Pharmacology and therapeutics

The therapeutic impact of HNP-1 in condyloma acuminatum

Shuyun Zhao1,*, MS, Hong Ying Zhou2,*, MD, Huijuan Li1, MS, Tao Yi1, MD, and
Xia Zhao1, MD

1
Biotherapy Laboratory of Gynecological
Oncology, Key Laboratory of Obstetric and
Gynecologic and Pediatric Diseases and
Birth Defects of Ministry of Education, West
China Second University Hospital, and
2
Department of Anatomy, School of
Preclinical and Forensic Medicine, Sichuan
University, Chengdu, China
Correspondence
Tao Yi, MD
Biotherapy Laboratory of Gynecological
Oncology
Key Laboratory of Obstetric and
Gynecologic and Pediatric Diseases and
Birth Defects of Ministry of Education
West China Second University Hospital
Sichuan University
Chengdu
Sichuan 610041
China
E-mail: yitao_yt@hotmail.com
*These authors contributed equally to this
work.
doi: 10.1111/ijd.12725
Abstract
Background Condyloma acuminatum is one of the most commonly occurring sexually
transmitted diseases. HNP1 is a small antimicrobial peptide that has been reported to have
antiviral activities.
Aim Using the condyloma acuminatum tissue culture to resemble the situation more
closely in vivo, we investigate the therapeutic effect of a recombinant plasmid encoding
HNP1 gene in condyloma acuminatum tissue.
Methods Recombinant plasmid DNA carrying HNP1 cDNA was constructed and identified.
Then the recombinant plasmid was transfected into a condyloma acuminatum tissue
fragment, and the HNP1 expression was determined on these tissue fragments by
immunohistochemistry. TUNEL staining and flow cytometry techniques were used to
examine cell apoptosis of condyloma acuminatum tissue. Relative real-time polymerase
chain reaction was used to validate antihuman papillomavirus therapeutics of the treatment
groups.
Results Transfected HNP1 gene was expressed mainly in the cytoplasmic granules of the
condyloma acuminatum tissues. Positive apoptotic cells were observed in condyloma
acuminatum tissues transfected with the HNP1 gene. In addition, the HPV expression was
lower in the HNP1 treatment tissues as compared to their corresponding control tissues.
Conclusion The results indicate that HNP1 can directly promote condyloma acuminatum
cell apoptosis and play an antivirus role in the condyloma acuminatum tissue by limiting
viral replication. These observations suggest a possible application for human HNP1 on
condyloma acuminatum therapy.

China. Traditional treatment for this disease is mainly

Introduction
Defensins are small, cationic proteins with 29–35 amino
acids in length and are members of the antimicrobial pep-
tide family, which play an important role in protecting
the host from the invasion of pathogens.1 Defensins have
antimicrobial activities,2 and their targeting includes some
bacteria, fungi, yeast, and virus.
Human papillomavirus (HPV) is one kind of nonenvel-
oped virus and can be classified into about 100 types, at
least 40 of which are sexually transmissible.3 Humans
infected by different types of HPV could suffer from dif-
ferent diseases, such as condyloma acuminatum and uter-
ine cervix cancer.4–7 Condyloma acuminatum is a kind of
sexually transmitted disease, and its incidence has risen
recently because of unsafe sexual intercourse. The preva-
lence of genital warts has been estimated to be 1% in the
sexually active population (15–49 years) in the United
States.8 The incidence of genital warts in Hong Kong was
estimated to be 203.7 per 100,000 person-years.9 The
prevalence of the diseases has also been increasing in
physical ablative methods, such as laser vaporization,
cryotherapy, electrodessication, and so on. We wondered
whether HNP1 could be applied for external use for dis-
eases associated with HPV infection, such as cervical can-
cer and condyloma acuminatum. In the present study, we
transfected condyloma acuminatum tissues using recombi-
nant HNP-1 eukaryotic expression plasmid as the vector
with liposome and then determined the effect of a-defen-
sin-1 on condyloma acuminatum to reveal the therapeutic
effectiveness.
Materials and methods
Plasmid DNA construction and preparation
The total RNA was prepared from human umbilical vein
endothelial cells using TRIzol reagent (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s instructions. Two
micrograms of total RNA were used as a template to amplify
the HNP1 gene by reverse transcription polymerase chain
reaction (RT-PCR) (Takara, Dalian, China). The primers of 1

ª 2015 The International Society of Dermatology International Journal of Dermatology 2015

2 Pharmacology and therapeutics Therapeutic impact of HNP-1
HNP1 gene were designed based on its cDNA sequence
(GenBank accession: NM_0040847) with upstream primer 50 -
CGC GGATCCATGAGGACCCTCGCCAT-30 and downstream
primer 50 -CCG CTC GAG TCA GCA GAA TGC CCA GA-30 .
RT-PCR was performed as follows: incubation at 50 °C for
30 min, and denaturation at 94 °C for 2 min, 94 °C for 30 s,
56 °C for 30 s, and 72 °C for 2 min. Glyceraldehyde 3-
phosphate dehydrogenase (GAPDH) was a housekeeping gene
and served as a control.
The amplified products were subcloned into pTango-zeo-
NEGFP (pTango) to construct the expression vectors pTango-
HNP1 (p-HNP1). As a control, pTango plasmid without HNP1
cDNA was used as an empty vector. The recombinant HNP1
was confirmed by restriction digestion and DNA sequencing.
Plasmids were purified by using two rounds of passage over
Endo-free columns (Qiagen, Chatsworth, CA, USA), as
previously reported.10
Tissue collection and culture
Condyloma acuminatum tissues were collected from female
patients of reproductive age in the Department of Gynecology
and Obstetrics (The Second West China Hospital of Sichuan
University). The tissues were excised under a clinical diagnosis
of condyloma acuminatum, which was confirmed by
histopathological study. Participation in the study was voluntary,
and a signed copy of the informed consent was approved by
the Ethics Committee of Second West China Hospital of
Sichuan University. The tissues were washed three times with
serum-free RPMI1640 containing 100 IU/ml of penicillin and
100 IU/ml of phytomycin. They were then cut into smaller
pieces about 1 mm3, put into six-well Petri dishes, incubated in
1 ml RPMI1640 containing with 10% FBS, 100 IU/ml penicillin.
One hundred IU/ml phytomycin and 5 mg/ml fluconazole at
37 °C, 5% CO2 incubated for 8–12 hours.
Transfected with the plasmid DNA
Condyloma acuminatum tissue particles were transfected with
plasmid P-HNP1 using the lipofectamine 2000 transfection
reagent as previously described.11 According to the
manufacturer’s instruction, the condyloma acuminatum tissue
particles were washed in serum-free medium then incubated
4 h at 37 °C in 5% CO2, 2 lg of constructed plasmid with
lipofectamine reagent (Life Technologies, Grand Island, NY,
USA) was transfected into the condyloma acuminatum tissue
particles in 24-well Petri dishes. In addition, the empty plasmid
and liposome alone were used as control agents. The cells
were harvested at 48 hours post-transfection.
Condyloma acuminatum tissue particles were assigned
randomly to one of the following groups: (a) untreated (un), only
2 ml medium; (b) lipofectamine (lipo), 10 ll of lipofectamine in
2 ml medium; (c) lipofectamine + empty plasmid (e-p), 2 lg of
empty plasmid/10 ll of lipofectamine in 2 ml medium; and (d)
lipofectamine + p-HNP1 complexes (p-HNP1/lipo), 2 lg of
International Journal of Dermatology 2015
Zhao et al.
p-HNP1/10 ll of lipofectamine in 2 ml medium, and each
repeated three times.
TUNEL assay for apoptotic cells
To detect apoptotic cells in condyloma acuminatum tissue
particles, terminal deoxynucleotidyl transferase-mediated dUTP
nick end-labeling (TUNEL) assay was performed to analyze
apoptotic cells using apoptotic cell kits according to the
manufacturer’s protocol (Promega, Madison, WI, USA).
Sections were observed under fluorescence microscopy (Nikon
80i, Tokyo, Japan). Darkly stained nuclei or nuclear fragments
with green fluorescence were defined as TUNEL-positive. The
apoptotic index was determined by calculating the average
percentage of TUNEL-positive cells and comparing it to the total
number of cancer cells in the same field at a magnification of
9400 in three representative sections from each group (five
high-power fields excluding areas of necrosis/section).
Flow cytometric analysis
In flow cytometric analysis, condyloma acuminatum cells were
stained by propidium iodide (PI) to evaluate the cellular
apoptosis. Briefly, after 48 hours of transfection with p-HNP1/
lipo complexes, tissue particles were collected, made into
unicells, and washed twice with cold phosphate-buffered saline.
Cells were resuspended in prediluted binding buffer and stained
with PI for 30 minutes at room temperature in the dark, and the
apoptosis of the cells was analyzed by flow cytometry
immediately.
Relative real-time polymerase chain reaction analysis
Total RNA from condyloma acuminatum tissues was obtained
using Trizol reagent (Invitrogen) according to the manufacturer’s
instructions. One lg of RNA was used to synthesize cDNA with
PrimeScriptTM RT Reagent Kit (TaKaRa). b-actin housekeeping
gene, evaluation of the mRNA levels, was used as reference for
normalization. Primers used for qRT-PCR assays are listed in
Table 1. PCR was performed with SYBR Premix Ex TaqTM Kit
(TaKaRa) using the CFX96TM Real-Time System (Bio-Rad,
Hercules, CA, USA). The cycling profile used for PCR
conditions was as follows: the thermal cycle conditions were
95 °C for 10 s for one cycle, followed by 40 cycles of 95 °C for
15 s, 58 °C for 15 s, and 72 °C for 30 s. The relative
expression level of each gene was calculated as the 2DDC T
method. Mean and standard deviation were determined with
data from three replicates.
Immunohistochemistry analysis
Immunohistochemical detection protein expression has been
described in previous studies.12 It was used to determine the
HNP1 expression in condyloma acuminatum tissues. After
dewaxing and rehydration, paraffin sections were treated with a
microwave oven twice for 5 min. Then the sections were
incubated with a mouse anti-HNP-1 (Cell Sciences, Canton,
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Zhao et al.
Table 1 The sequence of the primer
Gene Primer sequence
b-actin Forward: 50 -ACAGAGCCTCGCCTTTGCCG-30
Reverse: 50 -CACCATCACGCCCTGGTGCC-30
HPV11 Forward: 50 -AAA ATTAGCAGACGAGGCATT-30
Reverse: 50 -CCACCTTGTCCACCTCATC-30
HPV6 Forward: 50 -CCAAGGCGCGGTTCATAA-30
Reverse: 50 -GGGTCTGGAGGTTGCAGGT-30
HPV, human papillomavirus.
Mass) monoclonal antibody at 4 °C overnight. Condyloma
acuminatum sections were incubated with the biotinylated
secondary antibody at 37 °C for 30 min, and then stained with
streptavidin–biotin complex method, employing a commercially
available staining kit (Boster, Wuhan, China).
Statistical analysis
All numerical values were recorded as means  standard error
of the mean. Statistical analysis was performed using the SPSS
13.0 statistical software (SPSS Inc., Chicago, IL, USA), and
statistical significance was set a priori at P ≤ 0.05.
Results
Cloning HNP1 cDNA and plasmid construction
Extracting total RNA from human umbilical vein endo-
thelial cells, we obtained a fragment of approximately
285 bp in length by RT-PCR, as shown in Figure 1b. The
purified PCR product was double-digested with BamHI/
XhoI, and HNP1 was then cloned into the pTango vec-
tor. The identity of recombinant plasmid p-HNP1 was
confirmed by double digestion (Fig. 1c) and direct auto-
mated DNA sequencing.
Expression of HNP1 in condyloma acuminatum tissues
To investigate whether HNP1 was expressed in
condyloma acuminatum in vitro transfected by p-HNP1/
(b)
(a)
(c)
Figure 1 Construction and identification of recombinant plasmid p-HNP1. (a) Schematic illustration of construction of
recombinant plasmid p-HNP1. p-HNP1 was digested with restriction enzymes BamHI and XhoI, and cloned into the
expression vector pTango. (b) Identification of HNP1 cloned into expression vector pTango by polymerase chain reaction. (c)
Identification of plasmid p-HNP1 by BamHI/XhoI digestion
ª 2015 The International Society of Dermatology
Therapeutic impact of HNP-1 Pharmacology and therapeutics 3
liposome complexes, condyloma acuminatum tissues were
seeded in six-well plates and then transfected with
p-HNP1/lipo, e-p/lipo, lipo, and untreated group. After
48 hours of incubation, the in vitro expression of the
HNP1 gene in transfected condyloma acuminatum tissues
was confirmed by RT-PCR and Western blot analysis.
The expression of HNP1 in condyloma acuminatum tis-
sues transfected with p-HNP1/lipo could be detected,
whereas there was no expression of HNP1 in condyloma
acuminatum tissues from the untreated, e-p or lipo groups
(Fig. 2a).
Immunohistochemistry was used to detect the HNP1
expression after condyloma acuminatum tissues being
transfected with recombinant HNP1 expression vector.
As shown in Figure 2b, the expression of HNP1 in con-
dyloma acuminatum tissues transfected with p-HNP1/
lipo could be detected in the cytoplasm of the cells,
whereas there was no expression of the HNP1 in con-
dyloma acuminatum tissues from the untreated, e-p or
lipo groups.
HNP1 inducted apoptosis of condyloma acuminatum
tissues in vitro
To explore the role of HNP1 in the anti-condyloma acu-
minatum activity elicited by the recombinant HNP1
expression vector, we seeded the tissue particles in
six-well plates and incubated them with p-HNP1, e-p,
liposome, or medium alone, under appropriate culture
conditions, respectively. As compared with the controls,
some degree of TUNEL-positive nuclei was detected on
HNP1-treated sections, which were characterized by
pyknotic and fragmented nuclei. In contrast, treatment
with liposome, e-p, and blank control groups had no
significant TUNEL-positive nuclei (Fig. 3a).
Cellular apoptosis was further verified by flow cyto-
metric analysis using PI staining at 48 hours after treat-
ments. As shown in Figure 3b, the assay revealed a
markedly higher increase in apoptosis within condyloma
International Journal of Dermatology 2015
4 Pharmacology and therapeutics Therapeutic impact of HNP-1 Zhao et al.

(a)

(b)

Figure 2 Expression of HNP1 in condyloma acuminatum tissues. (a) Identification of the expression of HNP1 in transfected
condyloma acuminatum tissues by reverse transcription–polymerase chain reaction and Western blot analysis. HNP1 expression
was detected in condyloma acuminatum tissues treated with recombinant plasmid p-HNP1; by contrast, no expression of
HNP1 was observed in the control groups. The housekeeper gene GAPDH served as internal control. The production of HNP1
protein was further confirmed by Western blot analysis; the 37 kDa GAPDH served as internal control. (b) Expression of
HNP1 in condyloma acuminatum tissues. e-p, empty plasmid; lipo, lipofectamine; un, untreated

(a)

(b)

Figure 3 Apoptosis assays of HNP1 in condyloma acuminatum tissues. (a) TUNEL staining of condyloma acuminatum tissues
revealed that HNP1 induced a significant enhancement of apoptotic cells in contrast to control therapies. (b) Detection of
apoptosis in condyloma acuminatum tissues by flow cytometry. e-p, empty plasmid; lipo, lipofectamine; un, untreated

acuminatum tissues from the HNP1 treatment group when

compared with the controls: 72.0% (HNP1) versus 29.4%
(normal control), 34.1% (e-p), and 41.2% (liposome).
Human papillomavirus inhibition assay
We used HPV6/11 as target HPV type to screen the abil-
ity of various compounds to inhibit infection of HPV in
condyloma acuminatum. The HPV inhibited level was
detected by real-time PCR in different groups to reveal
the ability of HNP1 to inhibit HPV duplication. The
expression of HPV was significantly lower in the
p-HNP1-treated group as compared to the untreated, e-p,
liposome groups (Fig. 4).
Discussion
Condyloma acuminatum is one of the most commonly
occurring sexually transmitted diseases. Although high-
risk oncogenic HPV (HPV-16, 18, 33, 35, etc.) is also
reported to cause genital warts,13 about 90% of the ac-
uminata are related to HPV strains 6 and 11.14,15 In a
developing country, the infection rate and the incidence
rate of condyloma acuminatum and cervical cancer is
higher than the developed countries. Mostly, clinical
symptoms of genital warts occur as small, painless
bumps, others include itching, burning, and tenderness at
the wart site.16 Various surgical treatments of condyloma

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Zhao et al.
2.5
UN
Relative expression
2 Lipo
e-p
HNP-1
1.5
1
0.5
0 against influenza A virus by binding with surfactant pro-
1 2 3 4 5 6 7
Case number
Figure 4 Detection of quantities of HPV in the condyloma
acuminatum tissues by real-time polymerase chain reaction.
Samples of total RNA isolated from the HNP1-treated group
or e-p, lipofectamine, and untreated groups were used to
determine relative gene expression levels by quantitative real-
time polymerase chain reaction, done in duplicate, for each
gene  SD. e-p, empty plasmid; lipo, lipofectamine; un,
untreated
acuminata are available, including local excision, laser
therapy, cryotherapy, and electrosurgical excision.
However, traditional treatments have some flaws, such as
the unbearable pain induced by undertreatment. Overtreat-
ment can cause scarring or other complications, such as
high rates of recurrence, which demands some new treat-
ment or method to promote the therapeutic effectiveness.
As there is no way of culturing HPV in the laboratory,
the transcription and proliferation of HPV must depend
on the infected host. Therefore, we used the tissue culture
to resemble closely the situation in vivo, engineered a
plasmid DNA expressing HNP1 as a novel therapeutic
agent, and tested its efficacy in condyloma acuminatum
tissue.
To elucidate the anti-condyloma acuminatum mecha-
nism of HNP1, apoptosis analyses were performed by
TUNEL and flow cytometric techniques. Our data
showed that HNP1 resulted in abundant apoptosis in
condyloma acuminatum tissues compared with control
groups. Marisa Nia Madison reported that HNP1 could
decrease cellular infection via membrane pore formation,
leading to apoptosis in response to early Trypanosoma
cruzi infection as an innate immune mechanism.17 In
addition, HNP1 has proinflammatory and apoptotic
effects in human bronchial and alveolar epithelial cells,
which are concentration-dependent and may be associated
with endoplasmic reticulum activity.18 These data and
our findings on the HNP1 provide evidence that HNP1
expression can promote the cell apoptosis of condyloma
acuminatum.
Defensins expressed on the mammal cell surface have
been hypothesized to function as a biochemical barrier
ª 2015 The International Society of Dermatology
Therapeutic impact of HNP-1 Pharmacology and therapeutics 5
against microorganism infection by inhibiting coloniza-
tion of the epithelium by a wide range of pathogenic
microorganisms. In addition to their antibacterial and
antifungal activities, some a-defensins have in vitro activ-
ity against viruses. Hazrati et al.19 examined the HNP’s
abilities and demonstrated that HNPs act at multiple
steps in the HSV life cycle and support the development
of defensins or defensin-like peptides as microbicides.
HNPs also had significant viral neutralizing activity
tein D.20 a-defensins also had anti-HIV-1 function by
stimulating CD8 T-lymphocyte activity.21 As a-defensins
have been found to inhibit HIV-1, HPV, and herpes sim-
plex viruses, they could potentially function as broad-
spectrum topical microbicides to block multiple sexually
transmitted pathogens simultaneously.22,23
Protein–protein interactions of defensins and nonenvel-
oped (HPV and human adenovirus) must be critical for
defensins binding to non-enveloped viruses. For human
adenovirus, structural studies and a chimeric approach
have been used to identify capsid determinants of human
defensin 5 binding.24 Multiple types of papillomaviruses
were sensitive to HNP1-4; HNPs blocked the HPVs entry,
and inhibited nuclear localization of the HPV genome.25
Its inhibition on the replication of nonenveloped virus is
partly explained by the antivirus mechanism of defensins
to the nonenveloped virus.26 The relative quantities of
HPV in each group of condyloma acuminatum tissues
were tested by real-time PCR. Our results indicate that
the HPV was lower in the HNP1 treatment group
than the corresponding control groups. This result also
revealed that HNP1 could limit viral replication.
All these data and our findings on the HNP1 provided
evidence to support our hypothesis that HNP1 expression
directly promotes condyloma acuminatum cell apoptosis
and plays an antivirus role in the condyloma acuminatum
tissue. These studies may provide an alternative strategy
for condyloma acuminatum therapy through the induc-
tion of HNP1 against papillomavirus infection. This may
be important to the further exploration of the role for an-
tivirus therapy.
Acknowledgments
This work was supported by the National 973 Program
of China (2010CB529905) and the National Natural Sci-
ence Foundation of China (grant no. NSFC81071861).
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