The effect of alpha fetoprotein on NF-κB translocation in lipopolysaccharide induced monocyte-derived dendritic cell.

Background: Alpha fetoprotein (AFP) is a tumor-associated Ag that has a function in both ontogenic and oncogenic growth and its serum level is elevated in patients with hepatocellular carcinoma (HCC). A recent study showed that the immunoregulatory effect of AFP was through impairment of dendritic cell function as antigen presenting cell (APC), a mechanism that is known to hamper efficient antitumor response. However, the underlying intracellular mechanism of action of AFP required elucidation. As an initial step to determine the signaling pathway of AFP, we analyzed whether LPS induced NF-κB translocation occured in AFP-treated monocyte-derived dendritic cell (MDDC), which was induced by lipopolysaccharide (LPS). Methods: Monocytes were cultured in GM-CSF (800 ng/mL) and IL-4 (1000 ng/mL) containing medium and incubated for six days to generate immature MDDCs with or without the presence of AFP. Mature MDDC was generated by stimulation of the immature MDDC with LPS for another 30 minutes. The analysis of NF-κB translocation was measured by fluorescent microscopy. Results: Following activation of MDDC by LPS, the control group showed a marked nuclear staining of NF-κB. However, the AFP-treated group showed negative nuclear staining similar as observed in unactivated MDDC. Conclusion: This study demonstrated that AFP prevented the activation and nuclear translocation of NF-κB and subsequently might cause the impairment of MDDC function as APC. This finding provides a new insight on the role of AFP in the suppression mechanism of anti tumor immune response.

Evaluation of PCR and nested PCR assays currently used for detection of Coxiella burnetii in Japan.

Detection of Coxiella burnetii, the etiologic agent of Q fever, is important for diagnosis of Q fever. PCR-based methods have been widely used for the detection mostly because isolation of C. bumetii is time-consuming. Recent reports showed that PCR-positive rates of Q fever infection widely differed. We have evaluated the PCR and nested PCR assays currently used in Japan. The nested PCR assay detected as few as 6 microorganisms and was 10 times more sensitive than the regular PCR assay. The nested PCR assay did not show any non-specific bands with 12 other bacteria, whereas the PCR assay showed some extra bands for 5 of the 12 bacteria. These results suggest that the nested PCR is more sensitive and specific than the PCR in the detection of C. burnetii. However, nested PCR generally has a risk of cross-contamination during preparation of the 2nd PCR. Using blood specimens serially collected from an acute Q fever patient, the PCR and the nested PCR assays gave very similar results, suggesting that sensitivity of the PCR assay is at an achieved level of the detection for clinical specimens although the nested PCR assay is more sensitive. It is recommended that both the PCR and nested PCR assays should be performed for the detection of C. burnetii to obtain reliable results.