Effects of salmeterol on host resistance to Trichinella spiralis in rats.

Salmeterol is a long-acting beta2-adrenoreceptor agonist. The compound has previously been screened for immunotoxic potential in a repeated dose toxicity study in rats for 28 days. The total serum IgG levels were increased at dose levels of 2 and 10 mg/kg/day. Presently, salmeterol was studied in an immune function assay addressing the host resistance to Trichinella spiralis parasites. Rats were daily treated with salmeterol for 28 days at dose levels of 0, 2, 6 and 10 mg/kg/day. On day 29, the animals were infected with T. spiralis parasites. After six weeks, host resistance was examined. The numbers of T. spiralis muscle larvae in the tongue nor the inflammatory reactions around the encapsulated larvae were affected by salmeterol treatment. The yield of muscle larvae in the whole carcass was not changed either. The IgM, IgA and IgE antibody responses to T. spiralis were unaffected. Only at the highest dose level tested, the anti-T. spiralis IgG antibody response was decreased significantly. However, salmeterol's interference with the generation of anti-T. spiralis antibodies of the IgG subclass apparently did not adversely affect the resistance to infection.

Effects of prolonged exposure to morphine and methadone on in vivo parameters of immune function in rats.

In rats, two 6-week repeated dose oral toxicity studies were performed with morphine (250 and 500 mg/kg food) and methadone (200 and 400 mg/kg food), respectively. Alterations in immune function were studied by assessing primary and secondary immune responses to sheep red blood cells. In addition, the ability to resist challenge with infectious agents was measured in host resistance models employing the parasite Trichinella spiralis and the bacterium Listeria monocytogenes. The primary and secondary antibody responses to sheep red blood cells were not affected by treatment with either morphine or methadone. The clearance of L. monocytogenes bacteria in the spleen was not affected either. Prolonged treatment with morphine, however, resulted in a decrease in host resistance to T. spiralis infection, as indicated by a 1.5-fold increase in numbers of muscle larvae counted in the carcass, but did not affect the T. spiralis-specific IgM, IgG and IgE antibody responses. In contrast to morphine, the methadone-treated animals did not show a significant change in host resistance to T. spiralis. Total serum IgG levels, however, were increased in high-dose methadone-treated animals. Apparently, prolonged administration of morphine to rats resulted in immune suppression, mediating a slight, though biologically relevant, exacerbation of the T. spiralis infection, whereas methadone did not.

Heterogeneity of epithelial cells in the rat thymus.

The morphological heterogeneity of the thymic epithelium has been well documented both at the light and electron microscopic level. Immunohistochemistry has revealed four broad classes of epithelial cells (EC): subcapsule/perivascular, cortical, medullary EC, and medullary Hassall's corpuscles. Ultrastructural analysis has revealed further heterogeneity. In the cortex, four EC subtypes have been described ultrastructurally: subcapsular/perivascular, "pale," "intermediate," and "dark" EC. These subtypes are also present in the medulla. Two additional EC subtypes are restricted to the medulla: an undifferentiated subtype, and a subtype displaying signs of high metabolic activity. Based on the morphological features of the epithelium, it has been hypothetized that the thymic EC subtypes represent a process of differentiation.

Differential effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, bis(tri-n-butyltin) oxide and cyclosporine on thymus histophysiology.

Recent advances in the histophysiology of the normal thymus have revealed its complex architecture, showing distinct microenvironments at the light and electron microscopic level. The epithelium comprising the major component of the thymic stroma is not only involved in the positive selection of thymocytes, but also in their negative selection. Dendritic cells, however, are more efficient than epithelial cells in mediating negative selection. Thymocytes are dependent on the epithelium for normal development. Conversely, epithelial cells need the presence of thymocytes to maintain their integrity. The thymus rapidly responds to immunotoxic injury. Both the thymocytes and the nonlymphoid compartment of the organ can be targets of exposure. Disturbance of positive and negative thymocyte selection may have a major impact on the immunological function of the thymus. Suppression of peripheral T-cell-dependent immunity as a consequence of thymus toxicity is primarily seen after perinatal exposure when the thymus is most active. Autoimmunity may be another manifestation of chemically mediated thymus toxicity. Although the regenerative capacity of thymus structure is remarkable, it remains to be clarified whether this also applies to thymus function. In-depth mechanistic studies on chemical-induced dysfunction of the thymus have been conducted with the environmental contaminants 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and bis(tri-n-butyltin)oxide (TBTO) as well as the pharmaceutical immunosuppressant cyclosporine (CsA). Each of these compounds exerts a differential effect on the morphology of the thymus, depending on the cellular targets for toxicity. TCDD and TBTO exposure results in cortical lymphodepletion, albeit by different mechanisms. An important feature of TCDD-mediated thymus toxicity is the disruption of epithelial cells in the cortex. TBTO primarily induces cortical thymocyte cell death. In contrast CsA administration results in major alterations in the medulla, the cortex remaining largely intact. Medullary epithelial cells and dendritic cells are particularly sensitive to CsA. The differential effects of these three immunotoxicants suggest unique susceptibilities of the various cell types and regions that make up the thymus.

The utility of two rodent species in carcinogenic risk assessment of pharmaceuticals in Europe.

For the past 20-30 years, lifespan carcinogenicity studies for pharmaceuticals have been required to be carried out in two rodent species. Due to scientific progress, the necessity/justification of lifespan studies in two species for the assessment of carcinogenic risk of pharmaceuticals is currently under discussion. A study in one species (either rat or mouse) might suffice. To appraise the need for a study in a second species, a database was compiled of all pharmaceuticals tested for carcinogenicity for which a marketing authorization was applied for in Germany and The Netherlands since 1980. The incidence of treatment-related tumor findings was determined in either rat or mouse or in both. Tumor findings occurred for nearly 50% of all compounds, with the rat being more sensitive than the mouse. Specific attention was given to the question whether tumor findings in mice ever caused the regulatory authorities to refuse registration, to restrict the proposed therapeutic indication of a pharmaceutical, or to apply a cautionary label. It was found that no tumor findings in mice alone ever led to such a regulatory action. In addition, whether mouse studies had been important in interpreting the results of rat studies was determined. A negative mouse study (no tumors found) was rarely used to declare the rat findings irrelevant to humans. A mechanistic explanation was used as a much more important argument in the assessment of tumor findings in rats. In case of transspecies findings, the target organs were the usual ones, such as lung and liver, or the tumors occurred as a result of an exaggerated pharmacodynamic action expected from the pharmacology of the compound. The results of the database thus question the need of maintaining the requirement of rodent carcinogenicity studies in two species.

An immunotoxicity screening study on salmeterol in rats.

Salmeterol, a long-acting beta 2-adrenoreceptor agonist without known immunotoxicity, was studied in a 28-day repeated dose toxicity test in Wistar rats. Several immunotoxicity screening parameters were incorporated in the study protocol to investigate the immunotoxic potential of the compound. Male rats were orally treated with 0, 0.2, 2 and 20 mg salmeterol/kg body weight/day. At the 20 mg/kg/day dose level, intubation errors occurred because the animals tried to resist intubation. Some of these animals died intercurrently. Therefore, the magnitude of the dose was lowered to 10 mg/kg/day at day 9 of treatment. Body weight and bone marrow cellularity were not affected. Hematological parameters were not altered either, except for platelet counts, that were decreased at all dose levels. Also liver weights were decreased at all dose levels tested. Absolute thymic weights were decreased at the 2 and 20/10 mg/kg/day dose levels. No treatment-related (histo)pathological lesions were seen in the (non)lymphoid organs. Serum IgM levels were increased at the 0.2, and IgG at the 2 and 20/10 mg/kg/day dose levels, respectively. B cell numbers in the spleen were decreased at all dose levels tested. The data indicate that the test battery applied to salmeterol is able to detect low immunotoxic potential. Further research is needed to elucidate whether salmeterol interferes with immune responses in rats upon antigenic challenge.

Alterations of dendritic cells in the rat thymus without epithelial cell loss during cyclosporine treatment and recovery.

After cyclosporine treatment, dendritic cells disappear from the rat thymic medulla. The present study was undertaken to examine the ultrastructural alterations in the dendritic cell population during 14-day cyclosporine treatment and subsequent 6-week recovery. Four dendritic cell subtypes were defined ultrastructurally by a newly developed classification system. In addition, the potential effect of cyclosporine on six medullary epithelial cell subtypes was studied. During cyclosporine treatment, a prominent reduction of dendritic cells was seen at the ultrastructural level, whereas the total number of medullary epithelial cells remained largely unchanged. These findings were confirmed by immunohistochemistry. The number of mature dendritic cells declined later than the number of immature ones. A decrease in the antigen-processing capacity of remaining dendritic cells was suggested by the disappearance of Birbeck granules and the reduced number of tubulovesicular complexes. These findings support a disturbance of clonal deletion during cyclosporine treatment. The dendritic cell alterations appeared reversible 4 weeks after the restoration of the original architecture. During recovery, dendritic cells displaying lysosomal elements outnumbered those found in the normal uninvoluted thymus. This phenomenon probably reflects an enhanced turnover of cell organelles. No treatment-related effect on epithelial cell subtypes was seen.

Investigation of a screening battery for immunotoxicity of pharmaceuticals within a 28-day oral toxicity study using azathioprine and cyclosporin A as model compounds.

The authors have investigated a panel of parameters for immunotoxicity that may be incorporated in routine screening for toxicity of pharmaceuticals. This panel comprises serum immunoglobulin concentrations, cellularity of bone marrow, weights and histopathology of thymus, spleen, and lymph nodes, histopathology of Peyers' patches, and FACScan analysis of lymphocyte subpopulations in the spleen, in addition to parameters of toxicity to other systems. To study the value of these assays for pharmaceuticals, the authors used the immunosuppressants azathioprine (AZP) and cyclosporin A (CsA) as model compounds with known immunotoxic activity. In two separate experiments, rats were treated by daily gastric intubation with 0, 5, 12.5, and 25 mg AZP/kg body wt or 0, 1.25, 5, and 20 mg CsA/kg body wt. In the AZP study, the histopathology of the thymus and the spleen were valuable parameters of immunotoxicity, since these organs showed microscopic alterations at relatively low dose levels. In the CsA experiment, both the histopathology of the thymus and the data provided by FACScan analysis were sensitive indicators of immunotoxicity detecting effects at the lowest dose level employed. The data indicate that the lymphoid system is the most sensitive target of toxicity after AZP or CsA administration. The authors conclude that their test battery yielded immunotoxicity profiles of AZP and CsA in rats that were consistent with published findings in the literature, indicating the usefulness of the test battery employed.

Interspecies extrapolation in safety evaluation of human medicines in The Netherlands (1990-1992): practical considerations.

Doses used in animal toxicity studies can be extrapolated to therapeutic doses in man on the basis of body weight equivalence, metabolism equivalence, or toxicokinetics. The validity of extrapolation rules is a matter of debate. However, extrapolation on the basis of toxicokinetics is often preferred for human medicines, provided that adequate data are available. In registration dossiers on human medicines, often insufficient data are available to extrapolate on toxicokinetics only. This prompted us to study whether extrapolation on the basis of body weight equivalents or metabolism equivalents resembles the toxicokinetic way of extrapolation best. A survey was made of new chemical entities that have been applied for registration to the Medicines Evaluation Board in The Netherlands (College ter Beoordeling van Geneesmiddelen) between 1990 and 1992. In the majority of cases, larger margins of safety were achieved by extrapolation on the basis of body weight equivalents as compared to toxicokinetic data. Extrapolation on the basis of metabolism equivalents resembled toxicokinetic extrapolation factors better. The method of extrapolation using metabolism equivalents was rarely encountered in the registration dossiers on human medicines submitted to the Board.

The intrathymic target cell for the thymotoxic action of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Sublethal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces thymic atrophy and thymic lymphodepletion in all species investigated. Lymphodepletion is first seen in the cortex of the thymus, but also occurs in the medulla at later time points. This article surveys in vivo and in vitro studies on TCDD-induced thymus toxicity. It is concluded that the first intrathymic target cell for the action of TCDD is the cortical epithelial cell.

Ultrastructure of the cortical epithelium of the rat thymus after in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known for inducing cortical atrophy in the rat thymus. The present study was conducted to provide ultrastructural evidence for the cortical epithelium to be a target for TCDD in vivo. Juvenile male Wistar rats were orally intubated once with either 50 or 150 micrograms/kg TCDD and killed 4 or 10 days thereafter. Major changes were found in the cortical thymic epithelium. First, a relative shift occurred from "pale" to darker cortical epithelial cell types, as judged by their nuclear and cytoplasmic electron density. This effect was most prominent at 10 days after exposure to 150 micrograms/kg TCDD. The increased electron density of the cortical epithelium indicates an altered state of cellular differentiation. Secondly, at the 150 micrograms/kg dose level focal epithelial cell aggregates were seen both at day 4 and day 10 after administration. This aggregation may either be compound induced or represent a secondary event to the collapse of the thymic stroma. Thirdly, increased vacuolation of cortical epithelial cells was apparent. This effect is interpreted as a consequence rather than a cause of thymocyte depletion from the cortex. This study indicates that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Electron microscopy reveals that the differentiation of epithelial cells is altered. In addition, epithelial cell aggregates are formed.

The cortical epithelium of the rat thymus after in vivo exposure to bis(tri-n-butyltin)oxide (TBTO). An (immuno)histological and ultrastructural study.

Bis(tri-n-butyltin)oxide (TBTO) induces cortical atrophy in the rat thymus. We studied the potential involvement of the cortical epithelium in TBTO-induced thymotoxicity by (immuno) histology and electron microscopy. Juvenile male Wistar rats were orally intubated once with either 30 or 90 mg/kg TBTO and sacrificed 4 or 10 days later. A dose-dependent thymic atrophy occurred. Anti-keratin labelling showed epithelial cell aggregation in some animals at 10 days after exposure to 90 mg/kg TBTO, when recovery of the thymus was apparent. At the ultrastructural level, a relative shift was observed from "pale" to darker epithelial cell subtypes at the 30 mg/kg dose level, both at day 4 and day 10 after intubation. This phenomenon was not observed after exposure to 90 mg/kg TBTO. Both the altered keratin distribution and the increased electron density of the epithelium probably represent non-specific phenomena. The present morphological observations support the concept that TBTO affects the thymus via its action on lymphoid cells rather than on the epithelial compartment.

Interdigitating cells in the rat thymus during cyclosporin A treatment: ultrastructural observations.

Major Histocompatibility Complex (MHC) class II-positive cells are almost completely absent in the rat thymic medulla after cyclosporin A (CsA) exposure. This phenomenon has been related to alterations in the medullary interdigitating cells (IDCs), representing either down-modulation of RT1 class II antigen on persistent IDCs, or actual disappearance of IDCs. To resolve this issue, we performed an ultrastructural study of the rat thymic medulla during CsA treatment (15 mg/kg body weight/day CsA subcutaneously for 7 to 14 days). At day 7 after exposure, only very few IDCs could be identified. They showed an abnormal ultrastructural morphology as judged by inconspicuous membrane interdigitations and lack of Birbeck granules. At day 14, the number of IDCs was larger. At this time, the IDCs had a normal ultrastructure, except for the almost complete absence of Birbeck granules. We conclude that CsA causes an initial depletion of IDCs from the thymic medulla. The ultrastructural morphology of the few IDCs present suggests an immature character. New IDC immigrants appear in the medullary area already during CsA treatment, indicating early recovery.

Effects of subcutaneous versus intravenous administration of cyclosporin A on rat thymic histology and pharmacokinetics.

Cyclosporin A (CsA) was administered to rats by repeated subcutaneous (s.c.) or intravenous (i.v.) injections for 14 days. Changes in thymic histology were independent of the route of administration. Blood concentration--time profiles of CsA were similar at dose levels of i.v. 7.5 and s.c. 15 mg/kg/day, respectively. So, provided that the dose is reduced, i.v. injection can serve as an adequate alternative to s.c. injection, thereby preventing unwanted painful side-effects associated with the latter route of administration.

Alterations in the cortical thymic epithelium of rats after in vivo exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): an (immuno)histological study.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces thymic atrophy in rats. The present study was initiated to provide (immuno)histological data on the mechanism of action. Juvenile male Wistar rats were orally intubated once with 50 or 150 micrograms/kg TCDD. They were euthanized 4 or 10 days thereafter, or were allowed to stay alive until Day 20 or 26. Growth retardation occurred rapidly in all TCDD-treated animals. Lethality was demonstrated within 20-21 days after administration. At Days 4 and 10 after intubation, thymic atrophy was shown by reduction of thymic weight and cortex/medulla ratio. Staining patterns for T-cell markers in the atrophic thymuses coincided with the reduction of cortical areas. There was no evidence indicating that the effects were indirectly caused by stress. TCDD-induced thymic atrophy persisted until Day 26 after administration. Immunohistochemical analysis revealed prominent changes in the cortical thymic epithelium at the 150-micrograms/kg dose level. First, in the cortex epithelial cell aggregates were observed both at Day 4 and at Day 10 after administration. Apparently, the architecture of the epithelium had changed in these animals. Second, at 10 days after administration epithelial cells were found with the simultaneous expression of markers that in the normal uninvoluted thymus only occur either in the subcapsular/medullary area or in the cortex. This phenotype points to an unusual stage of differentiation. We conclude that TCDD exposure affects the cortical epithelium of the rat thymus at a high dose level. Apparently, it disturbs the epithelial network and interfers with the differentiation of epithelial cells.

Starvation-induced microautophagic vacuoles in rat myocardial cells.

During prolonged starvation the heart atrophies and loses protein mass. Debate lingers over the basic mechanisms in the production of negative cardiac protein balance during starvation. The extent to which cardiac proteolysis takes place within the lysosomal vacuolar system is unknown. The present communication examines the starvation-induced changes within the lysosomal system of rat myocardial cells, as studied by means of conventional electron-microscopic techniques. Special attention has been paid to the occurrence of microautophagic vacuoles. It is concluded that during prolonged starvation microautophagic vacuoles appear in rat myocardial cells, suggesting the induction of a microautophagic pathway of lysosomal proteolysis.

Quantitative changes in the lysosomal vacuolar system of rat hepatocytes during short-term starvation. A morphometric analysis with special reference to macro- and microautophagy.

Ultrastructural morphometric analysis was used to study time-dependent variations in macro- and microautophagy in rat hepatocytes. Except during periods of short-term starvation for up to 24 h, animals were kept under standardized conditions of food intake. In hepatocytes of meal-fed rats the volume fraction of macroautophagic vacuoles is significantly higher at 23:00 h, i.e., immediately before food intake, compared to 11:00 h, i.e., 12 h following feeding. During fasting, macroautophagy drops to a low level. Microautophagic vacuoles in hepatocytes of meal-fed rats, sacrificed at 11:00 or 23:00 h respectively, do not show any significant quantitative differences. However, during 12 h of starvation, the volume fraction of microautophagic vacuoles rises significantly, whereas the numerical density remains constant. Subsequently, during the second 12-h period of fasting, the volume fraction of microautophagic vacuoles remains unchanged, but the numerical density increases. Over a period of 24 h of starvation the volume fraction of the total lysosomal system does not change significantly, whereas the numerical density rises. The time-dependent changes of the macroautophagic vacuolar system correlate with the circadian, food-related variations in the protein content of individual hepatocytes from meal-fed animals. The increase in volume fraction and thereafter in number of microautophagic vacuoles, as observed during starvation, coincides with a large decrease in protein content of individual hepatocytes.