Development of apoptosis and changes in lymphocyte subsets in thymus, mesenteric lymph nodes and Peyer's patches of mice orally inoculated with T-2 toxin.

Development of apoptosis and changes in lymphocyte subsets were examined mainly by flow cytometer in thymus, mesenteric lymph nodes and Peyer's patches of mice up to 24 hours after oral inoculation with T-2 toxin (10 mg/kg). T-2 toxin attacked Peyer's patches first, then mesenteric lymph nodes, and finally thymus in relation to the course of enteric absorption of orally inoculated T-2 toxin. The degree of lymphocyte apoptosis was prominent in the thymus, moderate in the Peyer's patches, and somewhat mild in the mesenteric lymph nodes, suggesting the difference in lymphocyte population susceptible to T-2 toxin. As to the changes in lymphocyte subsets, CD4+ CD8+ T cells were most sensitive to T-2 toxin, and CD4+ CD8- T cells were more severely depressed than CD4- CD8+ T cells in the thymus. In the mesenteric lymph nodes, CD3+ cells was more clearly affected than CD19+ cells, and the numbers of CD4+ and CD8+ cells were similarly decreased. In the Peyer's patches, the numbers of CD3+, CD 19+, CD4+ and CD8+ cells were unexceptionally decreased. In addition, among IgM+, IgG+ and IgA+ B cells, the number of IA+ B cells which are more important in the mucosal immunity was most severely affected.

Apoptosis in mouse fetuses from dams exposed to T-2 toxin at different days of gestation.

T-2 toxin (2 mg/kg b.w.) was orally inoculated to pregnant mice at gestational day (GD) 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 15.5 and GD 16.5, respectively, and the fetuses were examined 24 hours later. The number and region of pyknotic or karyorrhectic cells varied according to inoculation date. In the GD 13.5-subgroup, a moderate to high number of pyknotic or karyorrhectic neuronal cells were observed in the central nervous system, peri-ventricular zone to subventricular zone, and pyknosis or karyorrhexis were also observed in a small number of chondroblasts and chondrocytes. In the GD 16.5-subgroup, a moderate to high number of pyknotic or karyorrhectic cells were observed in the thymus and renal subcapsular parenchyma. The nuclei of these pyknotic or karyorrhectic cells were strongly stained by the terminal deoxy nucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling method widely used for the in situ detection of apoptotic nuclei. In addition, a few fetuses from dams which were given T-2 toxin at GD 13.5 or GD 14.5 and killed at GD 17.5 showed skeletal abnormalities such as wavy ribs and short scapula. From the present findings and the well known fact that T-2 toxin readily crosses the rat placenta, it seems that T-2 toxin-induced apoptosis in the developing mouse fetuses might be a direct effect of T-2 toxin on fetuses.

Kinetics of apoptosis-related genes mRNA expression in the dorsal skin of hypotrichotic WBN/ILA-ht rats after topical application of T-2 toxin.

The expression of apoptosis-related genes mRNAs was examined in the dorsal skin of hypotrichotic WBN/ILA-Ht rats topically applied with T-2 toxin (10 microl of 0.5 microg/microl solution). The total mRNA was obtained from skin biopsy samples from each rat at 3, 6, 12 and 24 hours after T-2 toxin treatment (HAT), and RT-PCR was carried out with pairs of oligonucleotide primers corresponding to the cDNA sequences of rat p53, bcl-2, c-ki-ras, c-fos and c-jun oncogenes. The expression of c-fos mRNA markedly increased at 3 HAT, peaked at 6 HAT, and greatly decreased at 12 HAT. However it maintained a higher level, compared with the control level, even at 24 HAT. Although not prominent, the expression of c-jun mRNA also showed significant elevation from 3 to 12 HAT. On the other hand, there were no changes in the expression of p53, bcl-2 and c-ki-ras mRNAs throughout the observation period. Judging from the present results and our previous report that epidermal cells developed apoptosis at 12 HAT (Histol Histopathol 1999; 14: 337-342), the induction of c-fos and perhaps of c-jun mRNAs may be associated with T-2 toxin-induced epidermal cell apoptosis.

Esophageal adenosquamous carcinoma in a cat.

Esophageal carcinoma was observed in an eight-year-old, castrated male, Japanese domestic cat. Histologically, this neoplasm consisted of two different growth patterns, squamous cell carcinoma and adenocarcinoma. The results of immunohistochemical examination supported the fact that the two kinds of neoplastic cells have different characteristics. The tumor was, therefore, diagnosed as adenosquamous carcinoma. Esophageal tumors in the cat are very rare and, if any, neither adenocarcinoma nor adenosquamous carcinoma has been reported up to the present.

Kinetics and distribution of transforming growth factor (TGF)-beta 1 mRNA in the dorsal skin of hypotrichotic WBN/ILA-Ht rats following topical application of T-2 toxin.

Depression of basal cell proliferating activity and subsequent induction of basal cell apoptosis in the epidermis and infiltration of inflammatory cells including mast cells in the dermis were observed in the dorsal skin of hypotrichotic WBN/ILA-Ht rats following the topical application of T-2 toxin in our previous study (ALBARENQUE et al. 1999). In the present study, kinetics of TGF-beta 1 mRNA was investigated using the same experimental system. The level of TGF-beta 1 mRNA of the whole skin tissue measured by competitive RT-PCR method showed a slight elevation from 6 to 12 hours after treatment (HAT) and reached the significantly higher level at 24HAT compared with the control skin. The increase in signals of TGF-beta 1 mRNA detected by in situ hybridization method started at 3HAT in the epidermis and progressed thereafter both in the epidermis and in the dermis. These results suggest that the elevated level of TGF-beta 1 mRNA may have a close relation to the induction of epidermal basal cell apoptosis as well as to the intradermal infiltration of mast cells and fibroblasts following the topical application of T-2 toxin.

Cerebral amyloid angiopathy in an aged great spotted woodpecker (Picoides major).

A male great spotted woodpecker (Picoides major), which was at least 16 years old, died due to general weakening. Cerebral vascular walls, including capillaries, were positively stained with Congo red with green-gold birefringence, and some of which showed a severe deposition of the Congophilic materials resulting in a corona-like fibrillar radiating structure. The Congophilic materials were positive for beta amyloid protein, but negative for prion protein. Only a few senile plaque-like structures were observed in the cortex by PAM stain and beta amyloid immunostain. The present case is the first observation of cerebral amyloid angiopathy in avian species and will indicate the presence of such age-related cerebral lesions also in birds.

Apoptosis in the developing mouse embryos from T-2 toxin-inoculated dams.

T-2 toxin (3 mg/kg b.w.) was orally inoculated to pregnant mice at 11 days of gestation to examine the effect of T-2 toxin on the developing embryos. At 24 hours after T-2 toxin-inoculation, moderate pyknosis or karyorrhexis was generally observed in some layers of the central nervous system, caudal sclerotomic segment, caudal region of the tongue to pharyngeal- to laryngeal-mesenchyma, trachea and facial mesenchyma. These pyknotic or karyorrhectic nuclei were strongly stained by the modified TUNEL method widely used for the in situ detection of apoptotic nuclei and also showed ultrastructural changes characteristic for apoptosis. This is the first report of mycotoxin-induced apoptosis in embryos.

T-2 toxin-induced acute skin lesions in Wistar-derived hypotrichotic WBN/ILA-Ht rats.

Acute lesions in the dorsal skin topically applied with T-2 toxin (10 microliters of 0.5 mg/ml-solution to 1 cm2) were examined in Wistar-derived hypotrichotic WBN/ILA-Ht rats up to 24 hours after treatment (24HAT). In the epidermis, depression of basal cell proliferating activity was detected at 3HAT by immunostaining for proliferating cell nuclear antigen (PCNA), and the percentage of PCNA-positive basal cells decreased thereafter. At 12HAT, in addition to intracytoplasmic edema of spinous cells, acidophilic degeneration of basal cells characterized by shrinkage of cell body with acidophilic cytoplasm and pyknotic or karyorrhectic nuclei became prominent. Most of these nuclei were positive for TUNEL which is a widely used immunostaining for the in situ detection of fragmented DNA, i.e. apoptosis, and the percentage of TUNEL-positive basal cells increased thereafter. The nuclei of these basal cells also showed ultrastructural changes characteristic for apoptosis. On the other hand, in the dermis, infiltration of inflammatory cells including mast cells started at 3HAT and increased thereafter. In addition, capillary and small vessel endothelial degeneration developed at 6HAT and progressed thereafter. These results suggest that T-2 toxin directly affects the epidermis and produces apoptosis in basal cells.

Possible involvement of DNA methylation in 5-azacytidine-induced neuronal cell apoptosis.

Eight chemicals that are cytidine analogues or nucleosides (5-azacytidine (5AzC), 5-azadeoxycytidine, 6-azacytidine, 5-azacytosin, cytidine, 3-deazaadenine, 3-deazauridine and 6-azauridine) were examined for the ability to induce neuronal apoptosis. 5AzC and 5-azadeoxycytidine induced apoptosis in the brain and spinal cord of the fetuses at 24 hr after the injection to dams, while the other chemicals tested failed to induce apoptosis. In the system of PC12 cells, only 5AzC induced apoptosis, and other chemicals failed to provoke morphological and biochemical changes characteristic of apoptosis. 5AzC, 5-azadeoxycytidine and 6-azacytidine failed to induce apoptosis in C6 cells. Gel electrophoresis after MspI or HapII digestions revealed no apparent evidence of DNA demethylation after 5AzC-treatment in either fetal brains or PC12 cells. These results indicate that DNA demethylation is possibly involved in 5AzC-induced neuronal apoptosis although no direct evidence of DNA demethylation was obtained.

Changes in location and number of tartrate-resistant acid phosphatase (TRAP)-positive cells during the development of type II collagen-induced arthritis in DBA/1J mice.

The authors investigated changes in the location and number of osteoclasts and their precursors during the development of articular lesions in type II collagen-induced arthritis in mice using tartrate-resistant acid phosphatase (TRAP) staining. The limb joints were examined at 6 to 15 weeks after the second immunization. The number of TRAP-positive cells increased as the articular lesions progressed. TRAP-positive macrophage-like cells were found in the hyperplastic synovial tissue and bone marrow stroma in the early stage. In the advanced stage, in addition to many TRAP-positive osteoclasts on the bone surface, TRAP-positive macrophage-like cells were observed in the pannus apart from the bone surface in the pannus-joint junctions. The above mentioned TRAP-positive macrophage-like cells are considered to be osteoclast precursors.

T-2 toxin-induced apoptosis in hematopoietic tissues of mice.

We examined T-2 toxin-induced lesions in the bone marrow and splenic red pulp as many as 48 hr after oral inoculation with 10 mg/kg body weight of T-2 toxin in female ICR:CD-1 mice. Histopathologically, the bone marrow and splenic red pulp showed a significant hypocellularity. In the bone marrow, the number of myelocytes significantly decreased due to the loss of immature granulocytes, erythroblasts, and lymphocytes. The nuclei of the remaining cells showing pyknosis or karyorrhexis were positively stained by the TdT-mediated dUTP nick end labeling (TUNEL) method, and these TUNEL-positive cells showed ultrastructural characteristics of apoptosis. With agarose gel electrophoresis, DNA ladders were clearly detected in bone marrow samples. The number of TUNEL-positive cells in splenic red pulp increased earlier than it did in the splenic white pulp. Thus, T-2 toxin induced-lesions in the hematopoietic tissues and in the lymphoid tissues were brought about by apoptosis of component cells. We believe that damage to the hematopoietic microenvironment may also play an indirect role in the induction of apoptosis in the bone marrow.

Apoptosis in feline panleukopenia virus-infected lymphocytes.

Feline panleukopenia virus (FPLV) was shown to induce apoptosis to feline lymphoid cells and to reduce the expression of interleukin-2 receptor alpha on the cells. FPLV-induced apoptosis might be a key element in the pathophysiology of atrophy of lymphoid tissues associated with feline panleukopenia caused by FPLV.

Immunohistochemical study on type II collagen-induced arthritis in DBA/1J mice.

We performed immunohistochemical examinations on type II collagen-induced arthritis (CIA) mice, focusing attention on the changes in distribution of plasma proteins and extracellular matrix materials (ECM) and in expression of adhesion molecules. The limb joints of male DBA/1J mice immunized with bovine type II collagen were obtained at 6 to 20 weeks after the first immunization. In the early stage of CIA, deposition of fibrin, IgG, von Willebrand factor (vWF) and fibronectin was detected on the surface of the synovial lining layer and articular cartilage and in the articular cavity. In the stage of pannus formation, prominent proliferation of ICAM-1-positive capillaries and marked infiltration of LFA-1-positive neutrophils were observed in the pannus. The superficial portion of the pannus and basement membranes of proliferated capillaries were strongly positive for type IV collagen and laminin. In the late stage, the pannus invaded and destroyed articular cartilage and subchondral bone, and strongly positive immunostainabilities for both lysozyme and fibronectin were observed on the surface of the pannus and at the junctional portion between the pannus and the cartilage. The present immunohistochemical findings on the distribution of plasma proteins and ECM materials and the expression of adhesion molecules in CIA mice were similar to those in rheumatoid arthritis (RA) in many aspects. This suggests that CIA is a useful model for the investigation of RA.

Process of the development of T-2 toxin-induced apoptosis in the lymphoid organs of mice.

Female ICR:CD-1 mice orally treated with 10 mg/kg b.w. of T-2 toxin were killed at 1, 3, 6, 9, 12, 24 and 48 hr after treatment (HAT) and subjected to examination of the process of the development of T-2 toxin-induced apoptosis in the thymus and spleen. The early ultrastructural changes in lymphocytes characterized by shrinkage of the cell body and condensation of nuclear chromatin were detected at 3HAT in the thymus. The number of apoptotic lymphocytes observed by the in situ detection method for fragmented DNA increased drastically from 9 to 24 HAT in the thymus while it began to increase at 12 HAT in the spleen. The DNA ladder was first detected by agarose gel electrophoresis at 9 HAT and became clearer at 12 and 24 HAT in the thymus but was not clearly detected in the spleen throughout the observation period. Thus T-2 toxin-induced apoptosis developed earlier and was apparently severer in the thymus than in the spleen. Apoptotic was first detected by electron microscopy, then by the in situ detection method for fragmented DNA, and finally by DNA agarose gel electrophoresis.

T-2 toxin-induced apoptosis in intestinal crypt epithelial cells of mice.

The characteristics of T-2 toxin-induced cell damage in the intestinal crypt epithelia was investigated in mice. Following T-2 toxin-inoculation (0, 2.5, 5 and 10 mg/kg b.w.), dead cells showing pyknosis were sporadically observed in the crypt epithelia, and the nuclei of these cells were strongly stained by the modified TUNEL method which detects fragmented DNA in situ. Electron microscopically, the dead cells were characterized by shrinkage of the cell body and condensation of nuclear chromatin frequently along the nuclear membrane, and such nuclei were sometimes fragmented into small pieces. These morphological characteristics are well consistent with those of apoptosis. The mitotic index in the crypt epithelia drastically decreased at 6 hours after T-2 toxin-inoculation (6 HAI), but thereafter it recovered to almost the same value with that in control mice at 48 HAI. On the other hand, the apoptotic index in the crypt epithelia increased with the lapse of time. Clear mouse strain- and sex-differences were detected in the apoptotic index but not in the mitotic index. This is the first report that T-2 toxin caused apoptotic cell death in the intestinal crypt epithelial cells.

T-2 toxin-induced apoptosis in lymphoid organs of mice.

Lymphoid organs of male and female mice of 4 strains (ICR: CD-1, BALB/c, C57BL/6 and DBA/2) were histologically and biochemically examined at 24 hours after oral inoculation of T-2 toxin (0, 2.5, 5 and 10 mg/kg b.w.). Light microscopically, dose-dependent decrease in number of lymphocytes was observed in the thymic cortex and splenic follicles. The nuclei of lymphocytes showed pyknosis or karyorrhexis, and they were positively stained by the modified TUNEL method which detects fragmented DNA in situ. Electron microscopic characteristics of damaged lymphocytes were shrinkage of the cell body, nuclear chromatin condensation and fragmentation. Agarose gel electrophoresis of DNA extracted from the thymus showed DNA fragmentation into nucleosome units, i.e. ladder formation. The above-mentioned findings clearly showed that T-2 toxin could induce apoptotic cell death in the lymphoid organs of mice. These changes were more prominent in female BALB/c and C57BL/6 mice.