Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.
Immune restoration disease (IRD) associated with Mycobacterium tuberculosis parallels the reconstitution of a pathogen-specific Th1 response. However, it is not clear whether humoral responses to M. tuberculosis antigens also rise, or whether antibody levels predict IRD. Here, humoral immunity to M. tuberculosis antigens was investigated in four Asian cohorts.
Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with L-N(6)-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A(2) (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. L-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA(2) but not PI-3K-dependent fashion.
The aim of this study was to determine the effect of exogenous nitric oxide (NO) on the induction of murine splenic immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in vitro. BALB/c mice were immunized with A. actinomycetemcomitans LPS and a control group was sham-immunized. Spleen cells were obtained, cultured and stimulated with A. actinomycetemcomitans LPS with or without the presence of S-nitroso acetyl-penicillamine (SNAP), a NO donor, and carboxy-PTIO, an NO scavenger. Culture supernatants were assessed for inducible nitric oxide synthase (iNOS) activity, specific IgG subclass levels, and both IFN-gamma and IL-4 levels. The results showed that in A. actinomycetemcomitans LPS-stimulated cells, SNAP enhances iNOS activity but inhibits the levels of specific IgG2a and IFN-gamma suggesting a Th1 response. The effect of SNAP on these immune parameters was ablated by carboxy-PTIO. These results suggest that exogenous NO may suppress the Th1-like immune response of A. actinomycetemcomitans LPS-stimulated murine spleen cells.
Human osteoblasts induced by inflammatory stimuli express an inducible nitric oxide synthase (iNOS). The aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans lipopolysaccharide stimulates the production of nitric oxide (NO) by a human osteoblast-like cell line (HOS cells).
The aim of the present study was to determine whether or not lipopolysaccharide from Actinobacillus actinomycetemcomitans could stimulate arginase activity in a murine macrophage cell line (RAW264.7 cells).
Elevated nitric oxide (NO) has been associated with destructive periodontal disease. The aim of the present study was to test the hypothesis that exogenous NO may inhibit a protective immune response to Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) in a murine model.
Neurodegeneration is typically preceded by neuroinflammation generated by the nervous system to protect itself from tissue damage, however, excess neuroinflammation may inadvertently cause more harm to the surrounding tissues. Attenuating neuroinflammation with non‑steroidal anti‑inflammatory drugs can inhibit neurodegeneration. However, such treatments induce chronic side effects, including stomach ulcers. Madecassoside, a triterpene derived from Centella asiatica, is considered to be an alternative treatment of inflammation. In the present study, the anti‑neuroinflammatory properties of madecassoside were assessed in BV2 microglia cells, which were pre‑treated with madecassoside at a maximum non‑toxic dose (MNTD) of 9.50 µg/ml and a ½ MNTD of 4.75 µg/ml for 3 h and stimulated with 0.1 µg/ml lipopolysaccharide (LPS). The effect of madecassoside was assessed by determining reactive oxygen species (ROS) levels in all groups. Furthermore, the expression of pro‑ and anti‑neuroinflammatory genes and proteins were analyzed using reverse transcription‑quantitative polymerase chain reaction and western blotting, respectively. The results demonstrated that ROS levels in cells treated with the MNTD of madecassoside were significantly reduced compared with cells treated with LPS alone (P<0.05). The expression of pro‑neuroinflammatory genes, including inducible nitric oxide synthase, cyclooxygenase‑2, signal transducer and activator of transcription 1 and nuclear factor‑κB, were significantly downregulated in a dose‑independent manner following treatment with madecassoside. Conversely, the anti‑neuroinflammatory component heme oxygenase 1 was significantly upregulated by 175.22% in the MNTD‑treated group, compared with cells treated with LPS alone (P<0.05). The gene expression profiles of pro‑ and anti‑inflammatory genes were also consistent with the results of western blotting. The results of the present study suggest that madecassoside may be a potent anti‑neuroinflammatory agent. The antioxidative properties of madecassoside, which serve a major role in anti‑neuroinflammation, indicate that this compound may be a functional natural anti‑neuroinflammatory agent, therefore, further in vivo or molecular studies are required.
There is a need for identification of new infection markers against common Leptospira isolates in Malaysia. To achieve this goal, seven-day-old cultures of Leptospira interrogans serogroup Icterohemorrhagiae (L44) and Leptospira interrogans serogroup Javanica (L55) were used for antigen preparation by sequential extraction method using 40 mM Tris, 8M Urea and 2M thiourea. Immunoblot analysis of the antigens were performed using serum samples from 46 local patients with confirmed acute leptospirosis, 28 patients with other infections and 14 healthy controls. The patients serum samples used in this study contained heterologous antibody against a number of different leptospira serovars. A strong IgM reactivity to a broad diffuse band of 10-15 kDa was observed. Combining results using L44 and L55 antigens showed sensitivity of 80.4% and specificity of 95.2% for detection of leptospirosis. Proteinase K and periodate treatment indicated that the band is likely to be lipopolysaccharide (LPS) in nature. This study showed that the 10-15 kDa antigen could potentially be useful for serodiagnosis of acute leptospirosis in Malaysia.
In this paper, we describe the development of VCUSM2, a live metabolic auxotroph of Vibrio cholerae O139. Auxotrophy was achieved by mutating a house keeping gene, hemA, that encodes for glutamyl-tRNA reductase, an important enzyme in the C5 pathway for delta-aminolevulenic acid (ALA) biosynthesis, which renders this strain dependent on exogenous ALA for survival. Experiments using the infant mouse and adult rabbit models show that VCUSM2 is a good colonizer of the small intestine and elicits greater than a four-fold rise in vibriocidal antibodies in vaccinated rabbits. Rabbits vaccinated with VCUSM2 were fully protected against subsequent challenge with 1 x 10(11) CFU of the virulent wild type (WT) strain. Experiments using ligated ileal loops of rabbits show that VCUSM2 is 2.5-fold less toxic at the dose of 1 x 10(6) CFU compared to the WT strain. Shedding of VCUSM2 in rabbits were found to occur for no longer than 4 days and its maximum survival rate in environmental waters is 8 days compared to the greater than 20 days for the WT strain. VCUSM2 is thus a potential vaccine candidate against infection by V. cholerae O139.
Chronic subclinical systemic inflammation has a key role in stimulating several chronic conditions associated with cardiovascular diseases, cancer, rheumatoid arthritis, diabetes, and neurodegenerative diseases. Hence, developing in vivo models of chronic subclinical systemic inflammation are essential to the study of the pathophysiology and to measure the immunomodulatory agents involved. Male Sprague-Dawley rats were subjected to intraperitoneal, intermittent injection with saline, or lipopolysaccharide (LPS) (0.5, 1, 2 mg/kg) thrice a week for 30 days. Hematological, biochemical, and inflammatory mediators were measured at different timepoints and at the end of the study. The hearts, lungs, kidneys, and livers were harvested for histological evaluation. Significant elevation in peripheral blood leukocyte includes neutrophils, monocytes, and lymphocytes, as well as the neutrophils-to-lymphocyte ratio. The pro-inflammatory mediator levels [C-reactive protein, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and IL-8] along with the biochemical profile (alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, creatine kinase, creatinine, and urea) were increased significantly (P
Brucella melitensis is one of the leading zoonotic pathogens with significant economic implications in animal industry worldwide. Lipopolysaccharide, however, remains by far the major virulence with substantial role in diseases pathogenesis. Nonetheless, the effect of B. melitensis and its lipopolysaccharide on immunopathophysiological aspects largely remains an enigma. This study examines the effect of B.melitensis and its lipopolysaccharide on immunopathophysiological parameters following experimental infection using mouse model. Eighty four (n = 84) mice, BALB/c, both sexes with equal gender distribution and 6-8 weeks-old were randomly assigned into three groups. Group 1-2 (n = 72) were orally inoculated with 0.4 mL containing 109 CFU/mL of B. melitensis and its LPS, respectively. Group 3 (n = 12) was challenged orally with phosphate buffered saline and served as a control group. Animals were observed for clinical signs, haematological and histopathological analysis for a period of 24 days post-infection. We hereby report that B.melitensis infected group demonstrated significant clinical signs and histopathological changes than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1β and IL-6) and antibody levels (IgM and IgG) with varying degrees of predominance in LPS infected group than B. melitensis infected group. For hormone analysis, low levels of progesterone, estradiol and testosterone were observed in both B. melitensis and LPS groups throughout the study period. Moreover, in B. melitensis infected group, the organism was re-isolated from the organs and tissues of gastrointestinal, respiratory and reproductive systems thereby confirming the infection and transmission dynamics. This report is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in a mouse model after oral inoculation with B. melitensis and its lipopolysaccharide.
Brucella melitensis is one of the major zoonotic pathogens with significant economic implications worldwide. The pathogenicity is complex and not always well understood. Lipopolysaccharide (LPS) remains the major virulent factor of B. melitensis and responsible for the mechanism by which the pathogen causes its deleterious effects. In this study, 84 mice of 6-8 weeks old of both sexes were divided equally into 3 groups; namely Brucella melitensis infected group, lipopolysaccharide (LPS) infected group and control group. The former two groups contained 36 mice each with equal gender distribution. The control group consisted of 12 mice only. Animals in B. melitensis infected group, a single inoculum of 0.4 ml containing 10(9) of B. melitensis were intraperitoneally challenged while animals in LPS group, a single dose of 0.4 ml containing LPS extracted from the B. melitensis were intraperitoneally inoculated. Animals in control group received intraperitoneally, a single dose of 0.4 ml phosphate buffered saline (PBS) of pH7. Animals that were infected intraperitoneally with B. melitensis demonstrated significant clinical presentation; gross and histo-pathological evidence than LPS infected group. However, both infected groups showed elevated levels of interleukins (IL-1β and IL6), antibody levels (IgM an IgG) as early as 3 days post-infection with predominance in LPS infected group. In contrast, low levels of sex related hormonal changes in which LPS infected group showed the least concentration were also detected throughout the experimental period. In conclusion, B. melitensis can be transmitted via gastrointestinal, respiratory and reproductive tract. Moreover, LPS stimulated significantly the innate and acquired immune system without significant systemic dysfunction, suggesting potentiality of the protective properties of this component as alternative vaccine for brucellosis infection.
In this study, we developed a mouse model and characterized the effects of intranasal inoculation of virulent Brucella melitensis strain 16M and its lipopolysaccharide (LPS). The effects of the exposure were compared with respective control groups. Both Brucella melitensis-infected and LPS-infected groups showed no significant clinical presentation with minor relevance in the mortality associated with the infection. In Brucella melitensis-infected group, significant histopathological changes in comparison to the LPS infected group with increase bacterial burden in the lungs, reproductive and reticuloendothelial organs were observed. However, both infected groups showed elevated levels of pro-inflammatory cytokine expression (IL-1β and IL6) and antibody production (IgM an IgG) as early as 3 days post-infection with predominance in LPS infected group. In contrast, low levels of sex related hormonal changes was recorded in both infected groups throughout the experimental period. This is the first detailed investigation comparing the infection progression and host responses in relation to the immunopathophysiological aspects in mouse model after intranasal inoculation with B. melitensis and its lipopolysaccharide. The study revealed a significant difference between infected and control groups with overlap in clinical, pathological, and immunological responses as well as sex related hormonal changes resulting from the infections.
Circular RNAs (circRNAs) constitute a large class of RNA species formed by the back-splicing of co-linear exons, often within protein-coding transcripts. Despite much progress in the field, it remains elusive whether the majority of circRNAs are merely aberrant splicing by-products with unknown functions, or their production is spatially and temporally regulated to carry out specific biological functions. To date, the majority of circRNAs have been cataloged in resting cells. Here, we identify an LPS-inducible circRNA: mcircRasGEF1B, which is predominantly localized in cytoplasm, shows cell-type specific expression, and has a human homolog with similar properties, hcircRasGEF1B. We show that knockdown of the expression of mcircRasGEF1B reduces LPS-induced ICAM-1 expression. Additionally, we demonstrate that mcircRasGEF1B regulates the stability of mature ICAM-1 mRNAs. These findings expand the inventory of functionally characterized circRNAs with a novel RNA species that may play a critical role in fine-tuning immune responses and protecting cells against microbial infection.
Purified lipopolysaccharide (LPS) of Pasteurella multocida type 6:B, while toxic at higher doses, was protective at lower dose levels against experimentally-induced pasteurellosis in mice. However, the observed protection was abrogated if such LPS was digested with proteinase K prior to use in immunisation. The O-antigen polysaccharide side-chain (OS) of LPS did not appear to contribute to the observed protection as judged by the fact that immunisation of mice with purified OS or OS-protein conjugates, all of which were nontoxic, failed to confer protection against challenge with homologous virulent organisms. This was despite generation of significant levels of OS-specific antibodies, predominantly either of the IgM or IgG isotypes, in immunised mice.
Alternanthera sessilis, an edible succulent herb, has been widely used as herbal drug in many regions around the globe. Inflammation is a natural process of the innate immune system, accompanied with the increase in the level of proinflammatory mediators, for example, nitric oxide (NO) and prostaglandin (PGE2); cytokines such as interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor alpha (TNFα); and enzymes including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) via the activation and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) subunit p65 due to the phosphorylation of inhibitory protein, IκBα. Inflammation over a short period of time is essential for its therapeutic effect. However, prolonged inflammation can be detrimental as it is related to many chronic diseases such as delayed wound healing, cardiovascular disease, arthritis, and autoimmune disorders. Therefore, ways to curb chronic inflammation have been extensively investigated. In line with that, in this present study, we attempted to study the suppression activity of the proinflammatory cytokines and mediators as a characteristic of anti-inflammatory action, by using stem extract of A. sessilis in the lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophage cell line. The results showed that the extract has significantly inhibited the production of the proinflammatory mediators including NO and PGE2; cytokines comprising IL-6, IL-1β, and TNFα; and enzymes covering the iNOS and COX-2 by preventing the IκBα from being degraded, to inhibit the nuclear translocation of NF-κB subunit p65 in order to hinder the inflammatory pathway activation. These results indicated that the stem extract of A. sessilis could be an effective candidate for ameliorating inflammatory-associated complications.
Macrophage phagocytosis is the first line of defense of the innate immune system against malaria parasite infection. This study evaluated the immunomodulatory effects of BCG and recombinant BCG (rBCG) strains expressing the C-terminus of the merozoite surface protein-1 (MSP-1C) of Plasmodium falciparum on mouse macrophage cell line J774A.1 in the presence or absence of lipopolysaccharide (LPS) or LPS + IFN-γ. The rBCG strain significantly enhanced phagocytic activity, production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, nitric oxide (NO), and inducible nitric oxide synthase (iNOS) as compared with parental BCG strain, and these activities increased in the presence of LPS and LPS+IFN-γ. Furthermore, the rBCG strain also significantly reduced the macrophage viability as well as the rBCG growth suggesting the involvement of macrophage apoptosis. Taken together, these data indicate that the rBCG strain has an immunomodulatory effect on macrophages, thus strengthen the rational use of rBCG to control malaria infection.
HMP [3-(2-hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone] was evaluated for its ability to inhibit the synthesis of major proinflammatory mediators and cytokines in interferon-gamma (IFN-gamma)- and lipopolysaccharide (LPS)-induced RAW 264.7 cells and phorbol myristate acetate (PMA)-differentiated/LPS-induced U937 cells. HMP suppressed the production of nitric oxide (NO) with significant inhibitory effects at doses as low as 0.78 microM (P < 0.05). Prostaglandin E2 (PGE2) secretion was also inhibited at doses of 12.5 microM and above (P < 0.01). The secretion of both TNF-alpha and IL-6 were only inhibited at the highest dose used (25 microM; P < 0.001). IL-1beta secretion was also inhibited from 12.5 microM onwards (P < 0.01). This inhibition was demonstrated to be caused by down-regulation of inducible enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), without direct effect upon iNOS or COX-2 enzyme activity. HMP only inhibited iNOS (P < 0.001) and IL-1beta (P < 0.05) gene expression at the highest tested concentration. HMP did not affect the secretion of chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) and the anti-inflammatory cytokine IL-10. The most striking effect of HMP was its NO inhibitory activity and therefore we conclude that HMP is a selective inhibitor of iNOS.
Excessive free radical production by immune cells has been linked to cell death and tissue injury during sepsis. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death that has been identified in several pathological conditions. Caffeic acid phenethyl ester (CAPE) is an active component of honeybee products and exhibits antioxidant, anti-inflammatory, and immunomodulatory activities. The present study examined the ability of CAPE to scavenge peroxynitrite in RAW 264.7 murine macrophages stimulated with lipopolysaccharide/interferon-γ that was used as an in vitro model. Conversion of 123-dihydrorhodamine to its oxidation product 123-rhodamine was used to measure peroxynitrite production. Two mouse models of sepsis (endotoxemia and cecal ligation and puncture) were used as in vivo models. The level of serum 3-nitrotyrosine was used as an in vivo marker of peroxynitrite. The results demonstrated that CAPE significantly improved the viability of lipopolysaccharide/interferon-γ-treated RAW 264.7 cells and significantly inhibited nitric oxide production, with effects similar to those observed with an inhibitor of inducible nitric oxide synthase (1400W). In addition, CAPE exclusively inhibited the synthesis of peroxynitrite from the artificial substrate SIN-1 and directly prevented the peroxynitrite-mediated conversion of dihydrorhodamine-123 to its fluorescent oxidation product rhodamine-123. In both sepsis models, CAPE inhibited cellular peroxynitrite synthesis, as evidenced by the absence of serum 3-nitrotyrosine, an in vivo marker of peroxynitrite. Thus, CAPE attenuates the inflammatory responses that lead to cell damage and, potentially, cell death through suppression of the production of cytotoxic molecules such as nitric oxide and peroxynitrite. These observations provide evidence of the therapeutic potential of CAPE treatment for a wide range of inflammatory disorders.