Downregulation of ß-actin and its regulatory gene HuR affect cell migration of human corneal fibroblasts.

PURPOSE: In an earlier study, we showed that human antigen R (HuR) and ß-actin expression levels were downregulated in fibroblasts isolated from human keratoconus stroma compared to normal corneal stroma. To further extend the finding, we determined whether HuR expression affects ß-actin gene expression and in turn affects corneal fibroblast migration and wound healing. METHODS: Stromal keratocytes from normal human corneas were cultured in the presence of serum. Cells were transfected with siRNA specific for ß-actin or HuR. SiRNAs specific for GAPDH or a scrambled sequence were used as positive and negative controls (siCTR) for transfection, respectively. The effects of gene silencing were analyzed at the transcriptional and translational levels. Specific proteins were immunohistochemically localized using confocal imaging. The effects of gene silencing on cell migration and cell proliferation were analyzed using a modified Boyden chamber and with a wound healing assay, respectively. RESULTS: Reverse-transcription PCR (RT-PCR) and western blot analyses showed that when the HuR gene was silenced, ß-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the ß-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the ß-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced. CONCLUSIONS: The results show that downregulation of the HuR gene results in decreased ß-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased ß-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.

Differential epithelial and stromal protein profiles in keratoconus and normal human corneas.

The purpose of the study was to identify epithelial and stromal proteins that exhibit up- or down-regulation in keratoconus (KC) vs. normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluted the specificity of the proteome of each tissue, we selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, i.e., a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS)(2)] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS)(2) method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset. Relative to normal corneal epithelial proteins, six KC epithelial proteins (lamin-A/C, keratin type I cytoskeletal 14, tubulin beta chain, heat shock cognate 71 kDa protein, keratin type I cytoskeletal 16 and protein S100-A4) exhibited up-regulation and five proteins (transketolase, pyruvate kinase, 14-3-3 sigma isoform, phosphoglycerate kinase 1, and NADPH dehydrogenase (quinone) 1) showed down-regulation. A similar relative analysis showed that three KC stromal proteins (decorin, vimentin and keratocan) were up-regulated and five stromal proteins (TGF-betaig h3 (Bigh3), serotransferrin, MAM domain-containing protein 2 and isoforms 2C2A of collagen alpha-2[VI] chain) were down-regulated. The 2D-DIGE-mass spectrometry followed by Decyder software analysis showed that relative to normal corneas, the KC corneal epithelium exhibited up-regulation of four proteins (serum albumin, keratin 5, L-lactate dehydrogenase and annexin A8) and down-regulation of four proteins (FTH1 [Ferritin heavy chain protein 1], calpain small subunit 1, heat shock protein beta 1 and annexin A2). A similar relative analysis of stroma by this method also showed up-regulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in KC corneas. Together, the results suggested that the Nano-ESI-LC-MS(MS)(2) method was superior than the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up- or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling.

Inhibitory effect of a complementary peptide on ulceration in the alkali-injured rabbit cornea.

PURPOSE: Two tripeptide chemoattractants, acetyl-proline-glycine-proline (Ac-PGP) and methyl-proline-glycine-proline (Me-PGP), are the primary triggers for early neutrophil invasion into the alkali-injured cornea. In the present study the effectiveness of a complementary peptide designed to inhibit the PGP chemoattractants (arginine-threonine-arginine [RTR] tetrameric peptide) and an apo A-1 mimicking peptide (5F) was investigated in the alkali-injured rabbit eye. METHODS: (L)-RTR tetramer, (D)-RTR tetramer, and 5F were tested in vitro for their effects on neutrophil polarization. Synthetic 5F was also tested in vitro for its effect on the neutrophil respiratory burst. In the alkali-injured rabbit eye model, the right corneas of 48 rabbits were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of three groups: phosphate-buffered saline (PBS) control; 800 microM RTR (dextrorotatory) tetramer in PBS alternating each hour with 1.5 mM RTR (levorotatory) tetramer in PBS; and 12 microM 5F in PBS. One topical drop of each substance was administered hourly (14 times per day) for 33 days. The experiment was continued until day 42 with no additional drops administered. RESULTS: (L)-RTR tetramer and (D)-RTR tetramer inhibited neutrophil polarization activated by the PGP chemoattractants in vitro. Synthetic 5F did not inhibit neutrophil polarization in the presence of Ac-PGP or the respiratory burst of neutrophils in the presence of a metabolic stimulant derived from alkali-degraded corneas. During the entire animal experiment, statistically fewer ulcers occurred in the RTR tetramer group than in the PBS control group (43.8% vs. 87.5%, P = 0.0046). The frequency of ulceration in the 5F group (68.8%) was not significantly different from the PBS control group. CONCLUSIONS: The reduction in the frequency of corneal ulceration by the RTR tetramer possibly resulted from its complementary binding to Ac-PGP and Me-PGP in the cornea shortly after alkali injury, leading to a reduction in the early and late infiltration of neutrophils. RTR tetramer appears to hold enough promise to warrant additional study as a therapeutic drug for the alkali-injured eye.

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

PURPOSE: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Bioactivity of peptide analogs of the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

PURPOSE: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP. RESULTS: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition. CONCLUSIONS: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.

Injection of chemoattractants into normal cornea: a model of inflammation after alkali injury.

PURPOSE: The objective of this study was to establish and characterize the invasion of polymorphonuclear leukocytes (PMNs) into a normal cornea after intrastromal injection of the tripeptide chemoattractants generated from alkali-degraded corneas. METHODS: The following samples were injected into the midstroma of normal rabbit corneas: ultrafiltered tripeptide chemoattractants (N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline) generated from alkali-degraded corneas, synthetic N-acetyl-PGP, positive control (leukotriene B4 [LTB4]), or negative control (Hanks' balanced salt solution [HBSS]). Timed responses of PMN infiltration were established for effective concentrations of LTB4 or the ultrafiltered chemoattractants. RESULTS: All intrastromal injections resulted in the immediate development of an edematous disc that was 10 mm in diameter. The lesion essentially had cleared in the HBSS-injected eyes by 8 hours, and histologic sections revealed minimal numbers of PMNs in the cornea or limbal tissue. The injection of LTB4 or the ultrafiltered tripeptide chemoattractants induced peak numbers of PMNs within the stroma at 8 hours, subsiding by 16 hours. Seventy units of ultrafiltered chemoattractants yielded a strong PMN response, similar to 1 X 10(-5) M LTB4. The highest concentration of ultrafiltered chemoattractants (350 U) produced a severe PMN response that was characterized by a solid sheet of neutrophils surrounding the injection site. The injection of synthetic N-acetyl-PGP (2 X 10(-4) M) produced a marked PMN response. CONCLUSIONS: PMN invasion of the normal cornea after the injection of the ultrafiltered tripeptide chemoattractants or the synthetic N-acetyl-PGP mimicked early PMN infiltration in the alkali-injured eye, confirming the importance of this chemoattractant as an inflammatory mediator.

A new classification system predicting keratomalacia after trauma in vitamin A deficiency: sodium citrate does not prevent disease progression.

PURPOSE: The purpose of this study was to develop a classification system to predict keratomalacia after trauma in vitamin A-deficient eyes and to determine whether citrate impedes polymorphonuclear leukocyte infiltration into the cornea, thus preventing keratomalacia. METHODS: Preliminary classification studies showed that a 7.0-mm corneal epithelial scrape, before clinical findings of corneal xerosis, did not induce keratomalacia. Primary studies were conducted concurrently on the same animals to develop the classification system and test the effect of citrate in vitamin A deficiency. A 7.0-mm corneal epithelial scrape was performed on vitamin A-deficient eyes in various stages of corneal xerosis and treated as follows. Experiment 1: group 1, 10% citrate drops; group 2, phosphate buffer solution (PBS) drops; experiment II: group 3, drops and subconjunctival injection of 10% citrate; group 4, drops and subconjunctival injection of PBS. RESULTS: Corneal abrasion in eyes with 2+ corneal xerosis yielded keratomalacia in 50% of cases; the remainder healed with xerotic epithelium. Eighty-three percent of eyes with > 2+ xerosis developed keratomalacia after corneal abrasion, whereas only 7.1% of eyes with < 2+ xerosis advanced to this stage. In experiment I, 27% of citrate-treated eyes and 38% of PBS-treated eyes developed keratomalacia (not significant). In experiment II, two of six citrate-treated eyes perforated and one eye developed keratomalacia. One of six control PBS eyes perforated and four developed keratomalacia. CONCLUSION: We correlated the degree of corneal xerosis with the occurrence of keratomalacia after corneal trauma. This led to the development of a classification scale that is of research and clinical significance. Additionally, citrate did not significantly reduce keratomalacia or perforation in the vitamin. A-deficient eye.

Effect of synthetic metalloproteinase inhibitor or citrate on neutrophil chemotaxis and the respiratory burst.

PURPOSE: A topical synthetic metalloproteinase inhibitor (SIMP) has been reported to reduce the incidence of corneal ulcerations in the alkali-injured eye by the putative mechanism of collagenase inhibition. The current study was performed to determine whether SIMP has a direct inhibitory effect on the activation of neutrophils and to compare any such effect with that of citrate, a known inhibitor of polymorphonuclear leukocyte (PMNL) activity. METHODS: The effect of SIMP or citrate was tested on chemotaxis of PMNL and the respiratory burst in vitro. RESULTS: Synthetic metalloproteinase inhibitor (1 mM) or citrate (12 mM) produced significant inhibition of chemotaxis of PMNL and the accompanying behavioral characteristics of motility. Synthetic metalloproteinase inhibitor, but not citrate, generated a dramatic respiratory burst when incubated with resting PMNL. Both SIMP and citrate inhibited the respiratory burst of PMNL in the presence of opsonized zymosan. For a brief, initial period each substance enhanced the respiratory burst generated by the metabolic stimulant from alkali-degraded corneas, followed by a rapid decline in activity that was associated with cell death. CONCLUSIONS: Although SIMP has been shown to exhibit powerful collagenase inhibition, its inhibitory effect on chemotaxis of PMNL might be the primary mechanism in reducing the incidence of ulceration in the alkali-injured cornea. Very significant reduction in the accumulation of PMNL in SIMP-treated corneas supports this conviction. Activation by SIMP of the respiratory burst in resting PMNL is of concern, but overall its beneficial effect is favorable, as demonstrated in prior alkali-injured animal models. The dual effect of inhibition of chemotaxis of PMNL and activity of collagenase makes SIMP a potential drug for combating ulceration in the alkali-injured cornea.

An excess of topical calcium and magnesium reverses the therapeutic effect of citrate on the development of corneal ulcers after alkali injury.

Our purpose was to determine whether chelation of Ca2+ and Mg2+ is the mechanism by which sodium citrate inhibits corneal ulceration in the alkali-injured rabbit eye. The right eyes of 60 albino rabbits (2-2.5 kg) were alkali-injured by filling a 12-mm-diameter plastic well placed on the corneal surface with 0.4 ml of 1 N NaOH. After 35 s the alkali was aspirated, and the well was rinsed with physiological saline. Animals were randomly distributed to three treatment groups of equal size. Two drops of the following topical medications were administered on the hour (14 times per day) for 35 days: physiological saline, 10% citrate in saline, and 346 mM Ca2+, 346 mM Mg2+, and 10% citrate in saline. During the experiment, significantly fewer ulcerations occurred in the citrate-treated eyes (five of 20, 25%) than in the saline-treated eyes (13 of 20, 65%) or in the calcium-magnesium-citrate-treated eyes (15 of 20, 75%). When ulcerations did develop in the citrate group, they occurred significantly later and were less severe than those in the saline and calcium-magnesium-citrate groups. There was a significant increase in the number of eyes with signs of band keratopathy and translucent areas in the calcium-magnesium-citrate group when compared with the other two groups. As in previous studies, sodium citrate significantly inhibited the development of corneal ulcers after alkali injury. The annullment of the favorable effect of citrate on ulceration in the alkali-injured eye by the addition of calcium and magnesium shows that the mechanism of action of citrate is the chelation of these divalent cations.

A neutrophil chemoattractant is released from cellular and extracellular components of the alkali-degraded cornea and blood.

PURPOSE: A tripeptide chemoattractant(s) for neutrophils has been shown to release from alkali-degraded cornea. This study is designed to determine the source of the chemoattractant(s). METHODS: Whole corneas were degraded in 1 N NaOH for 10 minutes, 30 minutes, 1 hour, 4, 8, 24, and 48 hours to determine an optimal duration of alkali exposure for production of the chemoattractant. Whole cornea, cornea minus epithelium, cornea minus endothelium, and stroma alone were degraded in 1 N NaOH for 4 hours to determine the relative contribution of each corneal layer. In a separate experiment, epithelium alone, endothelium alone, cultured keratocytes alone, or bovine corneal collagen were treated separately in 1 N NaOH for 4 hours. Finally, human plasma, platelets, polymorphonuclear leukocytes (PMNL), and red blood cells were treated separately in 1 N NaOH for 4 hours to determine if a similar chemoattractant was released from alkali-treated noncorneal tissue. Neutralized suspensions of all samples were ultrafiltered and dialyzed. The chemotactic potential of each sample was determined in the polarization assay. RESULTS: Activation of the PMNL polarization response increased with the duration of exposure of corneal tissue to alkali, peaking at 6 hours. Release of the chemoattractant from alkali-degraded corneal tissue showed a significant decrease when the epithelium was removed from the stroma. All tissue layers showed a PMNL polarization response when treated with alkali. The response decreased from epithelium > endothelium > cultured keratocytes > collagen. Alkali degradation of human blood components, including plasma, showed significant polarization responses ranked in the following order: plasma > PMNL > tendon collagen > platelets = red blood cells. CONCLUSIONS: This study demonstrates that the tripeptide chemoattractant(s) is released from all layers of the cornea after alkali injury. The substance released from blood components is of similar size and biologic activity as that found in the cornea, but its exact molecular composition is yet to be determined. Timed response of alkali degradation determined the optimal conditions for generation of the chemoattractant(s) including clinically relevant time intervals.

Identification and synthesis of chemotactic tripeptides from alkali-degraded whole cornea. A study of N-acetyl-proline-glycine-proline and N-methyl-proline-glycine-proline.

PURPOSE: To identify and synthesize the polymorphonuclear leukocyte chemoattractant(s) released from alkali-degraded corneas. METHODS: Corneas were degraded in 1.0 N NaOH, neutralized, ultrafiltered, and dialyzed. The final active ultrafiltrate was subjected to high-performance liquid chromatography on a Protein PAK I-60 column. The most active fractions were further separated on a mu-Bondapak-C18 and I-60 column in sequence. RESULTS: Fraction 38 from the final I-60 column associated with a 210-nm absorption peak and elicited a polarization and chemotactic response from polymorphonuclear leukocytes. The loss of polarization activity in fraction 38 after exposure to prolidase suggests that this peptide contains a Pro-X (X = amino acid) peptide bond. The amino acid composition of fraction 38 was 35% glycine and 53% proline. Peptide sequence analysis was unable to establish a primary sequence even though Picotag analysis showed the presence of large amounts of the two amino acids. Mass spectrometry revealed only two molecular species of 312 MWt and 284 MWt. Tripeptides were synthesized using all possible amino acid permutations of 2 Pro and 1 Gly and tested in the polarization and chemotactic assays. These techniques demonstrated that n-acetyl-Pro-Gly-Pro, and to a lesser degree n-methyl-Pro-Gly-Pro, were the only synthetic tripeptides with activity similar to the purified chemoattractant. CONCLUSIONS: The data show that the chemotactic peptides, purified from alkali-degraded whole cornea and confirmed with identical synthetic tripeptides, are N-acetyl-Pro-Gly-Pro and N-methyl-Pro-Gly-Pro. Although a number of proteins contain the Pro-Gly-Pro sequence, large amounts of collagen in the cornea suggest this as a major source. The small size and hydrophilic nature of these chemoattractants are predictive of a high degree of diffusibility. These chemoattractants are likely to play a major role in the early neutrophil response after an alkali injury.

Characterization of chemical gradients in the collagen gel-visual chemotactic assay.

Chemical gradients developing in a collagen gel-visual chemotactic assay (CG-VCA) for PMNs were evaluated by theoretical and experimental methods. First, a video image analysis system was used to establish the diffusion coefficients of bromophenol blue (BPB) through the membrane (D1) and across the collagen gel (D2) in a capillary tube apparatus used only for this purpose. The diffusion coefficients of BPB and the geometry of the CG-VCA system were then used to develop a mathematical model, estimating theoretical gradients in the CG-VCA system. In addition, gradients in the CG-VCA system were characterized experimentally by employing BPB as the source and determining the concentration profiles of BPB in the chemotactic compartment by video image analysis. A relative error of approximately 21% exists between the theoretical and experimental gradients for both 0.5 and 1.0 mM source concentrations of BPB. This favorable comparison demonstrates reliability in predicting chemical gradients for the CG-VCA system. The mathematical model was then used to predict gradients using nanomolar and micromolar concentrations of low molecular weight chemoattractants. Analysis of these specific gradients showed that gradients were steep enough to be detected by PMNs in the collagen gel during the observation period used in previous experiments. The determination of BPB gradients in the CG-VCA system illustrates the utility of this system.

Ventral preputial onlay island flap and proximal flip-flap technique for the repair of epispadias: a case report.

Use of the ventral preputial island flap technique for repair of epispadias has yielded satisfying functional and cosmetic results. We report a modification of this technique in a patient with proximal penile epispadias without exstrophy. We used the Duckett modification of the onlay island flap using ventral preputial skin and coupled it with a proximal flip-flap of perimeatal skin without division of the urethral plate. This achieved an excellent result.

Alkali-degraded cornea generates a low molecular weight chemoattractant for polymorphonuclear leukocytes.

PURPOSE: The current study was designed to determine if a polymorphonuclear leukocyte (PMN) chemoattractant is derived from alkali-degraded whole cornea and to establish a range for its molecular weight. METHODS: We utilized a collagen gel-visual chemotactic assay to quantify the directional movement of PMN exposed to alkali-degraded corneas (30 min or 24 hr). In this experiment, the sample to be tested for chemotactic activity passed through a 14,000 molecular weight cutoff membrane into a collagen gel in which resting neutrophils were suspended in a random fashion. Cell movement was videotaped and subsequently tracked by digitizing the centroid of the cells at 30-sec intervals. Computer analysis of these tracks illustrated many behavioral characteristics, including directional movement. RESULTS: Alkali-degraded whole bovine corneas produced a chemotactic response in neutrophils within 5 min. Dilution of the 24-hr sample to 1:7 yielded a significant chemotactic response. The chemotactic response of both the 30-min and 24-hr samples followed a dose-response curve. CONCLUSIONS: This agent may be one of the inflammatory mediators that trigger the early neutrophil response after an alkali-injury to the eye.