Adherence of human lens epithelial cells to conventional poly(methyl methacrylate), heparin-surface-modified, and polyHema lenses.

We developed an in vitro model to assess the adherence of human lens epithelial cells to three types of intraocular lenses: poly(methyl methacrylate) (PMMA), heparin-surface-modified PMMA (HSM-PMMA), and polyHema. Lenses were incubated with a fixed number of human lens epithelial cells. Adherent cells were counted after 72 hours in culture. Scanning electron microscopy showed significantly fewer cells adhering to the HSM-PMMA and polyHema lenses than to the PMMA lenses (P < .01). Repeat experiments on cell lines established from different donors confirmed these findings.

Daunomycin as an inhibitor of human lens epithelial cell proliferation in culture.

Posterior capsule opacification is still a major complication of both extracapsular cataract extraction and phacoemulsification. We evaluated the effects of the anti-proliferative agent daunomycin on cultured human and bovine lens epithelial cell viability and proliferation. After ten minutes of exposure, low concentrations of the agent markedly inhibited the proliferation of both cell types. The calculated LD50 for the drug against human cells was 2.20 micrograms/ml and against the bovine cells was 0.38 microgram/ml. The bovine cells appeared to be slightly more susceptible to the drug's effects, although this difference was not marked. Our results indicate that daunomycin is a potent inhibitor of both human and bovine lens epithelial cells in the laboratory.

Growth characteristics of human lens epithelial cells in culture. Effect of media and donor age.

The effect of different concentrations of fetal calf serum (FCS) on the proliferative capacity of human and bovine lens epithelial cells in culture was evaluated. The effect of donor age on the maximum number of passages achieved using thirty eight individual cultures was also studied. The donor ages ranged from 1-88 years. Fifteen percent FCS was found to be the optimum concentration for both human and bovine cells. The two cell types demonstrated very similar responses across the spectrum of concentrations used. Correlation analysis revealed a significant (p < 0.05) negative correlation between donor age and maximum number of cell passages achieved.

Morphological appearances of human lens epithelial cells in culture.

A system for culturing human lens epithelial cells in the laboratory was developed. The morphological appearances of the cells was studied using phase contrast, scanning and transmission electron microscopy. Cell marker studies using monoclonal antibodies to cytokeratin, vimentin and epithelial membrane antigen were also performed. There was a marked increase in cell size as a function of time in culture. After 3 to 4 weeks cells showed early signs of ageing. By 6 to 8 weeks the majority of the cells had become very irregular in shape and demonstrated irregularities of the plasma membrane and intra-cytoplasmic vacuole formation. The cells stained strongly for vimentin and epithelial membrane antigen. Staining with cytokeratin was somewhat weaker. This culture technique provides us with a suitable model for studying the growth behavior of these cells.

Are the mast cells antigen presenting cells?

Mast cells have an important role in allergic reactions secreting histamine and other mediators of immediate hypersensitivity. In the present study we evaluated major histocompatibility complex (MHC) class II antigen expression in mast cells and their possible role in antigen presentation. In rats, 10% of mast cells isolated from the pleural cavity expressed MHC class II antigen; after incubation with gamma interferon (INF) 80% of the cells were positive. These findings suggest that mast cells, in addition to their secretory function in allergic reactions, may also function as antigen presenting cells.

Intrathyroidal mast cells express major histocompatibility complex class-II antigens.

We studied the expression of major histocompatibility complex (MHC) class-II antigen in mast cells of rat thyroid glands. In the normal rat thyroid, mast cells express MHC class-II antigen. In BioBreeding Wistar (BB/W) rats, the strain of animals prone to develop spontaneous autoimmune thyroiditis, the number of MHC class-II-positive mast cells was significantly higher than in normal rats (p less than 0.01). Furthermore, intrathyroidal mast cells in BB/W rats showed an increased MHC class-II expression before the appearance of circulating thyroid antibodies or the infiltration of tissue with mononuclear cells. These data suggest that mast cells in the rat thyroid gland may have a function as antigen-presenting cells.

Sequential presentation of a case of hyperthyroidism with autonomously functioning nodules and Graves' disease in the presence of IgG thyroid stimulators.

The rare occurrence of hyperthyroidism with an autonomously functioning nodule which following 131I therapy presented as toxic diffuse goitre (Graves' disease) is described in a 60 year old male. This progression was characterised by the presence of varying concentrations of IgG thyroid stimulators, thyroid stimulating immunoglobulins and thyroid growth stimulating immunoglobulins, as measured by cytochemical bioassay. It is postulated that the presence of the nodule and its associated hypersecretion of thyroid hormones may have protected the gland from the effects of IgG stimulators by bringing about inhibitory short-loop feedback on normal thyroid cells. It is further suggested that following therapeutic ablation of the nodule, normal thyroid cells became sensitive to the thyroid stimulators with the evolution of typical features of toxic diffuse goitre.

Evidence that adenosine 3',5'-monophosphate mediates stimulation of thyroid growth in FRTL5 cells.

Thyroid function, including growth, is TSH dependent, and most metabolic functions of TSH are thought to be mediated by cAMP. Recently, it has been suggested by several groups that growth may be an exception and that it may not be related to cAMP action. In addition, evidence has accrued indicating that the thyroid-stimulating antibody (TSAb) of Graves' disease, the metabolic actions of which are also cAMP mediated, may not be the goitrogenic agent in that syndrome. To evaluate these concepts, we used functioning rat thyroid cells (FRTL5) in monolayer culture and, as indices of growth, the incorporation of [3H]thymidine ([3H]Tdr) into DNA, the concentration of DNA measured directly, and the percentage of cells in S phase, as assessed by flow cytometry, all studied over 72 h of incubation. TSH, forskolin, and cholera toxin enhanced growth by each criterion and increased the concentration of cAMP in parallel; the effect on cAMP occurred rapidly and was maximal well in advance of influences on growth. In all instances, measures of growth promotion were minimal at 24 h and maximal at 48 h, except for [3H]Tdr incorporation, which was greater at 72 h than at 48 h. 3-Isobutyl-1-methylxanthine (IBMX) and (Bu)2 cAMP were also tested. Both enhanced all indices of growth and were as effective as TSH. Maximal responses to TSH were obtained at 100-200 microU/ml, maximal responses to both IBMX and (Bu)2cAMP occurred at 5 X 10(-4) M, and all three stimulators increased the DNA concentration and [3H]Tdr uptake and induced S phase in at least 20% of all cells in culture. The peak effect on DNA and S phase was consistently at 48 h. Epidermal growth factor (EGF) was shown to increase [3H]Tdr incorporation in a nondose-dependent fashion (10(-10) to 5 X 10(-9) M gave approximately 250% of control) over 1, 2, 3, 5, and 7 days, with no increase in DNA and a slight decrement in the concentration of cAMP. A laboratory standard TSAb-immunoglobulin G was shown to parallel TSH in both increasing cAMP (over 2 h of incubation) and growth stimulation (over 72 h). The data are entirely consistent with the view that TSH-stimulated thyroid growth is mediated by cAMP and that the established action of TSAb on adenylate cyclase is sufficient to explain goiter as well as hyperthyroidism in Graves' disease.

Association of thyroid-stimulating immunoglobulins and thyrotropin-releasing hormone responsiveness in women with euthyroid goiter.

Thyroid-stimulating immunoglobulins (TSI) were measured, using a highly sensitive cytochemical bioassay, in plasma from 26 euthyroid women with idiopathic diffuse or multinodular goiter selected on the basis of their serum TSH responses to TRH stimulation. Thirteen were chosen because they were previously identified to have impairment in TRH responsiveness and were compared with 13 consecutive patients who had normal responses to TRH. TSI were present in a significantly greater number of those who had subnormal TRH responses (11:13) compared to those who had normal responses (3:13) P less than 0.005. Although serum T4, T3, and basal TSH values were all within the normal range, mean serum T4 and T3 values were significantly higher and basal TSH significantly lower in the 14 patients who had TSI than in the 12 in whom TSI was absent. The coexistence of impaired TRH responsiveness and TSI was associated with a family history of thyroid disease. The data suggest that TSI in patients with euthyroid goiter cause a modest increase in thyroid secretion sufficient to blunt the TSH response to TRH but not to cause clinical hyperthyroidism.

Lysosomal membrane recovery following labilization by thyroid stimulators.

Lysosomal membrane permeability was assessed by measuring freely available naphthylamidase activity in intact preparations of guinea pig thyroid follicular cells following exposure of thyroid tissue to sequential stimulation by two thyroid stimulators, thyrotrophin (TSH) and thyroid stimulating immunoglobulins (TSI). These investigations showed that following labilization by TSH, the lysosomal membranes recovered and were capable of responding to a second thyroid stimulator (TSI). That such recovery represented restabilization of lysosomal membranes was confirmed by the finding that latent naphthylamidase activity was restored without a change in total activity of the enzyme.

Dose-response relationships in a cytochemical section bioassay for thyroid stimulators.

The thyroid stimulators TSH and thyroid stimulating immunoglobulins (TSI) have previously been distinguished in a cytochemical section bioassay (CBA) by the different times at which they caused maximum increase of lysosomal membrane permeability within guinea-pig thyroid follicular cells. The present study demonstrates that the times at which maximum stimulation occurs in the assay depends not only on the type (TSH or TSI) but also on the concentration of the stimulator. At higher concentrations of stimulator earlier times of maximum stimulation were observed for both TSH and TSI. When both stimulators were simultaneously present in plasma at higher concentrations a single peak of stimulatory activity occurred. Neutralization studies using specific antisera revealed that the single peak represented a merging of TSH and TSI peaks. These findings explain the lack of parallelism to the standard curve seen at higher concentrations of TSH or TSI in the CBA. They emphasize the necessity of measuring plasma samples at a variety of dilutions in order to achieve parallelism and to avoid intra-assay interference between TSH and TSI. When these conditions are observed CBA can be used to detect TSH and TSI with extreme sensitivity even when both are simultaneously present in plasma.

The prevalence of thyroid-stimulating antibodies in goitrous disease assessed by cytochemical section bioassay.

Thyroid-stimulating antibodies (TSAb) were detectable using a highly sensitive cytochemical section bioassay in plasma from all 56 hyperthyroid patients studied, including those who had either diffuse hyperplasia or nodular goiters. The maximum dilution at which TSAb was detectable ranged from 10(-2) - 10(-6). Six of these patients had scintigraphic evidence of single functioning nodules, and, surprisingly, TSAb was present in all. Of 27 patients who had nontoxic goiter, 14 (52%) had positive titers for TSAb ranging from 10(-2) - 10(-4). The mean serum T3 value in nontoxic goitrous patients who had TSAb was significantly higher than that in subjects in whom TSAb was absent; in contrast, mean serum T4 values were not significantly different in those two groups. It is concluded that idiopathic nontoxic goiter, toxic nodular goiter with functioning nodules (Plummer's disease), and toxic diffuse goiter (Graves' disease) share, in part, a common pathogenesis.