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1.  Short-Chain Fatty Acids from Periodontal Pathogens Suppress Histone Deacetylases, EZH2, and SUV39H1 To Promote Kaposi's Sarcoma-Associated Herpesvirus Replication 
Journal of Virology  2014;88(8):4466-4479.
Periodontal pathogens such as Porphyromonas gingivalis and Fusobacterium nucleatum produce five different short-chain fatty acids (SCFAs) as metabolic by-products. We detect significantly higher levels of SCFAs in the saliva of patients with severe periodontal disease. The different SCFAs stimulate lytic gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) dose dependently and synergistically. SCFAs inhibit class-1/2 histone deacetylases (HDACs) and downregulate expression of silent information regulator-1 (SIRT1). SCFAs also downregulate expression of enhancer of zeste homolog2 (EZH2) and suppressor of variegation 3-9 homolog1 (SUV39H1), which are two histone N-lysine methyltransferases (HLMTs). By suppressing the different components of host epigenetic regulatory machinery, SCFAs increase histone acetylation and decrease repressive histone trimethylations to transactivate the viral chromatin. These new findings provide mechanistic support that SCFAs from periodontal pathogens stimulate KSHV replication and infection in the oral cavity and are potential risk factors for development of oral Kaposi's sarcoma (KS).
IMPORTANCE About 20% of KS patients develop KS lesions first in the oral cavity, while other patients never develop oral KS. It is not known if the oral microenvironment plays a role in oral KS tumor development. In this work, we demonstrate that a group of metabolic by-products, namely, short-chain fatty acids, from bacteria that cause periodontal disease promote lytic replication of KSHV, the etiological agent associated with KS. These new findings provide mechanistic support that periodontal pathogens create a unique microenvironment in the oral cavity that contributes to KSHV replication and development of oral KS.
PMCID: PMC3993761  PMID: 24501407
2.  Variation in human β-defensin genes: new insights from a multi-population study 
Human β-defensin 2 (hBD-2) and hBD-3, encoded by DEFB4 and DEFB103A, respectively, have shown anti-HIV activity, and both genes exhibit copy number variation (CNV). Although the role of hBD-1, encoded by DEFB1, in HIV-1 infection is less clear, single nucleotide polymorphisms (SNPs) in DEFB1 may influence viral loads and disease progression. We examined the distribution of DEFB1 SNPs and DEFB4/103A CNV, and the relationship between DEFB1 SNPs and DEFB4/103A CNV using samples from two HIV/AIDS cohorts from the United States (n = 150) and five diverse populations from the Coriell Cell Repositories (n = 46). We determined the frequencies of 10 SNPs in DEFB1 by using a post-PCR, oligonucleotide ligation detection reaction-fluorescent microsphere assay, and CNV in DEFB4/103A by real-time quantitative PCR. There were noticeable differences in the frequencies of DEFB1 SNP alleles and haplotypes among various racial/ethnic groups. The DEFB4/103A copy numbers varied from 2 to 8 (median, 4), and there was a significant difference between the copy numbers of self-identified whites and blacks in the US cohorts (Mann-Whitney U test p = 0.04). A significant difference was observed in the distribution of DEFB4/103A CNV among DEFB1 -52G/A and -390T/A genotypes (Kruskal-Wallis p = 0.017 and 0.026, respectively), while not in the distribution of DEFB4/103A CNV among -52G/A_-44C/G_-20G/A diplotypes. These observations provide additional insights for further investigating the complex interplay between β-defensin genetic polymorphisms and susceptibility to, or the progression or severity of, HIV infection/disease.
PMCID: PMC3664661  PMID: 23194186
3.  HBD-3 structure motifs important in CXCR4 antagonism 
The FEBS journal  2013;280(14):10.1111/febs.12328.
Previously, we reported that hBD-3 can both antagonize CXCR4 function on T cells, and promote receptor internalization in the absence of activation. In the present study, we explored the important structural elements of hBD-3 that are involved in blocking CXCR4 activation by its natural ligand, stromal derived factor 1α (SDF-1α; CXCL12). Results from site-directed mutagenesis studies suggest that the ability of hBD-3 to inhibit SDF-1α/CXCR4 interaction, as assayed either by blocking SDF-1 binding to CXCR4 or antagonizing SDF-1 induced Ca2+ mobilization, is correlated with the presence of hBD-3 cysteine residues, specific surface-distributed cationic residues, and the electrostatic properties and availability of both hBD-3 termini. Specifically, hBD-3 activity against CXCR4 is reduced by: 1) substituting all six cysteine residues; 2) substituting the cationic residues with acidic ones in the N- and C- termini; 3) removal of the first 10 N-terminal residues; and 4) substituting surface-exposed basic residues K8, K32 and R36 with neutral ones. The hBD-3/CXCR4 interaction has potentially wide ranging implications for HIV-related biology as well as for a host of CXCR4-dependent activities including hematopoiesis, neurogenesis, angiogenesis, carcinogenesis, and immune cell trafficking. CXCR4 is highly expressed on T cells, monocytes, and epithelial cells. Therefore, understanding the structure-function relationship between hBD-3 and CXCR4 that accounts for the antagonistic interaction between the two molecules may provide new insights into HIV/HAART-related pathology as well as novel insights into the interaction between innate and adaptive immunity at mucosal sites.
PMCID: PMC3831652  PMID: 23659571
Defensin; HBD-3; CXCR4; structure; antagonism
4.  Dorsal penile nerve block for robot-assisted radical prostatectomy catheter related pain: a randomized, double-blind, placebo-controlled trial 
SpringerPlus  2014;3:181.
Following Robotic-Assisted Radical Prostatectomy (RARP) patients routinely have penile pain and urethral discomfort secondary to an indwelling urethral catheter. Our objective was to assess the effect of dorsal penile nerve block with bupivacaine on urethral catheter-related pain after RARP.
From 2012–2013, 140 patients with organ-confined prostate cancer were enrolled in an IRB approved double-blinded, randomized control trial comparing a dorsal penile nerve block of bupivacaine versus placebo after RARP performed by a single-surgeon. Patients were asked to complete questionnaires using the Wong-Bakers FACES Pain Rating scale while hospitalized and for 9 days post-operatively, until the catheter was removed. The primary end-points were: catheter-related discomfort, abdominal (incisional) pain, and bladder spasm-related discomfort. Secondary end-points included narcotic and other analgesic usage.
120 patients were randomized to placebo vs. bupivacaine dorsal penile nerve bock. The two arms (n = 56 bupivacaine and n = 60 placebo) did not differ in preoperative, perioperative, or pathological results. There was no difference in narcotic utilization between the two cohorts. Abdominal pain was slightly lower in the bupivacaine arm at 6 hours compared to the placebo arm, but there was no difference in abdominal pain at other time points, and there were no differences in reported catheter-related discomfort or bladder spasm-associated discomfort at any of the measured time points.
The data does not support the routine use of a dorsal penile nerve block with bupivacaine following RARP.
PMCID: PMC4004790  PMID: 24790826
Postoperative pain; Prostate neoplasm; Analgesia; Prostatectomy; Urinary catheterization
5.  Human β-Defensin 3 Peptide is Increased and Redistributed in Crohn's Ileitis 
Inflammatory bowel diseases  2013;19(5):942-953.
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 β-defensin 2 and 3 (hBD-3), remain poorly characterized in healthy and chronically inflamed intestine.
hBD-2 and hBD-3 peptide concentrations in serum and intestinal biopsies of ulcerative colitis, Crohn's Disease (CD), and healthy subjects were measured by ELISA. HBD-2 and hBD-3 mRNA was quantified by qPCR in biopsies from the terminal ileum (TI) of CD patients and healthy controls. HBD-3 peptide localization in the TI was visualized by confocal microscopy.
Immunoreactive hBD-3 peptide is present in the TI and colon in healthy subjects. In the TI of CD patients, hBD-3, but not hBD-2 peptide, is increased four-fold, whereas hBD-2 peptide is elevated in the serum. hBD-3 mRNA in the CD TI remains unchanged and does not correlate with hBD-3 peptide expression. 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.
hBD-3 peptide 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, while increased serum hBD-2 in CD implicates its systemic antimicrobial and immunomodulatory role.
PMCID: PMC3746836  PMID: 23511030
beta-defensins; inflammatory bowel disease; Crohn's Disease; immunomodulation; epithelium; innate immunity
6.  Isolation of T cells from mouse oral tissues 
Utilizing mouse models provides excellent immunological and experimental tools to study oral immune responses. However for functional assays, isolating T lymphocytes from the oral tissues has proved to be challenging due to the absence of reliable methods that yield viable cells with consistency. To study adaptive immune cell interactions in the oral mucosal tissues, it is necessary to isolate T cells with a good viability and study them at the single cell level.
We have established an improved method to isolate immune cells, including Tregs and Th17 cells from intra-epithelial niches and lamina propria of the tongue, gingival and palatal tissues in the oral mucosa of mice.
This new method of isolating immune cells from oral tissues will enable us to further our understanding of oral tissue immune cells and their role during oral infections and oral inflammation.
PMCID: PMC3984730  PMID: 24612879
Murine oral tissue; Leukocyte isolation and oral T cells; Treg; Th17; ILC
7.  Kaposi's Sarcoma-Associated Herpesvirus Induces Rapid Release of Angiopoietin-2 from Endothelial Cells 
Journal of Virology  2013;87(11):6326-6335.
Kaposi sarcoma-associated herpesvirus (KSHV) stimulates proliferation, angiogenesis, and inflammation to promote Kaposi sarcoma (KS) tumor growth, which involves various growth factors and cytokines. Previously, we found that KSHV infection of human umbilical vein endothelial cells (HUVECs) induces a transcriptional induction of the proangiogenic and proinflammatory cytokine angiopoietin-2 (Ang-2). Here, we report that KSHV induces rapid release of Ang-2 that is presynthesized and stored in the Weibel-Palade bodies (WPB) of endothelial cells upon binding to its integrin receptors. Blocking viral binding to integrins inhibits Ang-2 release. KSHV binding activates the integrin tyrosine kinase receptor signaling pathways, leading to tyrosine phosphorylation of focal adhesion kinase (FAK), the tyrosine kinase Src, and the Calα2 subunit of the l-type calcium channel to trigger rapid calcium (Ca2+) influx. Pretreatment of endothelial cells with specific inhibitors of protein tyrosine kinases inhibits KSHV-induced Ca2+ influx and Ang-2 release. Inhibition of Ca2+ mobilization with calcium channel blockers also inhibits Ang-2 release. Thus, the interaction between KSHV and its integrin receptors plays a key role in regulating rapid Ang-2 release from endothelial cells. This finding highlights a novel mechanism of viral induction of angiogenesis and inflammation, which might play important roles in the early event of KS tumor development.
PMCID: PMC3648120  PMID: 23536671
8.  Epithelial cell-derived antimicrobial peptides are multi-functional agents that bridge innate and adaptive immunity 
Periodontology 2000  2010;54(1):10.1111/j.1600-0757.2010.00373.x.
PMCID: PMC3816379  PMID: 20712640
9.  Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells 
Journal of proteome research  2012;11(11):5492-5502.
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 inter-individual 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.
PMCID: PMC3508721  PMID: 23035736
Primary human oral epithelial cells; proteomics; inter-individual variability; innate immunity; saliva
10.  Effect of Nadir CD4+ T Cell Count on Clinical Measures of Periodontal Disease in HIV+ Adults before and during Immune Reconstitution on HAART  
PLoS ONE  2013;8(10):e76986.
The contribution of HIV-infection to periodontal disease (PD) is poorly understood.  We proposed that immunological markers would be associated with improved clinical measures of PD.
We performed a longitudinal cohort study of HIV-infected adults who had started highly active antiretroviral therapy (HAART) <2 years. PD was characterized clinically as the percent of teeth with ≥1 site with periodontal probing depth (PPD) ≥5.0mm, recession (REC) >0mm, clinical attachment level (CAL) ≥4.0mm, and bleeding on probing (BOP) at ≥4 sites/tooth and microbiologically as specific periodontopathogen concentration. Linear mixed-effects models were used to assess the associations between immune function and PD.
Forty (40) subjects with median 2.7 months on HAART and median nadir CD4+ T-cell count of 212 cells/μl completed a median 3 visits. Over 24 months, CD4+ T-cell count increased by a mean 173 cells/µl (p<0.001) and HIV RNA decreased by 0.5 log10 copies/ml (p<0.001); concurrently, PPD, CAL and BOP decreased by a mean 11.7%, 12.1%, and 14.7% respectively (all p<0.001). Lower nadir CD4+ T-cell count was associated with worse baseline REC (-6.72%; p=0.04) and CAL (9.06%; p<0.001). Further, lower nadir CD4+ T-cell count was associated with a greater relative longitudinal improvement in PPD in subjects with higher baseline levels of Porphyromonas gingivalis (p=0.027), and BOP in subjects with higher baseline levels of Porphyromonas gingivalis or Treponema denticola (p=0.001 and p=0.006 respectively). Longitudinal changes from baseline in CD4+ T-cell count and level of HIV RNA were not independently associated with longitudinal changes in any clinical markers of PD.
Degree of immunosuppression was associated with baseline gingival recession. After HAART initiation, measures of active PD improved most in those with lower nadir CD4+ T-cell counts and higher baseline levels of specific periodontopathogens. Nadir CD4+ T-cell count differentially influences periodontal disease both before and after HAART in HIV-infected adults.
PMCID: PMC3795634  PMID: 24146949
11.  Mucosal Transmission of Human Immunodeficiency Virus 
Current HIV research  2012;10(1):3-8.
Since the beginning of the AIDS pandemic, and following the discovery of the human immunodeficiency virus (HIV) as the etiological agent of the disease, it was clear that the virus gains access to the human host predominantly through the mucosal tissue after sexual exposure. As a consequence, the female genital tract (vaginal and cervical), as well as the rectal, penile, and oral mucosae have been extensively studied over the last thirty years towards a better understanding of - and to develop strategies to prevent - sexual HIV transmission. This review seeks to describe the biology of the events leading to HIV infection through the human mucosa and introduce some of the approaches attempted to prevent the sexual transmission of HIV.
PMCID: PMC3744389  PMID: 22264040
12.  Comparison of epigenetic profiles of human oral epithelial cells from HIV-positive (on HAART) and HIV-negative subjects 
Epigenetics  2013;8(7):703-709.
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.
PMCID: PMC3781189  PMID: 23804146
oral epithelium; HIV; HAART; DNMTs; HDAC-1; hBD-2
13.  Nutlin-3 induces apoptosis, disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells 
Cell Cycle  2012;11(7):1393-1399.
Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G1 cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. these results suggest that Nutlin-3 might serve as a novel therapy for KS.
PMCID: PMC3350880  PMID: 22421142
Kaposi sarcoma (KS); nutlin-3; p53; cell cycle arrest; apoptosis; angiopoietin-2
14.  Copy Number Variation within Human β-Defensin Gene Cluster Influences Progression to AIDS in the Multicenter AIDS Cohort Study 
Study background
DEFB4/103A encoding β-defensin 2 and 3, respectively, inhibit CXCR4-tropic (X4) viruses in vitro. We determined whether DEFB4/103A Copy Number Variation (CNV) influences time-to-X4 and time-to-AIDS outcomes.
We utilized samples from a previously published Multicenter AIDS Cohort Study (MACS), which provides longitudinal account of viral tropism in relation to the full spectrum of rates of disease progression. Using traditional models for time-to-event analysis, we investigated association between DEFB4/103A CNV and the two outcomes, and interaction between DEFB4/103A CNV and disease progression groups, Fast and Slow.
Time-to-X4 and time-to-AIDS were weakly correlated. There was a stronger relationship between these two outcomes for the fast progressors. DEFB4/103A CNV was associated with time-to-AIDS, but not time-to-X4. The association between higher DEFB4/103A CNV and time-to-AIDS was more pronounced for the slow progressors.
DEFB4/103A CNV was associated with time-to-AIDS in a disease progression group-specific manner in the MACS cohort. Our findings may contribute to enhancing current understanding of how genetic predisposition influences AIDS progression.
PMCID: PMC3610425  PMID: 23543857
AIDS progression; β-Defensin; DEFB4; DEFB103A; MACS; X4 emergence
15.  A Prospective Cohort Study of Periodontal Disease Measures and Cardiovascular Disease Markers in HIV-Infected Adults 
AIDS Research and Human Retroviruses  2011;27(11):1157-1166.
The determinants of HIV-associated cardiovascular disease (CVD) are not well understood. Periodontal disease (PD) has been linked to CVD but this connection has not been examined in HIV infection. We followed a cohort of HIV-infected adults to ascertain whether PD was associated with carotid artery intima media thickness (IMT) and brachial artery flow-mediated dilation (FMD). We performed a longitudinal observational study of HIV-infected adults on HAART for <2 years with no known heart disease. PD was characterized clinically and microbiologically. Cardiovascular disease was assessed by IMT/FMD. Linear mixed models assessed cross-sectional and longitudinal associations between PD and FMD/IMT. Forty three HIV+ adults completed a median of 24 (6–44) months on the study. Defining delta to be the change in a variable between baseline and a follow-up time, longitudinally, on average and after adjusting for change in time, CVD-specific and HIV-specific potential confounding covariates, a 1-log10 increase in delta Porphyromonas gingivalis was associated with a 0.013 mm increase in delta IMT (95% CI: 0.0006–0.0262; p=0.04). After adjusting for the same potential confounding covariates, a 10% increase in delta gingival recession was associated with a 2.3% increase in delta FMD (95% CI: 0.4–4.2; p=0.03). In a cohort of HIV-infected adults, an increase in subgingival Porphyromonas gingivalis, a known periodontal pathogen, was significantly associated with longitudinal increases in IMT, while increased gingival recession, which herein may represent PD resolution, was significantly associated with longitudinal improvement in FMD. In the context of HIV infection, PD may contribute to CVD risk. Intervention studies treating PD may help clarify this association.
PMCID: PMC3206743  PMID: 21443451
16.  Bacterial Colonization and Beta Defensins in the Female Genital Tract in HIV Infection 
Current HIV research  2012;10(6):504-512.
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.
PMCID: PMC3427638  PMID: 22716110
HIV; Bacterial 16S rDNA; beta defensins; Cervicovaginal lavage
17.  Fusobacterium nucleatum and Human Beta-Defensins Modulate the Release of Antimicrobial Chemokine CCL20/Macrophage Inflammatory Protein 3α ▿ † 
Infection and Immunity  2011;79(11):4578-4587.
Cells of the innate immune system regulate immune responses through the production of antimicrobial peptides, chemokines, and cytokines, including human beta-defensins (hBDs) and CCL20. In this study, we examined the kinetics of primary human oral epithelial cell (HOEC) production of CCL20 and hBDs in response to Fusobacterium nucleatum, a commensal bacterium of the oral cavity, which we previously showed promotes HOEC induction of hBDs. HOECs secrete maximal levels of CCL20 at 6 h, following stimulation by F. nucleatum cell wall (FnCW). The kinetics of CCL20 release is distinct from that of hBD-2 and -3, which peaks after 24 h and 48 h of FnCW stimulation, respectively. FnCW-induced release of CCL20 by HOECs requires both transcriptional and translational activation. Release of CCL20 by HOECs is inhibited by brefeldin A, suggesting that it is secreted through a vesicle transport pathway. Other epithelium-derived agents that FnCW induces, such as hBD-2, hBD-3, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), are also able to release CCL20. By focusing on mitogen-activated protein kinases, we show that both extracellular signal-regulated kinase 1/2 and p38, but not JNK, are required for hBD-, TNF-α-, and IL-1β-induced secretion of CCL20 by HOECs. The ability of FnCW and its induced hBDs to produce proinflammatory cytokines and CCL20 suggests the broad role of F. nucleatum and human antimicrobial peptides in primary immune responses elicited by oral epithelium.
PMCID: PMC3257922  PMID: 21911466
20.  Do β-Defensins and Other Antimicrobial Peptides Play a Role in Neuroimmune Function and Neurodegeneration? 
The Scientific World Journal  2012;2012:905785.
It is widely accepted that the brain responds to mechanical trauma and development of most neurodegenerative diseases with an inflammatory sequelae that was once thought exclusive to systemic immunity. Mostly cationic peptides, such as the β-defensins, originally assigned an antimicrobial function are now recognized as mediators of both innate and adaptive immunity. Herein supporting evidence is presented for the hypothesis that neuropathological changes associated with chronic disease conditions of the CNS involve abnormal expression and regulatory function of specific antimicrobial peptides. It is also proposed that these alterations exacerbate proinflammatory conditions within the brain that ultimately potentiate the neurodegenerative process.
PMCID: PMC3346844  PMID: 22606066
21.  The Yin and Yang of Human Beta-Defensins in Health and Disease 
Rapidly evolving research examining the extended role of human beta-defensins (hBDs) in chemoattraction, innate immune-mediated response, and promotion of angiogenesis suggest that the collective effects of hBDs extend well beyond their antimicrobial mechanism(s). Indeed, the numerous basic cellular functions associated with hBDs demonstrate that these peptides have dual impact on health, as they may be advantageous under certain conditions, but potentially detrimental in others. The consequences of these functions are reflected in the overexpression of hBDs in diseases, such as psoriasis, and recently the association of hBDs with pro-tumoral signaling. The mechanisms regulating hBD response in health and disease are still being elucidated. Clearly the spectrum of function now attributed to hBD regulation identifies these molecules as important cellular regulators, whose appropriate expression is critical for proper immune surveillance; i.e., expression of hBDs in proximity to areas of cellular dysregulation may inadvertently exacerbate disease progression. Understanding the mechanism(s) that regulate contextual signaling of hBDs is an important area of concentration in our laboratories. Using a combination of immunologic, biochemical, and molecular biologic approaches, we have identified signaling pathways associated with hBD promotion of immune homeostasis and have begun to dissect the inappropriate role that beta-defensins may assume in disease.
PMCID: PMC3465815  PMID: 23060878
human beta defensins; epithelial cells; carcinoma; monocytes; gingiva
22.  Proteomic Signatures of Human Oral Epithelial Cells in HIV-Infected Subjects 
PLoS ONE  2011;6(11):e27816.
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.
PMCID: PMC3218055  PMID: 22114700
23.  Determinants of Protection among High Risk HIV Seronegative Persons – an overview 
The Journal of infectious diseases  2010;202(Suppl 3):S333-S338.
Both clinical experience and a growing medical literature indicate that there are persons who have been exposed to HIV infection who have remained uninfected. While in some instances this may represent good fortune, cohorts of uninfected persons have been reported where risks for infection are thought to be high. In these cohorts a variety of characteristics have been proposed as mediating protection but to date only the 32 base pair deletion in the CCR5 gene that results in complete failure of cell surface expression of this co-receptor has been associated with high level protection from HIV infection. With this in mind, there are likely numerous other factors that may individually or in combination provide some level of protection from acquisition of HIV infection. As some of these factors are likely incompletely protective or inconsistently active, identifying them with confidence will be difficult. Nonetheless, clarifying the determinants of protection against HIV infection is a high priority that will require careful selection of high risk uninfected cohorts to which targeted studies of plausible mediators and broad screening for unexpected determinants of protection should be applied.
PMCID: PMC3184646  PMID: 20887220
HIV infection; Exposed Seronegatives; High Risk Seronegatives; Interferon; Restriction factors; CCR5
24.  Multiplex Immunoassay of Lower Genital Tract Mucosal Fluid from Women Attending an Urban STD Clinic Shows Broadly Increased IL1ß and Lactoferrin 
PLoS ONE  2011;6(5):e19560.
More than one million new cases of sexually transmitted diseases (STDs) occur each day. The immune responses and inflammation induced by STDs and other frequent non-STD microbial colonizations (i.e. Candida and bacterial vaginosis) can have serious pathologic consequences in women including adverse pregnancy outcomes, infertility and increased susceptibility to infection by other pathogens. Understanding the types of immune mediators that are elicited in the lower genital tract by these infections/colonizations can give important insights into the innate and adaptive immune pathways that are activated and lead to strategies for preventing pathologic effects.
Methodology/Principal Findings
32 immune mediators were measured by multiplexed immunoassays to assess the immune environment of the lower genital tract mucosa in 84 women attending an urban STD clinic. IL-3, IL-1ß, VEGF, angiogenin, IL-8, ß2Defensin and ß3Defensin were detected in all subjects, Interferon-α was detected in none, while the remaining mediators were detected in 40% to 93% of subjects. Angiogenin, VEGF, FGF, IL-9, IL-7, lymphotoxin-α and IL-3 had not been previously reported in genital mucosal fluid from women. Strong correlations were observed between levels of TNF-α, IL-1ß and IL-6, between chemokines IP-10 and MIG and between myeloperoxidase, IL-8 and G-CSF. Samples from women with any STD/colonization had significantly higher levels of IL-8, IL-3, IL-7, IL-1ß, lactoferrin and myeloperoxidase. IL-1ß and lactoferrin were significantly increased in gonorrhea, Chlamydia, cervicitis, bacterial vaginosis and trichomoniasis.
These studies show that mucosal fluid in general appears to be an environment that is rich in immune mediators. Importantly, IL-1ß and lactoferrin are biomarkers for STDs/colonizations providing insights into immune responses and pathogenesis at this mucosal site.
PMCID: PMC3091877  PMID: 21572958
25.  Protein Modification by Dicarbonyl Molecular Species in Neurodegenerative Diseases 
Journal of Amino Acids  2011;2011:461216.
Neurodegeneration results from abnormalities in cerebral metabolism and energy balance within neurons, astrocytes, microglia, or microvascular endothelial cells of the blood-brain barrier. In Alzheimer's disease, β-amyloid is considered the primary contributor to neuropathology and neurodegeneration. It now is believed that certain systemic diseases, such as diabetes mellitus, can contribute to neurodegeneration through the effects of chronic hyperglycemia/insulin resistance resulting in protein glycation, oxidative stress and inflammation within susceptible brain regions. Here, we present an overview of research focusing on the role of protein glycation, oxidative stress, and inflammation in the neurodegenerative process. Of special interest in this paper is the effect of methylglyoxal (MGO), a cytotoxic byproduct of glucose metabolism, elevated in neurodegenerative disease, and diabetes mellitus, on cerebral protein function and oxidative stress. How MGO interacts with amino acid residues within β-amyloid, and small peptides within the brain, is also discussed in terms of the affect on protein function.
PMCID: PMC3276062  PMID: 22332001

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