Urologic Oncology-seminars and Original Investigations, 2014
Purpose-Currently, there is no reliable tool to predict response to intravesical bacillus Calmett... more Purpose-Currently, there is no reliable tool to predict response to intravesical bacillus Calmette-Guérin (BCG). Based on the fact that BCG is a Th1-polarizing immunotherapy, we attempt to correlate the pretreatment immunologic tumor microenvironment (Th1 or Th2) with response to therapy. Materials and methods-Bladder cancer patients with initial diagnosis of carcinoma in situ (Tis) were stratified based on their response to BCG treatment. A total of 38 patients met inclusion criteria (20 patients who responded and 18 patients who did not respond). Immunohistochemical (IHC) methods known to assess the type of immunologic microenvironment (Th1 vs. Th2) were performed on tumor tissue obtained at initial biopsy/resection: the level of tumor eosinophil infiltration and degranulation (Th2 response); the number of tumor-infiltrating GATA-3 + (Th2polarized) lymphocytes; and the number of tumor-infiltrating T-bet + (Th1-polarized) lymphocytes. Results obtained from these metrics were correlated with response to treatment with BCG immunotherapy. Results-The IHC metrics of the tumor immune microenvironment prior to BCG treatment were each statistically significant predictors of responders (R) vs. nonresponders (NR). Eosinophil infiltration and degranulation was higher for R vs. NR: 1.02±0.17 vs. 0.5±0.12 (P = 0.01) and 1.1±0.15 vs. 0.56±0.15 (P = 0.04), respectively. Ratio of GATA-3 + (Th2-polarized) lymphocytes to T-bet + (Th1-polarized) lymphocytes was higher for R vs. NR: 4.85±0.94 vs. 0.98±0.19 (P<0.001). The 3 markers were combined to create a Th2 signature biomarker, which was a statistically significant (P<0.0001) predictor of R vs. NR. All IHC markers demonstrated that a preexisting Th1 immunologic environment within the tumor was predictive of BCG failure. Conclusion-The Th1 vs. Th2 polarization of bladder tumor immune microenvironment prior to treatment with BCG represents a prognostic metric of response to therapy. If a patient has a preexisting Th1 immunologic response within the tumor, there is no value in using a therapy intended to create a Th1 immunologic response. An algorithm integrating 3 IHC methods provided a sensitive and specific technique that may become a useful tool for pathologists and urologists to predict response to BCG in patients with carcinoma in situ of the bladder.
The Journal of Allergy and Clinical Immunology, Jun 1, 2019
We acknowledge the expert assistance of Shirley ''Charlie'' Kern. We dedicate the science in this... more We acknowledge the expert assistance of Shirley ''Charlie'' Kern. We dedicate the science in this article to the late James J. Lee, PhD, who devoted his career to clarifying the role of eosinophils in human health.
Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is dete... more Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ À / À mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNβ, IFNλ2/3 and IFN-stimulated 2-5 0 oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ À / À mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include overproduction of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ À / À mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.
Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of ... more Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of the eosinophil granule protein eosinophil peroxidase (EPX) in mediating inflammation and itch was tested in a dermatitis mouse model. Mice were sensitized to trimellitic anhydride (TMA) and subsequently challenged chronically on the ear to establish dermatitis. Loss of EPX (in EPX (-/-) mice) or blocking EPX with the drug resorcinol significantly reduced dermatitis in mice exposed to TMA. Resorcinol also reduced levels of thymic stromal lymphopoietin protein (TSLP) in skin. Further studies showed that EPX increased different cytokines in keratinocytes in cell culture via two distinct mechanisms. EPX induced TSLP expression requires lysophosphatidic acid signaling while EPX induced expression of TNF-a, CSF2, CSF3, and IL1a required IL-1 signaling. We also showed that blocking IL-1 reduced inflammation in skin following TMA exposure in mice. Thus, EPX is an important mediator of inflammation and itch, that are mediated via at least two pathways. This suggests that both EPX and its' signaling pathways may provide novel therapeutic strategies in dermatitis. .
The Journal of Allergy and Clinical Immunology, Feb 1, 2016
RATIONALE: With increasing recognition of the heterogeneity of severe asthma, better understandin... more RATIONALE: With increasing recognition of the heterogeneity of severe asthma, better understanding of underlying endotypes is needed to identify patients for targeted therapies. By enrolling a broad population of moderate-to-severe uncontrolled asthma patients, we are able to analyze the prevalence of key Type 2 biomarkers in a population not enriched by biomarker or medical history. METHODS: We analyzed baseline distribution of key Type 2 biomarkers in pooled data from three Phase 2 lebrikizumab trials in 659 moderate-tosevere asthma patients (NCT00930163/NCT01545440/NCT01545453). Key eligibility criteria included treatment with ICS (200-2000 mg/day fluticasone propionate or equivalent) and > _1 additional controller, uncontrolled asthma (ACQ-5 > _1.5), pre-bronchodilator FEV 1 40-80% predicted and > _12% FEV 1 reversibility. Patients were not excluded based on history of exacerbations or biomarker values. RESULTS: The key Type 2 biomarkers and cutoff values assessed in this post hoc analysis were serum periostin (> _50 ng/mL), blood eosinophils (> _300 cells/mL) and FeNO (> _30 ppb). Based on these cutoffs , 46%, 35% and 33% of patients were high for periostin, blood eosinophils and FeNO, respectively. There were 68% patients high for any Type 2 biomarker, 23% high for any two, and 11% high for all three. Overall, 18% of asthma patients were high for serum periostin only, 9% high for blood eosinophils only and 7% high for FeNO only. CONCLUSIONS: In a pooled analysis of three trials designed to evaluate the effects of lebrikizumab treatment in moderate-to-severe uncontrolled asthma, 68% of patients had evidence of Type 2 inflammation using the specified cutoff values for periostin, blood eosinophils and FeNO.
The Journal of Allergy and Clinical Immunology, Feb 1, 2004
RationalePrevious studies have shown that eosinophils accumulate within many epithelial tumors. N... more RationalePrevious studies have shown that eosinophils accumulate within many epithelial tumors. Nonetheless, the basis of this accumulation or the specific localization of eosinophils within these tumors remains obscure.
The Journal of Allergy and Clinical Immunology, Feb 1, 2004
RATIONALE: The activation state of eosinophils and its relationship to allergen-induced pulmonary... more RATIONALE: The activation state of eosinophils and its relationship to allergen-induced pulmonary pathologies has remained unclear. This report describes a link between eosinophil CD69 cell surface levels in the lung, activation, and allergen-provoked pathologies in mice. METHODS: Purified blood (ie, CD69−) eosinophils were transferred (viz., intratracheal instillation) into the lungs of either naive or ovalbumin (OVA)-treated IL-5−/− mice and subsequent changes in CD69 cell surface expression were determined by ...
The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding ... more The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape changes, along with secretion of the granule-derived enzyme eosinophil peroxidase (EPX) in response to chemotactic stimuli including platelet-activating factor and CCL11 (eotaxin-1). Here, we describe the analysis of eosinophil shape change by confocal microscopy analysis and provide an experimental approach for comparing unstimulated cells with those that have been stimulated to undergo chemotaxis. In addition, we illustrate two different degranulation assays for EPX using OPD and an enzyme-linked immunosorbent assay technique and show how eosinophil degranulation may be assessed from in vitro as well as ex vivo stimulation.
Eosinophils and the release of cationic granule proteins have long been implicated in the develop... more Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1 2/2 /EPX 2/2 mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1 2/2 /EPX 2/2 mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1 2/2 /EPX 2/2 mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/ IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1 2/2 /EPX 2/2 mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.
The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EP... more The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL-gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (p=0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (p=0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (p=0.0007) and 0.78 (p=0.0017), respectively. There is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum.
Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patie... more Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these
The Journal of Allergy and Clinical Immunology, Apr 1, 2018
Background: The persistence of eosinophils in sputum despite high doses of corticosteroids indica... more Background: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. Objectives: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. Methods: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. Results: We report a ''polyclonal'' autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a T H 2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. Conclusion: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.
Eosinophils are generally linked to innate host defense against helminths, as well as the patholo... more Eosinophils are generally linked to innate host defense against helminths, as well as the pathologies associated with allergic diseases, such as asthma. Nonetheless, the activities of eosinophils remain poorly understood, which in turn, has prevented detailed definitions of their role(s) in health and disease. Homologous recombination in embryonic stem cells was used to insert a mammalianized Cre recombinase in the ORF encoding Epx. This knock-in strategy overcame previous inefficiencies associated with eosinophil-specific transgenic approaches and led to the development of a knock-in strain of mice (eoCRE), capable of mediating recombination of "floxed" reporter cassettes in Ͼ95% of peripheral blood eosinophils. We also showed that this Cre expression was limited exclusively to eosinophil-lineage committed cells with no evidence of Cremediated toxicity. The efficiency and specificity of Cre expression in eoCRE mice were demonstrated further in a cross with a knock-in mouse containing a "(floxstop-flox)" DTA cassette at the ROSA26 locus, generating yet another novel, eosinophil-less strain of mice. The development of eoCRE mice represents a milestone in studies of eosinophil biology, permitting eosinophil-specific gene targeting and overexpression in the mouse as part of next-generation studies attempting to define eosinophil effector functions.
Urologic Oncology-seminars and Original Investigations, 2014
Purpose-Currently, there is no reliable tool to predict response to intravesical bacillus Calmett... more Purpose-Currently, there is no reliable tool to predict response to intravesical bacillus Calmette-Guérin (BCG). Based on the fact that BCG is a Th1-polarizing immunotherapy, we attempt to correlate the pretreatment immunologic tumor microenvironment (Th1 or Th2) with response to therapy. Materials and methods-Bladder cancer patients with initial diagnosis of carcinoma in situ (Tis) were stratified based on their response to BCG treatment. A total of 38 patients met inclusion criteria (20 patients who responded and 18 patients who did not respond). Immunohistochemical (IHC) methods known to assess the type of immunologic microenvironment (Th1 vs. Th2) were performed on tumor tissue obtained at initial biopsy/resection: the level of tumor eosinophil infiltration and degranulation (Th2 response); the number of tumor-infiltrating GATA-3 + (Th2polarized) lymphocytes; and the number of tumor-infiltrating T-bet + (Th1-polarized) lymphocytes. Results obtained from these metrics were correlated with response to treatment with BCG immunotherapy. Results-The IHC metrics of the tumor immune microenvironment prior to BCG treatment were each statistically significant predictors of responders (R) vs. nonresponders (NR). Eosinophil infiltration and degranulation was higher for R vs. NR: 1.02±0.17 vs. 0.5±0.12 (P = 0.01) and 1.1±0.15 vs. 0.56±0.15 (P = 0.04), respectively. Ratio of GATA-3 + (Th2-polarized) lymphocytes to T-bet + (Th1-polarized) lymphocytes was higher for R vs. NR: 4.85±0.94 vs. 0.98±0.19 (P<0.001). The 3 markers were combined to create a Th2 signature biomarker, which was a statistically significant (P<0.0001) predictor of R vs. NR. All IHC markers demonstrated that a preexisting Th1 immunologic environment within the tumor was predictive of BCG failure. Conclusion-The Th1 vs. Th2 polarization of bladder tumor immune microenvironment prior to treatment with BCG represents a prognostic metric of response to therapy. If a patient has a preexisting Th1 immunologic response within the tumor, there is no value in using a therapy intended to create a Th1 immunologic response. An algorithm integrating 3 IHC methods provided a sensitive and specific technique that may become a useful tool for pathologists and urologists to predict response to BCG in patients with carcinoma in situ of the bladder.
The Journal of Allergy and Clinical Immunology, Jun 1, 2019
We acknowledge the expert assistance of Shirley ''Charlie'' Kern. We dedicate the science in this... more We acknowledge the expert assistance of Shirley ''Charlie'' Kern. We dedicate the science in this article to the late James J. Lee, PhD, who devoted his career to clarifying the role of eosinophils in human health.
Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is dete... more Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ À / À mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNβ, IFNλ2/3 and IFN-stimulated 2-5 0 oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ À / À mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include overproduction of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ À / À mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.
Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of ... more Eosinophils play an important role in mediating itch and inflammation in dermatitis. The role of the eosinophil granule protein eosinophil peroxidase (EPX) in mediating inflammation and itch was tested in a dermatitis mouse model. Mice were sensitized to trimellitic anhydride (TMA) and subsequently challenged chronically on the ear to establish dermatitis. Loss of EPX (in EPX (-/-) mice) or blocking EPX with the drug resorcinol significantly reduced dermatitis in mice exposed to TMA. Resorcinol also reduced levels of thymic stromal lymphopoietin protein (TSLP) in skin. Further studies showed that EPX increased different cytokines in keratinocytes in cell culture via two distinct mechanisms. EPX induced TSLP expression requires lysophosphatidic acid signaling while EPX induced expression of TNF-a, CSF2, CSF3, and IL1a required IL-1 signaling. We also showed that blocking IL-1 reduced inflammation in skin following TMA exposure in mice. Thus, EPX is an important mediator of inflammation and itch, that are mediated via at least two pathways. This suggests that both EPX and its' signaling pathways may provide novel therapeutic strategies in dermatitis. .
The Journal of Allergy and Clinical Immunology, Feb 1, 2016
RATIONALE: With increasing recognition of the heterogeneity of severe asthma, better understandin... more RATIONALE: With increasing recognition of the heterogeneity of severe asthma, better understanding of underlying endotypes is needed to identify patients for targeted therapies. By enrolling a broad population of moderate-to-severe uncontrolled asthma patients, we are able to analyze the prevalence of key Type 2 biomarkers in a population not enriched by biomarker or medical history. METHODS: We analyzed baseline distribution of key Type 2 biomarkers in pooled data from three Phase 2 lebrikizumab trials in 659 moderate-tosevere asthma patients (NCT00930163/NCT01545440/NCT01545453). Key eligibility criteria included treatment with ICS (200-2000 mg/day fluticasone propionate or equivalent) and > _1 additional controller, uncontrolled asthma (ACQ-5 > _1.5), pre-bronchodilator FEV 1 40-80% predicted and > _12% FEV 1 reversibility. Patients were not excluded based on history of exacerbations or biomarker values. RESULTS: The key Type 2 biomarkers and cutoff values assessed in this post hoc analysis were serum periostin (> _50 ng/mL), blood eosinophils (> _300 cells/mL) and FeNO (> _30 ppb). Based on these cutoffs , 46%, 35% and 33% of patients were high for periostin, blood eosinophils and FeNO, respectively. There were 68% patients high for any Type 2 biomarker, 23% high for any two, and 11% high for all three. Overall, 18% of asthma patients were high for serum periostin only, 9% high for blood eosinophils only and 7% high for FeNO only. CONCLUSIONS: In a pooled analysis of three trials designed to evaluate the effects of lebrikizumab treatment in moderate-to-severe uncontrolled asthma, 68% of patients had evidence of Type 2 inflammation using the specified cutoff values for periostin, blood eosinophils and FeNO.
The Journal of Allergy and Clinical Immunology, Feb 1, 2004
RationalePrevious studies have shown that eosinophils accumulate within many epithelial tumors. N... more RationalePrevious studies have shown that eosinophils accumulate within many epithelial tumors. Nonetheless, the basis of this accumulation or the specific localization of eosinophils within these tumors remains obscure.
The Journal of Allergy and Clinical Immunology, Feb 1, 2004
RATIONALE: The activation state of eosinophils and its relationship to allergen-induced pulmonary... more RATIONALE: The activation state of eosinophils and its relationship to allergen-induced pulmonary pathologies has remained unclear. This report describes a link between eosinophil CD69 cell surface levels in the lung, activation, and allergen-provoked pathologies in mice. METHODS: Purified blood (ie, CD69−) eosinophils were transferred (viz., intratracheal instillation) into the lungs of either naive or ovalbumin (OVA)-treated IL-5−/− mice and subsequent changes in CD69 cell surface expression were determined by ...
The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding ... more The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape changes, along with secretion of the granule-derived enzyme eosinophil peroxidase (EPX) in response to chemotactic stimuli including platelet-activating factor and CCL11 (eotaxin-1). Here, we describe the analysis of eosinophil shape change by confocal microscopy analysis and provide an experimental approach for comparing unstimulated cells with those that have been stimulated to undergo chemotaxis. In addition, we illustrate two different degranulation assays for EPX using OPD and an enzyme-linked immunosorbent assay technique and show how eosinophil degranulation may be assessed from in vitro as well as ex vivo stimulation.
Eosinophils and the release of cationic granule proteins have long been implicated in the develop... more Eosinophils and the release of cationic granule proteins have long been implicated in the development of the type 2-induced pathologies linked with respiratory inflammation. Paradoxically, the ablation of the two genes encoding the most abundant of these granule proteins, major basic protein-1 (MBP-1) and eosinophil peroxidase (EPX), results in a near collapse of eosinophilopoiesis. The specificity of this lineage ablation and the magnitude of the induced eosinopenia provide a unique opportunity to clarify the importance of eosinophils in acute and chronic inflammatory settings, as well as to identify potential mechanism(s) of action linked with pulmonary eosinophils in those settings. Specifically, we examined these issues by assessing the induced immune responses and pathologies occurring in MBP-1 2/2 /EPX 2/2 mice after 1) ovalbumin sensitization/provocation in an acute allergen-challenge protocol, and 2) crossing MBP-1 2/2 /EPX 2/2 mice with a double-transgenic model of chronic type 2 inflammation (i.e., I5/hE2). Acute allergen challenge and constitutive cytokine/chemokine expression each induced the accumulation of pulmonary eosinophils in wild-type controls that was abolished in the absence of MBP-1 and EPX (i.e., MBP-1 2/2 /EPX 2/2 mice). The expression of MBP-1 and EPX was also required for induced lung expression of IL-4/ IL-13 in each setting and, in turn, the induced pulmonary remodeling events and lung dysfunction. In summary, MBP-1 2/2 /EPX 2/2 mice provide yet another definitive example of the immunoregulatory role of pulmonary eosinophils. These results highlight the utility of this unique strain of eosinophil-deficient mice as part of in vivo model studies investigating the roles of eosinophils in health and disease settings.
The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EP... more The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL-gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (p=0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (p=0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (p=0.0007) and 0.78 (p=0.0017), respectively. There is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum.
Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patie... more Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these
The Journal of Allergy and Clinical Immunology, Apr 1, 2018
Background: The persistence of eosinophils in sputum despite high doses of corticosteroids indica... more Background: The persistence of eosinophils in sputum despite high doses of corticosteroids indicates disease severity in asthmatic patients. Chronic inflamed airways can lose tolerance over time to immunogenic entities released on frequent eosinophil degranulation, which further contributes to disease severity and necessitates an increase in maintenance corticosteroids. Objectives: We sought to investigate the possibility of a polyclonal autoimmune event in the airways of asthmatic patients and to identify associated clinical and molecular characteristics. Methods: The presence of autoantibodies against eosinophil peroxidase (EPX) and anti-nuclear antibodies was investigated in patients with eosinophilic asthma maintained on high-dose corticosteroids, prednisone, or both. The ability of sputum immunoglobulins to induce eosinophil degranulation in vitro was assessed. In addition, the associated inflammatory microenvironment in patients with detectable autoantibodies was examined. Results: We report a ''polyclonal'' autoimmune event occurring in the airways of prednisone-dependent asthmatic patients with increased eosinophil activity, recurrent pulmonary infections, or both, as evident by the concomitant presence of sputum anti-EPX and anti-nuclear antibodies of the IgG subtype. Extensive cytokine profiling of sputum revealed a T H 2-dominated microenvironment (eotaxin-2, IL-5, IL-18, and IL-13) and increased signalling molecules that support the formation of ectopic lymphoid structures (B-cell activating factor and B cell-attracting chemokine 1). Immunoprecipitated sputum immunoglobulins from patients with increased autoantibody levels triggered eosinophil degranulation in vitro, with release of extensive histone-rich extracellular traps, an event unsuppressed by dexamethasone and possibly contributing to the steroid-unresponsive nature of these eosinophilic patients. Conclusion: This study identifies an autoimmune endotype of severe asthma that can be identified by the presence of sputum autoantibodies against EPX and autologous cellular components.
Eosinophils are generally linked to innate host defense against helminths, as well as the patholo... more Eosinophils are generally linked to innate host defense against helminths, as well as the pathologies associated with allergic diseases, such as asthma. Nonetheless, the activities of eosinophils remain poorly understood, which in turn, has prevented detailed definitions of their role(s) in health and disease. Homologous recombination in embryonic stem cells was used to insert a mammalianized Cre recombinase in the ORF encoding Epx. This knock-in strategy overcame previous inefficiencies associated with eosinophil-specific transgenic approaches and led to the development of a knock-in strain of mice (eoCRE), capable of mediating recombination of "floxed" reporter cassettes in Ͼ95% of peripheral blood eosinophils. We also showed that this Cre expression was limited exclusively to eosinophil-lineage committed cells with no evidence of Cremediated toxicity. The efficiency and specificity of Cre expression in eoCRE mice were demonstrated further in a cross with a knock-in mouse containing a "(floxstop-flox)" DTA cassette at the ROSA26 locus, generating yet another novel, eosinophil-less strain of mice. The development of eoCRE mice represents a milestone in studies of eosinophil biology, permitting eosinophil-specific gene targeting and overexpression in the mouse as part of next-generation studies attempting to define eosinophil effector functions.
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