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ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 3  |  Page : 1036-1040

Serum granulysin as a possible key marker of activity of alopecia areata


1 Department of Dermatology, Andrology and STDs, Faculty of Medicine, Menoufia University, Shebeen El Kom, Egypt
2 Department of Medical Biochemist, Faculty of Medicine, Menoufia University, Shebeen El Kom, Egypt
3 Department of Dermatology and Andrology, Shebin El Kom Teaching Hospital, Shebeen El Kom, Egypt
4 Department of Andrology and STDs, Shebeen El Kom, Egypt

Date of Submission27-Jan-2019
Date of Decision09-Mar-2019
Date of Acceptance10-Mar-2019
Date of Web Publication30-Sep-2020

Correspondence Address:
Doaa M. A. El-Wareth Tolba
Department of Andrology and STDs, Menoufia Governorate, Shebeen El Kom
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_45_19

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  Abstract 


Objective
To study whether granulysin (GNLY) level, which is known to reflect the activity of cytotoxic immune response, is related to the disease activity of alopecia areata (AA).
Background
AA is a common type of hair loss or alopecia in humans. GNLY is an important mediator of damage in a variety of skin diseases.
Patients and methods
A case–control study was carried out on 72 individuals. A total of 36 patients experiencing AA circumscripta, selected from the Dermatology Outpatient Clinic, Menoufia University Hospital, and 36 healthy individuals as a control group were recruited during the period from October 2017 to March 2018. Detailed history, laboratory investigations, and dermatological examination were done.
Results
There was a statistically highly significant difference (P < 0.001) between both groups regarding serum GNLY level. Moreover, most patients (30 case, 83.3%) were complaining of alopecia without association with any other allergic diseases. Serum GNLY level (ng/ml) was highly significantly positively correlated (P < 0.001) with recurrence of AA and course of AA. Moreover, it showed a significant positive correlation (P < 0.05) with the extent of alopecia in the body and the association with other allergic diseases.
Conclusion
Serum GNLY level was significantly positively correlated with recurrence, course, extent of alopecia, and association with other allergic diseases. Serum GNLY levels in the acute phase can be a useful and novel marker for the disease activity and prognosis of AA.

Keywords: alopecia areata, disease activity, marker, serum granulysin


How to cite this article:
Maraee AH, El-Hefnawy SM, Mahrous EA, El-Wareth Tolba DM. Serum granulysin as a possible key marker of activity of alopecia areata. Menoufia Med J 2020;33:1036-40

How to cite this URL:
Maraee AH, El-Hefnawy SM, Mahrous EA, El-Wareth Tolba DM. Serum granulysin as a possible key marker of activity of alopecia areata. Menoufia Med J [serial online] 2020 [cited 2020 Oct 29];33:1036-40. Available from: http://www.mmj.eg.net/text.asp?2020/33/3/1036/296698




  Introduction Top


Alopecia areata (AA) is a common type of hair loss or alopecia in humans; it is an autoimmune disease with a variable, typically relapsing or remitting, course that can be persistent especially when hair loss is extensive. AA is the second most frequent nonscarring alopecia after male and female pattern alopecia [1]. In 1–2% of cases, the condition can spread to the entire scalp (alopecia totalis) or to the entire epidermis (alopecia universalis). AA has a reported incidence of 0.1–0.2% with a lifetime risk of 1.7%, with men and women being affected equally [2]. The etiology of AA has eluded investigators for years, and therefore, a multitude of associations have been proposed by researchers in the field of trichology. One of the strongest associations is with autoimmunity [3]. There are two types: [1] scarring alopecia, where there is fibrosis, inflammation, and loss of hair follicles (HFs), and [2] nonscarring alopecia, where the hair shafts are gone but the HFs are preserved, making this type of alopecia reversible [4].

AA is an organ-specific autoimmune disease against HFs. Anagen HFs are immunoprivilege sites with downregulation of major histocompatibility complex class I antigens as well as with local expression of potent immunoinhibitory signaling media and are escaping from attacks by cytotoxic T cells (CTLs) [5]. HFs also appear to downregulate the expression of ligands that stimulate the activation of natural killer (NK) cells, which are primed to recognize and eliminate major histocompatibility complex class I negative cells [6]. Granulysin (GNLY) is a cytolytic and proinflammatory molecule expressed by activated human CTLs and NK cells [7]. GNLY is found in cytolytic granules in CTL and NK. GNLY is made in a 15 kDa form that is cleaved into a 9 kDa form, which is broadly cytolytic against tumors, microbes, fungi, yeast, and parasites. GNLY is also a chemoattractant for T lymphocytes, monocytes, and other inflammatory cells and activates the expression of several cytokines including interleukin (IL)-10, IL-1, and IL-6. GNLY is implicated in a lot of diseases including infections, cancer, transplantation, autoimmunity, and skin [8]. GNLY is an important mediator of damage in a variety of skin diseases, including folliculitis, psoriasis, acne, lichen planus, and viral vesicles [9]. A collapse of the immunoprivilege and subsequent cell-mediated cytotoxicity to HFs has been considered essential to the pathogenesis of AA [7]. Accordingly, it has been reported that CD4+ T cells, CD8+ T cells, and NK cells, infiltrate around anagen HFs in active AA lesions. Activated CTLs and NK cells express GNLY, and the serum concentration of GNLY is considered to reflect the activity of these cells. GNLY is steady in the serum, and its half-life is longer than that of cytokines; therefore, evaluation of serum GNLY levels is useful for monitoring cell-mediated cytotoxic immune responses in vivo [10]. The aim of this study was to study whether serum GNLY level, which is known to reflect the activity of cytotoxic immune response, is related to the disease activity of AA.


  Patients and Methods Top


A case–control study was carried out on 72 individuals. A total of 36 patients having alopecia areata circumscripta (AAC), who were selected from the Dermatology Outpatient Clinic, Menoufia University Hospital, and 36 healthy individuals as a control group were recruited during the period from October 2017 to March 2018.

Ethical consideration

A written consent approval was obtained from Research Ethical Committee of Faculty of Medicine Menoufia University from every patient before the study initiation.

Patients included in the study were divided into two groups as follow: group A (cases) included 36 (15 male and 21 female) patients having AAC, and their age ranged between 11.0 and 47.0 years. Group B (control) included 36 (19 male and 17 female) healthy individuals, and their age ranged between 15.0 and 46.0 years.

Inclusion criteria

Patients of both sexes with AAC and no history of systemic (1 month) or topical (2 weeks) treatment of AAC were included.

Exclusion criteria

Any patient with one or more of the following was excluded from the study: patients with a history of systemic treatment (corticosteroids, phototherapy, or cyclosporine A) before 6 weeks of the study, patients treated with antihistamines or vascular drugs (e.g., vitamin K antagonist, heparin, aspirin, ticlopidine, clopidogrel, pentoxifylline, or diosmin) within 6 weeks of the study.

Cases who fulfill the eligibility criteria were subjected to the following

History taking

It included personal history such as name, age, and sex; history of the present illness, such as duration of the disease, number of episodes of AA, onset, course, and duration of alopecia lesions, either limited to the scalp, or in other sites of the body; family history of AA cases; current medications; previous treatment; associated diseases; and drugs.

General and dermatological clinical examination

General examination was done to exclude associated systemic diseases. Dermatological examination was done for determination of the site of AA and nail affection.

Specific investigation

Assessment of serum GNLY level in the two studied groups was done by enzyme-linked immunosorbent assay (ELISA) technique.

Blood sampling

Overall, 2 ml of venous blood was withdrawn from every patient and then were transferred into a plain tube, centrifuged for 10 min at 4000 rpm. The serum obtained was kept frozen at − 20°C till determination of human GNLY levels.

GNLY was assayed using Sunred ELISA kit (Shanghai, China) [9].

Test principle

The kit uses a double-antibody sandwich ELISA. The chroma of color and the concentration of the GNLY of sample were positively correlated.

Statistical analysis

Results were analyzed using SPSS, version 22 (SPSS Inc., Chicago, Illinois, USA). Two types of statistics were done: descriptive, for example, percentage, mean, median and SD, and analytical, including Student's t-test, which was used to collectively indicate the presence of any significant difference between the two groups for a normally distributed quantitative variable, independent t-test was used to compare two groups regarding quantitative variables (mean ± SD), χ2 and Wallis analysis of variance for comparing categorical data, and Spearman coefficient was used to correlate between two distributed abnormally quantitative variables. A value of P less than 0.05 indicated statistically significant.


  Results Top


The current study shows that age of patients ranged from 11 to 47 years with mean ± SD of 29.53 ± 9.35 years, whereas the age of controls ranged from 15 to 46 years, with mean ± SD of 28.94 ± 8.61 years. Most patients were females (21, 58.3%), with male: female ratio of 1: 1.4, whereas most of control group were males (19, 52.8%) with male: female ratio of 1.1: 1. Regarding family history about AA, 22 (61.1%) patients had negative family history of AA and 14 (38.9%) had positive. On the contrary, 19.4% (seven patients) of control group had positive family history and 80.6% (29 patients) had negative family history [Table 1]. There was statistically highly significant difference (P < 0.001) between both groups regarding serum GNLY level. Mean serum GNLY level among the patient group is significantly increased (85.869 ± 43.827 ng/ml) than the control group (30.624 ± 6.489 ng/ml) [Table 2].
Table 1: Sociodemographic data of the studied groups

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Table 2: Comparison between the two studied groups regarding serum granulysin level

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In addition, in this study, the duration of AA in cases group ranged from 1 to 9 weeks, with mean ± SD of 3.61 ± 2.56 weeks, whereas SALT score of AA (%) ranged from 0.0 to 100, with mean ± SD of 32.78 ± 31.22. Among the case group, 19 (52.8%) patients had first attack of AA, whereas 17 (47.2%) showed recurrent attacks. Regarding the course of AA among the case group, most patients (58.3%) had progressive course more than half of patients (21 patient, 58.3%) showing the patches of alopecia in the scalp. In addition, most patients (30 case, 83.3%) were complaining of alopecia without association with any other allergic diseases [Table 3].
Table 3: Distribution of the studied cases according to disease duration (weeks), SALT score of alopecia areata (%), recurrence, course, and site of alopecia, and other allergic diseases in cases group (n=36)

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Furthermore, serum GNLY level (ng/ml) was highly significantly positively correlated (P < 0.001) with recurrence of AA and course of AA. Moreover, it showed a significantly positive correlation (P < 0.05) with the extent of alopecia in the body and other association with other allergic diseases [Table 3].


  Discussion Top


This study comes in agreement with a study conducted by Ono et al. [10] who found that the mean age of patients with acute AA was 34.7 ± 10.9 years, of those with chronic AA was 35.3 ± 15.5 years, and of healthy controls was 32.1 ± 4.8 years. Male/female ratio was 5: 11, 6: 12, and 3: 11 for acute AA, chronic AA, and healthy controls, respectively. It was also in agreement with Dy and Whiting [11] who found that mean age of patients with AA was 33 ± 7.11 years and that of normal healthy group was 43.0 ± 16.03 years. Most patients were females (615, 67.8%), with male: female ratio of 0.47: 1, but most of the control group was male (13, 59.09%), with male: female ratio of 1.4: 1. In addition, Lin et al. [9] found that the age of patients ranged from 17 to 49 years, with mean ± SD age of 22.57 ± 11.64 years. Most patients were males (68, 68%). Moreover, male: female ratio was 2.03: 1. Furthermore, El-Morsy et al. [12] found that the mean ± SD ages of patients and controls were 24.51 ± 14.26 and 29.27 ± 12.07 years, respectively. Of the 39 patients, 28 (71.8%) were male and 11 (28.2%) were female, whereas 21 (56.8%) members of the control group were male and 16 (43.2%) were female. Seven (17.9%) of the 39 patients had associated lesions on other body sites. Only three (7.7%) patients had a positive family history of AA. Four (10.3%) patients had associated nail involvement. Moreover, in a study by Mehran et al. [13] where 94 (47 cases and 47 controls) patients completed the questionnaires, it was found that the mean age and score of all patients were 28.56 ± 8.34 (14–48) years old and 7.05 ± 6.58 (0–27), respectively. Male patients represented 54 (57.4%) and females 40 (42.6%) [Table 4].
Table 4: Relation between serum granulysin level (ng/ml) and different parameters in cases group (n=36)

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In contrast, a 2014 study with 513 patients showed that 18.7% of patients were over 50 years old when the disease first appeared [14]. However, Lazzarini et al. [6] found that only two patients with AA (6.7%) showed family history, whereas the literature shows that family cases are present in 10–25% of patients with AA [15]. The difference may be related to different factors, such as a missing diagnosis of the disease in the family.

Our result is in agreement with the study of El-Morsy et al. [12] who studied 39 patients presenting with AA of the scalp, with or without the involvement of other body sites and 37 healthy controls. They found that disease duration ranged from 2 to 20 years (mean ± SD duration: 1.22 ± 3.56 years). Patient SALT scores ranged from 0 to 100 (mean ± SD: 17.23 ± 26.59). Moreover, Mehran et al. [13] found that the duration of alopecia was 9.85 ± 1.79 months (range: 5–11). The distribution of alopecia in patients was as follows: head 30 (63.8%), beard 16 (34%), and body 1 (2.1%). Patients who had AA on the beard area had higher age in comparison with head area (31.25 ± 7.09 vs. 27.3 ± 9.01 years, P = 0.13). However, the study carried out by You and Kim [3] showed that disease duration was 15.89 ± 34.70 and 33.68 ± 64.66 months in the mild-to-moderate and severe groups, respectively; thus, the severe group showed disease duration twice that of the mild-to-moderate group (P < 0.001). The mild-to-moderate and severe AA groups had disease duration of greater than 1 year in 27.4 and 49.2% of patients, respectively (P < 0.001). This finding is consistent with previous reports that prognosis is improved with early treatment initiation [16].

The result of our study revealed that there was a statistically highly significant difference between both groups regarding serum GNLY level. In the same line with our results, Ono et al. [10] found that serum GNLY levels (mean ± SD) in patients with acute AA, patients with chronic AA, and healthy controls were 26.2 ± 21.3 (range: 0.02–78.1), 12.1 ± 5.8 (range: 4.6– 21.4), and 5.9 ± 4.4 ng/ml (range: 0.004–12.6), respectively. Remarkably, serum GNLY levels in both patients with acute and those with chronic AA were significantly higher than those in healthy control patients.

In this study, serum GNLY level (ng/ml) was highly significantly positively correlated (P < 0.001) with recurrence of AA and course of AA. Moreover, it showed a significant positive correlation (P < 0.05) with sites of the body and other allergic diseases. Our results are in agreement with Ono et al. [10] who found that a positive correlation between the percentage of bald skin areas and serum GNLY levels was observed in acute AA. They next classified patients with acute AA into two groups from the aspects of prognosis based on the bald skin areas. Patients with poorer prognosis (disease progression, AT, or AU) exhibited higher serum GNLY levels than patients with better prognosis (disease amelioration or remission) [17]. In addition, Bodemer et al. [18] found that serum GNLY levels in patients with AA with allergic disorder such as atopic dermatitis and asthma were higher than those in patients with AA without such associations. Serum GNLY levels were significantly associated with the broader bald skin areas, poorer prognosis in acute AA, and co-existence of allergic disorders in patients with AA; perifollicular GNLY-bearing cells, which were mainly consistent with CD8-T cells, were mainly detected in acute AA lesions [18]. Therefore, GNLY may represent cell-mediated cytotoxicity and disease activity of AA, and the serum GNLY level may be a useful and novel marker for the disease activity in the acute phase of AA. In cell-mediated cytotoxicity, apoptotic mechanisms involving the perforin/granzyme B system or the Fas–Fas ligand pathways have been indicated to mediate the persistence of severe chronic AA, though there are some contradicting reports [19].


  Conclusion Top


Serum GNLY level was significantly positively correlated with recurrence of AA, course of AA, site of alopecia, and other allergic diseases. Serum GNLY levels in the acute phase can be a useful and novel marker for the disease activity and prognosis of AA. There are few studies concerning the clinical characteristics, epidemiology, and prognosis of Egyptian patients with AA as well as serum GNLY level in AA. Hence, further studies with larger samples are needed to clarify the serum GNLY role as novel marker for the disease activity and prognosis of AA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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