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1.  Capacity of human β-defensin expression in gene-transduced and cytokine-induced cells✩ 
The purpose of this study was to determine the capacity of cells transduced with human β-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9–99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabe-Neo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFα. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.
doi:10.1016/j.bbrc.2005.11.020
PMCID: PMC3282591  PMID: 16298338
HBD-2; HBD-3; Lentiviral vectors; Retroviral vectors; IL-1; TNFα; Gingival epithelial cells
2.  A Model for Antimicrobial Gene Therapy: Demonstration of Human β-Defensin 2 Antimicrobial Activities In Vivo 
Human gene therapy  2002;13(17):2017-2025.
We transfected host cells with an antimicrobial peptide/protein-encoding gene as a way to enhance host defense mechanisms against infection. The human b-defensin 2 (HBD-2) gene was chosen as a model because its protein does not require cell type-specific processing. Using a retroviral vector carrying HBD-2 cDNA, we treated several mouse or human cell lines and primary cell cultures including fibroblasts, salivary gland cells, endothelial cells, and T cells. All transduced cells produced detectable HBD-2. In Escherichia coli gel overlay experiments, secreted HBD-2 from selected cell lines showed potent antimicrobial activity electrophoretically identical to that of purified HBD-2. We then used a mouse model (nonobese diabetic/severely compromised immunodeficient [NOD/SCID]) to test HBD-2 antimicrobial activities in vivo. HT-1080 cells carrying HBD-2 or control vector were implanted subcutaneously into NOD/SCID mice to allow tumor formation. Escherichia coli was then injected into each tumor mass. Tumors were resected after 16 hr and homogenized for bacterial colony-forming unit analysis. Compared with control tumors, HBD-2-bearing tumors contained only 7.8 6 3.3% viable bacteria. On the basis of this demonstration of HBD-2 in vivo antimicrobial activity, enhancement of antibacterial host defense by HBD-2 gene therapy may be feasible.
doi:10.1089/10430340260395875
PMCID: PMC1790959  PMID: 12489997
3.  Production of β-Defensin Antimicrobial Peptides by the Oral Mucosa and Salivary Glands 
Infection and Immunity  1999;67(6):2740-2745.
β-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human β-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected β-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether β-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1β (IL-1β) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased ∼16-fold with IL-1β treatment and ∼5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human β-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1β and LPS. Human β-defensins may play an important role in the innate defenses against oral microorganisms.
PMCID: PMC96577  PMID: 10338476
4.  Oral Antimicrobial Peptides and Biological Control of Caries 
BMC Oral Health  2006;6(Suppl 1):S13.
The presence of antimicrobial peptides (AMPs) in saliva may be a biological factor that contributes to susceptibility or resistance to caries. This manuscript will review AMPs in saliva, consider their antimicrobial and immunomodulatory functions, and evaluate their potential role in the oral cavity for protection of the tooth surface as well as the oral mucosa. These AMPs are made in salivary gland and duct cells and have broad antimicrobial activity. Alpha-defensins and LL37 are also released by neutrophils into the gingival crevicular fluid. Both sources may account for their presence in saliva. A recent study in middle school children aimed to determine a possible correlation between caries prevalence in children and salivary concentrations of the antimicrobial peptides human beta-defensin-3 (hBD-3), the cathelicidin, LL37, and the alpha-defensins. The levels of these AMPs were highly variable in the population. While levels of LL37 and hBD-3 did not correlate with caries experience, the mean alpha-defensin level was significantly higher in children with no caries than in children with caries (p < 0.005). We conclude that several types of AMPs that may have a role in oral health are present in unstimulated saliva. Low salivary levels of alpha-defensin may represent a biological factor that contributes to caries susceptibility. Our observation could lead to new ways to prevent caries and to a new tool for caries risk assessment.
doi:10.1186/1472-6831-6-S1-S13
PMCID: PMC2147588  PMID: 16934114
5.  Salivary Antimicrobial Peptide Expression and Dental Caries Experience in Children 
Dental caries is a major worldwide oral disease problem in children. Although caries are known to be influenced by dietary factors, the disease results from a bacterial infection; thus, caries susceptibility may be affected by host factors such as salivary antimicrobial peptides. This study aimed to determine a possible correlation between caries prevalence in children and salivary concentrations of the antimicrobial peptides human beta-defensin-3 (hBD-3), the cathelicidin LL37, and the alpha-defensins HNP1-3 (a mixture of HNP1, 2, 3). Oral examinations were performed on 149 middle school children, and unstimulated whole saliva was collected for immunoassays of the three peptides and for assay of caries-causing bacteria in saliva. The median salivary levels of hBD-3, LL37, and HNP1-3 were in the microgram/ml range but were highly variable in the population. While levels of LL37 and hBD-3 did not correlate with caries experience, the median HNP1-3 levels were significantly higher in children with no caries than in children with caries. Children with high caries levels did not have high levels of salivary Streptococcus mutans, and the HNP1-3 level was not correlated with salivary S. mutans. By immunohistochemistry we localized HNP1-3 in submandibular salivary duct cells. HNPs are also released by neutrophils into the gingival crevicular fluid. Both sources may account for their presence in saliva. Low salivary levels of HNP1-3 may represent a biological factor that contributes to caries susceptibility. This observation could lead to new ways to screen for caries susceptibility and to new means of assessing the risk for this common oral problem.
doi:10.1128/AAC.49.9.3883-3888.2005
PMCID: PMC1195389  PMID: 16127066
6.  Viruses and Salivary Gland Disease (SGD) 
Advances in Dental Research  2011;23(1):79-83.
Viral infections are often associated with salivary gland pathology. Here we review the pathogenesis of HIV-associated salivary gland disease (HIV-SGD), a hallmark of diffuse infiltrative lymphocytosis syndrome. We investigate the presence and contributions of viral diseases to the pathogenesis of salivary gland diseases, particularly HIV-SGD. We have detected BK viral shedding in the saliva of HIV-SGD patients consistent with viral infection and replication, suggesting a role for oral transmission. For further investigation of BKV pathogenesis in salivary glands, an in vitro model of BKV infection is described. Submandibular (HSG) and parotid (HSY) gland salivary cell lines were capable of permissive BKV infection, as determined by BKV gene expression and replication. Analysis of these data collectively suggests the potential for a BKV oral route of transmission and salivary gland pathogenesis within HIV-SGD.
doi:10.1177/0022034510396882
PMCID: PMC3144046  PMID: 21441486
Virus; salivary gland; HIV; DILS
7.  Functional salivary gland regeneration by transplantation of a bioengineered organ germ 
Nature Communications  2013;4:2498.
Salivary gland hypofunction, also known as xerostomia, occurs as a result of radiation therapy for head cancer, Sjögren’s syndrome or aging, and can cause a variety of critical oral health issues, including dental decay, bacterial infection, mastication dysfunction, swallowing dysfunction and reduced quality of life. Here we demonstrate the full functional regeneration of a salivary gland that reproduces the morphogenesis induced by reciprocal epithelial and mesenchymal interactions through the orthotopic transplantation of a bioengineered salivary gland germ as a regenerative organ replacement therapy. The bioengineered germ develops into a mature gland through acinar formations with a myoepithelium and innervation. The bioengineered submandibular gland produces saliva in response to the administration of pilocarpine and gustatory stimulation by citrate, protects against oral bacterial infection and restores normal swallowing in a salivary gland-defective mouse model. This study thus provides a proof-of-concept for bioengineered salivary gland regeneration as a potential treatment of xerostomia.
Salivary gland dysfunction as a result of diseases or ageing reduces the quality of life and causes various oral health problems. Here the authors show that the salivary gland function of mice can be recovered by orthotopic transplantation of a bioengineered salivary gland germ.
doi:10.1038/ncomms3498
PMCID: PMC3806330  PMID: 24084982
8.  Nuclear hBD-1 accumulation in malignant salivary gland tumours 
BMC Cancer  2008;8:290.
Background
Whereas the antimicrobial peptides hBD-2 and -3 are related to inflammation, the constitutively expressed hBD-1 might function as 8p tumour suppressor gene and thus play a key role in control of transcription and induction of apoptosis in malignant epithelial tumours. Therefore this study was conducted to characterise proteins involved in cell cycle control and host defence in different benign and malignant salivary gland tumours in comparison with healthy salivary gland tissue.
Methods
21 paraffin-embedded tissue samples of benign (n = 7), and malignant (n = 7) salivary gland tumours as well as healthy (n = 7) salivary glands were examined immunohistochemically for the expression of p53, bcl-2, and hBD-1, -2, -3.
Results
HBD-1 was distributed in the cytoplasm of healthy salivary glands and benign salivary gland tumours but seems to migrate into the nucleus of malignant salivary gland tumours. Pleomorphic adenomas showed cytoplasmic as well as weak nuclear hBD-1 staining.
Conclusion
HBD-1, 2 and 3 are traceable in healthy salivary gland tissue as well as in benign and malignant salivary gland tumours. As hBD-1 is shifted from the cytoplasm to the nucleus in malignant salivary gland tumours, we hypothesize that it might play a role in the oncogenesis of these tumours. In pleomorphic adenomas hBD-1 might be connected to their biologic behaviour of recurrence and malignant transformation.
doi:10.1186/1471-2407-8-290
PMCID: PMC2567991  PMID: 18840281
9.  Streptococcus mutans strains recovered from caries-active or caries-free individuals differ in sensitivity to host anti-microbial peptides 
Molecular oral microbiology  2011;26(3):187-199.
SUMMARY
Antimicrobial peptides (AMPs) are among the repertoire of host innate immune defenses. In the oral cavity, several AMPs are present in saliva and have antimicrobial activities against oral bacteria, including Streptococcus mutans, a primary etiologic agent of dental caries. In this study, we hypothesized that unique S. mutans strains as determined by DNA fingerprinting from sixty 13 year-old subjects with or without caries experience would have different susceptibilities to α-defensins-1-3 (HNP-1-3), β-defensins-2-3 (HBD-2-3) and LL-37. The salivary levels of these peptides in subjects also were measured by enzyme-linked immunosorbent assays (ELISA). We found that S. mutans strains from caries-active subjects showed greater resistance to salivary HNP-1-2, HBD-2-3 and LL-37 at varying concentrations than those from caries-free subjects. In addition, combinations of these peptides increased their antimicrobial activity against S. mutans either additively or synergistically. The salivary levels of these peptides were highly variable among subjects with no correlation to host caries experience. However, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. Our findings suggest that the relative ability of S. mutans to resist host salivary AMPs may be considered a potential virulence factor for this species such that S. mutans strains that are more resistant to these peptides may have an ecological advantage to preferentially colonize within dental plaque and increase the risk of dental caries.
doi:10.1111/j.2041-1014.2011.00607.x
PMCID: PMC3092152  PMID: 21545696
Dental caries; defensins; S. mutans; innate immunity; saliva
10.  Effect of human vasoactive intestinal peptide gene transfer in a murine model of Sjögren's syndrome 
Annals of the Rheumatic Diseases  2005;65(2):195-200.
Background
Sjögren's syndrome (SS), an autoimmune exocrinopathy mainly affecting lachrymal and salivary glands, results in ocular and oral dryness (keratoconjunctivitis sicca and xerostomia). The aetiology and pathogenesis are largely unknown; currently, only palliative treatment is available.
Objective
To determine whether gene transfer of vasoactive intestinal peptide (VIP), based on its immunomodulatory properties, might be useful in the management of SS.
Methods
A recombinant serotype 2 adeno‐associated virus encoding the human VIP transgene (rAAV2hVIP) was constructed and its efficacy tested in the female non‐obese diabetic (NOD) mouse model for SS after retrograde instillation in submandibular glands (SMGs). 1010 particles/gland of rAAV2hVIP or rAAV2LacZ (encoding β‐galactosidase; control vector) were administered at 8 weeks of age (before sialadenitis onset). Salivary flow rates were determined before vector delivery and at time of death (16 weeks). After death, saliva, serum, and SMGs were harvested. Salivary output, inflammatory infiltrates (focus scores), VIP protein expression, cytokine profile, and serum anti‐VIP antibodies were analysed.
Results
rAAV2hVIP significantly improved the salivary flow, increased SMG and serum expression of VIP, and reduced SMG cytokines interleukin (IL) 2, IL10, IL12 (p70), and tumour necrosis factor α, and serum RANTES, compared with the control vector. No difference in focus scores or apoptotic rates was found; neutralising antibodies were not detected.
Conclusions
Local delivery of rAAV2hVIP can have disease modifying and immunosuppressive effects in SMGs of the NOD mouse model of SS. The new strategy of employing VIP prophylactically may be useful for both understanding and managing the salivary component of SS.
doi:10.1136/ard.2005.038232
PMCID: PMC1798026  PMID: 15975969
vasoactive intestinal peptide; Sjögren's syndrome; gene transfer; adeno‐associated virus; autoimmune disease
11.  Human β-Defensin-1 mRNA Is Transcribed in Tympanic Membrane and Adjacent Auditory Canal Epithelium 
Infection and Immunity  1999;67(9):4843-4846.
The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human β-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.
PMCID: PMC96817  PMID: 10456939
12.  Sensitivity of Candida Albicans Biofilm Cells Grown on Denture Acrylic to Antifungal Proteins and Chlorhexidine 
Archives of oral biology  2009;54(6):588-594.
Objectives
Candida albicans cells form biofilms on polymeric surfaces of dentures and other prostheses introduced into the oral cavity. Many biofilm microorganisms exhibit resistance to antimicrobial agents; C. albicans cells may also develop resistance to naturally-occurring antifungal peptides in human saliva including histatins (Hsts) and defensins (hBDs). Therefore, we evaluated Hst 5 activity on C. albicans biofilm cells compared to planktonic cells and measured whether surface treatment of denture acrylic with Hst 5, hBD-3, or chlorhexidine gluconate could inhibit in vitro biofilm development.
Methods
Acrylic disks were preconditioned with 500 μl saliva for 30 min, and inoculated with C. albicans cells (106 cells/ml) for 1 h, at 37 °C. Non-adherent cells were removed by washing and disks and were incubated in YPD growth medium for 24, 48, and 72 h at 37 °C. Candidacidal assays were performed on 48-hour-biofilms and on planktonically-grown cells using Hst 5 (15.5 μM, 31.25 μM, 62 μM). Cell adhesion was compared on disks pre-coated with 0.12% chlorhexidine gluconate, 50 μM Hst 5, or 0.6 μM hBD-3 after 24 h, 48 h, and 72 h growth.
Results
No significant difference was observed in sensitivity to Hst 5 of biofilm cells compared to planktonic cells (p > 0.05). Pre-coating disks with hBD-3 did not inhibit biofilm development; however, Hst 5 significantly inhibited biofilm development at 72 h, while 0.12% chlorhexidine significantly inhibited biofilm development at all time intervals (p < 0.05).
Conclusions
C. albicans biofilm cells grown on denture acrylic are sensitive to killing by Hst 5. Surface coating acrylic with chlorhexidine or Hst 5 effectively inhibits biofilm growth and has potential therapeutic application.
doi:10.1016/j.archoralbio.2009.01.016
PMCID: PMC2693315  PMID: 19249746
Candida albicans; Biofilm; Denture acrylic; Histatin; Defensin; Chlorhexidine
13.  Antimicrobial Peptides in the Oral Environment: Expression and Function in Health and Disease 
The oral cavity is a unique environment in which antimicrobial peptides play a key role in maintaining health and may have future therapeutic applications. Present evidence suggests that α-defensins, β-defensins, LL-37, histatin, and other antimicrobial peptides and proteins have distinct but overlapping roles in maintaining oral health and preventing bacterial, fungal, and viral adherence and infection. The expression of the inducible hBD-2 in normal oral epithelium, in contrast to other epithelia, and the apparent differential signaling in response to commensal and pathogenic organisms, provides new insights into innate immunity in this body site. Commensal bacteria are excellent inducers of hBD-2 in oral epithelial cells, suggesting that the commensal bacterial community acts in a manner to benefit the overall innate immune readiness of oral epithelia. This may have major significance for understanding host defense in the complex oral environment.
doi:10.1093/jac/dki103
PMCID: PMC1479858  PMID: 16053246
14.  Current cell models for bioengineering a salivary gland: a mini-review of emerging technologies 
Oral Diseases  2013;19(3):236-244.
Saliva plays a major role in maintaining oral health. Patients afflicted with a decrease in saliva secretion (symptomatically, xerostomia) exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. Despite recent improvements in treating xerostomia (e.g., saliva stimulants, saliva substitutes, and gene therapy), there is a need of more scientific advancements that can be clinically applied toward restoration of compromised salivary gland function. Here we provide a summary of the current salivary cell models that have been used to advance restorative treatments via development of an artificial salivary gland. These models represent initial steps toward clinical and translational research, to facilitate creation of clinically safe salivary glands. Further studies in salivary cell lines and primary cells are necessary to improve survival rates, cell differentiation, and secretory function. Additionally, the characterization of salivary progenitor and stem cell markers are necessary. Although these models are not fully characterized, their improvement may lead to the construction of an artificial salivary gland that is in high demand for improving the quality of life of many patients suffering from salivary secretory dysfunction.
doi:10.1111/j.1601-0825.2012.01958.x
PMCID: PMC3477256  PMID: 22805753
cell line; progenitor cells; primary culture; salivary gland dysfunction
15.  Current cell models for bioengineering a salivary gland: a mini-review of emerging technologies 
Oral diseases  2012;19(3):236-244.
Saliva plays a major role in maintaining oral health. Patients afflicted with a decrease in saliva secretion (symptomatically, xerostomia) exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. Despite recent improvements in treating xerostomia (e.g., saliva stimulants, saliva substitutes and gene therapy), there is a need of more scientific advancements that can be clinically applied toward restoration of compromised salivary gland function. Here we provide a summary of the current salivary cell models that have been used to advance restorative treatments via development of an artificial salivary gland. These models represent initial steps toward clinical and translational research, to facilitate creation of clinically safe salivary glands. Further studies in salivary cell lines and primary cells are necessary to improve survival rates, cell differentiation, and secretory function. Additionally, the characterization of salivary progenitor and stem cell markers are necessary. Although these models are not fully characterized, their improvement may lead to the construction of an artificial salivary gland that is in high demand for improving the quality of life of many patients suffering from salivary secretory dysfunction.
doi:10.1111/j.1601-0825.2012.01958.x
PMCID: PMC3477256  PMID: 22805753
cell line; progenitor cells; primary culture; salivary gland dysfunction
16.  Single-Nucleotide Polymorphisms (SNPs) in Human β-Defensin 1: High-Throughput SNP Assays and Association with Candida Carriage in Type I Diabetics and Nondiabetic Controls 
β-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human β-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as ≤350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5′ untranslated region (C→G at position −44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C→G SNP at position −44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.
doi:10.1128/JCM.41.1.90-96.2003
PMCID: PMC149626  PMID: 12517831
17.  Reducing Xerostomia After Chemo-IMRT for Head and Neck Cancer: Beyond Sparing the Parotid Glands 
Purpose
To assess whether in addition to sparing parotid glands (PGs), xerostomia after chemo-IMRT of head and neck cancer is affected by reducing doses to other salivary glands.
Methods
Prospective study: 78 patients with stages III/IV oropharynx/nasopharynx cancers received chemo-IMRT aiming to spare the parts outside the targets of bilateral PGs, oral cavity (OC) containing the minor salivary glands, and contralateral submandibular gland (SMG) (when contralateral level I was not a target). Pretherapy and periodically through 24 months, validated patient-reported xerostomia questionnaires (XQ) scores and observer-graded xerostomia were recorded, and stimulated and unstimulated saliva measured selectively from each of the PGs and SMGs. Mean OC doses served as surrogates of minor salivary glands dysfunction. Regression models assessed XQ and observer-graded xerostomia predictors.
Results
Statistically significant predictors of the XQ score in univariate analysis included OC, PG, and SMG mean doses, as well as baseline XQ score, time since RT, and both stimulated and unstimulated PG saliva flow rates. Similar factors were statistically significant predictors of observer-graded xerostomia. OC, PG and SMG mean doses were moderately inter-correlated (r=0.47–0.55). In multivariate analyses, after adjusting for PG and SMG doses, OC mean dose (p < 0.0001), time from RT (p < 0.0001), and stimulated PG saliva (p < 0.0025) were significant predictors for XQ scores, and OC mean dose and time for observer-graded xerostomia. While scatter plots showed no thresholds, OC mean doses <40 Gy and contralateral SMG mean <50 Gy were each associated with low patient-reported and observer-rated xerostomia at almost all post-therapy time points.
Conclusion
PG, SMG and OC mean doses were significant predictors of both patient-reported and observer-rated xerostomia after chemo-IMRT, with OC doses remaining significant after adjusting for PG and SMG doses. These results support efforts to spare all salivary glands by IMRT, beyond the PGs alone.
doi:10.1016/j.ijrobp.2011.09.004
PMCID: PMC3288420  PMID: 22056067
head neck cancer; xerostomia; IMRT; submandibular glands
18.  Multiple components contribute to ability of saliva to inhibit influenza viruses 
Introduction
Saliva is a potentially important barrier against respiratory viral infection but its mechanism of action is not well studied.
Methods
We tested the antiviral activities of whole saliva, specific salivary gland secretions, and purified salivary proteins against strains of influenza A virus (IAV) in vitro.
Results
Whole saliva or parotid or submandibular/sublingual secretions from healthy donors inhibited IAV based on hemagglutination inhibition and neutralization assays. This differs from human immunodeficiency virus (HIV), for which only submandibular/sublingual secretions are reported to be inhibitory. Among purified salivary proteins, MUC5B, scavenger receptor cysteine-rich glycoprotein 340 (salivary gp-340), histatins, and human neutrophil defensins (HNPs) inhibited IAV at the concentrations present in whole saliva. In contrast, some abundant salivary proteins (acidic proline-rich proteins and amylase) had no activity, nor did several other less abundant salivary proteins with known activity against HIV (e.g. thrombospondin or serum leukocyte protease inhibitor). Whole saliva and MUC5B did not inhibit neuraminidase activity of IAV and viral neutralizing and aggregating activity of MUC5B was potentiated by the neuraminidase inhibitor oseltamivir. Hence, MUC5B inhibits IAV by presenting a sialic acid ligand for the viral hemagglutinin. The mechanism of action of histatins requires further study.
Conclusions
These findings indicate that saliva represents an important initial barrier to IAV infection and underline the complexity of host defense activity of oral secretions. Of interest, antiviral activity of saliva against IAV and HIV differs in terms of specific glandular secretions and proteins that are inhibitory.
doi:10.1111/j.1399-302X.2008.00468.x
PMCID: PMC2848456  PMID: 19121065
histatins; innate immunity; MUC5B
19.  α-Galactosidase A Expressed in the Salivary Glands Partially Corrects Organ Biochemical Deficits in the Fabry Mouse Through Endocrine Trafficking 
Human Gene Therapy  2010;22(3):293-301.
Abstract
Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated successfully with enzyme replacement therapy (ERT). Gene therapy has been proposed as an alternative to ERT due to the presumed advantages of continuous, endogenous production of the therapeutic enzyme. GLA production in the liver and its therapeutic efficacy in the Fabry mouse have been demonstrated previously with various viral vector systems. In consideration of the potential advantages of using the salivary glands as endogenous GLA biosynthesis sites, we explored the feasibility of this approach in the Fabry mouse. GLA −/0 or −/− mice received an adenoviral vector (2 × 1010 or 1 × 109 viral particles) expressing GLA to the right submandibular gland via oral cannulation of the submandibular duct. Four days later, animals were sacrificed; saliva, plasma, kidney, liver, and brain were collected and assayed using ELISA, Western blot, and a GLA enzymatic activity assay using both traditional fluorescence methods and isotope dilution mass spectrometry by following the U.S. EPA Method 6800. GLA activity was significantly elevated in the serum and liver of both treatment groups, and improvement in the kidney was marginally significant (P < 0.069) in the high-dose group. Notably, we found that liver and salivary gland produce different glycoforms of the GLA transgene. Only small numbers of adenoviral genomes were observed in the livers of treated animals, but in four of 14 in the high-dose groups, liver levels of adenovirus exceeded 20 copies/μg, indicating that the sequestration in the salivary gland was imperfect at high doses. Taken together, these results indicate that the salivary gland-based gene therapy for Fabry disease is promising, and further studies with advanced viral vector gene delivery systems (e.g., adeno-associated virus) for long-term treatment appear to be warranted.
Fabry disease is caused by an X-linked deficiency of the lysosomal enzyme α-galactosidase A (GLA) and has been treated with enzyme replacement therapy (ERT). Here, Passineau and colleagues report results from a gene therapy study in the GLA-knockout mouse intended to provide a proof-of-concept that the salivary glands might be targets for gene transfer to serve as a depot for delivery of enzyme in Fabry disease and possibly other lysosomal storage disorders.
doi:10.1089/hum.2010.069
PMCID: PMC3057212  PMID: 20858137
20.  Differential and coordinated expression of defensins and cytokines by gingival epithelial cells and dendritic cells in response to oral bacteria 
BMC Immunology  2010;11:37.
Background
Epithelial cells and dendritic cells (DCs) both initiate and contribute to innate immune responses to bacteria. However, much less is known about the coordinated regulation of innate immune responses between GECs and immune cells, particularly DCs in the oral cavity. The present study was conducted to investigate whether their responses are coordinated and are bacteria-specific in the oral cavity.
Results
The β-defensin antimicrobial peptides hBD1, hBD2 and hBD3 were expressed by immature DCs as well as gingival epithelial cells (GECs). HBD1, hBD2 and hBD3 are upregulated in DCs while hBD2 and hBD3 are upregulated in GECs in response to bacterial stimulation. Responses of both cell types were bacteria-specific, as demonstrated by distinctive profiles of hBDs mRNA expression and secreted cytokines and chemokines in response to cell wall preparations of various bacteria of different pathogenicity: Fusobacterium nucleatum, Actinomyces naeslundii and Porphyromonas gingivalis. The regulation of expression of hBD2, IL-8, CXCL2/GROβ and CCL-20/MIP3α by GECs was greatly enhanced by conditioned medium from bacterially activated DCs. This enhancement was primarily mediated via IL-1β, since induction was largely attenuated by IL-1 receptor antagonist. In addition, the defensins influence DCs by eliciting differential cytokine and chemokine secretion. HBD2 significantly induced IL-6, while hBD3 induced MCP-1 to approximately the same extent as LPS, suggesting a unique role in immune responses.
Conclusions
The results suggest that cytokines, chemokines and β-defensins are involved in interaction of these two cell types, and the responses are bacteria-specific. Differential and coordinated regulation between GECs and DCs may be important in regulation of innate immune homeostasis and response to pathogens in the oral cavity.
doi:10.1186/1471-2172-11-37
PMCID: PMC2912831  PMID: 20618959
21.  Effect of Glycyrrhizin on Pseudomonal Skin Infections in Human-Mouse Chimeras 
PLoS ONE  2014;9(1):e83747.
In our previous studies, peripheral blood lineage−CD34+CD31+ cells (CD31+ IMC) appearing in severely burned patients have been characterized as inhibitor cells for the production of β-defensins (HBDs) by human epidermal keratinocytes (NHEK). In this study, the effect of glycyrrhizin on pseudomonal skin infections was studied in a chimera model of thermal injury. Two different chimera models were utilized. Patient chimeras were created in murine antimicrobial peptide-depleted NOD-SCID IL-2rγnull mice that were grafted with unburned skin tissues of severely burned patients and inoculated with the same patient peripheral blood CD31+ IMC. Patient chimera substitutes were created in the same mice that were grafted with NHEK and inoculated with experimentally induced CD31+ IMC. In the results, both groups of chimeras treated with glycyrrhizin resisted a 20 LD50 dose of P. aeruginosa skin infection, while all chimeras in both groups treated with saline died within 3 days of the infection. Human antimicrobial peptides were detected from the grafted site tissues of both groups of chimeras treated with glycyrrhizin, while the peptides were not detected in the same area tissues of controls. HBD-1 was produced by keratinocytes in transwell-cultures performed with CD31+ IMC and glycyrrhizin. Also, inhibitors (IL-10 and CCL2) of HBD-1 production by keratinocytes were not detected in cultures of patient CD31+ IMC treated with glycyrrhizin. These results indicate that sepsis stemming from pseudomonal grafted site infections in a chimera model of burn injury is controllable by glycyrrhizin. Impaired antimicrobial peptide production at the infection site of severely burned patients may be restored after treatment with glycyrrhizin.
doi:10.1371/journal.pone.0083747
PMCID: PMC3907411  PMID: 24497916
22.  Activation of TLR2 by a Small Molecule Produced by Staphylococcus epidermidis Increases Antimicrobial Defense against Bacterial Skin Infections 
Production of antimicrobial peptides by epithelia is an essential defense against infectious pathogens. In this study we evaluated whether the commensal microorganism Staphylococcus epidermidis may enhance production of antimicrobial peptides by keratinocytes and thus augment skin defense against infection. Exposure of cultured undifferentiated human keratinocytes to a sterile nontoxic small molecule of < 10 kDa from S. epidermidis conditioned culture medium (SECM), but not similar preparations from other bacteria, enhanced human β-defensin 2 (hBD2) and hBD3 mRNA expression and increased the capacity of cell lysates to inhibit the growth of group A Streptococcus (GAS) and S. aureus. Partial gene silencing of hBD3 inhibited this antimicrobial action. This effect was relevant in vivo as administration of SECM to mice decreased susceptibility to infection by GAS. Toll-like receptor 2 (TLR2) was important to this process as a TLR2-neutralizing antibody blocked induction of hBDs 2 and 3, and Tlr2-deficient mice did not show induction of mBD4. Taken together, these findings reveal a potential use for normal commensal bacterium S. epidermidis to activate TLR2 signaling and induce antimicrobial peptide expression, thus enabling the skin to mount an enhanced response to pathogens.
doi:10.1038/jid.2010.123
PMCID: PMC2922455  PMID: 20463690
23.  Impaired Neurotransmitter Release from Lacrimal and Salivary Gland Nerves of a Murine Model of Sjögren's Syndrome 
Purpose
To determine whether lacrimal and salivary gland nerves of an animal model of Sjögren's syndrome, the MRL/lpr mouse, are able to release acetylcholine. The second purpose was to determine whether activation of the lacrimal gland nerves of the MRL/lpr mouse leads to protein secretion.
Methods
Total saliva was collected for 10 minutes from the oral cavity of male and female MRL/lpr and MRL/+ mice, after intraperitoneal stimulation with pilocarpine and isoproterenol. Lacrimal and salivary gland lobules prepared from 18-week-old MRL/lpr and MRL/+ mice were incubated in the presence of depolarizing KCl (75 mM) solution. Acetylcholine release and peroxidase secretion (a protein secreted by the lacrimal gland) were measured using a spectrofluorometric assay.
Results
Female, but not male, MRL/lpr mouse salivary glands were hyper-responsive to in vivo injection of secretagogues. These mice produced significantly higher amounts of saliva than did age-matched MRL/+ mice. Lacrimal and salivary gland nerves from 18-week-old MRL/+ mice released acetylcholine in response to a depolarizing KCl solution. In contrast, nerves in glands from 18-week-old MRL/lpr mice did not increase acetylcholine release in response to the depolarizing solution. Moreover, lacrimal glands from 18-week-old MRL/+ mice were able to secrete peroxidase in response to a depolarizing KCl solution, whereas those from 18-week-old MRL/lpr could not. This was not due to a defect in the secretory process, because addition of an exogenous secretagogue elicited peroxidase secretion from 18-week-old MRL/lpr as well as MRL/+ mice lacrimal glands.
Conclusions
The results show that activation of nerves of lacrimal and salivary glands infiltrated with lymphocytes does not increase the release of neurotransmitters, which results in impaired secretion from these glands. (Invest Ophthalmol Vis Sci.
PMCID: PMC3241007  PMID: 11274068
24.  Human β-Defensins 2 and 3 Demonstrate Strain-Selective Activity against Oral Microorganisms 
Journal of Clinical Microbiology  2004;42(3):1024-1029.
Human β-defensins 2 and 3 (HBD-2 and HBD-3) are inducible peptides present at sites of infection in the oral cavity. A few studies have reported broad-spectrum antimicrobial activity for both peptides. However, no comprehensive study has thoroughly investigated their potential against oral pathogens. The purpose of this study was to test the effectiveness of HBD-2 and HBD-3 against a collection of oral organisms (Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Peptostreptococcus micros, Actinomyces naeslundii, Actinomyces israelii, Streptococcus sanguis, Streptococcus mutans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, and Candida albicans). Radial diffusion assays were used to test HBD-2 and HBD-3 activities against at least three strains of each species. There was significant variability in MICs, which was strain specific rather than species specific. MICs ranged from 3.9 to >250 μg/ml for HBD-2 and from 1.4 to >250 μg/ml for HBD-3. HBD-3 demonstrated greater antimicrobial activity and was effective against a broader array of organisms. Overall, aerobes were 100% susceptible to HBD-2 and HBD-3, whereas only 21.4 and 50% of the anaerobes were susceptible to HBD-2 and HBD-3, respectively. HBD-2 and HBD-3 also demonstrated strain-specific activity against the Candida species evaluated. Interestingly, an association between HBD-2 and HBD-3 activities was noted. This suggests that the two peptides may have similar mechanisms yet utilize distinct pathways. The lack of activity against specific anaerobic strains and Candida warrants further investigation of the potential resistance mechanisms of these organisms. Finally, the significant variability between strains underlies the importance of testing multiple strains when evaluating activities of antimicrobial peptides.
doi:10.1128/JCM.42.3.1024-1029.2004
PMCID: PMC356847  PMID: 15004048
25.  Antimicrobial Peptides and Colitis 
Current pharmaceutical design  2013;19(1):40-47.
Antimicrobial peptides (AMPs) are important components of innate immunity. They are often expressed in response to colonic inflammation and infection. Over the last several years, the roles of several antimicrobial peptides have been explored. Gene expression of many AMPs (beta defensin HBD2-4 and cathelicidin) is induced in response to invasion of gut microbes into the mucosal barrier. Some AMPs are expressed in a constitutive manner (alpha defensin HD 5-6 and beta defensin HBD1), while others (defensin and bactericidal/permeability increasing protein BPI) are particularly associated with Inflammatory Bowel Disease (IBD) due to altered defensin expression or development of autoantibodies against Bactericidal/permeability increasing protein (BPI). Various AMPs have different spectrum and strength of antimicrobial effects. Some may play important roles in modulating the colitis (cathelicidin) while others (lactoferrin, hepcidin) may represent biomarkers of disease activity. The use of AMPs for therapeutic purposes is still at an early stage of development. A few natural AMPs were shown to be able to modulate colitis when delivered intravenously or intracolonically (cathelicidin, elafin and SLPI) in mouse colitis models. New AMPs (synthetic or artificial non-human peptides) are being developed and may represent new therapeutic approaches against colitis. This review discusses the latest research developments in the AMP field with emphasis in innate immunity and pathophysiology of colitis.
PMCID: PMC3662473  PMID: 22950497
Antimicrobial peptides; colitis; infection; microflora; protein; Crohn’s disease; ulcerative colitis

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