Detection of secretory IgM in tears of IgA deficient individuals.

Tears from normal (n = 5) and serum IgA deficient (n = 3) individuals were investigated for the presence of secretory Immunoglobulin A (sIgA), sIgM and free secretory component (SC) by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 10-15% gradient minigels (PhastSystem), followed by immunoblotting using various immunological probes. Tear samples were treated in denaturing (SDS) sample buffer under non-reducing as well as reducing conditions, prior to analysis. All normal tear samples contained sIgA as well as free SC (estimated MW: 82kD) but only traces of IgM. Tears from the three serum IgA deficient subjects lacked sIgA but did contain free SC. In two of them sIgM was clearly detected and after treatment of tears with reducing agent, IgM (mu) heavy chain fragments (estimated MW: 78kD) were identified and could be distinguished from other tear proteins after SDS-PAGE. These findings indicate lacrimal secretion of free secretory component, even in the absence of its ligand. On the ocular surface, sIgM may play a compensatory role in IgA deficiency.

Sialic acid in human tear fluid.

A simple assay for the determination of sialic acid (N-acetylneuraminic acid) in human tear fluid was evaluated. Sialic acid, terminally bound on carbohydrate side-chains of glycoproteins, was released after treatment with neuraminidase and measured by an enzymatic colorimetric test. Tear fluid samples were collected from ten healthy adults, using glass capillaries and cellulose sponges. Sialic acid levels in tears collected with sponges (0.8-1.8 mmol l-1) did not differ significantly from those found in capillary tears (0.9-1.8 mmol l-1). Sialic acid, expressed as mmol g-1 protein, was significantly lower in tears collected with sponges (0.18-0.32 mmol g-1) than with capillaries (0.19-0.42 mmol g-1). Recovery of sialic acid and protein after incubation of cellulose sponges with tears was more than 99%. Sialic acid levels in human tears, which had been centrifuged to remove insoluble material, remained unchanged. Furthermore, tear sialic acid activity did not pass a filter with a molecular weight cut-off point of 10,000. Our data indicate that with the assay used in this report, sialic acid in tears is not due to secretory immunoglobulin A (sIgA), lactoferrin and lysozyme. The fact that the major tear proteins do not contribute to the sialic acid levels detected in tears suggests that other as yet unknown soluble glycoproteins are involved.

Vitamin A intervention: short-term effects of a single, oral, massive dose on iron metabolism.

A group of 134 school children aged 3-9 y, with signs of conjunctival xerosis, from the rural area of the Sakorn Nakhon province in Northeast Thailand were selected for a controlled study on the short-term effect (2 wk) of a single, oral high dose of vitamin A on iron metabolism. After collection of the baseline data, children within villages were randomly assigned to receive the capsules (n = 65) or serve as control subjects (n = 69). Two weeks after supplementation significant increases of retinol, retinol-binding protein, hemoglobin, hematocrit, serum iron, and saturation of transferrin were found in the supplemented group. Ferritin concentrations did not change significantly. These short-term changes completely exclude seasonal effects and change in morbidity. This study provides further evidence of a causal association between vitamin A and iron metabolism. In areas where vitamin A deficiency is endemic, periodic massive vitamin A dose programs can also improve iron status of the population.

The effect of vitamin A supplementation on tear fluid retinol levels of marginally nourished preschool children.

Vitamin A has been determined in tear fluid and blood plasma of marginally nourished Thai children before and after supplementation with a single, oral dose of 110 mg retinyl palmitate. After two months a significant rise of tear fluid retinol levels of the supplemented group was observed as compared to the non-supplemented group, while after four months no difference could be found. Determination of vitamin A levels in tear fluid may be useful in clinical eye research, with special regard to xerophthalmia.

Recovery of protein from tear fluid stored in cellulose sponges.

Human tear fluid collected in glass capillaries was absorbed in cellulose sponges. After various time intervals the recovery was measured for total protein, amylase (AMY), lysozyme (LZM), immunoglobulin A (IgA) and lactoferrin (LF). After one day storage at 4 degrees C of moistened sponges of at least 92% recovery was observed. Storage for longer periods at 4 degrees C up to seven days showed a decreasing recovery for total protein, AMY and IgA. Tear collection with sponges and storage of wet sponges for maximally one day at 4 degrees C followed by centrifugation or storage of the fluid at -20 degrees C are reliable ways of sample treatment.

Fluorometric determination of retinol in human tear fluid using high-performance liquid chromatography.

A fast and sensitive high-performance liquid chromatographic (HPLC) method for the determination of vitamin A (all-trans retinol) in 50 microliter human tear fluid is described. After deproteinization with ethanol and extraction with n-hexane, retinol is separated from the extract on a straight-phase HPLC column and detected fluorometrically. Vitamin A can be determined in concentrations as low as 0.4 ng/ml. A single analysis can be completed in 12 min while the analysis of a series of 60 samples takes about 8 h. The within-assay and between-assay coefficients of variation were 3.1 and 4.2% resp. The between-assay analytical recovery of retinol added to tear fluid was 97.4 +/- 6.0% (mean +/- SD). Retinol was determined in tear fluid of nine adult volunteers. The amounts observed ranged from less than 0.4 - 10.6 ng/ml.

Comparison of tears and lacrimal gland fluid in the rabbit and guinea pig.

The excretory duct of the lacrimal gland of rabbits and guinea pigs was cannulated in situ for collection of pure lacrimal gland fluid, not contaminated by secretions from the Harderian gland or contributions of desquamating cells of the conjunctival and corneal epithelium. Tears as well as lacrimal gland fluid of both species showed a species-specific and molecular weight-dependent pattern after sodium dodecylsulphate-polyacrylamide (SDS-PAA) gradient slab gel electrophoresis. The most striking difference in both species was a protein corresponding to serum albumin present in tears and almost lacking in lacrimal gland fluid. Likewise, a variety of enzymes, total protein and PGE2 were measured in tears and lacrimal gland fluid. For rabbit tears the lacrimal gland is the primary tissue source of lysozyme (LZM), beta-hexosaminidase (beta-hex), angiotensin-converting enzyme (ACE), plasminogen activator (PA) and total protein, while lactate dehydrogenase (LDH) and the greater part of prostaglandin E2 (PGE2) are present in rabbit tears mainly as products from other ocular tissue sources. In guinea pig tears peroxidase (POD), ACE, PA and less PGE2 are exceted by the lacrimal gland, amylase (AMY), LDH and a substantial amount of PGE2 are added to the guinea pig tears by other ocular tissue sources. Beta-hex and total protein are released from the lacrimal gland and from other ocular tissue sources as well.

Effect of acetazolamide (Diamox) on tear secretion.

The side effects of acetazolamide (Diamox) on lacrimation were measured in rats by means of the cotton-thread tear test. After a daily oral 1-mg dose (administered for five days), comparable to the dose used for adult humans on a drug-to-bodyweight basis, tear production remained unaffected but the lacrimal peroxidase secretion decreased by 60% of the baseline level. After withdrawal of acetazolamide the peroxidase secretion returned to the baseline level.

Macrophage support and suppression in rabbit T cell mitogenesis.

The role of macrophages in mitogen-induced rabbit T cell proliferation has been investigated. The blastogenic response to the 3 mitogens, PHA, ConA and oxidative treatment by neuraminidase and galactose oxidase (NaGo) was tested. T cell proliferation was reduced by removal of low density or plastic adherent cells, including macrophages, and could be enhanced by the addition of peritoneal resident macrophages, indicating a macrophage requirement for rabbit T cell proliferation. However, PHA-induced proliferation could not be raised to the level expected. It was found that catalase and especially 2-ME could considerably enhance macrophage dependent proliferation, even at low macrophage concentrations. It is concluded therefore, that macrophages not only support but also suppress lymphocyte proliferation, namely by non-specific damage to lymphocytes through release of radicals and hydrogen peroxide. In addition, peritoneal, but not lymph node macrophages were found to suppress lymphocyte proliferation by prostaglandin production, although to a lesser extent. Experiments, done in the presence of blockers of macrophage-mediated suppression, showed that macrophages were able to magnify the PHA-induced T cell proliferation to the expected values. The experiments thus show that unactivated macrophages support and suppress lymphocyte proliferation at the same time.