Experimental methods for immunization and challenge in antigenicity studies in guinea pigs.

Optimal experimental methods for antigenicity studies in guinea pigs were investigated on: (1) the effects of different immunizing methods using complete or incomplete Freund's adjuvants (CFA or IFA), and various injection sites, the number of immunizations, the immunizing doses, and the immunizing periods, (2) the relationship between the severity of active systemic anaphylaxis (ASA) reactions and passive cutaneous anaphylaxis (PCA) titers, (3) positive control for oral administration, and (4) the effects of incubation mixture of drug and serum protein as the challenge for the ASA assay. The following results provided useful information for designing more appropriate methods for antigenicity studies: (1) The optimal immunization method for benzylpenicillin (PcG), cephaloridine, 2,4,6-trinitrobenzene sulfonic acid and adriamycin, which were selected as positive controls for low molecular medicines in this experiment, involved subcutaneous administration of 1 ml of a test substance in CFA (1st immunization) or IFA (2nd and 3rd immunizations) at two doses, 1 and 10 mg/animal, 3 times at 2-week intervals on the back of a guinea pig. Blood collection for PCA assay was needed 2 weeks after the last immunization, and ASA assay, 1 or 2 days after the blood collection. (2) The insensitivity of ASA reactions in bovine serum albumin-immunized animals with very high PCA titers was overcome by increasing the challenge antigen dose from 1 to 10 mg/animal. (3) Most animals administered lysozyme at 0.1, 1 or 10 mg/animal by gavage for 2 weeks or more showed ASA and PCA reactions. (4) Incubation of a mixture of 20 mg/ml of PcG and 2 mg/ml of guinea pig serum albumin for 4 hr was the most effective as challenge for the induction of ASA reaction in PcG-immunized guinea pigs.

A subchronic toxicity study of two inhaled aerosolized atropine sulfate formulations in rats and dogs.

The potential subchronic (21 days) toxicity of inhaled metered aerosol formulations (solution and suspension) of atropine sulfate was investigated in rats and dogs. The doses administered to rats were 0.78 and 2.5 mg/kg/day (solution) or 1.4 and 3.2 mg/kg/day (suspension). In the dog, the daily doses achieved were 0.5 and 1.3 mg/kg/day, regardless of formulation. In both species, sham control animals inhaled air only and vehicle control animals inhaled either placebo solution or placebo suspension. There was no mortality or other evidence of a toxic effect of atropine sulfate. The expected mydriatic effect of atropine sulfate was seen in both species and, similarly, the pupillary light reflex was impaired in rats and dogs receiving either formulation of atropine sulfate at both dose levels. Reduced salivation was also noted and ophthalmologic examinations in both species were unremarkable. In dogs, atropine sulfate (high-dose) caused tachycardia but there was no evidence of an adverse effect on the electrocardiogram or on systolic blood pressure. In both species, atropine sulfate did not alter body weight, food consumption or clinical pathology parameters. Necropsy observations and histopathological findings revealed no effect of atropine sulfate in either species although, in the rat, adrenal gland hypertrophy in both sexes followed inhalation of the suspension at both dose levels. With this possible exception, nothing but the expected pharmacological effects of atropine sulfate were seen in either rats or dogs.

Quantitative techniques in neuropathology.

In the future, quantitative techniques will probably be used in industry as part of Tier II studies for the evaluation of chemicals and drugs for their neurotoxic potential. Movement towards quantifying some structures or neuropathological changes will be made possible by advances in tissue preparation and computer technology. Emphasis will need to be placed on standardized techniques, good quality samples and sampling techniques in order to produce good quantitative data in a reasonable time. In this paper, different sampling techniques are evaluated using a cross section of rat sural nerve as the tissue for quantitative investigation.

Correlation between behavioral and pathological changes in the evaluation of neurotoxicity.

Recently the EPA has implemented new guidelines under Section 4 of TSCA to undertake neurotoxicity testing. It is expected that other agencies, both national and international, will follow suit. The evaluation of neurotoxicity will be based primarily on behavioral and morphological observations and particularly on the correlation between them. Initially, considerable information will have to be gathered from various laboratories to form the background data on which decisions of toxic effects can be made. This paper presents our experience to date with behavioral and neuropathological procedures for the evaluation of chemical compounds or dietary regimens in rats for potential neurotoxicity. Reference is made to acrylamide, 2,5-hexanedione, 3,3'-iminodipropionitrile, amphetamine, physostigmine, ethanol, triethyltin, food and water deprivation, and carbon tetrachloride. The question of whether every change induced by xenobiotics can be considered as a sign of toxicity is discussed.

Morphometric evaluation of hepatocellular proliferative lesions in the rat liver.

Classification of rat hepatocellular proliferative lesions can vary between pathologists as the many qualitative histologic criteria have not been satisfactorily evaluated and ranked for prognostic value. Computer-assisted morphometry offers an objective method to evaluate certain cellular features. The Solt-Farber resistant hepatocyte model was used in this study to produce populations of rats with a full range of hepatocellular proliferative lesions. Cellular features within the lesions were then measured morphometrically and the data were analyzed by animal age and by subjective lesion diagnosis. The nuclear/cytoplasmic ratio followed by the cell area and nuclear area were found to be the most important parameters for separating microscopic foci and islands of cellular alteration, an early hyperplastic lesion, from other hepatocellular proliferative lesions. The coefficient of variation, as a relative measure of heterogeneity, increased in a linear manner for cell, nuclear and nucleolar areas as the animals aged and was significantly higher for cell and nuclear area in hepatocellular carcinoma compared to other hepatocellular proliferative lesions. Hepatocyte nodules (representing primarily late hyperplastic lesions) and persistent hepatocyte nodules (lesions with similarities to hepatocellular adenoma) could not be satisfactorily separated within the limits of this study. As these borderline lesions show a continuum of cytologic change, other features, such as architectural change, are necessary to satisfactorily classify them on a subjective basis. An alternative approach is to use discriminant functions derived from morphometric studies.

Quantitative immunocytochemistry by digital image analysis: application to toxicologic pathology.

Recent advances in immunocytochemical techniques allow the localization of specific antigens in tissue sections. The work reported here attempts to evaluate the application of antibody-labeled, disease-related protein, followed by microscopy and computerized image analysis. Using an experimental anti-tumor, polyclonal antibody (anti-oncomodulin) as a model, various tissues were prepared for light microscope immunocytochemistry. Sections were incubated with primary antibody, then biotinylated secondary antibody. This was followed by incubation with avidin-biotin-peroxidase (ABC method). Marker was visualized by the presence of precipitated diaminobenzidine. Samples were evaluated using a Zeiss/Kontron IBAS I & II semi-automatic digital image analysis system. Statistical analyses were performed on output data. Results demonstrated the localization and determined optical density of immunolabel. Statistical comparisons showed significant differences between control and experimental sections. The practical application of these combined techniques provides the toxicologic pathologist with a powerful tool for accurate and objective determination of the location and relative amount of selected proteins in normal and abnormal tissues.

A subchronic inhalation toxicity study of a Matacil formulation in the albino rat.

The subchronic toxicity of a new formulation of Matacil (aminocarb) was assessed by exposing male and female Sprague-Dawley rats via a nose-only technique to a respirable (2.0- to 4.1-microns diameter) aerosol at chamber concentrations of 22.5, 45, and 90 micrograms of insecticide/liter of air for 2 hr/day for 30 consecutive days. Control groups were exposed to a vehicle aerosol or to room air. Randomly selected rats of each group were bled after 8, 15, and 30 days of treatment, and after a 30-day recovery period. Routine clinical laboratory investigations (hematology, blood chemistry, and urinalysis) were conducted during treatment. Other parameters measured included body weight, feed intake, plasma, red blood cell count, brain cholinesterase activity, and hepatic and renal carboxylesterase activities. Organ weights were recorded at necropsy and routine histopathological evaluation was performed. Mild muscle tremors were observed occasionally in the intermediate- and high-dose groups. Treated females, but not males, demonstrated a dose-dependent inhibition of cholinesterase activity, though within treatment groups, there were no differences associated with the number of days of treatment. Enzyme values had returned to baseline levels by 30 days post-treatment. Hepatic carboxylesterase activity was significantly reduced only in male rats at the highest dose. Lung weights were increased in vehicle and Matacil-treated groups. Histological studies indicated that these changes were a nonspecific tissue response to a heavy burden of an oil-based irritant, which was partially resolved by 30 days post-treatment. The results showed that, at the concentrations tested, the formulation produced little or no acute symptoms and minimal long-term sequellae.

Comparison of central and peripheral nervous system lesions caused by high-dose short-term and low-dose subchronic acrylamide treatment in rats.

The effects of high-dose subacute acrylamide treatment of up to 50 mg/kg/day for 4 or 10 d were compared to those of subchronic exposure, up to 12 mg/kg/day for 90 d. In the subacute study, Purkinje cells, long ascending tracts of the spinal cord, optic tract terminal or preterminal regions in superior colliculus, sensory ganglion cells, and distal large-caliber peripheral axons were severely affected. Purkinje cells and fasciculus gracilis changes were the earliest lesions. In the subchronic study, the dominant lesion was confined to the distal peripheral axon, with only minor changes occurring in spinal cord and medulla. Paranodal swellings with the characteristic appearance of neurofilament aggregations were not seen. This morphological study suggests a significant difference between high- and low-dose acrylamide-induced lesions. If there is a reduced tendency for long-term low-dose acrylamide exposure to produce CNS lesions, the risk of irreversible nervous system damage would be less than that predicted from subacute studies.

Peripheral and central nervous system lesions caused by triethyl- and trimethyltin salts in rats.

Both trimethyltin and triethyltin salts are known to produce toxic lesions in the central nervous system. Triethyltin intoxication has been associated with central intramyelin edema, while trimethyltin has been shown to produce neuronal necrosis in selected limbic and sensory regions of the brain. Only scant attention has been paid to peripheral nerves of animals treated with alkyltins. In this study, we have treated rats with 6 or 8 mg/kg trimethyltin, and 1, 2, 4, 6, or 8 mg/kg triethyltin (single or multiple exposure), and evaluated in detail at the light microscope level both central and peripheral nervous system lesions. In addition to the central neuron necrosis or myelin edema described previously, both compounds produced peripheral axon degeneration and chromatolysis of large spinal cord and brain stem neurons. Chromatolysis was seen in reticular neurons of the brain stem and ventral horn or spinal cord in rats receiving high doses (6 or 8 mg/kg) of triethyltin, and in these same areas plus mesencephalic trigeminal nucleus in animals treated with trimethyltin. Wallerian-like degeneration of peripheral axons was seen in sciatic and tibial nerve and ventral roots of animals receiving 3 injections of 4 mg/kg or single or multiple injections of 6 or 8 mg/kg triethyltin. Axon degeneration was also seen in sciatic and tibial nerves 21 days after a single exposure to 8 mg/kg trimethyltin. Since myelin edema is believed to be reversible, the axonal changes described here may be of greater clinical significance in relation to human exposure.

Quantitative methods in the study of the pathogenesis of haemoprotozoal diseases.

The study of pathogenicity of haemoprotozoa and the pathogenesis of the diseases they cause requires quantitative descriptions. Statistical and mathematical methods are introduced to describe infectivity, parasitaemia, total body parasitosis and the severity of diseases.

Experimental bovine trypanosomiasis (Trypanosoma vivax and T. congolense). III. Serum levels of immunoglobulins, heterophile antibodies, and antibodies to T. vivax.

Twenty-five steers were infected with T. vivax (EATRO 1721), 25 steers with T. congolense (EATRO 1800) and 25 steers kept as controls. Serum levels of immunoglobulins (IgG1, IgG2, IgM), natural antibodies to erythrocytes of chicken and sheep, and complement-fixing antibodies to T. vivax were measured. A significant decrease of serum IgM and natural antibodies to chicken erythrocytes occurred in the T. vivax group at day 9, i.e. at the decline of the first parasitemic wave. This was followed by a transient moderate increase of IgM accompanied by a transient decrease of IgG2. The T. congolense group had moderate decreases (less than 30%) of the mean IgG1 levels and moderate increases (less than or equal to 50%) of the mean IgG2, levels. It was concluded that there was little evidence for polyclonal activation of lymphocytes and that the decreased IgG1 levels in the T. congolense group might have been a reflection of immunosuppression. The complement-fixation test proved to be a sensitive tool for monitoring the antibody response to T. vivax.

Experimental Bovine Trypanosomiasis (Trypanosoma congolense). Lack of relation of the level of Parasitemia to the J blood group.

Twenty-five Holstein-Friesian steers were experimentally infected with Trypanosoma congolense EATRO 1721. They were tested for their J blood group status. Twelve steers were found to belong to Ja blood group and thirteen steers were either Js or Jcs. The level of parasitemia did not significantly differ between these two groups of cattle. It was concluded that the level of parasitemia is not influenced by J blood group antigens.

The effect of Trypanosoma congolense and T vivax infections on the antibody response of cattle to live rinderpest virus vaccine.

Infections with Trypanosoma congolense or T vivax did not significantly depress the neutralising antibody response of cattle to live rinderpest vaccine when vaccination was carried out eight or 25 days after infection.

Hemolytic complement and serum C3 levels in Zebu cattle infected with Trypanosoma congolense and Trypanosoma vivax and the effect of trypanocidal treatment.

Total hemolytic complement and C3 levels were found to drop to 6.25% and 50% of preinfection levels, respectively, during trypanosome infections. Chemotherapeutic elimination of the trypanosomes with Berenil led to recovery of preinfection levels within 7 days and 11 days when cattle infected with Trypanosome congolense and Trypanosoma vivax, respectively, were treated 37 days after onset of infection. Recovery was slower in T. vivax-infected cattle treated on day 50. Berenil treatment had no effect on complement levels in control animals. The possible causes and implications of these low complement levels in bovine trypanosomiasis are discussed.

Immunosuppression in bovine trypanosomiasis. The establishment of "memory" in cattle infected with T. congolense and the effect of post infection serum on in vitro (3H)-thymidine uptake by lymphocytes and on leucocyte migration.

Cattle infected with Trypanosoma congolense were intravenously immunized with Leptospira biflexa 15 days after trypanosomal infection. The primary immune response to L. biflexa was considerably reduced as compared to uninfected controls. The infected cattle mounted a secondary response when they were cured of trypanosomes by treatment with Berenil 25 days after infection and re-immunized 8 days later. The mean secondary response in these previously infected animals was lower tha, but not significantly different from that of the uninfected control animals. Serum collected 15, 20 and 25 days after infection was inhibitory to the migration of both autologous and isologous (control) peripheral blood leucocytes. The migration inhibitory activity was abolished by heating the serum at 56 degrees C for 30 minutes implying the involvement of a heat labile serum component(s). The same serum did not modify the mitogenic effect of PHA on autologous peripheral lymphocytes.

Experimental bovine trypanosomiasis (Trypanosoma vivax and T. congolense). II. Serum levels of total protein, albumin, hemolytic complement, and complement component C3.

Serum levels of total protein, albumin, activity of hemolytic complement, and complement component C3 were decreased in cattle infected with either T. congolense or T. vivax. The hemolytic complement activity was reduced most, i.e. to 20% (T. congolense) or 5% (T. vivax) of control levels. The development of hypocomplementemia was closely associated with the first peak of parasitemia.

Suppression of antibody response to Leptospira biflexa and Brucella abortus and recovery from immunosuppression after Berenil treatment.

Zebu cattle infected with either Trypanosoma congolense EATRO 1800 or Trypanosoma vivax EATRO 1721 had suppressed humoral immune responses to Leptospira biflexa injected intravenously and to attenuated Brucella abortus injected subcutaneously. T. congolense infections were more suppressive than T. vivax infections. In cattle infected with T. vivax, the suppression of immune responses to both bacterial immunogens was abrogated when the animals were treated with Berenil at the time of antigen administration. In cattle infected with T. congolense, simultaneous Berenil treatment at the time of vaccination abolished the suppression of immune response to L. biflexa, and lessened but did not abrogate the suppression of immune response to B. abortus.

Bovine granulocyte/macrophage and erythroid colony culture: characteristics of the colonies and the assay systems.

Bovine bone marrow granulocyte/macrophage colonies were cultured in vitro in methyl cellulose and in plasma clots using bovine endotoxin-stimulated serum as a source of colony stimulating activity. The endotoxin-stimulated serum was four times as potent as the control serum in the methyl cellulose cultures. No significant increase in the number of colony forming units was observed when bovine marrow cells were maintained in suspension cultures for various periods prior to plating in methyl cellulose. The percentage of glass/plastic adherent cells in bovine marrow cells was observed to be 43% +/- 12 (SD). Benzidine positive erythroid colonies appeared in plasma clot cultures on day 4 and disappeared by day 9. No second population of erythroid colonies appeared either as a function of time or as a function of erythropoietin concentration. The optimum erythropoietin concentration for bovine erythroid cultures was found to be 1.0 unit/mL. A significant difference was observed between animals in their marrow capacity to produce erythroid colonies in culture but no significant difference was observed within individual animals over a period of three months.