Antiviral activity of Bifidobacterium adolescentis SPM1005-A on human papillomavirus type 16.

BACKGROUND: Probiotic lactic acid bacteria (LAB) support a functional and balanced immune system, and contribute to immune modulatory effects in combatting microbial pathogens, including viruses. Most cervical cancers are associated with anogenital region infection with high-risk (HR) human papillomavirus (HPV). In this study, we analyzed the antiviral activity of Bifidobacterium adolescentis SPM1005-A in the SiHa cervical cancer cell line expressing HPV type 16. METHODS: We assessed the cellular toxicity of B. adolescentis SPM1005-A in SiHa cells by the Trypan blue dye exclusion assay. Cells (3.6 × 105) in culture plates with or without B. adolescentis SPM1005-A in the same type of medium, were incubated with HPV type 16 at a concentration of 5.1 × 107 cfu/ml. For antiviral analysis, we performed quantitative real-time PCR (qRT-PCR) for E6 and E7 oncogene expressions and observed protein levels by immunoblotting. RESULTS: The qRT-PCR results showed that E6 and E7 mRNA levels decreased simultaneously. Western blot analysis revealed that the E6 protein expression slightly decreased after 24 and 48 h, but the level of E7 protein expression appear unaffected compared with that in the control. Decreased HPV16 E6 and E7 mRNA transcript and protein levels were not associated with cell morphology or significant cytotoxic effects. CONCLUSIONS: This study showed that B. adolescentis SPM1005-A had antiviral activity through suppression E6 and E7 oncogene expression. The results suggest that B. adolescentis SPM1005-A could be potential applications of HPV-associated cervical cancer prevention.

Genome sequence of Lactobacillus ruminis SPM0211, isolated from a fecal sample from a healthy Korean.

Lactobacillus ruminis SPM0211 is a potential probiotic strain that shows antimicrobial activity against emerging pathogens. Here we present the draft genomic sequence of L. ruminis SPM0211, isolated from a fecal sample from a healthy Korean and describe both the common and unique features of this strain.

Inhibition of proliferation in colon cancer cell lines and harmful enzyme activity of colon bacteria by Bifidobacterium adolescentis SPM0212.

In this study, we assessed the anticancer activity and bacterial enzyme inhibition of Bifidobacterium adolescentis SPM0212. B. adolescentis SPM0212 inhibited the proliferation of three human colon cancer cell lines: HT-29, SW 480, and Caco-2. SPM0212 also dose-dependently inhibited TNF-á production and changes in cellular morphology. B. adolescentis SPM0212 inhibited harmful fecal enzymes, including â-glucuronidase, â-glucosidase, tryptophanase, and urease. Thus, B. adolescentis SPM0212 exerts an anticancer effect and inhibits harmful fecal enzymes.

Auranofin inhibits overproduction of pro-inflammatory cytokines, cyclooxygenase expression and PGE2 production in macrophages.

Auranofin (AF), a gold compound, is an orally active therapeutic agent used to treat rheumatoid arthritis (RA), a self-perpetuating inflammatory disease. RA is characterized by autoimmune-mediated proliferation of synovial cells that leads to inflammation, pain, and swelling in most major joints: However, the mechanism as to how AF relieves RA symptoms has not been fully elucidated. The object of this study was to examine the ability of AF to immunomodulate macrophages as antigen presenting cells (APCs). Macrophages are recognized as playing an important role in the pathogenesis of RA, in that there is a relative abundance of macrophage-derived cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in rheumatoid synovium. In this work, we tested whether AF (2.5-20 mM) could inhibit inflammatory activity in the macrophage cell line RAW 264.7. AF decreased production of nitric oxide (NO) and the pro-inflammatory cytokines, TNF-alpha, IL-1beta and IL-6 in macrophages. Furthermore, AF inhibited cyclooxygenase-2 (COX-2)-dependent prostaglandin E2 (PGE2) production in a concentration-dependent manner. In conclusion, these findings may provide an explanation for the clinical effects of AF in patients with RA.

Auranofin, an immunosuppressive drug, inhibits MHC class I and MHC class II pathways of antigen presentation in dendritic cells.

Auranofin (AF), an orally administered, gold-based, anti-arthritic agent, has emerged as a clinically useful therapeutic drug for the treatment of rheumatoid arthritis. In the present study, we examined the effects of AF on major histocompatibility complex (MHC)-restricted antigen presentation in dendritic cells (DCs), which are the most important accessory cells for the induction of T cell responses. A mouse dendritic cell line, DC2.4 cells, and DCs that were generated from mouse bone marrow cells by culturing with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 were each pretreated with AF for 2 hr, and then incubated with ovalbumin (OVA). After the 2-hr incubation, the DCs were fixed, and the amounts of OVA peptide-H-2Kb complexes were assessed using OVa-specific CD8+ T cells. AF inhibited MHC class I-restricted presentation of exogenous OVA. This inhibitory activity of AF appeared to be due not only to the inhibition of the phagocytic activity of DCs, but also to the suppression of MHC molecule expression on DCs. AF also inhibited MHC class II-restricted presentation of exogenous OVA. These results show that AF exerts immunosuppressive activity at least in part by inhibiting MHC-restricted antigen presentation in professional antigen-presenting cells.

Occurrence of the van genes in Enterococcus faecalis and Enterococcus faecium from clinical isolates in Korea.

Enterococcus faecalis and Enterococcus faecium isolated from Korea patients in 1998 and 2005 were tested for susceptibility to nine different antimicrobial agents, including vancomycin and teicoplanin. Polymerase chain reaction (PCR) was used for the detection of several vancomycin resistance genes such as vanA ('high level'), vanB ('moderate high level'), vanC1 and vanC2 ('low level'). Both E. faecalis and E. faecium exhibited a resistance of 80% and more than 60% to synercid and mupirocin, respectively. Moreover, an average of 76% of all isolates was resistant to ciprofloxacin, gentamicin, lincomycin, cefotaxime, and meropenem, confirming the multiple drug resistance of most of the isolates. No resistance to vancomycin or teicoplanin was observed in the 1998 E. faecalis and E. faecium isolates. However, the 2005 E. faecalis and E. faecium isolates exhibited resistance of 16% and 12% to vancomycin and teicoplanin, respectively. In addition, vanA gene was detected in 4 strains of 2005 E. faecium isolates, thus showing a high resistance to vancomycin. No other vancomycin resistance genes, including vanB, vanC1, and vanC2, were found in our isolates. In this study, we compared the occurrence of antimicrobial resistance and the presence of specific resistance genes in E. faecalis and E. faecium. The results showed that the 2005 isolates were usually more resistant than the 1998 isolates.

Identification of Lactobacillus ruminus SPM0211 isolated from healthy Koreans and its antimicrobial activity against some pathogens.

The intestinal microbiota are important to the host with regard to resistance they impart against bacterial infections and their involvement in mediating metabolic functions. Lactic acid producing bacteria such as Lactobacillus play an important physiological role in these matters. The aim of the present study was to isolate Lactobacillus sp. that inhibits enteric pathogens. Initially, 17 isolates from healthy Koreans were collected on Lactobacillus selective medium. Resistance of the isolates to antibiotics including rifampicin, streptomycin, clindamycin and vancomycin was measured. One of the isolate was identified as Lactobacillus ruminus on the basis of bacterial cell morphology, cultural characteristic and biochemical characteristics, 16S rRNA sequence analysis and PCR-RAPD. Antimicrobial activity of the bacterium against Vancomycin Intermediate Resistant Staphylococcus aureus (VISA) and Vancomycin-Resistant Enterococci (VRE) was measured. About 10(4) cells of VISA or VRE were mixed with 1, 5, and 9 mL of L. ruminus SPM 0211 and the final volume was adjusted to 10 mL with brain heart infusion (BHI) broth. The cell suspension was incubated for 3, 6, 9, and 24 h, serially diluted and then plated on BHI agar plates. As numbers of L. ruminus SPM 0211 were increased, viable cell count of VISA and VRE decreased. The strongest antimicrobial activity of SPM 0211 was observed after 9 h incubation in any mixture, almost completely inhibiting the growth of these two bacteria. The results suggest that the freshly isolated L. ruminus SPM 0211 may be used as a pro-biotic microbe that prevents the colonization of enteric pathogens and can thereby promote good gastrointestinal health.

Antimicrobial resistance patterns of vancomycin-resistant Streptococcus equinus isolated from animal foods and epidemiological typing of resistant S. equinus by microbial uniprimer kit.

Raw milk samples, and cow and chicken intestines were tested to isolate vancomycin-resistant, gram-positive bacteria. From these samples, we isolated seven vancomycin-resistant Streptococcus equinus, two vancomycin-resistant viridans Streptococcus and two vancomycin-resistant Enterococcus faecium. The MICs of several antibiotics, including vancomycin, against these strains were tested. Seven isolates of S. equinus showed high level resistance to vancomycin and teicoplanin (>100 microg/mL). The cell wall thickness of these strains was compared with that of the sensitive strain by TEM and no differences were obserbed between these strains. We compared the strains of vancomycin-resistant Streptococcus equinus using PCR with Microbial Uniprimer Kit. We concluded that it is necessary to combine other methods in order to cluster and identify all isolates of S. equinus.

Activation of murine macrophage cell line RAW 264.7 by Korean propolis.

Monocytes and macrophages play a major role in defense mechanism of the host response to tumor, in part through the secretion of several potent products and macrophage cytokines. Monocytes and tissue macrophages produce at least two groups of protein mediators of inflammation, interleukin 1 (IL-1) and tumor necrosis factor (TNF). Recent studies emphasizes that TNF and IL-1 modulate the inflammatory function of endothelial cells, leukocytes, and fibroblasts. In this study, our work is directed toward studying the in vitro effects of Korean propolis on the ability to induce cellular and secretory responses in murine macrophage cell line, RAW 264.7. It was found that Water Extract of Korean Propolis (WEP) could activate macrophages by producing cytokines. The production of the macrophage cytokines, IL-1 and TNF-alpha, by RAW 264.7 treated with WEP was examined from 2.5 microg/ml up to 25 microg/ml with dose dependent manner. Nitric oxide (NO) production was also increased when cells were exposed to combination of LPS and WEP from 2.5 microg/ml up to 25 microg/ml. At high dose of WEP (50 to 100 microg/ml) used to prescribe for anti-inflammatory and analgesic medicine showed inhibition of NO production in LPS-stimulated macrophage. Besides cytokine production, NO release, surface molecule expression and cell morphologic antigen expression were increased in response to the stimulation by WEP. These results suggested WEP may function through macrophage activation.

Metformin Down-regulates TNF-α Secretion via Suppression of Scavenger Receptors in Macrophages.

Obesity is consistently increasing in prevalence and can trigger insulin resistance and type 2 diabetes. Many lines of evidence have shown that macrophages play a major role in inflammation associated with obesity. This study was conducted to determine metformin, a widely prescribed drug for type 2 diabetes, would regulate inflammation through down-regulation of scavenger receptors in macrophages from obesity-induced type 2 diabetes. RAW 264.7 cells and peritoneal macrophages were stimulated with LPS to induce inflammation, and C57BL/6N mice were fed a high-fat diet to generate obesity-induced type 2 diabetes mice. Metformin reduced the production of NO, PGE2 and pro-inflammatory cytokines (IL-1ß, IL-6 and TNF-α) through down-regulation of NF-κB translocation in macrophages in a dose-dependent manner. On the other hand, the protein expressions of anti-inflammatory cytokines, IL-4 and IL-10, were enhanced or maintained by metformin. Also, metformin suppressed secretion of TNF-α and reduced the protein and mRNA expression of TNF-α in obese mice as well as in macrophages. The expression of scavenger receptors, CD36 and SR-A, were attenuated by metformin in macrophages and obese mice. These results suggest that metformin may attenuate inflammatory responses by suppressing the production of TNF-α and the expressions of scavenger receptors.

Metformin Suppresses MHC-Restricted Antigen Presentation by Inhibiting Co-Stimulatory Factors and MHC Molecules in APCs.

Metformin is widely used for T2D therapy but its cellular mechanism of action is undefined. Recent studies on the mechanism of metformin in T2D have demonstrated involvement of the immune system. Current immunotherapies focus on the potential of immunomodulatory strategies for the treatment of T2D. In this study, we examined the effects of metformin on the antigen-presenting function of antigen-presenting cells (APCs). Metformin decreased both MHC class I and class II-restricted presentation of OVA and suppressed the expression of both MHC molecules and co-stimulatory factors such as CD54, CD80, and CD86 in DCs, but did not affect the phagocytic activity toward exogenous OVA. The class II-restricted OVA presentation-regulating activity of metformin was also confirmed using mice that had been injected with metformin followed by soluble OVA. These results provide an understanding of the mechanisms of the T cell response-regulating activity of metformin through the inhibition of MHC-restricted antigen presentation in relation to its actions on APCs.

In vitro evaluation of antibacterial activities and anti-inflammatory effects of Bifidobacterium spp. addressing acne vulgaris.

The objective of this study was to evaluate the antibacterial and anti-inflammatory effect of Bifidobacterium spp. In the first part of the study, the antibacterial activities of live and sonicated cells, from a total of 23 Bifidobacterium species, on the growth of 5 different strain of Staphylococcus aureus. Six strains, of sonicated Bifidobacterium, exhibited antibacterial activity against staphylococci samples, and seven Bifidobacterium strains exhibited antibacterial activity on the growth of S. aureus S.P.-N2. In the second part of the study, we tested the antimicrobial activity, of Bifidobacterium against Propionibacterium acne KCTC3320, using the co-culture method. The loss of P. acnes viability, caused by B. adolescentis SPM0308 and B. longum SPM1207, was 84% and 75%, respectively (*p < 0.05). In the third part of the study, the anti-inflammatory activity of B. adolescentis SPM0308 and B. longum SPM1207 were assessed; nitric oxide (NO), and tumor necrosis factor-α (TNF-α), production were tested using the murine macrophage RAW 264.7 cell line. Treatment of RAW 264.7 cells, with Bifidobacterium, decreased production of NO and TNF-α rather than LPS (100 ng/mL) treatment. The results suggest that B. adolescentis SPM0308 could be used as an effective control for P. acnes KCTC3320, and S. aureus, and reduce the risk of acne vulgaris development. We suggest that B. adolescentis SPM0308 may be a useful probiotic microorganism, for prevention of acne vulgaris, without adverse effects.

Anti-inflammatory function of arctiin by inhibiting COX-2 expression via NF-κB pathways.

BACKGROUND: Arctiin, isolated from Forsythia suspensa has been reported to have anti-inflammatory, anti-oxidant, antibacterial, and antiviral effects in vitro. However, there has been a lack of studies regarding its effects on immunological activity. The aim of this study is to investigate the anti-inflammatory potential and possible mechanisms of arctiin in LPS-induced macrophages. METHODS: We investigated the mRNA and protein levels of proinflammatory cytokines through RT-PCR and western blot analysis, followed by a FACS analysis for surface molecule changes. RESULTS: Arctiin dose dependently decreased the production of NO and proinflammatory cytokines such as IL-1ß, IL-6, TNF-α, and PGE2, and it reduced the gene and protein levels as determined by RT-PCR and western blot analysis, respectively. The expression of co-stimulatory molecules such as B7-1 and B7-2 were also inhibited by arctiin. Furthermore, the activation of the nuclear transcription factor, NF-κB in macrophages was inhibited by arctiin. CONCLUSION: Taken together these results provide evidence of the bioactivity of arctiin in inflammatory diseases and suggest that arctiin may exert anti-inflammatory effect by inhibiting the pro-inflammatory mediators through the inactivation of NF-kB.

Immunostimulatory Effects of Cordyceps militaris on Macrophages through the Enhanced Production of Cytokines via the Activation of NF-kappaB.

BACKGROUND: Cordyceps militaris has been used in traditional medicine to treat numerous diseases and has been reported to possess both antitumor and immunomodulatory activities in vitro and in vivo. However, the pharmacological and biochemical mechanisms of Cordyceps militaris extract (CME) on macrophages have not been clearly elucidated. In the present study, we examined how CME induces the production of proinflammatory cytokines, transcription factor, and the expression of co-stimulatory molecules. METHODS: We confirmed the mRNA and protein levels of proinflammatory cytokines through RT-PCR and western blot analysis, followed by a FACS analysis for surface molecules. RESULTS: CME dose dependently increased the production of NO and proinflammatory cytokines such as IL-1beta, IL-6, TNF-alpha, and PGE(2), and it induced the protein levels of iNOS, COX-2, and proinflammatory cytokines in a concentration-dependent manner, as determined by western blot and RT-PCR analysis, respectively. The expression of co-stimulatory molecules such as ICAM-1, B7-1, and B7-2 was also enhanced by CME. Furthermore, the activation of the nuclear transcription factor, NF-kappaB in macrophages was stimulated by CME. CONCLUSION: Based on these observations, CME increased proinflammatory cytokines through the activation of NF-kappaB, further suggesting that CME may prove useful as an immune-enhancing agent in the treatment of immunological disease.

Antibiotic resistance and assessment of food-borne pathogenic bacteria in frozen foods.

One hundred ninety-three frozen food samples collected in Korea various public bazaars from October 2006 to September 2007. Staphylococci were detected in 21.8% of frozen food samples. Staphylococcus aureus was isolated from 17 (8.8%) samples. Other staphylococci isolates were identified as S. warneri (7.8%), S. epidermidis (2.1%), S. xylosus (1.6%), S. eguorum (1%), and S. vitulinus (0.5%). Additionally, the antimicrobial susceptibility of 42 staphylococcal isolates to ten different antimicrobial agents was determined. The staphylococcal isolates demonstrated antimicrobial resistance to mupirocin (31%) oxacillin (14.3%), gentamicin (9.5%), teicoplanin (7.1%) and ciprofloxacin (7.1%). Most of the staphylococcal isolates showed high-level resistance to mupirocin (MIC(90), >128 microg/mL). Fortunately, most of the isolates were susceptible to vancomycin. The total bacteria and Escherichia coli count were tested to investigate the microbiological quality of frozen foods. From 193 frozen food samples, 43 (22.3%), 34 (17.6%) and 19 (9.8%) samples were shown to be of unacceptable quality due to total bacteria, coliform and E. coli counts, respectively.

Cordycepin Suppresses Expression of Diabetes Regulating Genes by Inhibition of Lipopolysaccharide-induced Inflammation in Macrophages.

BACKGROUND: It has been recently noticed that type 2 diabetes (T2D), one of the most common metabolic diseases, causes a chronic low-grade inflammation and activation of the innate immune system that are closely involved in the pathogenesis of T2D. Cordyceps militaris, a traditional medicinal mushroom, produces a component compound, cordycepin (3'-deoxyadenosine). Cordycepin has been known to have many pharmacological activities including immunological stimulating, anti-cancer, and anti-infection activities. The molecular mechanisms of cordycepin in T2D are not clear. In the present study, we tested the role of cordycepin on the anti-diabetic effect and anti-inflammatory cascades in LPS-stimulated RAW 264.7 cells. METHODS: We confirmed the levels of diabetes regulating genes mRNA and protein of cytokines through RT-PCR and western blot analysis and followed by FACS analysis for the surface molecules. RESULTS: Cordycepin inhibited the production of NO and pro-inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha in LPS-activated macrophages via suppressing protein expression of pro-inflammatory mediators. T2D regulating genes such as 11beta-HSD1 and PPARgamma were decreased as well as expression of co-stimulatory molecules such as ICAM-1 and B7-1/-2 were also decreased with the increment of its concentration. In accordance with suppressed pro-inflammatory cytokine production lead to inhibition of diabetic regulating genes in activated macrophages. Cordycepin suppressed NF-kappaB activation in LPS-activated macrophages. CONCLUSION: Based on these observations, cordycepin suppressed T2D regulating genes through the inactivation of NF-kappaB dependent inflammatory responses and suggesting that cordycepin will provide potential use as an immunomodulatory agent for treating immunological diseases.

Role of Cordycepin and Adenosine on the Phenotypic Switch of Macrophages via Induced Anti-inflammatory Cytokines.

BACKGROUND: Chronic low grade inflammation is closely linked to type II diabetes, obesity, and atherosclerosis. Macrophages play a key role in the regulation of pro- or anti-inflammatory actions at the lesion sites of disease. Components of cordyceps militaris, cordycepin and adenosine, have been used for the modulation of inflammatory diseases. The effects of cordycepin in the modulation of macrophages have yet to be elucidated. We investigated the effects of cordycepin and adenosine on the morphological changes of macrophages under the inflammatory condition of LPS and an anti-inflammatory condition involving high concentrations of adenosine. METHODS: We confirmed the mRNA levels of the M1/M2 cytokine genes through RT-PCR and morphological change. RESULTS: LPS-activated macrophages returned to their inactivated original shape, i.e., they looked like naïve macrophages, through the treatment with high concentrations of cordycepin (40 microg/ml). LPS and adenosine activated macrophages also returned to their original inactivated shapes after cordycepin treatment; however, at relatively higher levels of cordycepin than adenosine. This change did not occur with relatively low concentrations of cordycepin. Adenosine down-regulated the gene expression of M1 cytokines (IL-1beta, TNF-alpha) and chemokines (CX3CR1, RANTES), as well as cordycepin. Additionally, M2 cytokines (IL-10, IL-1ra, TGF-beta) were up-regulated by both cordycepin and adenosine. CONCLUSION: Based on these observations, both cordycepin and adenosine regulated the phenotypic switch on macrophages and suggested that cordycepin and adenosine may potentially be used as immunomodulatory agents in the treatment of inflammatory disease.