Early and late phases of ocular anaphylaxis in actively immunized guinea pigs.

A model of ocular anaphylaxis with distinct early- and late-phase components was studied in actively immunized guinea pigs. Twenty guinea pigs were injected with dinitrophenylated (DNP) bovine gamma globulin emulsified in Freund's complete adjuvant and challenged topically with di-DNP-lysine. Clinical signs were monitored over a 48 h period. An early-phase reaction (EPR) characterized by conjunctival edema, conjunctival erythema, lid swelling, and lid redness was observed. This reaction peaked at 0.5 h after challenge and subsided to a low point at 3-4 h. Subsequently, a second episode of lid swelling and lid redness was observed at 4-8 h. All animals in both groups exhibited an EPR. In addition, 75% of the animals underwent an EPR and an LPR. No animals exhibited an isolated LPR. Of the animals that underwent a dual response, 47% were biphasic, 6% were prolonged and 47% were multiphasic. The development of an active model of ocular anaphylaxis exhibiting both EPR and LPR components will enable studies of mechanisms which regulate the frequency and magnitude of these ocular allergic responses.

Effect of fish-fat or beef-fat supplemented diet on immune complex-induced enteropathy in the rat.

In contrast to animals on a beef fat-supplemented diet (BFD), animals maintained on a fish fat-supplemented diet (FFD) incorporate increased amounts of eicosapentaenoic acid (EPA) into membrane phospholipids. Generation of lipid mediators from such tissues favors the formation of compounds with less pro-inflammatory activity than are derived from tissues poor in EPA. Nevertheless, the FFD has not had a uniformly beneficial effect on animal models of inflammatory diseases. We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine. In this study, we tested the effect of a BFD and FFD on immune complex-induced enteropathy. After six (Expt. 1) or eight weeks (Expt. 2) on the diet, rats were injected with IC and the severity of serosal hyperemia in the small intestine was scored. In some FFD, no lesions were seen under conditions which elicited moderate to severe lesions in BFD rats. In Expt. 1 involving 22 rats and in Expt. 2 involving 28 rats, those on the FFD had a significantly lower composite lesional score compared to those on the BFD, p less than 0.005 and p less than 0.005, respectively. These results indicate that the FFD had a beneficial effect on IC-induced enteropathy. It is suggested that this effect of the FFD may be mediated primarily by a reduction in availability of platelet-activating factor.

Abnormal lymphocyte function following long-term PUVA therapy for psoriasis.

The surface markers and function of peripheral blood lymphocytes were examined in patients on long-term therapy with methoxsalen and UV-A radiation (PUVA). Ten patients with psoriasis were selected because they had received a high exposure to PUVA therapy, i.e., more than 200 treatments over 2-6 years with cumulative exposure doses of 1700-6000 J/cm2 UV-A radiation. Results were compared to those obtained with lymphocytes from untreated patients and UV-B treated patients with psoriasis. The PUVA-treated patients had low levels of E rosette-forming cells in the peripheral blood and markedly impaired lymphocyte responses following stimulation with optimal and suboptimal doses of mitogens. The sensitivity of lymphocytes to in vitro treatment with PUVA was similar in the three groups of patients. The results of this study indicate that long-term PUVA therapy alters the function and cell-surface markers or distribution of lymphocytes.

Reduction of the fraction of circulating helper-inducer T cells identified by monoclonal antibodies in psoriatic patients treated with long-term psoralen/ultraviolet-A radiation (PUVA).

Ultraviolet radiation has been found to alter the distribution and function of human lymphocytes. To determine whether photochemotherapy (PUVA) alters circulating levels of T cell subset marker-bearing lymphocytes, cells from 9 patients with psoriasis undergoing PUVA therapy for several years (mean 4.6 +/- 1.4 yr), 17 patients with active untreated psoriasis, and 20 healthy volunteers were reacted with monoclonal antibodies to T cell surface markers, including OKT3 (all peripheral blood T cells), OKT4 (helper/inducer T cells), OKT6 (common thymocytes), and OKT8 (suppressor/cytotoxic T cells), and analyzed by flow cytometry. There were no differences in the distribution of T cell subsets between healthy volunteers and patients with active psoriasis. In contrast, the percentages of lymphocytes reacting with OKT3 and OKT4 were lower (by 16% and 12% percent respectively, p less than 0.0025) in the PUVA-treated patients compared to healthy volunteers or patients with active psoriasis that had not received PUVA therapy. There was no difference in the percentage of OKT8 and OKT6 bearing cells. Squamous cell carcinoma of the skin subsequently developed in 2 of 3 PUVA-treated patients with the lowest percentages of T4-bearing cells. These findings indicate that long-term PUVA therapy is associated with a reduction in circulating helper/inducer T cells. This reduction may have a role in the altered immune function reported in PUVA-treated patients.

In vitro assay for phototoxic chemicals.

The photosensitizing potential of chemicals known to produce photosensitivity in humans was compared to chemicals not considered to be photosensitizers in an in vitro assay. The assay involved exposure of human lymphoid cells to UVA (320-400 nm), and in some cases UVB (280-320 nm) radiation, in the presence of the chemicals and the assessement of phototoxicity as measured by the incorporation of 3[H]-thymidine into nuclear DNA. All known photosensitizers tested were found to be phototoxic, while the nonphotosensitizing agents, with the exception of retinoic acid, were not phototoxic. Peripheral blood mononuclear cells were compared to a T lymphoblastoid cell line as target cells; the latter were superior in terms of convenience, cost and reproducibility of results. This test system has potential as a predictive assay for detecting additional phototoxic chemicals.

Influence of PUVA and UVB radiation on delayed hypersensitivity in the guinea pig.

Exposure of guinea pigs to UVA (320--400 nm) radiation following administration of 8-methoxypsoralen by gavage (referred to by the acronym, PUVA) or exposure to UVB (290--320 nm) radiation, produced suppression of the cutaneous delayed hypersensitivity reaction at the site of exposure to radiation and at distant nonexposed sites. In these experiments, the animals were immunized by injection of dinitrophenyl-bovine gamma-globulin (DNP-BGG) in complete Freund's adjuvant and delayed hypersensitivity responses were provoked by intradermal injections of DNP-BGG, DNP and BGG on the flanks. Exposure to erythemogenic doses of either PUVA or UVB radiation for 7 days prior to immunization and for the 7 days between immunization and challenge (total period of radiation: 14 days) produced inhibiton of responses to each of the test substances. In addition, treatment with erythemogenic doses of PUVA either for 7 days prior to immunization or during the interval between immunization and challenge with DNP-BGG, inhibited the delayed hypersensitivity responses at the site of irradiation and at a nonexposed site. These findings suggest that in vivo exposure to nonionizing radiation leads to both local and systemic alteration of certain immune responses.

In vivo effects of PUVA on lymphocytes function.

Whole-body exposure to erythemogenic doses of PUVA in normal volunteer subjects resulted in a decrease in the proportion of circulating sheep erythrocyte rosette-forming and surface membrane immunoglobulin-positive lymphocytes and an increase in the proportion of null cells. Doses of PUVA resulting in minimal or no erythema had similar but less marked effects on lymphocytes. The changes appeared 30 min after exposure, were maximal at 12-16 h and returned to pre-treatment levels by 72 h post-irradiation. The response of lymphocytes to stimulation with phytohaemagglutinin was unaffected. No alteration occurred in the absolute numbers of circulating lymphocytes or polymorphonuclear leukocytes.

The influence of ultraviolet radiation on allergic contact dermatitis in the guinea-pig. II. Psoralen/UVA radiation.

Guinea-pigs were sensitized by percutaneous application of dinitrochlorobenzene and exposed to UVA (320-400 nm) radiation following systemic administration of methoxsalen. This treatment diminished the response to an elicitation dose of the hapten administered 14 days later within the site of irradiation. The site of elicitation of the allergic response had to be included in the field of exposure, an reduction of the allergic response was observed only when the exposure to radiation was commenced at the time of induction of contact allergy. Established contact skin sensitivity was not affected by radiation.

Intestinal uptake of macromolecules. III. Studies on the mechanism by which immunization interferes with antigen uptake.

The mechanism(s) whereby immunization interferes with the intestinal absorption of macromolecules has been examined. After repeated intraperitoneal injection of protein antigens emulsified in incomplete Freund's adjuvant into rats, everted gut sacs from these animals and controls were exposed to radiolabeled antigen in vitro. There was a significant decrease in absorption of antigen by gut sacs from immunized rats as compared to control rats. On further analysis, it was found that, in contrast to controls, gut sacs from immunized rats showed an increased initial adsorption of labeled antigen. Furthermore, gut sacs from immunized rats showed enhanced breakdown of labeled antigen on prolonged incubation. Following incubation, gut sacs were rinsed and the amount of radioactivity eluted was determined. More radioactivity was present in the first rinse fluid of gut sacs from immunized rats than in rinse fluid from controls. On density gradient ultracentrifugation of the former fluids, radioactivity was localized in the middle or bottom of the gradient, whereas radioactivity in the latter fluids was located at the top of the gradient. The rapidly sedimenting labeled antigen was co-precipitated in the reaction of rabbit anti-rat IgG1 and IgG1 protein, suggesting the presence of antigen-IgG1 antibody complexes in the rinse fluid. These findings suggest that in vitro, antigen becomes rapidly associated with antibodies present on the surface of the gut. Formation of antigen-antibody complexes seems to prevent binding of antigen to and subsquent pinocytosis by the intestinal epithelial cells. Antigen-antibody complexes retained in the muous coat of the gut may be degraded by local proteases. The findings in this study suggest an additional role for antibodies present on mucosal surfaces, i.e., protection against absorption of ingested proteins which have escaped normal digestion in the intestinal lumen.

Serum IgE levels during the potentiated reagin response to egg albumin in rats infected with Nippostrongylus brasiliensis.

Rats were immunized with 10 mug EA and alum; a low titer IgE anti-EA response was observed; subsequent infection with N. brasiliensis greatly potentiated this response. Serum from these rats was tested for its IgE content by a recently developed radioimmunoassay for rat IgE. The average serum level of IgE was 1.15 mug/ml; this level was not altered by immunization, but infection with N. brasiliensis was accompanied by an 80-fold increase in concentration of serum IgE. This increase could not be accounted for by IgE anti-EA or anti-worm antibodies. Infection with N. brasiliensis appears to have an adjuvant effect on IgE antibody responses to diverse environmental antigens as well as the experimentally induced IgE anti-EA response.