Measurement of mRNAs for TGFss and extracellular matrix proteins in corneas of rats after PRK.

PURPOSE: To assess the role of the transforming growth factor (TGF)ss system in formation of corneal haze after excimer laser photorefractive keratectomy (PRK), levels of mRNAs for three TGFss isoforms (TGFss1, TGFss2, and TGFss3), the TGFss type II receptor (TssRII), and extracellular matrix (ECM) genes including fibronectin (FN), collagen I, collagen III, and collagen IV were measured in rat corneas. METHODS: Corneas were graded for corneal haze at 0, 1.5, 7, 21, 42, and 91 days after PRK. Total RNA was isolated from pooled corneas, and the levels of mRNAs were measured using competition-based quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Severe corneal haze developed by day 42 and persisted to day 91. Levels of TGFss1 mRNA were high in rat corneas before PRK and remained relatively constant. In contrast, levels of TGFss2 and TGFss3 mRNAs were very low in normal corneas, increased 300-fold and 25-fold, respectively, on day 21, and remained elevated on day 91. Levels of mRNA for TssRII increased, with a peak elevation of 50-fold on day 42 after PRK. Levels of mRNAs for ECM proteins also increased. Fibronectin mRNA was nondetectable in normal corneas but rapidly increased to 675 copies/cell on day 7 and remained elevated to day 91. Collagen III mRNA levels peaked on day 21 with a 700-fold increase compared with a very low level of expression in normal cornea, and then decreased on day 91. Expression of collagen I mRNA lagged expression of collagen III mRNA and peaked at day 42 after PRK with a 1200-fold increase over normal cornea. In contrast, mRNA for collagen alpha(1)IV, a major component in basement membranes, remained relatively stable through day 21 and then increased slightly on days 42 and 91. CONCLUSIONS: The synchronized increase in mRNA synthesis for both the TGFss system and key ECM genes supports the hypothesis that TGFss is a key growth factor promoting stromal haze formation in corneas after PRK and suggests that limiting TGFss system may reduce corneal scarring after excimer laser ablation.

Comparison of different biomaterials for glaucoma drainage devices: part 2.

BACKGROUND: Inflammation associated with biomaterials may contribute to the failure of glaucoma drainage devices. OBJECTIVE: To compare the inflammatory reaction associated with the insertion of Krupin silicone, Molteno polypropylene, and Acrosof end plates in the subconjunctival space of rabbits. METHODS: Similar-sized glaucoma end plates made of 3 different biomaterials were sutured to the sclera in the superotemporal quadrant of the rabbit eye. Thirty eyes of 15 albino New Zealand rabbits were randomly assigned to the 3 groups. Conjunctival vascular hyperemia was graded in a masked fashion among the 3 groups. At the end of 3 weeks, the enucleated eyes were examined histologically and by scanning electron microscopy. RESULTS: Molteno polypropylene was associated with more inflammation both in clinical observations and based on histological grading. Silicone and Acrosof were associated with less intense inflammation. One polypropylene end plate was extruded on day 21. CONCLUSIONS: Polypropylene appears to be more inflammatory than silicone. Flexible biomaterials appear to be less inflammatory than rigid ones. CLINICAL RELEVANCE: Bleb failure following glaucoma drainage device implantation could be related to the biomaterial-associated inflammation. Choosing a biomaterial with the least inflammatory potential might enhance the success rate of the glaucoma drainage device. Arch Ophthalmol. 2000;118:1081-1084

Comparison of different biomaterials for glaucoma drainage devices.

OBJECTIVES: To compare the inflammatory reaction associated with the insertion of silicone and polypropylene endplates and endplates made of a new biocompatible polymer, Vivathane, in the rabbit subconjunctival space. METHODS: Similar-sized endplates made of 3 different biomaterials were sutured to the sclera in the superotemporal quadrant of the rabbit eye. Thirty eyes of 15 albino New Zealand rabbits were randomly assigned to the 3 groups. Conjunctival vascular hyperemia was graded in a masked fashion among groups. At the end of 3 weeks, the enucleated eyes were examined histologically and using scanning electron microscopy. RESULTS: Polypropylene and Vivathane were associated with significantly more inflammation in clinical observations and based on histological grading. Silicone was associated with the least amount of inflammation. Three polypropylene and 1 Vivathane plate were extruded between the second and third week. CONCLUSIONS: Silicone is the most inert of the 3 materials tested. Inflammation associated with biomaterials may contribute to the failure of the glaucoma drainage devices. CLINICAL RELEVANCE: Bleb inflammation may be related to the biomaterial being used as the endplate. Endplates should be handled carefully during surgery to avoid creating rough spots.

Interleukin-12-mediated tumoricidal activity of patient lymphocytes in an autologous in vitro ovarian cancer assay system.

This study was designed to examine if interleukin-12 (IL-12) can induce cytolytic function of lymphocytes from ovarian cancer patients against either an ovarian cancer cell line or their own autologous tumor cells. Lymphocytes were obtained from the peripheral blood or ascites of ovarian cancer patients and activated with IL-12 alone or concomitantly with interleukin 2 (IL-2) for 2 to 3 days. Activation of lymphocytes and assessment of tumoricidal function by a chromium release assay were performed directly in a standard control medium (RPMI 1640 containing 2 mM glutamine, 100 micrograms/ml streptomycin, 100 units penicillin, 5% heat-inactivated human AB serum, and 5 mM 4-(2-hydroxyethyl)-1-piperazinesulfonic acid) and in 50% ascitic fluid (50% by volume filter-sterilized ascites with 50% of the above-mentioned control medium). Target cells were added directly into the medium in which the lymphocytes were activated in order to more closely mimic in vivo conditions. Lymphocytes, activated by IL-12 in 50% ascitic fluid, were able to lyse autologous tumor cells in 3 of 6 assays and were able to lyse SKOV3 cells (an ovarian cancer cell line) in 5 of 7 assays. The results were not significantly different in the control medium. When both IL-2 and IL-12 were used to activate lymphocytes in 50% ascitic fluid, significant cytotoxicity was generated in 6 of 6 autologous assays and in all 7 patient assays using SKOV3 as a target (P < 0.05). Synergy between the two cytokines was seen in all 13 patient assays in ascitic medium compared to only 5 of 13 assays in control medium. Additionally, when lymphocytes were stimulated with both IL-2 and IL-12, significantly greater cytotoxicity was seen in the ascitic fluid medium compared to the control medium in 13 of 14 assays (P < 0.05). No significant tumoricidal activity was seen by lymphocytes maintained in either medium without the addition of IL-2 or IL-12. Ascitic fluid consistently potentiates the synergy between IL-2 and IL-12 in generating cytotoxicity against ovarian cancer cells but does not increase cytotoxicity induced by IL-12 alone. IL-12 by itself activates tumoricidal activity of lymphocytes in ascitic fluid; however, the addition of IL-2 increases the degree and consistency of this effect. These data support the possibility that IL-12 may warrant further investigation as a potential therapeutic agent in the treatment of advanced ovarian cancer.

Serum soluble interleukin-2 receptor alpha levels in patients with gynecologic cancers: early effect of surgery.

PROBLEM: To determine the early effect of abdominal cytoreductive surgery on serum soluble interleukin-2 receptor alpha (sIL-2R alpha) levels in patients with ovarian and cervical cancer, and to determine if the extent of cytoreduction correlated with the changes in serum sIL-2R alpha. METHOD: Thirty-nine patients with gynecologic cancer had serum sIL-2R alpha measured by enzyme-linked immunosorbent assay before abdominal cytoreductive surgery and once in the early postoperative period. RESULTS: Only patients with advanced epithelial ovarian cancer had elevated preoperative serum sIL-2R alpha levels. In 20 of 25 ovarian cancer cases (80%) and 10 of 14 cervical cancer cases (71.4%) the postoperative serum sIL-2R alpha levels exceeded the preoperative level (P = .003 and P = .011, respectively). Overall, the mean postoperative serum sIL-2R alpha level was greater than the preoperative level (P = .0001). CONCLUSION: Patients with early stage gynecologic cancer did not have elevated serum sIL-2R alpha levels before surgery. In the early postoperative period the serum sIL-2R alpha level was increased, which may be a nonspecific response to the trauma of surgery. Soluble IL-2R alpha may be one of the factors responsible for the immunosuppression in the early postoperative period, but may also herald a surge of activated T cells.

High serum and ascitic soluble interleukin-2 receptor alpha levels in advanced epithelial ovarian cancer.

This study was undertaken to determine if advanced epithelial ovarian cancer was associated with increased serum and ascitic levels of soluble interleukin-2 receptor alpha (sIL-2R alpha). Serum and ascitic fluid samples from 23 ovarian cancer patients were analyzed for sIL-2R alpha using an enzyme-linked immunosorbent assay and compared with the serum and peritoneal levels in 18 normal females. The samples were analyzed for CA-125 levels using a radioimmunoassay and the total protein was also measured. Normal individuals had low serum levels of sIL-2R alpha (367.5 +/- 44.6 U/mL), with similar levels of sIL-2R alpha in the normal peritoneal fluid (438.6 +/- 48.8 U/mL). In contrast, the serum and ascitic fluid levels in ovarian cancer patients were significantly higher (746.7 +/- 82.9 U/mL, P = .0006; 2,656.7 +/- 373.7 U/mL, P = .00002, respectively). The results for sIL-2R alpha were also significant when the levels were expressed per milligram of total protein. More importantly, in almost every ovarian cancer patient the ascitic sIL-2R alpha level far exceeded the serum level, a pattern also observed for CA-125. There was no correlation between the serum and ascitic sIL-2R alpha levels, or between the serum and ascitic CA-125 levels. Although the serum levels of sIL-2R alpha and CA-125 were elevated in the same patient, overall there was no correlation between the serum sIL-2R alpha and serum CA-125 levels, either when the levels were expressed in absolute units or per milligram of total protein. Similarly, there was no correlation between sIL-2R alpha and CA-125 levels in individual ascitic samples. While CA-125 levels may reflect an independent index of tumor burden, these results suggest that selective accumulation of sIL-2R alpha in the ascites may be one of the factors associated with the known nonresponsiveness of the infiltrating lymphocytes against ovarian carcinoma cells.