Analysis of acidic metabolites of biogenic amines in bovine retina and vitreous and aqueous humour by gas chromatography-negative ion chemical ionisation mass spectrometry.

Acidic metabolites of a number of biogenic amines have been identified and quantified by reaction with either acetic or propionic anhydride in the aqueous phase followed by extraction into ethyl acetate, esterification of carboxyl groups with ditrifluoromethylbenzyl bromide (DTFMBzBr), and then conversion of the remaining free hydroxyl groups to acetates. Subsequent analysis of these derivatives revealed that most (greater than 60%) of the ion current was carried by the ion resulting from the loss of DTFMBz from the molecular ion. This made the method highly specific and practical--limits of detection were established at approximately 200 pg with a potential limit of detection below the picogram level. This method establishes unequivocally that the metabolites of tyramine, dopamine, and adrenaline/noradrenaline (4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, and dihydroxymandelic acid, respectively) are present in bovine retina and in vitreous and aqueous humour. In addition, high concentrations of the dopamine metabolite homovanillic acid were found in retina and vitreous, but not in aqueous humour. p-Hydroxymandelic acid, the acidic metabolite of p-octopamine/p-synephrine, was identified in vitreous and in aqueous humour.

Ocular inflammatory effects of intravitreally injected tumor necrosis factor-alpha and endotoxin.

Intravitreal injection of human recombinant tumor necrosis factor-alpha (TNF) induced inflammation in the rabbit eye characterized by dilation of blood vessels in the iris, disruption of the blood-ocular barriers, infiltration of inflammatory cells into the anterior chamber, and accumulation of prostaglandin E in intraocular fluids. Inflammation first appeared on day 1, increased on day 2, and remained elevated on day 7. The inflammatory cell infiltrate in the anterior segment of the eye was largely monocytic on days 1 and 2; by day 7 large numbers of lymphocytes were also present. TNF-induced ocular inflammation therefore differed from that reported for intravitreally injected endotoxin in terms of time course and the types of inflammatory cells in the aqueous humor. In a series of experiments in which combinations of TNF and endotoxin were used, intravitreal injection of TNF, 24 h after a low dose of Escherichia coli endotoxin, produced no more inflammation than that produced by TNF following an injection of endotoxin vehicle. However, if TNF was injected 24 h before endotoxin, the resulting inflammation was greater than that observed in animals given TNF followed by endotoxin vehicle.

Expression of two molecular forms of the complement decay-accelerating factor in the eye and lacrimal gland.

Complement is present in ocular fluids, but the molecular mechanism(s) restricting its activation to exogenous targets and not to autologous ocular cells are currently unknown. To clarify how this control is achieved, monoclonal antibody (mAb)-based techniques were used to examine the eye, the lacrimal gland, and ocular fluids for the decay-accelerating factor (DAF), a membrane regulatory protein which protects blood cells from autologous complement activation on their surfaces. Immunohistochemical staining of tissue sections revealed DAF antigen on corneal and conjunctival epithelia, corneal endothelium, trabecular meshwork, and retina, as well as on lacrimal gland acinar cells and in adjacent lumens. By flow cytometry, cultures of conjunctival epithelium exhibited the highest DAF levels and levels on corneal epithelium greater than corneal endothelium greater than conjunctival fibroblasts. Biosynthetic labeling of corneal endothelium yielded de novo DAF protein with an apparent molecular weight (Mr) of 75 kD, approximating that of blood cell DAF protein, and digestions of conjunctival epithelium with phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme which cleaves glycoinositolphospholipid membrane anchors, released approximately 70% of the ocular surface DAF protein similar to leukocyte surface DAF protein. Quantitations of DAF by radioimmunometric assay employing mAbs against two DAF epitopes revealed 325 ng/ml (n = 12), 4.8 ng/ml (n = 10), and 22.0 ng/ml (n = 8) of soluble DAF antigen in tears, aqueous humor, and vitreous humor, respectively. Western blot analyses of the tear DAF antigen revealed two DAF forms, one with an apparent Mr of 72 kD resembling membrane DAF forms in other sites, and a second with an apparent Mr of 100 kD, which is previously undescribed. Since DAF activity is essential physiologically in protecting blood cells from autologous complement attack, the identification of DAF on the ocular surface, intraocularly, in the lacrimal gland, and in tears suggests that DAF-mediated control of complement activation is also required in these locations.

Tau fraction of transferrin is present in human aqueous humor and is not unique to cerebrospinal fluid.

In view of the many similarities between the aqueous humor and the cerebrospinal fluid (CSF), we investigated whether the tau fraction of transferrin, which is believed to be specific for CSF, is also present in aqueous humor. Samples of aqueous humor and CSF from clinically normal individuals were analyzed by SDS-PAGE and isoelectric focusing (IEF), and probed with anti-transferrin antiserum and the lectin Limax flavus agglutinin. Specific bands which corresponded to transferrin and its tau fraction were detected in both fluids. After isolation of these bands by affinity chromatography with anti-transferrin antiserum, we detected a single, diffuse fraction at an apparent molecular weight of 80 kDa in both aqueous humor and CSF. Peptide mapping revealed no detectable difference in this fraction between the two fluids. The detection of the tau fraction in human aqueous humor indicates that this molecule cannot be considered a specific cerebrogenic marker as had been thought previously. In view of the known growth-promoting properties of transferrin, its isolation from both ocular and cranial fluids attains special significance.

Ion-exchange liquid chromatographic determination of nitrate and nitrite in biological fluids.

A rapid, ion-exchange liquid chromatographic method for the determination of nitrate and nitrite in biological fluids is presented. Samples are deproteinated by ultrafiltration followed by removal of chloride using a silver form cation-exchange resin. Nitrate and nitrite are measured by ion-exchange liquid chromatography with conductivity detection. Recoveries from serum, ocular fluid, and water were determined for fortifications from 10 to 150 mg/L. Average recoveries ranged from 96 to 104% for nitrate and from 89 to 105% for nitrite. Pooled RSD values ranged between 1.5 and 1.9% for these analytes in all matrixes examined. The method of joint confidence hexagons was applied to the data to determine constant and relative bias of the method for each of the 3 matrixes in the study.

Aqueous humor contains transforming growth factor-beta and a small (less than 3500 daltons) inhibitor of thymocyte proliferation.

The anterior chamber of the eye is an immunologically privileged site in which allografts survive longer than at other body sites. In this regard, it is relevant that aqueous humor (AH) inhibits lymphocyte proliferation. In order to analyze AH for specific substances that inhibit thymocyte proliferation, samples of human AH, murine AH, and rhesus monkey AH were added to cultures of thymocytes stimulated by IL-1 or IL-2 in the presence of PHA. All samples of AH tested had potent inhibitory activity on thymocyte proliferation in this system. Inhibitory activity was lost by heating AH to 80 degrees C for 1 h. Dialysis of murine AH indicated that species smaller than 3500 Da were capable of mediating this activity; we have termed the factor(s) responsible for this "small inhibitory factor(s) of AH." Retentate, containing species larger than 3500 Da, retained inhibitory activity, but less than nondialyzed AH. Assays for PGE2 demonstrated that murine and human AH contained small quantities of PGE2. These quantities were insufficient to inhibit thymocyte proliferation in our assay system. Furthermore, AH from mice treatend with indomethacin had full inhibitory activity. Assays for transforming growth factor beta (TGF-beta) after acid activation demonstrated significant quantities of latent TGF-beta within human and murine AH which could be largely neutralized by antisera to TGF-beta. Active TGF-beta "activity" was also present without acid activation in samples of AH at a level approximately 20% that of latent TGF-beta. However, most of this "activity" could not be neutralized by antisera to TGF-beta. AH contains factors capable of limiting thymocyte proliferation.

Inhibition by indomethacin of the increased facility of outflow induced by adrenaline.

Topical adrenaline lowers intraocular pressure (IOP) in the rabbit largely due to an increase in facility of outflow of aqueous humour. This paper studies the inhibition by indomethacin or piroxicam of the adrenaline-induced rise in facility of outflow. Topical indomethacin is shown to reduce the acute IOP changes induced by adrenaline in conscious rabbits; both the early rise and the prolonged fall in pressure were inhibited. In anaesthetized rabbits, indomethacin pretreatment prevented the large rise in facility of outflow which normally follows topical adrenaline. Indomethacin did not block the mydriasis induced by adrenaline, nor did it significantly alter aqueous humour protein levels. Piroxicam, a cyclo-oxygenase inhibitor which, unlike indomethacin, does not block Ca2+ movements in some tissues, also blocked the adrenaline-induced rise in facility of outflow, suggesting that this increased facility depends on cyclo-oxygenase and not on Ca2+ movements. Verapamil, a drug which blocks Ca2+ channels, was shown to inhibit the brief ocular hypertensive effect of adrenaline in the conscious rabbit, but to leave the hypotensive phase unchanged. It is concluded that the hypotensive mechanism of adrenaline may depend on synthesis of a prostaglandin, since inhibition of the adrenaline-induced rise in facility is achieved by inhibitors of cyclooxygenase. Despite previous reports that a prostaglandin may be responsible for the brief hypertensive phase, the present evidence suggests that Ca2+ movements may be involved, perhaps in activation of the extraocular muscles.

[In vivo examination of aqueous humor for cells and protein in clinically silent anterior uveitis before surgical interventions]. / In-vivo-Untersuchungen des Kammerwassers auf Zellen und Eiweiss bei klinisch stummer Uveitis anterior vor operativen Eingriffen.

The laser flare cell meter is a new device for measuring flare and cells in the anterior chamber. In 100 eyes we used this device to measure flare and cell counts. We found that eyes with former uveitis and no clinical signs of acute or earlier uveitis have more flare and more cells in the aqueous humor. This is important for IOL implantation and follow-up studies to determine the best way to perform eye surgery.

Study of aqueous humour in anterior uveitis.

Aetiological diagnosis of anterior uveitis was made clinically and substantiated with relevant investigations. Aqueous humour obtained under aseptic conditions, was analyzed for the cells study, culture and protein profile, using polyacrylamide gel electrophoresis. The results were analysed with the help of known clinical facts. Culture and smears were invariably negative, while the lymphocytes were present in varying numbers, polymorphs and macrophages afforded a useful clue for confirmatory diagnosis. The electrophoretic pattern of the proteins was related to the duration of the disease and was same in a group while it was distinctive among different groups of anterior uveitis.

[The possibilities for predicting and preventing postoperative inflammatory complications in intraocular correction]. / Vozmozhnosti prognozirovaniia i profilaktiki posleoperatsionnykh vospalitel'nykh oslozhnenii pri intraokuliarnoi korrektsii.

Measurements of unsaturated fatty acid diene conjugates in the anterior chamber humor are recommended to predict postoperative inflammatory complications. If their levels are increased, antioxidants should be included in the therapeutic scheme along with antibiotics and corticosteroids.

[Clinical observations and calcium determinations in hypocalcemic cataract].

Among 38 cases of hypoparathyroidism, 32 patients were found to have hypocalcemic cataract. The morphology and mechanism of cataract formation were studied by clinical observation and biochemical calcium determinations, which indicated that the development of hypocalcemic cataract was related to the calcium level in blood but not to the duration of disease. The results suggest that early diagnosis and treatment of hypocalcemia may be useful in controlling the development of cataract. Calcium determinations showed that the blood calcium level was related to that in the aqueous humor. It is probable that the blood calcium content may reflect the calcium level in the aqueous humor, thus evaluating the tendency for cataract development.

Presence of epidermal growth factor in human tears.

Epidermal growth factor (EGF) is a polypeptide that stimulates the growth of various tissues, including the cornea. The presence of EGF in tears from normal volunteers and in aqueous humor from cataract patients was investigated via human EGF (hEGF)-specific radioimmunoassay. Immunoreactive hEGF was found to be present at similar concentrations in both reflex (ranging from 0.7 to 8.1 ng/ml) and non-reflex tears (ranging from 1.9 to 9.7 ng/ml), but was undetectable in aqueous humor. Immunoreactive EGF in human tears was indistinguishable immunologically, biologically and biochemically from urine EGF and standard hEGF.

Detection of glycated polypeptides in human aqueous humor by lectin-binding analysis.

The proteins from 30 samples of normal human aqueous humor were separated into major fractions by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with lectins to characterize the glycated polypeptides. The lectins used were derived from Canavalia ensiformis (concanavalin-A), Phaseolus vulgaris (erythroagglutinin), Arachis hypogaea (peanut agglutinin), and Ulex europaeus agglutinin-I. Using this microanalytical technique, the glycated polypeptides in the aqueous humor were separated into nine major fractions with apparent molecular weights of 17, 41, 50, 55, 61, 77, 80, 120 and 151 kDa. The predominant oligosaccharides detected on these fractions were complex chains usually with sialic acid residues. The possible roles of the glycated polypeptides in the aqueous humor in health and disease are discussed. Our investigation of these molecules in human aqueous humor provides baseline data for a rapid diagnostic analysis of microsamples of this fluid obtained from diseased eyes.

Imunoglobulinas e anti-corpos específicos IgG-IgM para T.Gondii no soro e no humor aquoso / Immunoglobulines and specific antibodies IgG-IgM in T.Gondii in aqueous humor and serum

No humor aquoso de pacientes com uveite posterior ativa (25 pacientes) foi encontrada uma concentraçäo de imunoglobulinas significativamente maior do que em pacientes com catarata senil (10 pacientes). No humor aquoso de mais de 90% de pacientes portadores de uveite, foram detectados anticorpos anti-T. gondii, enquanto que nos pacientes com catarata isto näo ocorreu em nenhum. Näo houve diferença significativa entre os títulos de anticorpos anti-T. gondii, detectados pela reaçäo de imunofluorescencia indireta, no soro dos pacientes com uveite e dos com catarata. A reaçäo de imunofluorescência indireta para toxoplasmose, realizada com conjugado anti-IgM, foi quase sempre negativa, näo tendo colaborado para o esclarecimento diagnóstico dos casos

Controvérsias sobre a fisiopatogenia da toxoplasmose ocular / Controversy on the physiopathogeny of ocular toxoplasmosis

Este trabalho tem como objetivo, abrir uma discussäo entre os resultados obtidos por vários autores, em relaçäo as reaçöes sorológicas para Toxoplasma gondii. A preocupaçäo dos autores foi o estudo da pesquisa de anti-corpos IgG e IgM anti-T. gondii, pelas técnicas de Elisa e Imunofluorescência Indireta, quer no soro como no humor aquoso, em pacientes portadores de uveíte posterior focal, granulomatosa e necrótica (supostamente toxoplásmica)

Characterized and predictable rabbit uveitis model for antiinflammatory drug screening.

A model has been developed for the screening of antiinflammatory ophthalmic drugs in rabbits. This simple and rapid method is reproducible and uses fewer animals than do some other methods. Rabbits are sensitized to bovine serum proteins, then challenged intravitreally to induce a uveitis. The basis of the inflammatory response is shown to be due primarily to an immunologic mechanism. At 24 hr postchallenge, animals are sorted into treatment groups of approximately equal "titer" as defined by the slit lamp examination iris score which measures the magnitude of the immunologic response. Topical ocular treatment (control or drug) is then initiated and continued for a total of four days. Iris inflammation is evaluated by daily slit lamp exams. Results indicate that this model has statistically narrower frequency distribution of iris ratings than another published method, and is faster than other equally accurate and precise methods that depend upon blood antibody titer to sort animals.

Analysis of post mortem aqueous humour chemistry in the horse, with particular reference to urea nitrogen and creatinine.

The concentrations of several post mortem aqueous humour chemical constituents were compared with ante mortem serum chemical values in the horse. Urea nitrogen and creatinine values in post mortem aqueous humour were good predictors of ante mortem serum values. Aqueous humour urea nitrogen increased only slightly and creatinine did not change significantly for up to 24 h after death. Formulae were derived for calculating estimated ante mortem serum urea nitrogen and creatinine from aqueous humour values obtained after death. These results from normal horses identify analytes that are accurate predictors of ante mortem serum values. Determination of post mortem aqueous humour urea nitrogen of creatinine may assist in interpretation of the functional significance of equivocal histological lesions in the kidney.