Antibacterial activity of Dioscorea bulbifera Linn. extract and its active component flavanthrinin against skin-associated bacteria.

BACKGROUND: Dioscorea bulbifera Linn. has been used for wound care in Thailand. However, a comprehensive evaluation of its antibacterial activity is required. This study aimed to investigate the antibacterial efficacy of D. bulbifera extract against skin-associated bacteria and isolate and characterize its active antibacterial agent, flavanthrinin. METHODS: Air-dried bulbils of D. bulbifera were pulverised and extracted with hexane, dichloromethane, ethyl acetate, methanol, ethanol, and distilled water; vacuum filtered; concentrated; freeze-dried; and stored at -20 ºC. Antibacterial activity of the extracts was assessed using microdilution techniques against several skin-associated bacteria. Thin-layer chromatography (TLC) bioautography was used to identify the active compounds in the extract, which were fractionated by column chromatography and purified by preparative TLC. The chemical structures of the purified compounds were analysed using nuclear magnetic resonance (NMR). The cytotoxicity of the extract and its active compounds was evaluated in Vero cells. RESULTS: The ethyl acetate extract exhibited distinct inhibition zones against bacteria compared to other extracts. Therefore, the ethyl acetate extract of D. bulbifera in the ethyl acetate layer was used for subsequent analyses. D. bulbifera extract exhibited antibacterial activity, with minimum inhibitory concentrations (MICs) of 0.78-1.56 mg/mL. An active compound, identified through TLC-bioautography, demonstrated enhanced antibacterial activity, with MICs of 0.02-0.78 mg/mL. NMR analysis identified this bioactive compound as flavanthrinin. Both D. bulbifera extract and flavanthrinin-containing fraction demonstrated potent antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and S. epidermidis. The flavanthrinin containing fraction demonstrated low cytotoxicity against Vero cells, showing CC50 values of 0.41 ± 0.03 mg/mL. These values are lower than the MIC value, indicating that this fraction is safer than the initial ethyl acetate extract. CONCLUSIONS: Dioscorea bulbifera extract and its bioactive component flavanthrinin demonstrated significant antibacterial activity against the skin-associated bacteria Staphylococci, including MRSA. Flavanthrinin has potential as a complementary therapeutic agent for managing skin infections owing to its potent antibacterial effects and low cytotoxicity.

Synergistic Antimicrobial and Antioxidant Properties of Coccinia grandis (L.) Voigt, Clerodendrum inerme (L.) Gaertn. and Acanthus ebracteatus Vahl. Extracts and Their Potential as a Treatment for Xerosis Cutis.

BACKGROUND: A common health condition among older persons is xerosis cutis. Topical corticosteroid treatments are -associated with side effects. There is an unmet need for her-bal treatment alternatives. Coccinia grandis, Clerodendrum inerme and Acanthus ebracteatus are used to treat skin con-ditions in Thai traditional medicine. This study aimed to investigate their antimicrobial and antioxidant properties, synergistic properties as well as their cytotoxicity. METHODS: -Ethanolic herbal extracts were used to perform minimal -inhibitory (MIC) and minimal bactericidal concentration (MBC) assays on common skin pathogens. Synergistic anti-microbial activity was evaluated by a chequerboard assay. Antioxidant and synergistic properties were assessed by a 1,1-diphenyl-2-picrylhydrazyl assay. Cytotoxicity was tested on normal adult human primary epidermal keratinocytes. RESULTS: All extracts showed an inhibitory effect on growth of all microorganisms tested. MIC and MBC values ranged from 0.0625 to 32 mg/mL and from 0.0625 to >256 mg/mL, respectively. A. ebracteatus extract markedly demonstrated bactericidal activity against an methicillin-resistant Staphylococcus aureus strain. Additive antimicrobial activity was observed (fractional inhibitory concentration index values: 0.75-1). All extracts possessed antioxidant properties (IC50 values: 0.12-0.25 mg/L). However, antagonism was observed with paired extract combinations (combination index values: 1.025-1.455). The cell viability assay confirmed that herbal extracts were not cytotoxic. CONCLUSIONS: Our results provide early findings of pharmacological activities to support a novel choice of herbal combinations as potential local skin treatment options for xerosis cutis.

Antibacterial activity of cuminaldehyde on food-borne pathogens, the bioactive component of essential oil from Cuminum cyminum L. collected in Thailand.

Background Cuminum cyminum L., commonly known as cumin, has been traditionally used in Thai traditional medicine and traditional food flavoring. The present study investigated the chemical composition, antimicrobial activity against all tested major food-borne pathogenic bacteria, and bioactive components of essential oil extracted from C. cyminum L. collected in Thailand. Methods The main components of the essential oil were investigated by gas chromatography-mass spectrometry (GC-MS) technique. Antibacterial activities against Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Salmonella Typhi were investigated by disk diffusion and microdilution method. The presence of the biologically active antibacterial components was also confirmed by the thin-layer chromatography (TLC)-bioautography. Results The main components of the essential oil investigated by GC-MS were cuminaldehyde (27.10%), beta-pinene (25.04%) and gamma-terpinene (15.68%). The essential oil exhibited antibacterial activity against B. cereus, S. aureus, E. coli and S. Typhi. The essential oil showed the strongest antimicrobial activity against B. cereus with a comparable inhibition zone to tetracycline. TLC confirmed the presence of biologically active antibacterial component in the essential oil against all tested food-borne bacteria. It is further demonstrated that cuminaldehyde was the most active compound in TLC-bioautography which inhibited all of tested bacteria. Conclusions Essential oil extracted from C. cyminum L. exhibited antibacterial activity against all tested major food-borne pathogenic bacteria. Cuminaldehyde is a major bioactive component. Our results suggest that the essential oil extracted from C. cyminum L. could be applied as an alternative natural preservative to control food-borne disease and have the potential for further development of new antibacterial agents.

Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.

Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.

Evidence for inducible recruitment of Wiskott-Aldrich syndrome protein to T cell receptor-CD3 complex in Jurkat T cells.

BACKGROUND: The engagement of the T cell receptor (TCR)-CD3 complex induces the formation of multiple signalling complexes, which are required for actin cytoskeletal rearrangement. The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polymerization that is recruited to the TCR activation site. Since WASp is a binding partner of adaptor protein Nck, which is recruited directly to the TCR CD3? subunit upon TCR ligation, therefore we proposed that the direct recruitment of Nck to TCR-CD3 may also bring WASp directly to TCR-CD3. OBJECTIVE: The aim of this present study was to assess the distribution of WASp, in relation to Nck, to the TCR-CD3ε complex. METHODS: Jurkat T cells were stimulated with anti-TCR antibody and then the cell lysates were immunoprecipitated with anti-CD3 antibody before immunoblotting with antibodies specific to WASp, Nck1, Nck2, SLP-76 and CD3ε molecules. RESULTS: WASp was recruited to SLP-76 and also directly to the TCR-CD3 complex upon TCR triggering. The inducible recruitment of WASp to the TCR-CD3 complex is partially dependent of tyrosine phosphorylation. CONCLUSIONS: The present findings provide an alternative mechanism of WASp recruitment to the site of TCR activation that may be involved in recruitment of Nck.

Changes in the NS1 gene of avian influenza viruses isolated in Thailand affect expression of type I interferon in primary chicken embryonic fibroblast cells.

The non-structural protein 1 (NS1) of avian influenza virus was defined as one of the virulent factors. To understand the effect of NS1 protein of influenza virus H5N1 isolated in Thailand on type I (α/ß) interferon (IFN) synthesis, five reverse genetic viruses were constructed and used as models. The viruses were generated using NS genomic segment from A/Peurto Rico/8/1934 (H1N1) and four avian influenza viruses isolated from the first outbreak in Thailand. All the viruses have the rest of the genome from A/Peurto Rico/8/1934 (H1N1). The constructed viruses were named (1) NS1 PR8/34, (2) NS1 wild type, (3) NS1 L15FD53G, (4) NS1 N171I and (5) NS1 E71K, respectively. The type I (α/ß) IFN gene expression in control and infected primary chicken embryonic fibroblast cells were analyzed by quantitative polymerase chain reaction. The results show that the inhibition of IFN-ß gene expression by NS1 wild type infected cells is stronger than NS1 N171I, NS1 E71K, NS1 PR8/34 and NS1 L15FD53G, respectively. The data suggest that the difference of amino acid sequence of NS1 protein contributes to the IFN-ß antagonist. In contrast, the difference of the NS1 protein does not influence in the IFN-α antagonistic activity.

Detection of distribution of avian influenza H5N1 virus by immunohistochemistry, chromogenic in situ hybridization and real-time PCR techniques in experimentally infected chickens.

Ten specific pathogen free (SPF) chickens were inoculated intranasally with avian influenza virus subtype H5N1. Evaluation revealed distribution of the virus in twelve organs: liver, intestine, bursa, lung, trachea, thymus, heart, pancreas, brain, spleen, kidney, and esophagus. Immunohistochemistry (IHC), chromogenic in situ hybridization (CISH), and real-time polymerase chain reaction (PCR) were developed and compared for detection of the virus from the organs. The distribution of avian influenza H5N1 in chickens varied by animal and detecting technique. The heart, kidneys, intestines, lungs, and pancreas were positive with all three techniques, while the others varied by techique. The three techniques can be used to detect avian influenza effectively, but the pros and cons of each technique need to be determined. The decision of which technique to use depends on the objective of the examination, budget, type and quality of samples, laboratory facilities and technician skills.

Comparison of neuraminidase activity of influenza A virus subtype H5N1 and H1N1 using reverse genetics virus.

Neuraminidase (NA) is an envelope surface glycoprotein of influenza A viruses. It cleaves alpha-(2,3) or alpha-(2,6) glycosidic linkage between a terminal sialic acid residue of the host cell receptor and hemagglutinin of the viral envelope, thus releasing viral progeny from the infected cell. In this study, a reassortant virus (H1N1-NA-H5N1) containing the NA gene from A/duck/Phitsanulok/ NIAH6-5-0001/2007 (H5N1) virus and seven remaining genetic segments from A/ Puerto Rico/8/1934 (H1N1) was constructed using reverse genetic technique. NA activity of H1N1-NA-H5N1 virus was lower than that of A/Puerto Rico/8/1934 (H1N1), and NA activity of A/duck/Phitsanulok/NIAH6-5-0001/2007 study (H5N1) was the lowest among them (p < 0.05). To our knowledge, this is the first comparative study of NA activity of H1N1 and H5N1 virus using reverse genetic technique. It also indicates that the NA gene may be expressed at a higher level in the H1N1 infected cell than the H5N1 infected cell.

Pregnancy immunology: decidual immune cells.

Human pregnancy is a complex process. Placental development depends on the function of secretory molecules produced by placental trophoblast cells as well as by maternal uterine immune cells within the decidua. These decidual immune cells are T cells, natural killer cells, macrophages and dendritic cells. The interactions between the trophoblast cells and the maternal immune cells have an impact on the outcome of the pregnancy. Knowledge about the phenotypes and functions of the maternal immune cells in normal and pathological pregnancies including recurrent spontaneous abortions, preeclampsia and hydatidiform moles may improve our understanding of the immunobiology of the normal pregnancy as a whole and may provide approaches for improving the treatment of pathological pregnancies.

Genetic characterization of nonstructural genes of H5N1 avian influenza viruses isolated in Thailand in 2004-2005.

The outbreak of highly pathogenic avian influenza (HPAI) viruses has severely disrupted poultry production and trade. Humans have been infected with HPAI H5N1 viruses and many have died. The nonstructural (NS) proteins of the virus are a factor that determines virulence. In this report, 80 NS genes of H5N1 HPAI viruses isolated from Thailand were completely sequenced and phylogenically analyzed. The percentages of identity and variable site NS1 genes were similar to NS2/nuclear export protein (NEP) genes. All NS1 genes from the samples were located in allelic group A. The NS1 and NS2/NEP proteins possess 225 and 121 amino acids, respectively. All NS1 protein samples had five amino acid deletions typical of avian influenza viruses isolated since 2002. An amino acid substitution at position 92 (G92E) of the NS1 protein, known to promote the inhibition of host immune responses, was also found in the samples.

Reduced interleukin-17 expression of Burkholderia pseudomallei-infected peripheral blood mononuclear cells of diabetic patients.

Burkholderia pseudomallei is the causative agent of melioidosis. One of the main risk factors for B. pseudomallei infection in endemic areas is diabetes mellitus. The present study investigated IL-17 mRNA and protein expression by peripheral blood mononuclear cells in response to B. pseudomallei infection in 10 diabetic patients in comparison to 10 healthy blood donors. The IL-17 expression in diabetic patients was significantly lower (p < 0.05) than in the controls. However, IL-23 mRNA expression of the 2 groups was comparable. The present findings suggest that melioidosis affects T cell IL-17 production and that patients with diabetes mellitus have a defective IL-17 production in response to this type of infection.

Whole genome sequences of H5N1 influenza a virus isolated from a little grebe in Thailand.

This is the first report of the whole genome sequence of influenza A virus in an aquatic resident bird of Thailand. It was categorized into genotype Z according to its characteristics of a 20 amino acid deletion in neuraminidase and a five amino acid deletion in the nonstructural protein. The indicator for a highly pathogenic trait of the virus is the presence of a polybasic amino acid sequence at the cleavage site of HA0. The feature of resistance to the antiviral drug amantadine is found at the 31st amino acid position of M2 (serine to asparagine). Phylogenic analyses revealed that virus A/little grebe/Thailand/Phichit-01/2004 (H5N1) is closely related to the chicken and human isolates recovered from Thailand. The high degrees of similarity among the sequences and phylogenic trees indicate there was no difference between the viruses isolated from poultry and aquatic birds in Thailand at the time of study. The results also suggest the source of H5N1 avian influenza virus in the little grebe and others in Thailand may have the same origin.

JEG-3 cell culture supernatants cause reduced interferon-gamma and interleukin-17 production in mixed-lymphocyte reactions.

PROBLEM: Immunoregulatory effects of choriocarcinoma-derived factors on leukocytes have been documented. The present study was designed to investigate the effect of JEG-3 culture supernatants on interferon-gamma (IFN-gamma), interleukin-17 (IL-17) and IL-1beta production in the mixed lymphocyte reactions (MLRs). METHOD OF STUDY: A human choriocarcinoma cell line JEG-3 was used to test the effects of its culture supernatants on the proliferation and cytokine production in the MLRs. The cell proliferation was assessed using the BrdU incorporation and the amounts of cytokines were measured using enzyme-linked immunosorbent assays. RESULTS: The JEG-3 culture supernatants caused significantly reduced IFN-gamma and IL-17 production in the MLRs. However, the supernatants did not influence MLR production of IL-1beta. CONCLUSION: IFN-gamma and IL-17 are mainly produced by activated T cells but IL-1beta is primarily produced by monocytes, thus suggesting that immunoregulatory factors of JEG-3 cells selectively inhibit cytokine production by activated T cells rather than that of the monocytes.

The effect of interleukin-17 on the proliferation and invasion of JEG-3 human choriocarcinoma cells.

PROBLEM: As there has been a study in mice showing the expression of IL-17 by decidual cells and the status of IL-17 receptor expression in human pregnancy is not known, we hypothesized that IL-17 may regulate human trophoblast proliferation and invasion. METHOD OF STUDY: JEG-3 cell line was used as a model for human trophoblast. Immunohistochemitry and reverse transcriptase polymerase chain reaction techniques were used to identify IL-17 receptor protein and mRNA, respectively. The effects of IL-17 on JEG-3 cell proliferation and invasion were tested using the BrdU incorporation and the Matrigel invasion assays, respectively. RESULTS: IL-17 increased the invasive capacity of JEG-3 cells but had no effect on the proliferation and multinucleated formation of JEG-3 cells. CONCLUSION: In this JEG-3 cell model of human trophoblast, the IL-17R and IL-17 may have a regulatory role in trophoblast invasion.