CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes ... more CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes virus entry mediator (HVEM) activates NK cells for cytokine production and cytolytic function via CD160. Fc-fusions are a well-established class of therapeutics, where the Fc domain provides additional biological and pharmacological properties to the fusion protein including enhanced serum t 1/2 and interaction with Fc receptor-expressing immune cells. We evaluated the specific function of HVEM in regulating CD160-mediated NK cell effector function by generating a fusion of the HVEM extracellular domain with human IgG1 Fc bearing CD16-binding mutations (Fc*) resulting in HVEM-(Fc*). HVEM-(Fc*) displayed reduced binding to the Fc receptor CD16 (i.e., Fc-disabled HVEM), which limited Fc receptor-induced responses. HVEM-(Fc*) functional activity was compared with HVEM-Fc containing the wild type human IgG1 Fc. HVEM-(Fc*) treatment of NK cells and PBMCs caused greater IFN-g production, enhanced cytotoxicity, reduced NK fratricide, and no change in CD16 expression on human NK cells compared with HVEM-Fc. HVEM-(Fc*) treatment of monocytes or PBMCs enhanced the expression level of CD80, CD83, and CD40 expression on monocytes. HVEM-(Fc*)-enhanced NK cell activation and cytotoxicity were promoted via cross-talk between NK cells and monocytes that was driven by cell-cell contact. In this study, we have shown that soluble Fc-disabled HVEM-(Fc*) augments NK cell activation, IFN-g production, and cytotoxicity of NK cells without inducing NK cell fratricide by promoting cross-talk between NK cells and monocytes without Fc receptor-induced effects. Soluble Fc-disabled HVEM-(Fc*) may be considered as a research and potentially therapeutic reagent for modulating immune responses via sole activation of HVEM receptors.
Background Gangliosides are glycosphingolipids highly enriched in the brain, with important roles... more Background Gangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson’s disease and Huntington’s disease. In models of both diseases and other conditions, administration of GM1—one of the most abundant gangliosides in the brain—provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration. Methods In vitro studies we...
Interleukin (IL)-15 overexpression in eosinophilic gastrointestinal disorders is reported, but IL... more Interleukin (IL)-15 overexpression in eosinophilic gastrointestinal disorders is reported, but IL-15 0 s role in promoting eosinophilic gastroenteritis is largely unknown. Therefore, we generated enterocyte-overexpressed IL-15 transgenic mice using Fabpi promoter. The Fabpi-IL-15 (iIL-15) transgenic mice showed induced IL-15 levels in the jejunum with a marked increase in jejunum eosinophils. However, no induction of eosinophilia in the blood or any other gastrointestinal segment was observed. Eosinophilia in the jejunum villus was substantially higher in iIL-15 mice compared to wild-type mice. In addition, goblet cell hyperplasia was also observed in the jejunum of iIL-15 mice. Furthermore, a significant correlation between induced IL-15 transcript and the IL-18 transcripts was observed. Therefore, to further understand the role of IL-18 in IL-15 mice associated gastrointestinal disorders, we generated iIL-15/IL-18Ra À/À mice. Using these mice, we found that IL-18 has an important role in promoting IL-15-induced eosinophilia. As intestinal IL-15 overexpression is reported in food intolerance, we examined OVA intolerance in iIL-15 mice. The OVA-sensitized and challenged iIL-15 mice experienced weight loss, diarrhea and eosinophilia in the jejunum. Taken together, our findings demonstrate that intestinal IL-15 overexpression induces IL-18-dependent eosinophilia and immunoglobulins in the intestine that promotes food allergic responses.
Journal of Allergy and Clinical Immunology, Apr 30, 2010
Background: In human basophils from different subjects, maximum IgE-mediated histamine release an... more Background: In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FceRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRb/FceRIa is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment. Objective: We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgEmediated histamine release and disproportionately alters the relative presence of FcRb and FceRIa. Method: Syk, FceRIa, and FcRb expression was monitored during the treatment of subjects with omalizumab. Results: Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRb/FceRIa ratio in patients treated with omalizumab. Conclusions: In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FceRI a and b subunits in basophils is influenced by factors extrinsic to the cell. (J Allergy Clin Immunol 2010;125:902-8.)
RATIONALE: Prescribing self-injectable epinephrine (SIE) is appropriate for patients with an incr... more RATIONALE: Prescribing self-injectable epinephrine (SIE) is appropriate for patients with an increased risk of anaphylaxis, but there is no current consensus regarding the use of SIE prescriptions for patients on immunotherapy (IT). This study investigates current practices among allergists and those in-training on writing SIE prescriptions for IT patients. METHODS: A survey was sent to allergist-immunologists by email. Demographic information was obtained as well as information on their experience with IT and the role of SIE prescriptions for IT patients. Approval was obtained from the Long Island College Hospital IRB. RESULTS: 248 surveys were returned and 5 were excluded because they were incomplete. The majority of respondents were out of training (90%). Only 34.9% prescribe SIE to all of their patients on IT. 14.4% do not prescribe SIE for any of their IT patients. Of the remaining 50.6% of respondents, the three largest categories of patients prescribed SIE include: those with a history of previous systemic reaction, those on Omalizumab or those on Omalizumab and venom IT. Respondents who prescribe SIE for all of their patients have fewer years in practice (avg 14.5 years, range 0-44 years) compared to those who do not prescribe (avg 22 years, range 1-40 years) SIE. Interestingly, of those who prescribe SIE to all their IT patients, 61% give IT even if the patient fails to bring their SIE to their appointment. CONCLUSIONS: Among this sample of allergists there was a wide range of practices. Specific SIE prescribing recommendations for patients on IT may be helpful to practicing allergists.
Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activat... more Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.
Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immuni... more Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein-coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RT-PCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a-induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c-kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c-kit failed to inhibit ASM cell-mediated enhancement of mast cell degranulation. However, dexamethasonetreated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a-induced mast cell degranulation when compared with untreated cells. These findings suggest that cell-cell interaction between ASM cells and mast cells, via a SCF-ckit-independent but dexamethasone-sensitive mechanism, enhances C3a-induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma.
Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activat... more Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.
In human basophils from different subjects, maximum IgE-mediated histamine release and the level ... more In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FcεRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRβ/FcεRIα is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment.We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgE–mediated histamine release and disproportionately alters the relative presence of FcRβ and FcεRIα.Syk, FcεRIα, and FcRβ expression was monitored during the treatment of subjects with omalizumab.Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRβ/FcεRIα ratio in patients treated with omalizumab.In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FcεRI α and β subunits in basophils is influenced by factors extrinsic to the cell.
tion, suggesting that the 2 methods of enhancement operate on the same pathway. Pulse-chase studi... more tion, suggesting that the 2 methods of enhancement operate on the same pathway. Pulse-chase studies corroborated this conclusion. In contrast, IL-3 and bafilomycin A synergistically increased p46, suggesting that IL-3 increased synthesis of Fc RI ␣ . Conclusions: Taken together, these results suggest that secretagogue stimulation results in an increase in p46 due to reversal of degradative pathways rather than increased synthesis of Fc RI ␣ . Nevertheless, a decrease in the degradation of Fc RI ␣ at an intermediate step in its processing by non-Fc RI-dependent stimulation may still influence expression of this important receptor.
Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stim... more Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stimulates chemokine production in human mast cells and what signaling pathway it activates is not known. In the present study, we demonstrate that CD34+ cell-derived primary mast cells and a human mast cell line LAD 2 express surface C3a receptors at similar levels. Furthermore, C3a caused approximately 50% internalization of cell surface C3a receptors in both cell types. We therefore used LAD 2 cells as a model to study C3a-induced biological responses and signaling in human mast cells. We found that C3a stimulated substantial degranulation and induced chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production in LAD 2 mast cells. C3a caused a rapid and sustained extracellular-signal-regulated kinase (ERK) phosphorylation and Akt phosphorylation in LAD 2 mast cells. Furthermore, U0126 and LY294002, ...
Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined... more Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined family of proteins, including resistin, Relm-β, and Relm-γ. Although resistin was initially defined based on its insulin-resistance activity, the family members are highly induced in various inflammatory states. Earlier studies implicated Relm-α in insulin resistance, asthmatic responses, and intestinal inflammation; however, its function still remains an enigma. We now report that Relm-α is strongly induced in the esophagus in an allergen-challenged murine model of eosinophilic esophagitis (EoE). Furthermore, to understand the in vivo role of Relm-α, we generated Relm-α gene-inducible bitransgenic mice by using lung-specific CC-10 promoter (CC10-rtTA-Relm-α). We found Relm-α protein is significantly induced in the esophagus of CC10-rtTA-Relm-α bitransgenic mice exposed to doxycycline food. The most prominent effect observed by the induction of Relm-α is epithelial cell hyperplasia, basal layer thickness, accumulation of activated CD4(+) and CD4(-) T cell subsets, and eosinophilic inflammation in the esophagus. The in vitro experiments further confirm that Relm-α promotes primary epithelial cell proliferation but has no chemotactic activity for eosinophils. Taken together, our studies report for the first time that Relm-α induction in the esophagus has a major role in promoting epithelial cell hyperplasia and basal layer thickness, and the accumulation of activated CD4(+) and CD4(-) T cell subsets may be responsible for partial esophageal eosinophilia in the mouse models of EoE. Notably, the epithelial cell hyperplasia and basal layer thickness are the characteristic features commonly observed in human EoE.
Eosinophilic esophagitis (EoE) is a recently recognized allergic disorder, characterized by eosop... more Eosinophilic esophagitis (EoE) is a recently recognized allergic disorder, characterized by eosophageal dysfunction, accumulation of ≥15 eosinophils/high-powered field, eosinophil microabssess, basal cell hyperplasia, extracellular eosinophilic granules in the esophageal epithelial mucosal biopsy and a lack of response to a 8-week proton pump inhibitor treatment. Despite the increased incidences and considerable progress made in understanding EoE pathogenesis, there are limited diagnostic and therapeutic options available for EoE. Currently, the only criterion for diagnosing EoE is repetitive esophageal endoscopic biopsies and histopathological evaluation. Antigen elimination or corticosteroid therapies are effective therapies for EoE but are expensive and have limitations, if continued in the long term. Hence, there is a great necessity for novel noninvasive diagnostic biomarkers that can easily diagnose EoE and assess effectiveness of therapy. Herein, we have provided an update on key molecules involved in the disease initiation, and progression and proposed novel noninvasive diagnostic molecules and strategies for EoE therapy.
Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potent... more Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esoph...
Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a mod... more Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a modifier of N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) cytotoxicity in mice. Adult Swiss male mice, 6 -8 weeks old, weighing about 25 g were exposed to mild WBH (39°C, 1 h) in a precision temperature controlled environmental chamber maintained at 50-60% relative humidity. Twenty-four hours after treatment, animals were administered with different doses of MNNG either by intraperitoneal (i.p.) injections or by feeding through drinking water and were monitored for survival. The studies revealed that the exposure of animals to mild WBH, 24 h prior to MNNG administration results in an increase in survival and recovery in mean body weight compared with those administered with MNNG only. This suggests that prior WBH treatment can effectively reduce the MNNG cytotoxicity in mice.
CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes ... more CD160 is highly expressed by NK cells and is associated with cytolytic effector activity. Herpes virus entry mediator (HVEM) activates NK cells for cytokine production and cytolytic function via CD160. Fc-fusions are a well-established class of therapeutics, where the Fc domain provides additional biological and pharmacological properties to the fusion protein including enhanced serum t 1/2 and interaction with Fc receptor-expressing immune cells. We evaluated the specific function of HVEM in regulating CD160-mediated NK cell effector function by generating a fusion of the HVEM extracellular domain with human IgG1 Fc bearing CD16-binding mutations (Fc*) resulting in HVEM-(Fc*). HVEM-(Fc*) displayed reduced binding to the Fc receptor CD16 (i.e., Fc-disabled HVEM), which limited Fc receptor-induced responses. HVEM-(Fc*) functional activity was compared with HVEM-Fc containing the wild type human IgG1 Fc. HVEM-(Fc*) treatment of NK cells and PBMCs caused greater IFN-g production, enhanced cytotoxicity, reduced NK fratricide, and no change in CD16 expression on human NK cells compared with HVEM-Fc. HVEM-(Fc*) treatment of monocytes or PBMCs enhanced the expression level of CD80, CD83, and CD40 expression on monocytes. HVEM-(Fc*)-enhanced NK cell activation and cytotoxicity were promoted via cross-talk between NK cells and monocytes that was driven by cell-cell contact. In this study, we have shown that soluble Fc-disabled HVEM-(Fc*) augments NK cell activation, IFN-g production, and cytotoxicity of NK cells without inducing NK cell fratricide by promoting cross-talk between NK cells and monocytes without Fc receptor-induced effects. Soluble Fc-disabled HVEM-(Fc*) may be considered as a research and potentially therapeutic reagent for modulating immune responses via sole activation of HVEM receptors.
Background Gangliosides are glycosphingolipids highly enriched in the brain, with important roles... more Background Gangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson’s disease and Huntington’s disease. In models of both diseases and other conditions, administration of GM1—one of the most abundant gangliosides in the brain—provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration. Methods In vitro studies we...
Interleukin (IL)-15 overexpression in eosinophilic gastrointestinal disorders is reported, but IL... more Interleukin (IL)-15 overexpression in eosinophilic gastrointestinal disorders is reported, but IL-15 0 s role in promoting eosinophilic gastroenteritis is largely unknown. Therefore, we generated enterocyte-overexpressed IL-15 transgenic mice using Fabpi promoter. The Fabpi-IL-15 (iIL-15) transgenic mice showed induced IL-15 levels in the jejunum with a marked increase in jejunum eosinophils. However, no induction of eosinophilia in the blood or any other gastrointestinal segment was observed. Eosinophilia in the jejunum villus was substantially higher in iIL-15 mice compared to wild-type mice. In addition, goblet cell hyperplasia was also observed in the jejunum of iIL-15 mice. Furthermore, a significant correlation between induced IL-15 transcript and the IL-18 transcripts was observed. Therefore, to further understand the role of IL-18 in IL-15 mice associated gastrointestinal disorders, we generated iIL-15/IL-18Ra À/À mice. Using these mice, we found that IL-18 has an important role in promoting IL-15-induced eosinophilia. As intestinal IL-15 overexpression is reported in food intolerance, we examined OVA intolerance in iIL-15 mice. The OVA-sensitized and challenged iIL-15 mice experienced weight loss, diarrhea and eosinophilia in the jejunum. Taken together, our findings demonstrate that intestinal IL-15 overexpression induces IL-18-dependent eosinophilia and immunoglobulins in the intestine that promotes food allergic responses.
Journal of Allergy and Clinical Immunology, Apr 30, 2010
Background: In human basophils from different subjects, maximum IgE-mediated histamine release an... more Background: In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FceRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRb/FceRIa is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment. Objective: We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgEmediated histamine release and disproportionately alters the relative presence of FcRb and FceRIa. Method: Syk, FceRIa, and FcRb expression was monitored during the treatment of subjects with omalizumab. Results: Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRb/FceRIa ratio in patients treated with omalizumab. Conclusions: In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FceRI a and b subunits in basophils is influenced by factors extrinsic to the cell. (J Allergy Clin Immunol 2010;125:902-8.)
RATIONALE: Prescribing self-injectable epinephrine (SIE) is appropriate for patients with an incr... more RATIONALE: Prescribing self-injectable epinephrine (SIE) is appropriate for patients with an increased risk of anaphylaxis, but there is no current consensus regarding the use of SIE prescriptions for patients on immunotherapy (IT). This study investigates current practices among allergists and those in-training on writing SIE prescriptions for IT patients. METHODS: A survey was sent to allergist-immunologists by email. Demographic information was obtained as well as information on their experience with IT and the role of SIE prescriptions for IT patients. Approval was obtained from the Long Island College Hospital IRB. RESULTS: 248 surveys were returned and 5 were excluded because they were incomplete. The majority of respondents were out of training (90%). Only 34.9% prescribe SIE to all of their patients on IT. 14.4% do not prescribe SIE for any of their IT patients. Of the remaining 50.6% of respondents, the three largest categories of patients prescribed SIE include: those with a history of previous systemic reaction, those on Omalizumab or those on Omalizumab and venom IT. Respondents who prescribe SIE for all of their patients have fewer years in practice (avg 14.5 years, range 0-44 years) compared to those who do not prescribe (avg 22 years, range 1-40 years) SIE. Interestingly, of those who prescribe SIE to all their IT patients, 61% give IT even if the patient fails to bring their SIE to their appointment. CONCLUSIONS: Among this sample of allergists there was a wide range of practices. Specific SIE prescribing recommendations for patients on IT may be helpful to practicing allergists.
Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activat... more Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.
Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immuni... more Growing evidence suggests that anaphylatoxins, C3a and C5a, play important roles in innate immunity and may also participate in the pathogenesis of asthma. Previous studies with animal models and immunohistochemistry analysis of lung tissue indicated that anaphylatoxins may regulate airway hyperresponsiveness (AHR) in asthma via the activation of their cell surface G protein-coupled receptors (C3aR and C5aR) in airway smooth muscle (ASM) cells. Using RT-PCR, flow cytometry, and confocal microscopy, we made the surprising observation that while C3aR and C5aR were expressed in human mast cells, they were not present in cultured primary human or murine ASM cells. Furthermore, we could not detect C3aR in smooth muscle-positive cells of human trachea or bronchus. Interestingly, incubation of human mast cells with ASM cells, but not its culture supernatant, caused a significant enhancement of C3a-induced mast cell degranulation. Although stem cell factor (SCF) and its receptor c-kit are constitutively expressed on ASM cells and mast cells, respectively, neutralizing antibodies to SCF and c-kit failed to inhibit ASM cell-mediated enhancement of mast cell degranulation. However, dexamethasonetreated ASM cells were normal for cell surface SCF expression but were significantly less effective in enhancing C3a-induced mast cell degranulation when compared with untreated cells. These findings suggest that cell-cell interaction between ASM cells and mast cells, via a SCF-ckit-independent but dexamethasone-sensitive mechanism, enhances C3a-induced mast cell degranulation, which likely regulates ASM function, thus contributing to the pathogenesis of asthma.
Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activat... more Previously, we reported that platelet-activating factor (PAF) stimulates higher G protein activation and a more robust Ca2+ mobilization in RBL-2H3 cells expressing carboxyl terminus deletion, phosphorylation-deficient mutant of PAF receptor (mPAFR) when compared with the wild-type receptor (PAFR). However, PAF did not provide sufficient signal for CC chemokine receptor ligand 2 (CCL2) production in cells expressing mPAFR. Based on these findings, we hypothesized that receptor phosphorylation provides a G protein-independent signal that synergizes with Ca2+ mobilization to induce CCL2 production. Here, we show that a mutant of PAFR (D289A), which does not couple to G proteins, was resistant to agonist-induced receptor phosphorylation. Unexpectedly, we found that when this mutant was coexpressed with mPAFR, it restored NF-kappaB activation and CCL2 production. PAF caused translocation of beta-arrestin from the cytoplasm to the membrane in cells expressing PAFR but not a phosphorylation-deficient mutant in which all Ser/Thr residues were replaced with Ala (DeltaST-PAFR). Interestingly, PAF induced significantly higher NF-kappaB and nuclear factor of activated T cells (NFAT)-luciferase activity as well as CCL2 production in cells expressing DeltaST-PAFR than those expressing PAFR. Furthermore, a Ca2+/calcineurin inhibitor completely inhibited PAF-induced NFAT activation and CCL2 production but not NF-kappaB activation. These findings suggest that the carboxyl terminus of PAFR provides a G protein-independent signal for NF-kappaB activation, which synergizes with G protein-mediated Ca2+/calcineurin activation to induce CCL2 production. However, receptor phosphorylation and beta-arrestin recruitment inhibit CCL2 production by blocking both NF-kappaB activation and Ca2+/calcineurin-dependent signaling pathways.
In human basophils from different subjects, maximum IgE-mediated histamine release and the level ... more In human basophils from different subjects, maximum IgE-mediated histamine release and the level of Syk protein expression correlate well. Recent studies suggest that in some patients treated with omalizumab, the response to stimulation with anti-IgE antibody increases. In unrelated studies there is also evidence that the composition of FcεRI in basophils differs among subjects. This observation raised the possibility that the stoichiometry of FcRβ/FcεRIα is not fixed to a 1:1 ratio and might be modifiable during changes in the basophil's environment.We sought to determine whether treatment with omalizumab results in increases in Syk expression and anti-IgE–mediated histamine release and disproportionately alters the relative presence of FcRβ and FcεRIα.Syk, FcεRIα, and FcRβ expression was monitored during the treatment of subjects with omalizumab.Treatment with omalizumab reduced histamine release from peripheral blood leukocytes stimulated with cat allergen in vitro, but histamine release stimulated with anti-IgE antibody increased 2-fold. Expression of Syk increased 1.86-fold. There was no change in the expression of c-Cbl, a signaling element that is sensitive to the presence of IL-3, and no increase in response to formyl-met-leu-phe (tripeptide), a response that also increases in the presence of IL-3. There was a 60% decrease in the FcRβ/FcεRIα ratio in patients treated with omalizumab.In the context of previous studies, these studies provide support for a proposal that Syk expression is modulated in vivo through an IgE-dependent mechanism and that the ratio of FcεRI α and β subunits in basophils is influenced by factors extrinsic to the cell.
tion, suggesting that the 2 methods of enhancement operate on the same pathway. Pulse-chase studi... more tion, suggesting that the 2 methods of enhancement operate on the same pathway. Pulse-chase studies corroborated this conclusion. In contrast, IL-3 and bafilomycin A synergistically increased p46, suggesting that IL-3 increased synthesis of Fc RI ␣ . Conclusions: Taken together, these results suggest that secretagogue stimulation results in an increase in p46 due to reversal of degradative pathways rather than increased synthesis of Fc RI ␣ . Nevertheless, a decrease in the degradation of Fc RI ␣ at an intermediate step in its processing by non-Fc RI-dependent stimulation may still influence expression of this important receptor.
Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stim... more Complement component C3a causes a robust degranulation in human mast cells. Whether C3a also stimulates chemokine production in human mast cells and what signaling pathway it activates is not known. In the present study, we demonstrate that CD34+ cell-derived primary mast cells and a human mast cell line LAD 2 express surface C3a receptors at similar levels. Furthermore, C3a caused approximately 50% internalization of cell surface C3a receptors in both cell types. We therefore used LAD 2 cells as a model to study C3a-induced biological responses and signaling in human mast cells. We found that C3a stimulated substantial degranulation and induced chemokine monocyte chemoattractant protein 1 (MCP-1/CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES/CCL5) production in LAD 2 mast cells. C3a caused a rapid and sustained extracellular-signal-regulated kinase (ERK) phosphorylation and Akt phosphorylation in LAD 2 mast cells. Furthermore, U0126 and LY294002, ...
Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined... more Resistin-like molecule (Relm)-α is a secreted, cysteine-rich protein belonging to a newly defined family of proteins, including resistin, Relm-β, and Relm-γ. Although resistin was initially defined based on its insulin-resistance activity, the family members are highly induced in various inflammatory states. Earlier studies implicated Relm-α in insulin resistance, asthmatic responses, and intestinal inflammation; however, its function still remains an enigma. We now report that Relm-α is strongly induced in the esophagus in an allergen-challenged murine model of eosinophilic esophagitis (EoE). Furthermore, to understand the in vivo role of Relm-α, we generated Relm-α gene-inducible bitransgenic mice by using lung-specific CC-10 promoter (CC10-rtTA-Relm-α). We found Relm-α protein is significantly induced in the esophagus of CC10-rtTA-Relm-α bitransgenic mice exposed to doxycycline food. The most prominent effect observed by the induction of Relm-α is epithelial cell hyperplasia, basal layer thickness, accumulation of activated CD4(+) and CD4(-) T cell subsets, and eosinophilic inflammation in the esophagus. The in vitro experiments further confirm that Relm-α promotes primary epithelial cell proliferation but has no chemotactic activity for eosinophils. Taken together, our studies report for the first time that Relm-α induction in the esophagus has a major role in promoting epithelial cell hyperplasia and basal layer thickness, and the accumulation of activated CD4(+) and CD4(-) T cell subsets may be responsible for partial esophageal eosinophilia in the mouse models of EoE. Notably, the epithelial cell hyperplasia and basal layer thickness are the characteristic features commonly observed in human EoE.
Eosinophilic esophagitis (EoE) is a recently recognized allergic disorder, characterized by eosop... more Eosinophilic esophagitis (EoE) is a recently recognized allergic disorder, characterized by eosophageal dysfunction, accumulation of ≥15 eosinophils/high-powered field, eosinophil microabssess, basal cell hyperplasia, extracellular eosinophilic granules in the esophageal epithelial mucosal biopsy and a lack of response to a 8-week proton pump inhibitor treatment. Despite the increased incidences and considerable progress made in understanding EoE pathogenesis, there are limited diagnostic and therapeutic options available for EoE. Currently, the only criterion for diagnosing EoE is repetitive esophageal endoscopic biopsies and histopathological evaluation. Antigen elimination or corticosteroid therapies are effective therapies for EoE but are expensive and have limitations, if continued in the long term. Hence, there is a great necessity for novel noninvasive diagnostic biomarkers that can easily diagnose EoE and assess effectiveness of therapy. Herein, we have provided an update on key molecules involved in the disease initiation, and progression and proposed novel noninvasive diagnostic molecules and strategies for EoE therapy.
Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potent... more Eosinophilic esophagitis (EoE) is a recently recognized inflammatory disorder that needs a potential therapeutic strategy. We earlier showed that iNKT cell-deficient mice are protected from allergen-induced EoE. Therefore, we now tested the hypothesis that iNKT cells are induced in the human EoE and is a novel possible target for the treatment of human EoE. Accordingly, we examine number of iNKT cells and eosinophils and expression of iNKT-associated cell surface receptors and chemokines by performing immunofluorescence, qPCR and ELISA in the esophageal biopsies and blood samples of normal subjects (comparison control) and EoE patients. Herein, we show that iNKT cell number, their receptor subcomponents Vα24 and Vβ11 expression, and associated chemokine CXCL16 levels (or expression) are induced significantly in EoE patients compared with normal individuals. In addition, we show that CXCL16 levels (or expression) correlate with the mRNA levels of Vα24 receptor but not well with esoph...
Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a mod... more Studies were carried out to ascertain the efficacy of mild whole body hyperthermia (WBH) as a modifier of N-methyl-N%-nitro-N-nitrosoguanidine (MNNG) cytotoxicity in mice. Adult Swiss male mice, 6 -8 weeks old, weighing about 25 g were exposed to mild WBH (39°C, 1 h) in a precision temperature controlled environmental chamber maintained at 50-60% relative humidity. Twenty-four hours after treatment, animals were administered with different doses of MNNG either by intraperitoneal (i.p.) injections or by feeding through drinking water and were monitored for survival. The studies revealed that the exposure of animals to mild WBH, 24 h prior to MNNG administration results in an increase in survival and recovery in mean body weight compared with those administered with MNNG only. This suggests that prior WBH treatment can effectively reduce the MNNG cytotoxicity in mice.
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Papers by Asifa Zaidi