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The goal of this study is to investigate mast cells role in inflammatory skin disorders that worsen by stress, like atopic dermatitis, chronic urticaria kai psoriasis. Specifically, human samples are used to analyze the expression and the effect of corticotropin releasing hormone (CRH) and its receptors on the stimulation of skin mast cells and the vascular permeability increase. The hypothesis is that in these diseases acute stress causes CRH release systematically or topically on the skin, which either alone or in combination with substance P (SP) or neurotensin (NT) release, causes mast cells activation, leading to vascular permeability increase and neurogenic inflammation.The hypothesis above is built on important preliminary results that our team has published in the past. CRH receptors expression was studied human skin biopsies from patients with atopic dermatitis, chronic urticaria and psoriasis, using quantitative Real Time-PCR (qRT-PCR), while CRH, NT and VEGF serum levels were measured using ELISA or milliplex technology. Finally, State-Trait Anxiety Inventory questionnaire (STAI) was used to draw correlations between findings and stress levels.Data show statistically significant serum CRH increase in atopic dermatitis and psoriasis, while there is a statistically significant decrease in corticotrophin releasing hormone receptor 1 (CRHR-R1) in lesion skin of the same patients. Statistical analysis of a small group of patients, which filled in STAI, showed correlation between stress level and serum CRH levels. Moreover, serum NT and VEGF levels were statistically significant higher. A possible explanation would be acute stress to cause serum CRH level increase, subsequent CRH-R1 down-regulation topically in the skin, following by synergistic action of CRH and NT to cause vascular permeability increase.

Psoriasis is characterized by keratinocyte proliferation and chronic inflammation, but the pathogenesis is still unclear. Dysregulated mitochondria (mt) could lead to reduced apoptosis and extracellular secretion of mtDNA, acting as "innate pathogen" triggering inflammation. Serum was obtained from healthy volunteers and psoriatic patients. Mitochondrial DNA was extracted from the serum and amplified with quantitative PCR (qPCR). Punch biopsies were obtained from lesional and non-lesional psoriatic skin (10 cm apart) and from healthy volunteers, were placed in RNA later and were stored at -80°C until RNA was extracted and cDNA was synthesized; gene expression of uncoupling protein 2 (UCP2), Dynamin-related protein 1 (Drp1) and calcineurin, involved in the regulation of mitochondria function, was detected with qPCR. Mitochondrial DNA was significantly increased (7s, P = 0.0496 and Cytochrome B, CytB, P = 0.0403) in the serum of psoriatic patients (n = 63) as compared to controls (n = 27). Gene expression was significantly reduced for UCP2 (P = 0.0218), Drp1 (P = 0.0001) and calcineurin (P = 0.0001) in lesional psoriatic skin, as compared to non-lesional or control skin. Increased serum extracellular mtDNA in psoriatic patients and decreased expression of mitochondrial regulatory proteins in psoriatic skin suggest increased inflammation and reduced keratinocyte apoptosis, respectively. Inhibitors of mtDNA secretion and/or UCP2 stimulants may be potential treatment options.

Objective Stress precipitates and worsens not only asthma and atopic dermatitis but also acute coronary syndromes (ACSs), which are associated with coronary inflammation. Evidence linking stress to ACS was reviewed and indicated that activation of coronary mast cells (MCs) by stress, through corticotropin-releasing hormone (CRH) and other neuropeptides, contributes to coronary inflammation and coronary artery disease. Data Sources PubMed was searched (2005–2013) for articles using the following keywords: allergies, anaphylaxis, anxiety, coronary arteries, coronary artery disease, C-reactive protein, cytokines, chymase, histamine, hypersensitivity, interleukin-6 (IL-6), inflammation, mast cells, myocardial ischemia, niacin, platelet-activating factor, rupture, spasm, statins, stress, treatment, tryptase, and uroctortin. Study Selections Articles were selected based on their relevance to how stress affects ACS and how it activates coronary MCs, leading to coronary hypersensitivity, inflammation, and coronary artery disease. Results Stress can precipitate allergies and ACS. Stress stimulates MCs through the activation of high-affinity surface receptors for CRH, leading to a CRH-dependent increase in serum IL-6. Moreover, neurotensin secreted with CRH from peripheral nerves augments the effect of CRH and stimulates cardiac MCs to release IL-6, which is elevated in ACS and is an independent risk factor for myocardial ischemia. MCs also secrete CRH and uroctortin, which induces IL-6 release from cardiomyocytes. The presence of atherosclerosis increases the risk of cardiac MC activation owing to the stimulatory effect of lipoproteins and adipocytokines. Conditions such as Kounis syndrome, mastocytosis, and myalgic encephalopathy/chronic fatigue syndrome are particularly prone to coronary hypersensitivity reactions. Conclusion Inhibition of cardiac MCs may be a novel treatment approach.

Psoriasis is a chronic inflammatory skin disorder characterized by keratinocyte hyperproliferation and increased epidermal vascularization, associated with high skin vascular endothelial growth factor (VEGF).1 Neuropeptides have been implicated in psoriasis,2 but their mechanism of action is not well understood. Neurotensin (NT), is a vasoactive peptide,3 which increases histamine release from rodent skin in a mast cell-dependent manner.4 NT also augments the effect of corticotrophin-releasing hormone (CRH), secreted under stress, to increase mast cell-dependent skin vascular permeability.5 We therefore investigated NT serum levels, as well as the expression of genes encoding NT and NT receptor-1 (NTR-1) in the skin of patients with psoriasis and controls. We also studied the effect of NT on VEGF release in human cultured mast cells.

There is increased serum CRH with decreased lesional skin CRHR-1 gene expression in psoriasis and atopic dermatitis, suggesting possible involvement in stress-induced worsening of symptoms.

Corticotropin-releasing hormone (CRH) is secreted under stress and regulates the hypothalamic-pituitary-adrenal axis. However, CRH is also secreted outside the brain where it exerts proinflammatory effects through activation of mast cells, which are increasingly implicated in immunity and inflammation. Substance P (SP) is also involved in inflammatory diseases. Human LAD2 leukemic mast cells express only CRHR-1 mRNA weakly. Treatment of LAD2 cells with SP (0.5-2 μM) for 6 hours significantly increases corticotropin-releasing hormone receptor-1 (CRHR-1) mRNA and protein expression. Addition of CRH (1 μM) to LAD2 cells, which are "primed" with SP for 48 hours and then washed, induces synthesis and release of IL-8, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) 24 hours later. These effects are blocked by pretreatment with an NK-1 receptor antagonist. Treatment of LAD2 cells with CRH (1 μM) for 6 hours induces gene expression of NK-1 as compared with controls. However, repeated stimulation of mast cells with CRH (1 μM) leads to downregulation of CRHR-1 and upregulation in NK-1 gene expression. These results indicate that SP can stimulate mast cells and also increase expression of functional CRHR-1, whereas CRH induces NK-1 gene expression. These results may explain CRHR-1 and NK-1 expression in lesional skin of psoriatic patients.

The peptide substance P (SP) has been implicated in inflammatory conditions, such as psoriasis, where mast cells and VEGF are increased. A relationship between SP and VEGF has not been well studied, nor has any interaction with the proinflammatory cytokines, especially IL-33. Here we report that SP (0.1–10 μM) induces gene expression and secretion of VEGF from human LAD2 mast cells and human umbilical core blood-derived cultured mast cells (hCBMCs). This effect is significantly increased by coadministration of IL-33 (5–100 ng/mL) in both cell types. The effect of SP on VEGF release is inhibited by treatment with the NK-1 receptor antagonist 733,060. SP rapidly increases cytosolic calcium, and so does IL-33 to a smaller extent; the addition of IL-33 augments the calcium increase. SP-induced VEGF production involves calcium-dependent PKC isoforms, as well as the ERK and JNK MAPKs. Gene expression of IL-33 and histidine decarboxylase (HDC), an indicator of mast cell presence/activation, is significantly increased in affected and unaffected (at least 15 cm away from the lesion) psoriatic skin, as compared with normal control skin. Immunohistochemistry indicates that IL-33 is associated with endothelial cells in both the unaffected and affected sites, but is stronger and also associated with immune cells in the affected site. These results imply that functional interactions among SP, IL-33, and mast cells leading to VEGF release contribute to inflammatory conditions, such as the psoriasis, a nonallergic hyperproliferative skin inflammatory disorder with a neurogenic component.

Summary A 38-year-old woman presented with a pronounced increase in symptoms and proliferation of urticaria pigmentosa (UP) after acute psychological stress, which was quantified using the Spielberger’s State–Trait Anxiety Inventory. Immunohistochemical examination of a skin biopsy from a new UP lesion showed a large number of activated mast cells expressing corticotrophin-releasing factor receptor-1 (CRF-R1) and there was high serum CRF. This is the first documented report to our knowledge of UP worsening associated with acute stress, possibly through activation of skin mast-cell CRF-R1.

Background and purpose: Allergic inflammation and autoimmune diseases, such as atopic dermatitis, psoriasis and multiple sclerosis (MS), involve both mast cell and T-cell activation. However, possible interactions between the two and the mechanism of such activations are largely unknown. Experimental approach: Human umbilical cord blood-derived cultured mast cells (hCBMCs) and Jurkat T cells were incubated separately or together, following activation with myelin basic protein (MBP), as well as with or without pretreatment with the flavonoid luteolin for 15 min. The supernatant fluid was assayed for inflammatory mediators released from mast cells and interleukin (IL)-2 release from Jurkat cells. Key results: MBP (10 μM) stimulates hCBMCs to release IL-6, IL-8, transforming growth factor (TGF)-β1, tumour necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), histamine and tryptase (n=6, P

Background Allergic rhinitis is a common problem involving activation of nasal mast cells and irritability. The hypothalamic-pituitary-adrenal (HPA) axis is stimulated in cases of emotional or environmental stress, and mast cells have been implicated in stress-induced immune responses. Objective To investigate whether intranasal challenge of patients allergic to a single antigen would stimulate the HPA axis. Methods Plasma corticotropin and cortisol levels were measured 20, 40, 60, 80, 100, and 120 minutes after intranasal antigen administration in healthy volunteers (n = 3) and in patients with rhinitis who are allergic to Parietaria (n = 10). Results Mean ± SD corticotropin levels were 24.43 ± 14.38 pg/mL in patients compared with 8.83 ± 5.02 pg/mL in controls, and this increase was statistically significant ( P = .049). Patient cortisol levels also increased to a mean ± SD of 8.87 ± 4.90 pg/mL (at 40 minutes) compared with 4.36 ± 1.72 pg/mL in controls ( P = .11 due to 1 outlier). Compared with individual patient prechallenge levels, corticotropin levels increased in 7 patients and cortisol levels increased in 5 at 40 minutes. Conclusion These results suggest that allergic rhinitis may activate the HPA axis. A larger study with additional controls is required for definitive conclusions.

Mast cells are involved in allergic reactions, where they secrete numerous vasoactive, inflammatory and nociceptive mediators in response to immunoglobulin E (IgE) and antigen. However, they have also been implicated in inflammatory conditions, such as painful bladder syndrome/interstitial cystitis (PBS/IC), irritable bowel syndrome (IBS) and migraines, all of which occur more often in women and are exacerbated during ovulation, but are suppressed during pregnancy. Mast cells express high affinity estrogen receptors and estradiol augments their secretion, while tamoxifen inhibits it. Here we report that progesterone (100 nM), but not the structurally related cholesterol, inhibits histamine secretion from purified rat peritoneal mast cells stimulated immunologically or by substance P (SP), an effect also documented by electron microscopy. These results suggest that mast cell secretion may be regulated by progesterone and may explain the reduced symptoms of certain inflammatory conditions during pregnancy.

Background Chronic Fatigue Syndrome (CFS) is a neuroimmunoendocrine disease affecting about 1% of the US population, mostly women. It is characterized by debilitating fatigue for six or more months in the absence of cancer or other systemic diseases. Many CFS patients also have fibromyalgia and skin hypersensitivity that worsen with stress. Corticotropin-releasing hormone (CRH) and neurotensin (NT), secreted under stress, activate mast cells (MC) necessary for allergic reactions to release inflammatory mediators that could contribute to CFS symptoms. Objective To investigate the effect of isoflavones on the action of polyinosinic:polycytidylic acid (poly(I:C)), with or without swim stress, on mouse locomotor activity and inflammatory mediator expression, as well as on human MC activation. Methods Female C57BL/6 mice were randomly divided into four groups: (a) control/no-swim, (b) control/swim, (c) polyinosinic:polycytidylic acid (poly(I:C))/no swim, and (d) polyinosinic:polycytidylic acid (poly(I:C))/swim. Mice were provided with chow low or high in isoflavones for 2 weeks prior to ip injection with 20 mg/kg poly(I:C) followed or not by swim stress for 15 minutes. Locomotor activity was monitored overnight and animals were sacrificed the following day. Brain and skin gene expression, as well as serum levels, of inflammatory mediators were measured. Data were analyzed using the non-parametric Mann-Whitney U-test. Results Poly(I:C)-treated mice had decreased locomotor activity over 24 hours, and increased serum levels of TNF-α, IL-6, KC (IL-8/CXCL8 murine homolog), CCL2,3,4,5, CXCL10, as well as brain and skin gene expression of TNF, IL-6, KC (Cxcl1, IL8 murine homolog), CCL2, CCL4, CCL5 and CXCL10. Histidine decarboxylase (HDC) and NT expression were also increased, but only in the skin, over the same period. High isoflavone diet reversed these effects. Conclusion Poly(I:C) treatment decreased mouse locomotor activity and increased serum levels and brain and skin gene expression of inflammatory mediators. These effects were inhibited by isoflavones that may prove useful in CFS. Electronic supplementary material The online version of this article (doi:10.1186/s12974-014-0168-5) contains supplementary material, which is available to authorized users.

To examine health plan compliance with essential drug benefit regulations in California and Massachusetts and at the federal level.Health plan formulary review and analysis.We analyzed formularies from the 3 largest small group plans in California and Massachusetts, including each state's benchmark plan. With respect to both federal and state regulations, for each health plan, we examined whether the drug was covered, the designated patient cost sharing tier of the drug, and which conditions of reimbursement were applied to the drug.Most drugs included in state and federal mandates are covered by both benchmark and non-benchmark plans. However, health plans are not fully compliant with state and federal regulations. Significant differences among plans relate more to cost sharing and conditions of reimbursement, such as prior authorization, step edits, and quantity limits, than to drug coverage. CONCLUSIONS/POLICY IMPLICATIONS: Because health plans in California and Massachusetts are not fully compliant with state and federal mandates, they will have to adjust their formularies to meet minimum requirements. State policy makers need to balance competing aims of comprehensiveness of coverage and drug affordability. They must consider: (1) choice of benchmark plan -choice of a more generous benchmark plan implies less leverage for negotiating lower prices; and (2) breadth of state mandates which, if they exceed federal mandates, must be paid for by the states.

Acetaminophen is a leading cause of acute liver failure (ALF). Genetic differences might predispose some individuals to develop ALF. In this exploratory study, we evaluated genotype frequency differences among patients enrolled by the ALF Study Group who had developed ALF either intentionally from a single-time-point overdose of acetaminophen (n = 78), unintentionally after chronic high doses of acetaminophen (n = 79), or from causes other than acetaminophen (n = 103). The polymorphisms evaluated included those in genes encoding putative acetaminophen-metabolizing enzymes (UGT1A1, UGT1A6, UGT1A9, UGT2B15, SULT1A1, CYP2E1, and CYP3A5) as well as CD44 and BHMT1. Individuals carrying the CYP3A5 rs776746 A allele were overrepresented among ALF patients who had intentionally overdosed with acetaminophen, with an odds ratio of 2.3 (95% confidence interval, 1.1–4.9; P = 0.034) compared with all other ALF patients. This finding is consistent with the enhanced bioactivation of acetaminophen by the CYP3A5 enzyme. Persons homozygous for the CD44 rs1467558 A allele were also overrepresented among patients who had unintentionally developed ALF from chronic acetaminophen use, with an odds ratio of 4.0 (1.0–17.2, P = 0.045) compared with all other ALF subjects. This finding confirms a prior study that found elevated serum liver enzyme levels in healthy volunteers with the CD44 rs1467558 AA genotype who had consumed high doses of acetaminophen for up to 2 weeks. However, both genetic associations were considered relatively weak, and they were not statistically significant after adjustment for multiple comparisons testing. Nevertheless, both CYP3A5 rs776746 and CD44 rs1467558 warrant further investigation as potential genomic markers of enhanced risk of acetaminophen-induced ALF.

Background Mast cells are involved in allergy and inflammation by the secretion of multiple mediators, including histamine, cytokines, and platelet-activating factor (PAF), in response to different triggers, including emotional stress. PAF has been associated with allergic inflammation, but there are no clinically available PAF inhibitors. Objective To investigate whether PAF could stimulate human mast cell mediator release and whether rupatadine (RUP), a dual histamine-1 and PAF receptor antagonist, could inhibit the effect of PAF on human mast cells. Methods Laboratory of allergic diseases 2 cultured mast cells were stimulated with PAF (0.001, 0.01, and 0.1 μmol/L) and substance P (1 μmol/L) with or without pretreatment with RUP (2.5 and 25 μmol/L), which was added 10 minutes before stimulation. Release of β-hexosaminidase was measured in supernatant fluid by spectrophotoscopy, and histamine, interleukin-8, and tumor necrosis factor were measured by enzyme-linked immunosorbent assay. Results PAF stimulated a statistically significant release of histamine, interleukin-8, and tumor necrosis factor (0.001–0.1 μmol/L) that was comparable to that stimulated by substance P. Pretreatment with RUP (25 μmol/L) for 10 minutes inhibited this effect. In contrast, pretreatment of laboratory of allergic diseases 2 cells with diphenhydramine (25 μmol/L) did not inhibit mediator release, suggesting that the effect of RUP was not due to its antihistaminic effect. Conclusion PAF stimulates human mast cell release of proinflammatory mediators that is inhibited by RUP. This action endows RUP with additional properties in treating allergic inflammation.

Vitiligo is a disorder of depigmentation, for which the pathogenesis is as yet unclear. Interleukin (IL)-8 is a key inflammatory chemokine. We investigated the regulation of IL-8 production in human melanocytes, and the IL-8 serum levels and skin gene expression in patients with vitiligo and in controls. Cultured melanocytes were stimulated for 24 h with tumour necrosis factor (TNF) 100 ng/mL and IL-1β 10 ng/mL, with or without pretreatment with luteolin 50 μmol/L for 30 min, and IL-8 release was measured by ELISA. Serum cytokines were measured by a microbead array. Skin biopsies were taken from healthy subjects (n = 14) as well as from marginal lesional and nonlesional skin from patients with vitiligo (n = 15). IL-8 gene expression was evaluated by quantitative reverse transcriptase PCR. Both TNF and IL-1β stimulated significant IL-8 release (P < 0.01) from melanocytes, whereas pretreatment with luteolin significantly inhibited this effect (P < 0.01). IL-8 gene expression was significantly increased in vitiligo compared with control skin (P < 0.05). IL-8 may be involved in vitiligo inflammation. Inhibition by luteolin of IL-8 release could be useful for vitiligo therapy.

Many children with Autism Spectrum Disorders (ASD) have either family and/or personal history of “allergic symptomatology”, often in the absence of positive skin or RAST tests. These symptoms may suggest mast cell activation by non-allergic triggers. Moreover, children with mastocytosis or mast cell activation syndrome (MCAS), a spectrum of rare diseases characterized by increased number of activated mast cells in many organs, appear to have ASD at a rate tenfold higher (1/10 children) than that of the general population (1/100 children). Mast cell activation by allergic, infectious, environmental and stress-related triggers, especially perinatally, would release pro-inflammatory and neurotoxic molecules. We speculate these could disrupt the gut–blood–brain barriers, thus contributing to brain inflammation and ASD pathogenesis. Increased mast cell responsiveness may define at least a subgroup of ASD subjects, who could benefit from inhibition of mast cell activation.

Autism spectrum disorders (ASD) are a group of pervasive neurodevelopmental disorders diagnosed in early childhood. They are associated with a set of "core symptoms" that include disabilities in social interaction skills, verbal and non-verbal communication, as well as repetitive and stereotypic behaviors. There is no definite pathogenetic mechanism or diagnostic tests. Many children with ASD also have "allergic-like" symptoms, but test negative implying mast cell activation by non-allergic triggers. We measured by Milliplex arrays serum levels of 3 neuropeptides that could stimulate mast cells in children with autistic disorder (n = 19; 16 males and 3 females; mean age 3.0 ± 0.4 years) and healthy, unrelated controls (n = 16; 13 males and 3 females; mean age 3 ± 1.2 years). Only neurotensin (NT) was significantly increased from 60.5 ± 6.0 pg/ml in controls to 105.6 ± 12.4 pg/ml in autistic disorder (p = 0.004). There was no statistically significant difference in the serum levels of β-endorphin or substance P (SP). NT could stimulate immune cells, especially mast cells, and/or have direct effects on brain inflammation and ASD.

Atopic dermatitis (AD) is a chronic highly pruritic inflammatory skin disease, which has increased considerably in industrialized countries.1 AD is characterized by allergic inflammation in the skin,2 and has a strong association with other atopic disorders such as asthma and allergic rhinitis.1 Thymic stromal lymphopoietin (TSLP) is a cytokine closely related to IL-7,3 mostly produced by epithelial cells and keratinocytes in response to infection, trauma and inflammation. TSLP mediates Th2 responses through activation of dendritic cells, and has emerged as an important regulator of allergic inflammation. TSLP also activates mast cells to release several cytokines, such as IL-5, IL-6 and IL-13, but only in the presence of IL-1 and TNF.4 One paper reported increased TSLP protein expression in lesional skin of AD patients.3 We investigated gene expression of TSLP in lesional skin biopsies and serum levels of AD patients (n=9, mean age ± 1 SD=41.33 ± 21.40; three males, six females) compared to controls (n=9, mean age ± 1 SD=42.8 ± 18.1; three males, six females). All 10 mm punch skin biopsies were obtained (abdomen, arm, back, hand, and neck; there was no apparent correlation between any of these sites and TSLP levels) from patients who had mild, localized, chronic AD, and from controls (back). No subject had received any anti-inflammatory medication for 15 days prior to the biopsy. Contact dermatitis, asteatotic eczema or other types of dermatitis were excluded. The Medical Ethics Committee of University of Athens Medical School approved this protocol. All participants gave their written informed consent according to the Declaration of Helsinki Principles. Patients were free of other major medical conditions except allergic rhinitis as noted. All biopsies were placed in RNAlater (Ambion Inc., Austin, TX) and stored at −20 °C. Total RNA from skin biopsies was extracted by TRIzol isolation method (Invitrogen, Carlsbad, CA). cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Quantitative PCR was performed using Taqman probes (Applied Biosystems, Foster City, CA) on a 7300 Real Time PCR System (Applied Biosystems). For qPCR, the cDNA was reverse-transcribed from 300 ng RNA from each sample. Relative mRNA levels were normalized to the GAPDH endogenous control. Data were analyzed with the non-parametric Mann-Whitney U test, since values did not conform to a normal distribution. Serum measurements are reported in pg/ml. Relative TSLP gene expression was significantly increased in the lesional AD skin samples (median=4.93; range=2.27–23.99) as compared to controls (median=0.84; range=0.49–1.85) (Fig. 1a, p

Findings obtained using animal models have often failed to reflect the processes involved in human disease. Moreover, human cultured cells do not necessarily function as their actual tissue counterparts. Therefore, there is great demand for sources of human progenitor cells that may be directed to acquire specific tissue characteristics and be available in sufficient quantities to carry out functional and pharmacological studies. A case in point is the mast cell, well known for its involvement in allergic reactions, but also implicated in inflammatory diseases. Mast cells can be activated by allergens, anaphylatoxins, immunoglobulin-free light chains, superantigens, neuropeptides, and cytokines, leading to selective release of mediators. These could be involved in many inflammatory diseases, such as asthma and atopic dermatitis, which worsen by stress, through activation by local release of corticotropin-releasing hormone or related peptides. Umbilical cord blood and cord matrix-derived mast cell progenitors can be separated magnetically and grown in the presence of stem cell factor, interleukin-6, interleukin-4, and other cytokines to yield distinct mast cell populations. The recent use of live cell array, with its ability to study such interactions rapidly at the single-cell level, provides unique new opportunities for fast output screening of mast cell triggers and inhibitors.

Background Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. Objective We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. Methods Human umbilical cord blood–derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. Results Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. Conclusion Human mast cell degranulation requires mitochondrial dynamics, also implicated in AD.

Tumor necrosis factor (TNF) is involved in T cell-dependent diseases such as psoriasis, and in T cell activation by mast cells, which can both store and de novo synthesize TNF. Yet, regulation of TNF secretion is unknown hampering our understanding of how mast cells regulate allergic, immune and inflammatory processes. Here we show that stimulated human LAD2 leukemic and umbilical cord-derived cultured mast cell (hCBMCs) by SP or IgE/anti-IgE induces degranulation and prestored TNF release, which is accompanied by mitochondrial fission. Images of Confocal microscopy and electronic transmitted microscopy show fragmented mitochondria and relocation from a perinuclear distribution to the cell surface. Moreover, treatment with a mitochondrial fission blocker inhibits degranulation, indicating the necessarily of mitochondria fission in mast cell secretion. Besides mitochondria morphological changes, SP stimulation of LAD2 and hCBMCs increased expression (8 hr) of the mitochondrial uncoupling protein 2 (UCP2), which regulates production of ROS and intracellular calcium, leading to inhibition of subsequent re-stimulation by SP, when is enough for mast cells to recover. Moreover, UCP2 gene expression was reduced, as compared to control normal skin, in affected psoriatic skin, an inflammatory diseases in which mast cells play important role. These findings indicate that mitochondria play a key regulatory role in mast cell TNF secretion and in psoriasis.

Mast cells are important effector cells in IgE- and Th2-mediated immune responses. Mast cells are located close to substance P (SP) containing nerves. SP can activate connective tissue-type mast cells, but not mucosal mast cells. Here we examined the effect of SP on (a) mediator release from human umbilical cord blood-derived cultured mast cells (hCBMCs) grown in the presence of stem cell factor (SCF), IL-6 plus IL-4; (b) IL-2 from activated T cells alone or together with mast cells. SP dose-dependently released histamine, tryptase and IL-8 (at 10 μM, release was 72.8 ± 7.42%, 5390 ± 954 ng/106 cells and 1140 ± 103 pg/106cells, respectively) compared to controls (6.3 ± 2.4 %, 397 ± 235 ng/106 cells and 583 ± 40 pg/106 cells, respectively). Addition of mast cells to T cells, activated by anti-CD3/anti-CD28, for 24 h stimulated IL-2 production from 38.4 ± 9.2 pg/ml to 1450 ± 63 pg/ml (p90%) both SP-induced hCBMCs activation and T cell IL-2 release. Mast cells can superactivate T cells and could constitute a new therapeutic target for diseases such as multiple sclerosis. Luteolin could be used to inhibit this interaction. This work was supported by Theta Biomedical Consulting and Development Co., Inc. (Brookline, MA).

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by difficulties in communication, cognitive and learning deficits, as well as stereotypic behaviors. For the majority of cases there are no reliable biomarkers or distinct pathogenesis. However, increasing evidence indicates ASD may be associated with some immune dysregulation, and may have a neuroimmune component. We recently showed that the peptide neurotensin (NT) is increased in autistic children. We now show that NT induces release of extracellular mitochondrial DNA (mtDNA) that could act as "autoimmune" trigger. We further show that serum from young autistic patients contains mtDNA (n = 20; cytochrome B, p = 0.0002 and 7S, p = 0.006), and anti-mitochondrial antibody Type 2 (n = 14; p = 0.001) as compared to normally developing, unrelated controls (n = 12). Extracellular blood mtDNA and other components may characterize an autistic endophenotype and may contribute to its pathogenesis by activating autoimmune responses.