**1. Introduction**

Warts are muco-cutaneous lesions caused by human papillomavirus (HPV) [1]. Warts are very common in the general population, with up to one third of the children being a ffected; the incidence decreases with age. Considering that immunity plays an important role in the development of warts, the incidence increases up to 45% among immunosuppressed individuals [2]. To this date, more than 200 HPV types have been identified [3]. Warts are commonly associated with HPV 1, 2, 4 and 7. In immunosuppressed patients, HPV 75, 76 and 77 were identified. Warts manifest as skin-colored papules with a keratotic surface. Most frequently, the lesions involve the hands and feet, but they can also appear on extension areas (elbows, knees) [4,5]. Transmission of the infection occurs through direct contact in the context of skin microlesions or more rarely, indirectly through contaminated objects [5]. The incubation period ranges from 1 to 20 months; there is no systemic spread of the virus. HPV infects host cells without integrating viral DNA into their genome [6].

Warts occur when HPV infects the upper layers of the skin or mucous membranes, resulting in abnormal and rapid cell growth. There are several conditions that contribute to the acquisition of HPV infection, including incomplete immune system development, CD4+ T-cell dysfunction, other causes of immunosuppression, and the alteration of the normal epithelial barrier [7–13]. Most often in children, warts resolve spontaneously, without treatment, but the lesions can be persistent in adults. However, recurrences are common in both groups [10,11]. There is no clear evidence of the etiopathogenic mechanisms of HPV infection. Multiple factors contributing to its pathogenesis have been suggested, such as the lack of e fficient protective cell mechanisms against the virus and the accumulation of toxic compounds which induce oxidative stress and inflammation [14–19]. Oxidative stress and inflammation are two interconnected processes which produce multiple e ffects on cell function [20].

The 45 kDa receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily, which contains an extracellular region of 320 amino acids (sequence 23–342), a 21-amino acid transmembrane hydrophobic region (sequence 343–363) and a cytoplasmic region of 41 amino acids (sequence 364–404). The extracellular region is composed of two C-type domains (C1 consisting of 124–221 amino acid residues and C2 consisting of 227–317 amino acid residues) and a V-type domain (sequence 23–116 amino acid residues). The V domain confers the ability to bind various ligands. The V and C1 domains are involved in the stability/specificity of the receptor-ligand complex. The C2 domain participates in the dimerization/oligomerization of receptor-ligand complexes. The receptor is anchored to the cell membrane through its transmembrane domain. The endocellular domain is essential for intracellular signaling [21–23].

Many studies have shown that RAGE has a large number of ligands, such as advanced glycation end products (AGEs), S100 calgranulin proteins, high motility group box 1 protein, amyloid fibrils, phosphatidylserine, macrophage-1 antigen, lipopolysaccharide from the outer membrane of Gram-negative bacteria, peptidoglycan (present in the majority of bacteria), lipoteichoic acid (a component of many Gram-positive bacteria), bacterial DNA, viral DNA/RNA, yeas<sup>t</sup> cell wall mannans, degraded extracellular matrix components, and modified fibronectin [15,18,24,25].

RAGE has been found during embryonic development, its expression being reduced in adulthood, excepting the lung and skin [17]. Other cells, such as monocytes/macrophages, smooth muscle cells, endothelial cells, fibroblasts, and neuronal cells, do not express physiologically detectable amounts, but receptor expression may be induced during cell stress in various instances including the accumulation of the ligands of RAGE, or when the transcriptional factors which modulate the expression of RAGE are activated [26,27]. Soluble RAGE (sRAGE) acts as a decoy for RAGE ligands, preventing their interactions with membrane RAGE (mRAGE) through a competitive mechanism, and consequently, sRAGE disrupts the generation of oxidative stress and inflammation [14]. Increased amounts of RAGE ligands induce the overexpression of sRAGE [27].

To the best of our knowledge, the role of sRAGE in the onset and progression of warts has not been previously investigated. To analyze the possible molecular mechanisms involved in the development of cutaneous warts, we hypothesized that sRAGE acts as an endogenous factor able to maintain/restore the cutaneous homeostasis. We have analyzed the interaction between circulating sRAGE and inflammatory response on the one hand and the relationship between sRAGE and oxidant/antioxidant status on the other hand in patients with palmoplantar warts (Figure 1).

**Figure 1.** The plan of investigation of sRAGE in association with oxidative stress and inflammation in patients with warts. sRAGE = soluble receptor for advanced glycation end products, TAS = total antioxidant status, TOS = total oxidant status, OSI = oxidative stress index, ESR = erythrocyte sedimentation rate, hs-CRP = high-sensitive C reactive protein, Il-6 = interleukin-6.

#### **2. Materials and Methods**

## *2.1. Study Participants*

The patients were selected from those who presented to the Clinic of Dermatology and were diagnosed with palmoplantar warts, the most common HPV-related lesions. All study participants gave their consent to the use of their biological samples in research studies. All the procedures and the experiments performed in the study respect the ethical standards in the Helsinki Declaration, as well as the national law. The study protocol was approved by the Ethics Committee of "Victor Babes Infectious and Tropical Diseases Hospital" (13050/31.07.2017).

Inclusion criteria: otherwise healthy adults, aged 18 years or above; adequate nutritional status; non-smokers; and with no treatment for warts.

Exclusion criteria from the study (conditions widely known as being able to alter, and therefore interfere with parameters of inflammation and oxidative stress): chronic alcohol use, drug abuse; treatment with corticosteroids, immunosuppressant agents and nutritional supplements; and pregnancy and breastfeeding.

Based on similar demographic characteristics, the study participants were divided into two groups: patients with palmoplantar warts (*n* = 24) and healthy subjects as controls (*n* = 28). We analyzed the serum levels of sRAGE, oxidative stress parameters and markers of inflammation compared to controls. We also evaluated the levels of sRAGE, oxidative stress parameters and markers of inflammation according to the number of the lesions and the duration of the disease. According to the number of the lesions we stratified the patients into three groups: less than 5 lesions (*n* = 11), between 5–10 lesions (*n* = 8) and more than 10 lesions (*n* = 5). The distribution of the patients with warts according to the duration of the disease divided them into three groups: with a history of less than 1 month (*n* = 6), between 1 and 6 months (*n* = 10) and a history longer than 6 months (*n* = 8).

#### *2.2. Laboratory Tests*

Biological samples were drawn from the patients and controls enrolled in the study under basal conditions using a holder-vacutainer system. Venous blood collected on anticoagulant (K3EDTA) was used to determine the blood count and erythrocyte sedimentation rate. The samples were processed immediately. The plasma obtained from venous blood collected on heparin was used for serum fibrinogen determination. Serum was obtained from venous blood collected in vacutainer without anticoagulant. The hemolyzed or lactescent samples were rejected.

sRAGE levels were measured by ELISA method; the sandwich variant and the results were expressed as pg/mL. In the wells of a polystyrene plate in which known antibodies were attached, the unknown antigen solution was added and then incubated. After washing, enzyme-labelled antibodies were added and fixed to the free epitopes of a polyvalent antigen. After incubation, the wells were washed again. The presence of the labelled complex was detected using a chromogenic substrate (BioVision reagents, TECAN analyzer). The absorbance of the resulted yellow product was measured. The intensity of the color of the resulted product is proportional to the amount of sRAGE in the sample. To determine the concentrations of sRAGE in the samples, a standard curve was used. The intensity of the color was measured at 450 nm.

The following parameters were used to assess oxidative stress: total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI).

TOS and TAS levels were determined by spectrophotometric method (Randox reagents, HumaStar 300 analyzer); results were expressed as μmol of H2O2 equivalent/<sup>L</sup> serum for TOS and as μmol Trolox equivalent/<sup>L</sup> serum for TAS. OSI value was calculated using the following formula:

$$\text{OSI} \left( \text{arbitrary units} \right) = \frac{\text{TOS} \left( \mu \text{mol H}\_2 \text{O}\_2 \text{Eq/L} \right)}{\text{TAS} \left( \mu \text{mol TrolxEq/L} \right)} \tag{1}$$

For the early detection of inflammation, the following determinations were used: high sensitivity C-reactive protein (hs-CRP, latex-immunoturbidimetric method), interleukin-6 (IL-6, ELISA method, sandwich variant, automatic reading method), the erythrocyte sedimentation rate (ESR, automatic reading method), and fibrinogen (coagulometric method).

## *2.3. Statistical Analysis*

The comparison of obtained experimental data between groups was carried out using t-test. When we compared more than 2 groups we used Kruskal-Wallis test. The relationship between pairs of two parameters was assessed by Spearman's correlation coefficient after adequate assessment of normality of data using Kolmogorov-Smirnov test. We chose a significance level (*p*) of 0.05 (5%) and a confidence interval of 95% for hypothesis testing.
