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Clinical Chemistry 53: 757-765, 2007. First published February 22, 2007; 10.1373/clinchem.2006.081430
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(Clinical Chemistry. 2007;53:757-765.)
© 2007 American Association for Clinical Chemistry, Inc.

Clinical Immunology

Quantification of Human ß-Defensin-2 and -3 in Body Fluids: Application for Studies of Innate Immunity

Santosh K. Ghosh1, Thomas A. Gerken2, Keith M. Schneider1, Zhimin Feng1, Thomas S. McCormick3 and Aaron Weinberg1,a

1 Department of Biological Sciences, Case School of Dental Medicine;
2 Departments of Pediatrics and Biochemistry, Case Western Reserve University, Cleveland, OH.
3 Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH.

aAddress correspondence to this author at: School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44106-4905. Fax 216-368-0145; e-mail aaron.weinberg{at}case.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Human ß-defensins (hBDs) are epithelial cell-derived antimicrobial and immunoregulatory cationic peptides. Our objective was to establish an analytical tool to quantify inducible hBD-2 and -3 in body fluids.

Methods: We developed sandwich ELISAs using commercially available capture and detection antibodies and determined optimal assay conditions (with 250 mmol/L CaCl2) to overcome masking by endogenous components of body fluids. We used recombinant hBD as calibrators and for recovery testing.

Results: hBD-2 and -3 detection limits were ~75 ng/L and ~3 µg/L, respectively. Mean (SD range) values in saliva samples from healthy donors (n = 60) were 9.5 (1.2–21) µg/L for hBD-2 and 326 (50–931) µg/L for hBD-3. We did not detect hBD-3 in suction blister fluid (BF; n = 10) or bronchoalveolar lavage (BAL; n = 5) from healthy participants. We detected low hBD-2 peptide concentrations in BF and BAL, 0.16 (0.03–0.32) and 0.04 (0–0.049) µg/g total protein, respectively. We observed no correlation of hBD-2 in BF and saliva or BAL and saliva from the same person. In vaginal swabs from healthy women (n = 2), mean hBD-2 and -3 concentrations were 3.42 and 103 µg/g total protein, respectively. Cervicovaginal lavage from the same women contained mean concentrations of 1.46 and 55.5 µg/g total protein.

Conclusion: These ELISA assays can measure inducible hBD peptide concentrations in body fluids by overcoming masking effects of anionic molecules. This approach may therefore be applicable for quantifying these peptides in health and disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antimicrobial peptides have been identified as key components in innate host defense and as important contributors in maintaining health at mucosal barriers. Human ß-defensins, a family of epithelial cell-derived cationic peptides (4–5 kDa), exhibit both antimicrobial and immunomodulatory properties (1)(2)(3). Four human ß-defensins have been identified. Human ß-defensin 1 (hBD-1)1 is constitutively expressed, whereas hBD-2 and -3 are inducible (2)(4)(5). All 3 peptides can be isolated from mucosal sites of the body. An hBD-4 transcript has been described (6), but the peptide has not yet been isolated.

hBDs have demonstrated activity against gram-positive and -negative bacteria, mycobacteria, fungi, and certain enveloped viruses at low micromolar concentrations (7)(8). We recently showed that hBDs have antiretroviral activity by inhibiting HIV-1 infectivity of immunocompetent cells (9)(10). In addition, hBDs can enhance adaptive immunity by acting as adjuvant and chemoattracting T cells, immature dendritic cells, B cells, neutrophils, and macrophages (11)(12)(13). With new information emerging about these pluripotent peptides and their role in mucosal protection, diagnostic tools to quantify hBD-2 and -3 in body fluids and tissues are essential to better associate hBD expression with disease predisposition and progression.

Previous methods of quantifying hBDs in body fluids involved acid extraction followed by slot blot assays (14)(15), semiquantitative Western analysis (16)(17)(18), or RIA(19). ELISA assays have become one of the most popular biomedical methods for quantifying proteins in biological samples because, in addition to their sensitivity and specificity, they are simpler and less costly than other analyses. The main objective of the present study was to develop a sensitive and reproducible analytical tool to measure hBD-2 and hBD-3 peptide concentrations and use it to quantify these peptides in saliva and other body fluids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
sample collection
Individuals who contributed saliva samples were chosen at random, with equal representation of male and female participants and reflective of all age groups, from infants to the elderly. We did not exclude anyone for smoking or taking medications. We collected unstimulated saliva samples from infants and young children by use of sterile disposable pipettes. Adults were asked to expectorate directly into sterile tubes. Saliva samples were then transferred into sterile vials, centrifuged at 10 000g at 4 °C for 20 min, and stored at –70 °C until use. We obtained blister fluids (BF) from the Skin Diseases Research Center (Department of Dermatology, Case School of Medicine and University Hospitals of Cleveland). Blisters were generated by applying suction blister cups onto forearm sites; after ~90–120 min, the vacuum was released and fluid was aspirated from each blister using a 23-gauge needle. Fluids were microcentrifuged (8000g) for 5 min, and the supernatants were frozen at –70 °C. We collected bronchoalveolar lavage (BAL) fluids from healthy participants as described (20). We collected female genital secretions from premenopausal women visiting the Metro Health Medical Center of Case Western Reserve University by 2 different procedures. In the first procedure (vaginal swab), we used sterile dry swabs to collect genital secretions on the endocervix. The swabs were gently applied on the cervical os, and a slight pressure was applied by partly rotating the swabs, without any mucosal trauma. The swab samples were rapidly inserted into 1 mL PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl; pH 7.2) and stored at –70 °C until use. After vaginal swab sampling, we collected cervicovaginal lavage (CVL) by use of a standardized 60-s vaginal washing with 10 mL PBS (pH 7.2) as described (21). Informed consent was obtained for all sample donors.

generation of recombinant hbd-2 and -3
We produced recombinant human BD-2 from the infection of Sf21 cells with baculovirus constructs (a gift from T. Ganz, UCLA) as described (22). We produced recombinant human BD-3 using an hBD-3–His tag fusion construct, generated by PCR and cloned into pET-30c (2). We confirmed the identity and purity of rhBD-2 and -3 by acid urea–polyacrylamide gel electrophoresis migration, N-terminal amino acid sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We have described the use of these peptides in previous work (9).

elisa
We coated 96-well immunoplates (MaxiSorpTM; Nunc) with 50 µL anti–hBD-2 or –hBD-3 antibodies from different vendors (see Table 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue4), diluted to 1 mg/L in 0.05 mol/L carbonate buffer, pH 9.6, 4 °C, for 18 h. Subsequently, we blocked the wells with 200 µL of 1% bovine serum albumin in PBS at room temperature for 10 min. After washing 3 times with 200 µL PBS containing 1 mL/L Tween 20, we incubated 100 µL/well of test samples at room temperature for 60 min. The plates were washed 3 times with PBS containing 1 mL/L Tween 20, and wells were incubated at room temperature with 50 µL secondary antibody diluted to 0.2 mg/L in PBS plus 1 mL/L Tween 20 for 30 min. Plates were washed 3 times with PBS plus 1 mL/L Tween 20 and filled with 50 µL/well streptavidin-peroxidase (Roche Diagnostics; 1:10 000 in PBS plus 1 mL/L Tween 20). Plates were then incubated at room temperature for an additional 30 min, washed 3 times as described above, and incubated with 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (Roche Diagnostics) in the dark at room temperature for 20 min. Absorbance was measured at 415 nm with a microplate reader (Bio-Rad Model 680).

Unless otherwise mentioned, during the validation and standardization process we performed ELISA assays in 1x PBS (pH 7.3) using antibody pairs from Peprotech. To measure defensin concentrations in body fluids, we performed ELISA using 250 mmol/L CaCl2 (final concentration). We quantified hBDs by simultaneous ELISA runs (in 250 mmol/L CaCl2) using recombinant hBDs as calibrators.

mucin isolation and modification
Ovine submaxillary gland mucin (OSM) was purified from frozen glands as described (23), omitting the hydroxyapatite chromatographic step and including protease inhibitors (Chelex 100 and phenyl-methane-sulfonyl-fluoride) in the initial stages of purification. Enzymatic desialylization of OSM (giving a-OSM) was performed as described by Gerken and Dearborn (23) using neuraminidase from Clostridium perfringens (Sigma). [13C]NMR spectra confirmed the purity of the isolated mucin and complete removal of the sialic acid after neuraminidase treatment (23). Note that native OSM contains exclusively the disaccharide {alpha}-NeuNAc 2–6 {alpha}-GalNAc-O-Ser/Thr and is therefore one of the most heavily sialylated mucins.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
evaluation of elisa for detection of hbd-2 and -3 by use of different capture and detection antibodies
We investigated the sensitivity of commercially available antibodies against hBD-2 and -3 (see Table 1 in the online Data Supplement). Affinity-purified polyclonal capture antibodies (antihuman BD-2 and BD-3; Peprotech) and biotinylated anti-hBD detection antibodies (–2 and –3; Peprotech) provided the highest sensitivity in our ELISA formats (data not shown). Using this pair of antibodies, absorbance at 415 nm against the respective calibrators (rhBD-2, 0.075–9.6 µg/L; rhBD-3, 3.0–192 µg/L) exhibited a linear correlation coefficient of r >0.99 for both defensins (Fig. 1 ). The limit of detection (limit of the blank) for the assay, defined as the mean of the buffer control (PBS) + 3SD, was 75 ng/L for hBD-2 and 3 µg/L for hBD-3. We observed no cross-reactivity between the 2 defensin peptides (Fig. 1 ).


Figure 1
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  		Figure 1. ELISA detection of hBD-2 (A) and -3 (B) in serially diluted saliva samples.

Saliva samples were serially diluted as indicated with PBS. For comparison of linearity, serially diluted recombinant hBDs were used. ELISA readings for hBD-3 using hBD-2 calibrators (A) and for hBD-2 using hBD-3 calibrators (B) are also incorporated (black squares).

detection of hbd-2 and -3 in saliva
We and others have detected hBD-1 and hBD-2 in saliva using Western blot analysis (14)(16). Here we demonstrate that Western blot analysis can detect hBD-3 in normal human saliva (Fig. 2 ).


Figure 2
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  		Figure 2. Western blot analysis of hBD-3 in saliva.

Whole saliva (100 µL) was centrifuged (10 000g, 4 °C, 10 min); supernatant was lyophilized, reconstituted in 20 µL of sample buffer, separated on 12% Tris-HCl gels (Bio-Rad) along with recombinant hBD-3 (50 ng; positive control), and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk in TBS containing 0.05% Tween for 1 h, followed by overnight incubation with anti-hBD-3 primary antibody (Peprotech). The membrane was washed, incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad), and visualized with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).

We analyzed samples from 3 healthy individuals. Both hBD-2 and -3 were detectable in all 3 samples. When serially diluted saliva samples were used, however, nonlinearity in ELISA readouts for both hBDs was observed (Fig. 1Up ). Linear correlation coefficient (r) values for both hBD-2 and hBD-3 were far lower than the values obtained with serially diluted recombinant hBDs (r >0.99). This nonlinearity led us to investigate the possibility of interference by other molecules present in saliva.

masking effect of saliva in the detection of hbd-2 and -3
We performed ELISA measurements of pooled (n = 3) serially diluted saliva (1:2 to 1:128), with or without addition of rhBD-2 (20 pg) or rhBD-3 (5 ng). We observed significantly lower detection of added hBDs in the presence of saliva compared with hBDs in PBS, even when the dilution was as low as 1:128. From the calculated percent masking, it became apparent that salivary masking of both hBDs decreased with increased dilution of the saliva, and that the percent masking of hBD-3 was greater than that of hBD-2 (Fig. 3 ). These results indicate that masking agents act differentially on the 2 defensin peptides.


Figure 3
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  		Figure 3. Masking effect of saliva in the detection of hBD-2 (A) and hBD-3 (B) using ELISA.

Serially diluted (1:2 to 1:128 with PBS) saliva was enriched with recombinant hBDs (20 pg hBD-2 or 5 ng hBD-3), and percent masking of the respective hBDs in our ELISA was measured from absorbance readings of saliva enriched with hBDs and hBDs alone. The formula used was % masking = [100% – (ASaliva+rhBDs – ASaliva)/(ArhBDs – Anegative control) x 100], where A is absorbance. The absorbance reading of PBS was the negative control.

role of mucin in masking hbd-2 and -3 elisa signals
Because large anionic glycoproteins, i.e., mucins, are abundantly present in saliva (24) and in other epithelial cell-derived body fluids (25)(26), we conducted ELISAs of fixed concentrations of hBD-2 and -3 in the presence of serially diluted purified salivary mucins to determine the involvement of these molecules in masking hBD detection. Because the sensitivity of the 2 defensin assays varied, we used different fixed amounts of each, 20 pg hBD-2 and 5 ng hBD-3, and added purified mucin (OSM) in wt:wt ratios with the defensins (Fig. 4A ). The results demonstrated concentration-dependent mucin-associated masking of hBD-2 and -3. To establish if anionic sialic acid residues of mucin are involved in the masking effect, we conducted ELISAs of hBDs in the presence of neuraminidase-treated mucin, i.e., asialo-mucin. Fig. 4B shows a decrease of ~25%–30% in masking of both hBDs by asialo-mucin compared with untreated mucin (Fig. 4A ), confirming a role for sialic acid residues in masking hBD signals in the ELISAs.


Figure 4
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  		Figure 4. Masking effect of mucins and asialo-mucins in the detection of hBDs using ELISA.

Serially diluted mucin in PBS (A) or serially diluted asialo-mucin in PBS (B) was enriched with 20 pg rhBD-2 and 5 ng rhBD-3, respectively. The absorbance (A) of the positive control (20 pg for rhBD2; 5 ng for rhBD3) was set at 100% detection. The percent masking by either mucin or asialo-mucin was calculated by the following formula: % masking = [100% – (ArhBDs+mucins – Anegative control)/(ArhBDs – Anegative control) x 100]. The absorbance reading of PBS was the negative control.

optimization of the elisa for detection of hbd-2 and -3 in body fluids
We explored different strategies to overcome the masking. We examined the effect of pH on the detection of hBDs in saliva by assaying a pooled saliva sample (n = 3) using a pH interval of 5.5–7.8 using 100 mmol/L sodium phosphate. Detection of the salivary hBDs was slightly better in acidic pH, but acidic pH alone did not improve hBD detection in saliva to any significant degree (see Fig. 1 in the online Data Supplement). We then examined the effect of monovalent and divalent cations at various ionic strengths on the ELISAs to detect hBDs in saliva. We found that divalent cations (Mg2+, Ca2+) were better than monovalent cations (Na+) (Fig. 5 ) and that 250 mmol/L CaCl2 optimized the detection of both hBD-2 and hBD-3 equally well (see Fig. 2 in the online Data Supplement). We further compared the ELISA signals from neuraminidase-pretreated saliva in PBS to the ELISA signals from saliva in 250 mmol/L CaCl2 and observed that 250 mmol/L CaCl2 was best at enhancing the hBD-2 and hBD-3 signals.


Figure 5
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  		Figure 5. (A,B), Effect of inorganic salts on ELISA readouts.

A saliva sample was mixed (1:1) with respective inorganic salts (final concentration 100 mmol/L) as shown, and 100 µL from each condition was used to screen hBD-2 and -3 concentrations by ELISA. Triplicate assays were performed and results are expressed as the mean and SE. (C), ELISA readouts for hBD-2 and hBD-3 preincubated with serially diluted mucin and assayed in the presence of 250 mmol/L CaCl2. HBDs and mucin were preincubated in 250 mmol/L CaCl2, followed by the ELISA assay. The y axis represents the percent of ELISA readout obtained when comparing absorbance for hBDs in the presence or absence of mucin.

Indeed, when purified mucin was incubated with the respective hBDs in the presence of 250 mmol/L CaCl2, we were able to detect hBD-2 and -3 to virtually 100% (Fig. 5CUp ). We therefore performed subsequent ELISA assays for detection of hBDs in body fluids in the presence of 250 mmol/L CaCl2.

intra- and interassay precision
Using the hBD-2 calibrator (1 µg/L) and pooled saliva (8.2 µg/L hBD-2), the intraassay CVs were 4.8% and 6.06%, respectively (n = 20), and interassay CVs were 5.81% and 7.63%, respectively (n = 8). With an hBD-3 calibrator (50 µg/L) and the pooled saliva (627 µg/L hBD-3), the intraassay CVs were 4.6% and 6.7%, respectively (n = 20), and the interassay CVs were 5.31% and 8.12%, respectively.

analytical recovery of the calibrator
Recoveries of exogenously added recombinant hBD-2 (50, 100, and 200 ng/L) from saliva, BF, BAL, and CVL samples ranged from 82%–107%, 88%–105%, 81%–107%, and 84%–103%, respectively. The percentage recoveries of exogenously added recombinant hBD-3 (2.5, 5, and 10 µg/L) from saliva, BF, BAL, and CVL samples ranged from 89% to 104%, 83% to 99%, 86% to 109%, and 87% to 107%, respectively.

measurement of hbd-2 and -3 concentrations in saliva from healthy individuals
Saliva from 60 healthy individuals was analyzed for the presence of hBD-2 and -3. Concentrations of hBD-2 ranged from 1.2 to 21.1 µg/L (mean, 9.48; median, 3.28), whereas concentrations of hBD-3 ranged from 50 to 931 µg/L (mean, 325.77; median, 253) (Fig. 6A ).


Figure 6
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  		Figure 6. (A), Box-plot representation of salivary hBD-2 and hBD-3 peptide concentrations in healthy participants (n = 60).

Each saliva sample was mixed (1:1) with CaCl2 (final concentration 250 mmol/L), and 100 µL from each sample was assayed for the presence of the respective hBD. Each sample was run in duplicate. Results were obtained by running hBD-2 and hBD-3 calibrators with each assay, respectively. *Outliers. (B), ELISA measurements of hBD-2 concentrations in BFs (n = 10) and matched saliva from healthy donors. BF (75 µL) from each sample was mixed with 25 µL CaCl2 (final concentration 250 mmol/L) and assayed for hBD-2. C, Determination of hBD-2 in BAL (n = 5) samples and matched saliva from healthy donors. BAL samples were lyophilized, dialyzed, and reconstituted in 250 mmol/L CaCl2, and 100 µL from each sample was assayed for hBD-2. Salivary hBD-2 was measured as described (A). Results are calculated as the ratio of hBD-2 peptide compared with total protein (measured by Bio-Rad DC Protein Assay) per sample.

identification of hbd concentrations in blister fluids, bal, vaginal swabs, and cvl from healthy individuals
We analyzed BF samples from healthy participants (n = 10) by ELISA for the presence of both hBD-2 and hBD-3 peptides. Although we could not detect hBD-3 in the BF samples (limit of detection of assay, 3.0 µg/L), we found hBD-2 in all the samples (30–320 ng/g total protein). The hBD-2 concentrations (in µg/g total BF proteins) in BF were then compared with salivary hBD-2 concentrations (in µg/g total salivary proteins) from corresponding samples (Fig. 6BUp ). BF concentrations of hBD-2 did not correlate with the salivary concentrations of hBD-2 peptides. Similar analysis of the hBDs in BAL from healthy participants (n = 5) showed the absence of hBD-3 and low concentrations of hBD-2 (0–49 ng/g total proteins). We also compared the concentration of hBD-2 in BAL with that in corresponding saliva samples (Fig. 6CUp ). BAL concentrations of hBD-2 also did not correlate with the salivary hBD-2 peptide concentrations. Unlike BF and BAL, we could detect hBD-3, along with hBD-2, in CVL and vaginal swabs from healthy women (n = 2; mean hBD-2 in CVL, 1.42 µg/g total proteins; in vaginal swab, 3.42 µg/g total proteins; mean hBD-3 in CVL, 55 µg/g total proteins; in vaginal swab, 103 µg/g total proteins).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed ELISAs for quantifying hBD-2 and hBD-3 in body fluid samples. During assay development, it became clear that salivary components partially mask the detection of both hBD-2 and -3, with the effect being greater for hBD-3. This difference may be due to stronger electrostatic interactions between negatively charged moieties and hBD-3, since hBD-3 is more positively charged (+11) than hBD-2 (+6) (27). Moreover, previous studies with hBD-1 (+4 net charge) (28) demonstrated that mucins also mask the detection of hBD-1 in slot-blot assays but not in Western blots (14). We therefore surmised that negatively charged mucins, found in mg% (mg/100 mL) quantities in saliva (24), are masking hBD-3 more than hBD-2 due to the greater net positive charge of this peptide. Moreover, hBD-3 includes a greater percentage of total surface area available for electrostatic interactions compared with hBD-2 (28). In addition, our calculation of polar surface area using GetArea 1.1 software (29) was greater for hBD-3 (40% of total surface area) than hBD-2 (30% of total surface area), which further supports the greater masking of hBD-3 vs hBD-2.

To assess further the contribution of a mucin-dependent electrostatic interaction in masking hBD detection, we found that asialo-mucin significantly diminished the masking effect. The sialic acid residue is generally located on the terminal position of carbohydrate chains of glycoproteins, and sialic acid-mediated antigen masking has been described (30). Moreover, sialic acid–mediated antigen masking by steric hindrance has been reported in the 3-fucosyl-N-acetyl-lactosamine antigen (31). Thus, in addition to the possible role of electrostatic and polar interaction of mucins with defensins, the role of the sialic acid residue itself in masking the ELISA signal cannot be excluded.

Mucin forms a viscoelastic gel that coats epithelial surfaces and is affected by pH (32) and ion content (33). We observed that pH changes and ionic strength affect the recovery of the ELISA signal for both hBD-2 and -3 in saliva. Recovery of signal is better in acidic pH and best in the presence of 250 mmol/L CaCl2. Moreover, the persistence of hBD masking in the presence of asialo-mucins, albeit at reduced levels, supports possible macromolecular organization or viscoelastic properties of mucin in contributing to hBD masking. We cannot rule out the possibility that other negatively charged salivary components, aside from mucins, are also involved. These could include, but are not limited to, calprotectin present in mg/L quantities (34). Nevertheless, recovery of recombinant hBDs in the presence of inorganic salts supports the involvement of electrostatic interactions, as we obtained the best recovery using divalent cations rather than monovalent cations. Electrostatic interactions alone cannot fully explain recovery of the hBD signal, since asialylation of saliva or use of other divalent cations other than calcium does not completely unmask the hBD signal in our ELISA—near-complete recovery of signal was obtained only with calcium. This unique calcium-dependent phenomenon could be due to calcium-induced changes in the intrinsic viscosity of saliva (35) and macromolecular contraction/folding of mucins (36), or calcium could be shielding the interactions between hBDs, sialic acid residues, and other negatively charged moieties.

The almost 40-fold difference between hBD-2 and hBD-3 concentrations in saliva from healthy oral cavities is consistent with our observations that hBD-3 is more highly expressed than hBD-2 in healthy oral epithelium (data not shown). The concentrations of salivary hBD-3 that we found are in line with those reported by others using extraction and slot blot procedures (15). The large standard deviations in our data and those reported by Tao et al. (15) suggest large interindividual variability in hBD-2 and -3 expression. This suggestion is consistent with findings that hBD genes are polymorphic in copy numbers and that high copy numbers correlate with high levels of mRNA (37). Finally, being able to quantify salivary hBD-2 and hBD-3, which primarily reflect release from the oral epithelium where they would most likely be expressed at high concentrations, could provide a means to determine the degree of fitness of the mucosal epithelium toward microbial challenges.

Results for normal BFs also demonstrated the presence of hBD-2, albeit at low concentrations. Ortega et al. (38) demonstrated the absence of hBD-2 in burned blister fluids. The presence of hBD-2 peptide in normal blister (this report) and its absence in BF from burned sites (38) support reverse-transcription PCR results showing reduced or no expression of hBD-2 mRNA in burned skin compared with unburned skin (39). In contrast to hBD-2, we could not detect hBD-3 in BFs, perhaps because of the low sensitivity of our ELISA for hBD-3 compared with hBD-2.

Previous semiquantitative Western analysis showed the presence of hBD-2 in BAL samples of patients with cystic fibrosis (~15 µg/L) or bronchiolitis obliterans (~1.3 µg/L), but not in healthy individuals (17)(18). Our ELISA method, however, can detect the presence of hBD-2 (~0.4 ng/L) in BAL samples from healthy individuals, a clear advantage over semiquantitative Western blots. Moreover, our ELISA is able to detect both hBD-2 and hBD-3 in cervicovaginal lavage fluids (1.46 and 55.5 µg/g total proteins) and vaginal swabs (3.42 and 103 µg/g total proteins) from healthy women. This is the first reported documentation of these peptides in female genital tract secretions.

With new and exciting information promoting hBDs as important agents in mucosal defense, these assays should help to determine if individuals expressing low amounts of these peptides may be inherently more susceptible to mucosal infections. Moreover, do infectious diseases have an affect on mucosal hBD peptide concentrations? Interestingly, Sun et al. (40) showed diminished hBD-2 peptide expression in HIV-positive oral mucosa compared with healthy controls. The ability to now measure inducible hBD peptide concentrations in body fluids, in a manner conducive for screening samples from multiple body sites, sets the stage for epidemiological assessment of the role of hBDs in numerous infectious diseases.


   Acknowledgments
 
We thank Dr. Richard Silver, Division of Pulmonary Critical Care, and Department of Medicine, University Hospitals of Cleveland and Case Western Reserve University, for providing bronchoalveolar lavage samples, and Dr. Richard Beigi, Department of Obstetrics and Gynecology, Metro Health Medical Center, Cleveland, for providing cervicovaginal lavage and vaginal swab samples. This work was supported by National Institutes of Health grants RO1DE16334 (A.W.), RO1 DE17334 (A.W.), RO1DE15510 (A.W.), R01CA78834 (T.A.G.), and P30AR39750 (T.S.M.).


   Footnotes
 
1 Nonstandard abbreviations: hBD, human ß -defensin; BF, blister fluid; BAL, bronchoalveolar lavage; CVL, cervicovaginal lavage; OSM, ovine submaxillary gland mucin.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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D. E. Dietrich, X. Xiao, D. V. Dawson, M. Belanger, H. Xie, A. Progulske-Fox, and K. A. Brogden
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