Does involucrin have a Role in Verruca Vulgaris? A Clinical and Immunohistochemical Study.

INTRODUCTION: Several modalities are used in the treatment of verrucae vulgaris; however, their side effects are common. Vitamin D3 has been recently used as a treatment in verruca vulgaris. AIM OF THE WORK: We aimed to assess the expression of involucrin in verrucae before and after intralesional injection of vitamin D3 and its correlation with clinical response. SUBJECTS AND METHODS: This study included 60 patients with verrucae vulgaris. These patients were subjected to intralesional injection of vitamin D3 at 3-week intervals for a maximum of five sessions. The pathological assessment was done by skin biopsies obtained from thirty patients before the first session and after the last session of injection and compared to skin biopsies from 30 healthy individuals.
RESULTS: The injected verrucae showed complete response in 39 patients (65%), partial response in 15 patients (25%), and no response in 6 patients (10%). Nonsmoker patients had a better response than smokers. Vitamin D3 injections also resulted in increasing involucrin expression and changing its pattern of expression.
CONCLUSIONS: Intralesional vitamin D3 is an effective treatment for verrucae vulgaris. Involucrin expression is modified in verrucae. Copyright:

Introduction

Verrucae are common skin infections caused by the human papillomavirus (HPV) that may affect up to 2% of the population.[1] Most of the modalities used for the treatment of verrucae are destructive and may develop scars with the possibility of recurrence.[2] Intralesional immunotherapy is used in the treatment of verruca with several noticed side effects as flu-like symptoms, pain, and secondary bacterial infection.[3]

Intralesional vitamin D injection has been recently used for the treatment of verrucae,[45] and many theories are explaining the mechanism of action of vitamin D in the treatment of verrucae.

Involucrin is a differentiation marker of keratinocytes and is expressed in normal skin in upper spinous and granular layers.[6] This differentiation of keratinocytes is disturbed by HPV.[7]

Aim of the study

We aimed to assess the expression of involucrin in verrucae before and after intralesional injection of vitamin D3 and its correlation with clinical response.

Subjects and Methods

This study was designed as a quasi-interventional study.

Ethical considerations

This study was conducted after the approval of the Ethics Committee on Research involving Human Subjects in the Faculty of Medicine, Benha University, Egypt. This study had been registered in ClinicalTrials.gov under number [NCT04793529].

This study included 60 patients with verrucae vulgaris in the clinical trial and skin samples from 30 normal individuals for pathological assessment.

This study was carried out from January 2019 to October 2019 in the Outpatient Clinic of Dermatology and Venereology at Benha University Hospitals and Department of Pathology at Faculty of Medicine, Menoufia University, Egypt.

Patients older than 18 years complaining of multiple verrucae vulgaris from both sexes were included in this study. They had a verruca size that was sufficient for taking a biopsy and intralesional injection (more than 4 mm). Participants excluded from this study were patients with a history of an allergic response to vitamin D, pregnant or lactating females, hepatic patients, renal patients, and patients with bleeding tendency, under chemotherapy or using immunosuppressive drugs, or those who had received any treatment for verruca in the last 2 months before the study.

Methods

A complete history was obtained from each patient including personal history (age, sex, and special habits of medical importance), duration, course, and site of the existing verrucae. All patients have been examined before each injection session with emphasis on the number, size, and shape of verrucae.

I- Clinical procedure:

The injected verruca was cleansed and sterilized with povidone-iodine. An anesthetic cream (EMLA®) was applied for half an hour before injection to decrease pain. Intralesional injection of 0.6 ml of vitamin D3 (Devarol-S of Memphis®, Egypt) which contains 200,000 IU, 5 mg/2 ml was done in the base of the verruca (the biggest verruca was chosen from each patient) using a 31-gauge insulin syringe. The injection was done every 3 weeks up to five sessions or until complete clearance of the verruca. After the last session of injection, patients were followed up for 6 months to record recurrence of verruca or any side effects. The clinical response was divided into three grades according to a decrease of the size of verruca: no response (unchanged or <50% in a decrease of verruca size), partial response (>50% decrease of verruca size but not complete absence), and complete response (complete absence of verruca).[8] In addition, the noninjected verrucae were also followed up during the study.

II- Pathological assessment:

Initially, an excisional skin biopsy of a verruca was taken from thirty patients selected from patients before intralesional injection of vitamin D3 under local anesthesia using 0.01 ml of lidocaine 2%. A second biopsy was taken from other injected verrucae at the end of the study after 2 weeks of the last intralesional injection of vitamin D3. A nonsun exposed skin biopsy was taken from 30 apparently healthy individuals undergoing plastic surgery as a control group. These biopsies were fixed in neutral formalin 10% and submitted to routine tissue processing ending with paraffin-embedded blocks. Two sections of 5 μm thickness were cut from these blocks. One section was stained with hematoxylin and eosin for evaluation of histopathological changes, while the other section was cut on poly-L-lysine coated slides for immunohistochemical staining using a monoclonal antibody against involucrin.

This immunostaining was a streptavidin-biotin immunoperoxidase amplified system and was done according to producer instructions (sc-21748 AC), 500 μg/ml (Santa Cruz Biotechnology®, Dallas, TX, USA). Tissue slides were subjected to deparaffinization and rehydration. The antigen retrieval was performed by boiling in a citrate buffer saline (pH 6), followed by cooling at room temperature. The endogenous peroxidase was blocked by incubation with H2O2 3%. The primary antibody was incubated overnight at room temperature, and then the secondary antibody was applied with (3, 3'-diaminobenzidine (DAB) as a chromogenic substrate and Mayer's hematoxylin as a counterstain. The normal human epidermis was used as a positive control for involucrin. Replacement of the primary antibody in the staining process with a blocking buffer was included as a negative control.

Interpretation of involucrin immunohistochemical staining was done by an independent pathologist who was blinded to clinical procedure. Positive involucrin expression was assigned cytoplasmic, membranous, or combined type. The degree of the involucrin expression was assessed subjectively according to the intensity of immunostaining into mild (+), moderate (++), or strong (+++).

Statistical analysis

The collected data were tabulated in a suitable form. Quantitative data were summarized in the form of mean and standard deviation, while qualitative data were in the form of frequency and percentage. Both parametric and nonparametric models were used for the corresponding type of data. P value ≤0.05 was considered statistically significant. The statistical analysis was done using Statistical Package for Social Sciences (SPSS®), version 23 for Windows 10® 64 bit.

Results

This study included 60 patients complaining of verruca vulgaris and skin samples from 30 healthy subjects, and their data are summarized in Table 1. All patients had completed the trial.

Table 1

Demographic data of patients and controls included in the study

	Patients (60)	Control (30)	Test	P
Gender
Female	26 (43.33%)	14 (46.67%)	χ2=0.09	0.764
Male	34 (56.67%)	16 (53.33%)
Age (year)	28±8	27±7	Z=0.582	0.562
Smoking
Yes	22 (36.67%)	12 (40%)	χ2=0.095	0.758
No	38 (63.33%)	18 (60%0
Duration of verruca (month)	8±3
Site of verruca	Hand (50%)
	Foot (19%)
	Leg (18%)
	Arm (13%)

χ2=Chi-square test, Z=Mann-Whitney. P≤0.05 is significant

The injected verrucae showed complete response in 39 patients (65%) [Figure 1], partial response in 15 patients (25%), and no response in 6 patients (10%), while the noninjected verrucae showed complete response in 7 patients (11.67%), partial response in 11 patients (18.33%), and no response in 42 patients (70%).

Figure 1

Patient with multiple verrucae before (a) and after (b) sessions of intralesional injection of vitamin D3 shows complete response

There was a significant difference in clinical response to vitamin D3 injection between smoker and nonsmoker patients (P = 0.01). All patients with no response were smokers. In addition, 11 patients (73.33%) with partial response were also smokers compared to only 2 (5.13%) in those with complete response were smokers [Table 2].

Table 2

Effect of different variables on response of injected verrucae

	Clinical response			Test	P
	Complete (39)	Partial (15)	No response (6)
Age (year)	25±5	31	28	KW=1.975	0.389
Gender
Female	18	6	2	FET=0.846	0.874
Male	21	9	4
Duration of verrucae (month)	7±2	8±4	6±4	Z=3.853	0.273
Site
Hand	17 (43.59%)	7 (46.66%)	3 (50%)	FET=3.180	0.274
Foot	9 (23.08%)	3 (20%)	2 (33.33%)	FET=2.574	0.312
Leg	7 (17.95%)	3 (20%)	1 (16.67%)	FET=0.359	1.0
Arm	6 (15.38%)	1 (6.68%)	0 (0%)	FET=2.167	0.426
Number of sessions	2±1	3±2	5	Z=17.709	<0.001
Smoking
Yes	2 (5.13%)	11 (73.33%)	6 (100%)	FET=9.333	0.01
No	37 (94.87%)	4 (26.67%)	0
If yes, number of sessions	3±2	4±1	5	Z=18.35	<0.001

FET=Fisher’s exact test, KW=Kruskal-Wallis test, Z=Mann-Whitney test. P≤0.05 is significant

There were differences between verruca before vitamin D3 injection and control skin samples in histopathologic features [Figure 2a and b] and involucrin expression [Figure 2c and d]. The degree of involucrin expression was significantly higher in control [Figure 2c] compared to verruca before vitamin D3 injection [Figure 2d] (P < 0.001), and also the pattern of expression of involucrin was mostly cytoplasmic in verruca before vitamin D3 injection [Figure 2d], while it was mostly membranous in normal skin [Figure 2c] (P = 0.0001).

Figure 2

Normal skin (a) and biopsy from verrucae before vitaminD3 injection is characterized by acanthosis, hypergranulosis, papillomatosis and hyperkeratosis (b) (hematoxylin and eosin x100 for A and x 200 for B). Normal skin showed moderate involucrin expression with membranous pattern in spinous and granular cell layers (c) (immunohistochemical staining x 100). Verrucae before vitaminD3 injection showed mild and cytoplasmic pattern expression of involucrin (immunohistochemical staining x200) (d)

After vitamin D3 injection, the epidermal thickness decreased [Figure 3a] and the expression of involucrin was significantly increased in degree and become more membranous type than before injection (P = 0.0001 and 0.0008, respectively) [Figure 3b]. In addition, there were no significant differences regarding involucrin between normal skin and verruca after vitamin D3 injection regarding degree and pattern of expression (P = 0.1333 and 0.331, respectively) [Table 3 and Figure 4].

Figure 3

Verrucae after third session of vitamin D3 injection showed a decrease in epidermal thickness (a) (hematoxylin and eosin × 100). Verrucae after third session of vitamin D3 showed a strong and membranous pattern of involucrin expression (b) (immunohistochemical staining × 100)

Table 3

Comparisons between verruca and normal skin regarding expression of involucrin

	Pattern of expression					Degree of expression
	Cytoplasmic	Membranous	Combined	Test (χ2)	P	Mild	Moderate	Strong	Test (χ2)	P
Comparative group
Normal skin	2	21	7	24.757	<0.001	7	10	13	18.267	0.0001
Verruca before injection	15	3	12		23	5	2
Normal skin	2	21	7	2.211	0.331	7	10	13	4.0302	0.1333
Verruca after injection	5	16	9		8	6	16
Verruca after injection	5	16	9	14.323	0.0008	8	6	16	18.238	0.0001
Verruca before injection	15	3	12		23	5	2

χ2=Chi-square test. P≤0.05 is significant

Figure 4

Expression of involucrin in normal skin and in verruca before and after intralesional injection of vitamin D3

The expression of involucrin was significantly varied between patients with complete clinical response and those with partial or no response to vitamin D3 injection. The expression was mostly of membranous pattern with a high grade in patients with complete response than those with partial response or without response (P = 0.0055 and 0.0103, respectively) [Table 4 and Figure 5].

Table 4

Comparisons between expression of involucrin after vitamin D3 injection regarding clinical response

	Pattern of expression					Degree of expression
	Cytoplasmic	Membranous	Combined	Test (χ2)	P	Mild	Moderate	Strong	Test (χ2)	P
Complete response (16)	1	13	2	14.652	0.0055	1	2	13	13.216	0.0103
Partial response (8)	1	2	5			3	3	2
No response (6)	3	1	2			4	1	1

χ2=Chi-square test. P≤0.05 is significant

Figure 5

Expression of involucrin regarding clinical response

Regarding side effects, there was a temporary pain noticed after injection, which was treated by a mild analgesic. After a follow-up of the patients, there was a recurrence of verruca in only two patients (0.033%). No serious side effects were reported during the study and the follow-up period.

Discussion

Verrucae are acquired after abrasion of the skin and exposure of the basal cell to HPV viral particles.[9] After this, the virus begins to replicate in the first stage at a slow rate in undifferentiated basal cells and a second rapid amplification in the upper cell layer cells of the epidermis.[10] This replication affects the delicate balance between proliferation and differentiation of keratinocytes.[11]

The previous studies[1213] showed that serum vitamin D level is an important factor to determine immune response against HPV.

Results of this study showed an effective response to intralesional injection of vitamin D3 in verruca vulgaris. In addition to the increase of involucrin expression, there is a change of its pattern of expression from cytoplasmic to membranous.

This efficacy of vitamin D3 in the treatment of verrucae was reported by previous studies[5814]; however, there are no previous studies about the role of vitamin D3 on the expression of involucrin in verrucae.

Vitamin D3 is the active form of vitamin D and acts through nuclear vitamin D receptor (VDR).[15] Vitamin D3-VDR interaction results in transcription of target genes of VDR response elements leading to a decrease of proliferation and an increase of differentiation of the keratinocytes[16] and expression of differentiation markers;[17] these cellular changes are interrupted by HPV infection.[18]

Besides the direct effect of vitamin D3 as a regulator of keratinocyte proliferation and differentiation, vitamin D3 has other functions in the skin. Vitamin D3 can increase innate immunity through the production of the antimicrobial peptides,[19] activation of toll-like receptor 2, 7,[20] restoring of epidermal barrier,[21] natural killer cell activation,[22] and adaptive immunity.[23] In addition, vitamin D3 suppresses telomerase responsible for DNA replication,[24] which is pathologically activated by HPV.[25] All these factors are important immune factors against HPV infection.[2627]

Involucrin is an important component of the keratinocyte cell envelope and affords a scaffold to which other differentiation proteins subsequently become cross-linked.[28] The expression of involucrin begins in early differentiation and continues to complete till the end of the final keratinocyte terminal stage.[29] This expression is adjacent to the cell membrane in the cornified-envelope structure. This expression is important to the attachment of involucrin with extracellular lipids that form the exterior surface of the cornified envelope.[30]

In this study, the expression of involucrin was decreased and mainly of a cytoplasmic pattern. This may be due to the ability of HPV to induce an increase of keratinocytes cell cycle reentry occurring in suprabasal cell layers of the epidermis and decrease expression of differentiation markers.[313233]

The response to vitamin D3 injection was decreased in a smoker than in nonsmoker patients in this study. A previous report showed a negative effect of smoking on the efficacy of vitamin D3 injections in verrucae.[34]

This negative effect of smoking in response to vitamin D3 injection may be due to a high level of nicotine that leads to disruption of keratinocyte differentiation and decrease of cell envelope formation.[35] In addition, smoking may interfere with the interaction of vitamin D3 and VDR resulting in a weak effect.[36] Also, smoking may decrease toll-like activity that had a role in the innate immune system against HPV.[3738]

The use of topical anesthetic cream in this study was shown to reduce pain and vasovagal attacks reported in other studies.[8]

The low recurrence rate noted in the follow-up period in this study was similar to previous studies,[816] and less than other immunotherapy intralesional injections.[3]

Conclusions

Intralesional vitamin D3 injection is a safe and effective treatment of verrucae. Involucrin expression in verruca is decreased and mostly is a cytoplasmic pattern and is increased after intralesional vitamin D3 injection with modification of its pattern of expression to be membranous type.

Limitations

This study did not include a placebo injection.

Statement of Ethics

All the participants in this study gave their written informed constant signed by themselves.

Authors' contribution

Category 1:

a) Conception and design: All authors

b) Analysis of data: All authors

c) Interpretation of clinical data: First, second, and fourth authors

d) Interpretation of pathological data: Third author.

Category 2:

a) Drafting the article: All authors

b) Revising it critically for important intellectual content: All authors.

Category 3:

Final approval of the version to be published: All authors.

All authors state that the material contained in this manuscript has not been published, has not been submitted, or is not being submitted elsewhere for publication. All authors in the manuscript were contributed for collecting the materials, revising the data, and putting the draft and intellectual revision.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.