Immortalized bovine pancreatic duct cells become tumorigenic after transfection with mutant k-ras.

Mutation of the K-ras gene is thought to be an early and important event in pancreatic carcinogenesis. In order to study the role of this molecular alteration in the transition from the normal to the neoplastic pancreatic cell, bovine pancreatic duct cells were first immortalized by SV40 large T antigen (Ag) complementary (c)DNA transfection and then transfected with a mutated K-ras gene. As did primary duct cells, the immortalized duct cells (more than 100 passages) expressed cytokeratins, carbonic anhydrase type-II, cystic fibrosis transmembrane conductance regulator (CFTR), and multidrug resistance (mdr). They grew as a single layer after transplantation under plastic domes and formed three-dimensional structures resembling ducts when grown on Matrigel. Cell growth was stimulated by insulin, epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, but cells did not respond to gastrin and CCK-8. They did not form colonies in soft agar nor did they form tumors in nude mice. Immortalized cells transfected with mutated K-ras acquired the ability to form tumors after orthotopic injection into the nude mouse pancreas. It is concluded that SV 40 immortalized bovine pancreatic

Increased in vivo mitogenicity of anti-TCR/CD3 monoclonal antibody through reduced interaction with Fcgamma receptors.

Since its initial clinical use in 1980, anti-TCR/CD3 monoclonal antibody (mAb) has been shown to be a potent immunosuppressive agent in the prevention of renal allograft rejections. However, toxic side effects caused by release of cytokines, predominantly from activated CD4+ T-cells, remain a major problem with the use of these reagents. Previous work has shown that this activation is mediated via antibody binding to Fcgamma receptors (FcgammaR) on host effector cells. In the present study, we have demonstrated in an in vivo mouse model that the anti-TCR/CD3 mouse mAb 7D6, as well as that from rat (17A2) and hamster (H57-597), induce a gradual depletion of host CD4+ T-cells without any apparent proliferative effects on the cells. In contrast, when treatment with these mAbs was combined with a mAb (2.4G2) that blocks the low-affinity Fcgamma receptors (FcgammaRII/III), we found that the in vivo actions of the anti-TCR/CD3 mAbs resulted in a significant expansion, rather than depletion, of CD4+ cells. The ability of 2.4G2 to reduce mAb 7D6-FcgammaR interaction was directly demonstrated in an in vitro assay system in which 2.4G2 partially suppressed 7D6-mediated T-cell responses. Taken together, our results have shown that some so-called "nonmitogenic" anti-TCR/CD3 mAbs in fact possess potent activating properties and that their mitogenic potential can be exposed by reducing their interaction with FcgammaR on host effector cells.

K-ras point mutations are rare events in premalignant forms of Barrett's oesophagus.

OBJECTIVE: In Barrett's adenocarcinomas, in contrast to squamous oesophageal carcinomas, K-ras point mutations are thought to be a frequent event. The frequency of K-ras point mutations in premalignant forms of Barrett's oesophagus (metaplasia, dysplasia) leading to adenocarcinoma with increased risk is currently not known. To establish the frequency of K-ras mutations in premalignant forms of Barrett's oesophagus, we investigated oesophageal biopsy specimens with Barrett's metaplastic and dysplastic epithelium for point mutations in the K-ras gene/codons 12, 13. DESIGN: A total of 412 biopsies from patients with Barrett's oesophagus were histologically classified into biopsies with metaplasia (n = 252), dysplasia (n = 105) and adenocarcinoma (n = 11), as well as biopsies distant from disease (normal, n = 37 and hyperplastic squamous epithelium, n = 7). METHODS: DNA from biopsy specimens was amplified by polymerase chain reaction (PCR) with a modified primer for generating a restriction site in the case of wild type in codon 12. Wild-type or point mutations in the K-ras gene/codons 12, 13 were detected by restriction fragment length analysis of the PCR products. RESULTS: Point mutations in K-ras/codon 12 were found in 9 biopsies (n = 1 in metaplasia, n = 4 in dysplasias, n = 4 in adenocarcinomas). All the other biopsies showed the wild type of K-ras/codon 12. No K-ras/codon 13 mutation (GGCgly-->GACasp) was observed. CONCLUSION: Mutations in K-ras/codon 12 were rarely found in premalignant forms of Barrett's oesophagus. Whereas the screening for K-ras point mutations in metaplastic sites of Barrett's epithelium seems not to be of practical value, the screening for mutations in dysplastic lesions might be helpful to estimate the individual risk for progression of Barrett's epithelium to adenocarcinoma. A further evaluation in larger numbers of patients is needed.

Expression and function of receptors for extracellular matrix proteins in human ductal adenocarcinomas of the pancreas.

Extracellular matrix proteins (ECM) may influence cellular differentiation via their receptors, the integrins. We recently presented evidence that ductal adenocarcinomas of the pancreas are able to produce ECM in vitro and in vivo (Br J Cancer 1994;49:144-51). This study examines whether pancreatic carcinoma cells are able to interact with ECM by expressing functionally active integrins. In eight human pancreatic tumor cell lines (AsPC-1, BxPC-3, CAPAN-1 and -2, PANC, PaTu-2, -3, and -44) and six xenografted tumors RNA and protein expression of integrin subunits alpha 5 (fibronectin receptor), alpha 6 (laminin receptor), and alpha V (vitronectin receptor) was investigated. In addition, alpha 1-alpha 6 and alpha V as well as beta 1-beta 4 were studied by fluorescence-activated cell sorter analysis. Integrin function was tested by attachment assays. alpha 2, alpha 3, alpha 5, alpha 6, and alpha V as well as beta 1, beta 3, and beta 4 were expressed, at both the RNA and the protein level, by all pancreatic tumors in vitro and in vivo. The tumor cell lines showed dose dependent adhesion to collagen, fibronectin, and laminin. Integrin expression could be modulated in part by serum depletion. None of the tumors showed alpha 1, alpha 4, and beta 2. Tumor cell differentiation or production of ECM was not correlated with integrin expression. Pancreatic ductal adenocarcinomas express a certain pattern of functionally active integrins that enable interaction with most matrix proteins, e.g., collagen, fibronectin, and laminin. Pancreatic adenocarcinomas possess both the ligands and the receptors of the extracellular matrix network. We speculate that through both classes of molecules, the tumor cells may bind to fibroblasts and probably stimulate their growth. However, it remains unclear whether and how integrin-ECM interaction exerts an influence on tumor cell differentiation.

[Restriction enzyme mismatch polymerase chain reaction in the demonstration of Ki-ras-oncogene mutation in carcinoma of the pancreas]. / Restriktionsenzym-Mismatch-Polymerase-Kettenreaktion zum Nachweis von Ki-ras-Onkogen-Mutationen beim Pankreaskarzinom.

A pilot study was undertaken to test whether combining the polymerase chain reaction with restriction fragment length polymorphism is suitable for the routine diagnosis of carcinoma of the pancreas. The method makes it possible to recognize point mutations in codon 12 of the Ki-ras oncogene. 60 cytological specimens from the pancreatobiliary tract, the bronchopulmonary system (by bronchoalveolar lavage or from pleural effusion) and ascites were tested. Results from nine pancreas carcinoma cell lines served as control. A PCR product was successfully amplified in all cell lines and 47 of the clinical specimens. In all eight samples in which a mutated Ki-ras oncogene was demonstrated, there was at least a suspicion of malignancy by cytological examination. A mutation was also found in four of five pancreas carcinomas. But no mutation was found in one, clinically certain, case of pancreas carcinoma. The described method is an elegant and, most of all, rapid means to complement and optimize any cytological diagnosis.

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins.

Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV > laminin = fibronectin = vitronectin > collagen type III > undulin > collagen type I) or protein level (collagen type IV = collagen type III > vitronectin > laminin > collagen type I = fibronectin > undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested.

Isolation, culture, and characterization of human pancreatic duct cells.

To establish a suitable control for pancreatic tumor cell lines, we have isolated and cultured primary human pancreatic duct cells from transplant donors. Duct cells were isolated by dissecting the main pancreatic duct and first-degree branches and enzymatic digestion. Aggregates of cells were cultured for 1 up to 5 weeks and monitored for changes in morphology and growth by phase contrast microscopy. Contaminating fibroblasts were mechanically removed from day 4 on and by cloning of epithelial cells. Cultured cells were characterized by phase contrast microscopy, electron microscopy, and immunofluorescence with antibodies against intermediate filaments (cytokeratins, vimentin, desmin), mucins (Du-Pan-2, CA 19-9), carbonic anhydrase II, acinar cell enzymes (amylase, lipase, trypsin), and islet cells. About 90% of the cultured cells could be identified as ductal epithelial cells by their expression of cytokeratins, mucins, and carbonic anhydrase II. These cells showed the ultrastructural features of duct cells. After 3-5 weeks of culture, most of the cultured cells showed co-expression of cytokeratins and vimentin in addition to duct cell markers. About 10% of cells were contaminating fibroblasts (vimentin positive, cytokeratin negative). The cultured normal human duct cells as the postulated cells of origin of the pancreatic adenocarcinoma may serve as a useful control for cultured pancreatic tumor cell lines.

[The detection of antibodies against D-penicillamine. 3. Detection of antibodies against D-penicillamine in the serum of patients with Wilson's disease treated with D-penicillamine--preliminary results]. / Zum Nachweis von Antikörpern gegen D-Penicillamin. 3. Mitteilung: Nachweis von Antikörpern gegen D-Penicillamin im Serum von Patienten mit Morbus Wilson unter der Behandlung mit D-Penicillamin - Vorläufige Ergebnisse.

121 serum samples from 54 patients with Wilson's disease were tested for antibodies against D-penicillamine by indirect immunofluorescence (DASS-system). IgG antibodies were found in 44 serum samples from 16 patients (31% of all patients). The incidence of serum antibodies was higher in patients with side effects during therapy with D-penicillamine (10 of 13 patients 77%) compared to 6 of 39 15% in patients without side effects. The titre of antibodies was higher in patients with side effects. The antibodies bound complement demonstrated by double immunofluorescence. These observations indicate that complement binding IgG antibodies to D-penicillamine are involved in pathogenesis of side effects during therapy with D-penicillamine.

[Detection of antibodies against D-penicillamine. 2. Experimental animal proof of production of antibodies against D-penicillamine using indirect immunofluorescence with a DASS system]. / Zum Nachweis von Antikörpern gegen D-Penicillamin. 2. Mitteilung: Nachweis tierexperimentell erzeugter Antikörper gegen D-Penicillamin durch indirekte Immunfluoreszenz mittels DASS-System.

Antibodies against D-penicillamine conjugates produced in animals be proved by indirect immunofluorescence by means of the DASS system (defined antigen substrate spheres). The artificial substrate (Sephadex G-25) has not been bound to the D-penicillamine conjugates used for immunization, but only to D-penicillamine, which justifies the assumption that antibodies against D-penicillamine could be registered and proved by the immunofluorescence test mentioned.

[The detection of antibodies against D-penicillamine. 1. Coupling of D-penicillamine to various carriers and generation of antibodies against D-penicillamine conjugate]. / Zum Nachweis von Antikörpern gegen D-Penicillamin. 1. Mitteilung: Kopplung von D-Penicillamin an verschiedene Träger und Erzeugung von Antikörpern gegen D-Penicillamin-Konjugate.

D-Penicillamin as 2-substituted 5,5-Dimethyl-thiazolidine-4-carbonic acid has been bound to poly-L-lysin, human serum albumin and bovine gamma globulin by means of a water-soluble carbodiimid. The anti-bodies generated at animal tests by immunization of guinea pigs had been proved by means of the radial immune diffusion acc. to Ouchterlony [10].

[Serum immunoglobulins and third complement component in and after acute non-A/non-B hepatitis (author's transl)]. / Serum-Immunglobuline und dritte Komplementkomponente während und nach akuter Non-A/Non-B-Hepatitis.

The serum levels of IgG, IgA, IgM, IgD and IgE and of the third complement component (C 3) were determined by the single radial immunodiffusion method in 69 serum samples of 34 female patients during (2nd and 4th week) acute non-A/non-B hepatitis and 2 years after infection. The levels were compared with those of circulating immune complexes measured by polyethylene glycol precipitation method. The levels of immunoglobulins and C 3 were similar to those of healthy persons. During the course of disease there were no significant relations with exception of an increase of IgD levels in patients with chronic course. The positive correlation of the levels of IgM and immune complexes at the first and second serum sample (r = 0,5914, r = 0,6366 respectively, p less than 0,001) could not be verified in patients with noncomplicated course (r = 0,203 8, n. s.) but it was highly significant in patients with chronic course (r = 0,9429, p less than 0,001). Determinations of immunoglobulins and immune complexes may therefore be prognostically helpful in patients with non-A/non-B hepatitis.

Storage of the complement components C4, C3, and C 3-activator in the human liver as PAS-negative globular hyaline bodies.

Liver biopsies of a 58-year-old clinically healthy patient with a hepatomegaly and intracisternal PAS-negative globular hyaline bodies were immunofluorescent-optically examined for the content of the complement components C 1 q, C 4, C 9, C 1-inactivator, C 3-activator. Further examinations were performed for fibrinogen, IgG, IgA, IgM, IgD, IgE, L-chain (type chi and lambda), alpha 1-antitrypsin, alpha 1-fetoprotein, alpha 1- and alpha 2-glycoprotein, cholinesterase, ceruloplasmin, myoglobin, hemopexin, HBsAg and HBsAg. Th inclusion bodies reacted with antisera against the complement components C 4, C 3 and C 3-activator, as also identified by double immunofluorescence. Probably this is a disturbance of the protein metabolism of the liver cell with abnormal complement storage in the presence of normal total complement and normal complement components in the serum.

[The evidence of antibodies against motor endplates of the skeletal muscle in myasthenia gravis with the double immune-fluorescence technique (author's transl)]. / Doppelimmunfluoreszenzoptischer Nachweis von Antikörpern gegen motorische Endplatten des Skeletmuskels bei Myasthenia gravis.

Studies were undertaken on sera of 66 patients for detecting antibodies to motor endplates using a double immunofluorescence method according to Sondag-Tschroots et al. Rat tongue was used as antigenic substrate. Strong positive results at a serum dilution of 1: 20 were found only in patients with myasthenia gravis (15 out of 42 patients approximately equal to 35.7%). Titres between 1: 20 and 1: 1,280 occurred. 3 of 8 patients with chronic active hepatitis and with circulating antibodies to smooth muscle and 2 of 4 patients with autoimmune disorders and weakly positive antibodies in their 1: 20 diluted serum. The used rat tongue as antigenic substrate seems to be more suitable than diaphragm for detecting of such and other antibodies.

[Standardization of indirect immunofluorescence for detection of autoantibodies (author's transl)]. / Ringversuch zum Nachweis von Autoantikörpern durch indirekte Immunfluoreszenz.

Seven laboratories made a chessboard titration with a serum containing antimitochondrial antibodies and two conjugates labeled with fluorescein isothiocyanate. The variation of the plateau titer and the titer of the serum as well as the plateau-end point was slight with the exception of one case. The standardization of indirect immunofluorescence needs a close contact between the laboratories, the availability of reference sera and conjugates, and similar microscopic equipment.

Ontogeny of the hypothalamo-neurohypophysial system in rats--an immunohistochemical study.

In fetal rats neurophysin has been visualized immunohistochemically first at the 16th gestation day in perikaryons of the supraoptic nucleus, followed by the median eminence and the neurohypophysis at the 17th day, and the paraventricular neurons at the 19th day. The external zone of the median eminence contains abundantly immunoreactive fibres at the first days post partum. In the perikaryons of the suprachiasmatic nucleus immunoreactive material appears after the 3rd day of postnatal development.