The effect of 5-aminolevulinic acid on canine peripheral blood mononuclear cells.

5-aminolevulinic acid (ALA) is a natural amino acid and a product of the first heme synthesis pathway in mitochondria. Its immunomodulatory effects have garnered recent attention for their potential application to cancer, inflammation, and autoimmune diseases in humans. A supplement containing ALA is now available in Japan to enhance ATP synthesis via mitochondrial activity. However, how ALA affects canine immunity is unclear. Here we studied the effects of ALA on peripheral blood mononuclear cells (PBMCs) from healthy dogs in vitro. Heme oxygenase-1 (HO-1) protein was expressed in Madin-Darby canine kidney (MDCK) cells and PBMCs treated with ALA and ferrous sodium citrate (SFC), which showed that ALA works in dogs as well as humans. ALA also induced concanavalin A (ConA)-stimulated PBMCs to produce significantly more interferon-gamma (IFN-γ). Next-generation RNA sequencing (RNA-seq) revealed that ALA enhanced T cell immunity among Th1, Th2, and Th17 subsets, especially the IL-17 signaling pathway. We then confirmed that ALA promoted interleukin (IL)- 17A production in ConA-stimulated PBMCs. Together, these findings indicate that ALA promotes heme synthesis in mitochondria and enhances ConA-induced T cell immune responses in canine PBMCs.

Improvement Effect of 5-Aminolevulinic Acid on Hyperlipidemia in Miniature Schnauzer Dogs: An Open Study in 5 Cases of One Pedigree.

This is the first study to examine the long-term effect of 5-aminolevulinic acid mainly on serum lipoproteins in dogs with hyperlipidemia. We studied 5 Miniature Schnauzer cases whose fasting serum total triglyceride and very-low-density lipoprotein of triglyceride levels were extremely high (635 ± 116 and 520 ± 92 mg/dL, respectively). Although the total cholesterol values were normal, the very-low-density lipoprotein of cholesterol level was high (49 ± 7 mg/dL). Each dog received a 5-aminolevulinic acid supplement (5 mg/day) orally for 6 months. The mean values of total triglyceride, very-low-density lipoprotein of both triglyceride and cholesterol decreased significantly after the treatment period (319 ± 29, 245 ± 18, and 27 ± 2 mg/dL, respectively, P < 0.05). Our present results may present evidence that 5-ALA administration contributes to improvement of hyperlipidemia in Miniature Schnauzer.

Cytologic, histologic, and immunohistochemical features of maxillofacial alveolar rhabdomyosarcoma in a juvenile dog.

A 15-month-old castrated male dog with a history of intermittent epistaxis and sneezing was admitted for the examination of a maxillofacial mass. An impression smear of a biopsy sample from the cauliflower-shaped gingival mass contained numerous round cells, 5-25 microm in diameter, which contained a moderate amount of clear to pale blue cytoplasm and resembled lymphoid cells. Mitotic figures were frequently observed. The mass was diagnosed as malignant round cell neoplasia. On histologic examination the tumor was composed of diffusely arranged, small, atypical round cells with a small amount of fibrovascular stroma. Immunohistochemically, the cells were negative for CD3, CD18, CD20, CD79alpha, cytokeratin, melan-A, chromogranin A, alpha-smooth muscle actin, and myoglobin but positive for vimentin and desmin. The cells also had strong positive nuclear staining for myogenin and MyoD1. A diagnosis of solid-pattern alveolar rhabdomyosarcoma was made on the basis of morphologic and immunohistochemical results. Alveolar rhabdomyosarcoma should be considered in the differential diagnosis of tumors in juvenile dogs, especially when cytologic findings reveal round, undifferentiated cells.

Assembly and intracellular localization of the bluetongue virus core protein VP3.

The bluetongue virus (BTV) core protein VP3 plays a crucial role in the virion assembly and replication process. Although the structure of the protein is well characterized, much less is known about the intracellular processing and localization of the protein in the infected host cell. In BTV-infected cells, newly synthesized viral core particles accumulate in specific locations within the host cell in structures known as virus inclusion bodies (VIBs), which are composed predominantly of the nonstructural protein NS2. However, core protein location in the absence of VIBs remains unclear. In this study, we examined VP3 location and degradation both in the absence of any other viral protein and in the presence of NS2 or the VP3 natural associate protein, VP7. To enable real-time tracking and processing of VP3 within the host cell, a fully functional enhanced green fluorescent protein (EGFP)-VP3 chimera was synthesized, and distribution of the fusion protein was monitored in different cell types using specific markers and inhibitors. In the absence of other BTV proteins, EGFP-VP3 exhibited distinct cytoplasmic focus formation. Further evidence suggested that EGFP-VP3 was targeted to the proteasome of the host cells but was dispersed throughout the cytoplasm when MG132, a specific proteasome inhibitor, was added. However, the distribution of the chimeric EGFP-VP3 protein was altered dramatically when the protein was expressed in the presence of the BTV core protein VP7, a normal partner of VP3 during BTV assembly. Interaction of EGFP-VP3 and VP7 and subsequent assembly of core-like particles was further examined by visualizing fluorescent particles and was confirmed by biochemical analysis and by electron microscopy. These data indicated the correct assembly of EGFP-VP3 subcores, suggesting that core formation could be monitored in real time. When EGFP-VP3 was expressed in BTV-infected BSR cells, the protein was not associated with proteasomes but instead was distributed within the BTV inclusion bodies, where it colocalized with NS2. These findings expand our knowledge about VP3 localization and its fate within the host cell and illustrate the assembly capability of a VP3 molecule with a large amino-terminal extension. This also opens up the possibility of application as a delivery system.

[Portal vein thrombosis associated with hepatic encephalopathy].

A 54-year-old man who had been administered chlormadinone acetate 3 months after prostatectomy for prostate cancer, acutely developed disorientation and memory disturbance. Six days later, he experienced high grade fever and epigastralgia. He was suspected to have hepatic encephalopathy, because the Fischer ratio was low although serum ammonia level remained normal. Further examinations including abdominal echography and CT scan disclosed a thrombus extending from the portal vein to the superior mesenteric vein together with abnormal collateral vessels originating from the portal vein. He was successfully treated with warfarin potassium, urokinase and heparin sodium. It was suggested that the patient developed hepatic encephalopathy due to portal-systemic circulation shunting secondary to portal vein thrombosis.