Early embryo loss is associated with local production of nitric oxide by decidual mononuclear cells.

In early embryo loss, the fetus may be considered to be an allograft and, therefore, may be rejected by maternal immunocytes. However, the cytotoxic mechanisms involved are still poorly understood. We have previously shown the involvement of natural killer (NK) cells and mononuclear cells expressing Mac-1 (CD11b) and F4/80 in resorbing compared to nonresorbing embryos. In this study, the role of nitric oxide (NO) in the mechanism of early embryo loss was studied. Pregnant CBA/J females mated with DBA/2 males (20-30% early embryo loss) and CD1 females mated with CD1 males (5-10% early embryo loss) were studied on days 8, 10, and 12 of gestation. Cells from the implantation sites of individual embryos were tested for the production of nitrite and nitrate with or without in vitro challenge with lipopolysaccharide (LPS) to determine whether decidual macrophages were primed in situ. On day 12 of gestation, when resorption was clearly visible, resorbing embryos showed more than a fivefold increase in both basal- and LPS-induced nitrite and nitrate production compared to nonresorbing embryos in both mouse strains tested, indicating that the decidual mononuclear cells were primed. Furthermore, more than 20% of CBA/J embryos showed a significant nitrate release on days 8 and 10 of gestation before any signs of embryo cytopathology. This percentage corresponded to the spontaneous resorption rate seen in CBA/J female X DBA/2 male matings. Similarly, 4% of the embryos from pregnant CD1 mice on days 8 and 12 of gestation produced a significant amount of nitrate, which again correlated with the low incidence of resorption observed in these mice. Using immunohistochemistry, the presence of inducible nitric oxide synthase (iNOS) was detected at implantation sites. Furthermore, decidual cells positive for both iNOS and the macrophage marker Mac-1 were demonstrated in implantation sites by double immunostaining. This strongly suggests that decidual macrophages could be the cellular source of NO production. Aminoguanidine, a selective inhibitor of the iNOS, inhibited the in vitro production of nitric oxide by cells isolated from individual implantation sites, and more strikingly, significantly reduced early embryo losses in CBA/J females mated by DBA/2 males when given orally or parenterally to the gravid females starting on day 6 of gestation. In addition, aminoguanidine-treated pregnant mice showed a significant increase in average litter size when the pregnancies were allowed to proceed to term.(ABSTRACT TRUNCATED AT 400 WORDS)

The immunology of the eye and its systemic interactions.

Advances in immunology have provided us with new insights about the ocular inflammatory response. Still, the etiology and pathophysiology of many uveitides remain unclear. Factors such as the blood-ocular barrier, sequestration of retinal antigens, local immunomodulators in the aqueous humor, and anterior chamber-associated immune deviation (ACAID) interact and render the eye an immunologically "privileged" site. On the other hand, human leukocyte antigen complex (HLA) interactions, the immunopathology of hypersensitivity reactions, T-cell mediated disease, and autoimmunity are associated pathogenic mechanisms of immune-related ocular disease. In this paper, we review some of the immunologic characteristics of the eye and their relationship to intraocular inflammation. Clinocopathologic correlations are presented. Also, perspectives of current and future research, as well as diagnostic and treatment strategies, are discussed.

Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF). I. Preparation and fractionation of labelled IgG.

Dichlorotriazinylaminofluorescein (DTAF), the product of the reaction of aminofluorescein with cyanuric chloride, is an effective reagent for conjugating fluorescein to immunnoglobulins. DTAF has absorption and emission properties nearly identical to fluoresceinisothiocyanate (FITC) and DTAF and FITC-labelled antibodies are similar in terms of preparation and specificity of immlnofluorescence. However, DTAF is superior to FITC with regard to cost, purity and stability. Also, DTAF-labelled rabbit IgG conjugates can by quickly and efficientyly fractionated by simple ammonium sulfate precipitation procedures to yield preparations free of both over- and under-conjugated material. In addition, over-conjugated protein can be readily removed from DTAF:IgG conjugates by appropriate adjustment of pH and temperature.

A rapid and efficient microtechnique for the analysis of functional transferrin receptors on tumor cells.

A simple and efficient method for the analysis of the affinity and number of functional transferrin receptors (TFR) on human tumor cells is described. The technique is designed to utilize microtitration equipment; and is suitable for easy comparison of up to 8 different cell preparations per assay. Using this technique, 5 established cell lines were evaluated for functional TFR expression. The control erythroleukemic cell line K562 possessed 3.28 X 10(5) functional TFR per cell (+/- 3.69 X 10(4), SEM) Kd = 9.0 X 10(-9) X M-1. Trypsin and heat-pretreated cells were compared to control erythroleukemic K562 cells from the same culture to determine both the effects of receptor removal and cell viability on the assay. Trypsin and heat pretreatment of these K562 cells severely decreased receptor function as indicated by Scatchard analysis as well as by time course and cold competition analysis respectively. Whereas the affinity of trypsin-treated receptors on cells was similar to control values, heat-killed cells displayed an altered cellular affinity for 125I-transferrin underscoring the importance of utilizing cells of high viability in receptor assays.

Quantitation of a whole blood assay for human natural killer cell activity.

A method is described for the measurement of human natural killer cell activity using heparinized whole blood in a 51chromium release assay. Fractionation-reconstitution experiments showed that the cytotoxic activity was abolished by removal of the Fc receptor bearing lymphocytes, but not by the elimination of monocytes and granulocytes. Autologous or pooled plasma was not found to possess inherent cytolytic activity. By analogy to an enzyme kinetic reaction, the results were expressed as kinetic lytic units (KLU) which were defined as the maximum number of target cells that could be lysed per unit time per milliliter of whole blood. The buffy coat cytotoxicity (BCC) assay is quick, easy to perform, and suitable for screening and monitoring of natural cytotoxicity. Since this methodology preserves the actual concentration of natural killer cells, it may represent a truer reflection of in vivo events.

Ocular hypersensitivity to thimerosal in rabbits.

Rabbits were immunized to thimerosal conjugates and challenged with specific antigen-sensitized contact lenses. The symptoms of acute ocular hypersensitivity observed included corneal edema, corneal infiltration and erosion, infiltration of the anterior chamber, iritis, conjunctival edema and hyperemia, and a significant increase in mucous production. Occasional evidence of corneal neovascularization and giant conjunctival papillae were seen. All these parameters were assessed on a five-point scale (0 to +4) and tabulated as an ocular index. The index of ocular hypersensitivity correlated with the titers of the serum antibodies to thimerosal. The major class of serum antibodies consisted of immunoglobulin (Ig) G, with IgA compromising approximately 5% of serum antibodies. During the ocular challenge, the total and IgG tear antibody titers increased as a result of increased vascular permeability. The tear IgA titers increased to a lesser extent than IgG. The influx of serum proteins directly into the tear film was confirmed by a protein-dye tracer technique. Histologic analysis showed that the ocular inflammatory response was accompanied by both polymorphonuclear and mononuclear cell infiltrates into the cornea and conjunctiva. In conclusion, thimerosal-immunized rabbits show an exquisite sensitivity to the minute quantities of thimerosal conjugates adsorbed to contact lenses. Both serum and tear antibodies correlate with the severity of the ocular inflammatory response. This model would appear to simulate an antibody-mediated immune complex or Arthus type of ocular hypersensitivity commonly seen to foreign antigens including preservatives.

Expression of cathepsin S antisense transcripts by adenovirus in retinal pigment epithelial cells.

PURPOSE: To show the production of sense or antisense transcripts by recombinant adenoviruses, to investigate whether the transcripts produced were suitable for downregulating the expression of the targeted gene, cathepsin S (CatS), and to examine the effect of antisense transcript production on the biologic function of retinal pigment epithelial (RPE) cells, including the regulation of endogenous aspartic protease expression. METHODS: Ad.MLP.CatSAS, Ad.RSV.CatSAS, and Ad.MLP.CatSS recombinant viruses were produced by homologous recombination. The recombinant viruses were tested by restriction enzyme digestion to confirm the orientation of the inserts. The expression of antisense transcripts was tested by northern blot analysis. Western blot analysis was used to study the regulation of the endogenous CatS protein in ARPE19 cells. The biologic effect of CatS downregulation in ARPE19 cells was tested by proliferation and phagocytosis assays, de novo cathepsin D (CatD) synthesis, and measurement of aspartic protease activity. RESULTS: After characterization of the recombinant adenovirus constructs, the production of antisense and sense CatS transcripts was shown in ARPE19 cells. The transcripts appeared at approximately 1.9 kb 48 hours after transduction, and the expression of the antisense transcripts was similar in constructs carrying either the MLP or the RSV promoter. Western blot analysis showed that ARPE19 cells transduced with Ad.MLP.CatSAS and Ad.RSV.CatSAS had no detectable CatS. In contrast, there was a strong signal appearing at 24 kDa in ARPE19 cells transduced with Ad.MLP.CatSS. ARPE19 cells were transduced to a high level. The transduction of ARPE19 cells with the recombinant adenoviruses did not affect the morphologic appearance of the cells, their proliferation, or their phagocytosing ability. However, ARPE19 cells transduced by Ad.MLP.CatSAS recombinant adenovirus showed a significant downregulation of de novo CatD synthesis and a twofold decrease in aspartic protease activity. CONCLUSIONS: Recombinant adenoviruses were shown to be suitable for producing antisense CatS transcripts to modulate endogenous CatS expression in RPE cells. It is proposed that CatS may play an important role, directly or indirectly, in the lysosomal digestion of outer segments through the regulation of other lysosomal enzyme activity, such as the expression of CatD.

Involvement of transferrin and transferrin receptors in human natural killer effector:target interaction.

In this current study one of the determinants of natural killer cell specificity in immunosurveillance against cancer, may be the recognition of transferrin receptors on neoplastic cells by the NK effectors. Human transferrin, when saturated with iron (FeTf), was found to inhibit human natural killer (NK) activity against K562 tumor cells, if included in assay mixtures at physiologically relevant levels. Whereas both FeTf and iron-free transferrin (ApoTf) inhibited initial conjugate formation at the level of the target cell, only FeTf inhibited NK cytolytic activity, as judged by release of chromium from the targets. This suggested a functional role for FeTf on either NK or tumor cells. When either targets or effector lymphocytes were pre-incubated with FeTf, inhibition of killing was strongest when the targets were first exposed to FeTf. The evidence suggested that NK-associated transferrin mediated the interaction with target cells through free target-associated transferrin receptors. The finding that rabbit anti-human transferrin antibody (RaHTf) inhibited killing, when reacted with effector lymphocytes but not with target cells, supported this hypothesis.

Morphometric analysis of the histology of spontaneous fetal resorption in a murine pregnancy.

Immunopathology of the spontaneous resorption phenomenon in the CBA x DBA/J murine model was explored using morphometric analysis. Accompanying the previously reported presence of natural killer (NK) cells in resorptive feto-placental units we find major changes in tissue morphology indicating that early infiltration of the feto-placental unit by maternal leukocytes plays a direct role with NK cells in fetal demise. Total number of cell nuclei per field and total nuclear area per field were significantly elevated in feto-placental units containing abnormally increased NK cell presence before detectable resorption as early as day 7 of gestation. This difference persisted throughout all stages of early gestation up to and including the final resorption event at day 10 to 12. Increases in cell density were also detected in areas of the embryonic unit not associated with NK infiltration. These results demonstrate that the spontaneous resorption phenomenon in this model involves: (i) Early (day 7-8) cellular infiltration of the decidual-ectoplacental cone junction associated with the presence in this area of NK cells. (ii) Late (day 8-9) cellular infiltration of the ectoplacental cone.

Augmented natural killer cell activity during neonatal graft-versus-host disease is of predominantly donor origin.

Natural killer cells have been postulated to play a role in the immunopathogenesis of graft-versus-host disease (GVHD). This is based on reports of augmented natural killer (NK) activity, both in animal models and in humans. It has not been elucitated from previous reports, however, whether the cells mediating this activity are of donor or recipient origin. We have investigated the origin of the effector cells using highly specific antisera. Our results indicate that the augmented lytic activity seen during the course of GVHD is mediated primarily by donor lymphocytes. Depletion with anti-Class I antisera followed by immuno-staining with anti-asialo GM-1 indicate that the effector cells are also asialo GM-1 positive. We report here that the augmented non-specific lytic activity seen during GVHD is mediated by asialo GM-1 positive lymphocytes predominantly of donor origin.

Impairment of the thymus-dependent humoral immune response by syngeneic or allogeneic pregnancy.

Pregnancy results in a diminished capacity of the gravid mouse to respond to thymus-dependent antigens. The IgM response to thymus dependent and independent antigens is relatively unaltered whereas the IgG response to thymus-dependent antigens is markedly suppressed in both syngeneic and allogeneic pregnancy. The impairment of the humoral immune response is not due to loss of antibody-forming precursor lymphocytes (AFPL) from the spleen since their number remains unaltered. During pregnancy the spleen becomes enlarged and the precursor frequency is diluted somewhat but the AFPLs retain their normal activity in vitro. This implies an alteration in their amplification by accessory cells (T cells or macrophages) is responsible for the impaired IgG responses.

Modulation of the natural killer cell activity in pregnant mice alters the spontaneous abortion rate.

Effector cells associated with an aborting fetus appear to be both thymus derived (T) and natural killer (NK) cells. In order to test the hypothesis that NK cells are a major effector mediating early spontaneous abortion (less than day 8-10), CBA female mice mated by DBA/2 males were treated with either polyinosinic/cytidylic acid (poly I:C) to boost NK activity, or rabbit anti-asialo GM1 (RaASGM1) to decrease NK activity. The results of the NK assays of the spleens of treated mice confirmed that the reagents had the expected effect on NK activity and an inspection of the uteri indicated a significant increase in aborted embryos after poly I:C and a marked decrease in spontaneous abortions after RaASGM1 treatment. Therefore, spontaneous abortions may be mediated in part by the cytotoxic activity of unregulated NK cells.

Evaluation of the role of exogenous pathogens on the incidence of embryo loss during early pregnancy in mice.

The mating of CBA/j female mice (H2k) by DBA/2j male mice (H2d) typically results in an elevated incidence of spontaneous embryo loss thus providing an ideal genetically controlled laboratory model for the study of the factors causing early embryo loss during pregnancy. There is now considerable data on the cells and factors involved in fetal resorption but little is known about the events which activate this process. While the activation of the maternal response to the fetal implant could have endogenous or genetic origins, a role for exogenous factors including microbial pathogens could also be involved. In order to investigate these possibilities, the reproductive success of CBA/j female x DBA/2j male matings in a conventional animal care facility were compared with matings in a specific pathogen free (SPF) animal facility. All animals housed under these conditions were routinely screened by immunoassay and culture, for the presence of a number of viral and bacterial pathogens of mice. The incidence of spontaneous embryo loss in specific pathogen free CBA female mice mated by DBA and other male strains was found to be virtually identical to that of CBA female mice infected with multiple viral pathogens and housed under otherwise identical conditions (non-SPF). However, the numbers of implantation per pregnancy was significantly greater in an SPF facility. Therefore, exposure of mating mice to exogenous viral and bacterial pathogens did not appear to alter the overall incidence of spontaneous embryo resorption. It was concluded that the immunomodulatory effects of infection by common murine pathogens neither augmented nor reduced post-implantation embryo losses.

Effects of pregnancy on the maternal lymphoid system in mice.

The effects of pregnancy on the lymphocyte populations in the mouse have been studied. Though cell numbers decreased in the thymus and increased in the lymph nodes draining the parauterine area, no significant changes were seen in the proportions of thymus derived (theta allo-antigen positive) and bone marrow derived (Fc receptor positive) lymphocytes. The humoral response to sheep erythrocytes was suppressed during both syngeneic and allogeneic pregnancies following a decline coincidental with that of thymic mitotic activity. Though the observed immunosuppression could be explained by a selective lymphocyte depletion, it appears more likely to be a nonspecific effect of the steroid milieu of pregnancy.

DNA index and S phase fraction in uveal malignant melanomas.

AIMS: To predict 5 year survival in patients with uveal malignant melanomas DNA indices were studied. METHODS: Using 45 paraffin embedded uveal malignant melanomas, the DNA index and S phase fraction of each tumour were the predictor variables recorded. RESULTS: Using the Cox proportional hazards model, aneuploid tumours and tumours which had an S phase fraction greater than 4% were significant predictors of early death. In order to demonstrate a biological gradient between a larger DNA index and shorter survival time, linear regression and transformed linear regression models were used. However, no such gradient could be demonstrated. CONCLUSION: Although this study shows promise for the use of DNA studies in the prognosis of uveal malignant melanoma, the exact role of these techniques remains to be determined.

Purification of immunoglobulin g (IgG).

Several immunological procedures can be successfully carried out using nonpurified antibodies, such as unfractionated antisera, or ascitic fluid/culture supernatant containing monoclonal antibodies (MAbs). However, a much "cleaner" result can often be obtained if some form of enrichment or isolation of immunoglobulin is employed. Some procedures, such as conjugation with isotopes, fluorochromes, or enzymes, and preparation of immunoaffinity columns cannot usually be efficiently performed with nonpurified immunoglobulin, and some procedures may yield artifactual results if whole antiserum or ascitic fluid is used as a source of antibody. Purification of immunoglobulins is therefore essential or, at least, useful for a range of immunological methods. This process may consist of purification of total IgG or subpopulations (e.g., subclasses) of IgG from antisera/ascitic fluid/culture supernatant, or the isolation of a particular antigen binding fraction from such fluids. The former can be achieved by biochemical procedures, whereas the latter usually requires some form of affinity purification.

Purification of immunoglobulin g (IgG).

Several immunological procedures can be successfully carried out using nonpurified antibodies, such as unfractionated antisera, or ascitic fluid/culture supernatant containing monoclonal antibodies (MAbs). However, a much "cleaner" result can often be obtained if some form of enrichment or isolation of immunoglobulin is employed. Some procedures, such as conjugation with isotopes, fluorochromes, or enzymes, and preparation of immunoaffinity columns cannot usually be efficiently performed with nonpurified immunoglobulin, and some procedures may yield artifactual results if whole antiserum or ascitic fluid is used as a source of antibody. Purification of immunoglobulins is therefore essential or, at least, useful for a range of immunological methods. This process may consist of purification of total IgG or subpopulations (e.g., subclasses) of IgG from antisera/ascitic fluid/culture supernatant, or the isolation of a particular antigen binding fraction from such fluids. The former can be achieved by biochemical procedures, whereas the latter usually requires some form of affinity purification.

Purification of murine monoclonal antibodies.

Hybridoma technology has made possible the production of highly specific, homogeneous antibodies with predefined binding characteristics, which can be produced in large amounts, from immortal cell lines. They probably represent the immunochemist's ideal as reagents, and monoclonal antibodies (MoAbs) are invaluable for the investigation and assay of the entire antigen spectrum. However, it must be remembered that, although MoAbs can be exquisitely specific, they are far from pure unless specific procedures are used for their isolation. If produced as ascitic fluid, they contain proteins derived from the host animal, whereas in vitro production as cell culture supernatant produces MoAbs contaminated with tissue culture additives, nonimmunoglobulin secretion products, and material derived from dead disrupted cells.

Thimerosal: an ophthalmic preservative which acts as a hapten to elicit specific antibodies and cell mediated immunity.

A rabbit model for the study of hypersensitivity to thimerosal was established in order to develop better techniques for screening patient sera and tears for specific antibodies to lens care re-agents. Thimerosal (sodium ethylmercury thiosalicylatelate) was successfully coupled to several protein carriers using a water soluble carbodiimide which linked the carboxyl group of thimerosal to free amino groups of the carrier proteins. Thimerosal was also shown to spontaneously react with proteins as detected by the irreversible binding of mercury to the protein carrier. Immunization of rabbits with the chemically coupled thimerosal resulted in the production of antibodies which specifically reacted with thimerosal. The rabbits also manifested delayed and immediate forms of hypersensitivity to the thimerosal conjugates. The ELISA assay for specific serum antibodies was found to be a sensitive, reliable and specific screening tool However, there was no immunological cross reactivity between the chemically coupled thimerosal and the spontaneously coupled thimerosal. Therefore, the epitopes produced by these two reaction mechanisms were probably immunochemically different even though both contained detectable thimerosal derived mercury.

Quantification of transforming growth factor-ß in aqueous humor.

The use of biological assays in research often creates complex Problems of data analysis. Due to the nature of bioassays which respond to the intricate interplay between multiple factors that may either increase or decrease the readout, simple arithmetic or standard linear regression analysis often fails to provide accurate values when extrapolation from a standard curve is employed. Non-linear polynomial analysis could provide a better way for expressing such complex relationships. Using a bioassay for transforming growth factor-beta (TGF-ß) employing the mink lung epithelium cell line CCL-64, the authors present a new way to analyze experimental data based on non-linear polynomial statistics to provide accurate quantitative results when other simpler techniques have failed. This technique for data analysis provided accurate consistent results with acceptably small experimental variance. Using this approach, the bioactive concentration of TGF-ß in normal rabbit aqueous humor was found to be 2.72 ± 0.61 ng/ml, that is, corresponding to 2. 72 ng of PURE TGF-ß2, when referring to a standard curve.