Immunohistochemical Characterization of Proliferative Lesions in the Thymus of Aging CD-1 Mice From Two Studies on the RITA Database, With Special Reference to the Perivascular Space.

Thymic lymphoid hyperplasia is a common age-related finding, which occurs particularly in female CD-1 mice. The main differential diagnoses are malignant lymphoma and thymoma. A systematic investigation of control groups from two carcinogenicity studies was performed including measurements of thymic size, and the immunohistochemistry (IHC) markers pan-Cytokeratin (pan-CK) for thymic epithelial cells; CD3 and CD45R/B220 for T and B lymphocytes, respectively; CD31 for endothelial cells; and F4/80 for macrophages. Thymoma can be differentiated by increased numbers of proliferating epithelial cells demonstrated by pan-CK IHC staining. Differentiation between lymphoid hyperplasia and lymphoma is more challenging as a mixture of B and T lymphocytes can be present in both findings. The present investigation showed that the thymic perivascular space is the compartment where the increased numbers of lymphocytes in hyperplasia are localized and not the medulla, as previously thought. The lymphoepithelial compartment is atrophic to the same extent in thymi diagnosed with age-related involution or lymphoid hyperplasia. Both diagnoses are thus related to variations in lymphoid cellularity of the nonepithelial perivascular space, which is continuous with the perithymic tissue. Likewise, lymphomas have a predilection to colonize the perivascular space and to spare the lymphoepithelial compartment.

Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies: a guide for investigators, authors, reviewers, and readers.

Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated. The objectives of this article are to illustrate a number of specific morphologic findings in commonly examined fish tissues (e.g., gills, liver, kidney, and gonads) that are frequently either misdiagnosed or underdiagnosed, and to address related issues involving the interpretation of histopathologic data. To enhance the utility of this article as a guide, photomicrographs of normal and abnormal specimens are presented. General recommendations for generating and publishing results from histopathology studies are additionally provided. It is hoped that the furnished information will be a useful resource for manuscript generation, by helping authors, reviewers, and readers to critically assess fish histopathologic data.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia.

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Reactive and proliferative changes of splenic reticulum cells of rats investigated with special staining methods and immunohistochemistry.

In the rat spleen, reactive and proliferative changes of the reticulum cells are rare events and seem to occur almost exclusively in the red pulp. The normal structure of the splenic reticulum cell and fiber lattice and examples of spontaneous and induced pathological alterations were investigated by immunohistochemistry (smooth muscle-actin, vimentin, S100 and proliferating cell nuclear antigen) and special stains for extracellular fibers (silver impregnation, azan). In response to congestion, systemic tumor growth or treatment with a hematotoxic compound, the scaffold cells increased either their contractile properties or their production of extracellular fibers. Primary focal hyperplasias of stromal cells which had developed without obvious cause were characterized by vanishing of sinuses, increased fiber content, increased expression of sm-actin or foci of lipomatosis. The borders of focal hyperplasias were indistinct and they did not infiltrate the white pulp compartments. Neoplasms of the stromal reticulum cells resembled soft tissue tumors in other organs. Specific tumor entities as described in other species have so far not been observed in the rat.

ESTP comments on the draft updated OECD test guideline 407.

The revision of the OECD TG 407 test guideline (repeated dose 28-day oral toxicity study in rodents) focuses on endpoints to detect endocrine activities of chemicals. The new endpoints are likely to influence other previously established core endpoints of this study type. An expert group of pathologists and toxicologists within the European Society of Toxicologic Pathologists (ESTP) has contributed to the scientific discussion of the draft guideline. The advantages and disadvantages of methodical changes as necropsy of all females in dioestrus, blood collection for clinical chemistry and haematology at the same cycle stage, weighing of the thyroid gland and separate weighing of ventral and dorsolateral lobes of the prostate are considered. Possible alternatives are pointed out covering scientific as well as practical aspects.

Regulatory aspects of immunotoxicology.

Since years, differences among the regulatory requirements on preclinical immunotoxicity testing for pharmaceuticals in the EU, Japan and US indicated a need for an internationally accepted approach. Requests for immunotoxicity investigations are also addressed by guidelines in non-drug areas. While some contain more detailed information in their requirements, other regulations comprise only vague descriptions for consideration of (non-intended) effects on the immune effects. Since 2002, the International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use put effort in the development of a harmonised approach for testing of immunosuppression and immunoenhancement. Consensus on the ICH S8 guideline on immunotoxicity testing for pharmaceuticals was achieved which now can be implemented into national regulations. The new concept contains in-depth testing, e.g., by functional tests in a concern/weight of evidence approach if the standard toxicity studies or other causes of concern give evidence of an immunotoxic potential or when the target populations are specifically vulnerable. It is expected that the progress on immunotoxicity testing reached by the ICH process will also have an impact on other regulatory areas. Additionally, the regulatory differences in testing requirements on immunotoxicity in other pharmaceutical areas including biotechnology-derived drugs, medicinal products and vaccines and in non-drug areas consisting of chemicals, agrochemicals or food additives are briefly highlighted.

Harmonization of immunotoxicity guidelines in the ICH process--pathology considerations from the guideline Committee of the European Society of Toxicological Pathology (ESTP) .

As part of the ICH process of harmonization of testing guidelines for immunotoxicity, the European Society of Toxicologic Pathology (ESTP) has contributed to the scientific discussion on methods and evaluation of immunotoxicity studies with technical and scientific recommendations on toxicologic pathology. The weighing and sampling of immune organs is discussed taking into consideration specifically the value of lymph node weighing and the selection of appropriate lymph nodes for the detection of local and systemic effects. The different techniques of bone marrow preparation are considered for routine and extended investigations. Criteria are given for the gross and histopathological detection of effects in Peyer's patches. For the histopathological evaluation it is strongly recommended that each compartment within the different lymphoid organs is investigated separately and semiquantitatively since this approach has shown to increase the sensitivity and specificity of immunohistopathology.

Bilateral noncystic renal dysplasia in a Wistar-rat.

In a young Wistar rat a bilateral renal malformation was observed microscopically. Clinical chemistry gave no evidence of impaired kidney function. The kidney weight was slightly elevated and the kidneys showed no gross pathological changes. The lesion was located in the inner cortex of both kidneys and consisted of multiple foci of abnormal renal parenchyma similar to fetal kidney. Three components could be distinguished in the foci: primitive glomerular/tubular structures, tubules resembling collecting ducts and mesenchyme. For further characterisation, histological stains (H&E, PAS, Novotny) and immunohistochemistry (vimentin, pan-cytokeratin, S 100, proliferating cell nuclear antigen, and terminal desoxyribosyl-transferase mediated dUTP nick end labelling) were applied. The glomerular and tubular structures were hyperplastic and positive for proliferating cell nuclear antigen and vimentin. The collecting duct-like tubules were positive for pan-cytokeratin and gave no evidence of proliferation. The two epithelial components of the foci were surrounded by mesenchymal cells which extended also between the normal cortical tubules so that no clear demarcation was discernible. The mesenchymal cells were uniformly spindle-shaped and associated with reticulin fibers. Immunohistochemically they were vimentin-positive and non-proliferative. With terminal desoxyribosyl-transferase mediated dUTP nick end labelling (TUNEL) and S 100 all components were nearly negative. Based on morphology and immunohistochemistry this malformation containing structures derived from the ureteric bud and from the metanephric blastema associated with oligonephronia probably represents a noncystic renal dysplasia. Transition to neoplasia was not observed. A specific cause of this unusual developmental anomaly which was not previously reported in rats could not be determined.

Revised guides for organ sampling and trimming in rats and mice--Part 3. A joint publication of the RITA and NACAD groups.

This is the third part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the urinary, nervous, musculoskeletal, cardiovascular, and lymphoreticular systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (BAHNEMANN et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum "end-product". The objectives of this work, as addressed in detail in the first part (Ruehl-Fehlert et al. 2003), are to standardize tissue sampling and trimming for comparison of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming (Crissman et al. 2004).

Revised guides for organ sampling and trimming in rats and mice--Part 2. A joint publication of the RITA and NACAD groups.

This is the second part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies, covering the respiratory, male and female genital, and the endocrine systems. The article is based on the experience of the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (Bahnemann et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and/or a macro-photograph showing the plane of section as well as a low magnification of the H&E stained slide demonstrating the optimum "end-product". The objectives of this work, as addressed in detail in the first part (Ruehl-Fehlert et al. 2003), are to standardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming. dardize tissue sampling and trimming, to improve the comparability of historical data obtained from different studies and different laboratories, ensure the presence of all relevant target sites for histopathological evaluation and provide technical advice for preparatory techniques during necropsy, fixation and trimming.

Revised guides for organ sampling and trimming in rats and mice--part 1.

This is the first part of a series of three articles on trimming instructions of rat and mouse protocol organs and tissues in regulatory type toxicity studies. It is based on the experience made in the European RITA and American NACAD working groups and is an extended revision of trimming guides published in 1995 (Bahnemann et al.). The optimum localization for tissue preparation, the sample size, the direction of sectioning and the number of sections to be prepared is described organ by organ. These descriptions are illustrated for each organ by a schematic drawing and a macro-photograph showing the plane of section as well as a low power view of the H&E stained slide demonstrating the optimum "end-product". This revision will improve the quality and efficiency of routine procedures and facilitate daily work in the histotechnical lab. It will promote intra- and inter-study reproducibility and comparability and thus lead to a further coherence within each study and improvement of the validity of historical control data.

Pathology working group review of histopathologic specimens from three laboratory studies of diclofenac in trout.

While the pathology peer review/pathology working group (PWG) model has long been used in mammalian toxicologic pathology to ensure the accuracy, consistency, and objectivity of histopathology data, application of this paradigm to ecotoxicological studies has thus far been limited. In the current project, the PWG approach was used to evaluate histopathologic sections of gills, liver, kidney, and/or intestines from three previously published studies of diclofenac in trout, among which there was substantial variation in the reported histopathologic findings. The main objectives of this review process were to investigate and potentially reconcile these interstudy differences, and based on the results, to establish an appropriate no observed effect concentration (NOEC). Following a complete examination of all histologic sections and original diagnoses by a single experienced fish pathologist (pathology peer review), a two-day PWG session was conducted to allow members of a four-person expert panel to determine the extent of treatment-related findings in each of the three trout studies. The PWG was performed according to the United States Environmental Protection Agency (US EPA) Pesticide Regulation (PR) 94-5 (EPA Pesticide Regulation, 1994). In accordance with standard procedures, the PWG review was conducted by the non-voting chairperson in a manner intended to minimize bias, and thus during the evaluation, the four voting panelists were unaware of the treatment group status of individual fish and the original diagnoses associated with the histologic sections. Based on the results of this review, findings related to diclofenac exposure included minimal to slightly increased thickening of the gill filament tips in fish exposed to the highest concentration tested (1,000 µg/L), plus a previously undiagnosed finding, decreased hepatic glycogen, which also occurred at the 1,000 µg/L dose level. The panel found little evidence to support other reported effects of diclofenac in trout, and thus the overall NOEC was determined to be >320 µg/L. By consensus, the PWG panel was able to identify diagnostic inconsistencies among and within the three prior studies; therefore this exercise demonstrated the value of the pathology peer review/PWG approach for assessing the reliability of histopathology results that may be used by regulatory agencies for risk assessment.

Comparison of zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) as test species in the Fish Sexual Development Test (FSDT).

Results are presented from a validation (with 5 laboratories) of the Fish Sexual Development Test (FSDT) developed to detect endocrine disrupters (EDs) and included in the OECD (Organisation for Economic Co-operation and Development) working program. The aromatase-inhibiting fungicide prochloraz was tested in zebrafish (Danio rerio) and fathead minnow (Pimephales promelas). The fish were exposed during sexual differentiation and development from 0 to 60 days post hatch (dph). After exposure, the vitellogenin (VTG) concentrations were quantified in head/tail homogenate and the sex ratio was determined (defined as female, male, intersex or undifferentiated). NOEC/LOEC and EC(x) designs were compared to optimize the test approach. Results show that both species are highly sensitive to prochloraz during sexual development. They respond by skewing of the sex ratio towards male phenotype and by a VTG decline in females. The NOEC/LOEC approach is preferred because sex ratio is difficult to analyze with a regression model. The mean NOEC/LOEC for prochloraz on the sex ratio was 43.3/134 µg/L and 101/293 µg/L for zebrafish and fathead minnow, respectively. The mean NOEC/LOEC on the decline in female VTG concentration was 65/110 µg/L and ~30/68 µg/L respectively. In conclusion, zebrafish and fathead minnow are suitable species in the FSDT and their sexual differentiation is equally labile to EDs.