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Hyperplastic and metaplastic lesions in the prostate gland of bulls are used as morphological evidence for the illegal administration of oestrogens or compounds with oestrogenic activity, like stilbenes. Exogenous and endogenous androgens can suppress the effects of these drugs, resulting in suspect or negative results. The detection of epithelial changes immunohistochemically with cytokeratin antibodies appeared to facilitate the diagnosis of hyperplasia and metaplasia in the prostate. A specific and sensitive method for detecting such lesions in formalin-fixed, paraffin-embedded prostate samples has been developed with the commercially available cytokeratin monoclonal antibody 34 beta E12, and applied to bulls treated with diethylstilbestrol-dipropionate. The highly reproducible staining pattern of 34 beta E12 can be used for the detection of hyperplasia and metaplasia in the prostate glands of animals treated illegally with oestrogens.

Aberrant or defective immune responses are known to be the result of primary immunodeficiency diseases, e.g., in the DiGeorge syndrome (thymic hypoplasia) in man, or in homozygous mice with the mut...

In this experiment 20 one year old bulls received a single intramuscular injection of the anabolic preparation diethylstilbestrol dipropionate (DES-DP) (an oil preparation or an emulsion). Four animals received a corresponding placebo. The application of DES-DP to bulls caused characteristic histological alterations in the peripheral glandular epithelium of the prostate, which could be observed until four weeks after treatment. The value of histological investigation as a screening method was, however, limited by the occurrence of only few metaplastic lesions and a rapid recovery. By contrast, immunohistochemistry using a polyclonal cytokeratin antiserum K40 appeared to be a specific and very sensitive method to detect oestrogen-induced lesions in the prostate. In only two animals, six weeks after injection with the DES-emulsion, false-negative results were obtained, demonstrating the potential value of this screening method. The excretion of DES in the urine and faeces was monitored using radioimmunoassay following chromatographic purification of the urine and faeces extracts. The excretion of DES in urine was faster for animals of the oil group. The DES content in urine decreased to the 1 microgram/l level after 42 days (emulsion group) or 70 days (oil group). The excretion in faeces was comparable to that in urine. After day 21 the excretion patterns of the two excreta were indistinguishable.

Rats were fed diets containing 0, 500, 1000, and 2000 mg HCB/kg during a 3-week period. Marked weight increases of spleen, popliteal and mesenteric lymph nodes and of the liver were found. Histologically, the white pulp in the spleen was enlarged because of an increase in size of marginal zones and follicles. In addition, there was an increase of extramedullary hemopoiesis. In the lymph nodes, the number of high endothelial venules was increased at all dose levels. The number of neutrophils, basophils and monocytes in the peripheral blood was significantly increased, whereas peripheral lymphocyte counts were slightly higher. Total serum IgM levels were markedly increased, but IgG concentrations were unaltered. On the basis of this experiment, the 1000 mg HCB/kg diet level was chosen for the different function studies that were carried out after a 3-weeks dietary regimen. Regarding the humoral immunity, IgM antibodies to LPS were unaltered, whereas primary and secondary IgM and IgG antibody titers to tetanus toxoid were increased approximately three-fold. HCB did not significantly alter the cell-mediated immunity, as shown by the following parameters: resistance to Listeria monocytogenes infection, rejection of skin transplants, and delayed-type hypersensitivity to tuberculin. The phagocytizing capacity of macrophages was studied by measuring the blood clearance of carbon particles. HCB did slightly depress the phagocytic index, but the difference with control animals was statistically not significant. The in vitro responsiveness of thymus cells to the mitogens PHA, Con A, and PWM was not changed by in vivo HCB-treatment. On a cell-for-cell basis, the responsiveness of spleen cells was increased when cultured in the presence of LPS. On a whole organ basis, the response to PHA, Con A, PWM, and LPS was markedly enhanced because of an increase in the number of nucleated spleen cells. Regarding peripheral lymphocytes, only the response to the mitogen Con A was higher. On the basis of these studies it is concluded that HCB stimulates the humoral immune response in the rat, enhances the in vitro responsiveness of spleen cells to the different mitogens mainly as a result of an increase in the number of splenic lymphocytes, but does not alter the cell-mediated immunity as shown with in vivo tests. This result contrasts with data in the literature that show that HCB suppresses the humoral and cell-mediated immunity in mice. Finally, HCB pretreatment only marginally increased the susceptibility of rats to endotoxin, whereas mice have been shown to be 20-fold more susceptible to the lethal effects of bacterial endotoxin.

Three-month-old male C57B1 6 mice were given single oral doses of 0, 100, 150, or 200 μg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg. The LD50 was 114 μ/kgg. In mice that died, depletion of the thymus and spleen were consistently found and edema and terminal hemorrhages occurred frequently. In a second experiment, 4-month-old male mice were dosed po with 0, 0.2, 1.0, 5.0 or 25 μg/kg, once a week for 2 or 6 weeks. Some deaths and growth retardation occurred in the 25 μg/kg dose group. Significantly increased liver and decreased thymus weights were found in the 1, 5 and 25 μg/kg dose groups. Total neutrophils were increased significantly, whereas hemoglobin values and mean corpuscular hemoglobin concentrations were decreased significantly after 6 doses of 25 μg/kg. Total serum protein and α-, β-, and γ-globulins were significantly decreased. TCDD was porphyrogenic. The hepatic porphyria was probably associated with liver damage. Degenerative and necrotic changes in the liver were essentially centrilobular and were accompanied by cellular infiltrates and ceroid pigment deposition. Proliferation of bile duct and bile duct epithelial cells occurred. Lipid accumulation was centrilobulary localized in the mice receiving 0.2 μg/kg, was more pronounced in the mice of the intermediate dose levels, and involved hepatocytes throughout the lobule in the 25 μg/kg dose group.

Rat IgM and IgG was determined by mechanized "sandwich" enzyme-linked immunosorbent assay (ELISA) using peroxidase labeled anti-rat-IgM and -IgG. Linear ranges in standard curves of a reference rat serum had a slope similar to the slopes found with sera of 25 rats of various age. IgM and IgG measurements by ELISA in these sera correlated well with results obtained by single radial immuno-diffusion (SRID). In addition, the precision of the enzyme immunoassay was the same as obtained with the SRID. Compared with SRID, ELISA is less time consuming and the amount of antiserum used in the macro-ELISA is one order of magnitude lower; and again 10 times lower in the mechanized micro-ELISA that is currently being developed. In conclusion, the ELISA is a specific, reliable, sensitive, and economic method for routine measurement of rat serum IgG and IgM e.g. in toxicity studies. In the second part of this study, ELISA and the passive hemagglutination test were compared to determine the primary and secondary antibody response to E. coli lipopolysaccharide (LPS) and tetanus toxoid in rats. In the ELISA, the antigens were bound to the wells of polystyrene microplates. Tetanus toxoid was coated directly, LPS after complexing with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled antiimmunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with the respective antigens. ELISA proved to be more sensitive than the hemagglution reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, ELISA is a convenient method for measuring both IgM and IgG antibodies. Finally, evidence is presented that in the rat, the humoral immune response to LPS is a thymus-independent phenomenon. Thus, by using the antibody response to LPS and tetanus toxoid in function studies of the immune system of the rat, insight can be obtained in the thymus-independent and thymus-dependent humoral immune response.

Chlorodibenzo-p-dioxins, especially 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), are among the most toxic compounds known. These compounds are found as contaminants of technical chlorophenols and their derivatives. A variety of pathologic condition have been associated with the ingestion of products containing chlorodibenzo-p-dioxins. Toxic fat, the cause of chick edema disease (1, 2) and a variety of pathologic manifestations in monkeys (3), has been found to contain chlor3dibenzo-p-dioxins, among which is TCDD (4, 5). TCDD has been implicated in a variety of other toxicoses, including outbreaks of chloracne in chemical workers (6), hepatonecrosis in rabbits (6), and hepatonecrosis and thymic atrophy in rats (7). However, only in the studies in which monkeys were fed toxic fat (3) and in which rats were given TCDD orally (7, 8) were substantial clinical pathologic analyses performed. Toxic fat caused in the monkeys, among other lesions, anemia, leukopenia and hypoproteinemia. The hypoproteinemia was primarily due to decreased serum albumin concentration. The blood urea nitrogen

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is reported to be one of the most toxic chemicals known. There has been variation in such reported toxic parameters as single oral LD50 dose, range of time interval from dosing until death, and toxic manifestations which an animal exhibits (1-4). It seemed most appropriate, therefore, that the mean values and ranges of these and other general biological parameters be determined under the experimental condition to which animals are exposed at this Institute.

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In a comparative study, the enzyme-linked immunosorbent assay, using peroxidase labeled anti-rat immunoglobulin M and immunoglobulin G, and the passive hemagglutination test were applied to determine the primary and secondary antibody response to lipopolysaccharide and tetanus toxoid in rats. In the enzyme-linked immunosorbent assay, the antigens were bound to the wells of polystyrene microplates, tetanus toxoid directly, and lipopolysaccharide after complexing it with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled anti-immunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with their respective antigens. The enzyme-linked immunosorbent assay proved to be more sensitive than the hemagglutination reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, the enzyme-linked immunosorbent assay is a convenient method for measuring both immunoglobulin M and immunoglobulin G antibodies. At low serum dilutions of lipopolysaccharide antisera, inhibition of the reaction in the enzyme-linked immunosorbent assay occurred. This phenomenon could be prevented by heating the sera at 56°C for 30 min. Lipopolysaccharide was immunogenic in rats over an extremely wide dose range (from 10 pg to 1 mg); the optimal immunogenic dose of lipopolysaccharide for young adult rats was 0.1 to 1,000 μg when administered intravenously, and that of tetanus toxoid was 5 to 10 lines of flocculation, as determined by the Ramon flocculation test.

After immunization of Wistar rats with different concentrations of ovalbumin (OA) emulsified in complete H37Ra adjuvant, the optimal antigen dose for specific IgM, IgG and IgE production and for delayed-type hypersensitivity appeared to be the same. It is therefore concluded that in the rat these function tests can be combined, which decreases the number of experimental animals needed. For the determination of IgM, IgG and IgE antibodies to OA, the enzyme-linked immunosorbent assay (ELISA) was used, which proved to be a specific method. In addition, using a multi-channel photometer, it is shown that specific antibody concentrations can be readily quantified by micro-ELISA instead of using end-point titrations.

Studies in various animal models indicate that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and approximate isostereomers produce a similar pattern of toxic responses, including thymic atrophy. Effects on the immune system include suppression of cell-mediated immunity. Also in man, exposure to TCDD-related halo-genated aromatic hydrocarbons has been shown to impair thymus-dependent immunity. Several investigations have been performed to clarify the thymus atrophy. TCDD is not directly toxic for thymocytes, nor does it act through an increase of serum corticosteroid levels or a decrease of growth hormone levels. In studies with murine inbred strains differing in sensitivity to TCDD it has been shown that thymic atrophy segregates with the Ah locus and thus appears to be mediated by the Ah receptor. Recent studies indicate that TCDD acts through a receptor in thymic epithelium. Treatment of monolayers of thymic epithelial cells with TCDD resulted in the suppression of epithelium-dependent responsiveness of co-cultured thymocytes to mitogens. These results provide direct evidence that thymic epithelium is a target for TCDD. These findings are consistent with the hypothesis that TCDD stimulates terminal differentiation of reticular thymic epithelial cells to a stage at which they have lost their ability to support thymocyte maturation, thus leading to depletion of cortical thymocytes.

Summary A total of 45 normal or enlarged thyroid glands of adult slaughter cattle were weighed and analyzed by thin‐layer chromatography for the presence of residues of the following thyreostatic compounds: thiouracil, methylthiouracil, propylthiouracil, phenylthiouracil, and methimazole. In 21 glands, mostly from imported animals, residues of methylthiouracil or methimazole were detected. These thyroids ranged in weight from 68–245 g (mean value 118 g). Glands in which no residues were found, varied in weight between 20 and 124 g (mean value 42 g). Taking an upper limit of 60 g as normal, 4 out of 22 were ‘false‐positive’ with respect to the 5 thyreostatic compounds screened for, while no false‐negative cases were scored. On the basis of these results the weight increase of the thyroid gland is proposed as a simple indirect parameter in the screening for the illegal use of thyreostatic compounds in slaughter cattle.

We studied the early events in athymic immunoincompetent rats after implantation with cultured thymic fragments (CIF) under the kidney capsule, with special emphasis on the settlement of lymphocytes and non-lymphnid RTl class II elements. At 2 weeks after grafting, tissue under the kidney capsule comprises strands of keratin-positive epithelial cells from the graft, without immigrant cells. At 3 weeks, the CTF graft is populated with lymphoeytes and with non-lymphoid RTl class II-positive cells expressing the recipient haplotype (allogeneic combinations). Part of these cells bear determinants recognized by an anti-rat dendritic cell antibody. At 4 weeks the graft exhibits a completely restored thymic architecture. Al the periphery, the first indications of T-cell competence generated after CTF implantation are observed 6 weeks after implantation. At 18 weeks. the peripheral thymus-dependent immune system is almost completely developed. This includes in vitro alloreactivity, even to the donor RTl haplotype of the graft. But skin grafts of the allogeneic CTF donor haplotype are not rejected. Thus, a state of in vivo tolerance is induced under the influence of grafted epithelium, which is not due to a specific deletion of alloreactive cells. We conclude that CTF regain their original thymic architecture between 2 and 4 weeks after implantation in (allogeneic) athymic nude recipients, and that only after this restoration does peripheral thymus-dependent immune competence start to develop.

Summary The anti-parasite response was investigated after oral infection of athymic nude (rnu/rnu) rats and heterozygous (+/rnu) littermates with 1000 muscle larvae of Trichinella spiralis. No IgM, IgG and IgE antibodies were detected in serum of rnu/rnu rats. Expulsion of adult worms from the small intestine was prolonged (worms were nearly all expelled at days 14 and 91 in +/rnu and rnu/rnu rats respectively). The yield of muscle larvae in the carcasses of nude rats at day 91 was 33 times higher than in +/rnu rats. In contrast to the strong inflammatory reaction in the parasitized tongue of +/rnu rats, no infiltration was observed in rnu/rnu rats. Using an immunoperoxide method with monoclonal anti-rat T-cell antibody, no T cells were identified in spleen, mesenteric lymph node and Peyer's patches. These data support earlier studies that the nude rat lacks functional T cells. As the counts of connective tissue mast cells (CTMC), intestinal mast cells (IMC) and globule leucocytes (GL) in small intestine of uninfected rnu/rnu rats were equal or higher than in +/rnu rats, it is concluded that the origin of these cells is thymus-independent. In contrast to +/rnu rats, infection of rnu/rnu rats induced no increase of CTMC, IMC or GL. Thus, these cells depend on T cells to undergo proliferation. Finally, results of this study were inconclusive whether IMC are precursors for GL, or that they represent independent cell populations.

The lymphoid tissue can be structurally divided in to primary and secondary lymphoid organs or tissues. In the primary organs, the antigen-independent proliferation of lymphocytes takes place. Generation of T-lymphocytes from stem cells occurs in the thymus. In birds, the bursa of Fabricius has been shown to be the primary organ that supports extensive lymphopoiesis of B cells (the mammalian equivalent of the bursa of Fabricius may exist in different sites, e.g. the bone marrow or as suggested by Hall in this volume, in the lower gut) The secondary lymphoid tissue, in which the complex cellular interactions take place which form the basis of the immune response, includes the spleen, lymph nodes and the mucosa associated lymphoid tissues in the alimentary and respiratory tracts.

We studied the thymus-dependent immune system in congenitally athymic (nude) rats of the WAG (RT-1a) strain, at the ages of 2, 3, 5, 9 and 17 months. This included the histology of spleen, lymph nodes, and Peyer's patches, immunohistochemistry on tissue sections, and immunocytology on cell suspensions using a panel of monoclonal antibodies to T-lymphocyte subpopulations. in vitro mitogen responsiveness, and in vivo responsiveness to ovalbumin immunization. The results were compared with those in euthymic immunocompetent litter-mates of the same age. The cell marker analysis, especially in nude animals, was hampered by the staining of non-T cells by some of the antibodies (staining of putative macrophages, natural killer cells, cells of H-lymphocyte lineage, and of myeloid lineage I. Despite this, with age an increase in cells with these markers was observed; for instance, in spleen suspensions, cells labelled by the pan-I reagent MRCOX-19 increased from 5% at 2 months to 19% at 17 months (value in euthymic animals 34%). In the other assays too a gradual increase of T-cell reactivity with age was observed, from almost absent in 2- and 3-month-old nude rats to values in 17-month-old animals of about hall the level round in euthymic rats. However, none of the nude animals responded to ovalbumin immunization. These results indicate that in nude animals processes occur which compensate for the absent intrathymic T-cell generation. This applies in particular to changes in T-cell phenotype and mitogen responsiveness, but not to antigen responsiveness.

There are several reports that congenitally athymic nude mice and rats have cells with T-cell markers in spleen and lymph nodes. For the nude mouse, the numbers of these cells increase with age.1 In athymic rats we found cells with T-cell markers in T-dependent areas of mesenteric lymph node and Peyer’s patches, especially at ages older than 35–40 weeks.2 This observation prompted us to study in detail the presence of cells with T-cell phenotypes in lymphoid organs of nude rats as a function of age. Lymphoid organs of 8, 12, 20, 36 and 68-week-old nude rats were analysed by both flow cytometry (FACS) on cell suspensions and by immunohistochemistry on tissue sections. A panel of monoclonal antibodies to T-cell subpopulations was applied. The proliferative response after mitogenic stimulation was simultaneously investigated. In these assays athymic nude rats were compared with age- and sex-matched immunocompetent littermates.

As a screening procedure to detect immunotoxic effects, different parameters are discussed. These criteria include growth, weight and histology of lymphoid and endocrine organs, haematology, as well as serum immunoglobulin concentrations which can be quantified using the enzyme-linked immunosorbent assay (ELISA).