Molecular characterization of the nonstructural 5A (NS5A) region of hepatitis C virus in Thai blood donors.

Direct acting antivirals (DAAs) have been developed for hepatitis C virus (HCV) therapy, and they are usually effective, however resistance to DAA regimens has also been reported to have a significant impact. Resistance associated substitutions (RASs) in the NS5A region are known to be correlated with failure of DAA therapy. HCV genotypes 3a and 1 are the most prevalent genotypes in Thailand. This study analyzed the type and frequency of RASs associated with DAA failure, focusing on the NS5A region. Serum samples of HCV genotype 3a, 1a, and 1b infection from Thai blood donors were selected. The NS5A region was amplified using reverse transcription-polymerase chain reaction (RT-PCR). A phylogenetic tree was constructed to identify the genotypes of HCV. Nucleotide sequencing and amino acid sequencing were conducted to determine the prevalence of RASs. Construction of the phylogenetic tree indicated that 29 samples were genotype 3a, 11 samples were genotype 1a, and 9 were genotype 1b. Both HCV genotypes 1a and 3a can be categorized into two subclades. Results showed that the NS5A substitutions A30V/K, A62T/V/I/M/P/S/L, and S98G were present in HCV genotype 3a. In HCV genotype 1a, only NS5A RASs H54Y was detected. NS5A amino acid substitutions Q54H and P58L were found in HCV genotype 1b. In conclusion, NS5A RASs at amino acid positions 30, 62, 54, 58, and 98 are present within HCV genotypes 3a and 1. While keeping in mind that additional information was not available on the anonymous blood donors tested in this study, these findings can contribute to understand the NS5A mutation. Further study with known patients under drug treatment is recommended.

The pomegranate-derived peptide Pug-4 alleviates nontypeable Haemophilus influenzae-induced inflammation by suppressing NF-kB signaling and NLRP3 inflammasome activation.

The respiratory pathogen nontypeable Haemophilus influenzae (NTHi) is the most common cause of exacerbation of chronic obstructive pulmonary disease (COPD), of which an excessive inflammatory response is a hallmark. With the limited success of current medicines there is an urgent need for the development of novel therapeutics that are both safe and effective. In this study, we explored the regulatory potential of pomegranate-derived peptides Pug-1, Pug-2, Pug-3, and Pug-4 on NTHi-induced inflammation. Our results clearly showed that to varying degrees the Pug peptides inhibited NTHi-induced production of IL-1ß, a pivotal cytokine in COPD, and showed that these effects were not related to cytotoxicity. Pug-4 peptide exhibited the most potent inhibitory activity. This was demonstrated in all studied cell types including murine (RAW264.7) and human (differentiated THP-1) macrophages as well as human lung epithelial cells (A549). Substantial reduction by Pug-4 of TNF-α, NO and PGE2 in NTHi-infected A549 cells was also observed. In addition, Pug-4 strongly inhibited the expression of nuclear-NF-κB p65 protein and the NF-κB target genes (determined by IL-1ß, TNF-α, iNOS and COX-2 mRNA expression) in NTHi-infected A549 cells. Pug-4 suppressed the expression of NLRP3 and pro-IL-1ß proteins and inhibited NTHi-mediated cleavage of caspase-1 and mature IL-1ß. These results demonstrated that Pug-4 inhibited NTHi-induced inflammation through the NF-κB signaling and NLRP3 inflammasome activation. Our findings herein highlight the significant anti-inflammatory activity of Pug-4, a newly identified peptide from pomegranate, against NTHi-induced inflammation. We therefore strongly suggest the potential of the Pug-4 peptide as an anti-inflammatory medicine candidate for treatment of NTHi-mediated inflammation.

A wild rice-derived peptide R14 ameliorates monosodium urate crystals-induced IL-1ß secretion through inhibition of NF-κB signaling and NLRP3 inflammasome activation.

Gout is an inflammatory arthritis initiated by the deposition of monosodium urate crystals (MSU) around the joints and surrounding tissues. MSU crystals activate the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome to the release of interleukin-1ß (IL-1ß). Gout can have a substantial impact on patient's quality of life, and currently available medicines are unable to meet all the clinical needs. This study explored anti-gout potentials of the Rice14 (R14) peptide, a peptide derived from leaves of wild rice Oryza minuta. The effects of R14 peptide on IL-1ß secretion in THP-1 macrophages with MSU crystals-induced inflammation were examined. Our results clearly showed that the R14 peptide significantly inhibited the secretion of IL-1ß in MSU crystals-induced macrophages, and the effects were dose-related. For safety testing, the R14 peptide did not show both cytotoxicity and hemolytic activity. In addition, the R14 peptide strongly suppressed the phospho-IκB-α and nuclear factor kappa-B (NF-κB) p65 proteins in NF-κB signaling pathway, reduced the NLRP3 expression and inhibited the MSU crystals-mediated cleavage of caspase-1 as well as mature IL-1ß. The R14 peptide also reduced MSU-triggered intracellular ROS levels in macrophages. Taken together, these results indicated that R14 peptide inhibited MSU crystals-induced IL-1ß production through NF-κB and NLRP3 inflammasome activation. Our findings demonstrated that R14 peptide, the newly recognized peptide from wild rice, possessed potent regulatory activity against IL-1ß production in MSU crystals-induced inflammation, and we therefore propose that the R14 peptide is a promising molecule with potential clinical application in the treatment of MSU crystals-induced inflammation.

Partial nonstructural 3 region analysis of hepatitis C virus genotype 3a.

BACKGROUND: The hepatitis C virus (HCV) is a major cause of illness around the world. HCV genotype 3a is the most prevalent genotype in Thailand. Direct-acting antiviral (DAA) drugs are available for treatment, and these drugs target the NS3, NS5A, and NS5b proteins of HCV. However, HCV variants that are resistant to NS3 protease inhibitors have been found during treatment. This resistance can be naturally occurring or in response to treatment. The purpose of this study is to analyze the codon positions of the main mutation of the partial NS3 gene region of HCV genotype 3a. METHODS: In order to detect mutations and confirm the genotype of HCV genotype 3a, the nucleotide sequencing and amino acid portion of NS3 were analyzed. RESULTS: Twenty-six samples were successfully sequenced and clustered within two sub-clades defined as 3a-1 and 3a-2. Through amino acid mutation analysis, the variations were detected at codon positions 122 (3.8%), 132 (84.6%), 168 (100%), 170 (92.3%), 174 (100%), and 175 (100%). CONCLUSIONS: In conclusion, mutations at positions 168, 170, 174, and 175 of the NS3 region are common within the HCV genotype 3a. This information should be useful in the development of effective anti-viral drugs that can successfully treat HCV infection.

Genomic analysis uncovers laccase-coding genes and biosynthetic gene clusters encoding antimicrobial compounds in laccase-producing Acinetobacter baumannii.

Laccases are multicopper oxidase family enzymes that can oxidize various substrates. In this study, we isolated laccase-producing Acinetobacter spp. from the environment, and one isolate of laccase-producing Acinetobacter baumannii, designated NI-65, was identified. The NI-65 strain exhibited constitutive production of extracellular laccase in a crude extract using 2,6-dimethoxyphenol as a substrate when supplemented with 2 mM CuSO4. Whole-genome sequencing of the NI-65 strain revealed a genome size of 3.6 Mb with 3,471 protein-coding sequences. The phylogenetic analysis showed high similarity to the genome of A. baumannii NCIMB8209. Three laccase proteins, PcoA and CopA, that belong to bacterial CopA superfamilies, and LAC-AB, that belongs to the I-bacterial bilirubin oxidase superfamily, were identified. These proteins were encoded by three laccase-coding genes (pcoA, copA, and lac-AB). The lac-AB gene showed a sequence similar to that of polyphenol oxidase (PPO). Gene clusters encoding the catabolized compounds involved in the utilization of plant substances and secondary metabolite biosynthesis gene clusters encoding antimicrobial compounds were identified. This is the first report of whole-genome sequencing of laccase-producing A. baumannii, and the data from this study help to elucidate the genome of A. baumannii to facilitate its application in synthetic biology for enzyme production.

4-methoxybenzalacetone, the cinnamic acid analog as a potential quorum sensing inhibitor against Chromobacterium violaceum and Pseudomonas aeruginosa.

The continuous increase in the incidence of infectious diseases and the rapid unchecked rise in multidrug-resistance to conventional antibiotics have led to the search for alternative strategies for treatment and clinical management of microbial infections. Since quorum sensing (QS) regulates numerous virulence determinants and pathogenicity in bacteria, inhibition of QS promises to be an attractive target for development of novel therapeutics. In this study, a series of cinnamic acid analogs and benzalacetone analogs were designed and synthesized, and their QS-inhibitory activities explored. We found that, among the test compounds, 4-methoxybenzalacetone (8) exhibited potent anti-quorum sensing property, as evidenced by inhibition of QS-controlled violacein production of Chromobacterium violaceum ATCC12472. The inhibitory activity of such a compound, which was the methyl keto analog of the corresponding cinnamic acid, was not only stronger than the parent cinnamic acid (1), but also superior to that of furanone, the reference drug. Based on our observations, its mechanism of quorum sensing inhibition is likely to be mediated by interference with N-acyl-homoserine lactones (AHL) synthesis. Moreover, 4-methoxybenzalacetone (8) also suppressed the production of pyocyanin, rhamnolipids and swarming motility of Pseudomonas aeruginosa, suggesting a broad spectrum of anti-QS activities of this compound. In terms of structure-activity relationship, the possible chemical substitutions on the scaffold of cinnamic acid required for QS inhibitory activity are also discussed. Since 4-methoxybenzalacetone (8) showed no toxicity to both bacteria and mammalian cells, our findings therefore indicate the anti-QS potential of this compound as a novel effective QS inhibitor.

Quercetin negatively regulates IL-1ß production in Pseudomonas aeruginosa-infected human macrophages through the inhibition of MAPK/NLRP3 inflammasome pathways.

Pseudomonas aeruginosa remains a leading cause of nosocomial and serious life-threatening infections, and contributes to increased mortality in immunocompromised individuals. P. aeruginosa infection triggers host immune response and often provokes potent inflammatory mediators, which do not necessarily eradicate the causative pathogen. On the other hand, it causes severe airway damage and eventually decreased lung function. Such unfavorable outcomes of inflammatory injury have necessitated the development of novel effective agents that can combat with P. aeruginosa-mediated inflammation. Herein, we investigated the potential of quercetin in regulating P. aeruginosa-induced inflammation, with particular emphasized on the interleukin-1ß (IL-1ß). Our results showed that quercetin exerted the potent inhibitory activity against the production of IL-1ß in macrophages infected by live P. aeruginosa PAO1, without exhibiting cytotoxicity. According to our settings, such the potent inhibitory activity of quercetin was clearly demonstrated through its ability to efficiently inhibit IL-1ß during P. aeruginosa infection, pre- or even post-infection. In addition, quercetin strongly suppressed MAPK signaling pathway by inhibiting phosphorylation of the p38 MAPK and JNK2, and molecular docking study supported well with this observation. Moreover, quercetin reduced the NLRP3 expression and inhibited the P. aeruginosa-mediated cleavage of caspase-1 as well as mature IL-1ß. These results thus indicated that quercetin inhibition of P. aeruginosa-induced IL-1ß production is mediated by suppressing the initial priming step and by inhibiting the NLRP3 inflammasome activation. Taken together, our findings demonstrated the promising regulatory activity of quercetin against IL-1ß production in P. aeruginosa-infected macrophages, and indicated that quercetin has the potential to be effective as a novel therapeutic agent for treatment of P. aeruginosa-induced inflammation.

A scorpion venom peptide derivative BmKn‒22 with potent antibiofilm activity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a leading cause of nosocomial and serious life-threatening infections and infections caused by this bacterium continue to pose a major medical challenge worldwide. The ability of P. aeruginosa to produce multiple virulence factors and in particular to form biofilms makes this bacterium resistant to all known antibiotics. As a consequence, standard antibiotic therapy are increasingly become ineffective to clear such infections associated with biofilms. In search for novel effective agents to combat P. aeruginosa biofilm infections, a series of the BmKn‒2 scorpion venom peptide and its truncated derivatives were synthesized and their antibiofilm activities assessed. Among the peptides tested, BmKn‒22 peptide, which was a modified peptide of the parental BmKn‒2 scorpion venom peptide, clearly demonstrated the most potential inhibitory activity against P. aeruginosa biofilms without affecting the bacterial growth. This peptide was not only capable of inhibiting the formation of P. aeruginosa biofilms, but also disrupting the established biofilms of P. aeruginosa. Additionally, BmKn‒22 peptide was able to inhibit the production of key virulence factor pyocyanin of P. aeruginosa. Our results also showed that BmKn‒22 peptide significantly reduced lasI and rhlR expression, and suggested that BmKn‒22 peptide-mediated inhibition of P. aeruginosa biofilms and virulence factors was achieved through the components of quorum-sensing systems. Combination of BmKn‒22 peptide with azithromycin resulted in a remarkable reduction P. aeruginosa biofilms. Since this peptide exhibited low toxicity to mammalian cells, all our results therefore indicate that the BmKn‒22 peptide is a promising antibiofilm agent against P. aeruginosa and warrant further development of this peptide as a novel therapeutic for treatment of P. aeruginosa‒associated biofilm infections.

Ethyl rosmarinate inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in alveolar macrophages.

In this study, a series of rosmarinic acid and analogs were investigated for their anti-inflammatory potential against LPS-induced alveolar macrophages (MH-S). Our results showed that, among the test compounds, ethyl rosmarinate (3) exhibited the most potent inhibitory effect on NO production in LPS-induced MH-S cells, with low cytotoxicity. Compound 3 exhibited remarkable inhibition of the production of PGE2 in LPS-induced MH-S cells. The inhibitory potency of compound 3 against LPS-induced NO and PGE2 release was approximately two-fold higher than that of dexamethasone. Compound 3 significantly decreased the mRNA and protein expression of iNOS and COX-2 and suppressed p65 expression in the nucleus in LPS-induced MH-S cells. These results suggested that compound 3 inhibited NO and PGE2 production, at least in part, through the down-regulation of NF-κB activation. Analysis of structure-activity relationship revealed that the free carboxylic group did not contribute to inhibitory activity and that the alkyl group of the corresponding alkyl ester analogs produced a strong inhibitory effect. We concluded that compound 3, a structurally modified rosmarinic acid, possessed potent inhibitory activity against lung inflammation, which strongly supported the development of this compound as a novel therapeutic agent for the treatment of macrophage-mediated lung inflammatory diseases, such as COPD.

Potential role of an antimicrobial peptide, KLK in inhibiting lipopolysaccharide-induced macrophage inflammation.

Antimicrobial peptides (AMPs) are attractive alternatives to antibiotics. Due to their immune modulatory properties, AMPs are at present emerging as promising agents for controlling inflammatory-mediated diseases. In this study, anti-inflammatory potential of an antimicrobial peptide, KLK (KLKLLLLLKLK) and its analogs was evaluated in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. The results herein demonstrated that KLK peptide as well as its analogs significantly inhibited the pro-inflammatory mediator nitric oxide (NO), interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) production in LPS-stimulated RAW 264.7 macrophages in dose-dependent manners, and such inhibitory effects were not due to direct cytotoxicity. When considering inhibition potency, KLK among the test peptides exhibited the most effective activity. The inhibitory activity of KLK peptide also extended to include suppression of LPS-induced production of prostaglandin E2 (PGE2). KLK significantly decreased mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as mRNA expression of IL-1ß and TNF-α. Moreover, KLK inhibited nuclear translocation of nuclear factor-κB (NF-κB) p65 and blocked degradation and phosphorylation of inhibitor of κB (IκB). Taken together, these results suggested that the KLK peptide inhibited inflammatory response through the down-regulation of NF-κB mediated activation in macrophages. Since peptide analogs with different amino acid sequences and arrangement were investigated for their anti-inflammatory activities, the residues/structures required for activity were also discussed. Our findings therefore proved anti-inflammatory potential of the KLK peptide and provide direct evidence for therapeutic application of KLK as a novel anti-inflammatory agent.

Enhanced Antibacterial Activity of Acinetobacter baumannii Bacteriophage ØABP-01 Endolysin (LysABP-01) in Combination with Colistin.

Endolysins are lytic enzymes produced by bacteriophages with their ability to degrade the cell wall of bacterial hosts. Endolysin (LysABP-01) from Acinetobacter baumannii bacteriophage ØABP-01 was cloned, overexpressed and characterized. Endolysin LysABP-01 has a globular structure consisting of lysozyme-like (N-acetyl-ß-D-muramidase) catalytic domain. It contains 185 amino acids which correspond to a 21.1 kDa protein. The lytic activity of the recombinant endolysin protein was determined by a plate lysis assay for its ability to lyse the autoclaved cell (crude cell wall) of the different bacterial species. LysABP-01 can degrade the crude cell wall of A. baumannii strains, Escherichia coli and Pseudomonas aeruginosa but not of Staphylococcus aureus. The antibacterial activity of LysABP-01 and its synergism with various antibiotics were tested. The results exhibited elevated antibacterial activity in a combination of the sub-MIC LysABP-01 and colistin. The checkerboard assay for measuring antibiotic synergy of LysABP-01 and colistin was performed. This combination was synergistic against various drug-resistant strains of A. baumannii (FIC index < 0.5). In summary, our study highlights the ability of LysABP-01 endolysin to hydrolyze the A. baumannii cell wall and its synergistic interaction with colistin.

In vitro interference of cefotaxime at subinhibitory concentrations on biofilm formation by nontypeable Haemophilus influenzae

Objective: To investigate the in vitro interference of cefotaxime at subinhibitory con-centrations [sub-minimal inhibitory concentrations (MIC)] on biofilm formation by nontypeable Haemophilus influenzae (NTHi). Methods: The interference of subinhibitory concentrations of cefotaxime on biofilm formation of the clinical strong-biofilm forming isolates of NTHi was evaluated by a microtiter plate biofilm formation assay. The effect of sub-MIC cefotaxime on bacterial cell-surface hydrophobicity was determined using a standard microbial adhesion to n-hexadecane test. Additionally, the effects on bacterial adherence to human fibronectin and expression of bacterial adhesins were also investigated. Results: Subinhibitory concentrations of cefotaxime, both at 0.1× and 0.5× MIC levels, efficiently reduced the NTHi biofilm formation, and this effect was independent of decreasing bacterial viability. Sub-MIC cefotaxime also decreased bacterial cell-surface hydrophobicity and reduced adherence to human fibronectin. Inhibition in the P2 and P6 gene expressions upon exposure to sub-MIC cefotaxime was also noted. Conclusions: Taken together, our results indicate that sub-MIC cefotaxime interferes with the formation of NTHi biofilm, and this effect is feasibly related to the interference with cell-surface hydrophobicity, fibronectin-binding activity as well as alteration of the P2 and P6 gene expression. The findings of the present study therefore provide a rationale for the use of subinhibitory concentrations of cefotaxime for treatment of NTHi-related diseases.

High prevalence of multi-drug resistant Streptococcus pneumoniae among healthy children in Thailand.

Antibiotic resistance in Streptococcus pneumoniae is an emerging health problem worldwide. The incidence of antimicrobial-resistant S. pneumoniae is increasing, and nasal colonization of S. pneumoniae in children increases the risk of pneumococcal infection. In this study, the prevalence of S. pneumoniae nasal colonization was studied in Thai children from three different districts. S. pneumoniae nasal colonization was found in 38 of 237 subjects (16.0%). The carriage rate indicated higher rates in two rural districts (18.2% and 29.8%) than in the urban district (2.8%). The antibiotic susceptibility pattern was determined using the disk diffusion method. Prevalence of multi-drug resistance S. pneumoniae (MDR-SP) was 31.6%. Resistance to commonly prescribed antibiotics was found for ampicillin (5.3%), azithromycin (26.3%), cefepime (2.6%), chloramphenicol (18.4%), clindamycin (18.4%), erythromycin (21.1%), oxacillin (44.7%), trimethoprim/sulfamethoxazole (78.9%) and tetracycline (15.8%). All isolates were sensitive to ceftriaxone. The pulsed-field gel electrophoresis pattern was used to compare genetic diversity of the S. pneumoniae isolates. PFGE demonstrated the variation in genotypes of S. pneumoniae from different areas. High prevalence of multi-drug resistance S. pneumoniae nasal colonization in healthy Thai children was indicated. Effective strategies for appropriate use of antibiotics are therefore needed in the community.

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water.

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.

A chalcone with potent inhibiting activity against biofilm formation by nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi), an important human respiratory pathogen, frequently causes biofilm infections. Currently, resistance of bacteria within the biofilm to conventional antimicrobials poses a major obstacle to effective medical treatment on a global scale. Novel agents that are effective against NTHi biofilm are therefore urgently required. In this study, a series of natural and synthetic chalcones with various chemical substituents were evaluated in vitro for their antibiofilm activities against strong biofilm-forming strains of NTHi. Of the test chalcones, 3-hydroxychalcone (chalcone 8) exhibited the most potent inhibitory activity, its mean minimum biofilm inhibitory concentration (MBIC50 ) being 16 µg/mL (71.35 µM), or approximately sixfold more active than the reference drug, azithromycin (MBIC50 419.68 µM). The inhibitory activity of chalcone 8, which is a chemically modified chalcone, appeared to be superior to those of the natural chalcones tested. Significantly, chalcone 8 inhibited biofilm formation by all studied NTHi strains, indicating that the antibiofilm activities of this compound occur across multiple strong-biofilm forming NTHi isolates of different clinical origins. According to antimicrobial and growth curve assays, chalcone 8 at concentrations that decreased biofilm formation did not affect growth of NTHi, suggesting the biofilm inhibitory effect of chalcone 8 is non-antimicrobial. In terms of structure-activity relationship, the possible substituent on the chalcone backbone required for antibiofilm activity is discussed. These findings indicate that 3-hydroxychalcone (chalcone 8) has powerful antibiofilm activity and suggest the potential application of chalcone 8 as a new therapeutic agent for control of NTHi biofilm-associated infections.

Immune enhancement activities of silk lutein extract from Bombyx mori cocoons.

BACKGROUND: Declining immune function poses an important clinical challenge worldwide and supplementation with natural products that possessing immune enhancing properties is a promising approach for preventing or delaying immune function decline. Cocoons from yellow silkworms are a significant source of lutein, and this unexplored silk extract could be a viable alternative source for dietary lutein. This study assessed immunomodulatory activities of the silk lutein extract. Female BALB/c mice orally received lutein, either as silk or marigold extracts (10 or 20 mg/kg daily), or vehicle only (1% tween 80 in PBS pH 7.4) for 4 weeks. Natural killer (NK) cell activity, specific antibody production, lymphocyte subpopulations, mitogen-induced lymphocyte proliferation, and cytokine production were examined. RESULTS: Silk lutein extract increased NK cell activity, and the effect was dose-related whereas marigold lutein extract was ineffective. Silk lutein extract dose-dependently enhanced antibody production in pre-immunized mice but marigold lutein extract had no effect. Feeding with silk lutein extract increased the populations of CD3+ and CD4 + CD3 + cells. Silk lutein extract also stimulated concanavalin A- and lipopolysaccharide-induced proliferations of T and B lymphocytes, respectively. Moreover, silk lutein extract increased IL-2 and IFN-γ production while the effect of marigold lutein extract was undetectable. CONCLUSIONS: Together, silk lutein extract enhanced both innate and adaptive immune functions. This preparation may prove to be an effective supplement for strengthened immunity.

Immune enhancement activities of silk lutein extract from Bombyx mori cocoons

BACKGROUND: Declining immune function poses an important clinical challenge worldwide and supplementation with natural products that possessing immune enhancing properties is a promising approach for preventing or delaying immune function decline. Cocoons from yellow silkworms are a significant source of lutein, and this unexplored silk extract could be a viable alternative source for dietary lutein. This study assessed immunomodulatory activities of the silk lutein extract. Female BALB/c mice orally received lutein, either as silk or marigold extracts (10 or 20 mg/kg daily), or vehicle only (1% tween 80 in PBS pH 7.4) for 4 weeks. Natural killer (NK) cell activity, specific antibody production, lymphocyte subpopulations, mitogen-induced lymphocyte proliferation, and cytokine production were examined. RESULTS: Silk lutein extract increased NK cell activity, and the effect was dose-related whereas marigold lutein extract was ineffective. Silk lutein extract dose-dependently enhanced antibody production in pre-immunized mice but marigold lutein extract had no effect. Feeding with silk lutein extract increased the populations of CD3+ and CD4 + CD3 + cells. Silk lutein extract also stimulated concanavalin A- and lipopolysaccharide-induced proliferations of T and B lymphocytes, respectively. Moreover, silk lutein extract increased IL-2 and IFN-γ production while the effect of marigold lutein extract was undetectable. CONCLUSIONS: Together, silk lutein extract enhanced both innate and adaptive immune functions. This preparation may prove to be an effective supplement for strengthened immunity.

Epitope-specific immune recognition of the nontypeable Haemophilus influenzae outer membrane protein 26.

Previous studies using rodent respiratory infection models of nontypeable Haemophilus influenzae (NTHi) infection have established the 26-kDa outer membrane protein of the bacterium, OMP26, as a potential vaccine antigen for NTHi. This study undertook a comprehensive immunological identification of OMP26 T- and B-cell epitopes. A series of OMP26 peptides were constructed and regions of the OMP26 antigen involved in recognition by lymphocyte receptors and induction of acquired immune responses were identified. The dominant T-cell epitopes for OMP26 were located toward the C-terminus between amino acid residues 95 and 197 (T3+T4 region) as mapped using antigen-specific lymphocyte proliferation assays. The newly identified T-cell epitopes exhibited strong capacity for efficient T-cell activation, suggesting that, compared with other OMP26 regions; epitopes within the T3+T4 region have the highest affinity for binding to major histocompatibility complex molecules. In contrast, the predominant B-cell epitopes of OMP26 were located more centrally within the molecule between amino acid residues 45 and 145 (T2+T3 region) as determined using enzyme-linked immunosorbent assay and surface plasmon resonance assays. The T2+T3 region was immunodominant in several species including chinchilla, mice and rats when assessed using both mucosal and parenteral immunization regimes. In addition, the antibodies directed against the T2+T3 region bound to intact NTHi cell surface, according to flow cytometry. Collectively, these results specifically locate the amino acid sequences containing the OMP26 T- and B-cell epitopes, which, as newly mapped antigenic epitopes for lymphocyte recognition, will be useful to improve existing NTHi vaccine strategies. Comprehensive definition of the minimum epitope length required for optimal B- and T-cell responses requires further study.

A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.

OBJECTIVE: The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance. METHODS: We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates. RESULTS: The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%. CONCLUSION: Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms.

A shift in the hepatitis C virus genotype dominance in blood donor samples from Thailand.

Hepatitis C virus (HCV) can be classified into six major genotypes. The HCV genotypes variability accounts for its geographical distribution, its responses to treatments and the clinical outcomes. The aim of this study was to determine the distribution of HCV genotypes among volunteer blood donors in Thailand. Samples from 135 anti-HCV positive blood donors were analyzed. HCV RNA and genotyping was carried out using nested polymerase chain reaction (PCR) and genotype-specific primer PCR for a portion of the core region. HCV RNA was detected in 109 samples (80.7%). Genotype analysis demonstrated four different genotypes. The most common was genotype 3a (36.7%), followed by genotype 6 (29.4%), 1a (19.3%), 1b (6.4%) and mixed infection (1.8%). Seven samples were untyped (6.4%) in the present study. In several previous reports, the prevalence found in Thailand was HCV genotypes 3, 1 and 6. The present results show an increasing importance of the genotype 6 in HCV infections. This study has also described for the first time in Thailand mixed infections of HCV genotypes.