Vitamin D has been discovered to show a modulatory and regulatory role in multiple processes involving immunity, host defense, inflammation and epithelial repair, in addition to its essential functions in the regulation of calcium homeostasis 1.
Vitamin D also plays a role in various infectious processes 2. Warts are a benign proliferation of the skin and mucosa caused by infection with human papillomavirus (HPV), which are the most frequently recurring infections 3.
An important mechanism for innate immune influence came with the discovery that vitamin D directly induces human cathelicidin expression via a vitamin D response element located in the promoter region of the cathelicidin gene 4. The anti-viral effects of vitamin D could be explained by cathelicidin (in the form of LL-37), human beta defensin 2, and perhaps through the release of reactive oxygen species. Similar interactions may occur with the lipid envelopes of viruses through envelope disruption. LL-37 is chemotactic and may also block viral entry 5.
Vitamin D is also a known inducer of autophagy 6 by which pathogen encapsulation followed by fusion with the lysosome occurs, followed by degradation 7.
Vitamin D3 derivatives have been shown to be effective for topical treatment of warts, through regulation of epidermal cell proliferation and differentiation as well as modulation of cytokine production 8.
When the skin damage reaches the basal cell layer, HPV can infect dividing keratinocytes (KC) (which are part of innate immune defense). The initial inflammatory response produced by tissue damage leads to infiltration of immune cells 9, through pathogen-associated molecular patterns binding to pattern recognition receptors such as Toll-like receptors 10. Most of these cell types can enhance a cytokine-mediated pro-inflammatory process, which links the innate with the adaptive immune response 11.
Basal KCs act as nonprofessional antigen presenting cells (APCs) 10 with later infiltration of professional APCs that uptake viral antigens initiating adaptive immunity by activating different T cell subsets 9. Cytotoxic T-lymphocytes secrete the proteolytic enzymes, granzyme, and perforin 12.
Natural killer (NK) cells constitutively express IFN-γ, granzyme, and perforin and so are effectively ‘primed’ to initiate an anti-viral immune response. After activation, they mediate their cytotoxicity by perforin-dependent mechanisms and granzyme that that stimulate caspases in target cells, triggering apoptosis 13.
Persistent HPV infection is caused by the paucity of NK cells in the epidermis in direct contact with infected KC 13, as well as the fact that NK cells can interact with APC and acquire MHC II molecules from them, inhibiting CD4 T-cells by presenting antigen without costimulation 14.
Regulatory T-cells (T regs) are CD4+CD25+FoxP3+ T-lymphocytes that have an immunosuppressive action, aiming at preventing the immune response to self-antigen 15. The presence of T regs in persistent HPV infections types 6 and 11 implicates that T regs are involved in active repression of the immune response to infection 13.
T regs can be induced and stimulated by vitamin D in an indirect pathway, via APCs, which stay in an immature state upon vitamin D treatment and therefore present less antigens 16.
Therefore, as T regs are involved in active repression of the immune response to infection with HPV types 6 and 11 13, and are additionally induced and stimulated by vitamin D 17, it raises a question as to whether vitamin D is actually protective in warts or increases susceptibility to infection. Hence, estimating vitamin D levels in cases of common and plantar warts would help clarify this perspective, hence this study.
Patients and methods
This case–control study was conducted on total 87 participants, comprising 40 patients with viral warts and 47 healthy controls (28 females and 59 males), all attending the Dermatology Outpatient Clinic, Kasr Al Ainy Hospital, Cairo University, during the period from December 2016 to February 2017. The study was approved by the Dermatology Research Ethical Committee, Faculty of Medicine, Cairo University, and informed written consents were obtained from all participants. Participants were recruited at approximately the same time to minimize the differences of seasonal changes in the value of vitamin D.
Inclusion criteria included Egyptian patients, of both sexes from 18 years to 40 years of age with warts. Exclusion criteria were systemic and topical treatment before the study; associated systemic diseases with altered serum vitamin D level, for example, kidney and liver disease; associated autoimmune diseases, for example, SLE and vitiligo; patients on vitamin D supplementation 18; drugs that affect vitamin D levels such as antiepileptics, glucocorticoids, anti-estrogens, anti-retroviral drugs, lipid-lowering agents and cytostatic agents 19; and pregnant and lactating females 20.
Controls collected were age, sex, socioeconomic status, skin type, and BMI matched.
Participants were subjected to the following.
Personal history included the following:
- Name, age, sex, marital status, residence, special habits of medical importance, and history of previous gestations.
- Frequency and duration of sun exposure (adequacy of sun exposure time in relation to the skin type) 21. Adequate sun exposure means the exposure sufficient for production of the vitamin D required by the body, which can be defined as 5–10 min of exposure of the arms and legs or the hands, arms and face, two or three times per week 22.
Present history included the following:
- Onset, course, and duration of the disease (in the patients group).
Past history included the following:
- For exclusion; drug intake, any systemic or dermatological disorders that may affect serum vitamin D level.
Family history included the following:
- Consanguinity and similar condition within family members.
- Cutaneous examination for common warts:
- Distribution of lesion: localized to one limb, bilateral corresponding limbs, and two different anatomical sites.
A 3-ml peripheral venous blood sample was taken from each participant under sterile conditions for assessment of serum levels of 25-hydroxyvitamin D (25-OHD). Blood samples were collected in labeled tubes, and then centrifuged, and the separated sera were stored at −20°C.
Vitamin D status was defined based on the following:
- Deficiency: 25-OHD concentration less than 50 nmol/l.
- Insufficiency: 25-OHD concentration 50–75 nmol/l.
- Sufficiency: 25-OHD concentration more than 75 nmol/l 23.
Method of vitamin D assessment
Vitamin D was measured in sera of all patients and controls using human 25-OHD enzyme-linked immunosorbent assay kit (Elabscience Company, Houston, Texas USA).
The kit uses a double-antibody sandwich enzyme-linked immunosorbent assay to assess the level of human 25-OHD in samples. 25-OHD was added to monoclonal antibody enzyme well, which is precoated with human 25-OHD. Incubation was done, and then, 25-OHD antibodies labeled with biotin were added, and combined with streptavidin–HRP to form immune complex. Thereafter, incubation and washing were carried out again to remove the uncombined enzyme. Then, chromogen solutions A and B were added, and the color of the liquid was changed into blue, and at the effect of acid, the color finally becomes yellow. The chrome of color and the concentration of the human substance 25-OHD of samples were positively correlated.
According to standards’ concentration and the corresponding optical density values, the standard curve was calculated out with linear regression equation, and then the optical density values of the samples were applied on the regression equation to calculate the corresponding sample’s concentration.
Data were coded and entered using the statistical package for the social sciences, version 24 (SPSS Inc., Chicago, Illinois, USA). Data were summarized using mean, SD, median, minimum and maximum in quantitative data, and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the nonparametric Mann–Whitney test 24. For comparing categorical data, χ2 test was performed. Exact test was used instead when the expected frequency is less than 5 25. Correlations between quantitative variables were done using Spearman’s correlation coefficient 26. Linear regression analysis was done to predict vitamin D 27. P values less than 0.05 were considered statistically significant.
This case–control study was conducted on 40 patients with viral warts and 47 normal healthy individuals serving as controls.
The patient group included 27.5% females and 72.5% males, whereas the control group included 36.2% females and 63.8% males. Mean±SD was 24.65±6.67 years and median=22.00 years in the patient group and was 25.66±5.84 in the control group. Regarding age and sex, there was no statistically significant difference between patients and controls regarding age or sex (P values were 0.292 and 0.388, respectively).
According to skin types, 65.0% had skin type 3, whereas 14 (35%) patients had skin type 4. Again, there was no statistical significance between patients with wart and controls regarding the skin type (P=0.604).
However, there was statistical significance with sun exposure between patients with viral warts and controls (P<0.045), whereby 85.0% of the patients reported adequate sun exposure and 15.0% reported inadequate sun exposure, whereas 97.9% controls had adequate sun exposure, whereas one (2.1%) individual had inadequate sun exposure.
Ten (25.0%) patients gave positive family history of viral warts, whereas 30 (75.0%) patients gave negative family history. Duration of the disease ranged from 0.1 to 3.00 years (mean±SD, 1.03±0.72 years), with a median of 1.00. Number of the lesions ranged from 1 to 20 lesions (mean±SD, 5.07±4.15) with a median of 4.50. Regarding wart types, 30 (75%) patients had common warts and 10 (25.0%) patients had plantar warts. Fifteen (50%) patients with common warts had primary lesions, whereas 15 (50%) patients had recurrent lesions.
Mean±SD serum vitamin D level was 55.21±42.79 in the patient group and 79.01±51.79 in the controls, resulted in a statistically significant difference between patients with viral warts and controls regarding serum vitamin D level and vitamin D status (P<0.001) (Figs. 1, 2 and Table 1).
On comparing between male and female patients regarding mean±SD serum vitamin D level, it was 57.81±49.30 and 48.36±16.23, respectively, and there was no significant difference (P=0.788); moreover, regarding vitamin D status, there was also no significant difference (P=0.425). Again the comparison between patients reporting adequate and those reporting inadequate sun exposure regarding serum vitamin D level revealed no significant difference (P=0.810) (Table 2). Regarding the comparison between patients with common warts and plantar warts regarding serum vitamin D, patients with common warts had lower serum vitamin D levels than patients with plantar warts. However, the difference did not reach statistical significance (P=0.914). Finally, comparison within common warts patients reporting primary lesions and recurrent lesions regarding serum vitamin D level, patients with common wart with primary lesions had higher serum vitamin D levels than those with recurrent lesions. Moreover, the difference did not reach statistical significance (P=0.116) (Table 2).
Vitamin D is assessed by measuring the prohormone 25-OHD, which is an indicator of supply rather than function. The most stable and plentiful metabolite of vitamin D in human serum, 25-OHD has a half-life of about 3 weeks, making it the most suitable indicator of vitamin D status 28.
The present study showed a statistically significant decrease in the serum 25-OHD levels in patients with viral warts when compared with controls. In addition, there was a statistically significant difference between patients and controls regarding vitamin D status, as patients with viral warts had a more deficient pattern of vitamin D status than controls. To our knowledge, this is the first study in which the serum 25-OHD levels were measured in patients diagnosed with cutaneous warts.
Although the exact mechanism of vitamin D activity against warts has not been identified, the effect of vitamin D was speculated to be derived from its immune-regulatory activities, its potential role in regulation of epidermal cell proliferation and differentiation and its modulation of cytokine production 8.
Serum 25-OHD is involved in the regulation of expression of antimicrobial peptides 2. Moreover, AMPs are chemotactic 5 and may block the initial phase of HPV infection through the inhibition of viral replication 29.
In addition, activation of Toll-like receptors of human macrophages upregulates the expression of vitamin D response and vitamin D1-hydroxylase genes, resulting in expression and secretion of antimicrobial peptides 8.
Furthermore, vitamin D makes the physical barriers such as skin, respiratory tract, and genitourinary tract more resistant to bacteria and viruses by upregulating the proteins that promote tight junctions, gap junctions, and adherens junctions 30.
To sum up, insufficient levels of vitamin D may lead to a higher prevalence of HPV infection by increasing vulnerability to HPV penetration and reducing the host’s ability to clear the virus 31. Apparently, the fact that vitamin D induces the differentiation of native T-cells to T reg 32, with persistent HPV infections being characterized by presence of T regs cells 33, did not take the upper hand in the immune response to HPV infection, and according to our results, vitamin D was more protective against HPV infection. This conclusion was supported by the fact that our study not only showed lower levels of vitamin D among patients than controls but also revealed that vitamin D was lower in patients with recurrent warts than those with primary lesions.
These findings are in accordance with a study conducted by Shim et al. 31 on 2353 sexually active US women, aged 20–59 years, reporting cervicovaginal HPV infection. Approximately 25.3% of the women had a vitamin D deficiency, whereas 10.2% of women had a severe vitamin D deficiency and ∼39.7% of women had an insufficient vitamin D level. The authors suggested that a lower level of serum 25-OHD 3 was generally associated with an increase prevalence of cervicovaginal HPV infection.
Two studies used intralesional vitamin D, one of which has shown that it lead to complete recovery (90%) in 60 patients with one or more warts refractory to treatment 34, whereas the other study was done on 20 patients with plantar warts with complete resolution in 80% of patients and no recurrence or adverse effects were observed 8.
There are numerous reports of vitamin D deficiency in other viral infections. HIV-infected individuals with vitamin D deficiency were shown to have an increase in disease progression 35–38. Interestingly, HCV-positive patients with low levels of vitamin D may have more advanced liver disease 39–41.
Regarding vitamin D status, only 12.5% of patients and 23.4% of controls had sufficient levels of vitamin D in this study. This may be explained by limited sun exposure owing to dress styles, cultural practices, low outdoor activity, air pollution, and inadequate vitamin D intake 42.
Our results showed that females had statistically insignificant lower 25-OHD levels than males in patients group. On the contrary, a study conducted in Dakahila in Egypt found that the prevalence of vitamin D insufficiency was significantly higher in elderly males than in females 43. These results are in concordance with Looker et al.44 who found that the mean serum 25-OHD concentrations were significantly lower in males as compared with females. In contrast, Abu Shady et al. 45 in a cross-sectional study among Egyptian prepubescent school children aged 9–11 years found that boys had higher levels of serum 25-OHD than girls who had a significantly increased risk of vitamin D deficiency, which is in agreement with Houghton et al.46 and Habibesadat et al.47. This suggests that lifestyle factors such as spending more time indoors and the coverage of skin by clothing, which are encountered more commonly amongst girls, may explain these differences as both factors could affect the actual amount of sun exposure required for cutaneous synthesis of vitamin D. Additionally, many studies have shown high prevalence of vitamin D deficiency in Egyptian female population associated with social and cultural factors (conservative dress of Egyptian women, low socioeconomic status, staying outside of the sun, low dietary intake, and decrease time spent outdoors) 18,42,48–50.
In our study, there was a statistically significant difference of reporting adequate and inadequate sun exposure detected in patients and controls. We found that the relation between serum 25-OHD level and sun exposure was not significant in patient group. We can therefore speculate that factors such as clothing style and decreased time spent outdoors have much more effect on production of vitamin D than sun exposure.
Sunlight source of the vitamin D synthesis is estimated to account for 90% of total vitamin D requirements. However, the exact amount of sun exposure required for optimal vitamin D levels is affected by several factors such as time of day, season, latitude, skin color, body fat, excessive use of sunscreens, old age, and air pollution 18.
Conclusion and recommendations
The present study revealed low levels of serum vitamin D in patients with warts, suggesting a possible role for vitamin D as a supplement treating patients with viral warts. Further studies on a larger scale are required to study the important role of vitamin D in warts. Moreover, controlled studies to determine whether treatment of patients with warts with vitamin D supplementation is of significant effect would represent an attractive area of research. In addition to that, further studies should be done in which tissue levels of vitamin D are measured, as serum levels may be decreased whereas tissue levels remain normal, owing to the ability of the body to maintain normal tissue vitamin D levels, in spite of low serum levels.
Conflicts of interest
There are no conflicts of interest.
1. Zdrenghea MT, Makrinioti H, Bagacean C, Bush A, Johnston SL, Stanciu LA. Vitamin D
modulation of innate immune responses to respiratory viral infections. Rev Med Virol 2016; 10:1–12.
2. Kearns MD, Alvarez JA, Seidel N, Tangpricha V. The impact of vitamin D
on infectious disease: a systematic review of controlled trials. Am J Med Sci 2015; 349:245–262.
3. Nofal A, Nofal E. Intralesional immunotherapy of common warts: successful treatment with mumps, measles and rubella vaccine. J Eur Acad Dermatol Venereol 2010; 24:1166–1170.
4. Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q, Liao J, et al. Cutting edge: 1, 25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immunol 2004; 173:2909–2912.
5. Beard JA, Bearden A, Striker R. Vitamin D
and the anti-viral state. J Clin Virol 2011; 50:194–200.
6. Wang J, Lian H, Zhao Y, Kauss MA, Spindel S. Vitamin D3 induces autophagy of human myeloid leukemia cells. J Biol Chem 2008; 283:25596–25605.
7. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature 2011; 469:323–325.
8. Aktaş H, Ergin C, Demir B, Ekiz Ö. Intralesional vitamin D
injection may be an effective treatment option for warts. J Cutan Med Surg 2016; 20:118–122.
9. Rosales R, Rosales C. Immune therapy for human papillomaviruses-related cancers. World J Clin Oncol 2014; 5:1002–1019.
10. Nestle FO, Meglio PD, Qin JZ, Nickoloff BJ. Skin immune sentinels in health and disease. Nat Rev 2009; 9:679–691.
11. Amador-Molina A, Hernández-Valencia JF, Lamoyi E, Contreras-Paredes A, Lizano M. Role of innate immunity against human papillomavirus (HPV) infections and effect of adjuvants in promoting specific immune response. Viruses 2013; 5:2624–2642.
12. Zhang N, Bevan MJ. CD8+ T cells: foot soldiers of the immune system. Immunity 2011; 35:161–168.
13. Hibma MH. The immune response to papillomavirus during infection persistence and regression. Open Virol J 2012; 6:241–248.
14. Nakayama M, Takeda K, Kawano, M, Takai T, Ishii N, Ogasawara K. Natural killer (NK)–dendritic cell interactions generate MHC class II-dressed NK cells that regulate CD4+ T cells. Proc Natl Acad Sci 2011; 108:18360–18365.
15. Loddenkemper C, Hoffmann C, Stanke J, Nagorsen D, Baron U, Olek S, et al. Regulatory (FOXP3+) T cells as target for immune therapy of cervical intraepithelial neoplasia and cervical cancer. Cancer Sci 2009; 100:1112–1117.
16. Shin DM, Yuk JM, Lee HM, Lee SH, Son JW, Harding CV, et al. Mycobacterial lipoprotein activates autophagy via TLR2/1/CD14 and a functional vitamin D
receptor signalling. Cell Microbiol 2010; 12:1648–1665.
17. Prietl B, Treiber G, Pieber TR, Amrein K. Vitamin D
and immune function. Nutrients 2013; 5:2502–2521.
18. El-Mongy NN, El-Nabarawy E, Hassaan SA, Younis ER, Shaker O. Serum 25-hydroxy vitamin D3 level in Egyptian patients with alopecia areata. J Egypt Women’s Dermatol Soc 2013; 10:37–41.
19. Gröber U, Kisters K. Influence of drugs on vitamin D
and calcium metabolism. Dermatoendocrinology 2012; 4:158–166.
20. Ogan D, Pritchett K. Vitamin D
and the athlete: risks, recommendations, and benefits. Nutrients 2013; 5:1856–1868.
21. Youl PH, Janda M, Kimlin M. Vitamin D
and sun protection: the impact of mixed public health messages in Australia. Int J Cancer 2009; 124:1963–1970.
22. Holick MF. Sunlight and vitamin D
for bone health and prevention of autoimmune diseases, cancers, and cardiovascular disease. Am J Clin Nutr 2004; 80:1678s–1688s.
23. Alshahrani F, Aljohani N. Vitamin D
: deficiency, sufficiency and toxicity. Nutrients 2013; 5:3605–3616.
24. Chan YH. Biostatistics 102: quantitative data – parametric & non-parametric tests. Singapore Med J 2003; 44:391–396.
25. Chan YH. Biostatistics 103: qualitative data –tests of independence. Singapore Med 2003; 44:498–503.
26. Chan YH. Biostatistics 104: correlational analysis. Singapore Med J 2003; 44:614–619.
27. Chan YH. Biostatistics 201: linear regression analysis. Singapore Med J 2004; 45:55–61.
28. Thacher TD, Clarke BL. Vitamin D
insufficiency. Mayo Clin Proc 2011; 86:50–60.
29. Iwasaki A. Antiviral immune responses in the genital tract: clues for vaccines. Nature Rev Immunol 2010; 10:699–711.
30. Hewison M. An update on vitamin D
and human immunity. Clin Endocrinol (Oxf) 2012; 76:315–325.
31. Shim J, Pérez A, Symanski E, Nyitray AG. Association between serum 25-hydroxyvitamin D level and human papillomavirus cervicovaginal infection in women in the United States. J Infect Dis 2016; 213:1886–1892.
32. Jeffery LE, Wood AM, Qureshi OS, Zheng Hou T, Gardner D, Briggs Z, et al. Availability of 25-hydroxyvitamin D3 to APCs controls the balance between regulatory and inflammatory T cell responses. J Immunol 2012; 189:5155–5164.
33. Cao Y, Zhao J, Lei Z, Shen S, Liu C, Li D, et al. Local accumulation of FOXP3+ regulatory T cells: evidence for an immune evasion mechanism in patients with large condylomata acuminata. J Immunol 2008; 180:7681–7686.
34. Raghukumar S, Ravikumar BC, Vinay KN, Suresh MR, Aggarwal A, Yashovardhana DP, et al. Intralesional vitamin D3 injection in the treatment of recalcitrant warts: a novel proposition. J Cut Med Surg 2017; 21:320–324.
35. Lake JE, Adams JS. Vitamin D
in HIV-infected patients. Curr HIV/AIDS Rep 2011; 8:133–141.
36. Griffin AT, Arnold FW. Review of metabolic, immunologic, and virologic consequences of suboptimal vitamin D
levels in HIV infection. AIDS Patient Care STDS 2012; 26:516–525.
37. Pinzone MR, Di Rosa M, Malaguarnera M, Madeddu G, Focà E, Ceccarelli G, et al. Vitamin D
deficiency in HIV infection: an underestimated and undertreated epidemic. Eur Rev Med Pharmacol Sci 2013; 17:1218–1232.
38. Ansemant T, Mahy S, Piroth C, Ornetti P, Ewing S, Guilland JC, et al. Severe hypovitaminosis D correlates with increased inflammatory markers in HIV infected patients. BMC Infect Dis 2013; 13:7.
39. Fisher L, Fisher A. Vitamin D
and parathyroid hormone in outpatients with noncholestatic chronic liver disease. Clin Gastroenterol Hepatol 2007; 5:513–520.
40. Arteh J, Narra S, Nair S. Prevalence of vitamin D
deficiency in chronic liver disease. Dig Dis Sci 2010; 55:2624–2628.
41. Lange CM, Bojunga J, Ramos-Lopez E, von Wagner M, Hassler A, Vermehren J, et al. Vitamin D
deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy. J Hepatol 2011; 54:887–893.
42. Botros RM, Sabry IM, Abdelbaky RS, Eid YM, Nasr MS, Hendawy LM. Vitamin D
deficiency among healthy Egyptian females. Endocrinol Nutr 2015; 62:314–321.
43. Aly WW, Hussein MA, Ebeid S, Mortagy AK. Prevalence of vitamin D
insufficiency among community dwelling elderly in Dakahlia as a representative of rural areas in Egypt. Aging Clin Exp Res 2014; 26:47–51.
44. Looker AC, Pfeiffer CM, Lacher DA, Schleicher RL, Picciano MF, Yetley EA. Serum 25-hydroxyvitamin D status of the US population: 1988–1994 compared with 2000–2004. Am J Clin Nutr 2008; 88:1519–1527.
45. Abu Shady MM, Youssef MM, Salah El-Din EM, Abdel Samie OM, Megahed HS, Salem SME, et al. Predictors of serum 25-hydroxyvitamin D concentrations among a sample of Egyptian schoolchildren. Sci World J 2016; 2016:8175768.
46. Houghton LA, Gray AR, Harper MJ, Winichagoon P, Pongcharoen T, Gowachirapant S, et al. Vitamin D
status among Thai school children and the association with 1, 25-dihydroxyvitamin D and parathyroid hormone levels. PloS One 2014; 9:e104825.
47. Habibesadat S, Ali K, Shabnam JM, Arash A. Prevalence of vitamin D
deficiency and its related factors in children and adolescents living in North Khorasan, Iran. J Pediatr Endocrinol Metab 2014; 27:431–436.
48. Amr N, Hamid A, Sheta M, Elsedfy H. Vitamin D
status in healthy Egyptian adolescent girls. Georgian Med News 2012; 210:65–71.
49. Fawzi MM, Swelam E, Said NS. Plasma levels of 25-hydroxyvitamin D and dress style in a sample of Egyptian female university students. Life Sci J 2012; 9:763–767.
50. El Rifai NM, Abdel Moety GAF, Gaafar HM, Hamed DA. Vitamin D
deficiency in Egyptian mothers and their neonates and possible related factors. J Maternal Fetal Neonatal Med 2014; 27:1064–1068.