A simple method for the isolation and culture of epithelial and stromal cells from benign and neoplastic prostates

Objectives. Current primary prostate cell culture techniques use an overnight digestion or extensive media preparation. In this report, we describe a method for the culture of benign and neoplastic cells from human prostatectomy specimens that is rapid and contains no undefined factors in the medium.Methods. Characterization of the human cultured prostate cells was performed using immunohistochemical methods and monoclonal antibodies AE1/AE3 and cytokeratin 8, as well as monoclonal antibodies against prostate-specific antigen (PSA). Polymerase chain reaction was used to measure the exclusive epithelial and stromal cell products, c-met and hepatocyte growth factor (HGF), respectively. Electron microscopy was performed to assess the cell junctions and morphologic features of epithelial cells. Optimum cell growth in different media was tested using a cell replication assay.Results. Microscopic evidence revealed that the cells demonstrate typical epithelial morphology, with polyhedral cells forming tight junctions in a continuous monolayer. Desmosomes were present in electron micrographs of epithelial cells. The cultured epithelial cells described in this report also demonstrate positive cytokeratin staining. The epithelial cells reacted positively with PSA antibody, indicating that the cells retain their secretory role in cell culture for a limited period. Epithelial cells expressed the HGF receptor, c-met; stromal cells secreted HGF. Insulin, transferrin, and selenium increased the growth of cells in the chemically defined media, compared with minimum essential media (MEM) and Ham's F12.Conclusions. In summary, essentially pure cultures of prostate stromal or epithelial cells have been established using simple isolation and culture methods. These cells will be useful for the investigation of related growth factors, such as insulin-like growth factor I and insulin-like growth factor II, and in understanding the basis for stromal-epithelial cell interactions.     

Interleukin 4 receptor targeted cytotoxicity: genetic construction and in vivo immunosuppressive activity of a diphtheria toxin-related murine interleukin 4 fusion protein.

The interleukin 4 (IL 4) receptor is expressed on various cells of the immune system, including T and B lymphocytes, macrophages and mast cells. We have constructed a recombinant protein, DAB389-mIL 4, that is composed of the enzymatically active and membrane translocation domains of diphtheria toxin fused to murine IL 4. We demonstrate that this fusion toxin selectively inhibits protein synsthesis in eukaryotic cells which express the murine IL 4 receptor. The cytotoxic potency of this fusion toxin is shown to be directly proportional to the reported number of IL 4 receptors on the surface of target cells. Since the action of DAB389-mIL 4 can be blocked with either excess mIL 4 or antibody to mIL 4, we conclude that its entry into target cells is mediated through the mIL 4 receptor. A mutant form of DAB389-mIL 4, DA(197)B389-mIL 4, in which the fragment A-associated ADP-ribosyltransferase is inactive, is not cytotoxic to murine IL 4 receptor-bearing cells. Finally, we demonstrate that DAB389-mIL 4 administered subcutaneously to DBA/2 mice results in suppression of delayed-type hypersensitivity (DTH); whereas, the non-toxic DA(197)B389-mIL 4 fails to dampen the DTH response.     

Shoulder instability: impact of glenohumeral arthrotomography on treatment

We used arthrotomography to study the glenoid labrum in 114 patients. Sixty-nine of the patients had anatomic instability of the shoulder (including recurrent dislocation and subluxation of the shoulder), and 45 patients had functional instability of the shoulder (denoted by chronic pain, clicking of the joint, and the sensation that an unstable condition exists without the objective signs of it). Labral tears were revealed arthrotomographically in 86% of the patients with anatomic instability, while only 40% of the patients with functional instability had labral abnormalities, and these were primarily of minor severity. Fifty-six patients (44 of whom had anatomic instability; 12, functional instability) required surgery. The surgical findings were correlated with the arthrotomographic findings, and no false-positive results were revealed. However, arthrotomography demonstrated only part of the pathologic condition of two patients. These results confirm that there is a strong correlation between labral pathologic conditions and anatomic instability of the shoulder. Arthrotomographic studies have a great impact on the selection of therapy in cases of both anatomic and functional instability of the shoulder.     

Kinetic immunodominance: functionally competing antibodies against exposed and cryptic epitopes of Escherichia coli beta-galactosidase are produced in time sequence.

The murine antibody response to Escherichia coli beta-galactosidase (GZ) was analyzed in vivo and in vitro by focusing on two families of antibodies that exert distinct conformational/functional activity on the antigen. Activating antibodies--defined by their capacity to increase the enzymatic activity of defective GZ produced by mutant strains of E. coli--are detected early after secondary challenge. Inhibiting antibodies, which interfere with antibody-mediated enzyme activation, appear later and cause the abrupt fall of activating titer, a scenario suggesting either idiotype/anti-idiotype interaction or opposite pulsions exerted on the antigen molecule. Supporting the latter mechanism, the confrontation of mAbs of the two families produced classical competitive inhibition curves when the readout was enzyme activation, although they recognize two different epitopes of the same molecule: the activating mAb a quaternary conformation-dependent site of wild-type GZ, the inhibiting mAb a sequential determinant exposed only in denatured or in defective enzyme. The different timing of generation of these antibodies during the response may depend on a processing step necessary for unfolding of native antigen and consequent display of certain cryptic epitopes before they can trigger specific B cells. A picture emerges where the response to the various epitopes of a complex antigen is sequentially connected and where the uptake by antigen-presenting cells of antigen complexed with antibodies specific for the exposed epitopes may favor revelation of the cryptic ones.