Hyun Woo Kim

Analysis of a new histological and molecular-based classification of canine mammary neoplasia.

Canine mammary tumors (CMTs) are morphologically and biologically heterogeneous, prompting several attempts to classify such tumors on the basis of their histopathological characteristics. Recently, molecular-based analysis methods borrowed from human breast cancer research have also been applied to the classification of CMTs. In this study, canine mammary neoplasms (n = 648) occurring in Korea from 2008 to 2011 were analyzed according to the histological classification and grading system proposed by Goldschmidt et al. Furthermore, randomly selected mammary carcinomas (n = 159) were classified according to the molecular subtype using immunohistochemical characteristics. Canine mammary neoplasia accounted for 52.6% (648/1250) of the tumors in female dogs, and 51.7% (340/648) of these were malignant. All of the carcinoma-anaplastic subtypes were grade III tumors (5/5, 100%), while most of the carcinoma-tubular subtypes (15/18, 83.3%) and carcinoma arising in a complex adenoma/mixed-tumor subtype (115/135, 85.2%) were grade I tumors. Tumor cell invasion into lymphatic vessels was most common in the comedocarcinoma, carcinoma-anaplastic, and inflammatory carcinoma subtypes. The most frequently occurring molecular subtype (70/159, 44%) was luminal A. However, the basal-like subtype was the most malignant and was frequently associated with grade III tumors and lymphatic invasion. The carcinoma-solid subtypes were also often of the basal-like subtype. Reclassification of CMTs using the newly proposed histopathological classification system and molecular subtyping could aid in determining the prognosis and the most suitable anticancer treatment for each case.

The Scaffold Protein Prohibitin Is Required for Antigen-Stimulated Signaling in Mast Cells

Targeting a scaffold protein delivered by intracellular granules in mast cells may inhibit allergic responses. How to Prohibit Mast Cell Activation Mast cells are the major effector cells of the allergic response. Binding of antigen to immunoglobulin E (IgE) molecules bound to the cell-surface, high-affinity IgE receptor FcεRI results in receptor clustering, mast cell activation, and degranulation, resulting in the release of factors that mediate the allergic response. Phosphorylation and activation of the Src family kinases Lyn and Syk are critical for mast cell activation. Kim et al. found that the scaffold protein prohibitin (PHB), which mediates functions such as mitochondrial biogenesis and transcriptional regulation (see commentary by Yurugi and Rajalingam), was abundant in intracellular granules in mouse mast cells. Stimulation of mast cells with antigen resulted in the translocation of PHB to plasma membrane lipid rafts, which was required for the association of FcεRI with Syk, the activation of Syk, and degranulation. Knockdown of PHB in mice inhibited mast cell activation and anaphylaxis, suggesting that targeting PHB therapeutically may reduce allergic responses. The protein prohibitin (PHB) is implicated in diverse cellular processes, including cell signaling, transcriptional control, and mitochondrial function. We found that PHB was abundant in the intracellular granules of mast cells, which are critical for allergic responses to antigens. Thus, we investigated whether PHB played a role in signaling mediated by the high-affinity receptor for antigen-bound immunoglobulin E (IgE), FcεRI. PHB-specific small interfering RNAs (siRNAs) inhibited antigen-mediated signaling, degranulation, and cytokine secretion by mast cells in vitro. Knockdown of PHB inhibited the antigen-dependent association of the tyrosine kinase Syk with FcεRI and inhibited the activation of Syk. Fractionation studies revealed that PHB translocated from intracellular granules to plasma membrane lipid rafts in response to antigen, and knockdown of PHB suppressed the movement of FcεRIγ and Syk into lipid rafts. Tyrosine phosphorylation of PHB by Lyn was observed early after exposure to antigen, and point mutations in PHB indicated that Tyr114 and Tyr259 were required for the recruitment of Syk to FcεRIγ and mast cell activation. In mice, PHB-specific siRNAs inhibited antigen-initiated mast cell degranulation, passive cutaneous anaphylaxis, and passive systemic anaphylaxis. Together, these results suggest that PHB is essential for FcεRI-mediated mast cell activation and allergic responses in vivo, raising the possibility that PHB might serve as a therapeutic target for the treatment of allergic diseases.

Interleukin-10–producing CD5+ B cells inhibit mast cells during immunoglobulin E–mediated allergic responses

When specialized B cells come into contact with mast cells, the B cells produce a cytokine that inhibits the mast cells, thereby blunting allergic responses. Limiting allergic responses with B cells B cells promote immune responses by producing antibodies; however, subsets of B cells secrete the anti-inflammatory cytokine interleukin-10 (IL-10) and have immunosuppressive properties. Kim et al. found that these B cells inhibited the activation of mast cells, immune cells that are critical regulators of allergic reactions. Indeed, mice lacking these special B cells had more severe symptoms of anaphylaxis. Mast cell inhibition required physical contact with the B cells, which stimulated the B cells to produce more IL-10. B cell–mediated inhibition of mast cells depended on IL-10, which inhibited tyrosine kinase signaling in mast cells. Thus, IL-10–producing B cells might provide a therapeutic target to treat allergic diseases. Subsets of B cells inhibit various immune responses through their production of the cytokine interleukin-10 (IL-10). We found that IL-10–producing CD5+ B cells suppressed the immunoglobulin E (IgE)– and antigen-mediated activation of mast cells in vitro as well as allergic responses in mice in an IL-10–dependent manner. Furthermore, the suppressive effect of these B cells on mast cells in vitro and in vivo depended on direct cell-to-cell contact through the costimulatory receptor CD40 on CD5+ B cells and the CD40 ligand on mast cells. This contact enhanced the production of IL-10 by the CD5+ B cells. Through activation of the Janus-activated kinase–signal transducer and activator of transcription 3 pathway, IL-10 decreased the abundance of the kinases Fyn and Fgr and inhibited the activation of the downstream kinase Syk in mast cells. Together, these findings suggest that an important function of IL-10–producing CD5+ B cells is inhibiting mast cells and IgE-mediated allergic responses.

Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity

BackgroundComplementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo.MethodsThe effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis.ResultsLERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells.ConclusionThis study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.

Suppression of IgE-mediated mast cell activation and mouse anaphylaxis via inhibition of Syk activation by 8-formyl-7-hydroxy-4-methylcoumarin, 4μ8C

ABSTRACT Mast cells trigger IgE‐mediated allergic reactions by releasing various allergic mediators. 8‐Formyl‐7‐hydroxy‐4‐methylcoumarin, also called 4&mgr;8C, was originally known as an inositol‐requiring enzyme 1 (IRE1) suppressant, but no study has examined its relationship with mast cells and allergic diseases. Therefore, the purpose of this study was to determine whether 4&mgr;8C is effective in suppressing allergic reactions in mast cells and in IgE‐mediated allergic animal model. 4&mgr;8C suppressed the degranulation of IgE‐mediated mast cells (IC50 = 3.2 &mgr;M) and the production of cytokines such as tumor necrosis factor‐&agr; (TNF‐&agr;) and interleukin‐4 (IL‐4) in a dose‐dependent manner. 4&mgr;8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50 = 25.1 mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4&mgr;8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4&mgr;8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4&mgr;8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE‐mediated signaling pathway. In summary, for the first time, this study shows that 4&mgr;8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4&mgr;8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis. HighlightsAnti‐allergic effect of 8‐formyl‐7‐hydroxy‐4‐methylcoumarin, 4&mgr;8C, was investigated in vitro and in vivo.4&mgr;8C reversibly inhibited the degranulation of mast cells by antigen.4&mgr;8C suppressed passive cutaneous anaphylaxis (PCA) in mice.The anti‐allergic effect of 4&mgr;8C was mediated by inhibition of Lyn and Fyn in mast cells.

Obesity, expression of adipocytokines, and macrophage infiltration in canine mammary tumors

Obesity influences the development, progression and prognosis of human breast cancer and canine mammary cancer (MC) but the precise underlying mechanism is not well-documented in the fields of either human or veterinary oncology. In the present study, the expression of major adipocytokines, including leptin, adiponectin, and leptin receptor (ObR) in benign (nu2009=u200928) and malignant (nu2009=u200970) canine mammary tumors was investigated by immunohistochemistry and on the basis of the subjects body condition score (BCS). To evaluate the relationship between obesity and chronic inflammation of the mammary gland, macrophages infiltrating within and around tumoral areas were counted. The mean age of MC development was lower in overweight or obese dogs (9.0u2009±u20091.8 years) than in lean dogs or optimal bodyweight (10.2u2009±u20092.9 years), and the evidence of lymphatic invasion of carcinoma cells was found more frequently in overweight or obese group than in lean or optimal groups. Decreased adiponectin expression and increased macrophage numbers in overweight or obese subjects were significantly correlated with factors related to a poor prognosis, such as high histological grade and lymphatic invasion. Leptin expression was correlated with progesterone receptor status, and ObR expression was correlated with estrogen receptor status of MCs, regardless of BCS. Macrophage infiltration within and around the tumor may play an important role in tumor progression and metastasis in obese female dogs and may represent a prognostic factor for canine MCs.

An indoxyl compound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice.

Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC50, ~3.8μM) and human mast cells (IC50, ~3.0μM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED50 27.9mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells.

Evaluation of Clinicopathological Characteristics and Oestrogen Receptor Gene Expression in Oestrogen Receptor-negative, Progesterone Receptor-positive Canine Mammary Carcinomas

The existence of the oestrogen receptor-negative (OR(-))/progesterone receptor-positive (PR(+)) phenotype in canine mammary carcinomas (CMCs) is not well understood, although this phenotype was reported consistently in previous studies. In the present study, immunohistochemistry (IHC) was performed to categorize CMCs with the OR(-)/PR(+) phenotype and compare their clinicopathological features with OR(+)/PR(+) tumours. Of a total of 305 CMCs, 36 (11.8%) were categorized as OR(-)/PR(+) and showed intermediate characteristics between those of OR(+)/PR(+) and OR(-)/PR(-) cases. OR mRNA levels were measured in formalin-fixed, paraffin wax-embedded samples using a novel branched-chain DNA assay method. Similar to the IHC result, one-way analysis of variance showed that the mean normalized OR mRNA level of OR(-)/PR(+) tumours (11.4 ± 16.34) was between that of the OR(-)/PR(-) (mean 4.7 ± 6.35) and OR(+)/PR(+) (mean 15.8 ± 11.95) (P = 0.033) tumours. Only three of the 36 OR(-)/PR(+) tumours completely lacked OR mRNA expression. The OR(-)/PR(+) tumours were not categorized as an independent group nor were they included in the other groups on post-hoc analysis. OR(-)/PR(+) tumours were associated with factors related to poor prognosis compared with OR(+)/PR(+) tumours, but OR(-)/PR(-) tumours were associated with the worst prognostic indicators. Further studies are required in order to determine the clinical significance of the OR(-)/PR(+) phenotype.

DJ-1 controls bone homeostasis through the regulation of osteoclast differentiation

Receptor activator of NF-kB ligand (RANKL) generates intracellular reactive oxygen species (ROS), which increase RANKL-mediated signaling in osteoclast (OC) precursor bone marrow macrophages (BMMs). Here we show that a ROS scavenging protein DJ-1 negatively regulates RANKL-driven OC differentiation, also called osteoclastogenesis. DJ-1 ablation in mice leads to a decreased bone volume and an increase in OC numbers. In vitro, the activation of RANK-dependent signals is enhanced in DJ-1-deficient BMMs as compared to wild-type BMMs. DJ-1 suppresses the activation of both RANK-TRAF6 and RANK-FcRγ/Syk signaling pathways because of activation of Src homology region 2 domain-containing phosphatase-1, which is inhibited by ROS. Ablation of DJ-1 in mouse models of arthritis and RANKL-induced bone disease leads to an increase in the number of OCs, and exacerbation of bone damage. Overall, our results suggest that DJ-1 plays a role in bone homeostasis in normal physiology and in bone-associated pathology by negatively regulating osteoclastogenesis.Osteoclasts are involved in arthritis, and their differentiation depends on RANKL signaling. The author show that the ROS-scavenging protein DJ-1 negatively regulates RANKL signaling and that its ablation increases osteoclast numbers and exacerbates bone damage in mouse models of arthritis.

JQ1, a BET inhibitor, controls TLR4-induced IL-10 production in regulatory B cells by BRD4-NF-κB axis

Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomain-containing protein 4 (BRD4) was associated with NF-κB on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with NF-κB on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer.