Oral microbiota and host innate immune response in bisphosphonate-related osteonecrosis of the jaw.

Bacterial biofilms have emerged as potential critical triggers in the pathogenesis of bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) or BRONJ. BRONJ lesions have shown to be heavily colonized by oral bacteria, most of these difficult to cultivate and presents many clinical challenges. The purpose of this study was to characterize the bacterial diversity in BRONJ lesions and to determine host immune response. We examined tissue specimens from three cohorts (n=30); patients with periodontal disease without a history of BP therapy (Control, n=10), patients with periodontal disease having history of BP therapy but without ONJ (BP, n=5) and patients with BRONJ (BRONJ, n=15). Denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments revealed less bacterial diversity in BRONJ than BP and Control cohorts. Sequence analysis detected six phyla with predominant affiliation to Firmicutes in BRONJ (71.6%), BP (70.3%) and Control (59.1%). Significant differences (P<0.05) in genera were observed, between Control/BP, Control/BRONJ and BP/BRONJ cohorts. Enzyme-linked immunosorbent assay (ELISA) results indicated that the levels of myeloperoxidase were significantly lower, whereas interleukin-6 and tumor necrosis factor-alpha levels were moderately elevated in BRONJ patients as compared to Controls. PCR array showed significant changes in BRONJ patients with downregulation of host genes, such as nucleotide-binding oligomerization domain containing protein 2, and cathepsin G, the key modulators for antibacterial response and upregulation of secretory leukocyte protease inhibitor, proteinase 3 and conserved helix-loop-helix ubiquitous kinase. The results suggest that colonization of unique bacterial communities coupled with deficient innate immune response is likely to impact the pathogenesis of ONJ.

Dendritic Cells (DC) Facilitate Detachment of Squamous Carcinoma Cells (SCC), While SCC Promote an Immature CD16(+) DC Phenotype and Control DC Migration.

In the inflammatory mucosal microenvironment of head and neck SCC (HNSCC), DC express CD16 and are usually in direct contact with tumor cells. Mucosal and inflammation-associated DC develop from monocytes, and monocyte-derived DC are used in HNSCC immunotherapy. However, beyond apoptotic tumor cell uptake and presentation of tumor antigens by DC, HNSCC cell interactions with DC are poorly understood. Using co-cultures of monocyte-derived DC and two established HNSCC cell lines that represent well- and poorly-differentiated SCC, respectively, we found that carcinoma cells induced significant increases in CD16 expression on DC while promoting a CD1a(+)CD86(dim) immature phenotype, similar to that observed in HNSCC specimens. Moreover, HNSCC cells affected steady-state and CCL21-induced migration of DC, and these effects were donor-dependent. The CCL21-induced migration directly correlated with HNSCC-mediated effects on CCR7 and CD38 expression on DC-SIGN-high DC. The dominant pattern seen in six out of nine donors was the increase in steady-state and CCL21-induced DC migration in co-cultures with HNSCC, while the reverse pattern, i.e., decreased DC migration in co-cultures with SCC, was identified in two donors. A split in migratory DC behavior, i.e. increase with one HNSCC cell line and a decrease with the second cell line, was observed in one donor. Remarkably, the numbers of live detached HNSCC cells were orders of magnitude higher in DC-HNSCC co-cultures than in parallel HNSCC cell cultures without DC. This study provides novel insights into the effects of DC-HNSCC interactions relevant to the tumor microenvironment.

Vesiculated alpha-tocopheryl succinate enhances the anti-tumor effect of dendritic cell vaccines.

Alpha tocopheryl succinate (alpha-TOS) is a non-toxic vitamin E analog under study for its anti-cancer properties. In an earlier study, we showed that alpha-TOS, when used in combination with non-matured dendritic cells (nmDC) to treat pre-established tumors, acts as an effective adjuvant. In this study, we have used vesiculated alpha-TOS (Valpha-TOS), a more soluble form of alpha-TOS that is relevant for clinical use, in combination with dendritic cells to treat pre-established murine tumors. We demonstrate that Valpha-TOS kills tumor cells in vitro and inhibits the growth of pre-established murine lung carcinoma (3LLD122) as effectively as alpha-TOS. The combination of Valpha-TOS plus non-matured or TNF-alpha-matured DC is more effective at inhibiting the growth of established tumors than Valpha-TOS alone. We also observed that Valpha-TOS induces expression of heat shock proteins in tumor cells and that co-incubation of non-matured DC with lysate derived from Valpha-TOS-treated tumor cells leads to DC maturation evidenced by up-regulation of co-stimulatory molecules and secretion of IL-12p70. This study therefore demonstrates the immunomodulatory properties of Valpha-TOS that may account for its adjuvant effect when combined with DC vaccines to treat established tumors.

Chemo-immunotherapy of breast cancer using vesiculated alpha-tocopheryl succinate in combination with dendritic cell vaccination.

In this study, we evaluated the efficacy of vesiculated alpha-tocopheryl succinate (Valpha-TOS) in combination with non-antigen pulsed, nonmatured dendritic cells (nmDC) to treat pre-established tumors of the highly metastatic murine mammary cancer cell line 4T1. We demonstrated that Valpha-TOS in combination with non-antigen pulsed nmDC significantly inhibits the growth of established tumors in vivo and prolongs survival of treated mice. In addition, when initiated after resection of the established primary tumor, the combination treatment dramatically inhibits residual metastatic disease. The clinical response achieved with the combination therapy was correlated with increased interferon-gamma and interleukin-4 (IL-4) production by splenic lymphocytes and draining lymph node cells. Interestingly, when used in combination with Valpha-TOS, nmDC were as effective as tumor necrosis factor-alpha matured DC at inhibiting the growth of pre-established tumors. Valpha-TOS-induced cellular factors collected by high-speed centrifugation of supernatant from Valpha-TOS-treated tumor cells caused maturation of DC as evidenced by the up-regulation of co-stimulatory molecules and secretion of IL-12p70. These results demonstrate the potential usefulness of Valpha-TOS + DC chemo-immunotherapy in treating established primary mammary tumors as well as residual metastatic disease.

alpha-Tocopheryl succinate sensitizes established tumors to vaccination with nonmatured dendritic cells.

PURPOSE: Dendritic cells (DCs) are considered potential candidates for cancer immunotherapy due to their ability to process and present antigens to T cells and stimulate immune responses. However, DC-based vaccines have exhibited minimal effectiveness against established tumors in mice and human cancer patients. The use of appropriate adjuvants can enhance the efficacy of DC-based cancer vaccines in treating established tumors. METHODS: In this study we have employed alpha-tocopheryl succinate (alpha-TOS), a nontoxic esterified analogue of vitamin E, as an adjuvant to enhance the effectiveness of DC vaccines in treating established murine Lewis lung (3LL) carcinomas. RESULTS: We demonstrate that locally or systemically administered alpha-TOS in combination with nonmatured DCs injected intratumorally (i.t.) or subcutaneously (s.c.) significantly inhibits the growth of preestablished 10-day tumors (mean tumor volume of 77.5 +/- 17.8 mm3 on day 30 post-tumor injection) as compared to alpha-TOS alone (mean tumor volume of 471 +/- 68 mm3 on day 30 post-tumor injection). Additionally, the adjuvant effect of alpha-TOS was superior to that of cyclophosphamide (CTX). The mean tumor volume on day 28 post-tumor injection in mice treated with CTX+DCs was 611 +/- 94 mm3 as compared to 105 +/- 36 mm3 in mice treated with alpha-TOS+DCs. Analysis of purified T lymphocytes from mice treated with alpha-TOS+DC revealed significantly increased secretion of IFN-gamma as compared to T cells from the various control groups. CONCLUSION: This study demonstrates the potential usefulness of alpha-tocopheryl succinate, an agent nontoxic to normal cell types, as an adjuvant to augment the effectiveness of DC-based vaccines in treating established tumors.

Transforming growth factor beta inhibits the antigen-presenting functions and antitumor activity of dendritic cell vaccines.

Dendritic cell (DC)-based vaccines have exhibited minimal effectiveness in treating established tumors, likely because of factors present in the tumor microenvironment. One such factor is transforming growth factor beta (TGF-beta), a cytokine that is produced by numerous tumor types and has been demonstrated to impair DC functions in vitro. We have evaluated the effect of TGF-beta on the immunostimulatory activities of DCs. We demonstrate that TGF-beta exposure inhibits the ability of DCs to present antigen, stimulate tumor-sensitized T lymphocytes, and migrate to draining lymph nodes. Neutralization of TGF-beta using the TGF-beta-neutralizing monoclonal antibody 2G7 enhanced the ability of DC vaccines to inhibit the growth of established 4T1 murine mammary tumors. Treatment of 4T1 tumors transduced with the antisense TGF-beta transgene (4T1-asT) with the combination of DC and 2G7 monoclonal antibody inhibited tumor growth and resulted in complete regression of tumors in 40% of the mice. These results demonstrate that neutralization of TGF-beta in tumor-bearing mice enhances the efficacy of DC-based vaccines.