Improved methods and standards for telomerase detection: quantitative histopathology using antibody staining.

Evaluation of telomerase as an early detection biomarker for cancer has been hindered by a lack of reliable methods and standards for in situ histochemical measurement. Improved histochemical methods for measuring telomerase could expedite the acceptance of telomerase as a biomarker for use in diagnostic and clinical applications. The lack of a crystal structure for telomerase coupled with high variability in the antibodies available for immunohistochemical analysis has led to confusion in the literature regarding the binding specificity of these antibodies. We have developed an automated fluorescence microscopy protocol to assess the specificity of three fluorescently labeled telomerase antibodies and to quantify telomerase in cultured human tumor cells and in human fibroblast cells as a control. Significant differences in staining intensity and distribution were observed. Fluorescence measurements in these cell lines were compared to telomerase measured by the telomerase repeat amplification protocol, reverse transcription-polymerase chain reaction, and flow cytometry. This combination of measurements ensured a more complete quantitation of telomerase levels in each of the cell lines and could also be used as a model for validation of other biomarkers for clinical use.

Osteoblast cell membrane hybrid bilayers for studying cell-cell interactions.

Osteoblast-like cells were grown on a surface that presents cell membrane components to the cells in culture. The culture surface was a bimolecular layer formed by the interaction of osteoblast plasma membrane vesicles with an alkanethiol monolayer. The potential of these osteoblast-membrane hybrid bilayers for promoting osteoblast adhesion, growth and differentiation was examined. UMR-106 osteoblast-like cells cultured on these surfaces are normal in appearance, and in the presence of serum, proliferate as well or better than on control surfaces. The level of alkaline phosphatase production in the presence and absence of serum suggests that the osteoblast-like cells retain their differentiated phenotype, and appear to respond to the cell surface ligands presented by the osteoblast-membrane biomimetic surface. These observations suggest that biomimetic membrane films prepared from osteoblast cell membranes support osteoblast cell growth, allow the cells to maintain their differentiation state and may be suitable as a model system to probe cell-cell interactions.

Effect of hyaluronidase on brain extracellular matrix in vivo and optic nerve regeneration.

In the rat, intracerebral injection of bacterial hyaluronidase resulted in the almost complete disappearance of hyaluronic acid (HA) and glial hyaluronate-binding protein (GHAP) from cerebral hemispheres, brain stem, and cerebellum (but not from optic nerves and chiasm) starting 2-3 hr after the injection. HA and GHAP reappeared throughout the brain in characteristic patches 2-3 days after the injection. The patches gradually became confluent and after 12 days the brain appeared virtually normal. In normal rat optic nerve, staining for HA and GHAP ceased abruptly in the region of the lamina cribrosa. The retina was completely negative. HA and GHAP disappeared from hyaluronidase-injected optic nerve, chiasm, and contralateral optic nerve. In hyaluronidase-injected crushed optic nerves, regenerated axons were able to grow for short distances (about 500 microns) into the distal stump undergoing Wallerian degeneration. No such growth was observed in saline-injected controls.

Extracellular matrix in regenerating rat sciatic nerve: a comparative study on the localization of laminin, hyaluronic acid, and chondroitin sulfate proteoglycans, including versican.

Using a glial hyaluronate-binding protein as a probe, monoclonal antibodies against versican and ABC digested chondroitin sulfate proteoglycans, and polyclonal antibodies against laminin, we localized these extracellular matrix (ECM) components in the endoneurium of the adult rat sciatic nerve. During Wallerian degeneration caused by nerve crushing, the staining pattern of these ECM elements changed dramatically. In the first stages and up to 5 days after injury, the tubular endoneurial structures remained the same as in control nerves. Ten days after crush, the bands of Büngner formed by proliferating Schwann cells in the distal stump of crushed nerves stained diffusely for hyaluronate, laminin, and chondroitin sulfate. Regenerating axons were demonstrated in this location by double-labeling experiments with neurofilament antibodies. Conversely, staining with antibodies against versican, a hyaluronate binding proteoglycan, was reduced and the bands of Büngner were not stained. After 30 days the endoneurial tubes made their reappearance and, as in normal nerve, they stained for hyaluronate, laminin, versican, and chondroitin sulfate. It is concluded that proliferating Schwann cells in peripheral nerve undergoing Wallerian degeneration are capable of producing several endoneurial ECM constituents, versican being a notable exception.

Effect of highly concentrated gels of sodium hyaluronate on early phases of regeneration in the transected and tubulized rat sciatic nerve.

Silicone regeneration chambers prefilled with sodium hyaluronate (HA) gels of different molecular weight (MW) and at different concentrations were used to tubulize the transected rat sciatic nerve. After two weeks, the tissue cables bridging the severed nerve ends within the silicone tubes were examined. Low MW HA gels had no significant effect while high MW HA gels reduced the diameter of the growing bridges.