Β-defensin Research Articles

Altered expression of antimicrobial molecules in cigarette smoke‐exposed emphysematous mice lungs

The natural history of COPD, a disease usually caused by cigarette smoking, is associated with frequent respiratory infections. Consistent with human COPD, bacterial clearance in the lungs has been reported to be impaired in mice exposed to cigarette smoke. In the airways, several antimicrobial molecules such as surfactant proteins (SP), beta-defensins (BD), secretory leucocyte protease inhibitor (SLPI) and lysozyme play important roles in the defence against invading pathogens. This study evaluated the expression of antimicrobial molecules in mice lungs with cigarette smoke-induced emphysematous changes. Six B6C3F1 mice were exposed to cigarette smoke (2 cigarettes/day/mouse for 6 months) or room air. Gene expression within the lungs of mice in both groups was assessed by RT-PCR. The expression of SP-A, BD2, BD3 and SLPI was significantly elevated in the lungs of cigarette smoke-exposed mice compared with air-exposed mice. BD1 expression decreased in the smoke-exposed mice and lysozyme expression was unchanged. Chronic cigarette smoke exposure did not suppress the expression of antimicrobial molecules in the lung. Altered expression of antimicrobial molecules in this mouse model does not explain the impaired host defence against respiratory microbes seen in patients with COPD.

Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

Expression of human β‐defensin ‐1, ‐2, and ‐3 in non‐inflamed pseudocyst, mucoceles

The expressions of human beta defensin-1 (HBD-1), -2 (HBD-2) and -3 (HBD-3) in non-inflamed pseudocysts such as mucoceles were investigated immunohistochemically in this study. Mucocele specimens were obtained from 21 patients. The expression of HBDs was studied immunohistochemically by using antibodies directed against HBD-1, -2, and -3. Statistical analyses were carried out on serial sections stained with antibodies. Cells expressing HBDs were found in mucoceles. The expression of HBD-2 was observed in floating cells in all the specimens, whereas HBD-1 and HBD-3-expressing cells were detected in 93% and 73% of the mucoceles, respectively. The HBD-2 signal was the most intense and the HBD-3 signal intensity was weaker than that of HBD-1. HBDs were expressed in neutrophils and in other floating cells. Interestingly, the signal intensity and the population of positive cells located close to the centers of cysts were higher than those located in the peripheral areas of cysts. The expression of HBDs was found even in non-inflamed pseudocysts such as mucoceles. These results suggest that an unknown mechanism not involved in biophylaxis for the expression of HBDs may exist.

Transgenic expression of S100A2 in hairless mouse skin enhances Cxcl13 mRNA in response to solar-simulated radiation

S100A2 is a homodimeric protein that undergoes oxidative cross-linking and translocation from the nucleus to the cytosol in the context of oxidative stress. Suggestive of a role for S100A2 in the cutaneous response to ultraviolet light, we found altered S100A2 immunostaining in photodamaged human skin, and crosslinking of S100A2 after ultraviolet A (UVA) irradiation of normal human keratinocytes (NHK). Skin from mice, rats, and rabbits did not contain S100A2 protein, whereas skin samples from pigs, frogs and humans were strongly positive. Survival after UVA irradiation was significantly greater in NHK compared to mouse keratinocytes, suggesting a protective role for S100A2. To test this hypothesis in vivo, we expressed S100A2 in SKH2/J hairless mice under the control of a bovine keratin 5 promoter, and compared responses of TG and WT mice from 1 to 7 days after a single dose (0.5-1 MED) of solar-simulated radiation (SSR) from UVA-340 bulbs. WT and TG mice manifested a similarly robust response to SSR, characterized by epidermal hyperplasia, marked induction of p21(WAF), and a twofold increase in p53. Thymine dimers (TD) were markedly increased in the epidermis and the dermis, but while over 95% of the epidermal TD were removed by 5-6 days, elevated dermal TD persisted nearly unchanged for 7 days. Global transcriptional profiling of WT and TG mice revealed strong induction of multiple transcripts, including keratins K6 and K16, defensin beta 3, S100A8, S100A9, Sprr2i and Sprr2f. However, the only S100A2-dependent difference we observed was an induction of Cxcl13 transcripts in TG, but not WT mice (4.4-fold vs. 0.7-fold, n = 3, P = 0.022). This finding was confirmed in an independent set of mice analyzed by quantitative RT-PCR (8.8-fold vs. 1.2-fold, n = 4, P = 0.001). The finding of persistent dermal DNA damage after suberythemal doses of SSR merits further study.

Antimicrobial peptides expression in activated human corneal keratocytes

Abstract Purpose The eye despite being a delicate structure,has evolved many protective mechanisms.Antimicrobial peptides(AMPs) and Toll‐like receptors(TLRs)have an important role in the ocular defense system.However during an infection or trauma, the innate defense system is breached.In this instance,keratocytes and fibroblasts aids in wound healing as well as provide a second line of defense.Little is known about AMP expression by TLR signalling pathways in corneal fibroblasts.This study therefore demonstrates the mRNA expression of both novel and known AMPs in various fibroblast phenotypes(myofibroblasts and keratocytes).In addition, AMPs were also shown in microbial ligand and/or pro‐inflammatory cytokine stimulated human corneal fibroblast cultures (HCFC). Methods Fresh Keratocytes and HCFC were cultured and collected at different stages of growth and/or after stimulation with microbial ligands. Total RNA was extracted and cDNA was synthesized for semi‐quantitative gene expression studies. Polymerase chain reaction (PCR) amplified product were analyzed on ethidium bromide stained 1% agarose gels. Results Constitutive expression of Thy‐1(CD‐90), vimentin, keratocan and alpha SMA was shown in all cultures.Keratocyte phenotype marker CD‐34 showed decreased expression from 1st to 4th passage.Positive expression of Human beta defensin 109, Liver‐expressed AMP(LEAP‐2) and cathelicidin/LL‐37 was demonstrated in cytokine and microbial ligand stimulated HCFC. Conclusion Reminiscent with previous studies,we have demonstrated the mRNA expression of novel and known AMPs in corneal fibroblast mediated by TLR dependent signalling mechanisms. This result warrants the further study to fully understand the role of AMPs in ocular wound healing.

Profile of antimicrobial peptides (AMPs) at the ocular surface

Abstract Purpose Purpose: Antimicrobial peptides are eukaryotic analogues of antibiotics and serve as early effectors of innate defence. Our aim was to profile the spectrum of AMPs at the ocular surface in health and disease. Methods Methods: Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR) and real time PCR techniques with primers for 21 known AMPs were employed to evaluate gene expression on the following human ocular surface (OS) samples: a) Impression cytology specimen of normals and patients suffering from bacterial, viral keratitis and acanthamoeba keratitis and dry eyes. b) OS cells from cadaver donors and c) cultured corneal epithelial cells from limbal explants. Over one hundred samples were thus studied. Results Results: AMP expression was observed in all the different groups of samples but was variable. Nine AMPs, namely Human Beta defensins (HBD) 1 to 4, Cathelicidin (LL37), Liver expressed antimicrobial peptides, LEAP‐1 and ‐2, DEFB‐109 and RNAse were detected. Of these, HBD3 was prominent in bacterial keratitis and LEAP 1 and LL37 in viral keratitis. LEAP 2 and LL37 showed an increased tendency of expression in dry eyes. DEFB‐109 was the only AMP found to show decreased expression in the inflammatory conditions studied. RNAse, the most potent of all AMPs was found in abundance at the OS. Conclusion Conclusions: The OS is endowed with a range of AMPs which contribute to defence against environmental microbes and also participate in other immune mediated, inflammatory and wound healing events. They hold promise as therapeutic agents against microbes and in modulation of inflammation and wound healing.

Expression pattern of antimicrobial peptides (AMPs) in acanthamoeba keratitis

Abstract Purpose Acanthamoeba keratitis (AK) is a sight‐threatening ocular infection, which is more likely in contact lens wearer. Antimicrobial peptides(AMPs)are cationic polypeptides with wide‐spectrum activity against pathogens. We studied(1)the profile of AMP mRNA expression in ex‐vivo impression cytology (IC) specimen from normals and 5 AK patients &(2)compared AMP gene expression between ex‐vivo and in‐vitro cell culture samples. Methods a) IC samples were collected for Total RNA extraction and cDNA synthesis. Quantitative gene expression(qPCR)was employed to evaluate expression of Defensins,Cathelicidin,& Liver‐expressed AMPs (LEAPs).b) Immortalised human corneal epithelial cell line (HuCL) was incubated with trophozoites of Acanthamoeba castellanii for 24 hours and the supernatant analysed by BD cytometric bead array for pro‐inflammatory cytokines.The cells were processed for qPCR analyses. Results Variable expression of AMPs was observed in AK patients.Eight AMPs,Human Beta defensins(HBD)1 to 3,Cathelicidin (LL37,LEAP 1 and 2 and DEF 109 were detected.Of these HBD 1 to 3 and DEF 109 were shown to be reduced and whereas LEAP 1 & 2 were induced in AK specimen. Similarly, increased mRNA expression of LEAP 1 & 2 was also demonstrated in Acanthamoebae treated HuCL cultures.Whereas, HBDs showed the baseline expression when compare to untreated HuCLs. Conclusion This is a first study demonstrating AMP gene expression in AK. Differential expression pattern of these AMPs during AK may have important implications in understanding the pathogenesis of this blinding infection. Modulation of AMP defense barrier at the ocular surface or use of the AMP as a contact lens preservative agent may help to reduce the AK infections

Expression of human beta‐defensin‐2 and ‐3 in verrucae vulgares and condylomata acuminata

The human beta defensins (hBDs) 2 and 3 contribute to the inducible antimicrobial peptides in human skin. Besides an important role in fighting bacteria, they may also exert antiviral function. Verrucae vulgares and condylomata acuminata are typical cutaneous viral diseases caused by different subtypes of the human papillomavirus. Their tendency for spontaneous regression could be caused by antiviral proteins like defensins. In a retoperspective study, we investigated lesions of verrucae vulgares and condylomata acuminata for the presence of hBD-2 and hBD-3 by immunohistochemistry. All of the specimens of verrucae vulgares (n = 20) were positive for hBD-2 and hBD-3. The specimens of condylomata acuminata (n = 15) exhibited expression for hBD-2 and hBD-3, with the exception of three and two samples, respectively. The expression levels in condyloma acuminata were lower for both defensins compared with verrucae vulgares. Normal skin tissue exhibited no staining of both hBD-2 and hBD-3 with the exception of two cases. Taken together, this is the first report of an increased expression of the hBD-2 and hBD-3 in cutaneous papillomavirus infections.

Human beta defensin 2 promotes intestinal wound healing in vitro

Limiting microbial threats, maintenance and re-establishment of the mucosal barrier are vital for intestinal homeostasis. Antimicrobial peptides have been recognized as essential defence molecules and decreased expression of these peptides has been attributed to chronic inflammation of the human intestinal mucosa. Recently, pluripotent properties, including stimulation of proliferation and migration have been suggested for a number of antimicrobial peptides. However, it is currently unknown, whether the human beta-defensin 2 (hBD-2) in addition to its known antimicrobial properties has further effects on healing and protection of the intestinal epithelial barrier. Caco-2 and HT-29 cells were stimulated with 0.1-10 microg/ml hBD-2 for 6-72 h. Effects on cell viability and apoptosis were monitored and proliferation was quantified by bromo-deoxyuridine incorporation. Migration was quantified in wounding assays and characterized by immunohistochemistry. Expression of mucins was determined by quantitative PCR and slot-blot analysis. Furthermore, anti-apoptotic capacities of hBD-2 were studied. Over a broad range of concentrations and stimulation periods, hBD-2 was well tolerated by IECs and did not induce apoptosis. hBD-2 significantly increased migration but not proliferation of intestinal epithelial cells. Furthermore, hBD-2 induced cell line specific the expression of mucins 2 and 3 and ameliorated TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis. In addition to its known antimicrobial properties, hBD-2 might have further protective effects on the intestinal epithelium. Results of this in vitro study suggest, that hBD-2 expression may play a dual role in vivo, i.e. in impaired intestinal barrier function observed in patients with inflammatory bowel disease.

Resveratrol Delays Age-Related Deterioration and Mimics Transcriptional Aspects of Dietary Restriction without Extending Life Span

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation, and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at 12 months of age. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started midlife.

Study on vaginal production of human defensins and the correlated pathogenetic factors of vulvovaginal candidiasis

To investigate the correlated pathogenetic factors and vaginal local immunity in vulvovaginal candidiasis (VVC). A case control study was conducted to compare VVC group (60 cases) with normal group (60 cases). All of the women filled up the specific questionnaires. Routine examination, pH test and bacterial culture were done on the vaginal discharge. Cytokines of the vaginal lavage were measured by enzyme linked immunosorbent assay. (1) Outcomes of the questionnaires: there was no significant difference between the two groups in educational background, knowledge of gynecologic infection, history of gynecologic infection, hygienic habit, sex life, or use of medicine (P > 0.05). The incidence of chronic cervicitis in normal group (43%, 26/60) was higher than in VVC group (22%, 13/60; P < 0.05). (2) There was no difference in vaginal pH between the two groups (P > 0.05). (3) Detection rate of candida albicans by vaginal discharge routine examination was 72% (43/60). (4) The concentrations of interleukin (IL) 2, and IL-4 in vaginal lavage did not show significant difference between the two groups (P > 0.05), but the concentrations of human defensin 5, human beta-defensin (HBD) 1, and HBD2 in VVC group [(0.94 +/- 0.44) mg/L, (3.1 +/- 0.4) microg/L, (10 +/- 6) microg/L] were higher than normal group (P < 0.05). VVC is a common vulvovaginitis. There is no significant correlation between the incidence of VVC and educational background, knowledge of gynecologic infection, history of gynecologic infection, hygienic habit, sex life, or use of medicine in the child-bearing period. Human defensin may be closely correlated with the pathogenesis of VVC.

Expression of genes encoding antimicrobial proteins and members of the toll‐like receptor/nuclear factor‐κB pathways in engineered human skin

Skin functions as a first line of defense against microbial invasion. Tissue-engineered cultured skin substitutes (CSS) are used to aid wound closure in massively burned patients, and have been used to facilitate safe and effective wound closure in adult patients with chronic wounds. Although they contain only two cell types at grafting, they can potentially contribute to innate defense against pathogens and stimulation of adaptive immunity. Gene microarrays were used to identify expression in cultured skin of genes involved in innate and adaptive immune responses, and to evaluate the effects of cytokine stimulation on expression levels. Cultured skin expressed multiple antimicrobial protein genes, including human beta defensins 1 and 2 and S100A12. In addition, the antiviral gene APOBEC3G, which was not previously identified in skin, was expressed in CSS and up-regulated by interleukin-1alpha and tumor necrosis factor alpha. Cathelicidin was not expressed in unstimulated CSS, but was induced by cytokine treatment. Further, genes encoding several proinflammatory cytokines and members of the toll-like receptor and nuclear factor kappa B pathways were expressed in CSS, suggesting that cells in CSS can mediate activation of inflammatory responses. The observed expression patterns indicate that engineered human skin utilizes innate defense mechanisms similar to those reported for native skin. Therefore, regulation of these pathways by cytokine stimulation may offer a mechanism for increasing innate immunity in CSS to combat wound infection after grafting onto patients.

Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells

BackgroundAll mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human β-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the β-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly established. We believe that this report is the first attempt to elucidate NTHi induced β-defensin expression in airway mucosa, which includes the middle ear.MethodsMonoclonal antibody blocking method was employed in monitoring the TLR-dependent NTHi response. Two gene knock down methods – dominant negative (DN) plasmid and small interfering RNA (siRNA) – were employed to detect and confirm the involvement of several key genes in the signaling cascade resulting from the NTHi stimulated β-defensin 2 expression in human middle ear epithelial cell (HMEEC-1). The student's t-test was used for the statistical analysis of the data.ResultsThe experimental results showed that the major NTHi-specific receptor in HMEEC-1 is the Toll-like receptor 2 (TLR2). Furthermore, recognition of NTHi component(s)/ligand(s) by TLR2, activated the Toll/IL-1 receptor (TIR)-MyD88-IRAK1-TRAF6-MKK3/6-p38 MAPK signal transduction pathway, ultimately leading to the induction of β-defensin 2.ConclusionThis study found that the induction of β-defensin 2 is highest in whole cell lysate (WCL) preparations of NTHi, suggesting that the ligand(s) responsible for this up-regulation may be soluble macromolecule(s). We also found that this induction takes place through the TLR2 dependent MyD88-IRAK1-TRAF6-p38 MAPK pathway, with the primary response occurring within the first hour of stimulation. In combination with our previous studies showing that IL-1α-induced β-defensin 2 expression takes place through a MyD88-independent Raf-MEK1/2-ERK MAPK pathway, we found that both signaling cascades act synergistically to up-regulate β-defensin 2 levels. We propose that this confers an essential evolutionary advantage to the cells in coping with infections and may serve to amplify the innate immune response through paracrine signaling.

Transfection of human beta defensin 2 gene into the lung by aerosol inhalation: experiment with rats

To investigate the feasibility to transfect human beta defensin 2 (hBD2) gene into the lung so as to enhance the endogenous hBD2 expression to defend infectious diseases. Recombinant plasmid containing hBD2 gene, pLXSN-hBD2, was mixed with a new cationic liposome prepared by film at different weight ratios. Sixteen Wistar rats were divided into 2 groups: experimental group (n = 11), undergoing aerosol inhalation of liposome/pLXSN-hBD2 complex, and control group (n = 5), undergoing aerosol inhalation of blank vector pLXSN. The tracheae were taken out from 5 rats of the experiment and control groups each 2 days later, and from 2 rats of the experimental group 6, 15, and 21 days later respectively so as to obtain the epithelial cells of trachea. DNA was extracted from the tracheal epithelial cells and PCR was used to examine the transfection and integration of hBD2. Western blotting was used to detect the protein expression of hBD2. Aerosolization impacted obviously the microcapsule structure of liposome/plasmid complexes, and there was the least structural destruction of complex at a ratio of 10:1 that suited for aerosolization best. After the inhalation the relevant plasmids were all successfully integrated into the epithelial cells in both groups. Protein expression of hBD2 was not detected in the control group and the hBD2 protein expression level 2 days after transfection of the experimental group was 4866.9 +/- 148.2, and then decreased gradually, and reached 3.2 +/- 1.5 twenty-one days after the transfection. The recombinant plasmid pLXSN-hBD2 can be transfect into the airway epithelial cells via aerosol inhalation and the expression of hBD2 sustains for a period of time.

Association of β-defensin 1 single nucleotide polymorphisms with Crohn's disease

Background: It has been suggested that deficient defensin expression is connected with the chronic inflammation of Crohn's disease. The regional localization of the Crohn's disease, ileal or colonic disease can be linked to different defensin profiles. As constitutive β-defensin 1 has a colonic expression, we considered it of interest to investigate single nucleotide polymorphisms (SNPs) of the β-defensin 1 gene (DEFB1) in Crohn's disease. Methods: Three SNPs of the DEFB1 gene DEFB1 G-20A (rs11362), DEFB1 C-44G (rs1800972) and DEFB1 G-52A (rs1799946) were genotyped either by Custom TaqMan® SNP Genotyping Assays or by restriction fragment length polymorphisms (RFLP) in 190 patients with Crohn's disease and 95 Hungarian controls. Results: Strong associations between the G-20A and C-44G SNPs and the colonic, and ileocolonic localization of the disease, respectively, but no association was detected as concerns the ileal localization. A significantly higher frequency of the GA genotype of G-20A was observed among patients with colonic localization (60%) as compared with the healthy controls, (39%), with OR 2.39. The GG genotype of C-44G SNP, which can be regarded as a protective genotype, was much less frequent (4%) among the patients than among the controls (12%), OR 3.367. Conclusions: These results indicate that genetic variations in the DEFB1 gene encoding constitutive human β-defensin 1 may associated to the risk for Crohn's disease and may determine disease phenotype, e.g. colonic localization.

Beta defensin 2 gene copy variations influence mRNA expression and protein release in whole blood cultures from healthy individuals

Beta defensin 2 gene copy variations influence mRNA expression and protein release in whole blood cultures from healthy individuals

Chewing Gum Containing Citric Acid Reduces the Burden of Periodontal Pathogens

Objective: In a preliminary study, we noted that citric acid has the potency to induce human beta defensins (hBD-2) in salivary secretions. As hBDs are thought to ameliorate periodontal disease by suppressing key periodontal pathogens we developed a citric acid-containing chewing gum (CA-gum) to test this hypothesis. Here we report, the effect of CA-gum on three major periodontal pathogens Prevotella intermedia (Pi), Tannerella forsythia (Tf), Treponema denti- cola (Td) and Fusobacterium species (Fb). Methods: A randomized, double-blind, placebo-controlled trial with 61 individuals, was conducted over 14 days. Each subject was allowed to chew CA-gum (31 subjects) or placebo gum (30 subjects) at irregular intervals, but was instructed to eat at least three pieces of gum a day. None had active caries, symptoms of gingivitis or periodontal disease, smoking history, or concurrent medication. To determine the levels of oral carriage of the putative pathogens, saliva was collected from each subject by paraffin chewing in a standard manner and, the number of total cultivative microorganisms (CFUs) determined. Five key periodontal pathogens, Pg, Pi, Tf, Td and Fb present in saliva were determined quantitatively using real-time polymerase chain reaction (PCR) (BML Inc., Saitama, JAPAN). Results: Compared with the placebo chewing gum group the CA-gum group had significantly lower levels of Prevotella intermedia, Tannerella forsythia, and Fusobacterium spp. in their saliva. (Wilcoxon t-test; p 0.05).

Human Beta Defensin 3 (HBD3) induces migration and activation of antigen presenting cells and acts as an immune enhancer

Beta Defensins (BDs) are small, cationic peptides, classified by their pattern of conserved cysteines, that are primarily known for their antimicrobial properties. They also act as chemoattractantagents for monocytes and dendritic cells in mammals The best characterized human BDs are HBD1, HBD2, and mouse BD1∼3 (MBD1∼3). HBD1∼2 and MBD2∼3 are chemotactic for immature dendritic cells (iDCs), memory T cells, and mast cells. HBD2 and MBD2 are capable of activating mast cells and DCs. Human β‐defensin 3 (hBD3) is a highly basic 45‐amino‐acid protein that acts both as an antimicrobial agent and as a chemoattractant molecule. The effects of HBD3 and its mouse ortholog, MBD14, on leukocytes are largely uncharacterized to date. Here we report that HBD3 simultaneously acts as a chemoattractant and activator of antigen presenting cells. Using synthetic and recombinant HBD3, we observed that HBD 3 induced migration of CD34 progenitor derived iDCs and CCR6 transfected HEK 293 cells in a dose dependent manner. HBD3 was also chemotactic for human monocytes and was able to induce calcium flux in or by monocytes. HBD3 also induced activation of monocytes by upregulating surface expression of costimulatory molecules like CD40, CD64, CD80, CD86, MHC class II and adhesion molucles like LFA3 and ICAM1. Furthermore HBD3 induced production of IL‐6, IL‐8, IL‐1beta and TNF alpha by monocytes. When injected into mouse peritoneal cavity, HBD3 also caused a marked recruitment of neutrophils and macrophages. Thus, HBD3 appears to be an important mobilizer and activator of innate inflammatory and adaptive immune responses.

Probiotic lactobacilli and VSL#3 induce enterocyte β-defensin 2

Recent evidence suggests that probiotic bacteria may stabilize gut barrier function via induction of anti-microbial peptides such as defensins. This study aimed to elucidate the induction mechanism of the human beta defensin-2 (hBD-2) gene by different probiotic lactobacillus strains. The expression of hBD-2 mRNA peaked at 6 h of incubation upon treatment of Caco-2 cells and increased with higher dosage of various probiotic bacteria. Deletion of nuclear factor (NF)-kappaB and activator protein-1 (AP-1) binding sites on the hBD-2 promoter resulted in a complete abrogation of promoter activation by probiotics. As revealed by the use of specific mitogen-activated protein kinase (MAPK) inhibitors the hBD-2 induction was dependent on the MAPK extracellular regulated kinase (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK), although to varying degrees. Several Lactobacillus strains and VSL#3, a probiotic cocktail of four lactobacilli, three bifidum and one streptococcus species, induced the secretion of the hBD-2 peptide into the culture media as shown by enzyme-linked immunosorbent assay (ELISA). Thus, the present study suggests that lactobacilli and the VSL#3 bacterial mixture strengthen intestinal barrier functions through the up-regulation of hBD-2 via induction of proinflammatory pathways including NF-kappaB and AP-1 as well as MAPKs.

Rat Cardiomyocytes Express a Classical Epithelial Beta-Defensin

Beta-defensins (BDs) are classical epithelial antimicrobial peptides of immediate importance in innate host defense. Since recent studies have suggested that certain BDs are also expressed in non-traditional tissues, including whole heart homogenate, and because effector molecules of innate immunity and inflammation can influence the development of certain cardiovascular disease processes, we hypothesized that BDs are produced by cardiomyocytes as a local measure of cardioprotection against danger signals. Here we report that at least one rat beta- defensin, rBD1, is expressed constitutively in cardiomyocytes specifically isolated using position- ablation-laser-microdissection (P.A.L.M. Microlaser Technologies). RT-PCR analysis showed expression of a single 318 bp transcript in adult rat heart (laser-excised cardiomyocytes) and H9c2 cells (neonatal rat heart myoblasts). Moreover, the full length cDNA of rBD1 was established, and translated into a putative peptide with 69 amino acid residues. The predicted amino acid sequence of the adult rat cardiac BD-1 peptide displayed 99% identity with the previously reported renal rBD1, and 88%, 53%, 53% and 50% identity with mouse, human, gorilla, and rhesus monkey BD1 respectively. Furthermore, structural analysis of the cardiac rBD1 showed the classical six- cysteine conserved motif of the BD family with an alpha-helix and three beta-sheets. Additionally, rBD1 displayed a significantly greater number of amphoteric residues than any of the human analogs, indicating a strong pH functional dependence in the rat. We suggest that rBD1, which was initially believed to be a specific epithelium-derived peptide, may be also involved in local cardiac innate immune defense mechanisms.