ABSTRACT
<b>Background and Objective:</b> Biofilm formation activity of Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is one of the crucial factors rendering this pathogenic bacterium difficult to be eradicated. It has been reported that lupinifolin, is a major phytochemical agent isolated from <i>Derris reticulata</i> Craib. stem possesses antibacterial activity against MRSA. This study aimed to investigate the effects of lupinifolin and its combinations with some antibacterial drugs, including ampicillin, cloxacillin or vancomycin, on the biofilm formation activity of MRSA. <b>Materials and Methods:</b> The crystal violet biofilm formation assay was performed to evaluate the biofilm formation activity. <b>Results:</b> Lupinifolin produced a significant inhibitory activity against MRSA biofilm formation with the median inhibitory concentration (IC<sub>50</sub>) of 7.96±3.05 µg mL<sup>1</sup> (n = 6) at 24 hrs incubation. Lupinifolin at the concentrations of sub-MICs (1, 2, 4 and 8 µg mL<sup>1</sup>) combined with the antibacterial drugs at their sub-MICs also exhibited substantial antibiofilm formation activities. The maximal antibiofilm activity was found with the combination of lupinifolin (8 µg mL<sup>1</sup>) and vancomycin (1 µg mL<sup>1</sup>) by the percentage inhibition of 102.39±0.89 (n = 8). The antibiofilm formation activities of the combinations between lupinifolin and the antibacterial drugs at various concentrations tested were also significantly higher than those of lupinifolin alone. <b>Conclusion:</b> These results indicated that lupinifolin can potentially be developed as an antibacterial enhancer for the management of biofilm-associated bacterial infections caused by MRSA, in which the current pharmacological treatment is still limited.
Subject(s)
Methicillin-Resistant Staphylococcus aureus , Vancomycin/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Microbial Sensitivity Tests , BiofilmsABSTRACT
<b>Background and Objective:</b> Antibacterial resistance is one of the top global public health problems. The use of natural substances, which can enhance the antibacterial activity of currently used medications, is a promising alternative to oppose antibacterial resistance. The pharmacological activities of lupinifolin, a prenylated flavanone isolated from stems of <i>Derris reticulata</i> Craib., against growth and biofilm formation of <i>Streptococcus mutans</i> and <i>Staphylococcus aureus</i> have been previously documented. Nonetheless, interactions between lupinifolin and other antibacterial agents have not been determined. This study aimed to investigate the effects of lupinifolin in combinations with some antibacterial agents, specifically ampicillin, cloxacillin or vancomycin, against <i>S. mutans</i>, Methicillin-Sensitive <i>S. aureus</i> (MSSA) and Methicillin-Resistant <i>S. aureus</i> (MRSA). <b>Materials and Methods:</b> The checkerboard assay was performed to determine the antibacterial activity of lupinifolin plus the testing antibacterial agents. The Fractional Inhibitory Concentration Index (FICI) was calculated to indicate the interaction between lupinifolin and the antibacterial agent tested. <b>Results:</b> Lupinifolin exerted the synergistic activity when using in combination with ampicillin or cloxacillin against MSSA with the FICIs of <u><</u>0.5. The potential synergistic effect was also observed with lupinifolin plus ampicillin or cloxacillin against MRSA. However, the combination of lupinifolin plus vancomycin resulted in no interaction against MRSA. The combined effects of lupinifolin and ampicillin or cloxacillin against <i>S. mutans</i> were somewhat ambiguous with the borderline values of FICI of 0.5156 and 0.5625, respectively. <b>Conclusion:</b> Lupinifolin potentially plays a role as an antibacterial intensifier against some pathogenic gram-positive bacteria, particularly MSSA and MRSA. Nonetheless, further experiments are required to explain the precise mechanism of synergy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Flavonoids/pharmacology , Staphylococcus aureus/drug effects , Ampicillin/pharmacology , Cloxacillin/pharmacology , Drug Synergism , Drug Therapy, Combination , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/growth & development , Microbial Sensitivity Tests , Staphylococcus aureus/growth & development , Vancomycin/pharmacologyABSTRACT
BACKGROUND AND OBJECTIVE: Streptococcus mutans is a dominant causative pathogen of dental caries, which is a major oral health problem affecting million people worldwide. Derris reticulata is a medicinal plant possessing antimicrobial activity against several Gram-positive pathogenic bacteria. None the less, its effects on growth and cariogenic properties of S. mutans has not been clearly established. This study aimed to investigate the antibacterial and anti cariogenic activities of the D. reticulata ethanolic stem extract. MATERIALS AND METHODS: The TLC analysis was performed to authenticate the D. reticulata sample. Minimum inhibition concentration and minimum bactericidal concentration were determined by using broth dilution and drop plate methods, respectively. Sucrose dependent and sucrose independent-adherences, biofilm formation and glycolytic pH drop assays were performed to evaluate the anticariogenic activity. RESULTS: The ethanolic stem extract of D. reticulata possessed the antibacterial activity against S. mutans with the MIC and MBC of 0.875±0.250 and 1.750±0.500 mg mL-1, respectively. The extract at the lower concentrations of sub-MIC also had significant inhibitory actions against the cariogenic properties of S. mutans, including surface adherence, biofilm formation and glycolytic acid production. CONCLUSION: The D. reticulata stem extract had a substantial anticariogenic activities and thus potentially be developed as an oral health care product for dental caries prevention in the near future.
Subject(s)
Anticarcinogenic Agents/pharmacology , Derris/chemistry , Ethanol/chemistry , Plant Extracts/pharmacology , Streptococcus mutans/drug effects , Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Dental Caries/drug therapy , Microbial Sensitivity Tests/methodsABSTRACT
DNA containing unmethylated deoxycytidylyl-deoxyguanosine (CpG) dinucleotides (CpG DNA) is a potent stimulator of immune responses through triggering of Toll-like receptor 9 (TLR9). In the present study, we synthesized cholesterol-modified CpG oligodeoxynucleotide (Chol-CpG ODN) and investigated its ability to form nanoparticles by self-assembling, then examined their immunostimulatory activity and potency to deliver antigens to antigen presenting cells (APCs). Chol-CpG ODN spontaneously formed particles in aqueous solutions. Cholesterol modification increased the stability of ODN in serum. Chol-CpG ODN was efficiently taken up by mouse macrophage-like RAW264.7 cells and induced a large amount of tumor necrosis factor-α compared with unmodified CpG ODN. Then, ovalbumin (OVA), a model antigen, was incorporated into Chol-CpG ODN nanoparticles. Cholesterol-modified GpC ODN (Chol-GpC ODN) was used to assess the importance of CpG motif on the antigen-specific immune response. Vaccination of mice with OVA/Chol-CpG ODN induced high level interferon-γ production from splenocytes. Furthermore, a high serum level of OVA-specific immunoglobulin G2a was observed in mice receiving OVA/Chol-CpG ODN. Neither CpG ODN nor Chol-GpC ODN was effective at all. These results indicate that self-assembling nanoparticles of Chol-CpG ODN are effective for inducing antigen-specific immune responses because of the high immunostimulatory activity, ability to incorporate antigens and tropism to APCs.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Antigens/administration & dosage , Cholesterol/administration & dosage , Nanoparticles/administration & dosage , Oligodeoxyribonucleotides/administration & dosage , Ovalbumin/administration & dosage , Adjuvants, Immunologic/chemistry , Animals , Antigens/immunology , Cell Line , Cholesterol/chemistry , Female , Immunization/methods , Immunoglobulin G/blood , Interferon-gamma/immunology , Mice , Mice, Inbred C57BL , Nanoparticles/chemistry , Oligodeoxyribonucleotides/chemistry , Ovalbumin/immunology , Spleen/drug effects , Spleen/immunology , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.
Subject(s)
DNA/chemistry , Dinucleoside Phosphates/chemistry , Doxorubicin/administration & dosage , Drug Carriers/chemistry , Hydrogels/chemistry , Adenocarcinoma/drug therapy , Adenocarcinoma/immunology , Adenocarcinoma/metabolism , Animals , Cell Line , Cell Line, Tumor , DNA/immunology , Dinucleoside Phosphates/immunology , Doxorubicin/chemistry , Drug Carriers/administration & dosage , Hydrogels/administration & dosage , Immunization , Luciferases, Firefly/metabolism , Macrophages , Male , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Microscopy, Electron, Scanning , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation.
Subject(s)
Adjuvants, Immunologic/chemistry , DNA/chemistry , DNA/immunology , Oligodeoxyribonucleotides/chemistry , Phosphorothioate Oligonucleotides/chemistry , Adjuvants, Immunologic/metabolism , Animals , Cell Line , Cell Membrane Permeability , DNA/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/immunology , Oligodeoxyribonucleotides/metabolism , Organophosphates/chemistry , Organophosphates/immunology , Organophosphates/metabolism , Phosphorothioate Oligonucleotides/immunology , Phosphorothioate Oligonucleotides/metabolism , Tumor Necrosis Factor-alpha/immunologyABSTRACT
DNA is one of the most important components for all living organisms and many species, including humans, use DNA to store and transmit genetic information to new generations. Recent advances in the handling of DNA have made it possible to use DNA as a building block of nano-sized materials with precisely designed architectures. Although various approaches have been proposed to obtain DNA assemblies with designed architecture in the nano- to micrometer range, there is little information about their interaction with biological components, including target molecules. Understanding the interaction between DNA assemblies and the body is highly important for successful pharmaceutical and biomedical applications. Here, we first review the basic aspects of externally administered DNA molecules, including the stability, permeability and delivery issues. Then, we discuss the unique responses observed in the interaction of structured DNA assemblies and cells expressing Toll-like receptor-9, the receptor responsible for the recognition of unmethylated CpG dinucleotides that are abundant in the DNA of invading pathogens, such as bacteria and viruses.
Subject(s)
DNA/therapeutic use , Nanoparticles/therapeutic use , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/therapeutic use , DNA/chemistry , DNA/genetics , Drug Carriers , Humans , Molecular Conformation , Nanoparticles/ultrastructure , Nanotechnology , Nucleic Acid Conformation , Oligodeoxyribonucleotides/therapeutic useABSTRACT
To achieve delivery of doxorubicin (DXR), a very commonly used anticancer agent, to tumor tissues, it was intercalated to plasmid DNA to obtain a plasmid DNA/DXR complex. The cytotoxic effects of DXR, DNA and their complex were examined in colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, co-cultured with RAW264.7 murine macrophage-like cells. Both CpG motif-containing plasmid DNA (CpG plasmid DNA) and DXR significantly inhibited the proliferation of colon26/Luc cells, but their complex was the most effective among those examined. Non-CpG plasmid DNA was less effective than the CpG plasmid DNA. When injected into mice bearing hepatic metastases of colon26/Luc cells, the CpG plasmid DNA/DXR complex produced a significant level of IL-12 in the serum and liver. The amount of DXR delivered to tumor tissues in the liver was greater when DXR was injected as a CpG plasmid DNA/DXR complex than as free DXR. The CpG plasmid DNA/DXR complex effectively inhibited the proliferation of colon26/Luc cells in the liver compared with free DXR, CpG plasmid DNA, or non-CpG plasmid DNA/DXR complex. These results indicate that CpG plasmid DNA is an effective polymer that inhibits tumor growth by delivering both a proinflammatory signal and anticancer agent to tumor tissues.
Subject(s)
Adenocarcinoma/drug therapy , Adjuvants, Immunologic/administration & dosage , Antibiotics, Antineoplastic/administration & dosage , Colonic Neoplasms/drug therapy , CpG Islands , Doxorubicin/administration & dosage , Liver Neoplasms/drug therapy , Plasmids/administration & dosage , Adenocarcinoma/genetics , Adenocarcinoma/immunology , Adenocarcinoma/secondary , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Chemistry, Pharmaceutical , Coculture Techniques , Colonic Neoplasms/genetics , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , CpG Islands/immunology , Endothelial Cells/drug effects , Endothelial Cells/immunology , Gene Transfer Techniques , Inflammation Mediators/metabolism , Injections, Intravenous , Interleukin-12/blood , Liver/drug effects , Liver/immunology , Liver/pathology , Liver Neoplasms/genetics , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Macrophages/drug effects , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Plasmids/immunology , Time Factors , Tumor Necrosis Factor-alpha/metabolismABSTRACT
DNA containing unmethylated CpG dinucleotides, or CpG motifs, (CpG DNA) has been explored as a therapeutic agent, owing to its potent immunostimulatory activity. A previous study showing that Y-shaped (Y-) CpG DNA has a high immunostimulatory activity compared with single- or double stranded CpG DNA suggests the possibility that CpG DNA in a more complicated structure is a stronger activator of the immune system. In the present study, dendrimer-like DNA (DL-DNA) was prepared by ligating Y-DNA monomers. The DL-DNA of the second or third generation with 12 or 24 highly potent CpG motifs in one unit, respectively, were designed and successfully prepared for the first time. These DL-DNAs induced greater amounts of tumor necrosis factor-alpha and interleukin-6 from RAW264.7 macrophage-like cells than did a mixture of Y-DNA with the same sequences as the corresponding DL-DNA. DL-DNA was more efficiently taken up by RAW264.7 cells than Y-DNA, but the increase was lower than that exhibited by the levels of cytokine release. These results suggest that the dendritic structure formation is a potential approach to increasing the immunostimulatory activity of CpG DNA without any modifications of the chemical structure of the natural phosphodiester DNA.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Cytokines/immunology , DNA/administration & dosage , DNA/chemical synthesis , Macrophages/immunology , Animals , Cell Line , CpG Islands , DNA/ultrastructure , Dendrimers/administration & dosage , Dendrimers/chemistry , Macrophages/drug effects , RatsABSTRACT
DNA containing unmethylated CpG dinucleotides (CpG DNA) is a potent activator of innate and acquired immune responses. Although the sequence-specific immunostimulatory activity of CpG DNA has been extensively explored, little information is available about the importance of the stereochemical properties of CpG DNA. In this study, Y-shaped oligodeoxynucleotides (Y-ODNs) were prepared using three ODNs with the halves of each ODN being partially complementary to a half of the other two ODNs. Y-ODN induced greater amounts of tumour necrosis factor-alpha and interleukin-6 from RAW264.7 macrophage-like cells than did conventional single-stranded ODN (ssODN) or double-stranded ODN (dsODN). The Y-ODN was less stable in serum than dsODN, but greater amounts of Y-ODN were taken up by macrophage-like cells compared with dsODN. A newly designed Y-ODN containing three potent CpG motifs generated significantly higher levels of cytokines compared with dsODN containing the identical sequences. These results indicate that the Y-shaped form of ODN is a novel, reproducible and reliable approach to enhancing the immunostimulatory activity of ODNs.