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Mast cells are classically thought to play an important role in protection against helminth infections and in the induction of allergic diseases; however, recent studies indicate that these cells also contribute to neovascularization, which is critical for tissue remodeling, chronic inflammation, and carcinogenesis. Here, we demonstrate that mast cells are essential for sprouting angiogenesis in a murine model of oxygen-induced retinopathy (OIR). Although mouse strains lacking mast cells did not exhibit retinal neovascularization following hypoxia, these mice developed OIR following infusion of mast cells or after injection of mast cell tryptase (MCT). Relative hypoxia stimulated mast cell degranulation via transient receptor potential ankyrin 1. Subsequent surges in MCT stimulated retinal endothelial cells to produce monocyte chemotactic protein-1 (MCP1) and angiogenic factors, leading to sprouting angiogenesis. Mast cell stabilizers as well as specific tryptase and MCP1 inhibitors prevented the development of OIR in WT mice. Preterm infants with early retinopathy of prematurity had markedly higher plasma MCT levels than age-matched infants without disease, suggesting mast cells contribute to human disease. Together, these results suggest therapies that suppress mast cell activity should be further explored as a potential option for preventing eye diseases and subsequent blindness induced by neovascularization.

In order to avoid stress in horses, it has to be known to what extent the animals perceive a challenge as stressful. In this study, salivary cortisol, heart rate, and heart rate variability parameters SDRR (standard deviation of the beat-to-beat interval) and RMSSD (root mean square of successive beat-to-beat differences) were determined in Shetland ponies (six stallions, five mares) in response to a flashlight, exercise without a rider, road transport, and a nontreatment control. Saliva was collected from 1 hour before to 24 hours after the tests, and cardiac activity was recorded from 1 hour before to 2 hours after tests. Salivary cortisol concentration increased in response to transport (P

Nerve growth factor (NGF) is a neurotrophic factor that is essential for the maintenance of peripheral and central neurons. While importance of such physiological activities as well as the potential of it as a stress marker in Equus caballus has been implicated, the sequence of equine NGF remained unknown. In this study, we identified the sequence of equine NGF from the mRNA expressed in the peripheral blood in seven Thoroughbreds and three warmblood horses. There were no polymorphisms among samples analyzed, and the homology is more than 90% in comparison to human, mouse, rat, dog, and cow. When the sequence corresponding to the biologically active β-nerve growth factor was expressed in CHO-K1 cells, they were stained with an anti-NGF antibody that recognizes human NGF. Thus, it provides a rationale for using antibodies that react to other species' NGF to measure the equine NGF for further studies.

Background: Abnormality in skin sensitivity may be responsible for unbearable itch in patients with atopic dermatitis (AD). Objectives: We evaluated reactivity of NC/Tnd mice, a model for human AD, against various experimental stimulations. Methods: Several behavioral tests were performed after external stimuli were applied to NC/Tnd mice. Transient receptor potential vanilloid subtype 1 (TRPV1) reactivity of neuronal cells collected from the dorsal root ganglions (DRG) was analyzed with a Ca++ influx test. Finally, we evaluated suppressive effect of capsaicin on atopic itch of NC/Tnd mice. Results: Pain responses to heat, acidic stimulation, and capsaicin injection, which are transduced through TRPV1, were decreased in NC/Tnd mice, when compared to two standard strains BALB/c and C57BL/6 mice. The reactivity of the primary neurons isolated from DRG to capsaicin was markedly reduced in NC/Tnd mice. Topical application of histamine evoked scratching in NC/Tnd mice as well as other two strains; however, the scratching intensities induced by nonhistamine pruritogens were significantly lower in NC/Tnd mice comparing to the two strains. In conventional NC/Tnd mice with AD, topical application of capsaicin reduced the scratching behavior. Conclusion: TRPV1 is associated with both pain and itch sensation; however, abnormalities in TRPV1 reactivity may involve in severe itch in NC/Tnd mice.

Although the mechanism of development of equine laminitis is unclear, involvement of endotoxemia has been proposed. Endotoxemia is one of the described pathways for laminitis but not the only one. Lipopolysaccharide (LPS) promotes the release of vasoconstrictors from platelets and endothelium through the activation of nuclear factor (NF)-κB in these cells. The vasoconstrictors induced by LPS may cause digital hypothermia with vasoconstriction and local ischemia, contributing to the development of laminitis. In the present study, we evaluated beneficial effect of NF-κB inhibition on digital hypothermia induced by LPS in adult horses. To generate a model for the local ischemia below the pastern, LPS was infused to the palmar lateral digital vein. As an index of blood circulation, hoof wall surface temperature (HWST) was measured. LPS infusion reduced HWST time dependently; however, the reduction of HWST was significantly prevented by pretreatment of a NF-κB inhibitor, IMD-0354. Our results clearly demonstrated that the inhibition of NF-κB prevented the digital hypothermia induced by LPS, suggesting that the NF-κB inhibitor might have a therapeutic effect on laminitis associated with endotoxemic conditions in horses.

Chronic inflammation due to the absorption of asbestos is an important cause of mesothelioma. Although the increased prevalence of mesothelioma is a serious problem, the development of effective chemotherapeutic agents remains incomplete. As the nuclear factor-κB (NF-κB) pathway contributes to malignant transformation of various types of cells, we explored NF-κB activity in three different pathological types of malignant mesothelioma cells, and evaluated the therapeutic potential of a recently reported NF-κB inhibitor, IMD-0354. NF-κB was constantly activated in MSTO-211H, NCI-H28, and NCI-H2052 cells, and the proliferation of these cell lines was inhibited by IMD-0354. D-type cyclins were effectively suppressed in mixed tissue type MSTO-211H, leading to cell cycle arrest at sub G1 /G1 phase. IMD-0354 reduced cyclin D3 in both epithelial tissue type NCI-H28 and sarcomatoid tissue type NCI-H2052. In a sphere formation assay, IMD-0354 effectively decreased the number and diameter of MSTO-211H spheres. Preincubation of MSTO-211H cells with IMD-0354 delayed tumor formation in transplanted immunodeficient mice. Furthermore, administration of IMD-0354 markedly rescued the survival rate of mice that received intrathoracic injections of MSTO-211H cells. These results indicate that a targeted drug against NF-κB might have therapeutic efficacy in the treatment of human malignant mesothelioma.

The effects of probiotics on horses are still controversial. The present study was a randomized, double-blinded, placebo-controlled crossover study designed to evaluate the ability of probiotics to improve intestinal conditions in adult horses. Fermented probiotics were administered to 10 healthy adult geldings for 28 days. The clinical condition of the horses was monitored daily, and the blood and feces were biochemically analyzed every 14 days. In the probiotic-treated group, the concentration of carboxylic acids in the feces was increased at days 14 and 28. In contrast to the fecal pH in the control group, which increased at days 14 and 28, the fecal pH in the probiotic-treated group did not increase. Additionally, the relative amounts of enteropathogenic bacterial DNA were diminished in the probiotic-treated group. These results suggest that probiotic bacteria proliferated in the equine intestine. No instances of abnormal clinical conditions or abnormal values in blood tests were observed throughout the study. Oral administration of fermented probiotics may have the ability to improve the intestinal environment biochemically and microbiologically without the risk of adverse effects.

Aims Scedosporium apiospermum sometimes causes serious infectious diseases on the skin of immunodeficient subjects. Antifungal effects of fatty acid salts in soap against S. apiospermum were investigated under different water conditions. Methods and Results Ultrapure soft water (UPSW) was generated by the water softener with cation-exchange resin. The calcium and magnesium ions were replaced with sodium ions in UPSW. Scedosporium apiospermum was incubated with different fatty acid salts that constituted soap in distilled water (DW), tap water (TW) and UPSW. After incubation, the number of fungi was counted. Among the fatty acids, palmitic acid salt (C16) reduced the number of S. apiospermum. UPSW enhanced the antifungal effect of C16 on S. apiospermum. The absence of both calcium and magnesium ions and the existence of sodium chloride in UPSW were responsible for its antifungal effect. In addition, repeated short-term treatment with UPSW and C16 decreased the number of S. apiospermum. Conclusions Antifungal effects of C16 on S. apiospermum were demonstrated. Moreover, the use of UPSW promoted the antifungal effect of C16. Significance and Impact of Study This study provides the preventive method for diseases associated with S. apiospermum infection using novel palmitic acid soap in UPSW.

Arthritis is a disabling health problem and commonly develops in the late stages of life; the condition is typically accompanied by chronic pain. For the assessment of pain severity and therapeutic effects of analgesic drugs, we recently developed a gait analysis system, which provides an index of pain severity based on walking stride disturbance. Using this system, we evaluated the therapeutic effect of topical nonsteroidal anti-inflammatory drugs (NSAIDs) in rat models of acute inflammation. We found that the gait analysis system is more sensitive than conventional evaluation methods, such as measurement of swelling or analgesia, which indicated the superiority of our system for drug screening. The approach also indicated that ketoprofen is superior to other NSAIDs for providing pain relief because of its higher skin permeability. To the best of our knowledge, this is the first report demonstrating the effectiveness of topical NSAIDs in experimental animal models of acute inflammation.

Myeloid cell leukemia sequence 1 (MCL1) is a potent antiapoptotic protein that plays a critical role in cell survival and drug resistance in various cancers. However, to the best of our knowledge, the role of MCL1 in mast cell tumors (MCTs) has not been investigated in dogs. Here, we detected increased MCL1 expression in MCT cell lines, regardless of the presence of a c-kit mutation. MCL1 expression increased when the cells were exposed to specific inhibitors of mitogen-activated protein kinase or Janus kinase-signaling pathways, thus protecting the cells from apoptosis, but not when KIT or phosphatidylinositol-3 kinase signaling cascades were inhibited. These results indicate that MCL1 expression may contribute to MCT survival and confer drug resistance.

Keywords: Dieckol; Ecklonia cava ; mast cell; passive cutaneous anaphylactic reaction

AIMS Skin colonization of Staphylococcus spp. critically affects the severity of dermatitis in humans and animals. We examined different types of fatty acid salts for their antibacterial activity against Staphylococcus spp. when used in ultrapure soft water (UPSW). We also evaluated their therapeutic effect on a spontaneous canine model of dermatitis. METHODS AND RESULTS UPSW, in which Ca(++) and Mg(++) were replaced with Na(+) , was generated using a water softener with cation-exchange resin. Staphylococcus aureus (Staph. aureus), Staphylococcus intermedius (Staph. intermedius), and Staphylococcus pseudintermedius (Staph. pseudintermedius) were incubated with various fatty acid salts in distilled water (DW) or UPSW and the number of bacteria was counted. Among the fatty acids, oleic acid salt and linoleic acid (LA) salt reduced the number of these bacteria. Also, UPSW enhanced the antibacterial effect of LA on Staph. spp. In spontaneously developed itchy dermatitis in companion dogs, shampoo treatment with liquid soap containing 10% LA in UPSW improved skin conditions. CONCLUSIONS LA salt showed antibacterial activity against Staph. spp. Treatment with soap containing LA with UPSW reduced clinical conditions in dogs with dermatitis. SIGNIFICANCE AND IMPACT OF THE STUDY Because colonization of Staph. spp. on the skin exacerbates dermatitis, the use of LA-containing soap in UPSW may reduce unpleasant clinical symptoms of the skin.

Mutations in the juxtamembrane and tyrosine kinase domains of the KIT receptor havebeen implicated in several cancers and are known to promote tumorigenesis. However,the pathophysiological manifestations of mutations in the extracellular domainremain unknown. In this study, we examined the impact of a mutation in theextracellular domain of KIT on mast cell tumorigenesis. A KIT mutant with anAsn508Ile variation (N508I) in the extracellular domain derived from a canine mastcell tumor was introduced into IC-2 cells. The IC-2N508I cellsproliferated in a cytokine-independent manner and showed KIT auto-phosphorylation.Subcutaneous injection of IC-2N508I cells into the dorsal area ofimmunodeficient BALB/c-nu/nu mice resulted in the formation of solid tumors,but tumor progression was abrogated by treatment with a tyrosine kinase inhibitor(STI571). In addition, the N508I mutant KIT protein dimerized in the absence of thenatural ligand, stem cell factor. Structure modeling indicates that the increasedhydrophobicity of the mutant led to the stabilization of KIT dimers. These resultssuggest that this extracellular domain mutation confers a ligand-independenttumorigenic phenotype to mast cells by KIT auto-dimerization that isSTI571-sensitive. This is the first report demonstrating the tumorigenic potentialof a mutation in the extracellular domain of KIT.

Since more than 75% of breast cancers overexpress estrogen receptors (ER), endocrine therapy targeting ER has significantly improved the survival rate. Nonetheless, breast cancer still afflicts women worldwide and the major problem behind it is resistance to endocrine therapy. We have previously shown the involvement of nuclear factor-κB (NF-κB) in neoplastic proliferation of human breast cancer cells; however, the association with the transformation of ER-positive cells remains unclear. In the current study, we focused on roles of NF-κB in the hormone dependency of breast cancers by means of ER-positive MCF-7 cells. Blocking of NF-κB signals in ER-negative cells stopped proliferation by downregulation of D-type cyclins. In contrast, the MCF-7 cells were resistant to NF-κB inhibition. Under estrogen-free conditions, the ER levels were reduced when compared with the original MCF-7 cells and the established cell subline exhibited tamoxifen resistance. Additionally, NF-κB participated in cell growth instead of the estrogen-ER axis in the subline and consequently, interfering with the NF-κB signals induced additive anticancer effects with tamoxifen. MMP-9 production responsible for cell migration, as well as the cell expansion in vivo, were suppressed by NF-κB inhibition. Therefore, we suggest that NF-κB is a master switch in both ER-positive and ER-negative breast cancers.

Mineral ions in tap water react with fatty acids in soap, leading to the formation of insoluble precipitate (metallic soap) on skin during washing. We hypothesised that metallic soap might negatively alter skin conditions. Application of metallic soap onto the skin of NC/Tnd mice with allergic dermatitis further induced inflammation with elevation of plasma immunoglobulin E and proinflammatory cytokine expression. Pruritus and dryness were ameliorated when the back of mice was washed with soap in Ca2+- and Mg2+-free ultra-pure soft water (UPSW). Washing in UPSW, but not tap water, also protected the skin of healthy volunteers from the soap deposition. Furthermore, 4 weeks of showering with UPSW reduced dryness and pruritus of human subjects with dry skin. Washing with UPSW may be therapeutically beneficial in patients with skin troubles.

Mast cell tumor (MCT) is the most common cutaneous tumor in dogs. We recently revealed that production of stem cell factor (SCF) contributes to the proliferation of neoplastic mast cells in an autocrine/paracrine manner. The aim of the present study was to determine the contribution of the mechanism in clinical MCTs. In consequence, high SCF expression (>10 times compared to HRMC cells) was observed in 5 of 7 MCT samples used in the study regardless of KIT mutation, which was confirmed in immunohistochemical analysis. In addition, production of SCF was observed in Ki-67-positive cells in the MCT xenograft. These results indicate the broad contribution of SCF autocrine/paracrine mechanism on clinical MCTs, providing the rationale for the clinical use of KIT inhibitors regardless of KIT mutation.

Elevated skin surface pH has been reported in patients with atopic dermatitis. In this study, we explored the role of skin pH in the pathogenesis of atopic dermatitis using the NC/Tnd murine atopic dermatitis model. Alkalinization of the skin of asymptomatic NC/Tnd mice housed in specific pathogen-free conditions induced kallikrein 5 and activated protease-activated receptor 2, resulting in thymic stromal lymphopoietin secretion and a cutaneous T-helper 2 allergic response. This was associated with increased transepidermal water loss and development of eczematous lesions in these specific pathogen-free NC/Tnd mice, which normally do not suffer from atopic dermatitis. Injection of recombinant thymic stromal lymphopoietin also induced scratching behavior in the specific pathogen-free NC/Tnd mice. Thymic stromal lymphopoietin production and dermatitis induced by alkalinization of the skin could be blocked by the protease-activated receptor 2 antagonist ENMD-1068. In contrast, weak acidification of eczematous skin in conventionally housed NC/Tnd mice reduced kallikrein 5 activity and ameliorated the dermatitis. Onset of the dermatitis was associated with increased epidermal filaggrin expression and impaired activity of the sodium/hydrogen exchanger 1, a known regulator of skin pH. We conclude that alterations in skin pH directly modulate kallikrein 5 activity leading to skin barrier dysfunction, itch, and dermatitis via the protease-activated receptor 2-thymic stromal lymphopoietin pathway.