Yongmoon Han

Effects of Sugar Additives on Protein Stability of Recombinant Human Serum Albumin during Lyophilization and Storage

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35°C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35°C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.

Rutin has therapeutic effect on septic arthritis caused by Candida albicans.

As of late, numerous reports have demonstrated the multiple biological activities of polyphenolic flavonoids. Amongst these reports, some indicate that the flavonoids play an important role in inflammation therapy. In this present study, we investigated the effect of rutin, a polyphenolic flavonoid, on septic arthritis due to Candida albicans, a major etiological agent that causes fungal arthritis. To induce septic arthritis, an emulsified mixture of C. albicans cell wall and Complete Freunds Adjuvant (CACW/CFA) was injected into BALB/c mice via hind footpad route once a day, everyday, for three days. In order to determine the effect of rutin, twenty-four hours after the final injection, mice having the swollen footpad were given the flavonoid (1 mg/dose/mouse) intraperitoneally every other day for three times. The footpad-edema was measured for a period of 17 days. Results showed that the rutin treatment reduced app. 45% of the edema at the peak day (day 11) of septic arthritis (P<0.05). In addition, 6 days after the peak, there was an app. 35% additional reduction of the edema (P<0.05). We found that this anti-arthritic activity was mediated by rutins ability to inhibit nitric oxide production from macrophages and T-cells proliferation. Furthermore, this flavonoid also inhibited the growth of C. albicans yeast cells (P<0.01) and resulted in no hemolysis. These data indicate that rutin, which has both anti-arthritic and antifungal effects, can safely be administered into the blood circulation for treatment of septic arthritis caused by C. albicans. Ultimately, it can be suggested that the dual effects of rutin, anti-arthritic and anti-candidal may be helpful as an all-in-one treatment for septic arthritis.

Liquiritigenin, a licorice flavonoid, helps mice resist disseminated candidiasis due to Candida albicans by Th1 immune response, whereas liquiritin, its glycoside form, does not

Licorice (the root of Glycyrrhizae plant) has been used as an oriental herbal medicine for thousands of years. The licorice flavonoid components are reported to possess immunomodulatory activities. In this present study, we investigated the immunomodulatory effects of liquiritigenin (LG) and liquiritin (LQ), licorice flavonoid components, against disseminated candidiasis due to Candida albicans, a dimorphic fungus, that causes severe disease via hematogenous dissemination and local diseases such as vaginitis and thrush. Results showed that direct interaction of LG or LQ with C. albicans yeast cells resulted in no growth-inhibition, in vitro. When tested in a murine model of disseminated candidiasis, mice given LQ intraperitoneally before intravenous challenge with live C. albicans yeast cells had similar mean survival times (MST) as untreated mice groups. On the contrary, mice given LG in the same manner as LQ above had longer MST than the untreated mice groups (P < 0.05). In one experiment, 3 out of 5 LG-treated mice survived during the entire period of the 55-day observation. Furthermore, the 3 survivors were cured -- shown by a lack of CFU (colony forming unit) in the kidneys. This protection was nulled when mice were pretreated with anti-CD4+ antibody before LG-treatment and challenge with the yeast. However, the protection was transferable by the CD4+ T cells isolated from LG-treated mice not infected with the yeast. In addition, mice given CD4+ T cells that were pre-treated with LG, in vitro were also protected against disseminated candidiasis. ELISA analysis revealed that in LG-treated mice IFNgamma and IL-2 were dominantly produced compared to IL-4 and IL-10. When LG-given mice were treated with anti-mouse IFNgamma, the protection was again nulled. Combined together, these results indicate that LG protects mice against disseminated candidiasis by the CD4+ Th1 immune response.

Chlorogenic acid, a polyphenolic compound, treats mice with septic arthritis caused by Candida albicans

There has been an increasing number of studies concerning the multiple biological activities of polyphenolic compounds. In this present study, we determined the effect of chlorogenic acid (CRA), a polyphenolic compound, on septic arthritis caused by Candida albicans, a major etiological agent that causes fungal arthritis. To induce septic arthritis, an emulsified mixture of C. albicans cell wall and Complete Freunds Adjuvant (CACW/CFA) was injected into BALB/c mice via hind footpad route once a day for 3 days. Twenty-four hours after the final injection, in order to determine CRA effect, mice having the swollen footpad were given CRA (1 mg/dose/mouse) intraperitoneally every other day three times. The footpad edema was measured for 15 days. Results showed that the CRA treatment reduced approximately 40% of the edema at the peak of septic arthritis (P<0.05). This anti-arthritic activity appeared to be mediated by a complete inhibition of nitric oxide (NO) production from macrophages and suppression of T-cells proliferation. Furthermore, CRA also inhibited growth of C. albicans yeast cells (P<0.01) and caused no hemolysis. These data indicate that CRA, which has antifungal and anti-arthritic effects, can safely be administered into the blood circulation for treatment of septic arthritis due to C. albicans. In addition, in respect to antiseptic arthritis, it can be suggested that the anti-candidal effect of CRA may be helpful as an all-in-one treatment of the candidal arthritis.

Induction of apoptosis by ginsenoside Rk1 in SK-MEL-2-human melanoma

AbstactGinsenosides are active compounds isolated from Panax ginseng Meyer. Among these ginsenosides, less polar ginsenosides such as ginsenoside Rg3 and ginsenoside Rh2 have been demonstrated to have tumor inhibitory effects because of their cytotoxicity. In this study, we evaluated the apoptotic effects of ginsenoside Rk1 in SK-MEL-2 human melanoma. Ginsenoside Rk1 isolated from red ginseng is one of the novel ginsenosides that shows strong cytotoxicity compared to ginsenoside Rg3 in dose- and time-dependent manners. The results of DNA fragmentation, 4′,6-diamidino-2-phenylindole staining, and flow cytometric analysis are corroborated that ginsenoside Rk1 induced apoptosis in SK-MEL-2 cells. Western blot analysis revealed up-regulation of Fas, FasL, and Bax protein expression and down-regulation of procaspase-8, procaspase-3, mutant p53 and Bcl-2 protein expression. These findings suggest that ginsenoside Rk1 might be a promising compound to induce apoptosis through both extrinsic and intrinsic pathways in SK-MEL-2 cells.

Utilization of ferroproteins byCandida albicans during candidastasis by apotransferrin

Many reports have stated that some of the pathogenic bacteria can obtain iron from ferroproteins, such as cytochrome C, ferritin, hemin, hemoglobin, and myoglobin. These reports prompted us to determine if an opportunistic pathogenic fungus,Candida albicans, can utilize ferroproteins to circumvent the iron-regulatory effect of transferrin. The following assays were carried out to measurein vitro growth stimulation by the ferroproteins: as an initial step,C. albicans was cultured in iron-free (pretreated with apotransferrin for 24 h) culture medium. OnceCandida albicans yeast cell growth reached stasis from iron starvation, individual ferro-proteins were added to the culture media. Results showed that hemin, hemoglobin, and myoglobin supported a partial growth recovery. Additional studies with haptoglobin, a serum protein that interacts with the globin moiety of certain ferroproteins, established thatC. albicans could obtain iron from the haptoglobin-ferroprotein complexes. These data indicate that the heme part of the ferroproteins is the source of iron. This implies that heme oxygenase, CaHMX1 might be involved in bringing about dissociation of heme-containing protein for iron-acquisition. In addition, anticandidal activity of transferrin takes place not only by the process of iron regulation, but also by direct interaction with the yeast cells.

18β-glycyrrhetinic acid induces immunological adjuvant activity of Th1 against Candida albicans surface mannan extract.

The aim of this study was to determine the immunological adjuvant effect of 18β-glycyrrhetinic acid (GA) isolated from Glycyrrhizae radix. In the experiments, BALB/c mice were immunized on days 1 and 22 intraperitoneally (i.p.) with an emulsion form of Candida albicans surface mannan extract (SM) mixed with either Incomplete Freunds Adjuvant [SM/IFA], or Complete Freunds Adjuvant [SM/CFA] or GA mixed with IFA [SM/GA/IFA]. One week after the second immunization, polyclonal sera were collected from these animals in order to determine IgG isotypes and cytokine profiles in the sera. After the collection, the spleen samples were collected to determine the degree of T cell proliferation. Additionally, the DTH (delayed type hypersensitivity) response was examined by measuring the footpad swelling of immunized mice. Data resulting from the T cell proliferation test showed that SM/GA/IFA enhanced the proliferation the most. The enhancement was about 85% more compared to SM/IFA (p<0.05). IgG isotypes and cytokine profiles displayed that SM/GA/IFA induced the most abundant production of total IgG with the highest IgG2a/IgG1 ratio (1.31) and greatest IFN-γ secretion. In contrast, SM/CFA resulted in an IgG2a/IgG1 ratio less than 1 and SM/IFA produced a dominant induction of IL-4, but almost no IFN-γ secretion. Together, these observations revealed that GA developed a greater Th1 immune response than Th2 response. The DTH determination confirmed that GA-addition induced dominant Th1 immunity - displaying the highest footpad-swelling followed by SM/CFA and BSA/IFA, respectively. All of this data indicates that GA has a Th1-immunological adjuvant activity, which would be beneficial in the treatment of Th1-disordered disease due to C. albicans.

Combination immunotherapy of MAb B6.1 with fluconazole augments therapeutic effect to disseminated candidiasis

We recently reported that IgM MAb B6.1, specific for β-1, 2-mannotriose on the cell wall of Candida albicans, is therapeutic to disseminated candidiasis due to C. albicans. In the current study, we examined if MAbB6.1 enhances therapeutic effect of fluconazole (FLC) to the disseminated disease. To assess the combination effect, determination by the kidneys-colony forming unit and survival times were used. Results showed that the therapeutic effect of FLC on mice with disseminated candidiasis was dose-dependent, but a FLC dose at 0.8 mg/kg body weight of mice was ineffective. To determine combination effect, mice treated intraperitoneally with a combination of FLC plus MAb B6.1 at 1 h post-infection — a condition of developing partial therapeutic activity — enhanced survival times beyond the effect by only antibody (p < 0.05). The resulting MST (mean survival times) value from the combinationreceived mice was almost the same as MST value from 3.2 mg FLC dose-given animals (p < 0.05). Another combination of 1.6 mg FLC dose and B6.1 reduced severity of the disseminated disease at almost the same rate as combination efficacy of 0.8 mg FLC dose plus B6.1. This data indicates that B6.1 acts in concert with FLC and that this combination therapy augments protection, which suggests a possibility of reducing FLC dose. The augmentation response was specific because an irrelevant IgM MAb S9 was not effective to the disseminated disease. Thus, our present studies demonstrate that this combination immunotherapy may be a way of solving the problem of limited antifungal drug choices caused by drug-resistant C. albicans.

Efficacy of combination immunotherapy of IgM MAb B6.1 and amphotericin B against disseminated candidiasis

We previously reported that the IgM MAb B6.1, specific for β-1,2-mannotriose on the surface of the cell wall of Candida albicans, prevents mice from disseminated candidiasis. The preventive activity of the antibody is mediated by enhanced phagocytosis that caused killing of the fungus and involvement of the complement system. In the present study, MAb B6.1 was tested if the antibody enhances therapeutic efficacy of amphotericin B (Amp B) in a murine model of disseminated candidiasis due to C. albicans. Determination by the kidneys-cfu (colony forming unit) and survival times was used to assess treatment. Mice treated intraperitoneally with MAb B6.1 at 1h post-infection with C. albicans (5 x 10⁵ yeast cells/mouse) developed fewer 28%cfu and prolonged survival rates than negative controls, whereas administration of B6.1 to mice at 2h post-infection was ineffective. Therapeutic effect of Amp B on mice with the disseminated disease was dose-dependent, but dosage of Amp B (0.5mg/kg body weight of mice) was not effective. A combination of MAb B6.1 given at 1h post-infection plus Amp B (0.5mg dose) enhanced survival times beyond the effect due to only antibody (P<0.05). The MST (mean survival times) value resulted from the combination therapy-received mice was as almost the same as MST value from 2mg dose of Amp B-given animals (P<0.05). In case mice given a combination of Amp B (0.5mg dose) plus MAb B6.1 at 2h post-infection - a condition of developing no therapeutic efficacy, the combination also reduced disease severity. All these data indicate that MAb B6.1 acts in concert with Amp B, the antibody enhances therapeutic efficacy of Amp B, and this combination therapy augments protection which implies a possibility of reducing Amp B dose. The augmentation of the response appeared to be specific because an irrelevant IgM carbohydrate-specific MAb was not protective. In conclusion, these studies show that Amp B combined with MAb B6.1 may be an option of solving the problem of limited antifungal drug choices due to drug-resistant C. albicans.

Immunoadjuvant activity of icariin that induces Th1-type antibody in mice.

The adjuvant effect of icariin from Epimedium koreanum on the immune responses to bovine serum albumin (BSA) in mice was examined. Mice were immunized on days 1 and 22 intraperitoneally (i.p.) with one of the following: an emulsion form of BSA mixed with Incomplete Freund’s Adjuvant (BSA/IFA) or with Complete Freund’s Adjuvant (BSA/CFA) or BSA plus icariin mixed with IFA (BSA/Icariin/IFA). One week after the booster, polyclonal sera were collected from these animals to determine IgG isotypes specific for BSA in the sera and then spleens of these animals were harvested to evaluate IFN-γ and IL-4 produced in the splenocyte cultures. In order to determine the DTH (delayed type hypersensitivity) response, BSA was administered into the footpads of mice that were immunized as described above and the degree of footpad-swelling was measured. Data from these experiments showed that the icariin combined with BSA (BSA/Icariin/IFA) provoked the most abundant of IgG production in mice and enhanced the Th1-lineage development of IgG2a and IFN-γ productions (p < 0.05), whereas BSA/IFA resulted in a highest ratio of IgG1 to IgG2 and most dominant IL-4 production, indicating a Th2 response. This pattern of immunity was confirmed by the DTH determination revealing that icariin-containing formula caused the highest footpad-swelling followed by BSA/CFA and BSA/IFA, respectively. In addition, hemolytic assay showed that icariin at a dose of 1000 μg/mL caused no hemolysis when compared with a water-treated mouse. All of these data indicate that icariin has the immunoadjuvant effect which may enhance Th1-immune response, suggesting that icariin as an adjuvant would be beneficial in the treatment of Th1-disordered diseases.