INTRODUCTION:

The heterogeneous nature of asthma with various environmental –genetic interaction¹ means that a “one shaft hitting all targets” moves towards diagnosis, classification, prognosis and pharmacotherapy is uncertain to be successful². Lately, biological therapies for asthma have been developed that use biomarkers to identify patients with certain mechanisms (i.e. endotypes)³. Recent study strongly suggests an important role of genetic polymorphism as a risk factor for developing asthma⁴. Together the clinical phenotypic information and biomarkers were utilized to select patients for novel anti-inflammatory drugs align to the accurate treatment policy thereby to optimize the benefit versus risk ratio.³

Periostin is an extracellular matrix protein contributes to thickening of bronchial airways sub- epithelial tissues and thus it reflects the airway inflammation⁵ as a consequence of airway remodeling and repair, The repair mechanisms that periostin establishes following allergen introduction or injury may exist far beyond the initial insult and contribute to the sustained or chronic inflammation in airways⁶. So it may predict the response to treatment in poorly controlled severe asthmatic patients⁷.

Periostin also assists in eosinophil recruitment and infiltration to sites of TH2 mediated inflammation in the airways and eosinophil-mediated fibrosis through increased eosinophil motility and adhesion at the site of inflammation^7,8,9,10,11. In bronchial brushings, periostin gene expression is upregulated in a subset of patients in which it was associated with the expression of Th2 cytokines¹².

MATERIAL AND METHODS:

This is a prospective case control study. It was conducted in the department of microbiology/collage of medicine/university of Baghdad and respiratory and allergic Diseases center/Ministry of health/Iraq. The study extended from February 2018 to April 2019.

The study involved forty four (44) Iraqi asthmatic patients, who attended specialist center for allergic and asthmatic diseases in Baghdad AL-Resafa, Their age ranged from 18-67 years who underwent a special questionnaire for asthma control assessment. Patients were compared to forty four (44) apparently healthy individuals who were non-smokers and clear from any allergic and/or autoimmune disease, as well as their family. Their ages and sexes were matched as a control group who were visiting Al-Karama teaching hospital /Baghdad.

Anyone from study cases who proved to have previous history of respiratory illness like chronic obstructive pulmonary disease (COPD), tuberculosis, pneumonia, or bronchitis, and any other co-morbid illness that need treatment like Aspirin (2-Acetoxybenzoic acid), Non-steroidal anti-inflammatory drugs (NSAIDs) and Aspegic (DL-Lysine Acetylsalicylate) or anyone with past medical history of autoimmune diseases was excluded from the study.

ETHICAL CONSIDERATIONS:

Ethical approval for the study was obtained from the ethical committee in Department of microbiology and Dept. of Medicine as well as from council of Collage of medicine/University of Baghdad/Iraq.

All patients received a written and verbal information sheet explaining the aim of the study. A signed written consent was taken from each individual participating in the study.

SAMPLE COLLECTION:

Samples were collected from study groups, who were currently attending specialist center for allergic and asthmatic diseases in Baghdad AL-Resafa, and Consultative Clinic for Allergic Diseases/Baghdad during the period from March to June, 2018 and for apparently healthy people, who were visiting Al-Karama teaching hospital/Baghdad/Iraq during the period from May to June, 2018. For each individual 4ml of blood was drawn from vein puncture, and then 2ml were put in EDTA tube and mixed for a few minutes, then complete blood count test was done by blood auto analyzer laboratory machine. The rest 2ml of blood was centrifuged under 1000 x g for 15 minutes then 0.5ml of serum was pipette in Epindroff tube that was transported with ice packs and stored at -20°C until used for ELISA detection technique of Periostin.

The used Kit was Human Periostin for the quantitative determination of human periostin/osteoblast specific factor 2 (POSTN) concentrations in serum, plasma, tissue homogenates ELISA Kit (Cusabio Catalog Number CSB-E16444h) China.

PRINCIPLE OF THE ASSAY:

The quantitative sandwich enzyme immunoassay technique was employed for this assay. Micro plate was pre-coated by antibody specific for POSTN. The wells were pipetted by Standards and samples and any POSTN present was bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for POSTN was added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) was added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution was added to the wells and color develops in proportion to the amount of POSTN bound in the initial step. The color development was stopped and the intensity of the color was measured.

STANDARDS:

The standard vial was centrifuged at 6000-10000rpm for 30s, and then it was reconstituted with 1.0ml of Sample Diluent and mixed to ensure complete reconstitution and allowed the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions.

250μl of Sample Diluent was Pipetted into each tube (S0-S6). The stock solution was used to produce a 2-fold dilution series table (1). Each tube was mixed thoroughly before the next transfer. The undiluted Standard was served as the high standard (400ng/ml) whiles the Sample Diluent was served as the zero standards (0 ng/ml).

Table (1) Sample Diluent of Periostin in each tube (S0-S6)

Tube	S7	S6	S5	S4	S3	S2	S1	S0
Ng/ml	400	200	100	50	25	12.5	6.25	0

ASSAY PROCEDURE:

All reagents and samples were brought to room temperature before use. The 100μl of standard and sample was added per well, covered with the adhesive strip provided and Incubated for 2 hours at 37°C. Then the liquid was removed from each well directly, without wash and 100μl of Biotin-antibody (1x) was added to each well, covered with another adhesive strip and Incubated for 1 hour at 37°C. After that, each well was Aspirated and washed, the process was repeated two times for a total of three washes by the auto washer machine. The 100μl of HRP-avidin (1x) was added to each well, covered the micro titer plate with a new adhesive strip and incubated for 1 hour at 37°C. The wash process was repeated for five times. The 90μl of TMB substrate was added to each well and incubated for 15-30 minutes at 37°C and Protected from light. Lastly, 50μl of Stop Solution was added to each well and gently tapped the plate to ensure thorough mixing.

The optical density of each well was determined within 5 minutes by using a micro plate reader set to 450nm.

CALCULATION OF RESULTS

The professional soft "Curve Expert 1.4" was used to make a standard curve, which was downloaded from Cusdio web.

A standard curve was created by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. The data may be linearized by plotting the log of the Periostin concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data.

STASTICAL METHODS:

The results were calculated by the use of Statistical-Package for the Social Sciences (SPSS) package for Windows version 24.0 software (IBM Corporation, Armonk, NY, USA).

The tests for normal distribution like Kolmogorov–Smirnov and Shapiro–Wilk, were done. Median with interquartile range (IQR) (Mann-Whitney U test) for comparison of two non-normally distributed data and Kruskal-Wallis H test for comparison of more than two of non normally distributed data in order to calculate the significance of the differences. Pearson’s correlation was used to measure the effect size and the correlation.

Receiver operating characteristic (ROC) curves to determine periostin concentrations and to detect the accuracy of the diagnosis test of Asthma and/ or Eosinophilic endotype of asthma based on the complete blood cell count and to provide optimal cutoff values when biomarkers were identified.

RESULTS:

Serum Periostin concentration between study groups

Table-2 demonstrates the median concentration of serum Periostin was (60.13ng/ml) among 44 study cases and was (25.71ng/ml) in 44 of control group, which were significantly higher in patients with asthma compared with healthy control subjects with high statistical significant difference was observed (P=0.001) among study groups.

The figure -1 and -2 explain an extreme differences in serum concentrations of Periostin that were not normally distributed among studied groups, the overall concentration was below (200ng/ml) in asthmatics whereas below (50ng/ml) in control group and for this reason the Mann-Whiteny test was used as a statistical methods to compare the results in between.

Table -2: The difference in median concentration of serum Periostin level between study groups

*Periostin* *(ng/ml)*	*Study group*		*P – Value*
*Periostin* *(ng/ml)*	Cases N= 44	Control N= 44	*P – Value*
*Mean ± SD*	83.0 ± 56.2	33.5 ± 25.1	0.001
*Range*	(17.9 – 275.6)	(0.9 – 86.6)
*Median*	60.1	25.7
*IQR (Q3-Q1)*	(41.5 – 112.2)	(14.13 – 43.49)
*Mean Rank*	58.69	30.31

Figure -1 Histogram of Periostin concentration in asthmatics with line of normality distribution

Figure -2: Histogram of Periostin level in control group with line of normality distribution

Clinical sensitivity of periostin level by (roc) curves to identify patients with asthma:

ROC curves were generated by plotting sensitivity against (1-specificity) for the performance of serum Periostin marker in the diagnosis of asthma after his efficiency was calculated among study groups as displayed in figure -3.

The marker would provide an almost good test. The area under the ROC curve (AUC) was 0.823 with strong significance (p value 0.0001) among study groups (cases and control).

The optimum cutoff level of Periostin was 38.3ng/ml with a sensitivity and specificity of 80% and 70% respectively, while 17.6ng/ml for the highest sensitivity = (100%) and 87.9ng/ml for highest specificity (100%).

Figure -3: Summary of receiver operating characteristic curves for serum Periostin among Study groups.

Periostin (roc) curves to identify patients with eosinophil endotype of asthma:

In order to examine the utility of serum periostin in association of the presence of eosinophilic asthma endotype (in addition of being already asthmatic), the ROC curves were generated after the statistical calculation was done among Asthmatic group, ROC was demonstrated by Figure -4, the optimum concentration of serum periostin cutoff level for eosinophilic asthma endotype was 74.63ng/ml with a sensitivity and specificity of 81.3% and 85.7% respectively and 54.42 ng/ml for the highest sensitivity = (100%) and 179.8 ng/ml for highest specificity = (100%).

The area under the ROC curve (AUC) was 0.882 (p value 0.00003), 0.835 (p value 0.000253) and 0.766 (p value= 0.004) of Periostin, Eosinophil and Total IgE respectively, while about PFT (FEV %) the (AUC) was 0.575 without significance correlation (p value= 0.414) among cases group as illustrated in table 3

Table -3 Area under the Curve of serum Periostin in relation to other variables among Cases group

Test Result	Area under curve	P value	95% CI
Test Result	Area under curve	P value	Lower Bound	Upper Bound
Periostin	0.882	0.00003	0.784	0.979
Eosinophil	0.835	0.000253	0.715	0.954
Total IgE	0.766	0.004	0.626	0.905
PFT (FEV1)	0.575	0.414	0.393	0.756

Figure -4 reveals significance Positive linear correlation between Log. Periostin concentration and Log. PFT (FEV1) among Asthmatic Patients with (R² = 0.123) Correlation = (12%) and P value = 0.031.

The Correlation equation (Y=1.27+0.21X) in Figure -4 clarifies that an increase in one unit of Periostin will lead to increase the FEV by 21%.

Figure -4 ROC curve analysis of serum periostin in relation to other tests among Asthmatic Patients.

Association of serum conc. of periostin with general characteristics of asthmatic patient:

The relationship of general characteristics of cases with the serum Periostin revealed that the only statistical significance was observed in serum conc. of Periostin with male's gender in a median conc. of (89.3 ng/ml) compared to females where the median conc. of periostin was (52.6 ng/ml) with P- value = 0.026, however no statistical significance difference were registered for serum conc. of Periostin in association with other studied factors like; Age onset of asthma, BMI and Smoking status. For detail table -4.

DISSCUSION:

Asthma is regarded as an allergic disease mediated through Th2 driven inflammation¹². The dramatic increase in the prevalence rate of asthma worldwide in next coming years are due to under investigation and because of genetic-environmental changes or might relate to adoption of Western lifestyles and urbanization ^13,14.

In this study, the average age of adult onset asthmatics was calculated at the beginning of the 40s, particularly in association with a significant serum periostin level. More importantly, numerical convergence between the sexes except for a slight increase of females relative to men among study groups, this agreed with^7,15,16. Male predisposition to asthma is mainly endorsed to Yentl’s syndrome which means the affected female was neglecting unless severe attack develops¹⁷

In adulthood age, the frequency of having asthma in both sex is nearly equals¹⁸. This alteration is always attributed to the hormonal fluctuation during this stage. The over expression of Progesterone receptors in the airways epithelium inhibits the thrash frequency of cilia and consequent mucus clearance¹⁹. Conversely, Estrogen, has numerous activities in promoting the IgE class switching towards stimulation of the mast cells and basophils degranulation²⁰_.

Accordingly, the reversed original ratio during adult period makes females become more at risk to get asthma than males. It was reported that asthma morbidity among working women was 1.66%²¹. Rather than hormones²², a number of factors may attribute to this interpretation. Firstly, over report asthma symptoms by females, while males usually deny those symptoms, and secondly, raised BMI predisposed females greater than males for asthma. Therefore, gender does not influence the asthma susceptibility discretely, but it may be play a role in a way corresponding to the age period^21,22.

The results of many studies in regards to the risk of smoking on asthma susceptibility are contradictory with majority of them outbalance a positive effect. Accordingly, many studies showed that smoking stimulate especial inflammatory cells such as neutrophils and macrophages as well as inflammatory cytokines such as IL-6 IL-1β and further induces airway inflammation²³.

Lams et al. reported an increase in eosinophils number and activity as well as neutrophils in the sub mucosa of small airways of smokers compared to non-smokers²⁴. Moreover; previous study which showed the risk of asthma episode associated with very obese of BMI ≥ 35 ²⁵.

This study may conflict with others who demonstrated that the negative significance correlation between Periostin and BMI on the pretext that serum periostin levels might be underestimated in obese patients with asthma when the degree of eosinophilic airway inflammation is parallel to that observed in non-obese subjects²⁶.

Various earlier studies have proved obesity to be one of the noticeable asthma phenotypes^28,27,29,30. A number of mechanisms have been hypothesized as possible interpretation for the impact of obesity on the asthma severity, including atypical respiratory mechanics, bronchial hyper-responsiveness, quality of life, and high frequency of obesity related comorbidities.^31,32,33,34

For further explanations, obesity results in a state of low grade systemic inflammation. As expanded adipose tissue and the space between adipocytes and capillaries increases, hypoxic death of a number of adipocytes gathers macrophages into the tissue. So that, Fatty acids exited from dead adipocytes leads to macrophages activation and production of many inflammatory cytokines, including IL-1b, TNFa, and IL-6 which escape into the systemic circulation and thus may affect the lung³⁵.

The median value of serum Periostin in this study shows that (60 ng/ml) of asthmatic group has a vast difference by more than double the median (25ng/ml) in the control group this significances agree with^36,37,38.

Serum periostin has been identified as the single best predictor of airway eosinophilia in patients with severe asthma^7,39. Thomson et al. performed a study of lebrikizumab (IL-13 mono clonal Abs) in adults with uncontrolled asthma despite treatment with ICS⁴⁰. Lebrikizumab treatment was effective in improving lung function in patients with high levels of serum periostin, which correlates with a study done by⁴¹. Thus, identification of the serum periostin helps in managing the Th2 high eosinophilic phenotype as they do not respond to the traditional first-line therapy, i.e. ICS with Long Acting Bronchodilator Agonists (LABA) combination and those patients might require anti-IL therapy for their improvement of chest symptoms^42,43.

Periostin cut off value according to ROC curve:

In the present study, (Receiver operation characteristic) ROC curve analysis showed acceptable cut off value for serum periostin (38.3ng/ml) and the significant sensitivity (80%) and specificity (70%) values made the biomarker periostin as an ideal non-invasive tool for diagnosis of Th2 high eosinophilic phenotypes asthma. It seems that our cut off-value is in between the lowest one 25 ng/ml reported by⁷ and highest cut off value that registered by James and Jodie studies who found that the best cutoff value for serum periostin calculated was 90 ng/ml^44,45. Whereas, other cutoff value for serum periostin was found by Makoto et al. which was 65ng/ml this discrepancy in cut- off values may be explained by the study number of the included patients with different ages and different assay techniques for serum periostin which was used in their study⁴⁶.

Effect of serum Periostin on asthma control and severity:

Increased markers of allergy, bronchial fibrosis, eosinophilic airway inflammation and sensitivity to Inhaled Corticosteroids (ICSs) are the indications of high subset in Th2⁴⁷.

It is important to distinguish asthma severity and the degree of disease control. In patients receiving treatment, asthma severity varied from the minimum level of treatment required to achieve good control and the intensity of exacerbations while receiving appropriate controller therapy.⁴²

In the present study, the significance increase of Periostin among Poorly Asthmatic control group compare with well and not well controlled group separately and according to their Pulmonary Function Test results and according to ⁴⁸ that consider the FEV ≥ 80% as mild, from 60%-80% as moderate and ≤ 60% as severe asthma.

These results are agreed with⁴⁹ as they found that many patients have inadequately controlled asthma despite understanding and adherence to the recommended treatment modalities for bronchial asthma. Dolan et al. also confirmed the present study as they found that the percentage of uncontrolled severe asthma appeared high, possibly owing to poor management of the disease⁵⁰.

Previous study also in agreement with our results as they found that serum periostin is a clinically useful marker showing that serum periostin concentrations correlated with an annual FEV1 decline, independent of the severity of asthma⁵¹. However, our results are disagreed with other researchers who observed that patients with higher serum periostin concentrations had lower FEV1 in spite of shorter duration of asthma^52,53, thus confirming the fact that periostin is a biomarker indicative of rapid decline in pulmonary function.

A higher significant statistical increase in serum periostin level among uncontrolled asthma patients than those with controlled asthma which was reported previously by¹⁶. It was suggested that serum periostin levels were similar or of non considerable effects among those with mild-to-moderate airway obstruction to those with severe asthma, this may be explained by the high-dose ICS therapy used by severe asthmatics that reduced the serum periostin with its down regulation, thus reducing its significance as a severity marker⁷.

Relation of Periostin with EØ , Monocytes and IgE:

There was significant strong statistical direct correlation between blood eosinophils and serum periostin level among the studied patients (P=0.003), and significant correlation was found between total serum IgE and serum periostin in the present study.

In support of the these findings, Rajanandh et al. who found that there was a progressive increase in airway wall thickness related to serum periostin levels who concluded that subepithelial fibrosis or airway remodeling is one factor that causes steroid resistance in asthma indicating that airway remodeling in some populations may be hyporesponsiveness to ICS and usefulness of measuring serum periostin to expect tissue remodeling⁴³.

Unfortunately, our results were not in agreement with Al-Adawya et al. study who recorded irrelevant changes in serum periostin level between eosinophilic and non eosinophilic subgroups, this may be attributed to the small number of included participants along with the variable disease features in their study¹⁶.

The serum Periostin level in our study have a significance correlation with Monocytes. Our results correspond with a lot of another study that revealed the direct and indirect inflammatory role of Monocytes in immunopathogenesis of asthma.

The resident Alveolar macrophages/monocytes principally provide to maintain lung homeostasis by suppressing inflammation, while immigrating monocytes primarily support allergic inflammation^{54,55,56,57,58}. If lung macrophages derived from recruited monocytes are indeed pathogenic^55,56, then blocking their recruitment could boost allergic inflammatory responses⁵⁶.

In addition, monocytes/macrophages can fight some invading pathogens or allergen, and they have an important function as antigen presenting cells (APCs). Monocytes/macrophages and, to a certain extent, dendritic cells (DCs) appear to play a role in inflammation during asthma exacerbations^59,60.

Furthermore; in this study the Periostin had significant Positive correlation with IgE. This come to agree with the explanation of the ability to present antigens that amplified by IgE bound to FCεRI receptors expressed on the surface of DCs. It has been shown that IgE sequester the allergens, supporting their presentation to memory Th2 lymphocytes⁶¹. FcεRI–IgE-dependent allergen presentation by DCs may critically decline the atopic individual’s threshold to mount allergen-specific T cell responses. In fact, the targeting of allergens to FcεRI via IgE leads to a 1000-fold increase in the activation of T cells⁶², hence high level of IgE was associated with disease severity⁶³.

In this regard, the therapeutic approach of omalizumab may be helpful which can down-regulate FcεRI on both myeloid and plasmocytoid DCs and, consequently, reduce the allergen-specific proliferative response of T cells. Besides, inhibit cytokines like IL-3, IL-5 which strongly affects the eosinophils, GM-CSF, and IL-13 which inferred by Periostin⁶¹.

Relation of asthma treatment with the studied periostin:

The achievement of the accurate medicine in managing asthma in clinical setting requires valid biomarkers detection as inflammation of bronchial airways⁶³. A wide range of biomarkers have been used clinically to predict steroid therapy response, and in the clinical practice to identify asthmatics that will respond to biologic therapies, however currently existing biomarkers are inadequate in number and precision⁶⁴.

ICS treatment inhibits airway periostin expression and in turn leads to its down regulation^44,11,64 thus high levels of serum periostin may represent a non invasive marker for persistent airway eosinophilia despite the ICS treatment in patients with uncontrolled asthma. Makoto et al. also supported the present study and found that ICS therapy was associated with a reduction in serum periostin level and was associated with decreased wall thickness, increased FEV1%, and FEV1/FVC% predicted and decreased sputum eosinophils⁴⁶.

In contrast, serum periostin levels had been shown to be increased in patients who were receiving a higher dose of ICS^65,36.

This discrepancy may explained that an increased periostin levels reflect a TH2-high profile; Asthmatic patients with a TH2-high profile and increased periostin levels had significant improvement in FEV1, whereas the TH2-low population did not^11,12.

even though some results are conflicting, study of sputum and blood eosinophils, periostin, FeNO, and immunoglobulin E (IgE) in severe asthmatic patients established that periostin was a potent predictor from all others biomarkers of sputum and tissue eosinophilia ⁷as well as high serum periostin level predicts the response to omalizumab therapy⁶⁶.

Inflammatory pathways in the airways can be enabled directly through induced sputum sampling, measurement of exhaled gases and bronchoscopy^42,47. However, they are expensive, time consuming, invasive and also they are not widely available in primary care settings. Thus, measurement of non-invasive biomarkers is beneficial to identify Th2-driven eosinophilic airway inflammation for targeted therapies^36,67.

Beyond all advantages of serum biomarkers, it is essential to bear in mind that peripheral blood screen often do not reflect airway biology, and for that reason peripheral blood biomarkers might not represent physiologic mechanisms in the airways⁶⁸.

ACKNOWLEDGEMENT:

The authors are grateful to asthmatic patients recruited in this study, lab. and paramedical staff for their cooperation in this study.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Ahirwar B, Ram A, Ahirwar D. Air pollution: A major Culprit of asthma. Research J. of Science and Tech. 2009;1(1):1-3.

2. Pukelsheim K, Stoeger T, Kutschke D, Ganguly K, Wjst M. Cytokine profiles in asthma families depend on age and phenotype. PLoS One 2010; 5:e14299. doi: 10.1371/journal.pone.0014299.

3. Medrek SK, Parulekar AD, Hanania NA. Predictive biomarkers for asthma therapy. Curr Allergy Asthma Rep. 2017;17(10):69.

4. Gaidan AM , Abbas AA , Hassan , Hashim HM , Al-Mayah QS. The Impact of IL-10 C-597A Gene Polymorphisms on Asthma Susceptibility. Research J. Pharm. and Tech. 2017; 10(9): 2867-2870. DOI: 10.5958/0974-360X.2017.00505.4

5. Blanchard C, Mingler MK, McBride M, Putnam PE, Collins MH, Chang G, et al. Periostin facilitates eosinophil tissue infiltration in allergic lung and esophageal responses. Mucosal Immunol. 2008; 1:289-96.

6. Takayama G, Arima K, Kanaji T, Toda S, Tanaka H, Shoji S, McKenzie A, Nagai H, Hotokebuchi T, Izuhara. Periostin: A novel component of subepithelial fibrosis of bronchial asthma downstream of IL-4 and IL-13 signals. Journal of Allergy and Clinical Immunology 2006; 118: 98-104.

7. Jia G, Erickson RW, Choy DF, Mosesova S, Wu LC, Solberg OD, et al. Periostin is a systemic biomarker of eosinophilic airway inflammation in asthmatic patients. J Allergy Clin Immunol. 2012;130(3):647–54.

8. Conway S, Izuhara K, Kudo Y, Litvin J, Markwald R, Ouyang G, Arron J, Holweg C, Kudo A. The role of periostin in tissue remodeling across health and disease. Cellular and Molecular Life Sciences 2014; 71(7): 1279-1288.

9. Corren J, Lemanske R, Hanania N, Korenblat P, Parsey M, Arron J, Harris J, Scheerens H, Wu L, Su Z, Mosesova S, Eisner M, Bohen S, Matthews J. Lebrikizumab treatment in adults with asthma. New England Journal of Medicine 2011; 365; 1088-1098

10. Sidhu S, Yuan S, Innes A, Kerr S, Woodruff P, Hou L, Muller S, Fahy J. Roles of epithelial cell-derived periostin in TGF-β activation, collagen production, and collagen gel elasticity in asthma. Proceedings of the National Academy of Sciences of the United States of America. 2010; 107(32): 14170-14175.

11. Woodruff PG, Boushey HA, Douglanov GM, Barker CS, Yang YH, Donnely S, et al. Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl Acad Sci USA 2007; 104(40):15858–15863.

12. Woodruff PG, Modrek B, Choy DF, Jia G, Abbas AR, Ellwanger A, et al. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med 2009; 180:388-95.

13. Naveen MR, Santhosh YL. Asthma: An Overview.Research J. of Pharm. and Tech. 2011;4(6):883-890.

14. Maurya PR, Joshi YM, Kadam VJ. A review of bronchial asthma. Res J Pharmacol Pharmacodynamics 2013; 5(4); 257-265

15. Yehia A, Badr M, El-Emery F, El-Zahabi M. Clinical significance of periostin in Egyptian asthmatic patients J Invest Biochem 2017; Vol 6 Issue 1.

16. Al-Adawya E R, Gomaaa A A, Mohamedb A M. Correlation between serum periostin biomarker, spirometric airflow limitation, and airway dimensions by multidetector computed tomography in bronchial asthma Egyptian Journal of Bronchology 2018; 12:160–172.

17. Kuhni CE, Sennhauser FH. The Yentl syndrome in childhood asthma: risk factors for undertreatment in Swiss children. Pediatr Pulmonol 1995; 19:156–160.

18. Schatz M and Camargo CA. The relationship of sex to asthma prevalence, health care utilization, and medications in a large managed care organization. Ann Allergy Asthma Immunol 2003; 91:553–558.

19. Jain R, Ray JM, Pan JH and Brody SL. Sex hormone-dependent regulation of cilia beat frequency in airway epithelium. Am J Respir Cell Mol Biol 2012; 46(4):446-53.

20. Bondsa RS and Midoro-Horiuti T. Estrogen effects in allergy and asthma. Curr Opin Allergy Clin Immunol 2013; 13(1): 92–99.

21. Kausar H, Ubaid-Ur-Rahman M. Working women suffering from various comorbidities- An Overview. Research J. of Pharmacology and Pharmacodynamics 2013;5(2):126-128.

22. Mokif TA, Hassan AJ, Nabat ZN. Evaluation of some physiological parameters in patients with asthma. Research J. Pharm. and Tech. 2019; 12 (5): 2425-2429.

23. Amin K, Ekberg-Jansson A, Lofdah CG and Venge P. Relationship between inflammatory cells and structural changes in the lungs of asymptomatic and never smokers: a biopsy study. Thorax 2003; 58(2): 135–142.

24. Lams BE, Sousa AR, John Rees P and Lee TH. Immunopathology of the small-airway submucosa in smokers with and without chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 158:1518–23.

25. Hjellvik V, Tverdal A and Furu K . Body mass index as predictor for asthma: a cohort study of 118,723 males and females Eur Respir J 2010; 35: 1235–1242.

26. Kimura H, Konno S, Makita H, Taniguchi N, Goudarzi H, Shimizu K, Suzuki M, Shijubo N, Shigehara K , Ono J , Izuhara K, Minagawa Y, Nishimura M. Serum periostin is associated with body mass index and allergic rhinitis in healthy and asthmatic subjects Allergology International 67. 2018; 357-363.

27. Kimura H, Konno S, Isada A, Maeda Y, Musashi M, Nishimura M. Contrasting associations of body mass index and measles with asthma and rhinitis in young adults. Allergy Asthma Proc 2015; 36:293-9.

28. Konno S, Hizawa N, Fukutomi Y, Taniguchi N, Kawagishi Y, Okada C, et al. The prevalence of rhinitis and its association with smoking and obesity in a nationwide survey of Japanese adults. Allergy 2012; 67:653-60.

29. Moore WC, Meyers DA, Wenzel SE, Teague WG, Li H, Li X, et al. Identification ofasthma phenotypes using cluster analysis in the Severe Asthma Research Program. Am J Respir Crit Care Med 2010; 181:315-23.

30. Haldar P, Pavord ID, Shaw DE, Berry MA, Thomas M, Brightling CE, et al. Cluster analysis and clinical asthma phenotypes. Am J Respir Crit Care Med 2008;178: 218-24.

31. Bates JH. Physiological mechanisms of airway hyper responsiveness in obese asthma. Am J Respir Cell Mol Biol 2016;54:618-23.

32. Farah CS, Salome CM. Asthma and obesity: a known association but unknown mechanism. Respirology 2012;17:412-21.

33. Maheshwari P. Somasundaram I. Health related quality of life measurement in asthma and chronic obstructive disease. Res. J. Pharm. and Tech. 2016; 9(5):518-520.

34. Sweeney J, Patterson CC, Menzies-Gow A, Niven RM, Mansur AH, Bucknall C, et al. Comorbidity in severe asthma requiring systemic corticosteroid therapy: cross-sectional data from the optimum patient care research database and the British Thoracic difficult asthma Registry. Thorax 2016;71:339-46.

35. McNelis JC, Olefsky JM. Macrophages, immunity, and metabolic disease. Immunity 2014;41:36-48.

36. Emprm V, Rajanandh MG , Negeswari AD : Periostin - A Novel Systemic Biomarker for Eosinophilic Airway Inflammation: A Case Control Study Journal of Clinical and Diagnostic Research. 2016 ; 10(2): OC01-OC04.

37. Inoue T, Akashi K, Watanabe M, Ikeda Y, Ashizuka S, Motoki T, et al. Periostin as a biomarker for the diagnosis of pediatric asthma. Pediatr Allergy Immunol 2016; 27:521-6.

38. Mohammed D R, Abdelnaby AY, El Zamran EA, Ibrahim IS. Role of serum periostin as a biomarker in diagnosis of bronchial asthma The Egyptian Journal of Chest Diseases and Tuberculosis, 2018; Vol. 67 No. 1, January-March.

39. Izuhara K, Arima K, Ohta S, Suzuki S, Inamitsu M, Yamamoto K. Periostin in allergic inflammation. Allergol Int. 2014;63:143-51.

40. Thomson NC, Patel M, Smith AD. Lebrikizumab in the personalized management of asthma. Biologics. 2012;6: 329-35.

41. Vatrella A, Fabozzi I, Calabrese C, Maselli R, Pelaia G. Dupilumab: a novel treatment for asthma. J Asthma Allergy. 2014; 7:123-30.

42. Ramaya A ,Geetha P, Nandhini M ,Manoj Kumar Raja M. the role of leukotriene receptor antagonist as an add on therapy to b2 agonist in acute asthma. Research J. Pharm. and Tech 2019; 12(4); 1974-78.

43. Rajanandh MG, Nageswari AD, Irshad PP, Ramasamy C. Does dose reduction of an inhaled corticosteroid with the addition of leukotriene antagonist is clinical significance in asthma patients? A aandomized clinical trial. World Appl Sci J. 2013; 24:276-81.

44. James F, Irene B, Justin T, Kudo S. Serum periostin in obstructive airways disease. Eur Respir J 2016; 47:1383–1391.

45. Jodie L, Simpson S, Ian A. Periostin levels and eosinophilic inflammation in poorly-controlled asthma. BMC Pulm Med 2016; 1:67–72.

46. Makoto H, Junichi O, Kenta A. Association of airway wall thickness with serum periostin in steroid-naive asthma. Allergy Asthma Proc 2016; 44:37–45.

47. Rajanandh MG, Nageswari AD, Ilango K. Assessment of various second-line medications in addition to inhaled corticosteroid in asthma patients: a randomized controlled trial. Clin Exp Pharmacol Physiol. 2014; 41:509-13.

48. Michael Schatz, Christine A. Sorkness, James T. Li, Philip Marcus, John J. Murray, Robert A. Nathan, Mark Kosinski, Trudy B. Pendergraft, and Priti Jhingran, Asthma Control Test: Reliability, validity, and responsiveness in patients not previously followed by asthma specialists. J Allergy Clin Immunol 2006; 117:549-56.

49. Stephen P, Petersa G, Fergusonb M. Uncontrolled asthma: a review of the prevalence, disease burden and options for treatment. Respir Med 2006; 100:1139–1151.

50. Dolan C, Fraher K, Bleecker E. Design and baseline characteristics of the epidemiology and natural history of asthma: outcomes and treatment regimens (TENOR) study: a large cohort of patients with severe or difficult to treat asthma. Ann Allergy Asthma Immunol 2014; 92:32–39.

51. Kanemitsu Y, Beasley R, Hurd S, Kudo E. Mepolizumab for prednisone dependent asthma with sputum eosinophilia. N Engl J Med 2009; 360:985–993.

52. Masako M, Hiroki K, Koichi F. Phenotype of asthma related with high serum periostin levels. Allergol Int 2015; 64:175–180.

53. Habernau M, Del Pozo A, Rodríguez V. Role of periostin in uncontrolled asthma. J Investig Allergol Clin Immunol 2017; 27:291–301.

54. Holt PG, Oliver J, Bilyk N, McMenamin C, McMenamin PG, Kraal G, et al. Downregulation of the antigen presenting cell function(s) of pulmonary dendritic cells in vivo by resident alveolar macrophages. J Exp Med. 1993; 177:397–407.

55. Zasłona Z, Przybranowski S, Wilke C, van Rooijen N, Teitz-Tennenbaum S, Osterholzer JJ, et al. Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma. J Immunol. 2014; 193:4245–53.

56. Lee YG, Jeong JJ, Nyenhuis S, Berdyshev E, Chung S, Ranjan R, et al. Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/CCl2, regulate airway inflammation and remodeling in allergic asthma. Am J Respir Cell Mol Biol. 2015; 52:772–84.

57. Westphalen K, Gusarova GA, Islam MN, Subramanian M, Cohen TS, Prince AS, et al. Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity. Nature. 2014; 506:503–6.

58. Careau E, Bissonnette EY. Adoptive transfer of alveolar macrophages abrogates bronchial hyperresponsiveness. Am J Respir Cell Mol Biol. 2004; 31:22–7.

59. Cherrie AH, Anderson K, Wertz GW, Openshaw PJM. Human cytotoxic T cells stimulated by antigen on dendritic cells recognize the N, SH, F, M, 22K, and 1b proteins of respiratory syncytial virus. J Virol 1992; 66:2102–2110.

60. Masten BJ, Lipscomb MF. Dendritic cells: pulmonary immune regulation and asthma. Monaldi Arch Chest Dis 2000; 55:225–230.

61. Schroeder JT, Bieneman AP, Chichester KL, Hamilton RG, Xiao H, Saini S, et al. Decreases in human dendritic cell-dependent T(H)2-like responses after acute in vivo IgE neutralization. J Allergy Clin Immunol. 2010; 125:896–901.

62. Khan SH, Grayson MH. Cross-linking IgE augments human conventional dendritic cell production of CC chemokine ligand 28. J Allergy Clin Immunol. 2010; 125:265–7.

63. Al-Zubaidy BS,AL-Mahdawi ZMM,AL-Azaawie AF. Detection of IgE, some Antioxident Parameter levels and their Association with Polymorphism of ADAM33 Genein Asthma Pateints 2019; Research J. Pharm. and Tech. 2017; 10(9): 2867-2870.

64. Tiotiu A. Biomarkers in asthma: state of the art. Asthma Res Pract. 2018; 4: 10.

65. Kanemitsu Y, Matsumoto H, Izuhara K, Tohda Y, Kita H, Horiguchi T, et al. Increased periostin associates with greater airflow limitation in patients receiving inhaled corticosteroids. J Allergy Clin Immunol 2013; 132:305-312.e3.

66. Tajiri T, Matsumoto H, Gon Y, Ito R, Hashimoto S, Izuhara K, et al. Utility of serum periostin and free IgE levels in evaluating responsiveness to omalizumab in patients with severe asthma. Allergy. 2016; 71:1472–1479.

67. Pavithra H. Dave, Preetha. Pathogenesis and Novel drug for treatment of asthma- A review. Research J. Pharm. and Tech. 2016; 9(9):1519-1523. DOI: 10.5958/0974-360X.2016.00297.3

68. Grayson MH, Feldman S, Prince BT, Patel PJ, Matsui EC, Apter AJ. Advances in asthma in 2017: mechanisms, biologics, and genetics. J Allergy Clin Immunol. 2018; 142:1423–1436.

Received on 20.09.2019 Modified on 18.11.2019

Research J. Pharm. and Tech. 2020; 13(7): 3103-3112.

DOI: 10.5958/0974-360X.2020.00550.8

Variables		Serum Conc.			P –Value
Variables		Mean ± SD	Range	Median (IQR)	P –Value
Age of Onset	< 18 (11)	88.2±50.8	(30.9-176.7)	67.4 (47.4-121.1)	0.542
Age of Onset	≥ 18 (33)	81.3±58.5	(17.9-275.6)	56.8 (39.5-103.4)	0.542
Gender	Male (21)	102.4 ± 13.9	(20.6 – 275.6)	89.3 (51.9 – 151.3)	0.026
Gender	Female (23)	65.27 ± 8.81	(17.9 – 189.2)	52.6 (37.2 – 66.8)	0.026
BMI level	Normal (8)	81.4 ± 18.1	(32.1 – 182.9)	64.2 (49.6 – 119.4)	0.513
	Over weight (14)	99.4 ± 17.7	(30.9 – 275.6)	96 (96.0 – 128.4)
	Obese (22)	73.2 ± 10.9	(17.9 – 189.2)	54.4 (40.4 – 95.5)
Smoking	Smoker (3)	89.6 ± 27.9	(56.4 – 145.1)	67.4 (56.4 – 106.3)	0.658
	Non-smoker (30)	86.3 ± 10.9	(20.6 – 275.6)	60.9 (40.4 – 116.6)
	Passive smoker (11)	72.2 ± 15.1	(17.9 – 189.2)	54.7 (41.0 – 93.4)

Variables	Serum Conc.			P – Value
Variables	Mean ± SD	Range	Median (IQR)	P – Value
Duration of Asthma (Years)
< 5(11)	102.4 ± 60.7	(32.1 – 189.2)	93.4 (41– 157.5)	0.284
≥ 5(32)	76.5 ± 54	(17.9 – 275.6)	56.83 (41.7 –102)	0.284
Severity of Asthma
Mild (1)	51.09	51.09	51.09	0.220
Moderate (12)	61.2±35.1	(20.6-136.7)	49.98 (36.96 –93)
Severe (31)	92.5±61.3	(17.9-275.6)	66.8 (47.5-145.1)
Level of asthma Control
Well (17)	45±14	17.9- 67.4	47.4 (36.8- 55.3)	0.0001
partly (9)	63.5±35.6	30.9- 145.1	54.7 (37.2- 78.1)
Poorly (18)	128.6±57.9	35.5- 275.6	118.1 (90.5-170.8)

Variables	Serum Conc.				P–Value
	Mean ± SD		Range	Median (IQR)
Eosinophil
Normal (19)		54.1 ± 33.16	(17.9 – 176.7)	51.1 (36.4- 64.5)	0.003
High (25)		104.99± 33.2	(20.6 – 189.2)	101.8 (52.4- 147.7)
Monocytes
Normal (28)		57.97±35.82	(17.9- 176.7)	48.9 (36.6- 65.2)	0.0003
High (16)		126.8±59.3	(54.7- 275.6)	109.5 (83.7- 166)
Total IgE
Normal (10)		50.04 ±23.5	(20.6- 102.8)	44.7 (34.7- 65.8)	0.023
High (34)		92.7±23.5	(17.9- 275.6)	65.9 (49.5- 138.8)

Drug used	Serum Conc.			P – Value
Drug used	Mean ± SD	Range	Median IQR	P – Value
Salbutamol Inhaler
Yes (20)	78.6 ± 54.4	(17.9 – 189.2)	54.5 (36.7 – 102.6)	0.423
No (24)	86.7 ± 58.5	(32.1 – 275.6)	64.2 (44.1 – 119.6)	0.423
Corticosteroid Inhaler
Yes (18)	94.9 ± 71.2	(17.9 – 275.6)	73.1 (36.8 – 146.7)	0.667
No (26)	74.7 ± 42.5	(20.6 – 168.8)	56.6 (46.0 – 105.1)	0.667
Montelukast
Yes (21)	86.1 ± 68.4	(17.9 – 275.6)	57.4 (37.4 – 120.3)	0.664
No (23)	80.2 – 43.6	(20.6 – 168.8)	62.9 (62.9 – 154.6)	0.664