Effects of daunomycin implants on filtering surgery outcomes in rabbits.

PURPOSE: To evaluate the effects of a novel daunomycin (DM) implant on intraocular pressure (IOP), bleb survival, and anterior segment complications when administered during filtering surgery on New Zealand White rabbits. METHODS: Implants were prepared by covalent coupling of DM to a high molecular weight hyaluronic acid (HA) and fabricated into solid implants containing 250, 65, and 25 microg of DM. Full thickness sclerostomies were performed, and rabbits received no implant (control), placebo implant containing HA, or an implant containing HA-DM conjugate at the time of surgery. Rabbits were then followed for 30 days to assess change in IOP, bleb survival, and anterior segment associated complications. RESULTS: In vitro, the release of DM from the implants was a first order process with a half-life of 51 h. In control rabbits, rabbits receiving placebo implant, and rabbits receiving HADM (250 microg) implants, the mean IOPs on day 3 were 11.1 +/-1.6, 10.8 +/- 2.7, and 14 +/- 0.98 mm of Hg, respectively. On days 5 through 9, IOP in the control and placebo-implanted groups returned to preoperative levels. However, in rabbits receiving 250 microg of conjugated DM, mean IOP on day 7 was reduced from preoperative levels by 11.8 +/- 3.2 mm of Hg (P < 0.05). This reduction in IOP was significantly different (P < 0.05) from both control and placebo implant groups, and IOPs remained at these levels until studies were terminated on day 30. In control and placebo-implanted rabbits, bleb size started decreasing on day 1, and by day 7, no blebs were observed. In rabbits receiving 250 microg HA-DM implants, mean bleb survival time was greater than 30 days. The DM-induced reduction in IOP and enhanced bleb survival was dose-dependent. In rabbits receiving 65 microg of conjugated DM, lOPs were significantly reduced through day 30; however, at times beyond day 19 there was a gradual rise in mean IOP as filtering procedures in individual animals began to fail. Mean bleb survival time was greater than 30 days for implants containing 65 microg of conjugated DM. In rabbits receiving HA-DM implant containing 25 microg of DM, IOPs were not significantly different from preoperative levels beyond day 9, and no significant enhancement in bleb survival was observed in these animals. Comparison of HA-DM conjugated implants to those containing equal doses of free DM demonstrated that the mean IOP change at 30 days were similar; however, there was a reduction in anterior segment complications associated with the use of HA-DM conjugated implants. CONCLUSIONS: This study provides evidence that the controlled release of DM when conjugated to HA can significantly improve the success of filtering procedures. The maintenance of ocular hypotension and bleb survival along with the reduction in anterior segment complications supports the idea that HA-DM conjugate will be more efficacious than the use of free DM in improving the success rate of filtering surgery.

Inhibition of cell proliferation on lens capsules by 4197X-ricin A immunoconjugate.

PURPOSE: To evaluate the cytotoxicity of immunotoxin 4197X-ricin A (4197X-RA) and its ability to inhibit protein synthesis and human lens epithelial cell (LEC) proliferation on the inner surface of the lens capsule. SETTING: Houston Biotechnology, Inc., The Woodlands, Texas. METHODS: A cell culture system was established using human LECs as a model for the proliferation of remnant LECs that occurs during posterior capsule opacification (PCO) after extracapsular cataract extraction. The LEC culture system was also used in vitro for testing compounds that might inhibit this process in vivo. Human LECs were cultured on the surface of the original lens capsule fixed to collagen. Variability was reduced by dissecting each lens capsule into equivalent halves and exposing the segments to immunotoxin 4197X-RA. RESULTS: Protein synthesis and LEC proliferation were almost completely inhibited at relatively low 4197X-RA concentrations after short exposure. The inhibitory effects persisted up to 3 weeks after withdrawal of the immunotoxin and after several media exchanges. CONCLUSION: Immunotoxin 4197X-RA may help prevent PCO after primary cataract surgery.

Ocular pharmacokinetics of lens epithelial cell-specific immunotoxin 4197X-RA.

The ocular clearance, stability and systemic distribution of a lens epithelial cell-specific immunotoxin were evaluated in adult Dutch Belted rabbits. Immunotoxin 4197X-RA was prepared from a murine monoclonal antibody conjugated by a disulfide linkage to ricin A. After intracameral (i.c.) injection, clearance of the immunotoxin from the eye followed a first-order process. The half-life (t1/2) of immunotoxin in the aqueous humor was 51 min. Accumulation of the immunotoxin in ocular tissues appeared to be associated with aqueous outflow tracts, as it was primarily confined to the iris and the limbal regions of the cornea. No acute metabolism of immunotoxin was observed in the anterior chamber. The immunotoxin was detected in the blood 20 min post-injection and achieved a steady-state level after 40 to 180 min. A slow decline in labeled protein was then observed from 180 min to 24 hr. Systemic clearance of the immunotoxin appeared to occur primarily by way of the kidneys. The present data indicate that immunotoxin 4197X-RA, like other large proteins, is passively cleared from the anterior chamber by aqueous flow, and that little or no acute breakdown occurs in the anterior chamber. It is anticipated that these data, along with results from cytotoxicity studies, will result in the determination of optimal doses and frequency of administration for the use of immunotoxin in human clinical trials.

Growth inhibitory effects of interferon-beta but not interferon-alpha on human glioma cells: correlation of receptor binding, 2',5'-oligoadenylate synthetase and protein kinase activity.

The antiproliferative effects of human recombinant interferon-alpha (rIFN-alpha A) and interferon-beta (rIFN-beta ser) were assessed in vitro against seven human glioma cell lines. Further analysis of one of these lines (EFC-2) in response to rIFN-alpha A demonstrated a minimum growth inhibition by day 6 of treatment, whereas a 50% inhibition of cell growth was observed with a dose of 50 U/ml of IFN-beta ser. No significant growth inhibition was seen by rIFN-alpha A at doses up to 500 U/ml. Addition of rIFN-alpha A to rIFN-beta ser-treated EFC-2 cells neither suppressed nor augmented the antiproliferative response to IFN-beta ser. The binding of 125I-labeled rIFN-alpha A or 125I-labeled rIFN-beta ser to EFC-2 cells was inhibited competitively by increasing concentrations of either unlabeled rIFN-alpha A or rIFN-beta ser. This suggests that the cellular receptors for both rIFN-alpha A and rIFN-beta ser appear to be intact and appear to bind both agents equally. Furthermore, incubation of EFC-2 cells for 72 h with either rIFN-alpha A or rIFN-beta ser resulted in an increase in 2',5'-oligoadenylate (2-5A) synthetase activity 5-fold with rIFN-alpha A and 50-fold with rIFN-beta ser. Similarly, the 68-kD IFN-induced protein kinase was induced substantially with rIFN-beta ser but only slightly induced with rIFN-alpha A treatment. These results suggest that EFC-2 human glioma cells demonstrate a differential sensitivity in terms of growth inhibition to rIFN-beta ser and to rIFN-alpha A which appears to correlate with a differential induction of both intracellular 2-5A synthetase and protein kinase activity. These results cannot be explained solely on the basis of surface receptor binding of rIFN-alpha A and rIFN-beta ser. These data do suggest that, for human glioma cells in culture, type I IFN receptors may display a subtle architectural variation that allows equivalent binding of both IFN-alpha and IFN-beta ser, but allows an enhanced signal transduction and biological effect only after binding a specific IFN subtype.

Growth inhibitory effect of recombinant alpha and beta interferon on human glioma cells.

Growth inhibitory activity of recombinant alpha and beta interferon on two human glioma cell lines, EFC-2 and KE cells, was determined by two different growth assays. Recombinant beta interferon showed a slight growth inhibitory effect on EFC-2 cells at day 3, and maximum inhibition was seen on day 6 with an ID50 of 50 U/ml. Recombinant alpha interferon showed no significant growth inhibition at any concentration. KE cells were resistant to both recombinant alpha and beta interferon. The growth inhibitory activity of recombinant beta interferon on EFC-2 cells was not blocked by recombinant alpha interferon, although recombinant alpha and beta interferons shared same receptors on EFC-2 cells. Addition of DFMO (alpha-difluoromethylornithine) to interferon in the media showed additive effect rather than synergistic effect in growth inhibition of glioma cells. Out of 7 glioma cell lines tested, 4 showed heterogeneous sensitivity to recombinant beta interferon, and all were resistant to recombinant alpha interferon. These results suggest a differential sensitivity of EFC-2 cells to recombinant beta interferon, as well as a heterogeneous sensitivity to recombinant beta interferon among different glioma cell lines.

In vivo sensitivity and resistance of chronic myelogenous leukemia cells to alpha-interferon: correlation with receptor binding and induction of 2',5'-oligoadenylate synthetase.

Fourteen patients with chronic myelogenous leukemia were treated with partially pure leukocyte interferon (HuIFN alpha). The binding of recombinant leukocyte clone A IFN and the induction of 2',5'-oligoadenylate synthetase (2,5A) in peripheral blood cells were studied to determine whether they correlate with clinical response to IFN therapy. The mean pretherapy binding of radiolabeled recombinant leukocyte clone A IFN to peripheral blood cells was 0.053 +/- 0.02 (SE) fmol (53 +/- 20 amol)/10(6) cells and 0.049 +/- 0.015 fmol/10(6) cells in sensitive and resistant patients, respectively. Twenty-four h after the first HuIFN alpha dose, the binding of recombinant leukocyte clone A IFN decreased 3- to 8-fold in both sensitive and resistant patients. The activity of 2,5A synthetase was induced approximately 100-fold in sensitive patients from a pretherapy mean of 3 +/- 2 nmol/mg to a maximum of 317 +/- 184 nmol/mg during therapy. In contrast, 2,5A synthetase was induced from a pretherapy mean of 0.9 +/- 0.9 nmol/mg to only 6.7 +/- 4.9 nmol/mg in resistant patients. In two patients originally sensitive to HuIFN alpha who developed resistance to therapy, receptors were present in both sensitive and resistant disease stages and appeared to down regulate with therapy regardless of response. In these two patients, 2,5A synthetase was significantly induced with therapy in the sensitive stage but not in the resistant stage. This study shows that lack of clinical response to interferon therapy may coincide with failure to induce 2,5A synthetase activity. This suggests that resistance to alpha-interferon therapy may be mediated by events beyond receptor binding resulting in a failure to induce enzymes responsible for mediation of interferon antiproliferative effects.

Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG.

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.

An antibody that inhibits fibronectin-independent adhesion of fibroblasts to extracellular matrix material.

Chinese hamster ovary (CHO) fibroblasts adhere to the extracellular matrix by both fibronectin-dependent and -independent mechanisms (Harper and Juliano, 1981a,b). Previous studies have suggested that a trypsin-sensitive, 265,000-dalton membrane glycoprotein (gp265) is involved in the fibronectin-independent adhesion process. Using a polyclonal antibody against soluble products obtained from trypsin-treated CHO cells, we have been able to further analyze this involvement. This antibody immunoprecipitates a trypsin-sensitive 265,000-dalton protein from detergent-solubilized cells. Incubation of AdvF11, a variant cell line that does not utilize fibronectin for adhesion, with this antibody blocks their adhesion to extracellular matrix material (ECM). The immunoglobulin fraction will also partially block adhesion of the parental cell line to ECM particularly when the ECM is first treated with an antifibronectin antibody. Taken together these results add support for the involvement of gp265 in fibronectin-independent adhesion and provide a methodology for further characterization.

Detection of proteases in polyacrylamide gels containing covalently bound substrates.

Conjugates have been prepared from glutaraldehyde-activated linear polyacrylamide and bovine serum albumin, casein, or gelatin. Incorporation of these conjugates into sodium dodecyl sulfate-polyacrylamide gels has provided a simple and general method for the analysis of proteases following electrophoresis. The conjugates did not migrate during electrophoresis or development, but remained susceptible to proteolytic action following regeneration of enzyme activity. The sensitivity of this procedure was such that 2 pg of trypsin or chymotrypsin, 39 ng of elastase, and 2 ng of thermolysin could be detected. Results obtained with trypsin and chymotrypsin are 5 to 10 times more sensitive than previously reported techniques for protease detection following electrophoresis.

Enhanced immunogenicity of line 10 guinea pig hepatocarcinoma cells after culture.

Line 10 hepatocarcinoma cells derived from ascites in a strain 2 guinea pig were tumorigenic when transferred intradermally. After they had been cultured in vitro for 20 days or more in medium enriched with 10% fetal bovine serum (FBS), they became immunogenic. Injections of immunogenic cells did not cause lethal tumors, and recipients were resistant to subsequent challenges with tumorigenic line 10 cells. Resistance was specific since growth of line 1 cells, a syngeneic but antigenically distinct tumor, was not affected. Cells cultured in medium enriched with 10% calf bovine serum or 10% normal guinea pig serum or in reduced concentrations of FBS were less effective in inducing resistance. When cultured line 10 cells were injected ip into normal guinea pigs, ascites tumors developed that were tumorigenic. The growth rate of line 10 cells in culture was considerably decreased as determined by reduced [3H]thymidine incorporation and mitotic indices. The mechanism(s) responsible for enhancement of immunogenicity in cultured line 10 cells is discussed but was not determined.

A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells.

A solid-phase radioimmunoassay to detect antibodies that react with antigens derived from human melanoma cells is described. A soluble preparation derived from Nonidet P-40 lysates of tissue-cultured melanoma cells was dried on the surfaces of wells of polyvinyl chloride microtiter plates and fixed with 0.02% glutaraldehyde. Antibody preparations were added and incubated for 18 h at 4 degrees C. The wells were washed and bound antibodies were detected using radioactive Staphyloccoccal protein A (125I-SpA). Optimal conditions are described for all the steps employed. Concentrations of antigen selected, the amount of 125I-SpA employed and the duration of incubation of antibodies with antigen were found to be critical. The assay was sensitive and reproducible, and lent itself to the simultaneous evaluation of many individual antibody samples in a short period of time. The assay was particularly valuable for rapid screening of hybridoma supernatants for antibodies to antigens derived from melanoma cells and from a panel of other tumor and normal cells.

The use of cellular immunoadsorbents to prepare polyclonal antibodies that distinguish between antigens derived from human melanoma cells and autologous lymphocytes.

Cellular immunoadsorbents were employed to isolate xenogeneic antibodies that reacted with a restricted group of antigens on human melanoma cells. Melanoma cells and autologous lymphoid cells were grown in tissue culture. Cellular immunoadsorbents were prepared by coupling formalin-treated melanoma and lymphoid cells to diethylaminoethyl cellulose. Rabbits were immunized with melanoma cells and antisera were passed sequentially through immunoadsorbents made of fetal bovine serum, and lymphoid cells. Unbound effluents were then passed through an immunoadsorbent prepared with melanoma cells. Antibodies binding to melanoma cells were eluted and their reactivity to melanoma-derived antigens was tested using a solid-phase radioimmunoassay. Antigens for this assay were NP-40 lysates of melanoma and lymphoid cells and fetal bovine serum. Radioactive Staphylococcal protein A was used to detect binding by the antibodies to the test antigens. The effects of formalin-fixation and storage of melanoma and lymphoid cells were studied. Storage of fixed melanoma cells for periods up to 4 months did not affect their capacity to bind antibodies. A single exposure of formalin-fixed cells to a low-pH elution buffer which was followed by neutralization did not affect binding by these cells. Antibodies isolated in this manner were of the IgG class and reacted with antigens derived from melanoma cells but not from autologous lymphocytes or fetal bovine serum. This study demonstrated the feasibility of using cellular immunoadsorbents to prepare xenogeneic polyclonal antibodies with a high degree of reactivity to antigens derived from human melanoma cells.