Peptide inhibitors of C3 activation as a novel strategy of complement inhibition for the treatment of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis due to the lack of CD55 and CD59 on affected erythrocytes. The anti-C5 antibody eculizumab has proven clinically effective, but uncontrolled C3 activation due to CD55 absence may result in opsonization of erythrocytes, possibly leading to clinically meaningful extravascular hemolysis. We investigated the effect of the peptidic C3 inhibitor, compstatin Cp40, and its long-acting form (polyethylene glycol [PEG]-Cp40) on hemolysis and opsonization of PNH erythrocytes in an established in vitro system. Both compounds demonstrated dose-dependent inhibition of hemolysis with IC50 ∼4 µM and full inhibition at 6 µM. Protective levels of either Cp40 or PEG-Cp40 also efficiently prevented deposition of C3 fragments on PNH erythrocytes. We further explored the potential of both inhibitors for systemic administration and performed pharmacokinetic evaluation in nonhuman primates. A single intravenous injection of PEG-Cp40 resulted in a prolonged elimination half-life of >5 days but may potentially affect the plasma levels of C3. Despite faster elimination kinetics, saturating inhibitor concentration could be reached with unmodified Cp40 through repetitive subcutaneous administration. In conclusion, peptide inhibitors of C3 activation effectively prevent hemolysis and C3 opsonization of PNH erythrocytes, and are excellent, and potentially cost-effective, candidates for further clinical investigation.

The presence of a side population and its marker ABCG2 in human deciduous dental pulp cells.

Stem cells have been identified using the DNA-binding dye Hoechst 33342 and flow cytometry (FCM) in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population cells in human deciduous dental pulp cells (DPCs). Flow cytometric identification revealed that 2% of human deciduous DPCs were SP cells and that this SP profile disappeared in the presence of verapamil. The SP marker ABCG2 protein was localized to DPCs in the cell membrane by immunofluorescence staining, and flow cytometric analysis demonstrated that 3.6% of DPCs were ABCG2-positive. Furthermore, quantitative real-time PCR proved that ABCG2 mRNA expression in DPCs isolated from human exfoliated deciduous teeth was higher than in DPCs from permanent teeth. Our findings demonstrate that DPCs from human exfoliated deciduous teeth contain a higher proportion of the SP phenotype than permanent teeth and that they may constitute a stem cell population.

The effect of octamer-binding transcription factor 4B1 on microRNA signals in human dental pulp cells with inflammatory response.

INTRODUCTION: Dental pulp surrounded by rigid dentin is vulnerable to inflammatory stress; because of this, the invaded bacteria could cause irreversible pulpitis and necrosis. Octamer-binding transcription factor 4B1 (Oct-4B1), a newly discovered Oct-4 spliced variant belonging to the class V of the POU transcription factor family, serves as a precursor of Oct-4B and an essential functional isoform of Oct-4. However, its specific role in the inflammatory response of dental pulp cells (DPCs) remains unknown. METHODS: To explore the effect of Oct-4B1 on the inflammatory response of DPCs, messenger RNA expression of Oct-4B1 and Oct-4B in DPCs with lipopolysaccharide (LPS) induction was examined by real-time polymerase chain reaction. The expression of Oct-4B1 in DPCs was knocked down by specific small interfering RNA (siRNA); cell proliferation and the apoptosis rate were detected by the Cell Counting Kit-8 (Tokyo, Dojindo, Japan) and Hoechst-propidium iodide staining. The microRNA (miRNA) expression profiles were examined by miRNA microarray and bioinformatic analysis. RESULTS: We showed the messenger RNA expression of Oct-4B1 and Oct-4B was up-regulated in DPCs with LPS stimulation, whereas the knockdown expression of Oct-4B1 led to down-regulation of Oct-4B and an increased number of apoptotic cells in DPCs with LPS stimulation. Moreover, a total of 38 miRNAs were differentially expressed (including 4 up-regulated and 34 down-regulated) in DPCs with Oct-4B1 knockdown. Six of them were confirmed by real-time polymerase chain reaction, among which the target genes of miR-221 were predicted to be enriched in 14 Kyoto Encyclopedia of Genes and Genomes pathways represented by mitogen-activated protein kinase, Wnt, and Toll-like signaling pathways. CONCLUSIONS: Oct-4B1 may play a critical role in the inflammatory response of DPCs through interaction with miRNAs.

Identification and characterization of side population cells from adult human dental pulp after ischemic culture.

INTRODUCTION: Stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the side population (SP). Because the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of dental pulp stem cells, this study sought to determine the existence of SP cells and the expression of ABCG2 in human dental pulp and evaluate whether such SP cells had features associated with stem cells. METHODS: First, we defined the localization of the SP in healthy and inflammatory human dental pulp. Then, SP cells were isolated from human dental pulp after ischemic culture with flow cytometry and the Hoechst 33342 dye efflux assay. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype, including the colony-forming capacity, the multilineage differentiation ability in vitro, and the expression of stem cell markers. We also evaluated the effect of long-term culture on the marker ABCG2. RESULTS: SP cells in human dental pulp possess mesenchymal stem cell characteristics such as colony-forming efficiency, self-renewal, and multilineage differentiation capabilities and are able to differentiate into odontoblast/osteoblast-like cells, adipocytes, neural-like cells, and endothelial cells. However, under the present conditions, ABCG2 expression decreased along with cell passage. CONCLUSIONS: SP cells in human dental pulp were enriched in stem cells compared with main population cells after ischemic culture, suggesting a potential use for these subfractions of human dental pulp stem/progenitor cells in tissue engineering, but the culture condition in vitro should be improved before tissue regeneration.

The dynamic changes of circulating OCN+ cells versus insulinlike growth factor-I during primary healing of orthognathic surgeries.

OBJECTIVE: The objective of this study was to determine the dynamic changes of circulating osteocalcin(+) (OCN(+)) cells and insulinlike growth factor-I (IGF-I) in peripheral blood during early primary repair of jaw bones in patients with orthognathic surgery. STUDY DESIGN: The expression of bone-related genes was detected by RT-PCR in circulating OCN(+) cells. The numbers of OCN(+) cells and serum level of IGF-I were determined by flow cytometry, immunocytochemical staining, and ELISA. RESULTS: OCN(+) cells significantly increased in peripheral blood, and reached the peak at 1 to 2 weeks after surgery (P < .05). IGF-I in patients significantly decreased 1 week after surgery (P < .05), and then returned gradually to the normal level. There was no significant correlation between the number of circulating OCN(+) cells and the level of IGF-I (P > .05). CONCLUSIONS: These findings suggested that circulating OCN(+) cells, at least in part, could be mobilized in response to bone injury, and contribute to bone repair in patients with orthognathic surgery.

Side population increase after simulated transient ischemia in human dental pulp cell.

INTRODUCTION: Dental pulp is often exposed to ischemia in case of injury or inflammation because of narrow vascular openings at the apex and poor blood circulation in dental pulp tissue. Resident stem cell populations are thought to contribute to the postischemic regeneration process. The aim of this study was to investigate the influence of simulated ischemia (serum deprivation and hypoxia) on side population (SP) stem cells of human dental pulp cells in order to provide a better understanding of the postischemic tissue repair and regeneration process. METHODS: The proliferation of dental pulp cells (DPCs) after exposure to ischemic culture conditions (2% O2, 2% serum) for 24 hours and 48 hours was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. The SP fraction was detected by Hoechst 33342 fluorescence flow cytometry, and the expression of SP marker ABCG2 was investigated by immunofluorescence. ABCG2 and OCT4 messenger RNA levels before and after transient ischemia were determined by real-time polymerase chain reaction. RESULTS: Proliferation rate of DPCs was lower in 24- and 48-hour ischemic groups than control from day 5 to day 7. SP proportion was significantly higher 24 and 48 hours after simulated ischemic treatment, and immunofluorescence staining of ABCG2 also verified the increasing trend of side population. ABCG2 and OCT4 messenger RNA levels increased more than three folds in 48 hours ischemic group compared with control group. CONCLUSIONS: Side population in dental pulp cells increase notably after transient simulated ischemic culture, suggesting that SP may participate in post-ischemic repair and regeneration process of dental pulp.