Simultaneous hypersensitivity reactions to trimethoprim-sulfamethoxazole and amoxicillin-clavulanate in a dog.

Antibiotics are known to be able to cause hypersensitivity reactions through various mechanisms. We present a case of drug-induced immune thrombocytopenia (DITP) and anaphylactic shock occurring simultaneously in a dog after the administration of two classes of antibiotics, namely trimethoprim-sulfamethoxazole (TMP-SMX) and amoxicillin-clavulanate (AMC). The patient recovered completely from DITP on discontinuation of TMP-SMX and the anaphylactic shock caused by AMC was treated with intensive care. DITP is a rare adverse drug reaction (ADR), and anaphylactic shock is a life-threatening ADR. This is the first case report of a dog manifesting two types of hypersensitivity reactions caused by two antibiotics.

IL-31RA and TRPV1 Expression in Atopic Dermatitis Induced with Trinitrochlorobenzene in Nc/Nga Mice.

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Interleukin 31 (IL-31), a novel cytokine in AD, causes pruritus, typically characteristic of AD patients. The transient receptor potential vanilloid type 1 (TRPV1) is a cation channel activated by diverse noxious stimuli that has been studied in a variety of pruritic skin diseases. In this study, the AD animal model was generated by administering the hapten, trinitrochlorobenzene (TNCB), to Nc/Nga mice, and the degree of expression of the IL-31 receptor alpha (IL-31RA) and TRPV1 in the skin of these atopic models was evaluated. The Nc/Nga mice were divided into 3 groups: control, TNCB 2-weeks treated, and TNCB 8-weeks treated. After inducing AD, the skin lesions in each group were scored and compared, and the histology of the skin lesions and the IL-31RA and TRPV1 expression for each group were evaluated by analyzing immunohistochemistry. The results show a significant difference in the skin lesion scores between the groups. The immunohistochemistry evaluation highlighted the remarkable expression of IL-31RA and TRPV1 in the nerve fibers of the TNCB 8-weeks-treated group. We thus confirmed that the long-term application of TNCB induced chronic atopic-like dermatitis and that IL-31RA and TRPV1 were overexpressed in the peripheral nerve fibers in this AD model.

Establishing veterinary graduation competencies and its impact on veterinary medical education in Korea.

Competencies are defined as an observable and assessable set of knowledge, skills, and attitudes. Graduation competencies, which are more comprehensive, refer to the required abilities of students to perform on-site work immediately after graduation. As graduation competencies set the goal of education, various countries and institutions have introduced them for new veterinary graduates. The Korean Association of Veterinary Medical Colleges has recently established such competencies to standardize veterinary education and enhance quality levels thereof. The purpose of this study is to describe the process of establishing graduation competencies as well as their implication for veterinary education in Korea. Graduation competencies for veterinary education in Korea comprise 5 domains (animal health care and disease management, one health expertise, communication and collaboration, research and learning, and veterinary professionalism). These are further divided into 11 core competencies, and 33 achievement standards, which were carefully chosen from previous case analyses and nation-wide surveys. Currently, graduation competencies are used as a standard for setting clear educational purposes for both instructors and students. Establishing these competencies further initiated the development of detailed learning outcomes, and of a list of basic veterinary clinical performances and skills, which is useful for assessing knowledge and skills. The establishment of graduation competencies is expected to contribute to the continuous development of Korean veterinary education in many ways. These include curriculum standardization and licensing examination reform, which will eventually improve the competencies of new veterinary graduates.

Serum Concentration of Inflammatory Cytokines in Dogs with Suspected Acute Pancreatitis.

Acute pancreatitis is an acute inflammatory process in the pancreas that is common in dogs. This study was designed to compare cytokines between healthy dogs and dogs with suspected acute pancreatitis. For the canine cytokine antibody array, three healthy dogs and three dogs with suspected acute pancreatitis were included. Interleukin (IL)-2, IL-6, IL-10, GM-CSF, and TNF-α were not detected in either group based on the results. Conversely, IL-8 (p = 0.035), Monocyte Chemoattractant Protein-1 (MCP)-1 (p = 0.0138), Receptor for Advanced Glycation Endproducts (RAGE) (p = 0.0079), and stem cell factor (SCF) (p = 0.034) were significantly increased in dogs with suspected acute pancreatitis. However, vascular endothelial growth factor (VEGF) (p = 0.6971) did not differ significantly between groups. For the canine serum Enzyme-Linked Immunosorbent Assay (ELISA), eight healthy dogs and eight dogs with suspected acute pancreatitis were included. ELISA revealed that IL-8 (p < 0.0001), MCP-1 (p < 0.0001), RAGE (p = 0.006), and SCF (p = 0.0002) were all significantly upregulated in the experimental group. We confirmed multiple patterns of cytokines in suspected acute pancreatitis of dogs via canine cytokine antibody array using a small quantity of serum. After this procedure, we reevaluated the cytokines, which were significantly increased in dogs with suspected acute pancreatitis, by ELISA, with more samples. Through this study, we confirmed that MCP-1, RAGE, and SCF were newly suggested factors in dogs with suspected acute pancreatitis.

Anti-Inflammatory and Immune Modulatory Effects of Synbio-Glucan in an Atopic Dermatitis Mouse Model.

Many trials have been conducted to treat atopic dermatitis (AD), but these therapies are generally unsuccessful because of their insufficiency or side effects. This study examined the efficacy of ß-glucan derived from oats with fermented probiotics (called Synbio-glucan) on an AD-induced mouse model. For the experiment, Nc/Nga mice were exposed to a house dust mite extract (HDM) to induce AD. The mice were placed in one of four groups: positive control group, Synbio-glucan topical treatment group, Synbio-glucan dietary treatment group, and Synbio-glucan topical + dietary treatment group. The experiment revealed no significant difference in the serum IgE concentration among the groups. Serum cytokine antibody arrays showed that genes related to the immune response were enriched. A significant difference in the skin lesion scores was observed between the groups. Compared to the control group tissue, skin lesions were alleviated in the Synbio-glucan topical treatment group and Synbio-glucan dietary treatment group. Interestingly, almost normal structures were observed within the skin lesions in the Synbio-glucan topical + dietary treatment group. Overall, the ß-glucan extracted from oats and fermented probiotic mixture is effective in treating atopic dermatitis.

Entacapone promotes hippocampal neurogenesis in mice.

Entacapone, a catechol-O-methyltransferase inhibitor, can strengthen the therapeutic effects of levodopa on the treatment of Parkinson's disease. However, few studies are reported on whether entacapone can affect hippocampal neurogenesis in mice. To investigate the effects of entacapone, a modulator of dopamine, on proliferating cells and immature neurons in the mouse hippocampal dentate gyrus, 60 mice (7 weeks old) were randomly divided into a vehicle-treated group and the groups treated with 10, 50, or 200 mg/kg entacapone. The results showed that 50 and 200 mg/kg entacapone increased the exploration time for novel object recognition. Immunohistochemical staining results revealed that after entacapone treatment, the numbers of Ki67-positive proliferating cells, doublecortin-positive immature neurons, and phosphorylated cAMP response element-binding protein (pCREB)-positive cells were significantly increased. Western blot analysis results revealed that treatment with tyrosine kinase receptor B (TrkB) receptor antagonist significantly decreased the exploration time for novel object recognition and inhibited the expression of phosphorylated TrkB and brain-derived neurotrophic factor (BDNF). Entacapone treatment antagonized the effects of TrkB receptor antagonist. These results suggest that entacapone treatment promoted hippocampal neurogenesis and improved memory function through activating the BDNF-TrkB-pCREB pathway. This study was approved by the Institutional Animal Care and Use Committee of Seoul National University (approval No. SNU-130730-1) on February 24, 2014.

Entacapone Treatment Modulates Hippocampal Proteins Related to Synaptic Vehicle Trafficking.

Entacapone, a reversible inhibitor of catechol-O-methyl transferase, is used for patients in Parkinson's disease because it increases the bioavailability and effectiveness of levodopa. In the present study, we observed that entacapone increases novel object recognition and neuroblasts in the hippocampus. In the present study, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were performed to compare the abundance profiles of proteins expressed in the hippocampus after entacapone treatment in mice. Results of 2-DE, MALDI-TOF mass spectrometry, and subsequent proteomic analysis revealed an altered protein expression profile in the hippocampus after entacapone treatment. Based on proteomic analysis, 556 spots were paired during the image analysis of 2-DE gels and 76 proteins were significantly changed more than two-fold among identified proteins. Proteomic analysis indicated that treatment with entacapone induced expressional changes in proteins involved in synaptic transmission, cellular processes, cellular signaling, the regulation of cytoskeletal structure, energy metabolism, and various subcellular enzymatic reactions. In particular, entacapone significantly increased proteins related to synaptic trafficking and plasticity, such as dynamin 1, synapsin I, and Munc18-1. Immunohistochemical staining showed the localization of the proteins, and western blot confirmed the significant increases in dynamin I (203.5% of control) in the hippocampus as well as synapsin I (254.0% of control) and Munc18-1 (167.1% of control) in the synaptic vesicle fraction of hippocampus after entacapone treatment. These results suggest that entacapone can enhance hippocampal synaptic trafficking and plasticity against various neurological diseases related to hippocampal dysfunction.

Two-weeks repeated-dose oral toxicity study of Pediococcus acidilactici J9 in a mice model.

BACKGROUND: Helicobacter pylori (H. pylori) is an important pathogen that causes chronic gastritis and peptic ulcer, and is related to the development of gastric carcinoma. Several chemicals, including antibiotics, have been used to eradicate H.pylori. However, more studies are yet requred to accomplish a sufficient therapy. Pediococcus acidilactici (P. acidilactici) J9 were studied for inhibition of binding of H.pylori binding to human gastric cell lines. This study was performed in order to investigate the repeated-dose toxicity of P. acidilactici J9 in male and female mice. RESULTS: C57BL/6 male and female Mus musculus were divided into four groups (n = 10 in each group). P. acidilactici J9 was administered daily by oral injection of vehicle control at dosage levels to a low-dose group (500 mg/kg/day), middle-dose group (1000 mg/kg/day), and high-dose group (2000 mg/kg/day) for 2 weeks. After 14 days of exposure, the blood biochemistry and hematology were investigated, along with a histopathology exam. There were no bacterial-related deaths or abnormal clinical signs in either gender of mouse. The data was observed during the period in terms of body weight, food intake, and water consumption. Also, no alterations in organ weights upon administration of P. acidilactici J9 alone were observed. The adhesion and growth of H. pylori were inhibited by a 24 h treatment of H. pylori and P. acidilactici J9 on adenocarcinoma gastric (AGS) cells, which are gastric cancer cells. Compared to the control group (AGS cell and H. pylori), the number of H. pylori analyzed by FACS significantly (p < 0.01) decreased after incubation of AGS cell with P. acidilactici J9 for 24 h. CONCLUSIONS: These results suggest that the oral application of P. acidilactici J9, up to a dosage level of 2000 mg/kg/day, causes no adverse effects in both male and female mice. P. acidilactici J9 inhibits the adhesion of H.pylori to AGS cancer cells. When used as probiotics, P. acidilactici J9 may help decrease the occurrence of gastritis and reduce the risk of H.pylori infection with promising safety issues.

Pyridoxine Deficiency Exacerbates Neuronal Damage after Ischemia by Increasing Oxidative Stress and Reduces Proliferating Cells and Neuroblasts in the Gerbil Hippocampus.

We investigated the effects of pyridoxine deficiency on ischemic neuronal death in the hippocampus of gerbil (n = 5 per group). Serum pyridoxal 5'-phosphate levels were significantly decreased in Pyridoxine-deficient diet (PDD)-fed gerbils, while homocysteine levels were significantly increased in sham- and ischemia-operated gerbils. PDD-fed gerbil showed a reduction in neuronal nuclei (NeuN)-immunoreactive neurons in the medial part of the hippocampal CA1 region three days after. Reactive astrocytosis and microgliosis were found in PDD-fed gerbils, and transient ischemia caused the aggregation of activated microglia in the stratum pyramidale three days after ischemia. Lipid peroxidation was prominently increased in the hippocampus and was significantly higher in PDD-fed gerbils than in Control diet (CD)-fed gerbils after ischemia. In contrast, pyridoxine deficiency decreased the proliferating cells and neuroblasts in the dentate gyrus in sham- and ischemia-operated gerbils. Nuclear factor erythroid-2-related factor 2 (Nrf2) and brain-derived neurotrophic factor (BDNF) levels also significantly decreased in PDD-fed gerbils sham 24 h after ischemia. These results suggest that pyridoxine deficiency accelerates neuronal death by increasing serum homocysteine levels and lipid peroxidation, and by decreasing Nrf2 levels in the hippocampus. Additionally, it reduces the regenerated potentials in hippocampus by decreasing BDNF levels. Collectively, pyridoxine is an essential element in modulating cell death and hippocampal neurogenesis after ischemia.

Comparison of the presentation of atopic dermatitis induced by trinitrochlorobenzene and house dust mite in NC/Nga mice.

BACKGROUND: Atopic dermatitis (AD) is a common chronic inflammatory skin disease. To understand AD, there have been many trials establishing AD animal models. Although various trials to establish AD animal models have been existed, even the mechanisms of AD in animal models are not enough clarified. OBJECTIVES: This study assessed AD characteristics induced in Nishiki-nezumi Cinnamon/Nagoya (Nc/Nga) mice following trinitrochlorobenzene (TNCB) treatment for different periods and house dust mite (HDM) treatment to compare each model's immunological patterns, especially with cytokine antibody array tool. METHODS: In this study, we exposed Nc/Nga mice to TNCB or HDM extract to induce AD. Nc/Nga mice were divided into 4 groups: control, TNCB 2 weeks-treated, TNCB 8 weeks-treated, and HDM-treated groups. After AD induction, all mice were evaluated by serum immunoglobulin E (IgE) concentration and serum cytokine antibody assays, scoring of skin lesions, scoring of scratching frequency, and histological analysis. RESULTS: The results showed significant differences between groups in serum IgE concentration, skin lesion scores, and scratching frequency. The analysis results for serum cytokine antibody arrays showed that in the TNCB 8 weeks- and HDM-treated groups, but not in the TNCB 2 weeks-treated group, expressions of genes related to the immune response were enriched. Among the histological results, the skin lesions in the HDM-treated group were most similar to those of AD. CONCLUSIONS: We confirmed that immunological pattern of AD mice was markedly different between HDM and TNCB treated groups. In addition, the immunological pattern was quietly different dependent on TNCB treated duration.

Effects of Pyridoxine Deficiency on Hippocampal Function and Its Possible Association with V-Type Proton ATPase Subunit B2 and Heat Shock Cognate Protein 70.

Pyridoxine, one of the vitamin B6 vitamers, plays a crucial role in amino acid metabolism and synthesis of monoamines as a cofactor. In the present study, we observed the effects of pyridoxine deficiency on novel object recognition memory. In addition, we examined the levels of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenethylamine (DA), 3,4-dihydroxyphenylacetic acid, and homovanillic acid and the number of proliferating cells and neuroblasts in the hippocampus. We also examined the effects of pyridoxine deficiency on protein profiles applying a proteomic study. Five-week-old mice fed pyridoxine-deficient diets for 8 weeks and showed a significant decrease in the serum and brain (cerebral cortex, hippocampus, and thalamus) levels of pyridoxal 5'-phosphate, a catalytically active form of vitamin-B6, and decline in 5-HT and DA levels in the hippocampus compared to controls fed a normal chow. In addition, pyridoxine deficiency significantly decreased Ki67-positive proliferating cells and differentiated neuroblasts in the dentate gyrus compared to controls. A proteomic study demonstrated that a total of 41 spots were increased or decreased more than two-fold. Among the detected proteins, V-type proton ATPase subunit B2 (ATP6V1B2) and heat shock cognate protein 70 (HSC70) showed coverage and matching peptide scores. Validation by Western blot analysis showed that ATP6V1B2 and HSC70 levels were significantly decreased and increased, respectively, in pyridoxine-deficient mice compared to controls. These results suggest that pyridoxine is an important element of novel object recognition memory, monoamine levels, and hippocampal neurogenesis. Pyridoxine deficiency causes cognitive impairments and reduction in 5-HT and DA levels, which may be associated with a reduction of ATP6V1B2 and elevation of HSC70 levels in the hippocampus.

Neuropathological changes in dorsal root ganglia induced by pyridoxine in dogs.

BACKGROUND: Pyridoxine (PDX; vitamin B6), is an essential vitamin. PDX deficiency induces various symptoms, and when PDX is misused it acts as a neurotoxicant, inducing severe sensory neuropathy. RESULTS: To assess the possibility of creating a reversible sensory neuropathy model using dogs, 150 mg/kg of PDX was injected subcutaneously into dogs for 7 days and body weight measurements, postural reaction assessments, and electrophysiological recordings were obtained. In addition, the morphology of dorsal root ganglia (DRG) and changes in glial fibrillary acidic protein (GFAP) immunoreactive satellite glial cells and ionized calcium-binding adapter molecule 1 (Iba-1) immunoreactive microglia/macrophages were assessed at 1 day, 1 week, and 4 weeks after the last PDX treatment. During the administration period, body weight and proprioceptive losses occurred. One day after the last PDX treatment, electrophysiological recordings showed the absence of the H-reflex in the treated dogs. These phenomena persisted over the four post-treatment weeks, with the exception of body weight which recovered to the pre-treatment level. Staining (CV and HE) results revealed significant losses of large-sized neurons in the DRG at 1 day and 1 week after PDX treatment cessation, but the losses were recovered at 4 weeks post-treatment. The Iba-1 and GFAP immunohistochemistry results showed pronounced increases in reactive microglia/macrophage and satellite glial cell at 1 day and 1 week, respectively, after the last PDX treatment, and thereafter, immunoreactivity decreased with increasing time after PDX treatment. CONCLUSIONS: The results suggest that PDX-induced neuropathy is reversible in dogs; thus, dogs can be considered a good experimental model for research on neuropathy.

Beta-nerve growth factor gene therapy alleviates pyridoxine-induced neuropathic damage by increasing doublecortin and tyrosine kinase A in the dorsal root ganglion.

Beta-nerve growth factor (ß-NGF) is known to be a major leading cause of neuronal plasticity. To identify the possible action mechanisms of ß-NGF gene therapy for sciatic nerve recovery, experimental dogs were randomly divided into control, pyridoxine, and pyridoxine + ß-NGF groups. We observed chronological changes of morphology in the dorsal root ganglia in response to pyridoxine toxicity based on cresyl violet staining. The number of large neurons positive for cresyl violet was dramatically decreased after pyridoxine intoxication for 7 days in the dorsal root ganglia and the neuron number was gradually increased after pyridoxine withdrawal. In addition, we also investigated the effects of ß-NGF gene therapy on neuronal plasticity in pyridoxine-induced neuropathic dogs. To accomplish this, tyrosine kinase receptor A (TrkA), ßIII-tubulin and doublecortin (DCX) immunohistochemical staining was performed at 3 days after the last pyridoxine treatment. TrkA-immunoreactive neurons were dramatically decreased in the pyridoxine group compared to the control group, but strong TrkA immunoreactivity was observed in the small-sized dorsal root ganglia in this group. TrkA immunoreactivity in the dorsal root ganglia was similar between ß-NGF and control groups. The numbers of ßIII-tubulin- and DCX-immunoreactive cells decreased significantly in the pyridoxine group compared to the control group. However, the reduction of ßIII-tubulin- and DCX-immunoreactive cells in the dorsal root ganglia in the ß-NGF group was significantly ameliorated than that in the pyridoxine group. These results indicate that ß-NGF gene therapy is a powerful treatment of pyridoxine-induced neuropathic damage by increasing the TrkA and DCX levels in the dorsal root ganglia. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University, South Korea (approval No. SNU-060623-1, SNU-091009-1) on June 23, 2006 and October 9, 2009, respectively.

Transient Fanconi Syndrome After Treatment with Firocoxib, Cefadroxil, Tramadol, and Famotidine in a Maltese.

Fanconi syndrome is a renal proximal tubulopathy characterized by excessive urinary loss of glucose, amino acids, several electrolytes, and bicarbonate. Here, we report the case of transient Fanconi syndrome in a dog following administration of firocoxib, cefadroxil, tramadol, and famotidine. A 10 mo old Maltese was presented with lethargy, anorexia, vomiting, and weight loss. Transient Fanconi syndrome without azotemia was associated with firocoxib, cefadroxil, tramadol, and famotidine treatment. The dog received supportive care including IV fluids, gastroprotectants, and oral nutritional supplements. Two months after initial diagnosis and treatment, the dog showed complete resolution of glucosuria and aminoaciduria. The unique features of Fanconi syndrome in this case emphasize the potential renal tubular toxicity of this widely used multiple-drug combination.

Effect of a poloxamer-based thermosensitive gel on rotator cuff repair in a rabbit model: a controlled laboratory study.

BACKGROUND: A common complication after rotator cuff repair is postoperative stiffness, which can be reduced by a simple application of an anti-adhesive agent. However, anti-adhesive agents may affect rotator cuff healing by preventing fibrosis. This experimental animal study evaluated the effect of the application of a poloxamer-based thermosensitive anti-adhesive gel and its influence on the healing of an acute rotator cuff repair in a rabbit model. METHODS: Acute rotator cuff repair (supraspinatus tendon) was performed using a transosseous suturing method. One shoulder on a randomly selected side was treated with a local application of the anti-adhesive agent (applied side), and saline was applied to the contralateral side (control side). Biomechanical testing and histological analyses were performed at 4 and 8 weeks postoperatively. Eight rabbits were included for each testing and time point, for a total of 32 rabbits. RESULTS: The failure load at 4 weeks was lower in the experimental group (95.2 ± 19.6 N vs. 110.0 ± 20.5 N; P = 0.017). Conversely, at 8 weeks, the failure load was higher in the experimental group (148.3 ± 16.2 N) than in the control group (122.4 ± 16.9 N; P = 0.002). Histological analyses revealed no statistically significant differences in the tendon maturing scores at 4 and 8 weeks between the two groups (all P > 0.05). The thickness of the fibrosis between the rotator cuff tendon and deltoid was thinner in the experimental group at both time points (0.50 ± 0.25 vs. 1.27 ± 0.47; P = 0.002 at 4 weeks, and 0.37 ± 0.35 vs. 1.39 ± 0.50; P = 0.003 at 8 weeks). CONCLUSIONS: Application of an anti-adhesive agent in this rotator cuff model confirmed the agent's effectiveness at reducing fibrosis in the subacromial space. The healing of the tendon showed interesting results, as the experimental group had poorer biomechanical strength at 4 weeks but superior strength at 8 weeks.

Decrease in glucose transporter 1 levels and translocation of glucose transporter 3 in the dentate gyrus of C57BL/6 mice and gerbils with aging.

In the present study, we compared the cell-specific expression and changes protein levels in the glucose transporters (GLUTs) 1 and 3, the major GLUTs in the mouse and gerbil brains using immunohistochemistry and Western blot analysis. In both mouse and gerbils, GLUT1 immunoreactivity was mainly found in the blood vessels in the dentate gyrus, while GLUT3 immunoreactivity was detected in the subgranular zone and the molecular layer of the dentate gyrus. GLUT1-immunoreactivity in blood vessels and GLUT1 protein levels were significantly decreased with age in the mice and gerbils, respectively. In addition, few GLUT3-immunoreactive cells were found in the subgranular zone in aged mice and gerbils, but GLUT3-immunoreactivity was abundantly found in the polymorphic layer of dentate gyrus in mice and gerbils with a dot-like pattern. Based on the double immunofluorescence study, GLUT3-immunoreactive structures in gerbils were localized in the glial fibrillary acidic protein-immunoreactive astrocytes in the dentate gyrus. Western blot analysis showed that GLUT3 expression in the hippocampal homogenates was slightly, although not significantly, decreased with age in mice and gerbils, respectively. These results indicate that the reduction in GLUT1 in the blood vessels of dentate gyrus and GLUT3 in the subgranular zone of dentate gyrus may be associated with the decrease in uptake of glucose into brain and neuroblasts in the dentate gyrus. In addition, the expression of GLUT3 in the astrocytes in polymorphic layer of dentate gyrus may be associated with metabolic changes in glucose in aged hippocampus.

Amelioration of Huntington's disease phenotypes by Beta-Lapachone is associated with increases in Sirt1 expression, CREB phosphorylation and PGC-1α deacetylation.

Huntington's disease (HD) is one of the most devastating genetic neurodegenerative disorders with no effective medical therapy. ß-Lapachone (ßL) is a natural compound obtained from the bark of the Lapacho tree and has been reported to have beneficial effects on various diseases. Sirt1 is a deacetylase of the sirtuin family and deacetylates proteins including the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which is associated with mitochondrial respiration and biogenesis. To examine the effectiveness of ßL on HD, ßL was orally applied to R6/2 HD mice and behavioral phenotypes associated with HD, such as impairment of rota-rod performance and increase of clasping behavior, as well as changes of Sirt1 expression, CREB phosphorylation and PGC-1α deacetylation were examined. Western blot results showed that Sirt1 and p-CREB levels were significantly increased in the brains of ßL-treated R6/2 mice. An increase in deacetylation of PGC-1α, which is thought to increase its activity, was observed by oral administration of ßL. In an in vitro HD model, ßL treatment resulted in an attenuation of MitoSOX red fluorescence intensity, indicating an amelioration of mitochondrial reactive oxygen species by ßL. Furthermore, improvements in the rota-rod performance and clasping score were observed in R6/2 HD mice after oral administration of ßL compared to that of vehicle control-treated mice. Taken together, our data show that ßL is a potential therapeutic candidate for the treatment of HD-associated phenotypes, and increases in Sirt1 level, CREB phosphorylation and PGC-103B1 deacetylation can be the possible underlying mechanism of the effects of ßL.

Age-dependent changes in vesicular glutamate transporter 1 and 2 expression in the gerbil hippocampus.

Glutamate is a major excitatory neurotransmitter that is stored in vesicles located in the presynaptic terminal. Glutamate is transported into vesicles via the vesicular glutamate transporter (VGLUT). In the present study, the age­associated changes of the major VGLUTs, VGLUT1 and VGLUT2, in the hippocampus were investigated, based on immunohistochemistry and western blot analysis at postnatal month 1 (PM1; adolescent), PM6, PM12 (adult group), PM18 and PM24 (the aged groups). VGLUT1 immunoreactivity was primarily detected in the mossy fibers, Schaffer collaterals and stratum lacunosum­moleculare. By contrast, VGLUT2 immunoreactivity was observed in the granule cell layer and the outer molecular layer of the dentate gyrus, stratum pyramidale, Schaffer collaterals and stratum lacunosum­moleculare in the hippocampal CA1­3 regions. VGLUT1 immunoreactivity and protein levels remained constant across all age groups. However, VGLUT2 immunoreactivity and protein levels decreased in the PM3 group when compared with the PM1 group. VGLUT2 immunoreactivity and protein levels were not altered in the PM12 group; however, they increased in the PM18 group. In addition, in the PM18 group, highly immunoreactive VGLUT2 cells were also identified in the stratum radiatum and oriens of the hippocampal CA1 region. In the PM24 group, VGLUT2 immunoreactivity and protein levels were significantly decreased and were the lowest levels observed amongst the different groups. These results suggested that VGLUT1 may be less susceptible to the aging process; however, the increase of VGLUT2 in the non­pyramidal cells in the PM18 group, and the consequent decrease in VGLUT2, may be closely linked to age­associated memory impairment in the hippocampus.

Hypothyroidism increases cyclooxygenase-2 levels and pro-inflammatory response and decreases cell proliferation and neuroblast differentiation in the hippocampus.

The present study investigated the effects of hypothyroidism on cyclooxygenase-2 (COX-2) and pro­inflammatory cytokines in the dentate gyrus to elucidate the roles of COX­2 in the hypothyroid hippocampus. Hypothyroidism was induced in rats by treating with 0.03% 2­mercapto­1­methyl­imidazole dissolved in drinking water for 5 weeks. The animals were sacrificed at 12 weeks of age. Hypothyroidism rats exhibited decreased triiodothyronine and thyroxine levels in the serum, while the levels of thyroid­stimulating hormone and the weight of thyroid glands were significantly higher in the hypothyroid rats compared with those in the vehicle­treated group. COX­2 immunoreactivity was significantly increased in the hippocampal CA2/3 region and the dentate gyrus compared with the vehicle­treated group. Levels of pro­inflammatory cytokines including interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α were significantly higher in the hippocampal homogenates of hypothyroid rats. Cell proliferation and neuroblast differentiation based on Ki67 and doublecortin immunohistochemistry were decreased in the dentate gyrus of hypothyroid rats compared with those in the vehicle­treated group. These results suggested that hypothyroidism­mediated COX­2 expression affected hippocampal plasticity by upregulating the levels of pro­inflammatory cytokines in the hippocampus. Therefore, COX­2 may be suggested as a candidate molecule for preventing hypothyroidism­induced neurological side effects.

Clinical Implications of Quantitative JAK2 V617F Analysis using Droplet Digital PCR in Myeloproliferative Neoplasms.

BACKGROUND: JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden. METHODS: Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed. RESULTS: Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P<0.001). Follow-up samples were available for 12 patients, including eight that were JAK2 V617F-positive. Of these, mutational burden reduction after treatment was observed in six patients (75%), consistent with trends of hematologic improvement. CONCLUSIONS: Quantitative analysis of the JAK2 V617F mutation using ddPCR was highly correlated with pyrosequencing data and may reflect the clinical response to treatment.