Defensin DEFB103 bidirectionally regulates chemokine and cytokine responses to a pro-inflammatory stimulus.

Human ß defensin DEFB103 acts as both a stimulant and an attenuator of chemokine and cytokine responses: a dichotomy that is not entirely understood. Our predicted results using an in silico simulation model of dendritic cells and our observed results in human myeloid dendritic cells, show that DEFB103 significantly (p < 0.05) enhanced 6 responses, attenuated 7 responses, and both enhanced/attenuated the CXCL1 and TNF responses to Porphyromonas gingivalis hemagglutinin B (HagB). In murine JAWSII dendritic cells, DEFB103 significantly attenuated, yet rarely enhanced, the Cxcl2, Il6, and Csf3 responses to HagB; and in C57/BL6 mice, DEFB103 significantly enhanced, yet rarely attenuated, the Cxcl1, Csf1, and Csf3 responses. Thus, DEFB103 influences pro-inflammatory activities with the concentration of DEFB103 and order of timing of DEFB103 exposure to dendritic cells, with respect to microbial antigen exposure to cells, being paramount in orchestrating the onset, magnitude, and composition of the chemokine and cytokine response.

Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay.

The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9mg/ml; SPLUNC1 concentrations ranged from 34.7ng/ml to 13.8µg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7ng/ml to 5.3µg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies.

Communication: Antimicrobial Activity of SMAP28 with a Targeting Domain for Porphyromonas gingivalis.

Antibiotic therapy is often used with mechanical therapy to treat periodontal disease. However, complications associated with antibiotic use can occur. A 'bacteria-specific' targeted approach would eliminate some of these complications and kill specific periodontopathogens without harming the commensal bacteria. One such approach is to couple antimicrobial peptides to a ligand, pheromone, or antibody specific for the periodontopathogen, Porphyromonas gingivalis. To assess the feasibility of this approach, we attached PQGPPQ, a peptide from proline-rich protein 1 to either the N-terminus of SMAP28 (peptide ZS37-37) or the C-terminus of SMAP28 (peptide ZS37-38) to see whether it has potential as a carrier ligand to deliver SMAP28 to the surface of P. gingivalis. For Escherichia coli and Aggregatibacter actinomycetemcomitans, the median minimal inhibitory concentration (MIC) of ZS37-37 was higher than the median of SMAP28 alone, although the median MIC of ZS37-38 was lower than that of SMAP28 alone. For P. gingivalis, there was no difference in the median MIC values. For S. aureus, the median MIC was higher for ZS37-37 and ZS37-38 compared to SMAP28 alone, particularly for ZS37-38. For Fusobacterium nucleatum, the median MIC values were equal for ZS37-37 and ZS37-38 and higher than the median MIC for SMAP28 alone. Attaching PQGPPQ to SMAP28 did not greatly increase the antimicrobial activity of ZS37-37 or ZS37-38 for P. gingivalis nor substantially decrease the antimicrobial activity of ZS37-37 or ZS37-38 for the four other microorganisms tested. This is an initial step to develop a selective antimicrobial agent that has 'targeted' antimicrobial activity without adverse reactions often associated with the use of broad-spectrum antibiotics.

Defensins attenuate cytokine responses yet enhance antibody responses to Porphyromonas gingivalis adhesins in mice.

AIM: Our aim is to assess the ability of human neutrophil peptide alpha-defensins (HNPs) and human beta-defensins (HBDs) to attenuate proinflammatory cytokine responses and enhance antibody responses to recombinant hemagglutinin B (rHagB) or recombinant fimbrillin A (rFimA) from Porphyromonas gingivalis 381 in mice. MATERIALS & METHODS: In the first study, C57BL/6 mice were given 10 microg rHagB or rFimA without and with 1 microg HNP1, HNP2, HBD1, HBD2 or HBD3. At 24 h, mice were euthanized and cytokine concentrations were determined in nasal wash fluid (NWF), bronchoalveolar lavage fluids, saliva and serum. In the second study, C57BL/6 mice were given 10 microg rHagB or rFimA without and with 1 microg HNPs or HBDs similarly on days 0, 7 and 14. At 21 days, mice were euthanized and rHagB- and rFimA-specific antibody responses were determined in NWF, bronchoalveolar lavage fluids, saliva and serum. RESULTS: Mice given rHagB + HNP2, rHagB + HBD1 and rHagB + HBD3 produced significantly lower (p < 0.05) IL-6 responses than mice given rHagB alone. Mice given rHagB + HNP1, rHagB + HNP2, rHagB + HBD1 and rHagB + HBD3 produced significantly lower (p < 0.05) keratinocyte-derived chemokine responses than mice given rHagB alone. Mice given rFimA produced very low levels of IL-6 and only moderate levels of keratinocyte-derived chemokine in NWF that were not attenuated by prior incubation of rFimA with any defensin. Mice given rHagB + HNP1 produced a significantly higher (p < 0.05) serum IgG antibody response than mice given rHagB alone and mice given rFimA + HNP2 produced a higher, but not significant, antibody response. CONCLUSION: The ability of HNPs and HBDs to attenuate proinflammatory cytokine responses in murine NWF and enhance IgG antibody responses in serum was dependent upon both the defensin and antigen of P. gingivalis.

Defensins as anti-inflammatory compounds and mucosal adjuvants.

Human neutrophil peptide alpha-defensins and human beta-defensins are small, well-characterized peptides with broad antimicrobial activities. In mixtures with microbial antigens, defensins attenuate proinflammatory cytokine responses by dendritic cells in culture, attenuate proinflammatory cytokine responses in the nasal fluids of exposed mice and enhance antibody responses in the serum of vaccinated mice. Although the exact mechanisms are unknown, defensins first start by binding to microbial antigens and adhesins, often attenuating toxic or inflammatory-inducing capacities. Binding is not generic; it appears to be both defensin-specific and antigen-specific with high affinities. Binding of defensins to antigens may, in turn, alter the interaction of antigens with epithelial cells and antigen-presenting cells attenuating the production of proinflammatory cytokines. The binding of defensins to antigens may also facilitate the delivery of bound antigen to antigen-presenting cells in some cases via specific receptors. These interactions enhance the immunogenicity of the bound antigen in an adjuvant-like fashion. Future research will determine the extent to which defensins can suppress early events in inflammation and enhance systemic antibody responses, a very recent and exciting concept that could be exploited to develop therapeutics to prevent or treat a variety of oral mucosal infections, particularly where inflammation plays a role in the pathogenesis of disease and its long-term sequelae.

Human beta-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response.

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro-inflammatory cytokine responses. Here, we asked whether human beta-defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro-inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)-6, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and tumor-necrosis factor-alpha (TNF-alpha) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal-regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro-inflammatory cytokine response and an ERK 1/2 response.

Mechanisms of cell death induced by histone deacetylase inhibitors in androgen receptor-positive prostate cancer cells.

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Tapasin decreases immune responsiveness to a model tumor antigen.

The T-cell response against cancer is dependent on the cell surface presentation of tumor-associated or tumor-specific peptides by major histocompatibility complex (MHC) class I molecules. We found that tapasin, a chaperone protein that normally assists in the assembly of MHC class I molecules, is undetectable in an unstimulated pancreatic tumor cell line, Panc02, and only very weakly expressed after gamma-interferon stimulation. Transfection of tapasin into the Panc02 cells did not quantitatively increase MHC class I surface expression or detectably affect MHC class I association with peptide and beta(2)-microglubulin (beta(2)m). However, we found that transfected tapasin downregulated immune reactivity against a model tumor antigen, MUC1. Although tapasin has been previously shown by others to increase immune recognition of particular antigens, our results suggest that tapasin has a negative impact on the presentation of an immunodominant epitope from a specific model tumor antigen.

Tumor-specific immunity in MUC1.Tg mice induced by immunization with peptide vaccines from the cytoplasmic tail of CD227 (MUC1).

PURPOSE: CD227 (MUC1), a membrane-associated glycoprotein expressed by many types of ductal epithelia, including pancreas, breast, lung, and gastrointestinal tract, is overexpressed and aberrantly glycosylated by malignant cells. We sought to define epitopes on MUC1 recognized by the different cell-mediated immune responses by an in vivo assay. Epitopes identified by this assay were evaluated for efficacy to protect mice transgenic for human MUC1 (MUC1.Tg) against MUC1-expressing tumor growth. METHODS: We investigated contributions of the tandem repeat (TR) and the cytoplasmic tail (CT) of MUC1 to the MUC1-specific immunological rejection of tumor cells. MUC1 cDNA constructs, in which the TR region was deleted or the CT was truncated, were transfected into two different murine tumor cell lines (B16 and Panc02), which were used to challenge mice and evaluate immunological rejection of the tumors. We used tumor rejection in vivo to define epitopes on the TR and CT of MUC1 recognized by T cell-mediated immune responses in a preclinical murine model. RESULTS: Our findings demonstrated that the TR and a portion of the MUC1 CT contributed to CD4+ T cell rejection of MUC1-expressing B16 tumor cells, but not rejection of MUC1-expressing Panc02 tumor cells. A separate epitope in the CT of MUC1 was necessary for CD8+ T cell rejection of Panc02 tumor cells. Based on these studies, we sought to evaluate the efficacy of immunizing mice transgenic for (and immunologically tolerant to) human MUC1 with peptides derived from the amino acid sequence of the CT of MUC1. Results showed that survival can be significantly prolonged in vaccinated MUC1.Tg mice challenged with MUC1-expressing tumor cells, without induction of autoimmune responses. CONCLUSIONS: These studies demonstrated that MUC1 peptides may be utilized as an effective anticancer immunotherapeutic, and confirmed the importance of immunogenic epitopes outside of the TR.

Contribution of the MUC1 tandem repeat and cytoplasmic tail to invasive and metastatic properties of a pancreatic cancer cell line.

MUC1 is a polymorphic, highly glycosylated, type I transmembrane protein expressed by ductal epithelial cells of many organs including pancreas, breast, gastrointestinal tract, and airway. MUC1 is overexpressed and differentially glycosylated by adenocarcinomas that arise in these organs, and is believed to contribute to invasive and metastatic potential by contributing to cell surface adhesion properties [via the tandem repeat (TR) domain] and through morphogenetic signal transduction [via the cytoplasmic tail (CT)]. The large extracellular TR of MUC1 consists of a heavily glycosylated, 20 amino acid sequence that shows allelic variation with respect to number of repeats. This portion of MUC1 may directly mediate adhesive or antiadhesive interactions with other surface molecules on adjacent cells and through these interactions initiate signal transduction pathways that are transmitted through the CT. We investigated the contribution of the TR domain and the CT of MUC1 to the in vivo invasive and metastatic potential, and the gene expression profile of the human pancreatic tumor cell line S2-013. Results showed that S2-013 cells overexpressing full-length MUC1 displayed a less invasive and metastatic phenotype compared with control-transfected cells and cells expressing MUC1 lacking the TR domain or CT. Clonal populations were analyzed by cDNA array gene expression analysis, which showed differences in the gene expression profiles between the different cell lines. Among the genes differentially expressed were several that encode proteins believed to play a role in invasion and metastasis.

Molecular requirements for CD8-mediated rejection of a MUC1-expressing pancreatic carcinoma: implications for tumor vaccines.

Previous studies have indicated that different effector cells are required to eliminate MUC1-expressing tumors derived from different organ sites and that different vaccine strategies may be necessary to generate these two different MUC1-specific immune responses. In this study, we characterized molecular components that are required to produce immune responses that eliminate Panc02.MUC1 tumors in vivo by utilizing mice genetically deficient in molecules related to immunity. A parallel study has been reported for a B16.MUC1 tumor model. We confirmed that a CD8(+) effector cell was required to eliminate MUC1-expressing Panc02 tumors, and demonstrated that T cells expressing TCR-alpha/beta and co-stimulation through CD28 and CD40:CD40L interactions played critical roles during the initiation of the anti-Panc02.MUC1 immune response. TCR-alpha/beta(+) cells were required to eliminate Panc02.MUC1 tumors, while TCR-gamma/delta(+) cells played a suppressive non-MUC1-specific role in anti-Panc02 tumor immunity. Type 1 cytokine interferon-gamma (IFN-gamma), but not interleukin-12 (IL-12), was essential for eliminating MUC1-expressing tumors, while neither IL-4 nor IL-10 (type 2 cytokines) were required for tumor rejection. In vitro studies demonstrated that IFN-gamma upregulated MHC class I, but not MHC class II, on Panc02.MUC1 tumor cells. Surprisingly, both perforin and FasL played unique roles during the effector phase of immunity to Panc02.MUC1, while lymphotoxin-alpha, but not TNFR-1, was required for immunity against Panc02.MUC1 tumors. The findings presented here and in parallel studies of B16.MUC1 immunity clearly demonstrate that different effector cells and cytolytic mechanisms are required to eliminate MUC1-expressing tumors derived from different organ sites, and provide insight into the immune components required to eliminate tumors expressing the same antigen but derived from different tissues.

MUC1-specific anti-tumor responses: molecular requirements for CD4-mediated responses.

MUC1 was first defined as a tumor antigen in the late 1980s, yet little is known about the types of immune responses that mediate rejection of MUC1(+) tumors in vivo. MUC1-specific antibodies, T(h) cells and cytotoxic T cells can be detected in patients with different adenocarcinomas, yet these tumors usually progress. Thus, there is a need to better understand the in vivo mechanisms of antigen-specific tumor rejection. To characterize the nature of MUC1-specific immune responses in vivo, rejection of a MUC1-expressing melanoma tumor line (B16.MUC1) was evaluated in mice lacking specific T cell subsets, cytokines, co-stimulatory molecules or molecular effectors of cytolytic pathways. Results demonstrated that rejection of the B16.MUC1 tumor cell line was primarily mediated by CD4(+) T cells, and required Fas ligand, lymphotoxin-alpha, CD40, CD40 ligand and CD28, but not perforin, gammadelta T cells, IL-4, IL-10, IL-12 or tumor necrosis factor receptor-1. Depletion of NK cells demonstrated that NK cells might also contribute to MUC1 immunity in the B16.MUC1 tumor model. These results demonstrated that the immune response generated against MUC1 does not fit the type 1 or 2 model described for many immune responses. Additionally, multiple cytolytic mechanisms are required for B16.MUC1 rejection.