[Heterotopic pancreas in gallbladder]. / Páncreas heterotopico en vesícula biliar, un hallazgo incidental.

UNLABELLED: Heterotopic pancreas in the gallbladder is uncommon.condition and its clinical presentation is as an intramural nodule near the cystic duct, with an incidental finding in most cases. OBJECTIVE: to update Heterotopic pancreas condition in the gallbladder. We reported a case of a 18-year-old female, suffering with biliar colic who was operated on with diagnosis of acute cholecystitis. Six mixed gallstones were found in the surgery and the gallbladder was distended and edematous with serosal exudate and the serosa appeared discolored and hemorrhagic. The wall was thickened with edema and hemorrhage. Microscopic findings were: congestive mucosa , edema, acute inflammatory cells and fibrin deposition on the wall and a subtle (myo)-fibroblastic proliferation. The nodule was diagnosed as macroscopic heterotopic pancreatic tissue in the gallbladder wall thickness, without neoplastic changes. Clinical presentation as acute cholecystitis has been rarely reported in the presence of heterotopic pancreatic tissue.

Localization and expression of integrin subunits in the embryoid bodies of F9 teratocarcinoma cells.

F9 embryonal carcinoma cells can differentiate in vitro into either parietal (PE) or visceral (VE) endoderm, depending upon specific retinoic acid (RA) treatment and growth conditions. In differentiated aggregates of F9 cells (EB), the VE is a polarized monolayer surrounding a core of undifferentiated cells. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins to form a basal lamina under the newly formed epithelium. All these changes are likely to involve integrin expression and organization. In this study we have analyzed the spatio-temporal changes in the pattern and level of expression of beta1, beta4, alpha5, alpha6A, and alpha6B integrin subunits. We found that the organization of the VE monolayer in F9 aggregates involves both qualitative and quantitative changes in integrin expression. beta1 is downregulated and accumulates in the forming epithelium. The same occurs for alpha5, although its location on the surface of the aggregate appears to be transient as in fully differentiated EB its distribution is uniform. beta4 and alpha6A are also mainly localized in the VE but they are undetectable in undifferentiated aggregates and their expression is induced by RA treatment. An important exception is represented by alpha6B whose distribution and expression remain almost unchanged throughout treatment.

In vitro and in vivo antisense-mediated growth inhibition of a mammary adenocarcinoma from MMTV-neu transgenic mice.

Oncogene-bearing transgenic mice develop various kinds of tumors depending on both the regulatory sequences and the specific oncogene used. These mice not only help to clarify the pathogenetic pathways leading to tumor formation, but can also be useful as models to test novel therapeutic strategies, including gene therapy. We have previously reported the establishment of an MMTV-neu (ErbB-2) transgenic mouse lineage, in which 100% of females develop breast tumors with many features similar to their human counterparts; these tumors are due to the over-expression of the activated rat neu oncogene in the mammary gland. From one such mouse we established a cell line of mammary adenocarcinoma named MG1361. We report here that the growth of this cell line can be inhibited in vitro and in vivo by transfection of a plasmid vector carrying an antisense anti-neu construct. This inhibitory effect is specific, as it is related to the expression of the antisense transgene (determined by RT-PCR), and to a reduction in neu mRNA and protein, as determined by Northern and Western blot analyses. Moreover, inoculation of cells carrying the antisense or the control vector in nude mice demonstrated that the morphological and biochemical effects elicited by the antisense construct resulted in a significantly slower rate of in vivo growth of tumor xenografts. Finally, significant mammary tumor growth inhibition was obtained after liposome-mediated direct inoculation of the same antisense vector in tumors spontaneously arising in MMTV-neu mice. Taken together, these findings suggest that targeting neu expression by an integrated large anti-neu antisense segment affects the in vivo growth of these tumors.

Regulated expression of fibronectin, laminin and related integrin receptors during the early chondrocyte differentiation.

We have investigated the expression and localization of fibronectin, laminin, and their receptors, and we used an in vitro chick chondrocyte differentiation model to define a time hierarchy for their appearance in early chondro-genesis and to determine their role in the cell condensation process. By serum fibronectin depletion/reconstitution, or GRGDSP peptide competition experiments, we show that fibronectin contributes to the initial cell-cell interactions that occur during condensation. In later stages, a down-regulation of both fibronectin and of its alpha5beta1 integrin receptor occur, as demonstrated by mRNA and protein kinetics. Immunolocalisation studies suggest that the reduction of fibronectin in discrete areas is involved in local activation of the cell differentiation program. Furthermore, we show that laminin is expressed during the in vitro cell condensation process in areas that are negative for fibronectin staining. The types of laminin as well as the timing of expression have been determined by northern blot and RT-PCR analyses. The highest levels of expression are coincident with maximal cell aggregation. The alpha3beta1 laminin receptor, highly expressed in dedifferentiated cells, follows later on the ligand trend. During in vitro chondrogenesis, a down-regulation in the B isoform, and an up-regulation of the A isoform, of the alpha subunit of the alpha6beta1 laminin receptor occurs. Immunolocalisation studies suggest that laminin is involved in the definition of differentiating areas as opposed to non differentiating areas of the condensed region, i.e. the periphery, which eventually gives rise to the perichondrium.

Modulation of alpha 6/beta 1 integrin expression during differentiation of F9 murine embryonal carcinoma cells to parietal endoderm.

In vitro differentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in beta 1 integrin expression, its maturation, and organization on the cell surface in association with alpha 6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the beta 1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of beta 1 expression during F9 cells differentiation occurs first at post-translational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of alpha 6B and its association to a high molecular weight protein, while alpha 6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the beta 1 and alpha 6B integrin subunits from cell-cell contacts to focal contacts where they colocalize with vinculin. On the contrary alpha 6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two alpha 6 isoforms.

Osteoblastic cells from rat long bone. II: Adhesion to substrata and integrin expression in primary and propagated cultures.

Propagation in vitro of rat tibial osteoblasts (ROB) is accompanied by increased expression of the early osteogenic marker alkaline phosphatase (AP) and maturation of the osteogenic phenotype. In order to establish the pattern of the integrin expressed in ROB during progression to the mature osteoblastic phenotype, we have used biosynthetic, immunoblotting and immunohistochemical assays. We immunoprecipitated from osteoblasts, expanded for 1.5- and 7.5-doubling, alpha 5 beta 1, alpha v beta 3, alpha 3 beta 1, alpha 6 beta 1 and alpha 1 beta 1 integrin heterodimers; furthermore beta 5, alpha 2 and alpha 4 chains were detected by immunoblots and indirect immunofluorescence. alpha v, alpha 1, alpha 6 subunits in most cells, and beta 3 and beta 1 subunits in a minority, were found to be associated with adhesion plaques in osteoblasts of 1.5-, 4.5- and 7.5-doubling grown in the presence of FCS, while all other subunits stained diffusely all the cells. Adhesion to fibronectin (FN), laminin (LN), collagen type I (COL I) and III(COL III) by ROB at different doubling (1.5-11) was dependent on substratum concentration, and after 2.5 h at 55 nM 60% of the cells adhered to all substrata. Arg-Gly-Asp-Ser (RGDS) containing peptides inhibited adhesion of cells differentially, according to substratum; no dependence on extent of progation in vitro was observed. In conclusion, ROB cultured in vitro for 1.5- to 11-doubling had an unchanged pattern of expression of integrin subunits, heterodimer association and cellular distribution. Adhesion specificity and affinity were also unchanged. These results suggest that the phenotypic maturation, detected as an increase in AP expression, is not accompanied by major changes in the potential for cell-matrix interactions, and does not correspond to changes in the type of integrin subunits expressed by osteoblasts.

NAD+-dependent internalization of the transmembrane glycoprotein CD38 in human Namalwa B cells.

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD+ (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium-releasing activity of its intermediate product cADPR. Incubation of CD38+ human Namalwa B cells with external NAD+ elicited extensive membrane down-regulation of CD38 and its internalization in non-clathrin-coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD+-dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.

Synergistic cytotoxic effects of recombinant human tumor necrosis factor and Etoposide (VP16) or Doxorubicin on A2774 human epithelial ovarian cancer cell line.

Recombinant human tumor necrosis factor (rHuTNF) is a cytokine, with some antitumor activity, released by stimulated monocytes-macrophages. In vivo and in vitro cytotoxicity studies testing the effectiveness of rHuTNF alone or in combination with chemotherapeutic agents have been carried out. We have evaluated the direct cytotoxic effect of rHuTNF on a human epithelial ovarian cancer cell line in vitro (A2774), alone or in combination with Etoposide (VP16) or Doxorubicin (Doxo), some topoisomerase II (Topo II) targeted drugs, or in combination with Cisplatin (CDDP), a not Topo II interactive drug. Our results suggest that rHuTNF is directly cytotoxic and that it is also able to induce a potentiation of VP16- or Doxo-cytotoxicity, but it is unable to potentiate CDDP-cytotoxicity. These data represent a reasonable basis for combining rHuTNF with Topo II inhibitors within phase I studies. The combination regimen could be tested in ovarian cancer patients.

8-methoxycaffeine-induced protein-associated DNA breaks in isolated L1210 cell nuclei.

8-methoxycaffeine (8-MOC) is a caffeine derivative more potent than its parental compound in inducing chromosomal aberrations. 8-MOC has been postulated to produce chromosomal aberrations by DNA topoisomerase II inhibition. The effect of 8-MOC on nuclear DNA were studied by alkaline elution experiments and compared with those of Ellipticine and Adriamycin (ADR). Like Ellipticine and ADR, 8-MOC induced single strand breaks (SSBs), double strand breaks (DSBs), and DNA-protein cross-links (DPCs) in a bell-shaped manner with respect to drug concentration. As in the case of ADR and Ellipticine, 8-MOC induced equal SSB and DPC frequencies. These results could suggest that 8-MOC induces DNA breaks by interacting with DNA topoisomerase II or with a similar DNA metabolism enzyme.