Clonal Heterogeneity in the Neuronal and Glial Differentiation of Dental Pulp Stem/Progenitor Cells.

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K(+) but not outward Na(+) currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

Isolation of distinct progenitor stem cell populations from dental pulp.

The present study compared the cellular characteristics of progenitor stem cell populations present in adult dental pulp, isolated by different methods utilizing 2 different features of stem cell biology. One population expressing high levels of beta1 integrin was isolated by preferential selection of adherent cells to fibronectin over 20 min. In an alternative approach, cells expressing the embryonic neural crest cell marker, low-affinity nerve growth factor receptor (LANGFR), were selected by magnetic-activated cell sorting. For each method, clonal cell lines were established and expanded in culture. One clone derived via the respective methods was examined for embryonic/progenitor cell markers by immunocytochemistry and RT-PCR. Both clonal populations demonstrated the expression of stro-1 and stained positive for vimentin, demonstrating mesenchymal lineage. Of note, cells selected for LANGFR cells demonstrated the additional expression of CD105 and Notch 2. For both clonal populations, expanded cultures demonstrated the ability to differentiate into osteoblasts, adipocytes and chondrocytes. These results would suggest the potential isolation of 2 progenitor cell populations exhibiting different cellular characteristics in terms of their embryonic nature. The potential for both cell populations to derive from a common origin is discussed.

Lipopolysaccharide alters decorin and biglycan synthesis in rat alveolar bone osteoblasts: consequences for bone repair during periodontal disease.

A prime pathogenic agent associated with periodontitis is lipopolysaccharide (LPS) derived from Porphyromonas gingivalis. This study investigated the effects of P. gingivalis LPS on osteoblasts, which are responsible for alveolar bone repair. Bone cells were obtained from explants of rat alveolar bone chips and cultured with 0-200 ng ml(-1) of P. gingivalis LPS. Porphyromonas gingivalis LPS significantly increased cell proliferation and inhibited osteoblast differentiation, as judged by reduced alkaline phosphatase activity. Analysis of biglycan mRNA and protein levels indicated that P. gingivalis LPS significantly delayed the normally high expression of biglycan during the early stages of culture, which are associated with cell proliferation and early differentiation of progenitor cells. In the presence of P. gingivalis LPS, decorin expression by the alveolar bone cells was reduced during periods of culture relating to collagen fibrillogenesis and mineral deposition. Analysis of glycosaminoglycan chains conjugated to these proteoglycans suggested that in the presence of P. gingivalis LPS, dermatan sulfate persisted within the matrix. This study suggests that P. gingivalis LPS influences the expression and processing of decorin and biglycan in the matrix, altering alveolar bone cell activity and osteoblast phenotype development. The consequences of this altered expression in relation to hindering bone repair as part of the cycle of events during periodontal disease are discussed.

HPV16 E6 29-38-specific T cells kill cervical carcinoma cells despite partial evasion of T-cell effector function.

Persistent human papillomavirus type 16 (HPV16) infection is associated with the development of more than 50% of cervical cancers. The HPV16 E6 and E7 oncoproteins are constitutively expressed in cervical carcinomas and are attractive targets for cytotoxic T lymphocyte (CTL)-based immunotherapy. However, cervical carcinomas may possess multiple evasion mechanisms for HPV16 E6/E7-specific CTL. In this study, we investigated whether HPV16(+) cervical carcinoma cell lines (CaCxCL) could evade all effector functions of HPV16 E6(29-38)-specific T cells. Such CD8(+) T cells were detected in the blood (4/10) or invaded lymph node (1/1) of cervical cancer patients using HLA-A*0201/HPV16 E6(29-38) tetramers after in vitro stimulation. T cells cultured from 3 different donors killed HPV16 E6(29-38) peptide-pulsed target cells but not HPV16(+) CaCxCL in (51)Cr release assays. The absence of killing correlated with limited T-cell degranulation against CaCxCL, but this was not due to antigen processing defects per se; CaCxCL could induce specific T-cell release of IFN-gamma and TNF-alpha, and CaCxCL could be killed in longer cytotoxicity assays (>20 hr). Interestingly, the 'slow' killing of CaCxCL could be partially inhibited by concanamycin A, a known perforin inhibitor. The results suggest that CaCxCL was only partially activating T cells, but this was still sufficient for slow killing. Overall, our results highlight the need to examine multiple T-cell effector functions in the context of endogenous antigen presentation by tumour cells. In this study, testing for cytotoxicity using short-term assays only would have ruled out a candidate epitope for immunotherapy.

Definition of an HPV18/45 cross-reactive human T-cell epitope after DNA immunisation of HLA-A2/KB transgenic mice.

Although human papillomavirus (HPV) types 16 and 18 are the most common types associated with cervical cancer worldwide, other related HPV types such as HPV 35, 45 and 58 have significant prevalence in geographically distinct populations. For development of global prophylactic and therapeutic vaccine strategies, it is important to study immune responses against these viruses and to define the degree of cross-reactivity between related HPV types. To investigate the potential for T cell cross-reactivity after vaccination, HLA-A2/Kb transgenic mice were immunised with DNA plasmid constructs containing HPV18 and 45 E6 and E7. Splenocytes from immunised mice were tested in direct ELIspot assays against overlapping pools of HPV 18 peptides. Immunisation with either HPV18 or HPV45 E6 DNA produced dominant T cell responses against an epitope (KCIDFYSRI) that was shared between HPV18 and HPV45. This peptide was shown to bind to HLA-A*0201 but not Db or Kb molecules on the cell surface. Furthermore this peptide was shown to be immunogenic in vitro to human T cells from 2 out of 3 HLA-A2+ healthy donors. Collectively, these results demonstrate that HPV 18 and 45 E6 DNA vaccines are immunogenic in mice and demonstrate that cross-reactive T cell responses against closely related HPV types can be induced in vivo. The use of the HLA-A2/Kb transgenic mice allowed definition of an HLA-A*0201 binding peptide epitope that would have been rejected on the basis of predicted major histocompatibility complex binding affinity.

Cross-typic specificity and immunotherapeutic potential of a human HPV16 E7-specific CTL line.

Cervical cancer (CaCx) is strongly associated with human papillomavirus (HPV) infection, particularly HPV types 16 and 18. The constitutive expression of HPV E6 and E7 proteins in CaCx makes them attractive targets for CTL based immunotherapy. However cervical carcinomas may have features, e.g., antigen processing defects, that limit the effectiveness of HPV specific CTL. Furthermore most vaccine development has concentrated on HPV type 16, and it is not clear whether such vaccines could induce CTL able to cross-react on related oncogenic HPV types, e.g., HPV31 and 52. To investigate these potentially important parameters in vitro, we used a CTL (D4) specific for HPV16 E7(11-20). D4 was able to kill a variety of HPV16+ CaCx cell lines including those with suspected (CaSki) or known antigen processing defects (C33A), and with low HPV DNA copy number (SiHa). D4 was also able to cross react on a related peptide from HPV52 E7 but not HPV31 E7. Further analysis suggested that D4 cross reactivity against related peptides was influenced both by TCR contact residues and a certain threshold for peptide binding. The HPV cross-reactivity was confirmed at the whole protein level as D4 was also able to recognize the endogenously processed forms of HPV16 and 52 E7 but not 31 E7. These results suggest that HPV16 E7(11-20) would be a useful epitope for immunotherapy in both HPV 16 and 52 tumours. Despite this, it is difficult to generate these CTL in response to vaccination, emphasizing the need for definition of novel epitopes and more efficient vaccination strategies.

Activation of CD40 in cervical carcinoma cells facilitates CTL responses and augments chemotherapy-induced apoptosis.

In this study, we describe the expression and function of CD40, a TNF receptor family member, in cervical carcinomas. CD40 was present at very low levels in normal cervical epithelium but was overexpressed in human papillomavirus-infected lesions and advanced squamous carcinomas of the cervix. The stimulation of CD40-positive cervical carcinoma cell lines with soluble CD40L (CD154) resulted in activation of the NF-kappaB and MAPK signaling pathways and up-regulation of cell surface markers and intracellular molecules associated with Ag processing and presentation. Concomitantly, the CD154-induced activation of CD40 in carcinoma cells was found to directly influence susceptibility to CTL-mediated killing. Thus, CD40 stimulation in cervical carcinoma cell lines expressing a TAP-dependent human papillomavirus 16 E6 Ag epitope resulted in their enhanced killing by specific CTLs. However, CD154 treatment of carcinoma cells expressing proteasome-dependent but TAP-independent Ags from the EBV-encoded BRLF1 and BMLF1 failed to increase tumor cell lysis by specific CTLs. Moreover, we demonstrate that chemotherapeutic agents that suppress protein synthesis and reverse the CD40-mediated dissociation of the translational repressor eukaryotic initiation factor 4E-binding protein from the initiation factor eukaryotic initiation factor 4E, such as 5-fluorouracil, etoposide, and quercetin, dramatically increase the susceptibility of cervical carcinoma cells to CD40L-induced apoptosis. Taken together, these observations demonstrate the functional expression of CD40 in epithelial tumors of the cervix and support the clinical exploitation of the CD40 pathway for the treatment of cervical cancer through its multiple effects on tumor cell growth, apoptosis, and immune recognition.