Eur J Oral Sci 2019; 1–6 © 2019 Eur J Oral Sci

DOI: 10.1111/eos.12664 European Journal of

Printed in Singapore. All rights reserved
Oral Sciences

Alexandra Aidoukovitch1,2, Sara

Antimicrobial peptide LL-37 and its €lt1, Daniel Nebel1,
Dahl1, Felicia Fa
Daniel Svensson1,3, Ellen
pro-form, hCAP18, in desquamated Tufvesson4, Bengt-Olof Nilsson1
1
Department of Experimental Medical
Science, Lund University, Lund, Sweden;
epithelial cells of human whole saliva 2
3
Folktandvarden Skane, Lund, Sweden;
Department of Women’s and Children’s
Health, Karolinska Institute, Solna, Sweden;
4
Department of Clinical Sciences Lund,
Respiratory Medicine and Allergology, Lund
Aidoukovitch A, Dahl S, F€ alt F, Nebel D, Svensson D, Tufvesson E, Nilsson B-O. University, Lund, Sweden
Antimicrobial peptide LL-37 and its pro-form, hCAP18, in desquamated epithelial
cells of human whole saliva.
Eur J Oral Sci 2019; 00: 1–6. © 2019 Eur J Oral Sci
The antimicrobial peptide LL-37 is active against oral bacteria and has been
demonstrated to be present in human saliva, but its distribution in different frac-
tions of saliva is not known. LL-37 is formed from its intracellular pro-form,
hCAP18, in an extracellular enzymatic reaction catalyzed by proteinase 3 and kal-
likrein 5. Here, we prepared cell-containing and cell-free fractions of unstimulated
human whole saliva by centrifugation after depolymerization of mucins with
dithiothreitol, and measured the levels of hCAP18/LL-37 in these fractions using
ELISA. Cellular expression of hCAP18/LL-37 was determined by western blotting
and immunocytochemistry. The ELISA analyses demonstrated that both cells and
cell-free saliva contained hCAP18/LL-37. Western blot analysis of cell-pellet
homogenates showed a strong band corresponding to hCAP18 at the correct
Bengt-Olof Nilsson, Department of
molecular weight and a weak band corresponding to LL-37. Phase-contrast and
Experimental Medical Science, Lund
light microscopy revealed that the cells consisted of desquamated epithelial cells. University, BMC D12, SE-221 84 Lund,
These cells expressed cytoplasmic immunoreactivity for hCAP18/LL-37. The Sweden
peripheral part of the cytoplasm, corresponding to the plasma membrane, was
E-mail: bengt-olof.nilsson@med.lu.se
particularly rich in hCAP18/LL-37 immunoreactivity. No immunoreactivity was
observed after omission of the primary antibody. We conclude that desquamated
epithelial cells of human whole saliva contain antimicrobial hCAP18/LL-37, sug- Key words: cathelicidin; host defense peptide;
gesting that these cells may take part in the innate immune system by harboring innate immunity; salivary fractions
and releasing these peptides. Accepted for publication September 2019

The human antimicrobial peptide, LL-37, is mainly
produced by epithelial cells and neutrophils and plays
an important role in innate immunity (1, 2). This pep-
tide is released by these cells in its 16-kDa protein pro-
form, hCAP18, which is then cleaved to LL-37 in an
extracellular reaction catalyzed by proteinase 3 and kal-
likrein 5 (3, 4). Indeed, gingival epithelial cells/ker-
atinocytes have been reported to express LL-37 both
in vivo and in vitro (5–7). LL-37 is thought to exert its
antimicrobial activity through permeabilization of the
bacterial cell wall, causing lysis of both gram-positive
and gram-negative bacteria, and also via binding of the
bacterial endotoxin, lipopolysaccharide (LPS) (8–11).
LL-37 has been demonstrated in human whole saliva
from both adults and children (12–15), and can also be
found in saliva isolated from human parotid and sub-
mandibular/sublingual glands, showing that hCAP18/
LL-37 is indeed produced by cells of the major salivary
glands (16). It has been reported that LL-37 possesses
antibacterial effects against many strains of oral bacte-
ria, including Streptococcus mutans and Porphyromonas
gingivalis which are associated with caries and
periodontitis (17–19). Patients with the congenital dis-
ease Kostmann’s syndrome, characterized by neutrope-
nia and frequent and severe infections, have either no
or extremely low levels of LL-37 in plasma and saliva
(20). Interestingly, these patients also suffer from gin-
givitis and periodontitis, suggesting that LL-37 is rele-
vant for the in-vivo situation, although it should be
considered that the low plasma and salivary concentra-
tions of LL-37 are secondary to the lack of neutrophils
(20–23).
Human whole saliva has been shown to contain pro-
teinase 3, suggesting that when hCAP18 is released
from oral cells, it can be cleaved to LL-37 extracellu-
larly in the saliva (24). Whole saliva contains numerous
desquamated epithelial cells and also neutrophils (25,
26). The number of salivary neutrophils in an individ-
ual is regarded to correlate with the degree of gingivitis
(26). Interestingly, desquamated epithelial cells isolated
from human saliva and cultured in keratinocyte growth
medium in vitro respond to the cutaneous bacterial
strain Staphylococcus aureus with enhanced production
of interferon-gamma (IFN-c) and interleukin-12
2 Aidoukovitch et al.
(IL-12), implying that these cells are both viable and
functional (27). We propose that desquamated oral
epithelial cells can harbor and release hCAP18/LL-37
and thus supply saliva with these antimicrobial peptides.
As mentioned before, epithelial cells produce hCAP18
and LL-37, indicating that desquamated oral epithelial
cells indeed contain and release hCAP18/LL-37. Fur-
thermore, desquamated oral epithelial cells are present
at a very high density in whole saliva, suggesting that
they contribute significantly to the content of LL-37 in
saliva upon cellular release of hCAP18 and extracellular
processing of hCAP18 to LL-37 in a reaction catalyzed
by salivary proteinase 3.
The contents and distribution of hCAP18/LL-37 in
the cellular and non-cellular fractions of human saliva
have not been reported before. The aim of the present
study was to determine the concentration and cellular
localization of LL-37 and its precursor, hCAP18, in the
cells and the cell-free compartment of human whole
saliva.
Material and methods
Collection and preparation of saliva
Unstimulated, whole saliva was collected from eight
healthy volunteers of both genders (age 22–65 yr). Saliva
was collected at 9 AM, and the subjects had not eaten or
drank for 1 h before the collection of saliva. Each volun-
teer rinsed the mouth once with 15 ml of tap water, and
then 2 ml of unstimulated saliva was collected from each
individual by spitting for about 1 min in 15 ml plastic
tubes (VWR, Radnor, PA, USA) placed on ice. Informed
consent was obtained from all donors, and the procedure
was approved by the Regional Ethical Review Board in
Lund, Sweden, and performed in accordance with the
World Medical Association Declaration of Helsinki. For
mucin depolymerization, salivary samples were diluted 1:4
in a 0.1% dithiothreitol (DTT; Sigma-Aldrich, St Louis,
MO, USA) solution prepared in PBS, corresponding to a
final concentration of 0.08% DTT. Depolymerization of
mucins with DTT makes the saliva less viscous and allows
salivary cells to be isolated and pelleted. The samples were
incubated on a bench roller for 15 min at 4°C, filtered
through a nylon net filter of 60 µm pore size (Merck Milli-
pore, Burlington, MA, USA), and then centrifuged gently
at 82 g (1,000 r.p.m.) for 5 min at 4°C to separate cells
from the cell-free fraction of saliva. We used slow-speed
centrifugation to keep the cells intact and to avoid cell
lysis. The supernatant (cell-free fraction) contained no
cells, as determined by phase-contrast microscopy (Olym-
pus CKX41; Olympus Europa, Hamburg, Germany).
ELISA
For ELISA analysis, the cell pellet was dissolved in a vol-
ume of 0.08% DTT/PBS equal to that of the supernatant
(cell-free fraction). Both the cell pellet and the supernatant
samples were sonicated twice, for 10 s each time, with 30 s
rest time between the two sonications and stored at 80°C
until analysis. The sonication procedure was performed on
ice at 4°C. The hCAP18/LL-37 protein concentration in
each sample was determined in duplicate using an ELISA
kit (Hycult Biotech, Uden, the Netherlands) according to
the manufacturer’s instructions. The ELISA analysis does
not discriminate between hCAP18 and LL-37. The total
protein concentration in each sample was determined using
a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA,
USA). For each sample, the hCAP18/LL-37 concentration
was normalized to both volume (ng ml 1) and total pro-
tein concentration (ng mg 1 of total protein).
Western blot
The cells of whole saliva were isolated and pelleted as
described above, lysed in an SDS (Sigma-Aldrich) sample
buffer (62.5 mM Tris/HCl, 2% SDS, 10% glycerol, 1 mM
phenylmethanesulfonyl fluoride) and sonicated for 10 s.
The samples were boiled for 5 min, centrifuged at 16,000 g
for 15 min at 4°C, and the supernatants were collected.
The total protein concentration was measured in all sam-
ples to ensure that an equal amount of protein was loaded
into each lane of the gel (Bio-Rad DC protein assay kit;
Bio-Rad). Before loading, samples were supplemented with
2-mercaptoethanol (5%, Sigma-Aldrich) and Bromophenol
Blue (10&, Sigma-Aldrich). An equal amount of protein
(60 µg) was loaded into each lane of Criterion TGX pre-
cast gels (Any kD; Bio-Rad), followed by separation of
proteins by SDS-PAGE and transfer to nitrocellulose
membranes using a Trans-Blot Turbo System (Bio-Rad).
After blocking in 0.5% casein in TRIS-buffered saline for
2 h at room temperature with shaking, membranes were
incubated overnight with a mouse monoclonal hCAP18/
LL-37 antibody diluted 1:2,000 (28) followed by incuba-
tion with horseradish peroxidase-conjugated anti-mouse
IgG diluted 1:5,000 (Cell Signaling Technology, Danvers,
MA, USA) and West Femto chemiluminescence reagent
(Thermo Fisher Scientific, Waltham, MA, USA). Synthe-
sized LL-37 (Bachem, Bubendorf, Switzerland) served as a
positive control. The hCAP18/LL-37 immunoreactive sig-
nal was visualized and analyzed using a LI-COR Odyssey
Fc instrument (LI-COR Biosciences, Lincoln, NE, USA).
Cell morphology and immunocytochemistry
For analysis of cell morphology and immunocytochem-
istry, cells of whole saliva were isolated as described
before, dissolved in PBS, counted in B€ urker chambers,
and then transferred to microscope slides using cytospin
centrifugation (5,000 cells per slide). Cells were fixed in
4% paraformaldehyde for 20 min. Cell morphology of
fresh, unfixed cells was assessed by phase-contrast micro-
scopy using an Olympus CKX41 microscope (Olympus)
before cytospin centrifugation, and of fixed cells (after
transfer of the cells to microscope slides) by light micro-
scopy using an Olympus BX60 microscope (Olympus).
Both microscopes were linked to a digital camera (DP72;
Olympus). For light microscopy, cells were stained with a
differential Kwik Diff kit (modified Giemsa staining) from
Thermo Fisher Scientific, according to the manufacturer’s
instructions.
For immunocytochemistry, the fixed cells were perme-
abilized with 0.2% Triton X-100 for 20 min, and then the
non-specific binding sites were blocked with 2% BSA.
After blocking, the cells were incubated overnight at 4°C
with a mouse monoclonal hCAP18/LL-37 antibody diluted
1:400 (28). For visualization of the hCAP18/LL-37
immunoreactive signal, cells were incubated for 1 h with
secondary anti-mouse Alexa Fluor 488 diluted 1:500

(Invitrogen, Waltham, MA, USA). Cover glasses were
mounted using the Fluoroshield mounting medium con-
taining DAPI (Sigma-Aldrich) in order to stain the nuclei.
The immunoreactive signal and the DAPI fluorescence
were analyzed using an Olympus BX60 fluorescence micro-
scope with appropriate filter settings. For negative con-
trols, the primary antibody was omitted.
Statistics
Values are presented as mean  standard error of the
mean. Statistical significance was calculated using the Stu-
dent´s two-tailed t-test for paired comparisons (cell pellet
vs. cell-free fraction for each individual). Values of
P < 0.05 were considered statistically significant. Each
experiment was repeated at least twice.
Results
hCAP18/LL-37 in cells and cell-free fractions of
whole saliva
In the first set of experiments, we prepared cell-contain-
ing and cell-free fractions of whole saliva from eight
individuals by centrifugation after depolymerization of
mucins with DTT. The contents of hCAP18/LL-37 in
the two fractions of saliva were determined by ELISA
and normalized to the total protein concentration in
each sample. Importantly, the ELISA detects both
hCAP18 and LL-37. The ELISA analysis showed that
the cell pellet and the cell-free fraction contained simi-
lar concentrations (P = 0.4623) of hCAP18/LL-37 (i.e.,
about 1.5 ng of hCAP18/LL-37 per mg of total protein
in both fractions) (Fig. 1A). Also, the ELISA data
expressed per ml of saliva demonstrated no difference
between cells and cell-free fractions (Fig. 1B). Thus,
these data show that hCAP18/LL-37 can be found in
both fractions and seems to be equally distributed
between the cells and the cell-free fraction of whole sal-
iva.
hCAP18 and LL-37 immunoreactive bands in the cell
pellet of whole saliva
In the next experiments, we investigated the expression
of hCAP18 and LL-37 in the cells of whole saliva. The
Fig. 1. The cells and the cell-free fraction of whole saliva contain similar amounts of hCAP18/LL-37. (A, B) The contents of
hCAP18/LL-37 in cells and cell-free saliva were analyzed by ELISA and either normalized to the total protein concentration (A)
or expressed per ml of saliva (B) in each of the eight individuals included in the study. Summarized data are presented as mean
 SEM of eight observations in each group
Salivary cells harbor hCAP18/LL-37 3
saliva was collected from three individuals included in
the study and the cell pellet was isolated as described
above. The cell pellet was subjected to analysis of
hCAP18 and LL-37 expression by western blotting. A
strong immunoreactive band for hCAP18, at the
expected molecular mass of 16 kDa, was detected in
the cells of all three individuals (Fig. 2A). For LL-37, a
weak band was observed at the same position as syn-
thesized LL-37 which acted as a positive control
(Fig. 2A). In order to improve visualization of the LL-
37 immunoreactive band, we overexposed the blot and
highlighted the area of the LL-37 band (Fig. 2B). In
the overexposed blot, the LL-37 band is clearly visible
in the samples of all three individuals (Fig. 2B). Thus,
the cells of whole saliva seem to harbor predominantly
hCAP18 but also LL-37.
Cytoplasmic immunoreactivity for hCAP18/LL-37 in
epithelial cells of whole saliva
The morphology of cells isolated from whole saliva was
assessed using phase-contrast microscopy for fresh and
unfixed cells (Fig. 3) and light microscopy for fixed and
Giemsa-stained cells (Fig. 4). All salivary cells showed
similar morphology, namely large in size (about 50–
100 µm in diameter) and flat, with a large cytoplasm
and a small nucleus, as demonstrated by both phase-
contrast (Fig. 3) and light (Fig. 4) microscopy. These
signs are typical for desquamated and aged epithelial
cells, as described by SOROKA et al. (29). No, or very
few other cell types with different morphology com-
pared with epithelial cells were observed.
Cellular expression of hCAP18/LL-37 was assessed
using immunocytochemistry. Immunoreactivity for
hCAP18/LL-37 was observed in the cytoplasm of the
salivary epithelial cells (Fig. 5A–C). The outer region
of the cytoplasm, corresponding to the plasma mem-
brane, showed particularly strong hCAP18/LL-37
immunoreactivity, whereas weaker hCAP18/LL-37
immunoreactivity was observed in the central parts of
the cytoplasm (Fig. 5A–C). In fact, hCAP18/LL-37
immunoreactivity showed a punctuate staining pattern
in the peripheral region of the cytoplasm that corre-
sponds to the plasma membrane. No immunoreactivity
was observed after omission of the primary hCAP18/
LL-37 antibody (Fig. 5D,E). We used the same
4 Aidoukovitch et al.

Fig. 3. Cells of whole saliva show morphological signs typical

Fig. 2. Western blot analysis of whole saliva cell pellets shows
that the cells express both hCAP18 and LL-37. (A, B) Whole sal-
iva was collected from three individuals (1, 2, 3) and cells were
isolated from the saliva by centrifugation. A strong immunore-
active band, consistent with hCAP18, was observed at the corre-
sponding molecular mass (16 kDa) of hCAP18 in cells from all
three individuals. A weak immunoreactive band, consistent with
LL-37, was observed for all three individuals at the same posi-
tion as synthesized LL-37 (1.1 ng) used as the positive control.
The molecular weight markers (MW) are shown in the far-right
lane. (B) Overexposure of the same blot as in panel A, with the
LL-37-immunoreactive band highlighted
for desquamated epithelial cells assessed by phase-contrast
microscopy. Cells of whole saliva were counted, and cell mor-
phology was instantly analyzed in these fresh and unfixed cells
by phase-contrast microscopy using an Olympus CKX41
phase-contrast microscope. The cells are large in size, flat, and
have a large cytoplasm and small nuclei, typical of desqua-
mated epithelial cells. Some debris, probably cell debris, can
be seen as small spots. The bar represents 100 µm. This image
is representative of cells obtained from saliva of four different
individuals
(14). Based on their findings, DAVIDOPOULOU et al. (14)
conclude that gingival tissues contribute to the secre-
tion of LL-37, which is in line with our findings show-
ing that salivary epithelial cells contain hCAP18/LL-37.

hCAP18/LL-37 antibody for both immunocytochem-

istry and western blot analysis. This antibody detects
both the hCAP18 and the LL-37 immunoreactive bands
in western blotting, as demonstrated in Fig. 2, and has
been thoroughly characterized in many previous studies
(16, 28). Hence, the immunoreactive signal observed in
the cytoplasm of the desquamated oral epithelial cells
may represent either hCAP18 or LL-37, or both.

Discussion
The western blotting and immunocytochemistry results
presented in this study show that desquamated epithe-
lial cells of whole saliva harbor the important human
antimicrobial peptide hCAP18/LL-37. This suggests
that salivary epithelial cells may play a role in innate
immunity through production and release of hCAP18/
LL-37, presumably in association with lysis of these
cells. Saliva is continuously renewed, and desquamated
epithelial cells are swallowed, indicating that LL-37,
produced by these cells, modulates innate immunity not
only in the oral cavity but also in more distal parts of
the gastrointestinal tract. Interestingly, the levels of LL-
37 in unstimulated human whole saliva of edentulous
subjects are much lower than those in healthy subjects
and in patients suffering from chronic periodontitis
Fig. 4. Cells of whole saliva show morphological signs typical
of desquamated fixed and Giemsa-stained epithelial cells
assessed by light microscopy. Cells were transferred to micro-
scope slides using cytospin centrifugation, fixed, and stained
using a modified Giemsa staining kit. Cell morphology was
assessed by light microscopy using an Olympus BX60 fluores-
cence/light microscope with appropriate filter settings. The
cells are large in size, flat, and possess a large cytoplasm and
small nuclei, representative of desquamated epithelial cells.
The bar represents 100 µm. This image is representative of
cells obtained from saliva of three different individuals
 Salivary cells harbor hCAP18/LL-37 5

A B C

D E

Fig. 5. Epithelial cells of whole saliva express hCAP18/LL-37 immunoreactivity. For immunocytochemistry, cells were transferred
to microscope slides using the cytospin technique. (A–C) The same oral epithelial cells stained for both the nuclear marker DAPI
(A, blue) and hCAP18/LL-37 immunoreactivity (B, green). (C) Overlay of DAPI and hCAP18/LL-37 staining. hCAP18/LL-37
immunoreactivity was visualized with an Alexa Fluor 488-conjugated secondary antibody. (D, E) Cells stained for DAPI (D, blue)
and the same cells incubated with Alexa Fluor 488-conjugated secondary antibody without primary hCAP18/LL-37 antibody rep-
resenting a negative control (omission control, E). The bar in panel C represents 100 µm for panels A–E. The fluorescence signal
was assessed using an Olympus BX60 fluorescence microscope with appropriate filter settings. These images are representative of
cells obtained from the saliva of three different individuals

Both isolated human parotid and submandibular/
sublingual saliva contain hCAP18/LL-37, and vascular
neutrophils of human parotid and submandibular
glands express hCAP18/LL-37, indicating that salivary
glands produce hCAP18/LL-37 and deliver it to human
whole saliva (16). However, the results of the present
study demonstrate that even desquamated oral epithe-
lial cells may provide saliva with hCAP18/LL-37. In
addition to the epithelial cells present in whole saliva,
salivary neutrophils should also be considered as possi-
ble producers and suppliers of hCAP18/LL-37 to the
saliva although there seem to be very low levels of
these cells (26). Our western blot data showed that the
cells of whole saliva express a very strong hCAP18
band but only a weak LL-37 band, indicating that most
hCAP18/LL-37 is stored intracellularly as hCAP18.
Although large numbers of epithelial cells are present
in saliva, we cannot exclude that neutrophils also pre-
sent in the salivary samples may contribute to the
hCAP18- and LL-37-immunoreactive bands of the
western blots.
We have demonstrated, by immunocytochemistry,
that salivary epithelial cells express cytoplasmic
hCAP18/LL-37 immunoreactivity; this probably reflects
hCAP18 rather than LL-37 as cleavage of hCAP18 to
LL-37 only occurs extracellularly (3, 4). Human saliva
contains proteinase 3, and thus an important enzyme
that catalyzes the production of LL-37 from hCAP18 is
indeed present in the saliva (24). Interestingly, the cen-
tral part of the cytoplasm showed only a weak
hCAP18/LL-37 signal, whereas the outer, peripheral
region of the cytoplasm expressed strong hCAP18/LL-
37 immunoreactivity organized in a punctuate staining
pattern. This suggests that hCAP18/LL-37 is mainly
confined to a peripheral part of the cytoplasm corre-
sponding to the plasma membrane. The localization of
hCAP18/LL-37 to the peripheral part of the cytoplasm
may indicate that this accumulation of hCAP18/LL-37
represents a readily releasable pool of the peptide. It is
important to note that the hCAP18/LL-37 antibody
used in the present study detects both hCAP18 and
LL-37 immunoreactivity, as clearly demonstrated by
the western blot analysis. Indeed, the western blot data
revealed a weak LL-37 band in the cell homogenate,
which probably represents LL-37 that is cleaved extra-
cellularly from its pro-form, hCAP18. Interestingly,
hCAP18 can be released from cells of the salivary
glands, salivary epithelial cells, and/or salivary neu-
trophils, converted to LL-37 in the extracellular space,
and then the LL-37 may adhere to cell surfaces. Thus,
in whole saliva, the precursor of LL-37, hCAP18, may
originate from glandular tissue cells and/or salivary
epithelial cells and neutrophils.
In summary, we have shown that epithelial cells of
human whole saliva act as reservoirs of the antimicro-
bial hCAP18/LL-37. We suggest that hCAP18 can be
released from salivary epithelial cells and cleaved to
yield antimicrobial LL-37 in the oral cavity, and per-
haps this process may also occur in more distal parts
of the gastrointestinal tract, such as the pharynx and
esophagus. Moreover, we demonstrate that the antimi-
crobial peptide hCAP18/LL-37 is present in both the
6 Aidoukovitch et al.
cells and the cell-free fraction of human whole saliva.
In fact, our data show that the cells and the cell-free
fraction contain equal amounts of hCAP18/LL-37,
both normalized to the total protein concentration and
expressed per ml of saliva.
Acknowledgements – This study was supported by grants from the
Research Funds for Oral Health Related Research by Region
Skane, Folktandv arden Sk
ane, the Royal Physiographic Society,
€ CA, TANNER AC, DUQUE C. Antimicrobial peptides in saliva
the Alfred Osterlund Foundation, the Swedish Dental Society,
and the Crafoord Foundation. The authors wish to thank Profes-
sor Birgitta Agerberth, Karolinska Institute and Karolinska
University Hospital, Stockholm, Sweden, for kindly providing the
hCAP18/LL-37 antibody and Mrs Katarzyna Kawka for excellent
technical assistance.
Conflicts of interest – The authors report no conflicts of interest.
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