Kook Jin Lim

HMGB1 Is Phosphorylated by Classical Protein Kinase C and Is Secreted by a Calcium-Dependent Mechanism

High-mobility group box 1 protein (HMGB1) has been studied as a key mediator of inflammatory diseases, including sepsis. Regulating secretion is important in the control of HMGB1-mediated inflammation. Previously, it was shown that HMGB1 needs to be phosphorylated for secretion. In this study, we show that HMGB1 is phosphorylated by the classical protein kinase C (cPKC) and is secreted by a calcium-dependent mechanism. For this study, RAW264.7 cells and human peripheral blood monocytes were treated with PI3K inhibitors wortmannin, LY294002, and ZSTK474, resulting in inhibition of LPS-stimulated HMGB1 secretion, whereas inhibitors of NF-κB and MAPKs p38 and ERK showed no inhibition. Akt inhibitor IV and mammalian target of rapamycin inhibitor rapamycin did not inhibit HMGB1 secretion. However, the PKC inhibitors Gö6983 (broad-spectrum PKC), Gö6976 (cPKC), and Ro-31-7549 (cPKC) and phosphoinositide-dependent kinase 1 inhibitor, which results in protein kinase C (PKC) inhibition, inhibited LPS-stimulated HMGB1 secretion. PKC activators, PMA and bryostatin-1, enhanced HMGB1 secretion. In an in vitro kinase assay, HMGB1 was phosphorylated by recombinant cPKC and by purified nuclear cPKC from LPS-stimulated RAW264.7 cells, but not by casein kinase II or cdc2. HMGB1 secretion was also induced by the calcium ionophore A23187 and inhibited by the Ca2+ chelators BAPTA-AM and EGTA. These findings support a role for Ca2+-dependent PKC in HMGB1 secretion. Thus, we propose that cPKC is an effector kinase of HMGB1 phosphorylation in LPS-stimulated monocytes and PI3K-phosphoinositide-dependent kinase 1 may act in concert to control HMGB1 secretion independent of the NF-κB, p38, and ERK pathways.

A direct sandwich ELISA to detect antibodies against the C-terminal region of merozoite surface protein 1 could be a useful diagnostic method to identify Plasmodium vivax exposed persons

Abstract. We expressed a C-terminal 108-aa region of the Plasmodium vivax merozoite surface protein 1 (PvMSP1c) excluding the C-end transmembrane region in Escherichia coli in order to evaluate the antibody level to MSP in Korean malaria patients. We optimized a direct sandwich enzyme-linked immunosorbent assay (ELISA) method to simultaneously determine the total antibody levels, including IgG and IgM, to PvMSP1c. If the cut-off for seropositivity was determined as the mean+3SD of the antibody levels of the negative control group, the antibody levels were positive in 99.5% of the patient group (sensitivity 199/200). The antibody levels were negative in 99.4% of the negative control group (specificity 504/507). The positive reactions in the negative control group came from non-specific reactions, as confirmed by a competition assay. This direct sandwich ELISA for PvMSP1c antibody could prove to be a useful tool for the diagnosis of malaria patients and for blood screening in blood banks.

Isolated protein of Astragalus membranaceus acts as an allergen by binding human immunoglobulin E on human sera

Allergy is nearly always triggered by protein molecules and the majority of individuals with documented immunologic reaction to foods or plants IgE reactions. However, proteins derived from herbal medicine act as an allergen is unknown. In this study, we aimed to confirm if herbal medicine, Astragalus membranaceus, at proteomic level may contain to trigger the immunoreactivity of IgE. If it does, what components of protein molecules in herbal medicine, Astragalus membranaceus, is react to a binding IgE. In order to resolve this hypothesis, we performed proteomic tools, SDS-PAGE stained coomassie blue and identified protein by LC-MS/MS. Also, we tested plasma IgE reactivity on an isolated protein of Astragalus membranaceus using western blot of human IgE and enzyme immunoassay panel, Allergy-Q test in 400 patients. Through the described performances, we could select four sera of positive candidate and identify Astragalus membranaceus IgE binding proteins. Our data suggested that herbal medicine, Astragalus membranaceus was possible to act an allergen by identify IgE binding protein with isolated protein of Astragalus membranaceus.

Diagnosis of vivax malaria using an IgM capture ELISA is a sensitive method, even for low levels of parasitemia

Although diagnosis of Plasmodium vivax malaria has been difficult when it is present at a low parasite density, it was recently revealed that an antibody assay was a good method of screening for malaria in blood banks. However, the use of this method for the diagnosis of malaria is limited due to the persistence of specific immunoglobulin (Ig) G. Therefore, we evaluated specific IgM antibody responses against the C-terminal region of the merozoite surface protein 1 of P. vivax (PvMSP1c) in sera obtained from patients with vivax malaria using various assays. The IgM capture enzyme-linked immunosorbent assay showed good sensitivity (97.7%; 308/315) and specificity (99.1%, 446/450). In addition, the results of this assay were not related to parasite density, and a high reactivity was observed when there was a low level of parasitemia. Furthermore, we found that patients with cases of malaria that had relapsed still had the IgM titers against PvMSP1c. Therefore, the use of IgM ELISA for the detection of specific IgM that was not involved in memorial immune activity could be an alternative tool for the diagnosis of malaria and blood screening, even in areas in which malaria is endemic.

Aptamer-functionalized capacitance sensors for real-time monitoring of bacterial growth and antibiotic susceptibility

To prevent spread of infection and antibiotic resistance, fast and accurate diagnosis of bacterial infection and subsequent administration of antimicrobial agents are important. However, conventional methods for bacterial detection and antibiotic susceptibility testing (AST) require more than two days, leading to delays that have contributed to an increase in antibiotic-resistant bacteria. Here, we report an aptamer-functionalized capacitance sensor array that can monitor bacterial growth and antibiotic susceptibility in real-time. While E. coli and S. aureus were cultured, the capacitance increased over time, and apparent bacterial growth curves were observed even when 10 CFU/mL bacteria was inoculated. Furthermore, because of the selectivity of aptamers, bacteria could be identified within 1h using the capacitance sensor array functionalized with aptamers. In addition to bacterial growth, antibiotic susceptibility could be monitored in real-time. When bacteria were treated with antibiotics above the minimum inhibitory concentration (MIC), the capacitance decreased because the bacterial growth was inhibited. These results demonstrate that the aptamer-functionalized capacitance sensor array might be applied for rapid ASTs.

Identification of anti-SF3B1 autoantibody as a diagnostic marker in patients with hepatocellular carcinoma

BackgroundTumor-associated (TA) autoantibodies, which are generated by the immune system upon the recognition of abnormal TA antigens, are promising biomarkers for the early detection of tumors. In order to detect autoantibody biomarkers effectively, antibody-specific epitopes in the diagnostic test should maintain the specific conformations that are as close as possible to those presenting in the body. However, when using patients’ serum as a source of TA autoantibodies the characterization of the autoantibody-specific epitope is not easy due to the limited amount of patient-derived serum.MethodsTo overcome these limits, we constructed a B cell hybridoma pool derived from a hepatocellular carcinoma (HCC) model HBx-transgenic mouse and characterized autoantibodies derived from them as tumor biomarkers. Their target antigens were identified by mass spectrometry and the correlations with HCC were examined. With the assumption that TA autoantibodies generated in the tumor mouse model are induced in human cancer patients, the enzyme-linked immunosorbent assays (ELISA) based on the characteristics of mouse TA autoantibodies were developed for the detection of autoantibody biomarkers in human serum. To mimic natural antigenic structures, the specific epitopes against autoantibodies were screened from the phage display cyclic random heptapeptide library, and the streptavidin antigens fused with the specific epitopes were used as coating antigens.ResultsIn this study, one of HCC-associated autoantibodies derived from HBx-transgenic mouse, XC24, was characterized. Its target antigen was identified as splicing factor 3b subunit 1 (SF3B1) and the high expression of SF3B1 was confirmed in HCC tissues. The specific peptide epitopes against XC24 were selected and, among them, XC24p11 cyclic peptide (-CDATPPRLC-) was used as an epitope of anti-SF3B1 autoantibody ELISA. With this epitope, we could effectively distinguish between serum samples from HCC patients (n = 102) and healthy subjects (n = 85) with 73.53% sensitivity and 91.76% specificity (AUC = 0.8731). Moreover, the simultaneous detection of anti-XC24p11 epitope autoantibody and AFP enhanced the efficiency of HCC diagnosis with 87.25% sensitivity and 90.59% specificity (AUC = 0.9081).ConclusionsELISA using XC24p11 peptide epitope that reacts against anti-SF3B1 autoantibody can be used as a novel test to enhance the diagnostic efficiency of HCC.

Development of an antibody-based diagnostic method for the identification of Bemisia tabaci biotype B

The whitefly Bemisia tabaci is a very destructive pest. B. tabaci is composed of various morphologically undistinguishable biotypes, among which biotypes B and Q, in particular, draw attention because of their wide distribution in Korea and differential potentials for insecticide resistance development. To develop a biotype-specific protein marker that can readily distinguishes biotypes B from other biotypes in the field, we established an ELISA protocol based on carboxylesterase 2 (COE2), which is more abundantly expressed in biotypes B compared with Q. Recombinant COE2 was expressed, purified and used for antibody construction. Polyclonal antibodies specific to B. tabaci COE2 [anti-COE2 pAb and deglycosylated anti-COE2 pAb (DG anti-COE2 pAb)] revealed a 3-9-fold higher reactivity to biotype B COE2 than biotype Q COE2 by Western blot and ELISA analyses. DG anti-COE2 pAb exhibited low non-specific activity, demonstrating its compatibility in diagnosing biotypes. Western blot and ELISA analyses determined that one of the 11 field populations examined was biotype B and the others were biotype Q, suggesting the saturation of biotype Q in Korea. DG anti-COE2 pAb discriminates B. tabaci biotypes B and Q with high specificity and accuracy and could be useful for the development of a B. tabaci biotype diagnosis kit for on-site field applications.