-1/ciP-1) and p27, cyclin-dependent kinase inhibitors, by northern blot analysis. The expression of p21(waf-1/cip-1 ) in the LEC rat liver was 3-fold higher than that of age-matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p21(waf-1/cip-1) in the nuclear matrix fraction of the LEC rat liver. Immunohistochemically, p21(waf-1/cip-1) was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.

BACKGROUND While the assessment of the malignancy of neoplasms is based on morphologic studies of cells and tissues, use of objective molecular markers is leading to a better understanding and more biologically meaningful classification of neoplasms. In recent years, changes in the expression of cell adhesion molecules, especially E-cadherin, catenin, and adenomatous polyposis coli (APC), in carcinomas have attracted the attention of researchers. However, little is known about desmosomes in the uterine endometrium or in endometrial carcinomas. In this study, we semiquantified the desmosomal components desmoplakin I and II and desmoglein, in tissue sections using confocal laser scanning microscopy (LSM), and examined their relationship to the pathological type, the occurrence of lymph node metastases, and the extent of myometrial invasion. METHOD. Frozen sections of 31 specimens of normal endometrium, 5 specimens of atypical hyperplasia, and 41 specimens of endometrial carcinoma were stained by the immunofluorescence method using antidesmoplakin I and II and antidesmoglein, and these markers were then semiquantified in tissue sections by LSM. RESULTS. The expression and location of desmoplakin I and II and desmoglein were similar, and their expression decreased with loss of differentiation. The expression was lower in cases of lymph node metastasis than in negative cases and was lower in the cases with > one-half myometrial invasion than in cases with < one-half myometrial invasion. CONCLUSIONS. Reduction of desmoplakin I and II and desmoglein expression may play an important role in the invasiveness and metastatic activity of human endometrial carcinoma. They can therefore be used as differentiation markers for endometrial carcinoma. Cancer 1996;78:461-70.

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26). Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes. The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis. In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Cx proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm. These results indicated the formation and degradation of gap junctions in the cultured hepatocytes.

術前の子宮内膜細胞診で腫瘍細胞が検出された腺線維腫 (adenofibroma) の1例を経験したので報告した. 症例は45歳女性で, 月経過多と下腹部痛を主訴とし, 腹部超音波検査で, 子宮腔内に, 漿液を入れた嚢胞を含む腫瘍が発見された. 経過中, 膣より淡黄色の漿液の流出を認め, その後腫瘍が縮小, 症状が軽減したことから, 腫瘍内嚢胞の穿破が生じたものと考えられた. その後の子宮内膜細胞診で, 一部に線毛を伴う, 異型性に乏しい高円柱上皮細胞のシート状集塊を認めた. 摘出された腫瘍は組織学的に, 子宮内膜および筋層と明瞭に境され, 一層の高円柱上皮細胞で覆われた線維性結合組織の葉状, 乳頭状の増生からなっていた. 上皮, 問質成分のいずれにも, 細胞異型, 細胞分裂像は認められなかった. われわれの検索した限りでは, 内膜細胞診で腫瘍細胞を認めた子宮adenofibromaの報告はみられない.

A sequential decrease in the number of hepatocyte tight junctions during the course of rat hepatocarcinogenesis was demonstrated by immunohistochemistry with a new 7H6 monoclonal antibody generated in our laboratory. Semiquantitative analysis by confocal laser scanning microscopy revealed that the expression of 7H6 antigen was reduced in hyperplastic foci, hyperplastic nodules and hepatocellular carcinomas (HCC) to 43%, 28% and 25%, respectively, compared to corresponding normal liver tissues. 7H6 antigen was scarce in HCC with a trabecular pattern, whereas it was expressed intensely at the apical and basolateral membrane of HCC with a glandular pattern. Immunoblot analysis of 7H6 expression in hepatocellular carcinomas showed a decrease roughly coincident with that shown by immunohistochemistry. These results indicated, for the first time, that tight junctions decrease progressively during carcinogenesis, leading to disruption of cellular polarity and cellular adhesiveness.

A 76-year-old man presented with fever of unknown origin. Diagnostic imaging showed a liver tumor measuring 3cm in maximum dimension. The tumor was subsequently resected, and histopathology showed a moderately differentiated adenocarcinoma. This showed a number of bile ductules with variable amounts of stroma, well circumscribed but not encapsulated, so the lesion was diagnosed as a cholangiocarcinoma. Within the tumor there was also a cholangiolocarcinoma-like lesion. In addition, cystically dilated ductules resembling bile duct hamartoma and bile duct adenoma adjacent to the tumor were found, but with no area of transition among them. In the Glisson's capsule around the tumor, there was also a bile duct hamartoma.

Pathological investigation of progressive anterior operculum syndrome has rarely been reported. We describe clinico-pathological findings in a patient with progressive anterior operculum syndrome. A 74-year-old right-handed man had noticed speech and swallowing difficulties 1 year previously. Neurological examinations showed no abnormality other than a slight limitation of upward gaze and slow tongue movement without fibrillation. We investigated the patient using neuroimaging and neuropsychological examinations and observed him for 2 years until his death, at which point we obtained pathological findings. The patient's facial and masseteric muscles seemed hypotonic with drooling, but he could laugh and yawn normally, showing automatic voluntary dissociation. Palatal and pharyngeal reflexes were normal. Magnetic resonance imaging showed cortical atrophy in the temporal lobes bilaterally. (123)IMP single photon emission computed tomography and positron emission tomography showed decreased blood flow and activity in the frontotemporal lobes, predominantly on the left side. Neuropsychological examinations showed no aphasia, dementia or other neuropsychological abnormality. Intubation fiberscopy, laryngoscopy and video fluorography showed no abnormality. After 6 months his anarthria and dysphagia became aggravated. He died of aspiration pneumonia 2 years after onset. Postmortem examination revealed neuronal degeneration with TDP-43-positive inclusions in the frontal, temporal and insular cortices, consistent with frontotemporal lobar degeneration with TDP inclusions (FTLD-TDP). However, neuronal loss with gliosis was more prominent in the inferior part of the motor cortices, bilaterally. Progressive anterior operculum syndrome could be classified as a variant of FTLD-TDP.

穿刺吸引細胞診によって術前診断が可能であった膵尾部原発の粘液性嚢胞腺癌の1例を経験したので報告する.症例は56歳, 女性.左上腹部腫瘤を主訴として来院し, 外傷の既往, 画像所見から臨床的には外傷性尿腫が疑われたが, 穿刺吸引細胞診では多量の粘液を背景とした高円柱状細胞の集塊に混在して, より異型の高度な腺癌細胞を認め, 粘液性嚢胞腺癌と診断された.その原発臓器については腫瘍の存在部位と推定組織型を勘案して, 膵臓が最も疑われた。摘出された腫瘍は, 肉眼的に膵尾部に連続して存在する多房性嚢胞性腫瘍で, 組織学的には大部分は異型に乏しい粘液性嚢胞腺腫の像で, その一部に乳頭状増殖の著しい粘液性嚢胞腺癌の成分を混在していた.すなわち, 細胞診における良性集塊と悪性集塊の混在性はこれを反映しているものと考えられた.本症例では, 穿刺吸引細胞診によって腫瘍の悪性度, 組織型のみならず, 原発臓器の推定も可能であった.これは腫瘍の臨床診断における細胞診の有用性を示すものといえよう.

腹水細胞診で興味深い所見が得られた卵管原発腺癌の1例を経験したので報告する症例は66歳, 女性.不正出血にて初発後, 1年2ヵ月で著明な腹水貯留を認めた.術前の腹水細胞診で, 砂粒体およびいわゆる偽線毛を伴う多数の腺癌細胞を認め, 卵巣原発漿液性嚢胞腺癌が疑われ, 化学療法施行後子宮全摘術および両側附属器切除術が施行された.開腹時, 腹腔内に広汎に結節状転移巣を認め, 術後の病理組織学的検索により, 卵巣および子宮には腫瘍を認めず, 左卵管内腔に卵管壁と連続して直径約1.5cmの結節状腫瘤を認め, 卵管原発腺癌と診断した.原発性卵管癌は細胞診においては特徴的所見に乏しく, 術前診断は困難であるとされている.砂粒体および偽線毛は卵巣原発漿液性嚢胞腺癌に特徴的な所見であるが, 原発性卵管癌にこれらが認められたとする報告はこれまでなく, 今回これが認められたことは, 同腫瘍の細胞診断および組織発生を考えるうえで興味深い.

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer-mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin-induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.

Occludin is a tight-junction-associated transmembrane protein, and previous observations suggested that occludin might play a crucial role in the formation and maintenance of organized tubular structures. Based on these observations, we explored the possible role of occludin immunostaining in the diagnosis of lung carcinomas. A total of 68 lung carcinomas and surrounding normal lung tissues were studied. A formalin-fixed, paraffin-embedded section from each tumor was stained with a new anti-occludin monoclonal antibody raised in our laboratory. In normal lung tissues, the anti-occludin antibody strongly stained the apicoluminal borders of the bronchial/bronchiolar epithelia and bronchial glands as a dot or short line. The antibody also stained the intercellular borders of alveolar epithelia. In cancer cells that faced lumina of all adenocarcinomas, regardless of grade, including bronchioloalveolar carcinomas, occludin showed an expression pattern identical to that of the normal bronchial and alveolar epithelia. Occludin reactivity was not noted in any cases of squamous cell carcinoma, large cell carcinoma, small cell carcinoma, or large cell neuroendocrine carcinoma. The results of the present study suggest that occludin can serve as an immunohistochemical indicator of the "true" glandular differentiation that forms tubulo-papillary structures in human lung carcinoma tissues.

The expression and localization of glial cell line-derived neurotrophic factor (GDNF) and its receptor GFR alpha1 in the testis were examined, because the blood-testis barrier is a well-known tissue barrier and we previously reported that GDNF reduced the endothelial permeability of the blood-brain barrier (BBB). Five minutes after intravenous injection of Evans blue (molecular weight, 960.6) or fluorescent dextran (molecular weight 10000 and 70000), Evans blue was observed outside microvessels of the testis, whereas the fluorescent dextran was not. Immunohistochemically, GDNF was detected in alpha-smooth muscle actin-positive cells around the seminiferous tubules and in microvessels. On the other hand, GFR alpha1 was detected in endothelial cells in the interstitial space, as well as in spermatocytes. Although occludin was positive in Sertoli cells and endothelium, claudin-5 was localized only in the endothelium of the microvessels. Thus, it became very clear that the microvessels in the testis possessed relatively impermeable tight junctions, and that the alpha-smooth muscle actin-positive cells secreted GDNF, which receptor was expressed in endothelial cells. Because this relation between GDNF and GFR alpha1 is similar to that observed in the BBB, we hypothesize that GDNF is a general regulator of tight junctions of the endothelium forming a blood-tissue barrier in a paracrine fashion.

Introduction Anaplastic large cell lymphoma is a rare type of non-Hodgkin lymphoma. The most common extranodal sites of anaplastic large cell lymphoma are skin, subcutaneous tissue, bone, lung, and gastrointestinal organs. The involvement of the skeletal muscle has been described rarely in extranodal anaplastic large cell lymphoma. Case presentation An 89-year-old Japanese man visited our hospital with a three-month history of swelling of his left thigh and slight fever. The swelling had rapidly enlarged and become painful within the previous three days. Magnetic resonance imaging scans revealed two soft-tissue tumors in the intramuscular layer between the vastus medialis muscle and the adductor muscle. Extensive peritumoral inflammatory edema was obvious. As the results of physical and radiological examinations were highly suggestive of abscess formation, we prescribed antibiotics for two weeks. However, our patient’s symptoms did not improve. Therefore, we suspected a soft-tissue sarcoma, and our patient underwent an incision biopsy. Histological analysis revealed that the atypical cells were positive for CD3 and CD30 but negative for anaplastic lymphoma kinase. A computed tomography scan of the thorax revealed mediastinal lymphadenopathy and bilateral pleural effusions, suggestive of extranodal involvement of skeletal muscle in anaplastic lymphoma kinase-negative anaplastic large cell lymphoma. We planned to give our patient systemic chemotherapy. However, rapid systemic dissemination occurred and our patient died of multiple organ failure five weeks after his first visit to our hospital. Conclusions Here, we present a case of anaplastic lymphoma kinase-negative anaplastic large cell lymphoma with extranodal involvement in the thigh muscle. The involvement of such a rare organ may lead to initial misdiagnosis and a delay in the onset of treatment.

Tight junctions of hepatocytes form the intercellular barrier between the blood circulation and bile flow. We focused on early stages of common bile duct ligation to observe changes in tight junctions without the irreversible changes seen after lengthy ligation. Common bile ducts of 12-week-old male rats were ligated for 6 h because, at this time point, no histological changes were observed. Serum bilirubin and bile acid levels began to increase 3 h after ligation and were restored to the control level immediately after surgical removal of the ligation. To examine the barrier of hapatocytes, horseradish peroxidase was injected via the femoral vein, and bile was collected for the first 10 min. A four-fold elevation of the secretion and concentration was observed in the bile of ligated rats compared with that of control animals. We next examined lanthanum permeability by perfusion fixation of the liver. At 6 h after ligation, both dilation of the bile canaliculi and partial loss of microvilli were commonly observed. There were dense deposits of lanthanum in almost all bile canaliculi of ligated rats. In control animals, neither dilation of the bile canaliculi nor loss of microvilli was detected, and only 44% of bile canaliculi exhibited deposits. An apparent increase of occludin mRNA expression was detected in livers after 6 h ligation, whereas the expression of claudin-1, -2, and -3 was not influenced by ligation. These results indicate that regulation of occludin gene expression is different from that of claudin-1, -2, and -3. The early phase of bile stasis employed in this study is thought to be an indispensable approach for understanding the precise regulation of tight junctions.

Retinoids are critical for differentiation of columnar epithelial cells and for preventing metaplasia of these cells into stratified squamous epithelial cells, in which tight junctions (TJs) are essentially absent. This implies that retinoids might play important roles in regulating the structures and functions of TJs of columnar epithelium. F9 murine embryonal carcinoma cells differentiate into epithelial cells resembling visceral endoderm bearing TJs, when grown in suspension as aggregates in the presence of retinoic acid (RA). We show that RA induces the TJ structure and expression of several TJ-associated molecules, such as ZO-1, occludin, claudin-6, and claudin-7, as well as a barrier function in the genetically engineered cell line F9:rtTA:Cre-ER(T) L32T2, which allows sophisticated genetic manipulations simply by addition of ligands (H. Chiba et al., 2000, Exp. Cell Res. 260, 334-339). Interestingly, our data indicate that a barrier for small substances is generated after that for large ones during de novo formation of TJs. We also compared the RA-induced expression of TJ components and barrier function in RXRalpha(-/-)-RARgamma(-/-) F9 cells with those in wild-type cells and show that the retinoid signals for transduction of these events are mediated by specific RXR-RAR pairs.

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.

Since a deep involvement of astrocytes, a kind of glial cells, in differentiation of the blood-brain barrier (BBB) has been suggested, we examined the relation of glial cell line-derived neurotrophic factor (GDNF) to the BBB. First, immunohistochemical examination of the cerebral cortex of rats revealed that glial cell line-derived neurotrophic factor receptor (GFRalpha1) was preferentially expressed on the cell membranes of capillary endothelial cells. Second, to elucidate the effects of GDNF on the BBB, capillary endothelial cells isolated from the porcine cerebral cortex were cultured and then changes in tight junction function of the endothelial cells were examined after addition of GDNF, in terms of transendothelial electrical resistance (TER) and permeability. GDNF at concentrations of 0.1 and 1 ng/ml significantly activated the barrier function of the endothelial cells in the presence of cAMP. Since GDNF is secreted from astrocytes sheathing capillary endothelial cells in the brain cortex, our results strongly suggest that GDNF enhances the barrier function of tight junctions of the BBB on the one hand, and also supports the survival of neurons on the other hand.

The tight junction is not a constitutional junctional apparatus in endothelial cells, but develops in a particular lineage of endothelia, such as the capillary endothelia in the brain and retina, and thus is considered to be pivotal for the maintenance of the blood–tissue barrier. Occludin is an integral membrane component of tight junctions, but the role of occludin in the endothelial cell function remains to be elucidated. We have cloned and transfected rat full-length occludin cDNA into a rat endothelial cell line (RLE) that expressed only a trace amount of occludin with no fine circumferential actin bundles at the cell border in native conditions. Occludin was expressed at the cell border of RLE cells, and circumferential fine actin bundles developed in close relation to the sites of occludin localization. Even under subconfluent culture conditions, fine circumferential actin bundles were formed at the sites where occludin-positive cell–cell contact was achieved. In immunoelectron microscopy, occludin was localized at distinct areas of the plasma membrane, always in association with the cytoplasmic actin filaments. On the other hand, actin bundles were not seen in occludin-negative juxtaposing plasma membranes. Collectively, these data strongly suggested a possible determinant function of occludin for the organization of actin in endothelial cells.

Actin filament organization may play an important role in the maintenance of differentiated functions in epithelial cells. We previously reported our success in inducing and maintaining gap junctions, which are two kinds of differentiated function, in primary rat hepatocytes cultured with 2% DMSO and 10-7 M glucagon. In the present study, we demonstrated the formation of actin filament networks in the hepatocytes cultured with 2% DMSO and 10-7 M glucagon. Actin filaments in hepatocytes cultured in medium with only 2% DMSO added from 96 h after plating were concentrated under the plasma membrane and were observed to be circumferential. In hepatocytes cultured in the medium with both 2% DMSO and 10-7 M glucagon added from 96 h, not only the circumferential actin filaments but also the formation of actin filament networks were observed and the networks developed well with time in culture. The networks were observed as a dome-like structure under the cell face and terminated at the circumferential actin filaments. They were composed of electron-dense star-like vertices connected by microfilament bundles of varying length and were also very sensitive to the actin disruptor cytochalasin B. However, during the network formation, there were no significant increases in the amounts of actin protein and mRNA. The actin filament networks of the hepatocytes in this culture system might be closely related to the maintenance of differentiated functions.

To study pathophysiological roles of mesothelial barrier functions in protection against cancer cell invasion, we isolated mesothelial cells from the rat abdominal cavity and then cultured them with 10(-6)M all-trans-retinoic acid (RA) for 10 days. Mesothelial barrier function assessed by measuring transcellular electrical resistance (TER) and the expression of 7H6 tight junction-associated antigen at the cell border were induced by the treatment (10.01 +/- 0.8 vs 6.05 +/- 0.7 omega cm2, without RA; mean +/- s.e.m., n = 10). Then we quantified the attachment and penetration of rat mammary cancer cells (SST-2 cells) into the mesothelial cell monolayer by prelabelling of the cancer cells with fluorescent dye and by observing optical sections at different heights using a laser confocal scanning microscope. When SST-2 cells were overlaid onto the mesothelial cell monolayer treated with RA, the number of cancer cells found at the basal level of the monolayer was significantly reduced. These results showed that enhanced mesothelial barrier function at least partially prevents the penetration of cancer cells into mesothelial cells and suggested that 7H6 antigen serves as a reliable immunocytochemical marker for monitoring mesothelial barrier function. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 7 Figure 8

A second protein-tyrosine kinase (PTK) of the focal adhesion kinase (FAK) subfamily, cell adhesion kinase beta (CAK beta), was identified by cDNA cloning. The rat CAK beta is a 115.7-kDa PTK that contains N- and C-terminal domains of 418 and 330 amino acid residues besides the central kinase domain. The rat CAK beta has a homology with mouse FAK over their entire lengths except for the extreme N-terminal 88 residues and shares 45% overall sequence identity (60% identical in the catalytic domain), which indicates that CAK beta is a protein structurally related to but different from FAK. The CAK beta gene is less evenly expressed in a variety of rat organs than the FAK gene. Anti-CAK beta antibody immunoprecipitated a 113-kDa protein from rat brain, 3Y1 fibroblasts, and COS-7 cells transfected with CAK beta cDNA. The tyrosine-phosphorylated state of CAK beta was not reduced on trypsinization, nor enhanced in response to plating 3Y1 cells onto fibronectin. CAK beta localized to sites of cell-to-cell contact in COS-7 transfected with CAK beta cDNA, in which FAK was found at the bottom of the cells. Thus, CAK beta is a PTK possibly participating in the signal transduction regulated by cell-to-cell contacts.

The heat shock proteins (hsp) are regarded as being immunogenic to the animal hosts. Although certain hsp are suggested to be expressed on the cell surface, further evidence for the cell surface expression of these proteins has been required. In this article we report the development of a MAb NT22. This antibody reacted with ATP-binding proteins (which contain a large amount of 70-kDa hsp family) of HeLa cells, and with purified bovine 70-kDa hsp. It did not react with the E. coli lysate, but clearly reacted with the recombinant rat hsc73. However, NT22 failed to react with hsp72. Furthermore, stress treatment of cells also indicated that considerable amounts of NT22-defined antigen translocated into the nucleus from the cell cytoplasm. These results suggest that NT22 is a novel MAb that reacts specifically to the mammalian hsc73. Moreover, this antibody could detect the constitutive and stress-induced cell surface expression of its relevant antigen. It is expressed preferentially on EBV-transformed B cell and certain epithelial cancer cell lines. However, resting B cells did not express this antigen on the cell surface. These data indicate that hsc73 could be expressed on the cell surface of certain cells, and suggest that hsc73 may interact with the host immune system.

Spontaneous hepatitis in the LEC rat is characterized by hyperbilirubinemia, increased levels of serum glutamic-oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) in laboratory examination and spotty coagulative necrosis of single hepatocyte without inflammatory cell response [1, 2]. Although a single autosomal recessive gene (hts) has been shown to be responsible for the hepatitis, the mechanism(s) of hepatocyte necrosis in the LEC rat remains obscure. The histological features of the liver reveal a certain resemblance between the hepatitis of LEC rats and the liver necrosis induced by the administration of D-galactosamine (GalN).