Journal of Immunology Research

Journal of Immunology Research / 2016 / Article

Research Article | Open Access

Volume 2016 |Article ID 8373819 | 12 pages |

Elevated Concentration of Defensins in Hepatitis C Virus-Infected Patients

Academic Editor: Ghislain Opdenakker
Received28 Jan 2016
Revised17 Apr 2016
Accepted27 Apr 2016
Published20 Jun 2016


Hepatitis C virus (HCV) is the major etiological agent of human non-A and non-B hepatitis, affecting around 180 million people worldwide. Defensins, small cysteine-rich cationic peptides, are shown to have potent antibacterial, antiviral, and antifungal properties. Defensins can be found in both normal and microbial infected patients, at variable concentrations. Notably, viral infections are often associated with elevated concentrations of defensins. The current study aimed to estimate the concentrations of total, α-, and β-defensins in serum taken from normal and HCV-infected patients. 12 healthy (noninfected) and 34 HCV-infected patients were enrolled. Standardized immunoassay kits were used to obtain serum concentrations of defensins. The obtained results were calibrated against kit standard reagents. Total defensin concentrations in HCV-infected patients were significantly higher (2- to 105-fold) compared to healthy individuals. The concentrations of α-defensins were also significantly elevated in the HCV-infected patients (31–1398 ng/50 μL). However, concentrations of β-defensins ranged from 44.5 ng/50 μL to 1056 ng/50 μL. The results did not reveal differences in serum defensin concentration between male and female HCV-infected patients. A-defensin concentration of ≥250 ng/50 μL was found to contain more β-defensins than total defensins and α-defensins. This study concludes, for the first time, that serum defensin levels are elevated in HCV-infected patients.

1. Introduction

Hepatitis C virus (HCV) is an enveloped, single positive-stranded RNA virus that belongs to the Flaviviridae family. Its genome consists of around 10,000 nucleotides and encodes a single polyprotein of 3010–3033 amino acids. HCV polyprotein is cleaved by both host cell and viral proteases into at least 10 distinct structural and nonstructural protein products. The major structural proteins are a core (C) protein, two envelope proteins, E1 and E2, and a short hydrophobic peptide p7 [1]. HCV is a major cause of parenterally transmitted non-A and non-B hepatitis worldwide [2], and infection with HCV is one of the leading causes of chronic liver disease worldwide [3, 4]. The prevalence of HCV infection has increased during recent years; it is estimated that over 180 million people worldwide are infected with HCV, representing 3% of the world’s population, while, in countries like Egypt, the incidence of HCV infection is as high as 15% [5]. Efforts to achieve a breakthrough in antiviral clinical research for chronic HCV are currently underway in Western countries [6] and Japan [7].

Today, there is no available vaccine against HCV, and the current treatment for HCV infection is limited to nonselective alpha-interferon (IFN) and ribavirin. However, the Food and Drug Administration has recently approved a list of novel anti-HCV compounds [8]. These emerging antivirals should increase treatment options, particularly for difficult-to-treat patients, such as those suffering from advanced liver diseases or other coinfections, who have poor response rates to current regimens. Although the currently clinically approved and cocktail of anti-HCV therapy is believed to cure more than 90% of infected patients, incidence of viral resistance, null responders, and treatment failure (in addition to poor side-effect profiles and large treatment costs) poses a major limitation that must be resolved. As an RNA virus, HCV very easily develops a resistance to antiviral treatments, due to its error-prone replication properties. Most entry inhibitors (a class of antiretroviral agents) target the host cell components required for HCV entry, such as receptors or key enzymes, and have high genetic barriers to resistance due to their conserved nature. Therefore, these inhibitors tend to not only have pan-genotypic activity against viral infection but also have a greater risk of simultaneously causing cellular toxicity [9].

Defensins belong to a diverse group of antimicrobial peptides with pronounced antimicrobial activity [1017]. These are short, cationic cysteine-rich polypeptides, which are well known for their high and broad antimicrobial properties [16, 17]. Originally isolated from human and rabbit neutrophils (the most abundant type of white blood cells in most mammals, accounting for 40–75% of white blood cells) [18], defensins have also been found in various other vertebrates [19], invertebrates [20], insects [21], and plants [22, 23]. These polypeptides play important roles in innate immunity against microbial and viral infections, are involved in adaptive immunity, and are also involved in inflammation, wound repair, expression of cytokines and chemokines, production of histamine, and enhancement of antibody responses [2326]. They are also able to induce and augment antitumour immunity when fused with the nonimmunogenic tumour antigens [27]. Defensins are also activated in signal transduction and regulation of the inflammatory effects, participate in wound healing and chemotaxis, control proliferation, and regulate the release of cytokines [2830]. Defensin concentrations are shown to be elevated following microbial infection [31]. Levels of defensins in HCV-infected patient have not been estimated yet, which was the main aim of current study.

2. Materials and Methods

2.1. Samples and Defensin Estimation Kits

HCV-infected patients (34 samples: 19 male and 15 female) and healthy volunteers (12 samples: 6 male and 6 female) participated in this study. The enrolment criteria were based on thorough history taking: patients were considered eligible if (1) no coinfection with HIV or hepatitis B virus (HBV) was present; (2) they suffered from HCV disease and underwent a complete clinical and laboratory evaluation, including tests for liver function; and (3) their serum contained HCV antibodies (confirmed by measuring serum HCV-RNA titre using quantitative real-time polymerase chain reaction (RT-PCR) and TaqMan technology) [32]. Only HCV genotype 4a-infected patients were enrolled in this study. Venous blood samples were collected from all participants. Blood samples were set to clot and sera were separated by centrifugation, collected, aliquoted, and then stored at −80°C prior to use. Finally, all subjects were informed of the aims of the study and oral consent to participation was given. The study protocol was approved by the local ethical committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Total, α-, and β-defensin estimation kits were purchased from MyBioSource (San Diego, California, USA).

2.2. Defensin Concentration Estimation Kits

Human total, α-, and β-defensin concentration estimation kits were used in accordance with the manufacturer’s instructions (MyBioSource, San Diego, California, USA) and standard laboratory enzyme-linked immunosorbent assay (ELISA) protocol, as previously described [3336]. In brief, 50 μL infected or noninfected serum samples, as well as the standard reagent, were pipette into microelisa strips (in duplicate) and incubated at 37°C for 60 min, followed by a wash with washing solution (3x). 100 μL horseradish peroxidase (HRP) reagent was added to each well and incubated for 60 min at 37°C, followed by wash with washing solution (4x). 50 μL of chromogen A and 50 μL chromogen B were added to each strip well, then gently mixed, and incubated for 15 min at 37°C, away from direct light, following which 50 μL stop solution was added to each well. Following an observed colour change from blue to yellow, the optical density (OD) was read at 450 nm within 15–30 min after adding the stop solution. The output reading results were calculated, as per manufacturer’s instructions (MyBioSource, San Diego, California, USA), as the average of the duplicate readings for each standard and sample by subtracting the average optical density of the blank/control ().

2.3. Statistical Analysis

Raw OD data was presented as mean ± SD. The data obtained was analysed using the unpaired -test. values of <0.05 were considered to be statistically significant.

3. Results

3.1. Defensin Concentrations in Noninfected Individuals

In total, 46 human sera were used to calculate human defensin concentrations (total, α-, and β-defensins) using a commercial ELISA (Section 2.2). The serum concentrations of total, α-, and β-defensins in noninfected samples ranged from 18.66 to 2.88 ng/50 μL with a mean concentration (±SD) of  ng/50 μL (Figures 14 and Tables 14), with no gender-related differences (data not presented). For total defensins, the calculated concentration was  ng/μL, corresponding to the kit standard number 1 (31.25 ng/50 μL) and showing a clear significant difference from other kit standards (62.5 to 1000 ng/50 μL). The differences between human α-defensin concentrations and the kit standards started from 31.2 ng/50 μL (Figure 1 and Table 1). Human β-defensins concentrations were much lower than any kit standards used.

VariablesANOVA one-way
GenderMeanSDConcentration (ng/50 μL)P value

Health sample6 M, 6 F0.085–0.1651218.66 ± 3.500
STD 31.2Standard0.02350.03231.200
STD 62.5Standard0.13600.04262.500
STD 125Standard0.18850.03212500
STD 250Standard0.42100.03225000
STD 500Standard0.85000.03250000
STD 1000Standard1.22450.032100000
Sample 1M0.02820.03437.44<0.05
Sample 2M1.06250.144867.701<0.005
Sample 3F2.26850.1141852.60<0.0001
Sample 4F0.15620.034103.58<0.05
Sample 5M1.97350.3541611.68<0.001
Sample 6M0.10320.03447.43<0.05
Sample 7F1.31100.0341070.64<0.0001
Sample 8M0.05200.03469.04<0.05
Sample 9M0.15120.034100.30<0.05
Sample 10F0.06550.03486.96<0.05
Sample 11M2.42350.0541979.180<0.00001
Sample 12M0.15220.034100.93<0.05
Sample 13F0.11170.03451.33<0.05
Sample 14M0.38170.034226.66<0.005
Sample 15M1.20970.194987.91<0.001
Sample 16F0.64600.044380<0.005
Sample 17F1.94450.0341587.99<0.0001
Sample 18F0.59270.034242.02<0.05
Sample 19M0.23350.034138.7<0.05
Sample 20F0.60950.034358.5<0.005
Sample 21M0.56020.034329.53<0.005
Sample 22M0.27850.274165.4<0.05
Sample 23M0.03600.03439.50<0.05
Sample 24F0.42400.064251.78<0.005
Sample 25F0.35320.034209.74<0.005
Sample 26M0.80320.074472.5<0.005
Sample 27M0.31870.034189.25<0.05
Sample 28M0.04550.03460.41<0.05
Sample 29F0.31350.034186.16<0.05
Sample 30M0.08700.084115.51<0.05
Sample 31M0.34100.064202.5<0.05
Sample 32F0.03400.03445.15<0.05
Sample 33F0.35820.064212.71<0.05
Sample 34F0.07850.064104.22<0.05

VariablesANOVA one-way
GenderMeanSDConcentration (ng/50 μL) value

Health sample6 M, 6 F0.0442–0.08971213.5 ± 1.200
STD 31.2Standard0.08700.01231.200
STD 62.5Standard0.15600.12262.500
STD 125Standard0.27900.12212500
STD 250Standard0.55100.12225000
STD 500Standard1.06000.12250000
STD 1000Standard1.72600.122100000
Sample 1M0.08720.05431.3<0.05
Sample 2M0.08700.03431.2<0.005
Sample 3F1.08720.104512.83<0.005
Sample 4F0.08720.03431.3<0.05
Sample 5M2.41300.1441398.03<0.0001
Sample 6M0.08700.08431.2<0.05
Sample 7F2.03750.1341180.5<0.0001
Sample 8M0.45500.104206.44<0.05
Sample 9M0.08700.02431.2<0.05
Sample 10F0.08700.02431.2<0.05
Sample 11M1.83500.1141063.15<0.00001
Sample 12M0.08800.09431.6<0.05
Sample 13F0.08720.06431.3<0.05
Sample 14M0.14050.10456.3<0.05
Sample 15M0.41900.104190.11<0.05
Sample 16F0.36800.104164.9<0.05
Sample 17F1.58500.114918.31<0.001
Sample 18F0.27200.104121.9<0.05
Sample 19M0.18050.10472.32<0.05
Sample 20F0.40900.104184.21<0.05
Sample 21M0.15550.10462.3<0.05
Sample 22M0.29420.104131.81<0.05
Sample 23M0.08720.02431.3<0.05
Sample 24F0.19300.10477.32<0.005
Sample 25F0.22720.104101.8<0.005
Sample 26M0.20620.10492.4<0.005
Sample 27M0.09370.09433.60<0.05
Sample 28M0.24220.104108.51<0.05
Sample 29F0.08720.02431.3<0.05
Sample 30M0.08720.08431.3<0.05
Sample 31M0.16550.10466.31<0.05
Sample 32F0.08700.01431.2<0.05
Sample 33F0.08720.03431.3<0.05
Sample 34F0.16250.10465.10<0.05

VariablesANOVA one-way
GenderMeanSDConcentration (ng/50 μL) value

Health sample6 M, 6 F0.0290–0.0385122.88 ± 0.1400
STD 31.2Standard0.04050.04231.200
STD 62.5Standard0.14250.06262.500
STD 125Standard0.27650.06212500
STD 250Standard0.49950.06225000
STD 500Standard0.89650.06250000
STD 1000Standard1.63950.062100000
Sample 1M0.05900.05445.45<0.05
Sample 2M0.75900.194379.9<0.005
Sample 3F1.73150.6641056.11<0.0001
Sample 4F0.19650.06486.2<0.05
Sample 5M1.27250.594776.15<0.001
Sample 6M0.12700.05455.70<0.05
Sample 7F1.38370.074843.97<0.001
Sample 8M0.18600.09481.6<0.05
Sample 9M0.31100.054155.7<0.05
Sample 10F0.11750.05451.53<0.05
Sample 11M0.12850.05456.35<0.05
Sample 12M0.24650.054111.44<0.05
Sample 13F0.22750.054102.8<0.05
Sample 14M0.41450.054207.5<0.05
Sample 15M1.04100.054634.95<0.001
Sample 16F0.70700.054394.31<0.005
Sample 17F0.22350.054101.1<0.0001
Sample 18F0.23300.054105.33<0.05
Sample 19M0.10150.05444.51<0.05
Sample 20F0.78100.054390.9<0.005
Sample 21M0.08350.05464.33<0.005
Sample 22M0.20300.05491.77<0.05
Sample 23M1.12800.654688.01<0.005
Sample 24F0.51650.054258.0<0.005
Sample 25F1.12750.064687.71<0.005
Sample 26M1.12500.094686.2<0.005
Sample 27M0.12650.05455.5<0.05
Sample 28M0.74100.054370.9<0.005
Sample 29F0.64350.054322.1<0.005
Sample 30M1.26400.074770.97<0.001
Sample 31M0.82700.054461.3<0.005
Sample 32F0.24100.054108.95<0.05
Sample 33F1.30050.064793.23<0.001
Sample 34F0.51600.054258.26<0.005

VariablesConcentration (ng/50 μL)
GenderTotal defensin-defensin-defensin

Health sample6 M, 6 F1218.66 ± 3.513.5 ± 1.22.88 ± 0.14
STD 31.2Standard231.231.231.2
STD 62.5Standard262.562.562.5
STD 125Standard2125125125
STD 250Standard2250250250
STD 500Standard2500500500
STD 1000Standard2100010001000
Sample 1M437.4431.345.45
Sample 2M4867.70131.2379.9
Sample 3F41852.60512.831056.11
Sample 4F4103.5831.386.2
Sample 5M41611.681398.03776.15
Sample 6M447.4331.255.70
Sample 7F41070.641180.5843.97
Sample 8M469.04206.4481.6
Sample 9M4100.3031.2155.7
Sample 10F486.9631.251.53
Sample 11M41979.1801063.1556.35
Sample 12M4100.9331.6111.44
Sample 13F451.3331.3102.8
Sample 14M4226.6656.3207.5
Sample 15M4987.91190.11634.95
Sample 16F4380164.9394.31
Sample 17F41587.99918.31101.1
Sample 18F4242.02121.9105.33
Sample 19M4138.772.3244.51
Sample 20F4358.5184.21390.9
Sample 21M4329.5362.364.33
Sample 22M4165.4131.8191.77
Sample 23M439.5031.3688.01
Sample 24F4251.7877.32258.0
Sample 25F4209.74101.8687.71
Sample 26M4472.592.4686.2
Sample 27M4189.2533.6055.5
Sample 28M460.41108.51370.9
Sample 29F4186.1631.3322.1
Sample 30M4115.5131.3770.97
Sample 31M4202.566.31461.3
Sample 32F445.1531.2108.95
Sample 33F4212.7131.3793.23
Sample 34F4104.2265.10258.26

3.2. Total Human Defensin Concentrations in HCV-Infected Patients

The serum concentrations of human defensins (total, α-, and β-defensins) in patients infected with HCV genotype 4a were significantly higher () compared to control. 5 out of 34 (14.71%) of infected patients had the highest concentrations (1589–1979 ng/50 μL) of total defensins (Figures 1 and 4 and Tables 1 and 4); 3 of the patients were female, without statistical significance. The mean concentrations of total defensins in female patients ( ng/50 μL) were not different from those in male patients ( ng/50 μL) (Figures 1 and 4 and Tables 1 and 4).

3.3. Human α-Defensins Concentrations in HCV-Infected Patients

All HCV-infected patients revealed significantly higher concentrations of human α-defensins (31.2–1398 ng/50 μL) in comparison to controls ( ng/50 μL). The majority of HCV-infected patients (70.8%) showed α-defensin concentrations below 250 ng/50 μL, which ranged from 31.2 ng/50 μL to 206.44 ng/50 μL (Figures 2 and 4 and Tables 2 and 4) in their serum. Patients 5, 11, and 17 had the highest α-defensin concentrations at 1398.03, 1063.15, and 918.31 ng/50 μL, respectively; two patients (5 and 11) were male and the third patient (17) was female. The concentration of α-defensins in both male ( ng/50 μL) and female ( ng/50 μL) patients was nearly equal (Figures 2 and 4 and Tables 2 and 4).

3.4. Human β-Defensins Concentrations in HCV-Infected Patients

Concentrations of β-defensins were significantly higher in HCV-infected patient sera (44.50–1056.11 ng/50 μL) compared to controls ( ng/50 μL). Approximately 50% of the patients showed β-defensin concentrations of >250 ng/50 μL, ranging from 258 to 1056 ng/50 μL (Figures 3 and 4 and Tables 3 and 4). The concentrations of β-defensins in both male ( ng/50 μL) and female ( ng/50 μL) patients were similar (Figures 3 and 4 and Tables 3 and 4). Concentrations of β-defensins of 500 ng/50 μL were more commonly found in males (5 patients) than females (3 patients; Table 4). Patients 3, 7, 33, 5, and 30 were found to have highest β-defensin concentrations at 1056.11, 843.97, 793.23, 776.15, and 770.97 ng/50 μL, respectively; three of these five patients were female (Figures 3 and 4 and Tables 3 and 4).

4. Discussion

Defensins are “magic” 28–42 amino acid cationic peptides, assumed to possess a conserved structural fold containing six highly conserved cysteine residues, which form three pairs of highly conserved intramolecular disulfide bonds [17, 3740]. Vertebrate defensins are classified as α-, β-, and γ-defensins, based on their cellular origin, the spacing between the cysteine residues, and the number and pattern (topology) of their disulfide bridges [17, 38, 40]. In mammals, barrier epithelial cells mostly generate β-defensins, whereas α-defensins are mainly stored in the azurophil granules of neutrophils [16]. In the mouse, Paneth cells and fibroblasts produce at least 17 α-defensins, whereas various epithelial cells and keratinocytes generate 4 β-defensins. The α- and β-defensins are present in different vertebrate species, where they are found in the granules of immune cells, epithelial tissue, body fluids, and mucosal surfaces [40].

In the current study, circulating concentrations of defensins in HCV patients were evaluated, for first time worldwide, to determine whether levels of defensins altered during HCV infection. Sera from 12 noninfected and 34 HCV-infected patients were harnessed in order to test this, using the commercial ELISA kits. The obtained results demonstrated that HCV-infected patients had significantly increased (–0.0001) levels of defensin (total, α-, and β-defensins) concentrations compared to the noninfected group. The majority of patients (70.8%) revealed α-defensin concentrations below 250 ng/50 μL. The concentrations of α-defensins in both male ( ng/50 μL) and female ( ng/50 μL) patients were virtually equal. The highest α-defensin concentrations were reported in patients 5, 11, and 17 (1398.03, 1063.15, and 918.31 ng/50 μL, resp.); two patients (5 and 11) were male and the third patient (17) was female. Approximately half the HCV-infected patients showed β-defensin concentrations of >250 ng/50 μL. Concentrations of β-defensins of 500 ng/50 μL were more commonly found in males (5 patients) than in females (3 patients). Three of the five HCV-infected patients showing the highest β-defensin concentrations were female, although gender did not seem to have a significant effect. The high defensins concentrations within these patients may be due to a comicrobial infection, and/or a patient’s infection was in the acute or after acute phase. The latter suggestion may agree with the results of Aceti et al. [41], where a high anamnestic response in defensin concentration was reported after in vitro stimulation of PBMCs from chronic HCV-infected patients with HCV proteins (see below).

These results are generally consistent with the only two studies currently available in literature [41, 42]. One report evaluated α- and β-defensin concentrations in human peripheral blood by measuring mRNA copy number [42], while the second evaluated α-defensin mRNA copy number in human peripheral blood mononuclear cells (PBMCs) of patients with chronic HCV infection, after in vitro induction with HCV C proteins [41]. Fang et al. [42] concluded that human peripheral blood β-defensins 1 and 2 (DEFB1 and DEFB2) genes were transiently expressed following induction with lipopolysaccharide or heat-inactivated bacterial cells, whereas α-defensins 1–3 (DEFA13) genes were constitutively transcribed while the β-defensin 3 (DEFB3) gene was not expressed. The inducible expression of DEFB1 and DEFB2 genes displayed interindividual variability; however, the study did not indicate serum concentrations of defensin peptides.

Aceti et al. [41], however, identified and quantified α-defensins in PBMCs using mass spectrometry, ELISA, antibacterial activity, and mRNA levels. PBMCs from 3 patients and controls were stimulated with HCV core protein and hepatitis B virus antigen in vitro as well as the α-defensin mRNAs level was quantified. The authors found that HCV C protein activates transcription of α-defensin in vitro, and α-defensin peptide levels were accordingly significantly increased in patients with chronic hepatitis C ( ng/106 cells) and chronic hepatitis B () compared to healthy controls (; ). In patients with chronic hepatitis C, levels of α-defensin and antibacterial activity correlated with the liver fibrosis. Aceti et al. [41] suggested that HCV induces α-defensin expression and that the high linear correlation of α-defensin levels with advancing fibrosis makes measuring these peptides a reliable marker of fibrosis stage.

Higher concentrations of both α- and β-defensins in our samples may indicate an immune response profile in these patients. The Th-1 immunity profile (IL-2, IL-12, TNF-α, and IFN-γ) is correlated with liver fibrosis in patients with chronic hepatitis C, whereas Th2 immunity profile (IL-4 and IL-10) cannot control viral clearance [43]. Defensins are considered to be inducers of proinflammatory cytokines (TNF-α and IFN-γ) and the Th1-skewed immune response [30]. Recently, patients with severe liver fibrosis presented lower frequency of circulating CD8+ T-cells, higher levels of proinflammatory cytokines, lower levels of IL-10, and higher levels of proinflammatory cytokines (TNF and IFN-γ) [44], in line with a previous report that found a linear correlation between α-defensin levels and advancing liver fibrosis [41].

In a study by Erhart et al. [45], the expressions of various α- and β-defensins in biopsy samples taken from 35 patients infected with genital (warts) papillomavirus were analysed. The authors found significantly higher expression of β-defensin hBD-1 (), hBD-2 (), and hBD-3 (), while α-defensins (HNPs 1–3) were scarcely detectable in normal and viral infected tissues [45].

Generally, humans express 6 α-defensins and multiple β-defensin peptides. A-defensins 1–3 are especially abundant in human neutrophils, constituting 30–50% of the total protein of their azurophil granules [4648]. Although plasma levels of α-defensins normally vary between 40 and 200 ng/mL, higher levels are found at sites of infection, and plasma levels >100 μg/mL may occur during sepsis, intrauterine infections, and bacterial meningitis [4755]. These plasma α-defensin concentrations are higher than those estimated in our study in normal serum, which may reflect the differences in the methods of measurement used.

Increased plasma and bronchoalveolar lavage levels of α-defensins have been reported in individuals with Mycobacterium avium-intracellulare infection and pulmonary tuberculosis [53, 54]. A-defensin concentrations were not correlated with infection with Mycobacterium tuberculosis and/or its multidrug resistant strain [56]. A-defensins are active against a variety of gram-positive and gram-negative bacteria as well as fungi and parasites [47, 57].

Defensins are considered as one of strongest types of central and peripheral defenders, especially in mucosal tissues. They also link the innate and adaptive immunity. There is a close correlation between increased concentrations of α- and β-defensins in vaginal tissues and fluids following infection. An enormous increase in α-defensins and their secretory neutrophils in the vaginal during endometritis was reported [52]. Fan et al. [58] have shown that concentrations of human β-defensin 2 and α-defensin 5 were increased in women with vaginosis, which the authors considered to be an immune response against bacterial invasion [58]. This was later confirmed [59]; the group showed that bacterial vaginosis was associated with lower vaginal concentrations of β-defensin 3, but not β-defensin 2 or α-defensins 1–3, in pregnant women. Baricelli et al. [60] added that β-defensin 2 was secreted in the milk of lactating women. Interestingly, levels of β-defensin 2 were found to be significantly higher in colostrum than in mature milk samples [60]. The increased concentrations of various types of defensins in clinical conditions compared to normal health are not limited to microbial infections but extend to different kinds of human diseases [31, 61, 62].

Human natural α-defensins (HNPs 1, 2, and 3) concentrations should only be compared with the total amount of HNPs 1–3, measured by radio- and immunoassays. Reportedly, HNPs 1–3 serum concentrations measured with RIA [63] were found to be 250 ng/mL in controls sera, while they were 500 to 1750 ng/mL in patients with various lung diseases. In another study, HNPs 1–3 concentrations in serum were measured with ELISA [64] and revealed as ±7 ng/mL in normal individuals and were significantly increased in colon cancer patients with a median concentration of around 15–29 ng/mL. When comparing the total amount of HNPs 1, 2, and 3, as measured using the developed assay, in serum from IC patients (an average of 1076 ng/mL), these levels complied with the increased concentrations found in the study by Mukae et al. [63], whereas the total measured amounts in the serum batches used for the selectivity assessment (an average of 309 ng/mL) were comparable with the concentrations in healthy controls in the same study [61]. More studies have reported HNPs 1–3 concentrations in plasma: around 200–400 ng/mL in healthy controls, when measured with RIA [31, 5355, 63, 65, 66], and around 40–100 ng/mL when measured with ELISA [51, 67, 68]. Recent measurements in different plasma batches, used for selectivity assessment, showed between 40 and 175 ng/mL and seem comparable with the concentrations measured using the ELISA method [69].

Since 1997, the worldwide pioneer in the defensins, Lehrer [70], raised fundamental questions regarding defensins concentrations in disease and other related issues. Although a 50 kg female or 70 kg male will produce at least 250–700 mg defensins daily (more when neutrophilic leukocytosis occurs), the low levels of defensins in normal plasma (254.8 pg/mL) account for 0.05% of baseline daily production. Where are the rest of these peptides? Are defensins rapidly degraded, and if so, where does this process occur? Do extracellular defensins leave the circulation as rapidly as they enter it (an easy question, from the standpoint of steady-state kinetics), and how rapidly do they enter it (a more challenging question)? How short is the half-life of plasma defensins and do they recirculate or traverse epithelial barriers? Do extracellular defensins permeate tissues such as the lungs and the gastrointestinal tract? Do they impregnate epithelial cells, basement membranes, and mucosal tissues? All of these crucial questions are yet to be comprehensively and satisfactorily answered.

Although there is a clear correlation between the results presented here and previous reports of human circulated defensin concentrations, the exact concentrations of defensins in health and disease remain to be determined.

To conclude, circulated defensins are measured at significantly higher concentration in HCV-infected patients compared to healthy individuals. Total, α-, and β-defensin concentrations are all elevated by tenfold in patients with HCV infections.


The current work is a part of the Ph.D. thesis of Mr. Ehab Hussein Mattar (Department of Biology, Faculty of Science, King Abdulaziz University).

Competing Interests

The authors declare that there are no competing interests.


The authors thank Professors Drs. Khaleid Aljhmdi and Usama Abuzenadh for their continued support.


  1. A. Varaklioti, N. Vassilaki, U. Georgopoulou, and P. Mavromara, “Alternate translation occurs within the core coding region of the hepatitis C viral genome,” The Journal of Biological Chemistry, vol. 277, no. 20, pp. 17713–17721, 2002. View at: Publisher Site | Google Scholar
  2. C. M. Rice, “Flaviviridae: the viruses and their replication,” in Fields Virology, B. N. Fields, D. M. Knipe, and P. M. Howley, Eds., pp. 931–956, Lippincott-Raven, Philadelphia, Pa, USA, 3rd edition, 1996. View at: Google Scholar
  3. N. Kato, M. Hijikata, Y. Ootsuyama et al., “Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis,” Proceedings of the National Academy of Sciences of the United States of America, vol. 87, no. 24, pp. 9524–9528, 1990. View at: Publisher Site | Google Scholar
  4. J. Nakabayashi, “A compartmentalization model of hepatitis C virus replication: an appropriate distribution of HCV RNA for the effective replication,” Journal of Theoretical Biology, vol. 300, pp. 110–117, 2012. View at: Publisher Site | Google Scholar | MathSciNet
  5. D. A. Saleh, F. Shebl, M. Abdel-Hamid et al., “Incidence and risk factors for hepatitis C infection in a cohort of women in rural Egypt,” Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 102, no. 9, pp. 921–928, 2008. View at: Publisher Site | Google Scholar
  6. T. Poynard, P. Marcellin, S. S. Lee et al., “Randomised trial of interferon α2b plus ribavirin for 48 weeks or for 24 weeks versus interferon α2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus,” The Lancet, vol. 352, no. 9138, pp. 1426–1432, 1998. View at: Publisher Site | Google Scholar
  7. T. Okanoue, Y. Itoh, M. Minami et al., “Interferon therapy lowers the rate of progression to hepatocellular carcinoma in chronic hepatitis C but not significantly in an advanced stage: a retrospective study in 1148 patients,” Journal of Hepatology, vol. 30, no. 4, pp. 653–659, 1999. View at: Publisher Site | Google Scholar
  8. FDA,
  9. X. Qian, Y.-Z. Zhu, P. Zhao, and Z.-T. Qi, “Entry inhibitors: new advances in HCV treatment,” Emerging Microbes & Infections, vol. 5, article e3, 2016. View at: Publisher Site | Google Scholar
  10. A. Izadpanah and R. L. Gallo, “Antimicrobial peptides,” Journal of the American Academy of Dermatology, vol. 52, no. 3, pp. 381–392, 2005. View at: Publisher Site | Google Scholar
  11. C. Beisswenger and R. Bals, “Functions of antimicrobial peptides in host defense and immunity,” Current Protein and Peptide Science, vol. 6, no. 3, pp. 255–264, 2005. View at: Publisher Site | Google Scholar
  12. K. A. Brogden, “Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?” Nature Reviews Microbiology, vol. 3, no. 3, pp. 238–250, 2005. View at: Publisher Site | Google Scholar
  13. Y. J. Gordon, E. G. Romanowski, and A. M. McDermott, “A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs,” Current Eye Research, vol. 30, no. 7, pp. 505–515, 2005. View at: Publisher Site | Google Scholar
  14. Y. Nie, X.-C. Zeng, Y. Yang et al., “A novel class of antimicrobial peptides from the scorpion Heterometrus spinifer,” Peptides, vol. 38, no. 2, pp. 389–394, 2012. View at: Publisher Site | Google Scholar
  15. O. Toke, “Antimicrobial peptides: new candidates in the fight against bacterial infections,” Biopolymers—Peptide Science Section, vol. 80, no. 6, pp. 717–735, 2005. View at: Publisher Site | Google Scholar
  16. M. E. Selsted and A. J. Ouellette, “Mammalian defensins in the antimicrobial immune response,” Nature Immunology, vol. 6, no. 6, pp. 551–557, 2005. View at: Publisher Site | Google Scholar
  17. M. Pazgier, D. M. Hoover, D. Yang, W. Lu, and J. Lubkowski, “Human β-defensins,” Cellular and Molecular Life Sciences, vol. 63, no. 11, pp. 1294–1313, 2006. View at: Publisher Site | Google Scholar
  18. T. Ganz, M. E. Selsted, D. Szklarek et al., “Defensins. Natural peptide antibiotics of human neutrophils,” The Journal of Clinical Investigation, vol. 76, no. 4, pp. 1427–1435, 1985. View at: Publisher Site | Google Scholar
  19. B. Ericksen, Z. Wu, W. Lu, and R. I. Lehrer, “Antibacterial activity and specificity of the six human α-defensins,” Antimicrobial Agents and Chemotherapy, vol. 49, no. 1, pp. 269–275, 2005. View at: Publisher Site | Google Scholar
  20. R. C. Rodríguez de la Vega and L. D. Possani, “On the evolution of invertebrate defensins,” Trends in Genetics, vol. 21, no. 6, pp. 330–332, 2005. View at: Publisher Site | Google Scholar
  21. J. A. Hoffmann and C. Hetru, “Insect defensins: inducible antibacterial peptides,” Immunology Today, vol. 13, no. 10, pp. 411–415, 1992. View at: Publisher Site | Google Scholar
  22. F. T. Lay and M. A. Anderson, “Defensins—components of the innate immune system in plants,” Current Protein and Peptide Science, vol. 6, no. 1, pp. 85–101, 2005. View at: Publisher Site | Google Scholar
  23. T. Ganz, “Defensins: antimicrobial peptides of innate immunity,” Nature Reviews Immunology, vol. 3, no. 9, pp. 710–720, 2003. View at: Publisher Site | Google Scholar
  24. T. Ganz, “Defensins: antimicrobial peptides of vertebrates,” Comptes Rendus Biologies, vol. 327, no. 6, pp. 539–549, 2004. View at: Publisher Site | Google Scholar
  25. T. Ganz, “Defensins and other antimicrobial peptides: a historical perspective and an update,” Combinatorial Chemistry and High Throughput Screening, vol. 8, no. 3, pp. 209–217, 2005. View at: Publisher Site | Google Scholar
  26. N. A. El-Baky, V. N. Uversky, and E. M. Redwan, “Human consensus interferons: bridging the natural and artificial cytokines with intrinsic disorder,” Cytokine and Growth Factor Reviews, vol. 26, no. 6, pp. 637–645, 2015. View at: Publisher Site | Google Scholar
  27. D. Yang, A. Biragyn, L. W. Kwak, and J. J. Oppenheim, “Mammalian defensins in immunity: more than just microbicidal,” Trends in Immunology, vol. 23, no. 6, pp. 291–296, 2002. View at: Publisher Site | Google Scholar
  28. C. Kim and S. H. E. Kaufmann, “Defensin: a multifunctional molecule lives up to its versatile name,” Trends in Microbiology, vol. 14, no. 10, pp. 428–431, 2006. View at: Publisher Site | Google Scholar
  29. J. Shi, “Defensins and Paneth cells in inflammatory bowel disease,” Inflammatory Bowel Diseases, vol. 13, no. 10, pp. 1284–1292, 2007. View at: Publisher Site | Google Scholar
  30. E. H. Mattar, H. A. Almehdar, H. A. Yacoub, V. N. Uversky, and E. M. Redwan, “Antimicrobial potentials and structural disorder of human and animal defensins,” Cytokine & Growth Factor Reviews, vol. 28, pp. 95–111, 2016. View at: Publisher Site | Google Scholar
  31. T. Ihi, M. Nakazato, H. Mukae, and S. Matsukura, “Elevated concentrations of human neutrophil peptides in plasma, blood, and body fluids from patients with infections,” Clinical Infectious Diseases, vol. 25, no. 5, pp. 1134–1140, 1997. View at: Publisher Site | Google Scholar
  32. J. D. Scott and D. R. Gretch, “Molecular diagnostics of hepatitis C virus infection: a systematic review,” The Journal of the American Medical Association, vol. 297, no. 7, pp. 724–732, 2007. View at: Publisher Site | Google Scholar
  33. E.-R. M. Redwan and M. K. AL-Awady, “Prevalence of tetanus immunity in the Egyptian population,” Human Antibodies, vol. 11, no. 1-2, pp. 55–59, 2002. View at: Google Scholar
  34. E.-R. M. Redwan, “Biochemical and immunological properties of four intravenous immunoglobulin G preparations,” Human Antibodies, vol. 11, no. 3, pp. 79–84, 2002. View at: Google Scholar
  35. E. M. Redwan and M. K. El-Awady, “Status of diphtheria immunity in the Egyptian population,” Annals of Tropical Medicine and Parasitology, vol. 99, no. 1, pp. 93–99, 2005. View at: Publisher Site | Google Scholar
  36. E.-R. M. Redwan, A. Khalil, and Z. Z. EL-Dardiri, “Production and purification of ovine anti-tetanus antibody,” Comparative Immunology, Microbiology and Infectious Diseases, vol. 28, no. 3, pp. 167–176, 2005. View at: Publisher Site | Google Scholar
  37. E. B. Mallow, A. Harris, N. Salzman et al., “Human enteric defensins. Gene structure and developmental expression,” The Journal of Biological Chemistry, vol. 271, no. 8, pp. 4038–4045, 1996. View at: Publisher Site | Google Scholar
  38. A. Ouellette and J. C. Lualdi, “A novel mouse gene family coding for cationic, cysteine-rich peptides. Regulation in small intestine and cells of myeloid origin,” The Journal of Biological Chemistry, vol. 265, no. 17, pp. 9831–9837, 1990. View at: Google Scholar
  39. M. E. Selsted, Y.-Q. Tang, W. L. Morris et al., “Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils,” The Journal of Biological Chemistry, vol. 268, no. 9, pp. 6641–6648, 1993. View at: Google Scholar
  40. Y.-Q. Tang, J. Yuan, G. Ösapay et al., “A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated α-defensins,” Science, vol. 286, no. 5439, pp. 498–502, 1999. View at: Publisher Site | Google Scholar
  41. A. Aceti, M. L. Mangoni, C. Pasquazzi et al., “α-Defensin increase in peripheral blood mononuclear cells from patients with hepatitis C virus chronic infection,” Journal of Viral Hepatitis, vol. 13, no. 12, pp. 821–827, 2006. View at: Publisher Site | Google Scholar
  42. X.-M. Fang, Q. Shu, Q.-X. Chen et al., “Differential expression of α- and β-defensins in human peripheral blood,” European Journal of Clinical Investigation, vol. 33, no. 1, pp. 82–87, 2003. View at: Publisher Site | Google Scholar
  43. E. Gigi, M. Raptopoulou-Gigi, A. Kalogeridis et al., “Cytokine mRNA expression in hepatitis C virus infection: TH-1 predominance in patients with chronic hepatitis C and TH1-TH2 cytokine profile in subjects with self-limited disease,” Journal of Viral Hepatitis, vol. 15, no. 2, pp. 145–154, 2008. View at: Publisher Site | Google Scholar
  44. S. de Souza-Cruz, M. B. Victória, A. M. Tarragô et al., “Liver and blood cytokine microenvironment in HCV patients is associated to liver fibrosis score: a proinflammatory cytokine ensemble orchestrated by TNF and tuned by IL-10,” BMC Microbiology, vol. 16, no. 1, article 3, 2016. View at: Publisher Site | Google Scholar
  45. W. Erhart, Ö. Alkasi, G. Brunke et al., “Induction of human β-defensins and psoriasin in vulvovaginal human papillomavirus-associated lesions,” Journal of Infectious Diseases, vol. 204, no. 3, pp. 391–399, 2011. View at: Publisher Site | Google Scholar
  46. M. Salvatore, A. Garcia-Sastre, P. Ruchala, R. I. Lehrer, T. Chang, and M. E. Klotman, “alpha-defensin inhibits influenza virus replication by cell-mediated mechanism(s,” The Journal of Infectious Diseases, vol. 196, no. 6, pp. 835–843, 2007. View at: Publisher Site | Google Scholar
  47. M. E. Klotman and T. L. Chang, “Defensins in innate antiviral immunity,” Nature Reviews Immunology, vol. 6, no. 6, pp. 447–456, 2006. View at: Publisher Site | Google Scholar
  48. R. I. Lehrer, “Primate defensins,” Nature Reviews Microbiology, vol. 2, no. 9, pp. 727–738, 2004. View at: Publisher Site | Google Scholar
  49. T. Ganz and R. I. Lehrer, “Defensins,” Pharmacology and Therapeutics, vol. 66, no. 2, pp. 191–205, 1995. View at: Publisher Site | Google Scholar
  50. F. A. Maffei, R. P. Heine, M. J. Whalen, L. F. Mortimer, and J. A. Carcillo, “Levels of antimicrobial molecules defensin and lactoferrin are elevated in the cerebrospinal fluid of children with meningitis,” Pediatrics, vol. 103, no. 5, pp. 987–992, 1999. View at: Publisher Site | Google Scholar
  51. A. V. Panyutich, E. A. Panyutich, V. A. Krapivin, E. A. Baturevich, and T. Ganz, “Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis,” The Journal of Laboratory and Clinical Medicine, vol. 122, no. 2, pp. 202–207, 1993. View at: Google Scholar
  52. H. C. Wiesenfeld, R. P. Heine, M. A. Krohn et al., “Association between elevated neutrophil defensin levels and endometritis,” Journal of Infectious Diseases, vol. 186, no. 6, pp. 792–797, 2002. View at: Publisher Site | Google Scholar
  53. J.-I. Ashitani, H. Mukae, Y. Arimura, A. Sano, M. Tokojima, and M. Nakazato, “High concentrations of α-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome,” Life Sciences, vol. 75, no. 9, pp. 1123–1134, 2004. View at: Publisher Site | Google Scholar
  54. J.-I. Ashitani, H. Mukae, T. Hiratsuka, M. Nakazato, K. Kumamoto, and S. Matsukura, “Plasma and BAL fluid concentrations of antimicrobial peptides in patients with Mycobacterium avium-intracellulare infection,” Chest, vol. 119, no. 4, pp. 1131–1137, 2001. View at: Publisher Site | Google Scholar
  55. J.-I. Ashitani, H. Mukae, T. Hiratsuka, M. Nakazato, K. Kumamoto, and S. Matsukura, “Elevated levels of α-defensins in plasma and BAL fluid of patients with active pulmonary tuberculosis,” Chest, vol. 121, no. 2, pp. 519–526, 2002. View at: Publisher Site | Google Scholar
  56. L.-M. Zhu, C.-H. Liu, P. Chen et al., “Multidrug-resistant tuberculosis is associated with low plasma concentrations of human neutrophil peptides 1-3,” International Journal of Tuberculosis and Lung Disease, vol. 15, no. 3, pp. 369–374, 2011. View at: Google Scholar
  57. H.-Y. Lee, A. Andalibi, P. Webster et al., “Antimicrobial activity of innate immune molecules against Streptococcus pneumoniae, Moraxella catarrhalis and nontypeable Haemophilus influenzae,” BMC Infectious Diseases, vol. 4, article 12, 2004. View at: Publisher Site | Google Scholar
  58. S. R. Fan, X. P. Liu, and Q. P. Liao, “Human defensins and cytokines in vaginal lavage fluid of women with bacterial vaginosis,” International Journal of Gynecology and Obstetrics, vol. 103, no. 1, pp. 50–54, 2008. View at: Publisher Site | Google Scholar
  59. C. Mitchell, M. L. Gottsch, C. Liu, D. N. Fredricks, and D. B. Nelson, “Associations between vaginal bacteria and levels of vaginal defensins in pregnant women,” American Journal of Obstetrics and Gynecology, vol. 208, no. 2, pp. 132–e7, 2013. View at: Publisher Site | Google Scholar
  60. J. Baricelli, M. A. Rocafull, D. Vázquez, B. Bastidas, E. Báez-Ramirez, and L. E. Thomas, “β-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria,” Jornal de Pediatria, vol. 91, no. 1, pp. 36–43, 2015. View at: Publisher Site | Google Scholar
  61. Y. Arimura, J.-I. Ashitani, S. Yanagi et al., “Elevated serum β-defensins concentrations in patients with lung cancer,” Anticancer Research, vol. 24, no. 6, pp. 4051–4057, 2004. View at: Google Scholar
  62. A. Weinberg, G. Jin, S. Sieg, and T. S. McCormick, “The Yin and Yang of human beta-defensins in health and disease,” Frontiers in Immunology, vol. 3, article 294, 2012. View at: Publisher Site | Google Scholar
  63. H. Mukae, H. Iiboshi, M. Nakazato et al., “Raised plasma concentrations of α-defensins in patients with idiopathic pulmonary fibrosis,” Thorax, vol. 57, no. 7, pp. 623–628, 2002. View at: Publisher Site | Google Scholar
  64. C. Melle, G. Ernst, B. Schimmel et al., “Discovery and identification of α-defensins as low abundant, tumor-derived serum markers in colorectal cancer,” Gastroenterology, vol. 129, no. 1, pp. 66–73, 2005. View at: Publisher Site | Google Scholar
  65. K. Shiomi, M. Nakazato, T. Ihi, K. Kangawa, H. Matsuo, and S. Matsukura, “Establishment of radioimmunoassay for human neutrophil peptides and their increases in plasma and neutrophil in infection,” Biochemical and Biophysical Research Communications, vol. 195, no. 3, pp. 1336–1344, 1993. View at: Publisher Site | Google Scholar
  66. N. Yamaguchi, H. Isomoto, H. Mukae et al., “Concentrations of α- and β-defensins in plasma of patients with inflammatory bowel disease,” Inflammation Research, vol. 58, no. 4, pp. 192–197, 2009. View at: Publisher Site | Google Scholar
  67. J. Albrethsen, C. H. Møller, J. Olsen, H. Raskov, and S. Gammeltoft, “Human neutrophil peptides 1, 2 and 3 are biochemical markers for metastatic colorectal cancer,” European Journal of Cancer, vol. 42, no. 17, pp. 3057–3064, 2006. View at: Publisher Site | Google Scholar
  68. S. Kanmura, H. Uto, M. Numata et al., “Human neutrophil peptides 1-3 are useful biomarkers in patients with active ulcerative colitis,” Inflammatory Bowel Diseases, vol. 15, no. 6, pp. 909–917, 2009. View at: Publisher Site | Google Scholar
  69. I. van den Broek, R. W. Sparidans, J. H. M. Schellens, and J. H. Beijnen, “Validation of a quantitative assay for human neutrophil peptide-1, -2, and -3 in human plasma and serum by liquid chromatography coupled to tandem mass spectrometry,” Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, vol. 878, no. 15-16, pp. 1085–1092, 2010. View at: Publisher Site | Google Scholar
  70. R. I. Lehrer, “Editorial response: questions and answers about defensins,” Clinical Infectious Diseases, vol. 15, pp. 1141–1142, 1997. View at: Google Scholar

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