Beta-defensin index: A functional biomarker for oral cancer detection.

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

Human ß-Defensin-3 is Associated With Platelet-Derived Extracellular Vesicles and is a Potential Contributor to Endothelial Dysfunction.

While platelets are the essential mediators of hemostasis, they are being increasingly recognized for their potential of contributing to host defenses. Here, using immunofluorescent microscopy, western blot, and ELISA, we found that human ß-defensin 3 (hBD-3), an important antimicrobial peptide produced by epithelial cells, can be detected in human platelets and megakaryocytes. Flow cytometry and immuno-electron microscopy revealed hBD-3 on the surface of thrombin activated platelets. Moreover, hBD-3 was also found in platelet derived extracellular vesicles (p-EVs), isolated from platelet poor plasma and from platelet supernatants following thrombin stimulation. Incubation of platelets with hBD-3 peptide resulted in modest platelet activation and pre-incubation of platelets with synthetic hBD-3 prior to exposure to thrombin appeared to increase hBD-3 content in platelet lysates as well as in p-EVs, suggesting that hBD-3 can be initially taken up by platelets, perhaps via their open canalicular system. Interestingly, in vitro exposure of primary human endothelial cells to either hBD-3 peptide or purified p-EVs, caused significant endothelial dysfunction as documented by diminished levels of phosphorylated endothelial nitric oxide synthase (eNOS), Krüppel like factor-2 (KLF-2), and elevated relative expression of von Willebrand Factor (vWF). Pre-incubation of platelets with hBD-3 appeared to augment endothelial dysfunction caused by p-EVs. Overall, the current study provides evidence that hBD-3 enriched EVs can be released by activated platelets and may play a role in positive feedback of platelet activation as well as in endothelial dysfunction. Theoretically, these effects could contribute to both cellular recruitment to the endothelium creating a pro-thrombotic vascular microenvironment which serve as a bridge between innate immunity and hemostasis.

HBD-2 binds SARS-CoV-2 RBD and blocks viral entry: Strategy to combat COVID-19.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19-related deaths and medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell-derived host defense peptide that has anti-viral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical measurements confirm that hBD-2 indeed binds to the CoV-2-receptor-binding domain (RBD) (KD ∼ 2µM by surface plasmon resonance), preventing it from binding to ACE2-expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSVG-mediated infection, of ACE2-expressing human cells with an IC50 of 2.8 ± 0.4 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as agents to prevent SARS-CoV-2 infection.

Ramping Up Antimicrobial Peptides Against Severe Acute Respiratory Syndrome Coronavirus-2.

Human-derived antimicrobial peptides (AMPs), such as defensins and cathelicidin LL-37, are members of the innate immune system and play a crucial role in early pulmonary defense against viruses. These AMPs achieve viral inhibition through a variety of mechanisms including, but not limited to, direct binding to virions, binding to and modulating host cell-surface receptors, blocking viral replication, and aggregation of viral particles and indirectly by functioning as chemokines to enhance or curb adaptive immune responses. Given the fact that we are in a pandemic of unprecedented severity and the urgent need for therapeutic options to combat severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), naturally expressed AMPs and their derivatives have the potential to combat coronavirus disease 2019 (COVID-19) and impede viral infectivity in various ways. Provided the fact that development of effective treatments is an urgent public health priority, AMPs and their derivatives are being explored as potential prophylactic and therapeutic candidates. Additionally, cell-based platforms such as human mesenchymal stem cell (hMSC) therapy are showing success in saving the lives of severely ill patients infected with SARS-CoV-2. This could be partially due to AMPs released from hMSCs that also act as immunological rheostats to modulate the host inflammatory response. This review highlights the utilization of AMPs in strategies that could be implemented as novel therapeutics, either alone or in combination with other platforms, to treat CoV-2-infected individuals.

Molecular dynamics simulations and functional studies reveal that hBD-2 binds SARS-CoV-2 spike RBD and blocks viral entry into ACE2 expressing cells.

New approaches to complement vaccination are needed to combat the spread of SARS-CoV-2 and stop COVID-19 related deaths and long-term medical complications. Human beta defensin 2 (hBD-2) is a naturally occurring epithelial cell derived host defense peptide that has antiviral properties. Our comprehensive in-silico studies demonstrate that hBD-2 binds the site on the CoV-2-RBD that docks with the ACE2 receptor. Biophysical and biochemical assays confirm that hBD-2 indeed binds to the CoV-2-receptor binding domain (RBD) (KD ~ 300 nM), preventing it from binding to ACE2 expressing cells. Importantly, hBD-2 shows specificity by blocking CoV-2/spike pseudoviral infection, but not VSV-G mediated infection, of ACE2 expressing human cells with an IC50 of 2.4± 0.1 µM. These promising findings offer opportunities to develop hBD-2 and/or its derivatives and mimetics to safely and effectively use as novel agents to prevent SARS-CoV-2 infection.

Human Beta Defensins and Cancer: Contradictions and Common Ground.

Human beta-defensins (hBDs, -1, 2, 3) are a family of epithelial cell derived antimicrobial peptides (AMPs) that protect mucosal membranes from microbial challenges. In addition to their antimicrobial activities, they possess other functions; e.g., cell activation, proliferation, regulation of cytokine/chemokine production, migration, differentiation, angiogenesis, and wound healing processes. It has also become apparent that defensin levels change with the development of neoplasia. However, inconsistent observations published by various laboratories make it difficult to reach a consensus as to the direction of the dysregulation and role the hBDs may play in various cancers. This is particularly evident in studies focusing on oral squamous cell carcinoma (OSCC). By segregating each hBD by cancer type, interrogating methodologies, and scrutinizing the subject cohorts used in the studies, we have endeavored to identify the "take home message" for each one of the three hBDs. We discovered that (1) consensus-driven findings indicate that hBD-1 and-2 are down- while hBD-3 is up-regulated in OSCC; (2) hBD dysregulation is cancer-type specific; (3) the inhibition/activation effect an hBD has on cancer cell lines is related to the direction of the hBD dysregulation (up or down) in the cancer from which the cell lines derive. Therefore, studies addressing hBD dysregulation in various cancers are not generalizable and comparisons should be avoided. Systematic delineation of the fate and role of the hBDs in a specific cancer type may lead to innovative ways to use defensins as prospective biomarkers for diagnostic/prognostic purposes and/or in novel therapeutic modalities.

Conceptual Perspectives: Bacterial Antimicrobial Peptide Induction as a Novel Strategy for Symbiosis with the Human Host.

Human beta defensins (hBDs) are small cationic peptides, expressed in mucosal epithelia and important agents of innate immunity, act as antimicrobial and chemotactic agents at mucosal barriers. In this perspective, we present evidence supporting a novel strategy by which the oral bacterium Fusobacterium nucleatum induces hBDs and other antimicrobial peptides (AMPs) in normal human oral epithelial cells (HOECs) and thereby protects them from other microbial pathogens. The findings stress (1) the physiological importance of hBDs, (2) that this strategy may be a mechanism that contributes to homeostasis and health in body sites constantly challenged with bacteria and (3) that novel properties identified in commensal bacteria could, one day, be harnessed as new probiotic strategies to combat colonization of opportunistic pathogens. With that in mind, we highlight and review the discovery and characterization of a novel lipo-protein, FAD-I (Fusobacterium Associated Defensin Inducer) associated with the outer membrane of F. nucleatum that may act as a homeostatic agent by activating endogenous AMPs to re-equilibrate a dysregulated microenvironment. FAD-I has the potential to reduce dysbiosis-driven diseases at a time when resistance to antibiotics is increasing. We therefore postulate that FAD-I may offer a new paradigm in immunoregulatory therapeutics to bolster host innate defense of vulnerable mucosae, while maintaining physiologically responsive states of inflammation.

Kaposi's sarcoma-associated herpesvirus infection promotes differentiation and polarization of monocytes into tumor-associated macrophages.

Tumor associated macrophages (TAMs) promote angiogenesis, tumor invasion and metastasis, and suppression of anti-tumor immunity. These myeloid cells originate from monocytes, which differentiate into TAMs upon exposure to the local tumor microenvironment. We previously reported that Kaposi's sarcoma-associated herpes virus (KSHV) infection of endothelial cells induces the cytokine angiopoietin-2 (Ang-2) to promote migration of monocytes into tumors. Here we report that KSHV infection of endothelial cells induces additional cytokines including interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-13 (IL-13) that drive monocytes to differentiate and polarize into TAMs. The KSHV-induced TAMs not only express TAM-specific markers such as CD-163 and legumain (LGMN) but also display a gene expression profile with characteristic features of viral infection. More importantly, KSHV-induced TAMs enhance tumor growth in nude mice. These results are consistent with the strong presence of TAMs in Kaposi's sarcoma (KS) tumors. Therefore, KSHV infection of endothelial cells generates a local microenvironment that not only promotes the recruitment of monocytes but also induces their differentiation and polarization into TAMs. These findings reveal a new mechanism of KSHV contribution to KS tumor development.

When Mr. Fap Meets the Gals.

Fusobacteria are found to be overrepresented in the colorectal tumor microenvironment. In this issue of Cell Host & Microbe, Abed et al. (2016) describe a novel homing mechanism by which fusobacteria localize to tumors by recognizing a host polysaccharide (Gal-GalNAc) on cancer cells using a fusobacterial lectin, Fap2.

Suppression of KSHV-induced angiopoietin-2 inhibits angiogenesis, infiltration of inflammatory cells, and tumor growth.

Kaposi's sarcoma (KS) is a highly angiogenic and inflammatory neoplasia. The angiogenic and inflammatory cytokine angiopoietin-2 (Ang-2) is strongly expressed in KS due to Kaposi's sarcoma-associated herpesvirus (KSHV) infection. In the present study, we determined how Ang-2 contributes to development of KS by using telomerase-immortalized human umbilical vein endothelial cells (TIVE) as a model, which become malignantly transformed and express increased levels of Ang-2 following KSHV infection. Ang-2 released from TIVE-KSHV cells induces tyrosine phosphorylation of Tie-2 receptor from both human and mouse endothelial cells and promotes angiogenesis in nude mice. Functional inhibition or expressional "knock-down" of Ang-2 in these cells blocks angiogenesis and inhibits tumor growth. Ang-2 suppression also reduces the numbers of infiltrating monocytes/macrophages in tumors. In transwell-based cell migration assays, Ang-2 indeed enhances migration of human monocytes in a dose-dependent manner. These results underscore a pivotal role of KSHV-induced Ang-2 in KS tumor development by promoting both angiogenesis and inflammation. Our data also suggest that selective drug targeting of Ang-2 may be used for treatment of KS.

Human papillomavirus oncogenic E6 protein regulates human ß-defensin 3 (hBD3) expression via the tumor suppressor protein p53.

Human ß-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer.

Antimicrobial Properties of Mesenchymal Stem Cells: Therapeutic Potential for Cystic Fibrosis Infection, and Treatment.

Cystic fibrosis (CF) is a genetic disease in which the battle between pulmonary infection and inflammation becomes the major cause of morbidity and mortality. We have previously shown that human MSCs (hMSCs) decrease inflammation and infection in the in vivo murine model of CF. The studies in this paper focus on the specificity of the hMSC antimicrobial effectiveness using Pseudomonas aeruginosa (gram negative bacteria) and Staphylococcus aureus (gram positive bacteria). Our studies show that hMSCs secrete bioactive molecules which are antimicrobial in vitro against Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumonia, impacting the rate of bacterial growth and transition into colony forming units regardless of the pathogen. Further, we show that the hMSCs have the capacity to enhance antibiotic sensitivity, improving the capacity to kill bacteria. We present data which suggests that the antimicrobial effectiveness is associated with the capacity to slow bacterial growth and the ability of the hMSCs to secrete the antimicrobial peptide LL-37. Lastly, our studies demonstrate that the tissue origin of the hMSCs (bone marrow or adipose tissue derived), the presence of functional cystic fibrosis transmembrane conductance regulator (CFTR: human, Cftr: mouse) activity, and response to effector cytokines can impact both hMSC phenotype and antimicrobial potency and efficacy. These studies demonstrate, the unique capacity of the hMSCs to manage different pathogens and the significance of their phenotype in both the antimicrobial and antibiotic enhancing activities.

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6.

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

Modification of ß-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro.

BACKGROUND: Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human ß-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO). METHODS: The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells. RESULTS: MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg22 and Arg23 near the C-terminus, and the N-terminal glycine (Gly1). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO. CONCLUSIONS: Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.

Epithelial innate immune response to Acinetobacter baumannii challenge.

Currently, Acinetobacter baumannii is recognized as one of the major pathogens seriously threatening our health care delivery system. Aspects of the innate immune response to A. baumannii infection are not yet well understood. Human ß-defensins (hBDs) are epithelial cell-derived cationic antimicrobial peptides (AMPs) that also function to bridge the innate and adaptive immune system. We tested the induction of hBD-2 and -3 by A. baumannii on primary oral and skin epithelial cells and found that A. baumannii induces hBD-3 transcripts to a greater extent than it induces hBD-2 transcripts on both types of cells. In addition, we found that A. baumannii is susceptible to hBD-2 and -3 killing at submicromolar concentrations. Moreover, hBD-3 induction by A. baumannii was found to be dependent on epidermal growth factor receptor (EGFR) signaling. Inhibition of mitogen-activated protein kinase resulted in reduced expression of both hBD-2 and -3. Lastly, a disintegrin and metalloprotease 17 (ADAM17; also known as TACE) was found to be critical for hBD-3 induction, while ADAM10 and dual oxidase 1 (Duox1) were not required for hBD-3 induction. Induction of AMPs is an important component of innate sensing of pathogens and may play an important role in triggering systemic immune responses to A. baumannii infection. Further studies on the interactions between epithelial cells and A. baumannii will help us understand early stages of infection and may shed light on why some individuals are more vulnerable to A. baumannii infection.

Comparison of epigenetic profiles of human oral epithelial cells from HIV-positive (on HAART) and HIV-negative subjects.

HIV-infected subjects on highly active antiretroviral therapy (HAART) are susceptible to comorbid microbial infections in the oral cavity. We observed that primary oral epithelial cells (POECs) isolated from HIV+ subjects on HAART grow more slowly and are less innate immune responsive to microbial challenge when compared with POECs from normal subjects. These aberrant cells also demonstrate epigenetic differences that include reduction in histone deacetylase 1 (HDAC-1) levels and reduced total DNA methyltransferase (DNMT) activity specific to enzymes DNMT1 and DNMT3A. The DNMT activity correlates well with global DNA methylation, indicating that aberrant DNMT activity in HIV+ (on HAART) POECs leads to an aberrantly methylated epithelial cell phenotype. Overall, our results lead us to hypothesize that, in patients with chronic HIV infection on HAART, epigenetic changes in key genes result in increased vulnerability to microbial infection in the oral cavity.

Human ß-defensin 3 peptide is increased and redistributed in Crohn's ileitis.

BACKGROUND: Antimicrobial peptides (AMPs) maintain a sterile environment in intestinal crypts, limiting microbial colonization and invasion. Decreased AMP expression is proposed to increase the risk for inflammatory bowel disease. Expression and function of inducible AMPs, human ß-defensin 2 and 3 (hBD-2 and hBD-3), remain poorly characterized in healthy and chronically inflamed intestine. METHODS: Peptide concentrations of hBD-2 and hBD-3 in serum and intestinal biopsies of subjects with ulcerative colitis and Crohn's disease (CD), and those of healthy subjects were measured by ELISA. Messenger RNA of hBD-2 and hBD-3 was quantified by quantitative PCR in biopsies from the terminal ileum (TI) of patients with CD and healthy controls. Peptide localization of hBD-3 in the TI was visualized by confocal microscopy. RESULTS: Immunoreactive hBD-3 peptide is present in the TI and colon in healthy subjects. In the TI of patients with CD, hBD-3, but not hBD-2 peptide, is increased 4-fold, whereas hBD-2 peptide is elevated in the serum. Messenger RNA of hBD-3 in the CD TI remains unchanged and does not correlate with hBD-3 peptide expression. However, hBD-3 is localized to Paneth cell granules and the apical surface of the healthy columnar epithelium. In CD, hBD-3 peptide location switches to the basolateral surface of the columnar epithelium and is diffusely distributed within the lamina propria. CONCLUSION: The peptide hBD-3 throughout the healthy gastrointestinal tract suggests a role in maintaining balance between host defenses and commensal microbiota. Increased and relocalized secretion of hBD-3 toward the lamina propria in the CD TI indicates possible local immunomodulation during chronic inflammation, whereas increased serum hBD-2 in CD implicates its systemic antimicrobial and immunomodulatory role.

Proteomic and bioinformatic profile of primary human oral epithelial cells.

Wounding of the oral mucosa occurs frequently in a highly septic environment. Remarkably, these wounds heal quickly and the oral cavity, for the most part, remains healthy. Deciphering the normal human oral epithelial cell (NHOEC) proteome is critical for understanding the mechanism(s) of protection elicited when the mucosal barrier is intact, as well as when it is breached. Combining 2D gel electrophoresis with shotgun proteomics resulted in identification of 1662 NHOEC proteins. Proteome annotations were performed based on protein classes, molecular functions, disease association and membership in canonical and metabolic signaling pathways. Comparing the NHOEC proteome with a database of innate immunity-relevant interactions (InnateDB) identified 64 common proteins associated with innate immunity. Comparison with published salivary proteomes revealed that 738/1662 NHOEC proteins were common, suggesting that significant numbers of salivary proteins are of epithelial origin. Gene ontology analysis showed similarities in the distributions of NHOEC and saliva proteomes with regard to biological processes, and molecular functions. We also assessed the interindividual variability of the NHOEC proteome and observed it to be comparable with other primary cells. The baseline proteome described in this study should serve as a resource for proteome studies of the oral mucosa, especially in relation to disease processes.

Bacterial colonization and beta defensins in the female genital tract in HIV infection.

Beta defensins are antimicrobial peptides that serve to protect the host from microbial invasion at skin and mucosal surfaces. Here we explore the relationships among beta defensin levels, total bacterial colonization, and colonization by bacterial vaginosis (BV)-related bacteria and lactobacilli in the female genital tract in HIV infected women and healthy controls. Cervicovaginal lavage (CVL) samples were obtained from 30 HIV-infected women and 36 uninfected controls. Quantitative PCR assays were used to measure DNA levels of bacterial 16S ribosomal DNA (reflective of total bacterial load), and levels of three BV-related bacteria, three Lactobacillus species (L. crispatus, L. iners and L. jensenii), and total Lactobacillus levels in CVL. Levels of human beta defensins (hBD-2 and hBD-3) were quantified by ELISA. In viremic HIV+ donors, we found that CVL levels of bacterial 16S rDNA were significantly increased, and inversely correlated with peripheral CD4+ T cell counts in HIV+ women, and inversely correlated with age in both HIV+ women and controls. Although CVL DNA levels of BV-associated bacteria tended to be increased, and CVL levels of Lactobacillus DNAs tended to be decreased in HIV+ donors, none of these differences was significant. CVL levels of hBD-2 and hBD-3 were correlated and were not different in HIV+ women and controls. However, significant positive correlations between hBD-3 levels and total bacterial DNA levels in controls were not demonstrable in HIV+ women; the significant positive correlations of hBD2 or hBD-3 and three Lactobacillus species in controls were also not demonstrable in HIV+ women. These results suggest that HIV infection is associated with impaired regulation of innate defenses at mucosal sites.

Proteomic signatures of human oral epithelial cells in HIV-infected subjects.

The oral epithelium, the most abundant structural tissue lining the oral mucosa, is an important line of defense against infectious microorganisms. HIV infected subjects on highly active antiretroviral therapy (HAART) are susceptible to comorbid viral, bacterial and fungal infections in the oral cavity. To provide an assessment of the molecular alterations of oral epithelia potentially associated with susceptibility to comorbid infections in such subjects, we performed various proteomic studies on over twenty HIV infected and healthy subjects. In a discovery phase two Dimensional Difference Gel Electrophoresis (2-D DIGE) analyses of human oral gingival epithelial cell (HOEC) lysates were carried out; this identified 61 differentially expressed proteins between HIV-infected on HAART subjects and healthy controls. Down regulated proteins in HIV-infected subjects include proteins associated with maintenance of protein folding and pro- and anti-inflammatory responses (e.g., heat-shock proteins, Cryab, Calr, IL-1RA, and Galectin-3-binding protein) as well as proteins involved in redox homeostasis and detoxification (e.g., Gstp1, Prdx1, and Ero1). Up regulated proteins include: protein disulfide isomerases, proteins whose expression is negatively regulated by Hsp90 (e.g., Ndrg1), and proteins that maintain cellular integrity (e.g., Vimentin). In a verification phase, proteins identified in the protein profiling experiments and those inferred from Ingenuity Pathway Analysis were analyzed using Western blotting analysis on separate HOEC lysate samples, confirming many of the discovery findings. Additionally in HIV-infected patient samples Heat Shock Factor 1 is down regulated, which explains the reduced heat shock responses, while activation of the MAPK signal transduction cascade is observed. Overall, HAART therapy provides an incomplete immune recovery of the oral epithelial cells of the oral cavity for HIV-infected subjects, and the toxic side effects of HAART and/or HIV chronicity silence expression of multiple proteins that in healthy subjects function to provide robust innate immune responses and combat cellular stress.