VAMP7 knockdown in secretory granules impairs CCL2 secretion in mast cells.

Mast cells (MCs) possess numerous potent inflammatory mediators and undergo differential regulation in response to antigen (Ag) stimulation. Among the regulatory systems governing secretory responses, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in facilitating granule-plasma membrane fusion and subsequent secretion. Our previous investigation documented the involvement of vesicle-associated membrane protein 3 (VAMP3) in regulating cytokine secretions in RBL-2H3 cells, a model for MC IgE-mediated responses. In addition to VAMP3, VAMP7 is expressed in MCs, but its functional role remains elusive. The present study seeks to explore VAMP7-specific regulatory mechanisms in MCs, shedding light on one of the mechanisms governing heterogeneous secretory responses in these cells. Murine bone marrow-derived mast cells (BMMCs) were examined to analyze the subcellular distribution of inflammatory mediators, specifically TNFα, CCL2, and histamine, and VAMPs (i.e., VAMP3, VAMP7, and VAMP8). Immunocytochemistry and the transient expression of fluorescent protein-conjugated target proteins were used to discern the distribution of various inflammatory mediators and VAMP7 through confocal laser scanning microscopy. Each inflammatory mediator (TNFα, CCL2, and histamine) was found in secretory granules of different sizes within BMMCs. VAMP7 exhibited a distinct distribution compared to VAMP3 in these granules. Notably, an overlapping distribution was observed between VAMP7 and CCL2, but not between VAMP7 and TNFα or VAMP7 and histamine. This suggests that CCL2 resides within VAMP7-expressing granules and is subject to VAMP7-dependent secretory regulation. Consistently, BMMCs with VAMP7 knockdown showed markedly reduced CCL2 secretion after Ag stimulation. These observations underscore the heterogeneity of MC secretory responses and unveil a novel VAMP7-dependent CCL2 secretion mechanism within MCs. This discovery might pave the way for the development of more precise therapeutic strategies to modulate MC secretion in allergic conditions.

Ephedrine Alkaloid-Independent High-Affinity Immunoglobulin-E Receptor (FcεRI) Internalization Results in CCL2 Production without Inducing Mast Cell Degranulation.

Mast cells (MCs) play an important role in allergies, leading to the development of MC-targeted therapies. Ephedra herb (Mao) has potent anti-allergic activity, but contains ephedrine alkaloids (EAs); therefore, its hazardous effects are taken into consideration during its clinical use. We previously reported that Mao attenuates robust MC degranulation by an allergen through high-affinity immunoglobulin E (IgE) receptor (FcεRI) internalization, in which an EA-independent mechanism was suggested to be at play. This study aimed to deepen our understanding of the potential of Mao against FcεRI internalization using two strains with different EA contents. Mao extracts were administered to bone marrow-derived MCs (BMMCs), and their cellular responses, including FcεRI internalization, were analyzed. In addition, physiological events were evaluated using a passive cutaneous anaphylactic (PCA) reaction mouse model. BMMCs mediate the production of diverse inflammatory mediators. Among these, the potent chemokine CCL2 is thought to be differentially regulated from other pro-inflammatory mediators. We found that Mao significantly induces CCL2 expression in BMMCs despite suppressing robust degranulation through FcεRI internalization. Importantly, this was a distinctly EAs-independent response. In the PCA reaction, local MC activation following allergen challenge was suppressed by Mao treatment, which strengthened the view that Mao sufficiently decreased the rapid activation of MCs and promoted CCL2 secretion. Collectively, these observations provide additional insights into the mechanism of Mao-induced silent FcεRI internalization in MCs and the complex and heterogeneous secretory responses operating in MCs.

Vertebral Osteomyelitis Caused by Mycobacterium arupense Mimicking Tuberculous Spondylitis: First Reported Case and Literature Review.

Mycobacterium arupense is a slow-growing, nontuberculous mycobacterium widely found in the environment and is known to cause tenosynovitis and osteomyelitis, mainly in the hands and wrists. We present the first case of vertebral osteomyelitis caused by M arupense in a 78-year-old man with renal cell carcinoma. The patient had a history of tuberculous pleuritis in childhood. Although the nucleic acid amplification test of the vertebral tissue for Mycobacterium tuberculosis was negative, we initiated tuberculosis treatment based on the history and pathological findings of auramine-rhodamine-positive organisms and epithelioid cell granulomas. Subsequently, the isolated mycobacterium was identified as M arupense by genome sequencing. Accordingly, the treatment regimen was changed to a combination of clarithromycin, ethambutol, and rifabutin. Owing to a subsequent adverse event, rifabutin was switched to faropenem, and the patient was treated for a total of 1 year. In previous literature, we found 15 reported cases of bone and soft tissue infections caused by M arupense, but none of them had vertebral lesions. Physicians should be aware that M arupense can cause vertebral osteomyelitis mimicking tuberculous spondylitis. In addition, molecular testing of isolated mycobacteria is essential for diagnosis, even if tuberculous spondylitis is suspected.

Leukotriene D4 accelerates antigen-mediated mast cell responses via the cysteinyl leukotriene 1 receptor.

Cysteinyl leukotrienes (CysLTs), released from mast cells (MCs), are important mediators in allergy. Type 1 receptors for CysLTs (CysLT1R) are involved in accelerating IgE-mediated MC activation. In this study, we aimed to elucidate the mechanisms underlying CysLT1R-mediated MC activation. The CysLT1R agonist/antagonist was applied to two types of major MC models-RBL-2H3 cells and bone marrow-derived MCs (BMMCs). The use of CysLT1R and CysLT2R inhibitors revealed that CysLT1R plays a major role in the acceleration of MC activation. The administration of the CysLT1R agonist leukotriene D4 upregulated IgE-mediated Akt and ERK phosphorylation and subsequently enhanced TNF-α expression, suggesting that CysLT1R regulates the downstream pathway of MC activation. However, these observations were not corroborated by CysLT1R knockdown using shRNA, suggesting a differential regulatory mechanism between the temporal and constitutive inhibitions of CysLT. In conclusion, CysLT1R enhances MC activation by accelerating IgE-induced signal transduction, which enables the co-regulation of rapid degranulation and delayed synthesis of inflammatory mediators in MCs.

Ecthyma gangrenosum caused by Staphylococcus aureus in hematological malignancies: Case reports and literature review.

RATIONALE: Ecthyma gangrenosum (EG) is a potentially life-threatening, systemic infection generally caused by Pseudomonas aeruginosa. Data on EG caused by Staphylococcus aureus in patients with hematological malignancies are scarce. The present case report aimed to describe the clinical features of EG caused by S. aureus in patients with hematological malignancies and to provide a comprehensive review of previous studies on the topic. PATIENT CONCERNS: The first patient was a 61-year-old man with acute myeloid leukemia who presented fever and multiple lesions during chemotherapy. The second patient was a 47-year-old man with myelodysplastic syndrome who developed progressive erythematous necrotic plaques on his extremities and face. DIAGNOSIS: Both cases were diagnosed as EG caused by S. aureus. While the first patient had concurrent methicillin-resistant S. aureus (MRSA) bacteremia, the second patient had positive results only for tissue culture of the skin lesion isolated methicillin-sensitive S. aureus. INTERVENTIONS: Vancomycin was initiated with critical care to the first patient. Cefazolin was administered to the second patient for 3 weeks, followed by cephalexin for 1 week. OUTCOMES: The first patient died of a brain hemorrhage and multiple organ failure. The second patient was cured without relapse. LESSONS: Of 18 patients in the previous and current studies with EG caused by S. aureus, 6 (33%) had an underlying hematological malignancy, and 10 (56%) had EG caused by MRSA. While 28% of the patients had positive blood cultures, all tissue cultures were positive. All 3 fatalities had concurrent bacteremia (MRSA caused two). EG caused by MRSA with concurrent bacteremia can be fatal, especially in patients with hematological malignancies. Although S. aureus-associated EG in patients with hematological malignancies is relatively uncommon, tissue cultures with an initial gram stain smear are essential for selecting appropriate empirical antimicrobials, including the coverage of S. aureus.

Augmented Self-Association by Electrostatic Forces in Thienopyrrole-Fused Thiadiazoles that Contain an Ester instead of an Ether Linker.

During the self-assembly of π-conjugated molecules, linkers and substituents can potentially add supportive noncovalent intermolecular interactions to π-stacking interactions. Here, we report the self-assembly behavior of thienopyrrole-fused thiadiazole (TPT) fluorescent dyes that possess ester or ether linkers and dodecyloxy side chains in solution and the condensed phase. A comparison of the self-association behavior of the ester- and ether-bridged compounds in solution using detailed UV-vis, fluorescence, and NMR spectroscopic studies revealed that the subtle replacement of the ether linkers by ester linkers leads to a distinct increase in the association constant (ca. 3-4 fold) and the enthalpic contribution (ca. 3 kcal mol-1 ). Theoretical calculations suggest that the ester linkers, which are in close proximity to one another due to the π-stacking interactions, induce attractive electrostatic forces and augment self-association. The self-assembly of TPT dyes into well-defined 1D clusters with high aspect ratios was observed, and their morphologies and crystallinity were investigated using SEM and X-ray diffraction analyses. TPTs with ester linkers exhibit a columnar liquid crystalline mesophase in the condensed phase.

Ephedra Herb, Mao, Inhibits Antigen-Induced Mast Cell Degranulation by Induction of the Affinity Receptor for IgE Internalization.

PURPOSE: Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis. METHODS: Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated. RESULTS: Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs. CONCLUSIONS: Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.

Ginger Increases ALDH1A1 Expression and Enhances Retinoic Acid Signaling in a Human Colonic Epithelial Cell Line.

Aldehyde dehydrogenase 1A1 (ALDH1A1) in intestinal epithelial cells (IECs) plays a critical role in regulating immune responses through the production of retinoic acid (RA). However, little is known about its regulation by dietary components. We previously demonstrated that kakkonto, a Japanese traditional herbal medicine, and its constituent puerarin induce the expression of ALDH1A1 mRNA in colonic IECs and thereby attenuate food allergy symptoms in mice. This study aims to investigate the cellular responses of IECs to ALDH1A1 expression as a result of natural food components. The seven medicinal herbs that compose kakkonto were used to treat cultured an IEC line: Caco-2 cells. Expressions levels of ALDH1A1 were analyzed in Caco-2 cells by quantitative RT-PCR, immunocytochemistry and western blotting. Ginger increased the expression levels of ALDH1A1 mRNA and protein in Caco-2 cells. In addition, ginger significantly upregulated the gene expression of retinoic acid receptor (RAR) alpha (RARA), thereby enhancing RA signaling. Furthermore, ginger downregulated the expression of histone deacetylase (HDAC)2 (HDAC2) and HDAC3 in Caco-2 cells. The present study suggests the possibility that food ingredients such as a ginger modulate vitamin A metabolism in the gut through the regulation of RA synthesis, which may contribute to RA-mediated regulation of immune responses and the regulation of allergic inflammation.

Vascular smooth muscle cell polyploidization involves changes in chromosome passenger proteins and an endomitotic cell cycle.

Vascular smooth muscle cell polyploidization occurs during normal development and is enhanced under physiologic stress, but the mechanism of this cell cycle has not been explored. We show via time-lapse video imaging and immunofluorescence analyses that primary vascular smooth muscle cells (VSMC) undergo an endomitotic-type cell cycle, including a normal progression through part of mitosis. Mononuclear polyploid cells are generated by defects in sister chromatid separation and/or segregation, and cellular binucleation occurs by reversal of cytokinesis. To obtain further leads to regulators involved, we examined the chromosomal passenger proteins, Aurora B, inner centromere protein and Survivin, and concluded that Aurora B and inner centromere protein are normally colocalized in centromeres, the midzone, and the midbody during mitosis. Survivin, however, is dim and diffused; it does not colocalize with either Aurora B or inner centromere protein in VSMC, which could account for defects in sister chromatid separation and/or segregation and reversal of cytokinesis. In accordance with the reported dependency of Aurora B activity on Survivin, the Aurora B substrate, vimentin, is not phosphorylated during cytokinesis. Finally, the data show that ectopically expressed Survivin inhibits polyploidization in vascular smooth muscle cells. Hence, aberrant chromosome passenger protein activity and endomitosis are associated with VSMC polyploidization.

Aberrant quantity and localization of Aurora-B/AIM-1 and survivin during megakaryocyte polyploidization and the consequences of Aurora-B/AIM-1-deregulated expression.

Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.

Proplatelet formation of megakaryocytes is triggered by autocrine-synthesized estradiol.

A matured megakaryocyte releases thousands of platelets through a drastic morphological change, proplatelet formation (PPF). The megakaryocyte/erythrocyte-specific transcription factor, p45 NF-E2, is essential for initiating PPF, but the factor regulating PPF has not been identified. Here we report that estradiol synthesized in megakaryocytes triggers PPF. We demonstrate that a key enzyme for steroid hormone biosynthesis, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), is a target of p45 NF-E2, and rescues PPF of p45 NF-E2-deficient megakaryocytes. We also show that estradiol is synthesized within megakaryocytes, and that extracellular estradiol stimulates PPF, inhibition of 3beta-HSD activity blocks PPF, and estrogen receptor antagonists inhibit platelet production in vivo. We conclude that autocrine estradiol action regulates platelet production by triggering PPF.

Thrombopoietin-induced CXC chemokines, NAP-2 and PF4, suppress polyploidization and proplatelet formation during megakaryocyte maturation.

BACKGROUND: We previously reported that the expressions of two CXC chemokines, neutrophil activating peptide-2 (NAP-2) and platelet factor-4 (PF-4), were induced by megakaryocyte-specific cytokine thrombopoietin (TPO) in mouse bone marrow megakaryocytes. The roles of these chemokines on megakaryocyte maturation/differentiation processes, including polyploidization and proplatelet formation (PPF) remain unresolved. RESULTS: NAP-2 and PF-4 suppressed the PPF of mature megakaryocytes freshly prepared from mouse bone marrow as well as that of the megakaryocyte progenitors, c-Kit+CD41+ cells, isolated from mouse bone marrow and cultured with TPO. NAP-2 and PF-4 inhibited polyploidization of c-Kit+CD41+ cells in the presence of TPO, and also inhibited the proliferation of c-Kit+CD41+ cells. CONCLUSIONS: NAP-2 and PF-4 produced by TPO stimulation in megakaryocytes suppress megakaryocyte maturation and proliferation as a feedback control.

Isolation and characterization of mouse homolog of the neutrophil activating peptide-2.

In the presence of thrombopoietin (TPO), megakaryocytes mature by polyploidization and cytoplasmic maturation, and the matured megakaryocytes induce drastic morphological change and proplatelet formation and release a number of platelets. However, the regulatory mechanism of this unique differentiation process is still obscure. We therefore attempted to identify the factors, expression of which is induced by TPO stimulation in mouse bone marrow megakaryocytes. We isolated the mouse homolog of the neutrophil activating peptide-2 (NAP-2). Mouse NAP-2 cDNA encodes a predicted sequence of 113 amino acids and contains the Cys motif (CXC) found in other members of the alpha-chemokine family. At the amino acid level, the predicted mouse NAP-2 has 50.4%, 51.8%, and 72.6% identity with the predicted human, pig, and rat NAP-2, respectively. Northern blot analysis demonstrates that mouse NAP-2 is expressed only in spleen. Furthermore, the RT-PCR technique shows that the mouse NAP-2 gene is clearly upregulated by TPO stimulation in mouse megakaryocytes.