Human placental extract suppresses mast cell activation and induces mast cell apoptosis.

BACKGROUND: Human placental extract (HPE) has been documented to facilitate the healing of certain disorders including allergy. However, the effects of HPE on the functionality of mast cells, a critical cell type in allergic diseases, have not been reported. METHODS: To investigate the effects of HPE on the regulation of allergy with respect to the biological functions of mast cells, the mast cell line C57 or HMC-1 cells were treated with HPE followed by the assessment of cell proliferation, apoptosis, activation, chemotaxis and phagocytosis. Mouse peritoneal mast cells were also investigated for their responses to induction of apoptosis by HPE in vivo. Furthermore, the effect of HPE on mast cell degranulation was confirmed using the passive cutaneous anaphylaxis (PCA) assay, an acute allergy model. RESULTS: HPE was capable of suppressing mast cell proliferation and inducing mast cell apoptosis. Mast cell degranulation in response to compound 48/80- or anti-DNP IgE and DNP-mediated activation was suppressed. In addition, treatment with HPE compromised the production of cytokines by mast cells and cell chemotaxis. These observations were consistent with the dampened passive cutaneous anaphylaxis (PCA) assay following treatment with HPE. CONCLUSION: This study revealed a suppressive effect of HPE on overall mast cell activities, suggesting a potential regulatory role of HPE on the alleviation of allergic diseases through mast cells.

FcγR-dependent apoptosis regulates tissue persistence of mucosal and connective tissue mast cells.

Rodent mast cells can be divided into two major subtypes: the mucosal mast cell (MMC) and the connective tissue mast cell (CTMC). A decade-old observation revealed a longer lifespan for CTMC compared with MMC. The precise mechanisms underlying such differential tissue persistence of mast cell subsets have not been described. In this study, we have discovered that mast cells expressing only one receptor, either FcγRIIB or FcγRIIIA, underwent caspase-independent apoptosis in response to IgG immune complex treatment. Lower frequencies of CTMC in mice that lacked either FcγRIIB or FcγRIIIA compared with WT mice were recorded, especially in aged mice. We proposed that this paradigm of FcγR-mediated mast cell apoptosis could account for the more robust persistence of CTMC, which express both FcγRIIB and FcγRIIIA, than MMC, which express only FcγRIIB. Importantly, we reproduced these results using a mast cell engraftment model, which ruled out possible confounding effects of mast cell recruitment or FcγR expression by other cells on mast cell number regulation. In conclusion, our work has uncovered an FcγR-dependent mast cell number regulation paradigm that might provide a mechanistic explanation for the long-observed differential mast cell subset persistence in tissues.

[Detection of memory T cells, follicular T cells and relevant cytokines in the peripheral blood of patients with myasthenia gravis during treatment].

Objective The variations in T cell subset composition and levels of inflammatory cytokines and acetylcholine receptor (AChR) antibody during the interval for myasthenia gravis (MG) patients were evaluated to investigate regulatory roles of in the pathogenesis of MG. Methods Thirty patients with MG and 20 healthy controls (HCs) were recruited. Flow cytometry was employed to detect the frequencies of CD4+ T cells, CD8+ T cells, central memory T cells (CD4+CD45RO+CCR7+, Tcm), effector memory T cells (CD4+CD45RO+CCR7-, Tem), follicular helper T cells (CD4+CXCR5+, Tfh) and follicular regulatory T cells (CD4+CXCR5+ FOXP3 +, Tfr) in the peripheral blood. The levels of interleukin 2(IL-2), IL-4, IL-12, IL-17 and interferon γ (IFN-γ) in the peripheral blood were determined by cytometric beads array (CBA). The levels of IL-7 and anti-AChR antibody were measured with ELISA. Results No obvious differences were observed in the frequencies of Tfh, Tem, CD8+ T cells and CD4+ T cells, and the ratio of CD4/CD8 in the peripheral blood of MG patients, compared with HCs. MG subjects presented notably decreased frequencies of Tcm and increased frequencies of Tfr compared with HCs. In addition, elevated levels of IL-2, IL-4, IL-12, IL-17 and IFN-γ and lowered levels of IL-7 were observed in the plasma of MG individuals, compared with HCs. No significant correlations were found among the levels of cytokines and frequencies of T cell subsets. No significant changes were found in AChR antibody levels. Conclusion The results suggest a unique spectrum of the memory T cells and follicular T cells, together with a unique cytokine profile in the MG individuals during treatment.

Mutational analysis of compound heterozygous mutation p.Q6X/p.H232R in SRD5A2 causing 46,XY disorder of sex development.

BACKGROUND: Over 100 mutations in the SRD5A2 gene have been identified in subjects with 46,XY disorder of sex development (DSD). Exploration of SRD5A2 mutations and elucidation of the molecular mechanisms behind their effects should reveal the functions of the domains of the 5α-reductase 2 enzyme and identify the cause of 46,XY DSD. Previously, we reported a novel compound heterozygous p.Q6X/p.H232R mutation of the SRD5A2 gene in a case with 46,XY DSD. Whether the compound heterozygous p.Q6X/p.H232R mutation in this gene causes 46,XY DSD requires further exploration. METHODS: The two 46,XY DSD cases were identified and sequenced. In order to identify the source of the compound heterozygous p.Q6X/p.H232R mutation, the parents, maternal grandparents, and maternal uncle were sequenced. Since p.Q6X mutation is a nonsense mutation, p.H232R mutation was transfected into HEK293 cells and dihydrotestosterone (DHT) production were analyzed by liquid chromatography-mass spectrometry (LC-MS) for 5α-reductase 2 enzyme activities test. Apparent michaelis constant (Km) were measured of p.H232R mutation to analyze the binding ability change of 5α-reductase 2 enzyme with testosterone (T) or NADPH. RESULTS: The sequence results showed that the two 46,XY DSD cases were the compound heterozygous p.Q6X/p.H232R mutation, of which the heterozygous p.Q6X mutation originating from maternal family and heterozygous p.H232R mutation originating from the paternal family. The function analysis confirmed that p.H232R variant decreased the DHT production by LC-MS test. The Km analysis demonstrated that p.H232R mutation affected the binding of SRD5A2 with T or NADPH. CONCLUSIONS: Our findings confirmed that the compound heterozygous p.Q6X/p.H232R mutation in the SRD5A2 gene is the cause of 46,XY DSD. p.H232R mutation reduced DHT production while attenuating the catalytic efficiency of the 5α-reductase 2 enzyme.

S100A4 Is Critical for a Mouse Model of Allergic Asthma by Impacting Mast Cell Activation.

Background: The calcium-binding protein S100A4 demonstrates important regulatory roles in many biological processes including tumorigenesis and inflammatory disorders such as allergy. However, the specific mechanism of the contribution of S100A4 to allergic diseases awaits further clarification. Objective: To address the effect of S100A4 on the regulation of mast cell activation and its impact on allergy. Methods: Bone marrow-derived cultured mast cells (BMMCs) were derived from wild-type (WT) or S100A4-/- mice for in vitro investigation. WT and S100A4-/- mice were induced to develop a passive cutaneous anaphylaxis (PCA) model, a passive systemic anaphylaxis (PSA) model, and an ovalbumin (OVA)-mediated mouse asthma model. Results: Following OVA/alum-based sensitization and provocation, S100A4-/- mice demonstrated overall suppressed levels of serum anti-OVA IgE and IgG antibodies and proinflammatory cytokines in serum, bronchoalveolar lavage fluid (BALF), and lung exudates. S100A4-/- mice exhibited less severe asthma signs which included inflammatory cell infiltration in the lung tissue and BALF, and suppressed mast cell recruitment in the lungs. Reduced levels of antigen reencounter-induced splenocyte proliferation in vitro were recorded in splenocytes from OVA-sensitized and challenged mice that lacked S100A4-/-. Furthermore, deficiency in the S100A4 gene could dampen mast cell activation both in vitro and in vivo, evidenced by reduced ß-hexosaminidase release and compromised PCA and PSA reaction. We also provided evidence supporting the expression of S100A4 by mast cells. Conclusion: S100A4 is required for mast cell functional activation, and S100A4 may participate in the regulation of allergic responses at least partly through regulating the activation of mast cells.

Dysregulated T Cell Activation and Aberrant Cytokine Expression Profile in Systemic Lupus Erythematosus.

Accumulating evidence indicates a critical role for T cells and relevant cytokines in the pathogenesis of systemic lupus erythematosus (SLE). However, the specific contribution of T cells together with the related circulating cytokines in disease pathogenesis and organ involvement is still not clear. In the current study, we investigated relevant molecule expressions and cytokine levels in blood samples from 49 SLE patients and 22 healthy control subjects. The expression of HLA-DR and costimulatory molecules on T cells was evaluated by flow cytometry. Concentrations of serum C-reactive protein, erythrocyte sedimentation rate, anti-double-stranded DNA (anti-dsDNA) antibody, total lgG, complement 3, and complement 4 were measured. Serum cytokines and chemokines were measured by a cytometric bead array assay. Elevated frequencies of HLA-DR+ T cells and ICOS+ T cells were observed in SLE patients with positive anti-dsDNA antibodies compared with those in healthy controls (P < 0.001). The expression of HLA-DR+ T cells was positively correlated with SLEDAI (r = 0.15, P < 0.01). Furthermore, levels of serum IL-6, MCP-1, TNFRI, IL-10, IL-12, and CCL20 were higher in SLE patients compared with healthy controls. In addition, patients with hematologic manifestations displayed elevated frequencies of HLA-DR+ T cells and ICOS+ T cells. Patients with renal manifestations had a decreased frequency of TIGIT+ T cells. These results suggested a dysregulated T cell activity and cytokine expression profiles in SLE subjects. We also developed a chemokine and cytokine profiling strategy to predict the activity of SLE, which has clinical implication for better monitoring the flares and remission during the course of SLE and for assessing therapeutic interventions.

FcγRIIIA Negatively Impacts Humoral Immune Responses but Not Overall Lung Inflammation in an Ovalbumin-Induced Allergic Asthma Mouse Model.

BACKGROUND: Fcγ receptors (FcγR) play substantial immune regulatory roles both positively and negatively in pathophysiological processes including allergy and asthma. Compared with FcγRIIB which is classically defined as an inhibitory receptor, mouse FcγRIIIA and its functional human homologue FcγRIIA have been assumed to be activating receptors. However, evidence demonstrating inhibitory regulation by mouse FcγRIIIA has recently been emerging. OBJECTIVE: To dissect the contributory roles of mouse FcγRIIIA (human FcγRIIA) in parallel with FcγRIIB in an ovalbumin (OVA)-induced mouse model of asthma and to preliminarily assess the correlation of the respective FcγR with circulating IgE levels in human asthma patients. METHODS: Wild-type, FcγRIIB-/-, and FcγRIIIA-/- mice were used in an OVA-induced asthma model followed by assessment of the allergic pathology focused on the lung tissues. Expression levels of FcγRIIB, FcγRIIA, and FcγRIIIA on peripheral blood mononuclear cells (PBMC) together with the circulating IgE levels in the serum from patients with allergic asthma were analysed. RESULTS: Although enhanced humoral immune responses typically represented by augmented OVA-specific IgG and IgE levels in serum were observed in the absence of FcγRIIIA in the mouse asthma model, no overall regulation by FcγRIIIA, especially in terms of those parameters measuring lung tissue inflammation, was recorded. As expected, in the absence of FcγRIIB, augmented immune responses measured as serum antibody levels as well as those in various regulatory pathways in this mouse asthma model were observed. The expression levels of human FcγRIIB but not FcγRIIA were negatively correlated with serum levels of IgE in human asthma patients. CONCLUSION: We did not find major evidence demonstrating an immune inhibitory role of mouse FcγRIIIA in this OVA-induced mouse asthma model. As asthma is a complex disease and the immune regulatory responses involve sophisticated components and pathways, the exact roles of FcγRIIIA as well as its human functional homologue FcγRIIA in asthma still await further clarification using other mouse asthma models as well as clinical studies.