Increasing Sphingolipid Synthesis Alleviates Airway Hyperreactivity.

Impaired sphingolipid synthesis is linked genetically to childhood asthma and functionally to airway hyperreactivity (AHR). The objective was to investigate whether sphingolipid synthesis could be a target for asthma therapeutics. The effects of GlyH-101 and fenretinide via modulation of de novo sphingolipid synthesis on AHR was evaluated in mice deficient in SPT (serine palmitoyl-CoA transferase), the rate-limiting enzyme of sphingolipid synthesis. The drugs were also used directly in human airway smooth-muscle and epithelial cells to evaluate changes in de novo sphingolipid metabolites and calcium release. GlyH-101 and fenretinide increased sphinganine and dihydroceramides (de novo sphingolipid metabolites) in lung epithelial and airway smooth-muscle cells, decreased the intracellular calcium concentration in airway smooth-muscle cells, and decreased agonist-induced contraction in proximal and peripheral airways. GlyH-101 also decreased AHR in SPT-deficient mice in vivo. This study identifies the manipulation of sphingolipid synthesis as a novel metabolic therapeutic strategy to alleviate AHR.

Comparative systems pharmacology of HIF stabilization in the prevention of retinopathy of prematurity.

Retinopathy of prematurity (ROP) causes 100,000 new cases of childhood blindness each year. ROP is initiated by oxygen supplementation necessary to prevent neonatal death. We used organ systems pharmacology to define the transcriptomes of mice that were cured of oxygen-induced retinopathy (OIR, ROP model) by hypoxia-inducible factor (HIF) stabilization via HIF prolyl hydroxylase inhibition using the isoquinolone Roxadustat or the 2-oxoglutarate analog dimethyloxalylglycine (DMOG). Although both molecules conferred a protective phenotype, gene expression analysis by RNA sequencing found that Roxadustat can prevent OIR by two pathways: direct retinal HIF stabilization and induction of aerobic glycolysis or indirect hepatic HIF-1 stabilization and increased serum angiokines. As predicted by pathway analysis, Roxadustat rescued the hepatic HIF-1 knockout mouse from retinal oxygen toxicity, whereas DMOG could not. The simplicity of systemic treatment that targets both the liver and the eye provides a rationale for protecting the severely premature infant from oxygen toxicity.

Mast cells in the periprosthetic breast capsule.

BACKGROUND: Symptomatic capsular contracture occurs in about 10 % of primary breast augmentations and in more than double that rate in reconstruction after mastectomy, especially in the setting of radiation. Mast cells, traditionally associated with immune response and inflammation, secrete profibrotic mediators and may play a role in capsule formation and contracture. We analyzed the mast cell and fibroblast populations in breast capsule tissue from patients who underwent capsular excision. METHODS: Capsule tissue was collected from patients who underwent exchange of tissue expanders for permanent implants, revision of reconstruction, or revision augmentation. Breast capsule tissues were prepared for histological analyses of mast cells, fibroblasts, and collagen. Mast cells and fibroblasts were isolated from capsule tissue and screened for mediators and receptor expression. RESULTS: In breast capsule tissue, the average numbers of mast cells and fibroblasts were 9 ± 1/mm(2) and 33 ± 10/mm(2), respectively. There were significantly more mast cells on the posterior side than on the anterior side of the capsule tissue (12 ± 2 vs. 6 ± 1/mm(2), p < 0.01). Baker grade IV capsules had an increased number of fibroblasts compared to Baker grade I capsules (93 ± 9 vs. 40 ± 19/mm(2), p < 0.001). In breast capsule tissue, mast cells contained renin, histamine, and TGF-ß, and their respective receptors, AT1R, H1R, and TGF-ßRI were expressed by fibroblasts. CONCLUSION: These data indicate that within breast capsule tissue mast cells contain mediators that may activate neighboring fibroblasts. Understanding the role of mast cells in pathologic periprosthetic breast capsule formation may lead to novel therapies to prevent and treat capsular contracture. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266.

Gut bacteria-derived serotonin promotes immune tolerance in early life.

The gut microbiota promotes immune system development in early life, but the interactions between the gut metabolome and immune cells in the neonatal gut remain largely undefined. Here, we demonstrate that the neonatal gut is uniquely enriched with neurotransmitters, including serotonin, and that specific gut bacteria directly produce serotonin while down-regulating monoamine oxidase A to limit serotonin breakdown. We found that serotonin directly signals to T cells to increase intracellular indole-3-acetaldehdye and inhibit mTOR activation, thereby promoting the differentiation of regulatory T cells, both ex vivo and in vivo in the neonatal intestine. Oral gavage of serotonin into neonatal mice resulted in long-term T cell-mediated antigen-specific immune tolerance toward both dietary antigens and commensal bacteria. Together, our study has uncovered an important role for specific gut bacteria to increase serotonin availability in the neonatal gut and identified a function of gut serotonin in shaping T cell response to dietary antigens and commensal bacteria to promote immune tolerance in early life.

Mast cell isolation from the immature rat brain.

Mast cells are immune cells of hematopoietic origin that circulate as precursor cells prior to migration into vascularized tissues where they mature and undergo terminal differentiation in response to different cytokines within the local environment. Mast cells are well known as important regulators of inflammatory processes in peripheral tissues and recent studies support the involvement of mast cells in mediating the inflammatory response to cerebral hypoxia-ischemia in both the neonatal and adult brain. To better study mast cell function in vivo, it is important to be able to identify their environment-specific phenotype, as well as to study their interaction with other neural cells in vitro. Previous such studies of mast cells have relied on mast cells isolated from gut or bone marrow, or on a number of mast cell lines, all of which may behave differently from brain mast cells. The purpose of this study was to develop a technique for the isolation of mast cells from neonatal rat brain and to characterize these cells following hypoxia and hypoxia-ischemia. We adapted a previously described technique of coupling an antibody to the mast cell-specific FcεR1 receptor to a MACS microbead for the selective removal of intact mast cells from a neonatal brain preparation. We have isolated toluidine blue-positive brain mast cells that provide substrate for both protein analysis and in vitro studies. These cells express proteins previously used to specifically identify microglia in the brain, Iba-1 and coronin-1a. A subpopulation of mast cells in vivo also expresses Iba-1. Thus, we report a novel method for isolation of brain mast cells suitable for the study of mast cell phenotype under a variety of conditions. Further, we suggest that the use of proteins such as Iba-1 for the identification of microglia in the brain includes the caveat that mast cells may also be detected.

The mast cell exosome-fibroblast connection: A novel pro-fibrotic pathway.

Introduction: In addition to the traditional activation of resident receptors by release of local mediators, new evidence favors the existence of exosomes in cell-to-cell communication that mediates delivery of specific cargo to modulate recipient cell function. We report that mast cell exosomes are an additional source of pro-fibrotic substances and constitute a unique pathway for the generation of excess collagen. Methods: We use primary human lung fibroblasts (HLFs) to demonstrate the uptake of labeled exosomes isolated from the human mast cell line HMC-1 (MC-EXOs), previously shown to contain protein cargo in common with human mast cell exosomes. Results: The MC-EXO uptake by HLF is to the cytosol and increases both proline hydroxylation in HLF lysate and secreted collagen, within 24 h, which is sustained over 72 h, the same time required for transforming growth factor-ß (TGF-ß) to activate collagen synthesis in the HLFs. Unlike TGF-ß, MC-EXO uptake does not induce fibrillar gene activation or invoke the Smad-nuclear transcription pathway. We show that MC-EXO uptake and TGF-ß have an additive effect on collagen synthesis in HLF and postulate that MC-EXO uptake by HLFs is a contributing factor to excess collagen synthesis and represents a unique paradigm for understanding fibrosis. Discussion: It is known that, in the lungs, mast cells are more activated and increase in number with inflammation, injury and viral infection associated with fibrosis. With the reported increased incidence of post-COVID-pulmonary fibrosis (PCPF), data from patients with severe COVID-19 are presented that show an increase in the mast cell number in lung parenchyma, the site of PCPF. Our findings provide a rationale for targeting multiple fibrogenic pathways in the management of lung fibrosis and the use of mast cell exosomes as a biomarker for the prognostic and diagnostic management of evolving fibrotic lung disease.

Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model.

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.

Aldehyde dehydrogenase activation prevents reperfusion arrhythmias by inhibiting local renin release from cardiac mast cells.

BACKGROUND: Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia. METHODS AND RESULTS: We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC (two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillation duration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A(2b)/A(3) receptors and activation and inhibition of protein kinase Cepsilon (PKCepsilon) mimicked and prevented, respectively, the anti-RAS effects of IPC. Moreover, activation of A(2b)/A(3) receptors or activation of PKCepsilon prevented degranulation and renin release elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehyde dehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects of IPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1. Notably, PKCepsilon and ALDH2 were both activated by A(2b)/A(3) receptor stimulation in HMC-1, and PKCepsilon inhibition prevented ALDH2 activation. CONCLUSIONS: The results uncover a signaling cascade initiated by A(2b)/A(3) receptors, which triggers PKCepsilon-mediated ALDH2 activation in cardiac mast cells, contributing to IPC-induced cardioprotection by preventing mast cell renin release and the dysfunctional consequences of local RAS activation. Thus, unlike classic IPC in which cardiac myocytes are the main target, cardiac mast cells are the critical site at which the cardioprotective anti-RAS effects of IPC develop.

IL-10 suppresses calcium-mediated costimulation of receptor activator NF-kappa B signaling during human osteoclast differentiation by inhibiting TREM-2 expression.

Induction of effective osteoclastogenesis by RANK (receptor activator of NF-kappaB) requires costimulation by ITAM-coupled receptors. In humans, the TREM-2 (triggering receptor expressed on myeloid cells 2) ITAM-coupled receptor plays a key role in bone remodeling, as patients with TREM-2 mutations exhibit defective osteoclastogenesis and bone lesions. We have identified a new rapidly induced costimulatory pathway for RANK signaling that is dependent on TREM-2 and mediated by calcium signaling. TREM-2-dependent calcium signals are required for RANK-mediated activation of calcium/calmodulin-dependent protein kinase (CaMK)II and downstream MEK and ERK MAPKs that are important for osteoclastogenesis. IL-10 inhibited RANK-induced osteoclastogenesis and selectively inhibited calcium signaling downstream of RANK by inhibiting transcription of TREM-2. Down-regulation of TREM-2 expression resulted in diminished RANKL-induced activation of the CaMK-MEK-ERK pathway and decreased expression of the master regulator of osteoclastogenesis NFATc1. These findings provide a new mechanism of inhibition of human osteoclast differentiation. The results also yield insights into crosstalk between ITAM-coupled receptors and heterologous receptors such as RANK, and they identify a mechanism by which IL-10 can suppress cellular responses to TNFR family members.

Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction.

We previously reported that mast cells express renin, the rate-limiting enzyme in the renin-angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157. Local generation of angiotensin (ANG II) from mast cell renin elicited bronchial smooth muscle contraction mediated by ANG II type 1 receptors (AT(1)R). In a guinea pig model of immediate type hypersensitivity, anaphylactic mast cell degranulation in bronchial rings resulted in ANG II-mediated constriction. As in rat bronchial rings, bronchoconstriction (BC) was inhibited by a renin inhibitor, an AT(1)R blocker, and a mast cell stabilizer. Anaphylactic release of renin, histamine, and beta-hexosaminidase from mast cells was confirmed in the effluent from isolated, perfused guinea pig lung. To relate the significance of this finding to humans, mast cells were isolated from macroscopically normal human lung waste tissue specimens. Sequence analysis of human lung mast cell RNA showed 100% homology between human lung mast cell renin and kidney renin between exons 1 and 10. Furthermore, the renin protein expressed in lung mast cells was enzymatically active. Our results demonstrate the existence of an airway renin-angiotensin system triggered by release of mast-cell renin. The data show that locally produced ANG II is a critical factor governing BC, opening the possibility for novel therapeutic targets in the management of airway disease.

Interaction between sensory C-fibers and cardiac mast cells in ischemia/reperfusion: activation of a local renin-angiotensin system culminating in severe arrhythmic dysfunction.

Renin, the rate-limiting enzyme in the activation of the renin-angiotensin system (RAS), is synthesized and stored in cardiac mast cells. In ischemia/reperfusion, cardiac sensory nerves release neuropeptides such as substance P that, by degranulating mast cells, might promote renin release, thus activating a local RAS and ultimately inducing cardiac dysfunction. We tested this hypothesis in whole hearts ex vivo, in cardiac nerve terminals in vitro, and in cultured mast cells. We found that substance P-containing nerves are juxtaposed to renin-containing cardiac mast cells. Chemical stimulation of these nerves elicited substance P release that was accompanied by renin release, with the latter being preventable by mast cell stabilization or blockade of substance P receptors. Substance P caused degranulation of mast cells in culture and elicited renin release, and both of these were prevented by substance P receptor blockade. Ischemia/reperfusion in ex vivo hearts caused the release of substance P, which was associated with an increase in renin and norepinephrine overflow and with sustained reperfusion arrhythmias; substance P receptor blockade prevented these changes. Substance P, norepinephrine, and renin were also released by acetaldehyde, a known product of ischemia/reperfusion, from cardiac synaptosomes and cultured mast cells, respectively. Collectively, our findings indicate that an important link exists in the heart between sensory nerves and renin-containing mast cells; substance P released from sensory nerves plays a significant role in the release of mast cell renin in ischemia/reperfusion and in the activation of a local cardiac RAS. This culminates in angiotensin production, norepinephrine release, and arrhythmic cardiac dysfunction.

Pressure activates epidermal growth factor receptor leading to the induction of iNOS via NFkappaB and STAT3 in human proximal tubule cells.

Ureteral obstruction leads to increased pressure and inducible nitric oxide synthase (iNOS) expression. This study examined the involvement of epidermal growth factor (EGF) receptor (EGFR), nuclear factor-kappaB (NFkappaB), and signal transducers and activators of transcription 3 (STAT3) in iNOS induction in human proximal tubule (HKC-8) cells in response to pressure or EGF. HKC-8 cells were subjected to 60 mmHg pressure or treated with EGF for 0-36 h. iNOS was more rapidly induced in response to EGF than pressure. The addition of EGFR, NFkappaB, and STAT3 inhibitors significantly suppressed pressure- or EGF-stimulated iNOS mRNA and protein expression. Analysis of the activated states of EGFR, NFkappaB p65, and STAT3 after exposure to both stimuli demonstrated phosphorylation within 2.5 min. Anti-EGF antibody inhibited iNOS induction in pressurized HKC-8 cells, providing evidence that endogenous EGF mediates the response to pressure. In ureteral obstruction, when pressure is elevated, phosphorylated EGFR was detected in the apical surface of the renal tubules, validating the in vitro findings. These data indicate that EGFR, NFkappaB, and STAT3 are required for human iNOS gene induction in response to pressure or EGF, indicating a similar mechanism of activation.

Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release, and arrhythmias in ischemia/reperfusion.

Having identified renin in cardiac mast cells, we assessed whether its release leads to cardiac dysfunction. In Langendorff-perfused guinea pig hearts, mast cell degranulation with compound 48/80 released Ang I-forming activity. This activity was blocked by the selective renin inhibitor BILA2157, indicating that renin was responsible for Ang I formation. Local generation of cardiac Ang II from mast cell-derived renin also elicited norepinephrine release from isolated sympathetic nerve terminals. This action was mediated by Ang II-type 1 (AT1) receptors. In 2 models of ischemia/reperfusion using Langendorff-perfused guinea pig and mouse hearts, a significant coronary spillover of renin and norepinephrine was observed. In both models, this was accompanied by ventricular fibrillation. Mast cell stabilization with cromolyn or lodoxamide markedly reduced active renin overflow and attenuated both norepinephrine release and arrhythmias. Similar cardioprotection was observed in guinea pig hearts treated with BILA2157 or the AT1 receptor antagonist EXP3174. Renin overflow and arrhythmias in ischemia/reperfusion were much less prominent in hearts of mast cell-deficient mice than in control hearts. Thus, mast cell-derived renin is pivotal for activating a cardiac renin-angiotensin system leading to excessive norepinephrine release in ischemia/reperfusion. Mast cell-derived renin may be a useful therapeutic target for hyperadrenergic dysfunctions, such as arrhythmias, sudden cardiac death, myocardial ischemia, and congestive heart failure.

Immediate hypersensitivity elicits renin release from cardiac mast cells.

BACKGROUND: We recently reported that murine and cavian heart mast cells are a unique extrarenal source of renin. Ischemia/reperfusion releases this renin leading to local angiotensin formation and norepinephrine release. As mast cells are a primary target of hypersensitivity, we assessed whether anaphylactic mast cell degranulation also results in renin and norepinephrine release. METHODS: Hearts isolated from presensitized guinea pigs were challenged with antigen. RESULTS: Cardiac anaphylaxis was characterized by mast cell degranulation, evidenced by beta-hexosaminidase release and associated with renin and norepinephrine release. Mast cell stabilization with cromolyn or lodoxamide markedly attenuated the release of beta-hexosaminidase, renin and norepinephrine. Renin inhibition with BILA2157 did not affect mast cell degranulation, but attenuated norepinephrine release. CONCLUSIONS: Our findings disclose that immediate-type hypersensitivity elicits renin release from mast cells, activating a local renin-angiotensin system, thereby promoting norepinephrine release. As renin is stored in human heart mast cells, allergic reactions could initiate renin release, leading to local angiotensin formation and hyperadrenergic dysfunction.

Histamine H3-receptor signaling in cardiac sympathetic nerves: Identification of a novel MAPK-PLA2-COX-PGE2-EP3R pathway.

We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.

Activation of a renin-angiotensin system in ischemic cardiac sympathetic nerve endings and its association with norepinephrine release.

We had reported that in the ischemic heart, locally formed bradykinin (BK) and angiotensin II (Ang II) activate B2- and AT1-receptors on sympathetic nerve terminals (SNE), promoting reversal of the norepinephrine (NE) transporter in an outward direction (i.e., carrier-mediated NE release). Although both BK and Ang II contribute to ischemic NE release, Ang II is likely to play a more important role. Since BK is formed by ischemic SNE, we questioned whether cardiac SNE also contribute to local Ang II formation, in addition to being a target of Ang II. SNE were isolated from surgical specimens of human right atrium and incubated in ischemic conditions. These SNE released large amounts of endogenous NE via a carrier-mediated mechanism, as evidenced by the inhibitory effect of desipramine on this process. Moreover, two renin inhibitors, pepstatin-A and BILA 2157 BS, the ACE inhibitor enalaprilat and the AT1-receptor antagonist EXP3174 prevented ischemic NE release. Western blot analysis revealed the presence of renin in cardiac SNE. Renin abundance increased more than three-fold during ischemia. Thus, renin is present in cardiac SNE and is activated during ischemia, eventually culminating in Ang II formation, stimulation of AT1-receptors and carrier-mediated NE release. Our findings uncover a novel autocrine mechanism, by which Ang II, formed at SNE in myocardial ischemia, elicits carrier-mediated NE release by activating prejuntional AT1-receptors.

Skin of patients with large/giant congenital melanocytic nevi shows increased mast cells.

Nevocytes (NC) and mastocytes (MC) have different progenitors but share stem cell factor as regulator/activator of NC and for differentiation/proliferation of MC. Both cell types express stem cell factor receptor CD117. We hypothesize that large/giant congenital melanocytic nevi (L/GCMN) may associate with MC hyperplasia. Forty-nine L/GCMN were examined, 12 samples from uninvolved skin of L/GCMN patients and 6 control skin samples studied with Giemsa and immunohistochemistry for CD117 and MC-tryptase. Picrosirius red (PR) was used to assess fibrosis. Digital images were used to count MC/mm(2) using ImageJ software. Western blot (WB) for MC-tryptase in 12 GCMN and 12 non-nevus samples was performed. Analysis of variance (Tukey) and Pearson statistical tests were applied. Increased MCs were observed in nevus tissue (75.1 ± 35.3 MCs/mm(2)) and in uninvolved skin (53.74 ± 27.7 MC/ mm(2)). P  =  0.109 from patients with L/GCMN, compared with controls from individuals without L/GCMN (28.74 ± 8.4 MC/mm(2)); P  =  0.001 supported by results of WB analysis for tryptase. A positive trend toward correlation of MC numbers with fibrosis, assessed by PR staining fell short of statistical significance (r  =  0.245; P  =  0.086); no difference in fibrosis was found between nevus and non-nevus skin from patients with L/GCMN (P  =  0.136). We found a higher density of MC, both in normal-appearing skin and nevus areas of L/GCMN patients, compared with control skin samples from individuals without nevi. Given the abnormal wound healing and allergic reactions described in L/GCMN patients, these findings suggest a potential role for MC in the biology of L/GCMN, making them a potential target for therapeutic intervention.

Ratio imaging: practical considerations for measuring intracellular Ca2+ and pH in living cells.

This chapter discusses the use of ratiometric fluorescent probes for measuring intracellular pH (pHi) and Cai(2+) concentration at the single cell level. The development of sensitive and stable probes for monitoring pHi and Cai(2+) in living cells has provided the scientists with invaluable tools for studying a multitude of cellular processes. These probes afford a noninvasive and semiquantitative assessment of pHi and Cai(2+), eliminating the need to impale cells with microelectrodes. The development and availability of membrane permeant Cai(2+)- and pH-specific fluorescent probes coupled to major advances in the technology and design of low-light-level charge-coupled devices geared toward biological applications, and improved microscope optics, have made it possible to visualize a two-dimensional fluorescence signal that is related to Cai(2+) and pHi. The chapter describes the basis for using dual excitation ratio imaging and tries to provide a framework for understanding and developing the technique for investigating the roles of Cai(2+) and pHi in cellular processes. The technique of quantitative ratio imaging for the measurement of pHi and Cai(2+) has revolutionized the field of cell physiology. Using the proper equipment and choosing the right dyes for the experimental needs should provide reliable and reproducible results. More importantly, the amount of data produced from each experiment, when analyzing pHi and Cai(2+) on an individual cell basis, yields valuable information on the heterogeneity of cellular responses.

Impaired sphingolipid synthesis in the respiratory tract induces airway hyperreactivity.

Asthma is a clinically heterogeneous genetic disease, and its pathogenesis is incompletely understood. Genome-wide association studies link ORM (yeast)-Like protein isoform 3 [corrected] (ORMDL3), a member of the ORM gene family, to nonallergic childhood-onset asthma. Orm proteins negatively regulate sphingolipid (SL) synthesis by acting as homeostatic regulators of serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme of de novo SL synthesis, but it is not known how SPT activity or SL synthesis is related to asthma. The present study analyzes the effect of decreased de novo SL synthesis in the lung on airway reactivity after administration of myriocin, an inhibitor of SPT, and in SPT heterozygous knockout mice. We show that, in both models, decreased de novo SL synthesis increases bronchial reactivity in the absence of inflammation. Decreased SPT activity affected intracellular magnesium homeostasis and altered the bronchial sensitivity to magnesium. This functionally links decreased de novo SL synthesis to asthma and so identifies this metabolic pathway as a potential target for therapeutic interventions.