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Biomedical subjects

G Graff

Publications and source records attributed to G Graff.

At least 19 recordsLinked to original sources
PubMed2006

Evidence for a clathrin-mediated recycling of albumin in human term placenta.

During human pregnancy, the trophoblast layer is in direct contact with maternal albumin. In contrast to immunoglobulins, albumin does not cross the placental barrier. However, albumin affects the trophoblast placental lactogen and chorionic gonadotroph secretion. The present study investigated the interaction between albumin and syncytiotrophoblast using human term placental explants. Bovine serum albumin, labeled with either 125I or fluorescein isothio-cyanate, was taken up rapidly by placental explants. This process was temperature-sensitive. The internalized labeled BSA quickly outflowed from the tissue at the maternal side, largely without any major modification in molecular weight. Colchicine (1 mM), which disrupts the microtubule network, or cytochalasin B (40 microM), which disassembles filamentous actin, did not interfere with the placental transmembrane movements of labeled BSA. Megalin, clathrin, and caveolin 1 are three membrane proteins associated with albumin endocytosis in other tissues, but only megalin and clathrin were detected in the syncytiotrophoblast layer by immunohistochemistry. The uptake of labeled BSA into placental explants was not modified by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (1 mM) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM), two pharmacological tools known to disturb megalin-mediated albumin endocytosis. By contrast, methyl-beta-cyclodextrin (10 mM) and chlorpromazine (1.4 mM), both of which disrupt the clathrin-mediated endocytotic system, significantly reduced the uptake of labeled BSA. These data suggest, to our knowledge for the first time, that maternal albumin is actively internalized into the human trophoblast according to an apical recycling pathway. This temperature-sensitive process does not depend on an intact cytoskeleton, but it is associated with a clathrin-mediated endocytotic system.

Caveolin 1↗
PubMed2004

Colloidal effect of albumin on the placental lactogen and chorionic gonadotrophin releases from human term placental explants.

Albumin has been reported to stimulate the release of placental lactogen and chorionic gonadotrophin from human term placental explants within physiological concentrations. This study aimed at characterizing further its effect on the placental hormonal secretion. The placental lactogen and chorionic gonadotrophin secretory response of incubated explants to 5% albumin was reproduced by colloidal agents, i.e., dextran (4.5%) and polygelin (4%), indicating that a rise in colloidal osmotic pressure can elicit hormonal release from the syncytiotrophoblast. Their secretory effects were not modified by the absence of extracellular calcium or the presence of verapamil in the medium. The three agents also provoked a marked increase in (45)calcium outflow from preloaded and perifused explants that persisted in absence of extracellular calcium. These data indicate that the triggering effect of albumin on placental lactogen and chorionic gonadotrophin release can be partly reproduced by colloidal agents and is independent of extracellular calcium.

Albumins↗
PubMed2002

Suppression of post-vitrectomy lens changes in the rabbit by novel benzopyranyl esters and amides.

This study reports for the first time a therapeutic modality for the suppression of posterior subcapsular cataract (PSC) formation in an animal model (rabbit) of vitrectomy. This therapeutic modality may also have the potential to attenuate/prevent the high incidence of loss of vision due to cataract formation in patients that undergo vitrectomy. Unilateral, partial vitrectomy was performed on 2.5 month old Dutch Belted rabbits with vitreous replaced by either commercially available BSS((R)) or BSS PLUS((R)) (n=16). Alternatively, vitreous was replaced with a proprietary, modified BSS PLUS((R)) irrigating solution containing 1.25 microM AL-8417 (n=12), 5.0 microM AL-12615 (n=5) or 5.0 microM AL-17052 (n=9). Age matched, non-operated rabbits were used as controls (n=16). Lenses were analysed by correlative structural (light, scanning electron microscopic and three-dimensional computer-assisted drawings) and optical (low power helium-neon laser scan) quality analysis 6 months following surgery. Results demonstrate that vitreous replacement with an irrigating solution that contains the ester-linked benzopyran, AL-8417, the amide-linked benzopyran pro-drug, AL-17052, or its active metabolite, AL-12615, prevented abnormal post-vitrectomy lens growth, or fiber formation. Focal length variability (FLV) assessments (sharpness of focus) confirmed the beneficial drug effects detected morphologically, with FLV being essentially equal to that of age-matched, non-surgical controls. In contrast, lenses of animals with vitreous replaced solely with BSS((R)) or BSS PLUS((R)) exhibited significantly higher FLV than both age-matched controls and animals that underwent vitrectomy with drug-containing irrigating solutions. The ability of AL-8417, AL-17052 and its active metabolite, AL-12615, to suppress vitrectomy-induced posterior lens fiber changes appears to reside in their unique pharmacological profile, acting as antioxidant, anti-inflammatory and cytostatic agents.

Animals↗
PubMed2001

The effect of partial vitrectomy on blood-ocular barrier function in the rabbit.

PURPOSE: To compare ocular vascular permeability in the rabbit after vitrectomy as assessed by contrast-enhanced magnetic imaging (CE-MRI) and measurements of aqueous and vitreous humor protein concentration. METHODS: Partial vitrectomies were performed, irrigating with BSS or BSS PLUS. Post-operative vascular leakage was determined by CE-MRI following intravenous administration of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). Aqueous and vitreous protein concentrations were quantified by standard biochemical assay. ERG evaluations were performed on postoperative days 1, 3, and 7. RESULTS: Using BSS as irrigant, breakdown of the inner blood-retinal barrier (BRB) occurred in 4/7 eyes on post-operative day 1. The rate of Gd-DTPA leakage was significantly greater on postoperative day 1 than that in unoperated, control eyes, but declined approximately 50% by day 3. At both time points, outer BRB breakdown was restricted to the sclerotomy wounds. No BRB leakage was detectable in control eyes. Blood-aqueous barrier (BAB) leakage was bilateral on day 1. Significantly greater Gd-DTPA leakage occurred in the operated eye than in the nonsurgical contralateral eye. On day 3, approximately 40% bilateral reduction in leakage indicated resolution of BAB leakage. Notably, Gd-DTPA leakage of the BAB and BRB was significantly reduced in the BSS PLUS treated group. In contrast to MRI assessments, protein concentrations of the aqueous and vitreous in the surgical eye showed no detectable differences between BSS and BSS PLUS. Concurrent with the transient loss of ocular barrier function, ERG responses also declined. However, by day 7 greater than 90% recovery was noted in BSS PLUS treated animals but not in the BSS treatment group. CONCLUSIONS: CE-MRI is capable of detecting subtle changes in vascular permeability following ocular surgery. Advantages of using BSS PLUS compared to BSS as the irrigating solution can be detected using this technique. BSS PLUS's protection of barrier function is consistent with a rapid recovery in retinal function not observed in BSS treated eyes.

Animals↗
PubMed2001

Physiological concentrations of albumin stimulate chorionic gonadotrophin and placental lactogen release from human term placental explants.

This study investigates whether albumin, a major plasma protein in direct contact with the trophoblast in vivo, can modulate human chorionic gonadotrophin (HCG) and human placental lactogen (HPL) releases from placental explants. Incubating explants with a near physiological, i.e. 5%, concentration of human or bovine albumin during 30 min increased HCG and HPL release by at least 150%. This albumin effect was not mediated by any difference in hormone adsorption onto glass surfaces. In contrast to the sustained stimulation of hormone releases elicited by the addition of 10 mmol/l extracellular calcium, the albumin-mediated secretory responses were transient. However, the albumin- and calcium-stimulatory effects were abolished at 4 degrees C, depressed by 0.36 mmol/l cycloheximide or 1 mmol/l colchicine and potentiated by 40 micromol/l cytochalasin B. Moreover, the stimulatory effect of albumin on the hormone releases was not modified in the absence of Ca(2+) or in the presence of 1 or 10 mmol/l Ca(2+) in the extracellular milieu. These data suggest that albumin is involved, at physiological concentration, in the secretion of HCG and HPL by human placenta. The cellular mechanism(s) underlying the albumin-mediated secretory responses may be partly different from those involved during the calcium-mediated stimulation.

Animals↗
PubMed2000

The relationship between rabbit lens optical quality and sutural anatomy after vitrectomy.

This study was conducted first, to characterize structural changes in rabbit lenses after vitrectomy; and second, to assess whether such changes correlate with a quantifiable compromise in optical function. Unilateral, partial vitrectomies were performed on 2.5 month old Dutch Belted rabbits (n = 64). Age matched non-operated rabbits (n = 32) were used as controls. Lenses were analysed by correlative structural (light, scanning electron microscopic and three-dimensional computer-assisted drawings) and optical (low power helium-neon laser scan) analysis at 1.5, 3, 6 and 12 months post-surgery (n = 16 lenses from operated animals and n = 8 lenses from non-operated controls at each time point). Results demonstrate that in rabbits lens growth, or fiber formation, is compromised after vitrectomy. From 1.5 to 12 months after surgery, lenses had progressively more crooked posterior line sutures with sub-branches of increasing size and number in successive growth shells. Quantification of lens optical quality specifically along and/or between these atypical suture branches and sub-branches revealed a significant increase in focal length variability (sharpness of focus) after vitrectomy. A peripheral zone of fibers with abnormal posterior ends was produced surrounding the pre-surgical lens mass. This additional zone of aberrant fibers was associated with a quantifiable degradation in lens optics. Studies on the prevention of post-vitrectomy lens changes in this rabbit model may yield useful information applicable to the human condition.

Animals↗
PubMed2000

Nepafenac, a unique nonsteroidal prodrug with potential utility in the treatment of trauma-induced ocular inflammation: II. In vitro bioactivation and permeation of external ocular barriers.

Nepafenac, the amide analog of the NSAID amfenac, was examined in vitro for its bioactivation by ocular tissue components and its ability to permeate external ocular barriers. Rabbit tissues catalyzed a concentration-dependent conversion of nepafenac to amfenac. The order of specific hydrolytic activity is retina/choroid >> iris/ciliary body. Corneal tissue showed only minimal activity. Similarly, in human ocular cadaver tissue the specific activity of iris/ciliary body was greater than cornea. Continued perfusion of the corneal epithelium demonstrated a nearly six-fold greater permeation coefficient for nepafenac (k(p) = 727 x 10(-6) min(-1)) than for diclofenac (k(p) = 127 x 10(-6) min(-1)). Superior permeation of conjunctival and scleral tissue by nepafenac (k(p) = 128 x 10(-6) min(-1)) compared to diclofenac (k(p) = 80 x 10(-6) min(-1)) was also evident. Short term perfusion (5 min) of the corneal surface with 0.1% nepafenac resulted in sustained flux of drug across the cornea for 6 h. Under identical conditions only 3.3 microM of diclofenac accumulated on the corneal endothelial side compared to 16.7 microM nepafenac. The enhanced permeability of nepafenac, combined with rapid bioactivation to amfenac by the iris/ciliary body and retina/choroid, make it a target specific NSAID for inhibiting prostaglandin formation in the anterior and posterior segments of the eye.

Animals↗
PubMed2000

Nepafenac, a unique nonsteroidal prodrug with potential utility in the treatment of trauma-induced ocular inflammation: I. Assessment of anti-inflammatory efficacy.

Nepafenac, the amide analog of 2-amino-3-benzoylbenzeneacetic acid (amfenac), was examined in preclinical models for its potential utility as a topical ocular anti-inflammatory agent. Diclofenac was selected as the reference compound. In contrast to diclofenac (IC50 = 0.12 microM), nepafenac exhibited only weak COX-1 inhibitory activity (IC50 = 64.3 microM). However, amfenac was a potent inhibitor of both COX-1 (IC50 = 0.25 microM) and COX-2 activity (IC50 = 0.15 microM). Ex vivo, a single topical ocular dose of nepafenac (0.1%) inhibited prostaglandin synthesis in the iris/ciliary body (85-95%) and the retina/choroid (55%). These levels of inhibition were sustained for 6 h in the iris/ciliary body and 4 h in the retina/choroid. Diclofenac (0.1%) suppressed iris/ciliary body prostaglandin synthesis (100%) for only 20 min, with 75% recovery observed within 6 h following topical dosing. Diclofenac's inhibition of prostaglandin synthesis in the retina/choroid was minimal. Nepafenac's inhibitory efficacy and longer duration of action was confirmed in a trauma-induced rabbit model of acute ocular inflammation monitoring protein or PGE2 accumulation in aqueous humor. Results warrant further assessment of nepafenac's topical ocular efficacy in the treatment of postoperative ocular pain, inflammation, and posterior segment edema.

Animals↗
PubMed2000

A comparison of the effects of olopatadine and ketotifen on model membranes.

Olopatadine is a human conjunctival mast cell stabilizer with antihistaminic activity. Ketotifen is an older molecule that possesses antihistaminic activity and is reported to have additional pharmacological properties. The interactions of these two compounds with model membranes (i.e., monolayers of 1-stearoyl-2-oleoyl-sn-glycerophosphocholine at the argon-buffer interface), and natural (i.e., erythrocyte) membranes were compared in an effort to understand the differences in their biological activities. Drug-lipid interaction with monolayers was determined by monitoring the surface pressure as a function of the drug concentration in the aqueous phase supporting the monolayer. Drug interaction with erythrocyte membranes was determined by monitoring changes in the permeability of the membranes to hemoglobin and 6-carboxyfluorescein as a function of drug concentration in the medium. Olopatadine and ketotifen are both intrinsically surface active and both interact with phospholipid monolayers. However, in both the presence and absence of lipid monolayers, the changes in surface pressure induced by olopatadine are lower than those caused by ketotifen. The effects of these two drugs on cell membranes were dramatically different. Exposure of bovine erythrocytes to increasing concentrations of ketotifen (1-10 mM) resulted in complete hemolysis of the cells, whereas olopatadine (1-10 mM) caused only minimal hemolysis (< 8%). Consistent results were obtained in experiments measuring the leakage of 6-carboxyfluorescein from erythrocyte ghosts as a more sensitive marker of membrane perturbation. Olopatadine treatment (0.1-10 mM) minimally perturbed the cell membrane while ketotifen (1-10 mM) caused a concentration dependent release of the fluorescent marker. These data demonstrate fundamental differences between the two drugs in their effects on cell membranes. Moreover, the differences are consistent with the surface activities of the two compounds measured in monolayers and with reported differences in their pharmacological activities. These findings offer an explanation for the biphasic non-specific cytotoxic effect of ketotifen on histamine release from mast cells and may account for the nonlytic mast cell stabilizing activity of olopatadine.

Animals↗
PubMed2000

Noninvasive ventilation in respiratory failure due to cystic fibrosis.

BACKGROUND: Noninvasive positive-pressure ventilation (NIPPV) is increasingly used as an effective means of avoiding endotracheal intubation and mechanical ventilation in patients with respiratory insufficiency or failure. METHODS: We retrospectively reviewed our experience with NIPPV to treat respiratory failure in five patients with cystic fibrosis (CF). RESULTS: Despite chronic lung disease related to CF, none of our cases were end-stage. All patients had recent pulmonary function tests showing a forced expiratory volume in 1 second (FEV1) of more than 30% predicted for age. All patients had progressive atelectasis, hypoxemia, and impending respiratory failure related to an acute pulmonary exacerbation or upper abdominal surgical procedure (open gastrostomy tube placement). Respiratory rates decreased, oxygen saturation increased, fraction of inspired oxygen (FiO2) requirement decreased, transcutaneous CO2 decreased, and atelectasis resolved with NIPPV. CONCLUSIONS: Use of NIPPV provides effective respiratory support while avoiding the need for endotracheal intubation. The applications of NIPPV, reports of its use in patients with CF, and the equipment required are reviewed.

Adolescent↗
PubMed1999

Novel esters and amides of nonsteroidal antiinflammatory carboxylic acids as antioxidants and antiproliferative agents.

A series of phenolic antioxidant ester and amide derivatives of the nonsteroidal antiinflammatory drug naproxen was designed to have both antiinflammatory and cytoprotective activity. Compounds were evaluated in vitro both for antioxidant activity, as assessed indirectly by thiobarbituric acid reactive substance (TBARS) formation in a membrane lipid peroxidation assay, and for antiproliferative activity, as indexed by the inhibition of DNA synthesis in cultured human vascular endothelial cells. Compounds of this series exhibited potent antioxidant activity, with IC50 values (1.6-11.63 microM) 2-6-fold lower than that of Trolox (6-hydroxy-2,5, 7,8-tetramethylchroman-2-carboxylic acid) and 400-1300-fold lower than that of vitamin E. Structural modifications of the ester or amide substructure (5a and 6a) did not affect antioxidant activity, but methylation of the 6-hydroxy substituent resulted in compound 6f which was devoid of antioxidant activity. Although indistinguishable in antioxidant activity, the amide derivatives tended to be more potent as antiproliferative agents than the corresponding esters. The IC50's for the amide derivatives (3, 5a-e, 8) ranged from 2 to 7 microM, while the IC50's for the structurally related esters (1, 2a-c, 6a-e) ranged from 9 to 22 microM. Moreover, studies with compound 6a indicate that the observed inhibition of DNA synthesis is reversible, suggesting that the antiproliferative activity is due to a cytostatic rather than cytotoxic activity of the compounds. Thus, the antioxidant-naproxen derivatives represent a novel series of agents that both protect against free-radical damage and possess cytostatic activity in vascular endothelial cells. Studies are in progress to assess the utility of these compounds as potential components of an ocular irrigating solution.

Amides↗
PubMed1999

Pharmacological validation of a feline model of steroid-induced ocular hypertension.

OBJECTIVE: To validate pharmacologically the feline model of steroid-induced ocular hypertension. METHODS: Serial studies were conducted in domesticated adult female cats trained to accept topical ocular drug administration and pneumotonometry. To establish intraocular pressure (IOP) values for each study, measurements were performed at the same time of day for 6 consecutive days. Beginning on day 7, cats received either steroid or vehicle administered topically to both eyes three times a day for approximately 28 days. The IOP measurements were performed daily. RESULTS: After 5 to 7 days of treatment with 0.1% dexamethasone or 1.0% prednisolone acetate, IOP began to increase, reaching peak values within 2 weeks. These values were sustained throughout dosing but declined rapidly to baseline upon cessation of treatment. Maximum IOPs for the dexamethasone- and prednisolone-treated groups averaged 4.5 +/- 0.3 mm Hg (n = 12) greater than the mean IOP value obtained in vehicle-treated cats. Cats treated with 0.25% fluorometholone, 1.0% loteprednol etabonate, and 1.0% rimexolone exhibited increases of 0.6, 1.2, and 1.7 mm Hg, respectively. These values were significantly lower than those observed following treatment with dexamethasone or prednisolone. CONCLUSIONS: The ocular hypertensive effects of selected anti-inflammatory topical ocular steroids in this model are consistent with clinical findings. CLINICAL RELEVANCE: This feline model is a useful tool for assessing the potential IOP liability of novel anti-inflammatory steroids.

Administration, Topical↗
PubMed1999

Molecular analysis of Mycobacterium avium isolates by using pulsed-field gel electrophoresis and PCR.

Genetic relationships among 46 isolates of Mycobacterium avium recovered from 37 patients in a 2,500-bed hospital from 1993 to 1998 were assessed by pulsed-field gel electrophoresis (PFGE) and PCR amplification of genomic sequences located between the repetitive elements IS1245 and IS1311. Each technique enabled the identification of 27 to 32 different patterns among the 46 isolates, confirming that the genetic heterogeneity of M. avium strains is high in a given community. Furthermore, this retrospective analysis of sporadic isolates allowed us (i) to suggest the existence of two remanent strains in our region, (ii) to raise the question of the possibility of nosocomial acquisition of M. avium strains, and (iii) to document laboratory contamination. The methods applied in the present study were found to be useful for the typing of M. avium isolates. In general, both methods yielded similar results for both related and unrelated isolates. However, the isolates in five of the six PCR clusters were distributed among two to three PFGE patterns, suggesting that this PCR-based method may have limitations for the analysis of strains with low insertion sequence copy numbers or for resolution of extended epidemiologic relationships.

Electrophoresis, Gel, Pulsed-Field↗
PubMed1998

Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis.

Previously described models of endotoxin-induced uveitis quantify neutrophil influx into the eye using biochemical or direct cell count methods that result in an underestimation of ocular leukocyte accumulation following the inflammatory stimulus. We have optimized the rat model of endotoxin-induced uveitis by first overcoming interference in the biochemical assay of myeloperoxidase due to endogenous ocular reductants and cellular constituents containing free thiol functional groups. This was accomplished by simultaneously 1) extensively diluting soluble, interfering substances and 2) blocking tissue sulfhydril functional groups during tissue homogenization. Uveitis was induced in rats by subplantar injection of endotoxin. Twenty-four hours later, eyes were enucleated, homogenized, fractionated, and myeloperoxidase activity of neutrophils sedimenting with the membranous pellet was extracted. Previously published extraction procedures yielded only 40% of total assayable myeloperoxidase activity. Optimal recovery of myeloperoxidase activity (>twofold increase) was achieved only with two sequential extractions using 50 mM phosphate buffer (pH 7.4) containing 10 mM N-ethylmaleimide, and subsequent solubilization of myeloperoxidase activity by extraction with 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0). This modified extraction procedure and optimized myeloperoxidase assay conditions (300 microM hydrogen peroxide and 1.5 mM o-dianisidine) were then used to enhance the uveitis model. Maximum ocular neutrophil accumulation was observed at endotoxin doses of 100-200 microg. Total ocular neutrophil infiltrations ranged from 250,000 to 800,000 cells/globe. This leukocyte influx was inhibited dose-dependently by topical ocular administration of dexamethasone, with half-maximal inhibition observed at a concentration of 0.01%, w/v. Further validated by the correlation of biochemical results with histological evaluation, the refined methodology described in this report has application in assessing the ophthalmic therapeutic potential of antiinflammatory agents.

Administration, Topical↗
PubMed1998

Transient loss of prostaglandin synthetic capacity in rabbit iris-ciliary body following anterior chamber paracentesis.

The trauma-induced acute ocular inflammatory response has been characterized by investigating the kinetics of blood-aqueous barrier (BAB) breakdown, prostaglandin (PG) accumulation in the aqueous humor, and cyclooxygenase (PGH synthase) activity of the iris-ciliary body (ICB) following paracentesis in the NZA rabbit. BAB breakdown was assessed by quantifying plasma protein extravasation into the anterior chamber. PGE2 and 6-keto-PGF(1alpha) concentrations in the aqueous humor were quantified by radioimmunoassay. The capacity of ICB tissue homogenates to generate eicosanoids from exogenously supplied [I-14C]-arachidonic acid was assessed radiometrically by HPLC. Paracentesis resulted in a rapid and dramatic increase in aqueous humor PGE2 concentrations. Within 10 minutes, PGE2 concentrations increased 937-fold, from 6.2+/-4.9 pg/ml to maximal concentrations of 5810+/-3829 pg/ml. PG synthesis was followed temporally by an increase in aqueous humor protein, with peak levels (53.1 mg/ml) achieved within 30 minutes post paracentesis. Both PGE2 and protein levels gradually declined to near baseline levels 48 hours after trauma. ICB homogenates from naive animals produced significant amounts of eicosanoids (total PG=2.95 nmol/ 10 min/100 mg tissue). HHT (12 hydroxy-heptadecatrienoic acid) was produced in the greatest quantity, followed by PGE2alpha, PGI2, and TXB2/ PGF2 . Notably, following paracentesis, eicosanoid synthesis by the isolated ICB was observed to diminish abruptly. Formation of all eicosanoids was uniformly reduced by approximately 40% five minutes following paracentesis, with an 81% decrease in synthetic activity at 15 minutes. Eicosanoid synthetic capacity was only restored to baseline 48 hours post paracentesis. These findings suggest that, following ocular trauma, temporal changes occur in ICB PG synthetic activity that may impact on the selection of an optimal dosing paradigm for efficacy testing of topically administered NSAIDs.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Secretion of proinflammatory cytokines by human conjunctival epithelial cells.

The production of cytokines by human conjunctival epithelial cells following stimulation was investigated. Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression. Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals. Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF). With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels. IL-1ra was detected in both culture supernatants and cell lysates under basal conditions. In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion. After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged. None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells. These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines. Further evaluation of these cells as targets of therapy is warranted.

Calcimycin↗
PubMed1996

The in vitro and in vivo ocular pharmacology of olopatadine (AL-4943A), an effective anti-allergic/antihistaminic agent.

Olopatadine (AL-4943A; KW-4679) [(z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2 acetic acid hydrochloride] is an anti-allergic agent which inhibits mast cell mediator release and possesses histamine H1 receptor antagonist activity. Studies were conducted to characterize the in vitro and in vivo pharmacological profile of this drug relevant to its topical ocular use. AL-4943A inhibits histamine release in a concentration-dependent fashion (IC50 = 559 microM) from human conjunctival mast cell preparations in vitro. Histamine release was not stimulated by AL-4943A at concentrations as high as 10 mM. In contrast, ketotifen stimulated histamine release at concentrations slightly higher than effective inhibitory concentrations. AL-4943A did not display any in vitro cyclooxygenase or 5-lipoxygenase inhibition. Topical ocular application of AL-4943A effectively inhibits antigen- and histamine-stimulated conjunctivitis in guinea pigs. Passive anaphylaxis in guinea pig conjunctiva was attenuated by AL-4943A applied 30 min prior to intravenous or topical ocular antigen challenge (ED50 values 0.0067% and 0.0170%, w/v, respectively). Antihistaminic activity in vivo was demonstrated using a model of histamine-induced vascular permeability in guinea pig conjunctiva. AL-4943A applied topically from 5 min to 24 hrs prior to histamine challenge effectively and concentration-dependently inhibited the vascular permeability response, indicating the compound has an acceptable onset and a long duration of action. Drug concentrations 5-fold greater than those effective against histamine-stimulated conjunctival responses failed to inhibit vascular permeability responses induced with either serotonin or Platelet-Activating-Factor. These data indicate that the anti-histaminic effect observed with AL-4943A is specific. These anti-allergic/antihistaminic activities of AL-4943A observed in preclinical model systems have been confirmed in clinical trials in allergic patients.

Administration, Topical↗