A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

BACKGROUND: Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells. METHOD: A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting. RESULTS: We found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids. CONCLUSIONS: We developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Immunohistochemical markers for Barrett's esophagus and associations to esophageal Z-line appearance.

BACKGROUND: Data from previous studies on intestinal metaplasia at the gastroesophageal junction have been conflicting, which makes the diagnosis of Barrett's esophagus less obvious. This may partly be due to the lack of a reliable classification of the Z-line appearance. We previously proposed such a classification (the ZAP classification) that was shown to correlate with the prevalence of intestinal metaplasia. The use of different immunohistochemical techniques has increased in the study of intestinal metaplasia. In the present study our aim was to 1) evaluate the impact of different antibodies, namely cytokeratin (CK) 7, 13, and 20, CaCO3/73, and FBB2/29, in order to differentiate between Barrett's esophagus and cardia intestinal metaplasia, and 2) explore the staining patterns in different ZAP grades. METHODS: Thirty-nine specimens with intestinal metaplasia were compared--9 from Barrett's esophagus, 6 from cardia, and 24 from the Z-line. The Z-line specimens were evaluated with respect to ZAP grade. RESULTS: No differences were encountered regarding staining patterns for CK13 and CaCO3/73 in Barrett's esophagus and cardia. The staining pattern of CK7/20 was significantly different between Barrett's esophagus and cardia. CK7/20 showed a rising frequency of Barrett's esophagus staining pattern with rising ZAP grade. CONCLUSION: CK7/20 is a feasible marker for Barrett's esophagus. Intestinal metaplasia in different ZAP grades differs regarding expression of immunohistochemical markers.

p53 expression in correlation to clinical outcome in patients with renal cell carcinoma.

OBJECTIVE: The aim of this study was to evaluate the role of p53 as prognostic factor in renal cell carcinoma (RCC) and its relation to clinicopathological factors. MATERIAL AND METHODS: The nuclear accumulation of p53 protein was determined by immunohistochemical analysis in RCC specimens from 90 patients and was correlated with clinical stage, grade, DNA ploidy, S-phase fraction and cancer-specific survival. RESULTS: p53 overexpression was observed in 17 of 90 (19%) tumours. There was a significant correlation to stage (p = 0.016) and grade (p = 0.020) but not to DNA ploidy or S-phase. Patients with high p53 immunoreactivity had shorter cancer-specific survival (p = 0.003) than those with normal p53 protein expression. This difference was found in papillary and chromophobe tumour types (p < 0.0001) but not in conventional RCC. CONCLUSIONS: In patients with RCC, significant correlations between p53 protein expression and tumour stage, grade and survival time were observed. For patients with chromophobe and papillary tumour types, but not in conventional RCC, p53 immunoreactivity gave prognostic information, suggesting that the prognostic differences in p53 immunoreactivity might be due to disparate genetic abnormalities in the different RCC types.

[Meningeal melanocytoma]. / Meningealis melanocytoma.

The authors report on a case of melanocytoma surgically removed from the craniocervical meninges of a 59-year-old man. Although the excision had been incomplete, the patient showed a disease-free course extending well over ten years. On histology, the tumor consisted of moderately cellular arrays of spindle-shaped melanocytes with a vaguely angiocentric whorling tendency, and without evidence of infiltrative growth. Electron microscopy identified tumor cells as ones bearing dendritic processes with complex melanosomes. The latter showed histochemical properties of melanosomal melanin, as well as immunoreactivity for the melanosome-associated markers HMB-45, and MELAN-A. Hallmarks of meningial differentiation were, at the same time, absent. The MIB-1 proliferation rate of the lesion, as assessed in a simultaneous testing of a panel including primary and metastatic central nervous system melanomas, as well as a uveal melanoma remained inferior to 1.5 percent. The data presented and a critical review of the literature suggest that meningeal melanocytoma is a mostly benign nevus-like lesion of neural crest cells with a very limited, although not discountable, margin for aggressive growth.

Human colorectal cancers with an intact p16/cyclin D1/pRb pathway have up-regulated p16 expression and decreased proliferation in small invasive tumor clusters.

A systematic spatial heterogeneity with high proliferative activity at the luminal border and low activity at the invasive margin is an unexpected behavior that has been observed in colorectal cancer (CRC). To clarify this phenomenon and possible underlying regulatory mechanisms, we have by immunohistochemistry elucidated the proliferative activity and the expression of G1/S regulatory proteins in small and large tumor cell clusters at the invasive margin in 97 CRCs. By identifying small tumor clusters at the tumor front, actually invading cancer cells could be characterized and analyzed separately. These cells could then be compared with the main tumor mass represented by the larger tumor clusters. The proliferation was significantly lower in small tumor clusters compared with larger clusters (P < 0.001) and the decrease in proliferation was correlated with a p16 up-regulation (r(s) = -0.41, P < 0.001). Interestingly, CRCs lacking p16 expression (18%) or tumors with other aberrations in the p16/cyclin D1/pRb pathway had a less pronounced decrease in proliferation between large and small clusters (P < 0.001), further strengthening the association between p16 and ceased proliferation at the invasive margin. This contrasts to tumors with low p27 or abnormal p53 levels showing sustained proliferation in small tumor clusters. Our findings imply that invading CRC cells generally have low proliferative activity, and this phenomenon seems to be mediated through p16 and the p16/cyclin D1/pRb pathway.

Cloacogenic adenocarcinoma of the vulva.

Primary adenocarcinoma of the vulva is rare, and cloagocenic adenocarcinoma of the vulva is extremely rare. Here we report a vulvar tumor characterized by columnar cells with prominent brush border and the presence of goblet cells and endocrine cells, presenting the tubulovillous pattern and mucin histochemistry of enteric adenocarcinoma. Electron microscopy verified a colon-like pattern. We suggest that cloacogenic carcinoma of the vulva arises from embryonic tissue, normally present in the vulvar introital area. In our case, a wide local excision was sufficient for radical cure.

Pharyngeal dysphagia caused by isolated myogen dystrophy of musculus cricopharyngeus.

Five patients suffering from idiopathic cricopharyngeal dysfunction (without Zenker's diverticulum) were treated surgically. Together with cricopharyngeomyotomy biopsies were taken at the level of the cricopharyngeus. Histological, enzyme hystochemical and electronmicroscopic examinations were performed on all patients. In two cases the histology revealed myogen dystrophy (presence of necrosis, myophagocytosis, abnormal fiber structure, basophilic fibers, fibrosis, mild cellular reaction and predominancy of fiber type I). Since the complete patient evaluation (clinical features, electromyography, serum creatinin phosphokinase level, etc.) could rule out any general, muscle disorders, the cause of the idiopathic pharyngeal dysfunction must have been in these two cases an isolated myogen dystrophy of the cricopharyngeus.

Ultrastructural characteristics of glial fibrillary acidic protein expression in epoxy resin-embedded human brain tumors.

Thirteen surgically removed, epoxy resin (Durcupan ACM or Epon 812)-embedded human brain tumors were examined for glial fibrillary acidic protein (GFAP) content in semithin and ultrathin sections with the immunogold-silver staining method. Mild aldehyde fixation and the hydrophobic resin embedding did not interfere with the antigenicity, since silver intensification of the immunogold marker provided excellent visualization of the reaction on both light microscopic and ultrastructural levels. The GFAP reaction was usually localized on the glial intermediate filament bundles, usually correlating well with the amount of filaments. The unstained filamental regions of two ependymomas might correspond to the vimentin expression revealed by double labeling in semithin sections. Occasional GFAP immunopositivity without filamental appearance was observed in one of the oligodendrogliomas, as patchy electron-dense cytoplasmic corpuscules, in Rosenthal fibers and in some mainly necrobiotic tumor cells, reflecting a possible connection with glial filaments.

Ultrastructural investigations in late infantile type of ceroid lipofuscinosis (Jansky-Bielschowsky).

Ultrastructural findings of biopsy materials of four gipsy first cousin infants suffering from late infantile type of ceroid lipofuscinosis (Jansky-Bielschowsky) were investigated. The diagnostic significance of the conjunctival biopsy is emphasized. The pericytes and the vascular smooth muscle cells of the arterioles proved to be the main inclusion storing cells.

Double and triple immunocytochemical labelling at the light microscope level in histopathology.

Double and triple immunocytochemical detection methods for routine use in histopathology were investigated. For double immunostaining, the combinations of immunogold-silver staining (IGSS, black) with an immunoperoxidase method (3-amino-9-ethylcarbazole, red-brown) or with an immunoalkaline phosphatase method (Fast Red TR, red) proved very useful. These were followed by a Haematoxylin counterstain. An alternative approach using immunoperoxidase (red-brown) and immunoalkaline phosphatase (Fast Blue, BB, blue) methods was also successful, particularly for frozen sections of unfixed tissue and for the establishment of intermediate filament coexpression in tumours. The coexistence of desmin with vimentin in rhabdomyosarcoma, and of glial fibrillary acidic protein with vimentin in ependymoma, could be demonstrated directly by means of non-crossreacting murine and rabbit antibodies in the above combinations. The black (IGSS), red-brown (immunoperoxidase) and blue (immunoalkaline phosphatase) colours gave excellent contrast in triple immunostaining. The side-by-side demonstration of helper and suppressor T lymphocytes during renal allograft rejection, of kappa and lambda light chains in B-immunoblastic lymphoma, and of T and B lymphocyte populations in non-Hodgkin's lymphomas provided immediate information on the topography and cellular organization of the tissues.

[Cricopharyngeal achalasia caused by isolated muscular dystrophy]. / Izolált izomdystrophia okozta cricopharyngealis achalasia.

The authors--in connection with two surgically successful treated cases--discuss the diagnosis and the surgical treatment of the cricopharyngeal achalasia. By means of histological methods it was verified that in the background of this disease stads an isolated muscle dystrophy localized to the upper esophageal sphincter.

Effect of tiacrilast, a mast cell mediator-release inhibitor, on murine experimental contact dermatitis.

The effect of tiacrilast, a mast cell mediator-release inhibitor, was studied in dinitrofluorobenzene-induced allergic and croton oil- or dimethyl sulfoxide-induced irritant murine contact dermatitis. At 1% concentration, the compound significantly reduced the ear swelling in both allergic and irritant dermatitis and preserved the mast cell architecture on histopathology. These findings suggest that mast cells participate in the elicitation of murine contact dermatitis.

The mouse erythrocyte binding receptor is expressed on human keratinocytes.

Immunocytes and different types of epidermal cells share cell surface antigen characteristics e.g. keratinocytes react specifically with the Leu 11 (CD16) monoclonal antibody and they express MHC class II and OKM5 (CD36) antigens after gamma-interferon stimulation. The present study provides evidence that a subpopulation of human keratinocytes most probably at a certain stage of differentiation, possesses the mouse erythrocyte binding receptor the marker of a human B lymphocyte subset. This finding is a further evidence for the relationship between the epidermis and the immune system.

[Imprint cytology of intracranial tumors]. / Intracraniális tumorok imprint cytológiája.

Imprints of haematoxilin-eosin and Giemsa-stained, 33 surgically removed different intracranial tumors were examined. In case of sufficient number of cells and adequate clinical data, the cytological examination was suitable to quick intraoperative diagnosis in majority of cases. Its demonstration on GFAP smear facilitates the diagnosing of gliomas.

New monoclonal antibody that specifically recognizes murine interdigitating and Langerhans cells.

A new monoclonal antibody, M1-8, that recognizes murine interdigitating cells (IDC) and Langerhans cells was obtained from a hybridoma prepared by fusion of SP2/0 mouse myeloma cells with splenic cells of rats immunized with IDC-rich cell suspension obtained from lymph nodes of athymic nude mice (BALB/c nu/nu). The specificity was assessed immunohistochemically on frozen sections of lymph nodes and epidermal sheets from both nude and normal mice. M1-8 reacted with paracortical IDC, veiled cells of the marginal sinus, and epidermal Langerhans cells in both normal and nude mice. In simultaneous staining by M1-8 and nonspecific esterase or anti-Ia or anti-Thy-1,2 antibody, the same epidermal dendritic cells were positive for all these antigens except Thy-1,2. Immunoelectron microscopy of the lymph node suspension using gold colloid particles revealed the attachment of gold particles to the cell membrane of IDC. Analysis by flow cytometry of the lymph node cell suspension showed 14 or 6% of M1-8-positive cells in nude or normal mouse, respectively. Immunohistochemical analysis showed that M1-8 also reacted with dendritic cells in the thymus and spleen and had a different distribution from F4/80. M1-8 also reacted with monocytes in bone marrow and peripheral blood, alveolar macrophages, and thioglycollate-stimulated peritoneal exudate macrophages. The antibody belongs to the immunoglobulin M class, reacts immunochemically with a glycoprotein in the cell membrane, and has a molecular mass of approximately 15 kDa.

Adherence of cells to myelin basic protein. II. Adherence of red blood cells of SJL mice with chronic relapsing EAE.

Adherence of red blood cells from SJL mice suffering of chronic relapsing experimental allergic encephalomyelitis was studied to myelin basic protein coated microtiter plates. Control animals received either bovine serum albumine or "protein-antigen free" adjuvant using the same immunization protocol. Characteristic changes in adherence were found in bovine or human myelin basic protein injected animals compared to the bovine serum albumine immunized group. After a nonspecific increase in adherence between Days 2 to 6 observed in all 3 groups, in the encephalitogen challenged animals on Days 13-14 a marked decrease in red blood cell adherence was detected which maintained at this decreased level during the clinically active stage of the disease and reappeared with the relapse of EAE. No such decreased adherence of red blood cells was observed in BSA immunized animals or in adherence of cells from myelin basic protein injected animals to other basic type protein such as histone. Thus, decreased adherence of red blood cells in animals with EAE appears to be an interestingly unique measure of the disease activity.

Immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) in nasal gliomas.

Using the Sternberger method (Immunoluk Histoset KIT) GFAP (glial fibrillary acidic protein) was demonstrated immunohistochemically in 4 nasal gliomas. In these histologically complex tumour-like lesions mesenchymal, epithelial, and neuroglial tissues as well as small groups of scattered glial elements could be differentiated specifically by the highly sensitive GFAP immunoperoxidase technique. GFAP was present in astrocytes and astrocyte-like differentiations. The reactivity of cell processes was essentially lower. The GFAP immunostain does not always correlate with Mallory's phosphotungstic acid hematoxylin (PTAH) stain and Gallyas' silver impregnation method for astrocytes. Additionally the immunohistochemical investigation of semithin sections prepared by the so-called pop off technique after Bretschneider et al. (1981) allows the correct localization of GFAP in astrocytes and their modulations. Furthermore, in this study, the intimate connection of epithelium and glial cells as well as astrocytes containing hemosiderin granules could be demonstrated. The latter findings suggest a possible phagocytotic activity of astrocytes. Our results show that the demonstration of GFAP by the Sternberger method is a valuable aid in establishing astrocytic glial differentiations and modulations in complex tumour-like lesions such as nasal gliomas.

Myocardial lipid peroxidation in rats after chronic alcohol ingestion and the effects of different antioxidants.

The effects of chronic intake of dietary alcohol on myocardial peroxidation (measured as formation of diene conjugates), reduced glutathione content, and morphology and the protective actions of different antioxidant compounds (vitamin E and (+)-cyanidanol-3) were studied in rats. Alcohol, comprising more than 30% of the dietary calorie content, was administered to rats for six weeks. Compared with the controls, the left ventricle of the alcoholic animals had an increased diene conjugate content (5.4(0.5) vs 4.3(0.6) optical density X g wet weight-1) and a slightly, but not significantly, decreased glutathione content (1.62(0.05) vs 1.66(0.07) mumol X g wet weight-1). Simultaneous administration of antioxidants (vitamin E or (+)-cyanidanol-3) prevented the pathological changes in diene conjugates and significantly increased the glutathione content compared with the alcoholic rats. Electron microscopy showed remarkably few ultrastructural abnormalities in the myocardium of alcoholic animals fixed by vascular perfusion. The data are consistent with the hypothesis that reactive oxygen radicals are involved in the ethanol induced biochemical changes and that the antioxidants could prevent the increased formation of peroxides in the myocardium.