Sputum Protein Biomarkers in Airway Diseases: A Pilot Study.

Background: Chronic mucous hypersecretion (CMH or chronic bronchitis) per se or when associated with chronic inflammatory airway diseases such as asthma or chronic obstructive pulmonary disease (COPD) has several adverse clinical consequences. The sputum fluid phase has several candidate proteins including mucins which have the potential of being therapeutic targets, but has not yet been explored in-depth. This study aimed at exploring the profile of sputum proteins in various airway diseases. Methods: Sputum from thirty-one patients with various airway diseases was collected and the fluid phase analyzed by LC-MS/MS and subsequently by sequential window acquisition of all theoretical fragments ion spectra (SWATH) (n = 15) for protein quantitation. Hierarchical clustering and functional grouping were performed. Results: A total of 185 proteins were quantitated by SWATH of which 21 proteins were identified which could distinguish between the clinical phenotypes by hierarchical clustering. Functional protein clustering revealed 4 groups: those that are inflammation related, oxidative stress related, mucin related and a cytoskeletal and calcium related group. The levels of eight proteins (Azurocidin1, Neutrophil defensin 3, Lactotransferrin, Calmodulin 3, Coronin1A, Mucin 5B, Mucin 5AC and BPI fold containing family B1) were significantly altered (relative to mean) in exacerbator prone subjects compared to nonexacerbators. Another simple but useful metric which emerged from this study was total protein concentration in sputum which was significantly higher in frequent exacerbators. Conclusion: Sputum proteins can detect the various airway disease clinical phenotypes. Total protein concentration and eight other proteins are biomarkers for frequent exacerbators. The clinical and therapeutic implications of the functional groups of proteins need further evaluation.

Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE-cross-reactivity.

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

Pilot-Scale Study Of Human Plasma Proteomics Identifies ApoE And IL33 As Markers In Atopic Asthma.

BACKGROUND: The pathobiology of atopic asthma is complex and the symptoms similar to other respiratory diseases. As such, identification of biomarkers of atopic asthma is of prime importance for better diagnosis and control of the disease. OBJECTIVES: We sought to study the changes in plasma proteome and cytokine-expression profile across healthy and atopic asthmatics for identifying biomarkers and exploring aberrant pathways for atopic asthma. METHODS: A pilot-scale study in humans was performed to identify differentially expressed proteins in blood plasma of healthy controls (n=5) and treatment-naïve atopic asthma patients (n=5) using quantitative label-free liquid chromatography-tandem mass spectrometry proteomics and ELISA. RESULTS: Mass spectrometry-based proteomic analysis revealed ApoE to be significantly downregulated in atopic asthmatics compared to healthy volunteers. Decreased expression of ApoE in atopic asthmatics was validated by immunoblotting (50.74% decrease). Comparison with atopic asthmatics and COPD patients showed that ApoE was decreased (36.33%) in atopic asthma compared to COPD. IL33 was significantly upregulated in atopic asthmatics compared to healthy subjects (3.84-fold). CONCLUSION: ApoE was downregulated and IL33 upregulated in atopic asthma patients compared to healthy volunteers. These two proteins' profiles were distinct in atopic asthma from healthy and COPD plasma samples. Differential expression of these proteins could serve as a probable candidate for a two-protein classifier-based prognostic biomarker of atopic asthma.

Cari p 1, a Novel Polygalacturonase Allergen From Papaya Acting as Respiratory and Food Sensitizer.

Papaya has been reported to elicit IgE-mediated hypersensitivity via pollen inhalation and fruit consumption. Certain papaya sensitive patients with food allergy were found to experience recurrent respiratory distresses even after quitting the consumption of fruits. This observation prompted us to investigate the allergens commonly present in fruits and pollen grains of papaya. A discovery approach consisting of immunoproteomic detection followed by molecular characterization led to the identification of a novel papaya allergen designated as Cari p 1. This allergen was detected as a 56 kDa IgE-reactive protein from pollen as well as fruit proteome through serological analysis. The protein was identified as an endopolygalacturonase by tandem mass spectrometry. Full length Cari p 1 cDNA was isolated from papaya pollen, cloned in expression vector, and purified as recombinant allergen. The recombinant protein was monomeric and displayed pectinolytic activity. Recombinant Cari p 1 reacted with IgE-antibodies of all the papaya sensitized patient sera. In addition to IgE-reactivity, rCari p 1 displayed allergenic activity by stimulating histamine release from IgE-sensitized granulocytes. CD-spectroscopy of rCari p 1 revealed the presence of predominantly ß-sheet characters. The melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational IgE-epitopes characteristic of inhalant allergens in addition to the linear IgE-epitopes of food allergens. The expression of this allergen in papaya fruits was detected by immunoblot with anti-Cari p 1 rabbit IgG and reconfirmed by PCR. In an in vivo mouse model, rCari p 1 exhibited a comparable level of inflammatory responses in the lung and duodenum tissues explaining the dual role of Cari p 1 allergen in respiratory sensitization via pollen inhalation and sensitization of gut mucosa via fruit consumption. Purified rCari p 1 can be used a marker allergen for component-resolved molecular diagnosis. Further immunological studies on Cari p 1 are warranted to design immunotherapeutic vaccine for the clinical management of papaya allergy.

Spectrum of Allergens and Allergen Biology in India.

The growing prevalence of allergy and asthma in India has become a major health concern with symptoms ranging from mild rhinitis to severe asthma and even life-threatening anaphylaxis. The "allergen repertoire" of this subcontinent is highly diverse due to the varied climate, flora, and food habits. The proper identification, purification, and molecular characterization of allergy-eliciting molecules are essential in order to facilitate an accurate diagnosis and to design immunotherapeutic vaccines. Although several reports on prevalent allergens are available, most of these studies were based on preliminary detection and identification of the allergens. Only a few of these allergen molecules have been characterized by recombinant technology and structural biology. The present review first describes the composition, distribution pattern, and natural sources of the predominant allergens in India along with the prevalence of sensitization to these allergens across the country. We go on to present a comprehensive report on the biochemical, immunological, and molecular information on the allergens reported so far from India. The review also covers the studies on allergy- related biosafety assessment of transgenic plants. Finally, we discuss the allergen-specific immunotherapy trials performed in India.

Sputum cell counts to manage prednisone-dependent asthma: effects on FEV1 and eosinophilic exacerbations.

BACKGROUND: Prednisone dependence in asthma is usually described based on clinical and spirometric characteristics. It is generally believed that these patients have frequent exacerbations and lose lung function rapidly because of uncontrolled airway eosinophilia. OBJECTIVES: The objectives of this study are to report the effect on asthma exacerbations and the change in lung function over time in prednisone-dependent asthma when severe asthma is managed using a protocol that aims to maintain normal sputum cell counts. METHODS: A retrospective survey of patients prospectively assessed in a university tertiary care asthma clinic. RESULTS: 52 patients (30 males, mean age 51 years, 64% non-atopic) were followed for a median period of 5.4 years (min-max: 0.2-35.2). Monitoring with the aim of keeping sputum eosinophils below 3% resulted in higher doses of corticosteroids (median daily dose of prednisone was 10 mg and for inhaled corticosteroids was 1500 µg of fluticasone equivalent) than at baseline and this was associated with predictable adverse effects. Despite the disease severity, 10 patients (19%) did not require LABA for symptom control. Most importantly, over the period of follow-up, there were only 0.3 eosinophilic exacerbations/patient/year. Overall, there was an increase in FEV1 over the period of follow-up (mean +84.6 ml/year) rather than an expected decline. CONCLUSIONS: Monitoring of eosinophils in sputum enables to maintain symptom control and preserve FEV1 in patients with severe prednisone-dependent asthma.

The use of cellular and molecular biomarkers to manage COPD exacerbations.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) exacerbations are a common cause of respiratory morbidity and mortality, and have various etiologies. Multiple cellular and molecular biomarkers have been associated with exacerbations. Quantitative sputum cell counts are able to identify the presence and type of bronchitis, which is an important contributor to exacerbations. Their utility to monitor bronchitis and to help treat exacerbations has been evaluated, yet they are not used in routine clinical practice. Areas covered: This review will provide a brief summary of biomarkers utilized in COPD, with a focus on the application of cellular markers for the management of exacerbations. A case study will demonstrate the application of these methods. With quantitative sputum cell counts, the presence of eosinophilic bronchitis predicts corticosteroid-responsiveness, while neutrophilic bronchitis identifies infection and suggests the need for antibiotics. Gastroesophageal reflux-related aspiration and heart failure can also be identified by examining sputum. Expert commentary: Quantitative sputum cytometry is an essential tool in the management of exacerbations of COPD, particularly those prone to frequent exacerbations. Treatment based on sputum cell counts is superior to current guideline-based recommendations to prevent future exacerbations and hospitalizations in observational and single-centre controlled trials. Large multicentre clinical trials are necessary to confirm this.

Epitope Mapping of Rhi o 1 and Generation of a Hypoallergenic Variant: A CANDIDATE MOLECULE FOR FUNGAL ALLERGY VACCINES.

Efficacy of allergen-specific immunotherapy is often severely impaired by detrimental IgE-mediated side effects of native allergen during vaccination. Here, we present the molecular determinants for IgE recognition of Rhi o 1 and eventually converting the allergen into a hypoallergenic immunogen to restrain health hazards during desensitization. Rhi o 1 is a respiratory fungal allergen. Despite having cross-reactivity with cockroach allergen, we observed that non-cross-reactive epitope predominantly determined IgE binding to Rhi o 1. Denaturation and refolding behavior of the allergen confirmed that its IgE reactivity was not essentially conformation-dependent. A combinatorial approach consisting of computational prediction and a peptide-based immunoassay identified two peptides ((44)TGEYLTQKYFNSQRNN and (311)GAEKNWAGQYVVDCNK) of Rhi o 1 that frequently reacted with IgE antibodies of sensitized patients. Interestingly, these peptides did not represent purely linear IgE epitopes but were presented in a conformational manner by forming a spatially clustered surface-exposed epitope conferring optimal IgE-binding capacity to the folded allergen. Site-directed alanine substitution identified four residues of the IgE epitope that were crucial for antibody binding. A multiple mutant (T49A/Y52A/K314A/W316A) showing 100-fold lower IgE binding and reduced allergenic activity was generated. The TYKW mutant retained T-cell epitopes, as evident from its lymphoproliferative capacity but down-regulated pro-allergic IL-5 secretion. The TYKW mutant induced enhanced focusing of blocking IgG antibodies specifically toward the IgE epitope of the allergen. Anti-TYKW mutant polyclonal IgG antibodies competitively inhibited binding of IgE antibodies to Rhi o 1 up to 70% and suppressed allergen-mediated histamine release by 10-fold. In conclusion, this is a simple yet rational strategy based on epitope mapping data to develop a genetically modified hypoallergenic variant showing protective antibody response for immunotherapeutic applications.

Anti-Neutrophil Cytoplasmic Antibody (ANCA)-Negative Small Vessel Vasculitis: A Rare Cause of Pulmonary Renal Syndrome.

Pulmonary renal syndrome (PRS) is characterized by both diffuse alveolar haemorrhage and glomerulonephritis as pathological features. Several immunologic and non-immunologic mechanisms including anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis, anti-glomerular basement membrane disease, and systemic lupus erythematosus are commonly involved in the pathogenesis of the syndrome. We herein present a 60-year-old woman, non-smoker, who presented with fever, polyarthralgia, and the spreading of generalized purpuric rashes. The patient displayed rapid deterioration over the following two weeks marked by progressive declining renal function followed by haemoptysis. The patient was subsequently diagnosed with PRS, which was confirmed by the radiological evidence of alveolar haemorrhage and the histopathological evidence of pauci-immune glomerulonephritis. All immune markers including ANCA were negative. The patient was successfully treated with hemodialysis and immunosuppressive therapy. ANCA-negative vasculitis is a rare entity and even more rare as an etiology of PRS. An early diagnosis of this disease and its timely intervention is crucial.

Changing Pattern of Sputum Cell Counts During Successive Exacerbations of Chronic Obstructive Pulmonary Disease.

BACKGROUND: Chronic Obstructive Pulmonary Disease exacerbations are associated with worsening of airway inflammation, the nature of which may be neutrophilic, eosinophilic, or both. OBJECTIVE: The primary objective was to examine the cellular nature of airway inflammation in successive COPD exacerbations in order to ascertain if they changed in individual patients. The secondary objective was to estimate the relative risk indicating the extent to which a particular type of exacerbation changed as a function of the most recent exacerbation. DESIGN: This was a retrospective survey performed on a computerised sputum cell count database of a referral respiratory service in Hamilton, Canada. Recurrent event analyses were used to model the incidence of exacerbations and subtypes of exacerbations. RESULTS: 359 patients and 148 patients had sputum examined during stable condition and during exacerbations, respectively. It was found 65 patients had sputum examined during both situations. The exacerbations were eosinophilic in 15.9%, neutrophilic in 18%, combined in 2.6%, of unknown clinical significance in 19.6% and normal in 19.6%. There were missing counts for 24.3% samples. In 85.2% of patients, a different subtype of bronchitis was noted in successive exacerbations. The relative risk of a subsequent neutrophilic or eosinophilic exacerbation was 6.24 (p = 0.02) and 2.8 (p = 0.24) when the previous exacerbation was neutrophilic or eosinophilic respectively. CONCLUSIONS: This non-intervention study suggests that the cellular nature of bronchitis is largely unpredictable and needs to be examined at each COPD exacerbation This has important implications in choosing the appropriate therapy. Future intervention studies would provide further evidence.

Reference equation for spirometry interpretation for Eastern India.

INTRODUCTION: Spirometry measurements are interpreted by comparing with reference values for healthy individuals that have been derived from multiple regression equations from earlier studies. There are only two such studies from Eastern India, both by Chatterjee et al., one each for males and females. These are however single center and approximately two decades old studies. AIMS: (1) to formulate a new regression equation for predicting FEV1 and FVC for eastern India and (2) to compare the results to the previous two studies by Chatterjee et al. MATERIALS AND METHODS: Healthy nonsmokers were recruited through health camps under the initiative of four large hospitals of Kolkata. Predicted equations were derived for FEV1, FVC and FEV1/FVC in males and females separately using multiple linear regression, which were then compared with the older equations using Bland-Altman method. RESULTS: The Bland-Altman analyses show that the mean bias for females for FVC was 0.39 L (95% limits of agreement 1.32 to -0.54 L) and for FEV1 was 0.334 L (95% limits of agreement of 1.08 to -0.41 L). For males the mean bias for FEV1 was -0.141 L, (95% limits of agreement 0.88 to -1.16 L) while that for FVC was -0.112 L (95% limits of agreement 0.80 to -1.08 L). CONCLUSION: New updated regression equations are needed for predicting reference values for spirometry interpretation. The regression equations proposed in this study may be considered appropriate for use in current practice for eastern India until further studies are available.

The effects of rituximab on serum IgE and BAFF.

BACKGROUND: There are few treatment options for patients with severe atopic asthma. Antagonism of IgE is an effective strategy. We investigated, by utilizing serum samples from a clinical trial of Rituximab in patients with Idiopathic Thrombocytopenic Purpura, if B cell depletion would decrease serum IgE and therefore be a potential therapeutic option. FINDINGS: In a placebo-controlled randomized clinical trial of Rituximab, an anti-CD20 molecule, there were no significant differences in serum levels of IgE or BAFF levels between the two treatment groups at 3 or 6 months irrespective of the baseline serum IgE levels. CONCLUSIONS: Since Rituximab did not significantly decrease serum IgE levels, this proof of concept study suggests that Rituximab may not be a useful treatment strategy for patients with severe IgE mediated disease.

Targeted therapy of bronchitis in obstructive airway diseases.

Guidelines for the management of obstructive airway diseases do not emphasize the measurement of bronchitis to indicate appropriate treatments or monitor response to treatment. Bronchitis is the central component of airway diseases and contributes to symptoms, physiological and structural abnormalities. It can be measured directly and reliably by quantitative assay of spontaneous or induced sputum. The measurement is reproducible, valid, and responsive to treatment and to changes in disease status. Bronchitis may be eosinophilic, neutrophilic, mixed, or paucigranulocytic (eosinophils and neutrophils not elevated). Eosinophilic bronchitis is usually a Th2 driven process and therefore a sputum eosinophilia of greater than 3% usually indicates a response to treatment with corticosteroids or novel therapies directed against Th2 cytokines such as IL-4, IL-5 and IL-13. Neutrophilic bronchitis which is a non-Th2 driven disease is generally a predictor of response to antibiotics and may be a predictor to therapies targeted at pathways that lead to neutrophil recruitment such as IL-8 (eg anti-CXCR2), IL-17 (eg anti-IL17) etc. Paucigranulocytic disease may not warrant anti-inflammatory therapy. Several novel monoclonals and small molecule antagonists have been evaluated in clinical trials with variable results and several more are likely to be discovered in the near future. The success of these agents will depend on appropriate patient selection by accurate phenotyping or characterization of bronchitis.

A kit to facilitate and standardize the processing of sputum for measurement of airway inflammation.

BACKGROUND: The use of inflammometry has been shown to be effective for managing asthma. However, sputum processing can be time consuming. Furthermore, methods of sputum processing can vary among facilities. To help with standardization and to simplify the procedure for laboratory staff, a novel, commercially available processing device (Accufilter, Cellometrics Inc, Canada) has been developed. OBJECTIVE: To assess the validity of the Accufilter device and kit for recovery of treated specimens, and for quantitative sputum inflammatory cell counts by comparing intrasample measurements with those using the same procedure without the Accufilter device and kit. METHODS: The present study was a wet laboratory comparison of induced sputum cell counts obtained from sputum processed with versus without the device and kit. Comparisons of each sputum specimen were performed by the same technologist in random order. RESULTS: A total of 39 samples were processed using both the standard method and the Accufilter device. The intraclass correlation coefficients were high for the weight of the filtrate, and for eosinophil and neutrophil differential counts. CONCLUSION: A good degree of agreement of results was apparent when the two methods were compared. The differences noted between both methods were minimal and did not modify clinical interpretation. The use of the Accufilter device and kit can be used in place of the standard method for sputum quantitative analysis, especially in centres with large sample loads.

Asthma associated with incontinentia pigmenti and Fanconi anemia: variable airflow limitation without cellular bronchitis.

Airway inflammation is considered a central component of asthma and, therefore, international guidelines recommend antiinflammatory medications. We describe the clinical history of a 34-year-old woman with airway hyperresponsiveness and asthma who had a reduced ability to mount an inflammatory response due to two unrelated and rare genetic conditions: Fanconi anemia and incontinentia pigmenti. Absence of eosinophils in blood and sputum led to a successful reduction in the dose of corticosteroids without loss of asthma control demonstrating the clinical utility of monitoring treatment using biomarkers and the importance of recognizing the components of airway diseases that contribute to symptoms.

When are biomarkers useful in the management of airway diseases?

Biomarkers are characteristics that are objectively measured and evaluated as indicators of biological or pathogenic processes, or responses to therapeutic interventions, and may provide information on the prognosis or progression of the disease and response to treatment. They are likely to be helpful in the management of airway diseases because of the heterogeneity of their pathobiology. Most biomarkers have been developed and evaluated to assess the airway inflammation (or bronchitis) associated with airway diseases. These include quantitative cell counts in sputum, fraction of nitric oxide in exhaled breath, and various metabolites in exhaled breath. This review provides a brief description of these biomarkers with a particular emphasis on how eosinophil and neutrophil counts in sputum could be used to manage airway diseases such as asthma, chronic obstructive pulmonary disease, and chronic cough.