Reduced induction of human ß-defensins is involved in the pathological mechanism of cutaneous adverse effects caused by epidermal growth factor receptor monoclonal antibodies.

Epidermal growth factor receptor inhibitors (EGFRIs) frequently cause cutaneous adverse effects such as papulopustular eruptions. However, the mechanism of the reactions remains unclear. To assess the pathological mechanism of cutaneous adverse reactions caused by EGFRIs, we investigated whether EGFRIs have an influence on the innate immune response of the skin. Levels of human ß-defensins (hBDs), which serve as the first line of defence against infection by pathogenic microorganisms, in the stratum corneum samples of patients treated with EGFR. monoclonal antibodies were measured before and after starting therapy. There were no obvious trends in hBD production in patients without eruptions, whereas a significant decrease in hBD1 and hBD3 production and a nonsignficant decrease in hBD2 production were observed in patients who developed papulopustular eruptions. Our results suggest that a reduction in hBD contributes to the increased incidence of papulopustular eruptions.

Water-based extracts of Zizania latifolia inhibit Staphylococcus aureus infection through the induction of human beta-defensin 2 expression in HaCaT cells.

Zizania latifolia is a perennial herb belonging to the family Gramineae that has been used as a health food in Asian countries. In this study, we investigated the antimicrobial effect of Z. latifolia, which increased human beta-defensin 2 (hBD2) expression in HaCaT cells. hBD2 expression was further increased in cells treated with Z. latifolia extracts and subsequently infected with Staphylococcus aureus. Inversely, S. aureus infection decreased after treatment. The induction of hBD2 in HaCaT cells was mediated by the Toll-like receptor 2 (TLR2) signaling pathway, including the activation of extracellular signal-regulated kinase (ERK) and activator protein 1 (AP-1). Further study using siRNA revealed that hBD2 played an important role in the inhibition of S. aureus infection in HaCaT cells. Our data suggest that Z. latifolia extracts can be used as an antimicrobial ingredient for skin treatment formulas.

Effect of cytostatic agents on expression levels of human beta-defensins-1-4 in A431 and MCF-7 cell lines.

The aim of the study was to analyze an effect of cytostatic agents of different mechanism of action on expression levels of human beta-defensins-1-4 (hBD-1-4) in cultured human cancer cell lines. MATERIALS AND METHODS: Expression levels of hBD-1-4 mRNA were assessed using qPCR in human epidermoid carcinoma A431 cells and human breast adenocarcinoma MCF7 cells treated with cisplatin, methotrexate, doxorubicin or vincristine at the IC20 concentrations. RESULTS: The cytostatic agents with different mechanisms of action affected differently expression of hBDs, dependent on the cell line. Mostly, cytostatic agents suppressed significantly expression of hBDs. In contrast, vincristine caused significant up-regulation of hBD-1 (12 fold, p < 0.05) and hBD-4 (2 fold, p < 0.05) in MCF7, and doxorubicin significantly enhanced expression of hBD-3 (2 fold, p < 0.05) and hBD-4 (> 10 fold, p < 0.05) in A431 cells. CONCLUSION: The results of this pilot study show that expression levels of hBD-1-4 may be altered upon treatment with cytostatic agents depending on nature of cells.

Flagellin Induces ß-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli.

Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human ß-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8.

Influence of Th2 Cytokines on the Cornified Envelope, Tight Junction Proteins, and ß-Defensins in Filaggrin-Deficient Skin Equivalents.

Atopic dermatitis is a chronic skin condition with complex etiology. It is characterized by skin barrier defects and T helper type 2 (Th2)-polarized inflammation. Although mutations in the filaggrin gene are known to be prominent genetic risk factors for the development of atopic dermatitis, the interdependency between these and an altered cytokine milieu is not fully understood. In this study, we evaluated the direct effects of filaggrin deficiency on the cornified envelope, tight junction proteins, and innate immune response, and report the effects of Th2 cytokines in normal and filaggrin-deficient skin equivalents. Supplementation with IL-4 and IL-13 led to distinct histologic changes and significantly increased skin surface pH, both of which were enhanced in filaggrin knockdown skin equivalents. We detected a compensatory up-regulation of involucrin and occludin in filaggrin-deficient skin that was dramatically disturbed when simultaneous inflammation occurred. Furthermore, we found that a lack of filaggrin triggered an up-regulation of human ?-defensin 2 via an unknown mechanism, which was abolished by Th2 cytokine supplementation. Taken together, these results indicate that defects in the epidermal barrier, skin permeability, and cutaneous innate immune response are not primarily linked to filaggrin deficiency but are rather secondarily induced by Th2 inflammation.

Ellagic acid modulates the expression of oral innate immune mediators: potential role in mucosal protection.

BACKGROUND: Ellagic acid (EA) found in various fruits such as pomegranates, blackberries, raspberries, strawberries, and walnuts has different pharmacological functions including antioxidant, antitumor, antiallergic, anti-inflammatory, antibacterial, and antiviral activities. It is not known, however, if EA could enhance mucosal innate immunity. Our goal was to determine the effects of EA on the expression of innate immune mediators produced by oral epithelial cells. METHODS: Culture of primary human gingival epithelial cells (HGEs) was performed in duplicate, and after the primary HGEs had been treated with EA at a concentration ranging from 12.5 to 100 µM for 18 h the cells and supernatants were harvested. The expression of innate immune mediators including human ß-defensin 2 (hBD2), secretory leukocyte protease inhibitor (SLPI), and various cytokines and chemokines was measured at both transcriptional and translational levels by using quantitative real-time PCR, ELISA, and Luminex assay. RESULTS: In the presence of EA, the expression of hBD2-and SLPI mRNA was 3.7-folds and 2.6-folds greater than untreated controls, respectively, and consistent with their secreted protein levels. For cytokines and chemokines, increased expression of RANTES, IL-2, and IL-1ß was found in response to EA. In contrast, EA decreased the expression of IL-6, IL-8, and TNF-α. CONCLUSIONS: This study demonstrated that oral innate immunity is affected by EA found in fruits. Thus, it may play some roles in mucosal innate immunity. The potential of EA for modulating the innate immune mediators may lead to developing a new topical agent to treat and/or prevent immune-mediated oral diseases.

mRNA expression and localization of LPS-induced ß-defensin isoforms in rat salivary glands.

ß-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of ß-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat ß-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. ß-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. ß-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce ß-defensins.

Green tea extract and its major constituent, epigallocatechin-3-gallate, induce epithelial beta-defensin secretion and prevent beta-defensin degradation by Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Antimicrobial peptides, such as beta-defensins, secreted by gingival epithelial cells, are thought to play a major role in preventing periodontal diseases. In the present study, we investigated the ability of green tea polyphenols to induce human beta-defensin (hBD) secretion in gingival epithelial cells and to protect hBDs from proteolytic degradation by Porphyromonas gingivalis. MATERIAL AND METHODS: Gingival epithelial cells were treated with various amounts (25-200 µg/mL) of green tea extract or epigallocatechin-3-gallate (EGCG). The secretion of hBD1 and hBD2 was measured using ELISAs, and gene expression was quantified by real-time PCR. The treatments were also carried out in the presence of specific kinase inhibitors to identify the signaling pathways involved in hBD secretion. The ability of green tea extract and EGCG to prevent hBD degradation by proteases of P. gingivalis present in a bacterial culture supernatant was evaluated by ELISA. RESULTS: The secretion of hBD1 and hBD2 was up-regulated, in a dose-dependent manner, following the stimulation of gingival epithelial cells with a green tea extract or EGCG. Expression of the hBD gene in gingival epithelial cells treated with green tea polyphenols was also increased. EGCG-induced secretion of hBD1 and hBD2 appeared to involve extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Lastly, green tea extract and EGCG prevented the degradation of recombinant hBD1 and hBD2 by a culture supernatant of P. gingivalis. CONCLUSION: Green tea extract and EGCG, through their ability to induce hBD secretion by epithelial cells and to protect hBDs from proteolytic degradation by P. gingivalis, have the potential to strengthen the epithelial antimicrobial barrier. Future clinical studies will indicate whether these polyphenols represent a valuable therapeutic agent for treating/preventing periodontal diseases.

Human ß-defensin 2 may inhibit internalisation of bacillus Calmette-Guérin (BCG) in bladder cancer cells.

OBJECTIVE: To investigate whether secretion of human ß-defensin 2 (HBD-2) is induced by bacillus Calmette-Guérin (BCG) and to determine whether HBD-2 affects BCG internalisation in bladder cancer cells. MATERIALS AND METHODS: Reverse transcription-polymerase chain reaction analysis was used to determine whether HBD-2 mRNA increases after incubation with BCG. HBD-2 proteins in 5637 and T24 human bladder cancer cell lines were assayed by enzyme-linked immunosorbent assay. The internalisation rate was evaluated by double immunofluorescence assay and confocal microscopy to test the optimal dose of HBD-2 for BCG internalisation. We also investigated the difference in internalisation rates and cell viability between recombinant HBD-2 protein, anti-HBD-2 antibody, and HBD-2 plus anti-HBD-2 antibody pretreatments. RESULTS: BCG induced HBD-2 mRNA expression and HBD-2 production dose and time-dependently in bladder cancer cells and affected BCG internalisation. Pretreatment with recombinant HBD-2 protein lowered internalisation of BCG dose-dependently. Moreover, anti-HBD-2 antibody prevented the effect of HBD-2 on BCG internalisation in bladder cancer cells. The internalisation rate of BCG pretreated with anti-HBD-2 antibody was higher than that in the control in 5637 (P < 0.01) and T24 cells (P < 0.05). The BCG internalisation rate in cells pretreated with anti-HBD-2 antibody plus recombinant HBD-2 protein was higher than that in the control in 5637 (P < 0.01) and T24 cells (P < 0.05). Mycobacterium bovis BCG decreased bladder cancer cell viability, and anti-HBD-2 antibody prevented the inhibitory role of HBD-2 on the anti-proliferative effects of M. bovis BCG in bladder cancer cells CONCLUSION: Bladder cancer cells produce HBD-2 when they are infected by BCG to defend themselves against BCG internalisation, which plays an important role during the initiation and propagation of the immunotherapeutic response in bladder cancer cells.

Keratinocyte growth factor phage model peptides can promote human oral mucosal epithelial cell proliferation.

OBJECTIVE: The objective of this study was to find keratinocyte growth factor (KGF) mimic peptides by a phage display library screening and to analyze their effects on proliferation of human oral mucosal epithelial cells (HOMECs). STUDY DESIGN: A phage display library was screened by anti-KGF antibody. ELISA was performed to select monoclonal phages with higher binding activity. The promotion of the phage model peptides on HOMEC proliferation were analyzed by MTT and their cell affinities were confirmed by immunofluorescence assay. Their effect on KGFR, human beta-defensin 3, c-Fos, and c-Jun in HOMEC were analyzed by quantitative real-time PCR. RESULTS: Two model peptides with higher affinity with HOMEC were found to have promotive activity on cell proliferation, similar to that of KGF. These 2 model peptides have no KGF-like promotion effect on the expression of c-Fos and c-Jun. CONCLUSIONS: The 2 phage model peptides can promote the proliferation of HOMEC in vitro without tumorigenic effects, which suggests their possible usages in oral mucosal wound healing.

HBD-1 and hBD-2 expression in HaCaT keratinocytes stimulated with nicotine.

OBJECTIVE: The impact of nicotine on the local innate immune response in the oral cavity is unclear. The aim of the present study was to evaluate the possible effects of nicotine on the gene expression of human beta-defensin-1 and -2 in HaCaT keratinocytes. MATERIALS AND METHODS: HaCaTs were cultured in six-well plates in Dulbecco's minimum essential medium (DMEM) supplemented with 10% FBS at a density of ×10(6). Cells were pretreated with 10 µg/ml nicotine (12 h), and then stimulated with 50 ng/ml TNF-α (during the following 12 h); or were pretreated with 50 ng/ml TNF-α, and then stimulated with 10 µg/ml nicotine; or were not pretreated but only stimulated with either nicotine or TNF-α, or a combination of both. Total RNA was extracted and analysed by real-time RT-PCR for human beta-defensins-1-, -2-, and interleukins IL-1ß- and IL-6-, as well as GAPDH-mRNA. The obtained data were analysed using Tukey's B multiple comparison test for post hoc analysis. RESULTS: Pretreatment with nicotine caused a significant 2.5-fold inhibition of TNF-α-stimulated hBD-2 mRNA expression compared to TNF-α alone (p = 0.004). Simultaneous treatment with TNF-α and nicotine caused a significant 2-fold inhibition of hBD-2 mRNA compared to TNF-α alone (p = 0.041). CONCLUSION: The present results suggest that the pre-exposition to nicotine seems to reduce a stimulating effect of TNF-α on the gene expression of hBD-2.

Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers.

BACKGROUND: Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet. FINDINGS: 26 women aged 18-21 (median 19) years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing Bifidobacterium lactis Bb12® (109 in 200 ml) for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184) and returned to normal after cessation of probiotic yoghurt intake. CONCLUSIONS: Bifidobacterium lactis Bb12® increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.

Human beta-defensin-3 (hBD-3) upregulated by LPS via epidermal growth factor receptor (EGFR) signaling pathways to enhance lymphatic invasion of oral squamous cell carcinoma.

OBJECTIVE: In this study, the hypothesis that hBD-3 is upregulated by LPS via epidermal growth factor receptor (EGFR) signaling pathways to enhance metastasis in oral squamous cell carcinoma (OSCC) was tested. STUDY DESIGN: hBD-3 expression in human tissue specimens was evaluated by RT-qPCR and immunohistochemical staining. The presence of hBD-3 peptide in the culture supernatants of each type of treated cells was evaluated by enzyme-linked immunosorbent assay. The chemotaxis response to LPS or hBD-3 protein of SCC-25 cells or siRNA-hBD-3 transfected cells were also measured by chemotaxis assay. Paired, 2-tailed Student t test and analysis of variance was used to assess the statistical significance between 2 groups or many groups. RESULTS: hBD-3 is highly expressed and associated with lymphatic invasion of OSCC. hBD-3 expression and EGFR phosphorylation were markedly upregulated when SCC-25 cells were treated with LPS. When SCC-25 cells were preincubated with EGFR inhibitor or TLR4-neutralizing Ab before LPS stimulation, a decrease in the expression of hBD-3 was observed. hBD-3 markedly enhanced cancer metastasis, and the chemotaxis response to LPS of SCC-25 cells was partly blocked by siRNA target hBD-3. CONCLUSION: These findings indicate that hBD-3 is upregulated by LPS via EGFR signaling pathways to enhance lymphatic invasion of OSCC.

Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Human gingival epithelial cells (HGECs) are continually exposed to oral bacteria and to other harmful agents. Their responses to stimuli are critical in maintaining periodontal homeostasis. The aim of this study was to investigate the modulating effect of cigarette smoke extract (CSE) on the innate immune responses of HGECs. MATERIAL AND METHODS: Toll-like receptor (TLR) expression of HGECs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of CSE or nicotine on the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) and the pro-inflammatory cytokine interleukin (IL)-8 in stimulated HGEC cultures was evaluated by RT-PCR and enzyme-linked immunosorbent assay. RESULTS: The HGECs expressed mRNA of TLRs 1, 2, 3, 5, 6, 9, 10, and minimally of TLR4, but not of TLRs 7 or 8. Stimulation of HGECs with highly purified TLR2, 3 or 5 ligands led to expression of hBD-2 and of IL-8. Enhancement of hBD-2 and IL-8 was observed in HGECs after combined stimulation with Porphyromonas gingivalis lipopolysaccharide (TLR2 ligand) and tumour necrosis factor-alpha, compared with stimulation using either agent alone. After CSE exposure, hBD-2 expression was markedly reduced in stimulated HGEC cultures, whereas IL-8 expression was markedly increased. These effects were also observed, but were markedly attenuated, upon nicotine treatment. CONCLUSION: Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

Role of human beta-defensin-2 during tumor necrosis factor-alpha/NF-kappaB-mediated innate antiviral response against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) constitutes a highly pathogenic virus that infects lung epithelial cells to cause a wide spectrum of respiratory diseases. Our recent studies have revealed the existence of an interferon-alpha/beta-independent, innate antiviral response against RSV that was dependent on activation of NF-kappaB. We demonstrated that NF-kappaB inducing pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) confers potent antiviral function against RSV in an NF-kappaB-dependent fashion, independent of interferon-alpha/beta. During our efforts to study this pathway, we identified HBD2 (human beta-defensin-2), a soluble secreted cationic protein as an antiviral factor induced during NF-kappaB-dependent innate antiviral activity in human lung epithelial cells. Our results demonstrated that HBD2 is induced by TNF and RSV in an NF-kappaB-dependent manner. Induction of HBD2 in infected cells was mediated by the paracrine/autocrine action of TNF produced upon RSV infection. HBD2 plays a critical role during host defense, because purified HBD2 drastically inhibited RSV infection. We also show that the antiviral mechanism of HBD2 involves blocking of viral cellular entry possibly because of destabilization/disintegration of the viral envelope. The important role of HBD2 in the innate response was also evident from loss of antiviral activity of TNF upon HBD2 silencing by short interfering RNA. The in vivo physiological relevance of HBD2 in host defense was apparent from induction of murine beta-defensin-4 (murine counterpart of HBD2) in lung tissues of RSV-infected mice. Thus, HBD2 functions as an antiviral molecule during NF-kappaB-dependent innate antiviral immunity mediated by the autocrine/paracrine action of TNF.

Effects of AV119, a natural sugar from avocado, on Malassezia furfur invasiveness and on the expression of HBD-2 and cytokines in human keratinocytes.

AV119 is a patented blend of two sugars from avocado that can induce human beta-defensin-2 production by normal human keratinocytes. In this study, we analysed the effect of AV119 on growth and invasiveness of Malassezia furfur, a dimorphic, lipid-dependent yeast that is part of the normal human cutaneous commensal flora. The ability to modulate the expression of the proinflammatory and immunomodulatory cytokines in normal human keratinocytes was also investigated. Microbiological assay demonstrated that this sugar induced the aggregation of yeast cells and inhibited the invasiveness of M. furfur, without affecting its growth. Real-time PCR analysis demonstrated that AV119 was able to modulate the HBD-2 response in treated keratinocytes, reaching a maximum after 48-h treatment, and to induce the recovery of a satisfactory proinflammatory response in human keratinocytes. As AV119 can induce aggregation of yeast cells, thus inhibiting their penetration into the keratinocytes, the sugar could be used in the preparation of cosmetics or pharmacological drugs to inhibit colonization of the skin by pathogenic strains of M. furfur.

Changes in vaginal morphology, steroid receptor and natural antimicrobial content following treatment with low-dose mifepristone.

BACKGROUND: We have previously shown that the antigestagen mifepristone is contraceptive when given in a daily dose of 5 mg, po. Epidemiological studies suggest that gestagen-only contraceptives may increase the risk of transmission of human immunodeficiency virus (HIV) due to effects on the vaginal defenses to infection. We investigate the effects of mifepristone on vaginal thickness, steroid receptor and natural antimicrobial content and pharmacokinetics of mifepristone. METHODS: In a pilot study, eight women were given mifepristone 5 mg/day for an average of 33 days. Ovarian function was assessed by measurement of estradiol and progesterone in blood and their metabolites in urine and by serial ultrasound of their ovaries. Vaginal biopsies were collected before (late proliferative) and after taking mifepristone. RESULTS: All subjects showed a similar pattern of descending serum concentrations of mifepristone. The elimination phase half-life was 18+/-5.1 h (mean+/-SD). Mean Cmax measured at 1 h was 641.7 nmol/L (range, 502-740 nmol/L). All eight women reported amenorrhea for the duration of treatment and seven of eight women showed biochemical and ultrasound evidence of anovulation. There was no significant change in vaginal thickness following treatment [342+/-40 microm pretreatment, 303+/-69 microm posttreatment (mean+/-SEM); p>.05]. Estrogen (ERalpha, ERbeta) and androgen receptor were expressed in both vaginal epithelium and subepithelial stroma, whereas progesterone receptor was expressed predominantly in the subepithelial stroma. There was no change in receptor content and distribution following mifepristone treatment. Natural antimicrobial mRNA [secretory leukocyte protease inhibitor, human beta defensins mRNA (HBD1, HBD2, HBD3, HBD5), granulysin and elafin] was extracted from the vaginal tissues, and the content was unaffected by mifepristone treatment. CONCLUSION: The absence of changes in vaginal thickness, steroid receptor and natural antimicrobial content and its distribution in this preliminary study suggests that in contrast to other estrogen-free contraceptives, mifepristone is unlikely to be associated with the increased risk of transmission of HIV and other sexually transmitted infections.

Effects of picolinic acid on the antimicrobial functions of host macrophages against Mycobacterium avium complex.

Picolinic acid (PA) potentiates macrophage (MPhi) antimicrobial activity against intracellular Mycobacterium avium complex (MAC). Here, we studied the mechanisms of this phenomenon using human THP-1 MPhis. First, when PA-treated MAC-infected MPhis were cultured in the presence or absence of reactive oxygen intermediate (ROI) scavengers, nitric oxide synthase (NOS) inhibitors or phospholipase A(2) (PLA(2)) inhibitors, none of these agents blocked the activity of PA in potentiating MPhi anti-MAC activity. Second, when PA was added to the in vitro anti-MAC bactericidal system consisting of either ROIs, reactive nitrogen intermediates (RNIs) or free fatty acid (FFA) molecules, which are the major MPhi antimicrobial effectors, PA inhibited the activity of ROIs and conversely potentiated the activity of RNIs; PA did not affect the activity of FFAs. Third, PA reduced mRNA expression of NADPH oxidase and beta-defensin-1 by MAC-infected MPhis, whilst neither cytosolic PLA(2) nor CAP37 mRNA expression was affected. Notably, inducible NOS and secretory PLA(2) mRNA expression was not detected for MAC-infected MPhis even when given PA treatment. These findings suggest that ROIs, RNIs, FFAs and beta-defensin-1 do not play important roles in the PA-induced potentiation of MPhi anti-MAC activity.

ESP13.2, a member of the beta-defensin family, is a macaque sperm surface-coating protein involved in the capacitation process.

Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the beta-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the beta-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.