Abstract: In our previous study, we found that cycloheximide (CHX) induces hepatocellular necrosis as well as hepatocellular apoptosis. This article evaluates the role of Kupffer cells on cycloheximide-induced hepatic injury using gadolinium chloride (GdCl(3)) for the inhibition of Kupffer cells. One group of rats was treated with CHX (CHX group), and another was treated with GdCl(3) before being treated with the same dose of CHX (GdCl(3)/CHX group). The necrotic change in the GdCl(3)/CHX group was exacerbated under the induction of hepatocellular apoptosis by the CHX treatment. A substantial diminution of the number of ED1- or ED2-positive cells was demonstrated in the GdCl(3)/CHX group compared to the CHX group. In addition, the degree of decrease in ED2-positive cells was more apparent than that in ED1-positive cells. Increases in the mRNA levels of IL-10 and Stat3 were observed in the CHX group, but not in the GdCl(3)/CHX group. On the other hand, the hepatic mRNA levels of chemokines and adhesion molecules such as Ccl20, LOX-1, and E-selectin were significantly increased only in the GdCl(3)/CHX group. Thus, Kupffer cell inactivation by the GdCl(3) treatment leads to a loss of the capacity to produce IL-10, supposedly resulting in the enhancement of pro-inflammatory cytokine activities such as tumor necrosis factor (TNF) signaling. These events are suggested to be a factor of the inflammatory exacerbation in the livers of the GdCl(3)/CHX group. In conclusion, Kupffer cells may play a role in protecting hepatic necroinflammatory changes by releasing anti-inflammatory cytokines following the hepatocellular apoptosis resulting from CHX treatment.

Abstract: Male F344 rats were intravenously treated with 6 mg/kg cycloheximide (CHX), and microarray analysis was conducted on their livers 1, 2 and 6h after the CHX treatment. The histopathological examination and serum chemistry results indicated a mild hepatic cell death 2 and 6h after the CHX treatment, respectively. Multi-focal hepatocellular necrosis with slight neutrophil infiltration was observed 6h after the CHX treatment. The TUNEL staining results showed that the number of apoptotic hepatocytes was the highest 2h after the CHX treatment. Dramatic increases in the mRNA levels of ATF3 and CHOP genes, both of which were reported to play roles in the ER stress-mediated apoptosis pathway, were observed from 1h after the CHX treatment. In addition, increase of GADD45, p21 and p53 mRNA levels also suggested a time course-related stimulation of hepatocellular apoptotic signals. These results suggest that the hepatocyte apoptosis induced by the CHX treatment is triggered by ER stress. The hepatic mRNA levels of proinflammatory genes, such as TNFalpha, IL-1alpha and beta, were also increased 1 and 2h after the CHX treatment, supposedly mediated by the activated Kupffer cells engulfing the apoptotic hepatocytes.

Abstract: C/EBP homologous protein (CHOP) is a transcriptional factor and is induced under conditions such as the unfolded protein response or amino acid starvation. A previous study showed that the transcriptional level of CHOP was highly increased in rat liver in which hepatocellular apoptosis was induced by cycloheximide (CHX) treatment. Here, we investigated the relationship between hepatocellular apoptosis and CHOP-mediated apoptotic pathway, and studied the mechanisms of induction of CHOP gene in the liver of rats treated with CHX. Male F344 rats were treated intravenously with 6mg/kg CHX, and sacrificed at 1, 2 and 6h after the treatment. In the gene expression assay using quantitative RT-PCR, the genes related to CHOP-mediated apoptosis such as the C/EBPbeta, ATF3 and ATF4 genes were significantly increased corresponding to the induction of hepatocellular apoptosis in rats treated with CHX. However the GRP78/Bip gene, which serves as a representative marker for the unfolded protein response, did not change after the treatment. Toxicoproteomics using two-dimensional difference gel electrophoresis and mass spectrometry indicated that GRP78/Bip was inactivated by the CHX treatment. Furthermore, the CHX-treated animals exhibited a significant decrease of phosphorylated Akt/PKB (protein kinase B). These results indicate that the protein synthesis inhibition by CHX induces the CHOP gene through a pathway similar to that of amino acid starvation, and that Akt/PKB inactivation enhances the CHOP-mediated hepatocellular apoptosis.

Abstract: The relationship between the canine distemper virus (CDV) infection and apoptosis in the canine lymphoid tissues was investigated using immunostaining for single stranded DNA (ssDNA), TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method, and electron microscopy. Twenty-six lymphoid tissues from 8 spontaneously CDV-infected dogs and 1 non-infected dog were used, and lesions were classified into 4 groups according to frequency of the CDV-antigen. Histologically, the degree of lymphoid depletion tended to depend on amount of CDV antigen. The numbers of ssDNA- and TUNEL-labeling cells were significantly high in the lymphoid tissues with abundant viral antigen. However, ssDNA- and TUNEL-positive lymphocytes were also frequently found even in the lymphoid tissues where there was only a small amount of CDV-antigen in sinus histiocytes. The incidence and distribution of apoptotic cells in the CDV-antigens-negative lymphoid tissues from infected dogs were equal to those from a non-infected dog. Double labeling immunostaining using a ssDNA and a CDV nucleocapsid protein (CDV-NP) antibody revealed that there were ssDNA positive but CDV-NP negative cells besides those stained doubly positive. Ultrastructurally, lymphocytes in the CDV-infected lymphoid tissues frequently had characteristic morphological features of apoptosis such as apoptotic bodies. All these results suggest that CDV leads to lymphocytic apoptosis directly or indirectly, resulting in severe lymphoid depletion and immunosuppression in acute or subacute phase of CDV infection.

Abstract: Three canine gastrointestinal stromal tumors (GISTs) were examined. Histopathologically, the tumor mass in the jejunum (Case 1) consisted of the proliferation of epithelioid cells with abundant eosinophilic or vacuolated cytoplasm. Gangliocyte-like or multinucleated giant cells were scattered. The tumor cells exhibited neural natures mimicking human gastrointestinal autonomic nerve tumors, which were immunopositive for several neuronal markers. Another jejunal mass (Case 2) was composed by a solid proliferation of spindle-shaped cells, arranging in interlacing fascicles and occasional storiform pattern. The tumor seemed to be classified undifferentiated GISTs, that showed no apparent neural or muscular features by ultrastructural and immunohistochemical examinations. In the pyloric mass (Case 3), the spindle cells having eosinophilic processes and elongated nuclei were arranged in sheets. Immunohistochemically, the tumor cells showed muscular natures as regards alpha smooth muscle actin and desmin expression.