Histopathologic approaches to detect changes indicative of immunotoxicity.

Toxicologic pathology is crucial in the identification and characterization of health effects following exposure to xenobiotics, mainly in toxicity experiments in rodents. Regarding regulatory toxicology, histopathology of lymphoid organs and tissues is a cornerstone in the identification of immunotoxic compounds. A 2-tier testing system is usually employed in which the first tier is a general screen for (immuno)toxicity and the second tier consists of specific immune function studies, including host resistance tests or mechanistic studies. The role attributed to histopathology of lymphoid organs in the updated Organisation for Economic Cooperation and Development and Food and Drug Administration guidelines requires improvement and standardization of the histopathology procedures. Optimalization and standardization was started in an international collaborative immunotoxicity study (ICICIS). However, several problems were left unaddressed, mostly because of the few compounds tested in this study. Based on the results of the ICICIS study and the morphologic changes induced by immunotoxic/immunomodulatory compounds observed in other investigations, suggestions are given to further improve the identification and (semi)quantification of histopathologic changes in lymphoid organs and tissues.

Approaches to the identification and recording of findings in the lymphoreticular organs indicative for immunotoxicity in regulatory type toxicity studies.

Recent validation studies showed that histopathological examination of the hematopoietic and lymphoreticular system is one of the most sensitive tools in the evaluation of non-specific immune stimulatory or immune suppressive effects and hazard identification of potential immunotoxicity in routine safety tests. Most immunotoxic effects have a classical dose-response relationship and an immediate effect (i.e. do not need an induction phase). The findings associated with a specific immunomodulatory response are generally not detected morphologically in routine sections of the immune system in safety studies, but may be detected, because of their effects on other organs such as skin (contact dermatitis) or joints and kidneys (immune complex deposits). Careful detailed examination of the immune system may give valuable clues for the possible mechanism of action of the test material, as was also demonstrated in these validation studies.

Treatment of experimental DMBA induced mammary carcinoma with Cetrorelix (SB-75): a potent antagonist of luteinizing hormone-releasing hormone.

Cetrorelix, (Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10)-LHRH (SB-75) is a new highly potent antagonist of LH-RH. In the model of DMBA-induced mammary carcinoma, this antagonist was very effective in reducing tumor mass. A rapid decrease in tumor weights to levels below 0.1 g total tumor mass was achieved with 300 micrograms/kg given sc. daily for 14 days. The weights of uteri and ovaries were reduced to about 40-50% of control values. In all treated rats the estrus cycle was interrupted and the animals remained in a state of anestrus. Microscopically, the effects of Cetrorelix on the tumors were characterized by a loss of mitotic activity, marked regression with apoptosis, an increase of stroma and differentiation towards a normal mammary architecture. On the basis of a dose-response curve, a dose of 100 micrograms/kg/d of Cetrorelix was determined as sufficient for a full antitumor response. Large DMBA-tumors with total tumor mass of about 6 g could also be treated very effectively with a dose of 100 micrograms/kg/d. To achieve a complete tumor regression, the treatment had to last 34 days. After the cessation of treatment with 100 micrograms/kg/d and regrowth of the tumors the animals were treated with the agonist Decapeptyl (Trp6-LHRH) using a dose of 50 micrograms/rat/d for 14 days. Again, the tumors responded well and regressed within 10 days. The treatment with an overlapping dose schedule of Cetrorelix and Decapeptyl showed a continuous antitumor response. A transient stimulation of tumor growth by the LH-RH agonist was not observed under these experimental conditions. In ovariectomized rats bearing DMBA-tumors, treatment with Cetrorelix and estradiol, produced no tumor growth inhibition as compared to estradiol control group, indicating that there is no estrogen nullifying effect of this antagonist on tumor cells in this model. On the basis of these results, Cetrorelix is a highly effective antitumor agent in this breast cancer model, which might also be useful under clinical conditions.

The evaluation of HBDH and LDH isoenzymes in cardiac cell necrosis of the rat.

The total LDH, HBDH and LDH isoenzyme activities were assessed in a number of rat tissues. HBDH did not correlate with LD1 and LD2 isoenzyme activity in tissues with high HBDH activity. HBDH therefore cannot be used as a marker for cardiac necrosis in the rat. In rats treated with isoprenaline and SK&F 94120 at doses producing myocardial necrosis, only the LD1 isoenzymes showed any significant change but only within the first 24 h of treatment. No statistically significant differences were seen in the plasma AST, CK, CK-MB and total LDH activities.

Gastric neuroendocrine cell hyperplasia after treatment with the long-acting, potent H2-receptor antagonist SK&F 93479.

The time course and dose response of the neuroendocrine cell hyperplasia in the oxyntic mucosa of the rat was examined after treatment with the potent, long-acting H2-receptor antagonist SK&F 93479 at doses of 0 and 1000 mg/kg orally for 1, 3, 7, and 14 days and at doses of 0, 40, 200, and 1000 mg/kg orally for 1 and 6 months. The number of oxyntic neuroendocrine cells (chromogranin-positive) increased after 7 days of treatment. In the 1- and 6-month studies with doses of 1000 mg/kg, the grading for the number of oxyntic chromogranin-positive cells was 2.5 to 3 times the control levels, and they were distributed mostly throughout the mucosa, whereas at lower doses, which did not produce carcinoid tumours at 2 years, the neuroendocrine cells were distributed in the lower half of the mucosa with 1.5- to 2-fold increases in grades for cell numbers. Increases in cell numbers and cell distribution may be useful factors in the evaluation of the neuroendocrine cell hyperplasia found in, for example, the Zollinger-Ellison syndrome and chronic atrophic gastritis, in which hypergastrinaemia and fundic neuroendocrine cell hyperplasia are present.

Cardiotoxicity of a new inotrope/vasodilator drug (SK&F 94120) in the dog.

The cardiotoxicity and haemodynamic changes induced by IV administration of high doses of SK&F 94120, a novel positive inotrope/vasodilator, were studied in the dog. Inotropism was observed at a dose of 0.375 mg/kg. Mean blood pressure was reduced in a dose-related manner, reaching a nadir of 43.7 mm Hg at 24 mg/kg. Heart rate was increased at doses of 1.5 mg/kg and above. ECG examination revealed a shortening of PR interval and an increase in T-wave amplitude. At doses of 15 mg/kg and above, occasional AV dissociation with accrochage were sometimes seen shortly after the start of the infusion. Ventricular extrasystoles were only seen at doses of 120 mg/kg and above. A dose-related increase in severity and incidence of haemorrhages, deposition of haemosiderin and fibroplasia were seen in the left ventricular endocardium and atrioventricular valves at doses of 15 mg/kg and above. Focal myocardial necrosis, predominantly of the left ventricular papillary muscle, and mild periarteritis of medium-sized extramural arteries, mostly in the right atrium, were seen at doses of 45 mg/kg and above. Intimal proliferation of intra- and extramural coronary vessels was observed. A necrotising peri/panarteritis of these arteries was noted at doses of 200 and 400 mg/kg. The cardiotoxicity observed was considered to be due to the exaggerated pharmacologic effects seen at these high dose levels.

Pathogenicity of Isospora suis in gnotobiotic and conventionalised piglets.

Isospora suis is unequivocally a primary pathogen of swine. Inoculation of I suis in conventionalised and germ-free piglets caused a biphasic disease course with marked diarrhoea, villous atrophy and necrosis of the intestinal epithelium at four to six and eight to 10 days after inoculation. The presence of a normal bacterial flora markedly (P less than 0.05) influenced the survival rate of piglets but did not appear to affect the histopathological changes observed. Mild limited focal necrosis and bile stasis were present in the liver at eight to 10 days after inoculation. In this period there was also ectasia of lymph vessels in the intestinal lymph nodes.

Life cycle of Isospora suis in gnotobiotic and conventionalized piglets.

Isospora suis had 3 asexual and 1 sexual intra-intestinal conventional life cycle. The first asexual generation was most prominent at 2 days p.i. (post inoculation) and produced 2-7 merozoites. The second-generation meronts were prevalent at 3-4 days p.i. and produced 2-12 large merozoites. At 4-5 days p.i. the third generation meronts were prominent and produced 4-24 small crescent shaped merozoites. Mature sexual stages were most prominent at 5-6 days p.i. The stages were most numerous in the distal half of the small intestine. At 8-9 days p.i. stages morphologically similar to the second generation of meronts reappeared, followed by the further development into third generation merozoites and sexual stages. This was reflected in a prepatent period of 5 days and a biphasic patent period of 5-8 or 9, and 11-14 days p.i. Intraperitoneal injection of liver/spleen and intestinal lymph node homogenates, respectively, from piglets infected 24 and 48 h, previously with high doses of oocysts, resulted in a patent infection 10-12 days post inoculation of the donor piglets. No differences in the life cycle of I. suis were observed between conventionalized and germ-free piglets. An extra-intestinal life cycle of I. suis related to the second patent period was postulated.

Isospora suis infection in piglets. A review.

Recently several reports have been published on the association of Isospora suis with diarrhea in piglets. A review is given of I. suis infection in piglets, in which the taxonomy, life cycle, pathogenicity, immunity, and treatment of I. suis and related organisms are discussed.

Ectopic spleen tissue in the pancreas of Himalayan rabbits.

Ectopic foci of splenic tissue were observed in the pancreas of nine of 48 Himalayan rabbits. One of 24 males and eight of 24 females were affected.

The use of biotyping of Enterobacteriaceae as an evaluation of the isolation efficacy of laboratory animals.

2 sets of data are given of biotyping the Enterobacteriaceae from the faecal flora of SPF rats and cats housed with different isolation arrangements. The method is presented as an evaluation of the isolation efficiency of laboratory animals. Biotypes which were determined on almost every occasion belonged to the resident flora of the animals while those found occasionally represented contamination. Contaminating bacteria are cleared from an SPF breeding unit by colonization resistance and the continuous efflux of animals from the unit. A parameter of isolation efficacy is the number of different biotypes determined in time. With optimal isolation arrangements a stable bacterial flora of permanently colonized biotypes is obtained in the animals.

The haemoglobin types of mice.

The haemoglobin types of 27 inbred strains of mice have been determined. Besides the common types, Hbb-s and Hbb-p, 2 strains were found with Hbb-p. Affinity between strains with the Hbb-p type and asiatic mice seems likely. The influence of storage on the polymerisation of Hbb-d and Hbb-p, the possibility of distinguishing between homo- and heterozygote Hbb types, and the possible existence of a 4th haemoglobin type, are discussed.