Search

Allergic rhinitis (AR), one of the most common inflammatory diseases, is caused by immunoglobulin E (IgE)-mediated reactions against inhaled allergens. AR involves mucosal inflammation driven by type 2 helper T (Th2) cells. Previously, it was shown that the

Atopic dermatitis (AD) is an inflammatory disease driven in part by type 2 helper T (Th2) cytokines and skin barrier disruption alleviating the entry of allergens. Thymic stromal lymphopoietin (TSLP), an epithelial cell‒derived cytokine, is known to aggravate AD symptoms by activating Th2. In addition, regulatory T cells (Tregs) inhibit inflammatory cells such as Th2. However, the relationship between TSLP and Tregs in AD is unclear. A murine dermatitis model was induced by applying oxazolone to the ear skin of mice. Prophylactic and therapeutic responses were analyzed by immunizing mice intranasally with a pneumococcal pep27 mutant (Δpep27 mutant), attenuated strain by reducing the virulence of a pathogen. Intranasal immunization with a pneumococcal pep27 mutant could elicit anti-inflammatory Treg-relevant factors and epithelial barrier genes (loricrin, involucrin, filaggrin, and small proline-rich repeat proteins). Thus, pneumococcal pep27-mutant immunization suppressed epidermal collapse, IgE, TSLP, and upregulation of Th2 expression by upregulating Treg activity. In contrast, Treg inhibition aggravated AD symptoms through the upregulation of TSLP and Th2 and the repression of epithelial barrier function compared with that of the noninhibited pneumococcal Δpep27-mutant group. Taken together, immunization with pneumococcal Δpep27 mutant upregulated Treg and epithelial barrier function and inhibited TSLP and Th2 to relieve AD symptoms.

Inflammatory bowel disease (IBD) is a chronic gut inflammatory disease characterized by extensive colitis and remission of the symptoms. The incidence rate and prevalence of IBD are increasing worldwide; IBD affects millions of people, has poorly defined etiology, and often results in a failure of pharmacological interventions. Regardless of the cause, mucosal healing is indispensable for the regeneration of inflamed mucosa to ensure intestinal homeostasis. Intranasal immunization with the pneumococcal pep27 mutant (Δpep27) has been reported as an avirulent and live vaccine that has been proposed to suppress immune-regulated disorders, eliciting long-lasting immunity. The dose-dependent activity of Δpep27 in the lungs was measured by transcriptome analysis to investigate the long-lasting immunogenic response against IBD. Novel therapeutic targets based on the modulation of Wnt signaling and T regulatory cells interconnected with other signaling cascades in the context of IBD were investigated by qPCR and immunoblotting. M1/M2 macrophages were quantified by FACS analysis. Dextran sulfate sodium-induced colitis induced significant upregulation of Th2 and Th17 as well as noncanonical Wnt5, which subsequently inhibited regulatory T (Treg) expression. In contrast, Δpep27 immunization significantly attenuated the levels of Wnt5, proinflammatory cytokines, oxidative stress parameters, and infiltration of inflammatory cells and enhanced barrier integrity via T helper cell homeostasis and upregulation of M2 macrophages. The data of the present study suggested that Δpep27-elicited Tregs were able to repress Wnt5a expression, assisting with the restoration of immunological tolerance and providing a robust regenerative and antioxidant milieu.

Under specific conditions, white adipose tissue (WAT) depots are readily converted to a brown-like state, which is associated with weight loss. However, whether diet-derived factors directly induce browning of white adipocytes has yet to be established. Thus, we investigated the effects of allicin, one of the major components of garlic, on brown-like adipocyte formation in inguinal WAT (iWAT), and prevention of obesity and related complications in animal models. Allicin significantly increased mRNA and/or protein expression of brown adipocyte markers including uncoupling protein 1 (UCP1) in differentiated mouse embryonic fibroblast cell line 3T3-L1 and differentiated iWAT stromal vascular cells (SVC), suggesting that allicin induced brown-like adipocyte formation in vitro. Concomitantly, allicin markedly enhanced the protein expression of KLF-15 and its interaction with UCP-1 promoter region. Such changes were absent in cells lacking KLF-15, suggesting the critical role of KLF15 in allicin action. Allicin also induced brown-like adipogenesis in vivo along with the appearance of multilocular adipocytes, increased UCP1 expression and increased lipid oxidation. In summary, our data suggest that allicin potentially prevents obesity and associated metabolic disorders such as type 2 diabetes mellitus by enhancing the expression of brown adipocyte-specific genes, including UCP-1, through KLF15 signal cascade.

Aims Streptococcus pneumoniae infection in acute bacterial meningitis can lead to widespread brain damage and mortality. Inflammatory responses by immune cells in the brain are thought to determine the degree of brain injury. Yet, the mechanisms underlying host responses to pneumococcal meningitis are largely unknown. To explore host responses as a potential therapeutic target for preventing brain injury after pneumococcal meningitis. Methods We evaluated signaling mechanisms that minimize neuronal damage caused by pneumococcal infection; specifically, we assessed pathways related to neuronal survival after enhancing estrogen receptor-β (ER-β) expression using a natural therapeutic substance known as ginsenoside Rb1 and Rg3 enhanced ginseng. Results Tissue damage caused by bacterial infection was reduced in Rb1/Rg3-treated mice as a result of microglial activation and the inhibition of apoptosis. Furthermore, Rb1 upregulated the expression of brain-derived neurotrophic factor (BDNF) as well as anti-apoptotic factors including Bcl-2 and Bcl-xL. Using BV2 microglial cells in vitro, Rb1 treatment inhibited microglial apoptosis in a manner associated with JAK2/STAT5 phosphorylation. Conclusion After S. pneumoniae infection in mice, particularly in female mice, Rb1-containing ginseng increased bacterial clearance and survival. These findings inform our understanding of the host immune response to pneumococcal meningitis.

Streptococcus pneumoniae (pneumococcus) is responsible for significant morbidity and mortality worldwide. It causes a variety of life-threatening infections such as pneumonia, bacteremia, and meningitis. In bacterial physiology, the metabolic pathway of branched-chain amino acids (BCAAs) plays an important role in virulence. Nonetheless, the function of IlvC, one of the enzymes involved in the biosynthesis of BCAAs, in S. pneumoniae remains unclear. Here, we demonstrated that downregulation of BCAA biosynthesis by ilvC ablation can diminish BCAA concentration and expression of pneumolysin (Ply) and LytA, and subsequently attenuate virulence. Infection with an ilvC mutant showed significantly reduced mortality and colonization in comparison with strain D39 (serotype 2, wild type), suggesting that ilvC can potentiate S. pneumoniae virulence due to adequate BCAA synthesis. Taken together, these results suggest that the function of ilvC in BCAA synthesis is essential for virulence factor and could play an important role in the pathogenesis of respiratory infections.

Pneumonia is an inflammatory disease of the lung, responsible for high morbidity and mortality worldwide. It is caused by bacteria, viruses, fungi, or other microorganisms. Streptococcus pneumoniae, a gram-positive bacterium with over 90 serotypes, is the most common causative agent. Moreover, comorbid factors including heart failure, renal disease, and pulmonary disease could increase the risk of pneumococcal pneumonia. Since the advent of the pneumococcal vaccine in the 1980s, the incidence of pneumonia has decreased significantly. However, current vaccines confer only limited protection against serotypes included in the vaccine. Thus, to overcome this limitation, new types of pneumococcal vaccines have been sought and under clinical trials. In this review, we discuss pneumonia and summarize the various types of pneumococcal vaccines in progress.

Asthma is an allergic airway disease (AAD) characterized by eosinophilic inflammation, mucus hypersecretion, and airway hyper responsiveness, and it is caused by dysregulated immune responses. Conversely, regulatory T cells (Tregs) control aberrant immune responses and maintain homeostasis. Recent evidence suggests that Streptococcus pneumoniae, including its components as well as a live attenuated mutant, and pneumococcal infection induce Tregs and can thus potentially be harnessed therapeutically for asthma treatment. Previously, a pep27 deletion mutant (Δpep27) demonstrated a significantly attenuated virulence in a sepsis model, and Δpep27 immunization induced serotype-nonspecific protection against S. pneumoniae infection, as well as influenza virus, possibly via an immune tolerance mechanism. Here, the potential of Δpep27 immunization for asthma protection was studied. Mice were immunized intranasally with Δpep27 before or after ovalbumin sensitization and subsequent challenge. Δpep27 immunization suppressed hallmark features of AAD, including antigen-specific type 2 helper T cell cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, and goblet cell hyperplasia. Thus, a Δpep27 vaccine may be highly feasible as a preventive or therapeutic agent for asthma.

Obesity is a serious global health problem. Natural substances that could be effective remedies for treatment of obesity, and which are relatively safe, are desired. The aim of this study was to examine the anti-obesity effect and the mechanism of ramalin in 3T3-L1 preadipocytes and high fat diet (HFD)-induced obese mice. In this study, 3T3-L1 cells were treated with various concentrations of ramalin (1, 5, and 10 μg/ml). Ramalin reduced the accumulation of intracellular lipid droplets in 3T3-L1 cells. In addition, ramalin inhibited 3T3-L1 adipocyte differentiation by blocking adipogenic gene expression including CCAAT enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptors γ (PPARγ), adipocyte fatty acid-binding protein (aP2), and leptin. The suppression of adipogenesis by ramalin was mediated through the inhibition of MAPK pathways. Ramalin also reduced the secretion of TNF-α and IL-6 in 3T3-L1 adipocytes. Oral administration of ramalin (50 and 100 mg/kg) to HFD-fed mice reduced body weight gain and abdominal fat accumulation without changes in food intake. Ramalin also attenuated organ weight and basal serum level by inhibiting Iiver X receptors (LXRs), sterol regulatory element-binding protein-1c (SREBP-1c), and lipoprotein lipase (LPL) mRNA expression in HFD-fed mice. Taken together, these results indicate that ramalin inhibits adipogenesis in 3T3-L1 preadipocytes and prevents HFD-induced obesity. The present study also provides insight into the mechanisms underlying the anti-obesity activity of ramalin and suggests that ramalin has the potential to prevent obesity.

Streptococcus pneumoniae is a well-known pathogenic bacterium with a high mortality rate. Currently, a 23-valent pneumococcal polysaccharide vaccine (PPV23) and protein-conjugate vaccines (PCVs) are available on the market. However, both of these vaccines have limitations; specifically, PPV23 produces weak antibody responses in children younger than 2 years and PCVs only partially protect against secondary infection. Previously, we showed serotype-nonspecific protection by Δpep27 vaccine, but the reversion of Δpep27 to the wild type serotype during immunization cannot be excluded. To ensure the safety of the Δpep27 vaccine, comD, an important protein that activates competence, was inactivated, and the transformability of the double mutant (Δpep27ΔcomD) was determined. The transformation ability of this double mutant was successfully abolished. Δpep27ΔcomD immunization significantly increased the survival time after heterologous challenge(s), and diminished colonization levels independent of serotype, including a non-typeable strain (NCC1). Moreover, the double mutant was found to be highly safe in both normal and immunocompromised mice. In conclusion, this pneumococcal Δpep27ΔcomD vaccine appears to be a highly feasible and safe vaccine to prevent various types of pneumococcal infections.

Streptococcus pneumoniae is responsible for significant mortality and morbidity worldwide and causes invasive pneumococcal diseases including pneumococcal meningitis. Pyroptosis is caspase-1-dependent inflammatory cell death and is known to be induced by various microbial infections. In the present study, we investigated the molecular mechanisms that regulate pyroptosis induced by S. pneumoniae in microglia. Our results revealed that S. pneumoniae induced pyroptosis through caspase-1 activation and IL-1β production. We also found that the activation of caspase-1 and the maturation of IL-1β and IL-18 in the S. pneumoniae-triggered pyroptotic cell death process were mediated by NLRP3 inflammasome. In addition, pneumococcal infection increased the expression of autophagy-related genes and induced autophagosome formation. We also showed that the inhibition of autophagy promoted pneumococcus-induced pyroptosis. Furthermore, ROS was generated by pneumococcal infection and inhibited caspase-1 activation within 4 h of infection. However, in the late phase of infection, IL-1β secretion and caspase-1-dependent cell death were induced by ROS. These results suggest that autophagy induction transiently delay pyroptosis induced by S. pneumoniae in microglia. Our study also revealed that the activation of caspase-1 and the production of IL-1β were induced by pneumolysin and that pneumolysin triggered pyroptosis in microglial cells. Similar to the in vitro results, S. pneumoniae induced caspase-1 activation and caspase-1-dependent cytokine maturation in the mouse meningitis model. Thus, the present data demonstrate that S. pneumoniae induces pyroptosis in murine microglia and that NLRP3 inflammasome is critical for caspase-1 activation during the process. Furthermore, the induction of autophagy could transiently protect microglia from pyroptosis.

Macrophage-associated nitric oxide (NO) production plays a crucial role in the pathogenesis of tissue damage. However, negative factors that regulate NO production remains poorly understood despite its significance of NO homeostasis. Here, we show that activating transcription factor 3 (ATF3), a transcriptional regulator of cellular stress responses, was strongly induced in activated macrophages and its depletion resulted in pronounced enhancement of inducible nitric oxide synthase (iNOS) gene expression and subsequently the induction of high levels of NO production. In response to lipopolysaccharide (LPS) and IFN-γ, ATF3 inhibited transcriptional activity of NF-κB by interacting with the N-terminal (1-200 amino acids) of p65 and was bound to the NF-κB promoter, leading to suppression of iNOS gene expression. In addition, inhibitory effects of ATF3 on iNOS and NO secretion were suppressed by inhibitor of casein kinase II (CK2) activity or its knockdown. Moreover, the levels of ATF3 were highly elevated in established cecal ligation and puncture or LPS-injected mice, a model of endotoxemia. ATF3 is also elevated in peritoneal macrophages. Collectively, our findings suggest that ATF3 regulates NO homeostasis by associating with NF-κB component, leading to the repression of its transcriptional activity upon inflammatory signals and points to its potential relevance for the control of cell injuries mediated by NO during macrophage activation.

Activating transcription factor-3 (ATF3) in the ER stress pathway induces cytokine production and promotes survival during gram-positive bacterial infection. IL-17A is a critical cytokine that is essential for clearance of Streptococcus pneumoniae. However, the mechanism by which ATF3 induces IL-17A production remains unknown. Here, we show that ATF3 induces IL-17A production via NLRP3 inflammasome-dependent IL-1β secretion. Survival rates were comparable in IL-17A-depleted and ATF3 KO mice but were lower than in WT mice treated with isotype control, indicating that ATF3 positively regulated IL-17A production. Indeed, ATF3 KO mice showed a marked reduction in IL-17A protein and mRNA expression compared to levels in WT mice. Moreover, mitochondrial IL-1β production by bone marrow-derived macrophages was significantly reduced in ATF3 KO mice as a result of the disruption of cellular ROS and Ca2+ homeostasis. Accordingly, ATF3 KO mice displayed diminished survival and bacterial clearance following S. pneumoniae infection. Taken together, these data suggest a mechanism in which macrophage ATF3 promotes IL-17A production in γδ T cells to rapidly induce host defenses during early S. pneumoniae infection.

Streptococcus pneumoniae (pneumococcus), the major pathogen for pneumonia, commonly colonizes the lung, but the mechanism underlying the coordination of virulence factors during invasion via the host protein remains poorly understood. Bacterial lysis releases the components of the cell wall, and triggers innate immunity and the subsequent secretion of pro-inflammatory cytokines. Previously, the virulence of the pep27 mutant was shown to be attenuated as a feasible candidate for vaccine development. However, the role of pep27 gene, belonging to the vancomycin-resistance locus (vncRS operon), in virulence, is largely unknown. This study demonstrates that transferrin in the host serum reduces the survival of the host during S. pneumoniae infections in mice. The exposure of the pneumococcal D39 strain to lactoferrin induced the vncRS operon, lysis, and subsequent in vivo cytokine production, resulting in lung inflammation. However, these responses were significantly attenuated in pneumococci harboring a mutation in pep27. Mechanistically, the VncS ligand, identified as lactoferrin, induced the vncRS operon and increased the in vivo mortality rates. Thus, serum-induced activation of vncRS plays an essential role in inducing pneumonia.

Previously we showed that conjugation of pneumococcal surface protein A (PspA) to Vi capsular polysaccharide from Salmonella Typhi enhanced the anti-PspA response without the need to add adjuvant. In the current study conjugates consisting of the α helical regions of PspA families 1 or 2 bound to Vi were used to vaccinate mice to test their ability to protect against a lethal intravenous challenge of a range of various strains of Streptococcus pneumoniae. Conjugate vaccine containing PspA family 1 provided good protection from PspA family 1 challenge strains but offered very little protection against PspA family 2 challenge strains. Similarly, PspA family 2 conjugates provided good protection from PspA family 2 challenge strains and poor protection against PspA family 1 challenge strains. This observation was supported by the low levels of cross-reactivity of PspA antibodies seen in ELISA plates coated with the heterologous PspA family. Cytokine profiles showed a mixed Th1/Th2 response to Vi and the Vi-PspA conjugates. IgG subclass analysis of the anti-Vi response showed a shift from predominantly IgG2a/3 to IgG1 after conjugation to PspA was consistent with other polysaccharide conjugate vaccines. The results demonstrate that conjugation of the α helical region of PspA to Vi enhances its capacity to induce a protective immune response and that a vaccine based on the α helical region of PspA should contain PspA from both families 1 and 2 to achieve broad cross-protection.

Obesity is characterized by hypertrophy and/or by the differentiation or adipogenesis of pre-existing adipocytes. In this study, we investigated the inhibitory effects of theobromine, a type of alkaloid in cocoa, on adipocyte differentiation of 3T3-L1 preadipocytes and its mechanisms of action. Theobromine inhibited the accumulation of lipid droplets, the expression of PPARγ and C/EBPα, and the mRNA expression of aP2 and leptin. The inhibition of adipogenic differentiation by theobromine occurred primarily in the early stages of differentiation. In addition, theobromine arrested the cell cycle at the G0/G1 phase and regulated the expressions of CDK2, p27, and p21. Theobromine treatment increased AMPK phosphorylation and knockdown of AMPKα1/α2 prevented the ability of theobromine to inhibit PPARγ expression in the differentiating 3T3-L1 cells. Theobromine reduced the phosphorylation of ERK and JNK. Moreover, the secretion and the mRNA level of TNF-α and IL-6 were inhibited by theobromine treatment. These data suggest that theobromine inhibits adipocyte differentiation during the early stages of adipogenesis by regulating the expression of PPARγ and C/EBPα through the AMPK and ERK/JNK signaling pathways in 3T3-L1 preadipocytes.

Background Previous studies have focused on colonization resistance of the gut microbiota against antibiotic resistant strains. However, less research has been performed on respiratory colonization resistance. Methods Because respiratory colonization is the first step of respiratory infections, intervention to prevent colonization would represent a new approach for preventive and therapeutic measures. The Th17 response plays an important role in clearance of respiratory pathogens. Thus, harnessing the Th17 immune response in the mucosal site would be an effective method to design a respiratory mucosal vaccine. Results In this study, we show that intranasal Δpep27 immunization induces noncanonical Wnt and subsequent interleukin (IL)-17 secretion, and it inhibits Streptococcus pneumoniae, Staphylococcus aureus, and Klebsiella pneumoniae colonization. Moreover, IL-17A neutralization or nuclear factor of activated T-cell inhibition augmented bacterial colonization, indicating that noncanonical Wnt signaling is involved in pulmonary colonization resistance. Conclusions Therefore, Δpep27 immunization can provide nonspecific respiratory colonization resistance via noncanonical Wnt signaling and IL-17A-related pathways.

Adipocyte differentiation is a critical adaptive response to nutritional overload and affects the metabolic outcome of obesity. Sinigrin (2-propenyl glucosinolate) is a glucosinolate belong to the glucoside contained in broccoli, brussels sprouts, and black mustard seeds. We investigated the effects of sinigrin on adipogenesis in 3T3-L1 preadipocytes and its underlying mechanisms. Sinigrin remarkably inhibited the accumulation of lipid droplets and adipogenesis by downregulating the expression of CCAAT-enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), leptin and aP2. Sinigrin arrested cells in the G0/G1 phase of the cell cycle and increased the expression of p21 and p27. CDK2 expression was suppressed by sinigirn in MDI-induced adipocytes. Sinigrin increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and acetyl-CoA carboxylase (ACC) in the early stage of adipocyte differentiation, suggesting that sinigrin has anti-adipogenic effects through AMPK, MAPK and ACC activation. Sinigrin also inhibited the production of pro-inflammatory cytokines including tumor necrosis factor -alpha (TNF-α) and interleukin (IL)-6, IL-1β and IL-18. Taken together, these data suggest that sinigrin inhibits early-stage adipogenesis of 3T3-L1 adipocytes through the AMPK and MAPK signaling pathways.

More than 50% of sepsis cases are associated with pneumonia. Sepsis is caused by infiltration of bacteria into the blood via inflammation, which is triggered by the release of cell wall components following lysis. However, the regulatory mechanism of lysis during infection is not well defined. Mice were infected with Streptococcus pneumoniae D39 wild-type (WT) and lipase mutant (ΔlipA) intranasally (pneumonia model) or intraperitoneally (sepsis model), and survival rate and pneumococcal colonization were determined. LipA and autolysin (LytA) levels were determined by qPCR and western blotting. S. pneumoniae Spd_1447 in the D39 (type 2) strain was identified as a lipase (LipA). In the sepsis model, but not in the pneumonia model, mice infected with the ΔlipA displayed higher mortality rates than did the D39 WT-infected mice. Treatment of pneumococci with serum induced LipA expression at both the mRNA and protein levels. In the presence of serum, the ΔlipA displayed faster lysis rates and higher LytA expression than the WT, both in vitro and in vivo. These results indicate that a pneumococcal lipase (LipA) represses autolysis via inhibition of LytA in a sepsis model.

During influenza pandemics, secondary pneumococcal infections cause excessive mortality. However, the current 13-valent pneumococcal conjugate vaccine, PCV13, provides only limited protection against secondary infection. Therefore, a more effective pneumococcal vaccine is required to protect against secondary pneumococcal infections. Here, intranasal immunization with an attenuated pneumococcal pep27 mutant provides protection from influenza virus infection, and also from secondary pneumococcal challenge. These results indicate that mucosal immunity might be an effective way to reduce the morbidity and mortality due to secondary pneumococcal infections during influenza pandemics.

Activating transcription factor-3 (ATF3) acts as a negative regulator of cytokine production during Gram-negative bacterial infection. A recent study reported that ATF3 provides protection from Streptococcus pneumoniae infection by activating cytokines. However, the mechanism by which S. pneumoniae induces ATF3 after infection is still unknown. In this study, we show that ATF3 was upregulated via Toll-like receptor (TLR) pathways in response to S. pneumoniae infection in vitro. Induction was mediated by TLR4 and TLR2, which are in the TLR family. The expression of ATF3 was induced by pneumolysin (PLY), a potent pneumococcal virulence factor, via the TLR4 pathway. Furthermore, ATF3 induction is mediated by p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Thus, this study reveals a potential role of PLY in modulating ATF3 expression, which is required for the regulation of immune responses against pneumococcal infection in macrophages.

In the current study pneumococcal surface protein A (PspA) was conjugated to Vi capsular polysaccharide from Salmonella Typhi to make available a vaccine against typhoid fever that has the potential to also provide broad protection from Streptococcus pneumoniae. High yielding production processes were developed for the purification of PspAs from families 1 and 2. The purified PspAs were conjugated to Vi with high recovery of both Vi and PspA. The processes developed especially for PspA family 2 could readily be adapted for large scale production under cGMP conditions. Previously we have shown that conjugation of diphtheria toxoid (DT) to Vi polysaccharide improves the immune response to Vi but can also enhance the response to DT. In this study it was shown that conjugation of PspA to Vi enhanced the anti-PspA response and that PspA was a suitable carrier protein as demonstrated by the characteristics of a T-cell dependent response to the Vi. We propose that a bivalent vaccine consisting of PspA from families 1 and 2 bound to Vi polysaccharide would protect against typhoid fever and has the potential to also protect against pneumococcal disease and should be considered for use in developing countries.

The standard opsonophagocytosis killing assay (OPKA) for antibodies to pneumococcal capsular polysaccharide was modified to permit an evaluation of the protection-mediating antibodies to pneumococcal surface protein A (PspA). We found that by increasing the incubation time with the complement and phagocytes from 45 min to 75 min, the protective activity was readily detected. In another modification, we used a capsule type 2 target strain that expressed PspA but not pneumococcal surface protein C (PspC). With these modifications separately or in combination, rabbit antisera to the recombinant α-helical or proline-rich domains of PspA mediated >50% killing of the target strain. The ability of normal human sera to mediate the killing of pneumococci in this modified OPKA correlated with their levels of antibodies to PspA and their ability to protect mice against fatal infection with a type 3 strain. Passive protection of mice against pneumococci and killing in the modified OPKA were lost when normal human sera were adsorbed with recombinant PspA (rPspA) on Sepharose, thus supporting the potential utility of the modified OPKA to detect protective antibodies to PspA. In the standard OPKA, monoclonal antibodies to PspA were strongly protective in the presence of subprotective amounts of anti-capsule. Thus, the currently established high-throughput OPKA for antibodies to capsule could be modified in one of two ways to permit an evaluation of the opsonic efficacy of antibodies to PspA.

Senescence is an irreversible proliferation arrest that is induced by various stress stimuli including genotoxin. Pneumolysin (PLY) is a pathogenicity factor unique to Streptococcus pneumoniae that is important in pneumococcal-induced diseases such as otitis media, meningitis and pneumonia. However, the cell fate response to the toxin is mechanistically unclear. We investigated the effect of PLY on cellular senescence in BV-2 microglial cells. Exposure to PLY resulted in changes in the expression of phospho-p53, p21, p16, pRb and CDK2 and increased the number of senescence associated β-gal positive cells. PLY-treatment also increased PAI-1 expression and cell proliferation arrest in concentration- and time-dependent manners. PLY induced NF-κB activation and phosphorylation of SIRT-1, ERK1/2, JNK, and p38 MAPK. In addition, PLY increased the production of reactive oxygen species. Overall, the results suggest that PLY regulates microglial cellular senescence by enhancing production of reactive oxygen species, activation of MAPK and NF-κB, and phosphorylation of SIRT-1.

Sinigrin (2-propenyl glucosinolate) is found mainly in broccoli, brussels sprouts, and black mustard seeds. Recently, sinigrin has received attention for its role in disease prevention and health. This study investigated the effect of sinigrin on macrophage function, including the activity of Nod-like receptor protein 3 (NLRP3) inflammasome. In a concentration-dependent manner, sinigrin inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production and the expression of COX-2 and prostaglandin E2 (PGE2) in RAW 264.7 cells. In addition, sinigrin significantly suppressed the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 via suppression of MAPK phosphorylation and nuclear factor-kappa B (NF-κB) activity. Treatment with sinigrin decreased IL-1β and IL-18 production and concurrently suppressed NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 expression in LPS/ATP-stimulated cells, suggesting that the blocking of NLRP3 inflammasome activation prevented the production of both cytokines. Collectively, these results suggest that sinigrin has immunomodulatory effects by suppressing the production of inflammatory mediators, possibly by inhibiting the NF-κB/MAPK pathways or NLRP3 inflammasome activation. Our findings also provide evidence that the pharmacological modulation of sinigrin could have an anti-inflammatory effect.

Background information Adipose tissue regulates energy metabolism by means of adipocyte hypertrophy and/or the differentiation of pre-existing adipocytes. Excessive production of some cytokines in adipose tissue is known to be a negative regulator of adipocyte differentiation, and the resulting impaired adipogenesis contributes to disorders like insulin resistance. IFN-α is a key immunoregulatory cytokine in the development of type 1 diabetes, lipid disorders and insulin resistance; however, its effect on adipogenesis remains unknown. Method We examined the effect of IFN-α on adipocyte differentiation and its mechanisms. The effect of IFN-α on adipogenesis was evaluated by Western blotting, qRT-PCR, flow cytometric analysis and Oil Red O staining. We also investigated the role of STAT1 in adipogenesis using gene silencing analysis. Results IFN-α inhibited the accumulation of lipid droplets and the expression of adipogenesis related genes. The inhibition of adipocyte differentiation by IFN-α occurred in the early stages of differentiation. IFN-α arrested the cell cycle at the G0/G1 phase and regulated the expression of CDK2 and p21. These results were confirmed in MEF cells. Treatment with IFN-α increased STAT1 phosphorylation, and STAT1 siRNA or inhibitor prevented IFN-α from inhibiting the expression of PPARγ and C/EBPα as well as cell cycle progression in 3T3-L1 cells. Conclusion We suggest that IFN-α inhibits adipocyte differentiation during the early stage of adipogenesis by regulating the expression of PPARγ and C/EBPα as well as the cell cycle through JAK/STAT1 signaling pathways. General significance Our study provides new insights into possible mechanisms of the anti-adipogenetic effects of IFN-α.

In the presence of normal serum, complement component C3 is deposited on pneumococci primarily via the classical pathway. Pneumococcal surface protein A (PspA), a major virulence factor of pneumococci, effectively inhibits C3 deposition. PspA’s C terminus has a choline-binding domain that anchors PspA to the phosphocholine (PC) moieties on the pneumococcal surface. C-reactive protein (CRP), another important host defense molecule, also binds to PC, and CRP binding to pneumococci enhances complement C3 deposition through the classical pathway. Using flow cytometry of PspA+ and PspA− strains, we observed that the absence of PspA led to exposure of PC, enhanced the surface binding of CRP, and increased the deposition of C3. Moreover, when the PspA− mutant was incubated with a pneumococcal eluate containing native PspA, there was decreased deposition of CRP and C3 on the pneumococcal surface compared with incubation with an eluate from a PspA− strain. This inhibition was not observed when a recombinant PspA fragment, which lacks the choline-binding region of PspA, was added to the PspA− mutant. Also, there was much greater C3 deposition onto the PspA− pneumococcus when exposed to normal mouse serum from wild-type mice as compared with that from CRP knockout mice. Furthermore, when CRP knockout mouse serum was replenished with CRP, there was a dose-dependent increase in C3 deposition. The combined data reveal a novel mechanism of complement inhibition by a bacterial protein: inhibition of CRP surface binding and, thus, diminution of CRP-mediated complement deposition.

Atherosclerosis is a chronic inflammatory disease and the expression of adhesion molecules on vascular smooth muscle cells (VSMCs) contributes to the progress of the disease. Diosgenin, a precursor of steroid hormones, has been shown to have a variety of biological activities including anti-inflammatory activity; however, its molecular mechanisms are poorly understood. This study examined the effect of diosgenin on the expression of adhesion molecules induced by TNF-α in cultured mouse VSMC cell line, MOVAS-1. Preincubation of VSMCs for 2h with diosgenin (0.1-10 μM) dose-dependently inhibited TNF-α-induced adhesion of THP-1 monocytic cells and mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Diosgenin abrogated TNF-α induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38, ERK, JNK and Akt. Diosgenin was also shown to inhibit NK-κB activation induced by TNF-α. Furthermore, diosgenin inhibited TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NF-κB. Our results indicate that diosgenin inhibits the adhesive capacity of VSMC and the TNF-α-mediated induction of ICAM-1 and VCAM-1 in VSMC by inhibiting the MAPK/Akt/NF-κB signaling pathway and ROS production, which may explain the ability of diosgenin to suppress inflammation within the atherosclerotic lesion and modulate immune response.

Helicobacter pylori is one of the main causes of atrophic gastritis and gastric carcinogenesis. Gastritis can also occur in the absence of H. pylori as a result of bile reflux suggesting the eradication of H. pylori by bile acids. However, the bile salts are unable to eradicate H. pylori due to their low solubility and instability at acidic pH. This study examined the effect of a highly soluble and acid stable ursodeoxycholic acid (UDCA) formula on H. pylori-induced atrophic gastritis. The H. pylori infection decreased the body weight, mitochondrial membrane potential and ATP level in vivo. Surprisingly, H. pylori-induced expression of malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, at both the protein and mRNA levels. However, the UDCA formula repressed MDH expression and increased the membrane potential thereby increasing the ATP level and body weight in vivo. Moreover, UDCA scavenged the reactive oxygen species (ROS), increased the membrane potential, and inhibited apoptosis in AGS cells exposed to H(2)O(2) in vitro through the mitochondria-mediated pathway. Taken together, UDCA decreases the MDH and ROS levels, which can prevent apoptosis in H. pylori-induced gastritis.

Multidrug resistance (MDR) is a major problem in cancer chemotherapy. It was previously reported that a red ginseng saponin, 20(S)-ginsenoside Rg3 could modulate MDR in vitro and extend the survival of mice implanted with ADR-resistant murine leukemia P388 cells. This study examined the cytotoxicity of Rg3 on normal and transformed cells, along with its effect on the membrane fluidity. The cytotoxicity study revealed that 120 microM of Rg3 was cytotoxic against a multidrug-resistant human fibroblast carcinoma cell line, KB V20C, but not against normal WI 38 cells in vitro. Flow cytometric analysis using rhodamine 123 as the artificial substrate showed that Rg3 promoted the accumulation of rhodamine 123 in ADR-resistant murine leukemia P388 cells in vivo. Fluorescence polarization studies using the hydrophilic fluorescent probe, DPH, and hydrophobic probe, TMA-DPH, showed that 20 microM Rg3 induced a significant increase in fluorescence anisotropy in KB V20C cells but not in the parental KB cells. These results clearly show that Rg3 decreases the membrane fluidity thereby blocking drug efflux.

Streptococcus pneumoniae is comprised of more than 90 serotypes and is the major causative agent of pneumonia, which results in over 1million deaths worldwide every year. Currently available injectable vaccines can protect against only 13-23 serotypes, and result in decrease of colonization against vaccine serotypes. However, they are neither effective for inhibition of non-vaccine serotypes colonization nor inhibition against initial colonization in the nasopharynx against various serotypes. Thus, development of a vaccine conveying broader protection at the colonization stage is required. This study examined whether the Δpep27 mutant could provide protection at the nasopharynx against a broad range of serotypes. Δpep27 immunization stimulated secretion of IL-4, IL-10, TNF-α, INF-γ and IL-17, and significantly increased secretory-IgA levels in bronchoalveolar lavage fluid. Colonization and opsonophagocytosis assays demonstrated that Δpep27 immunization could protect against many heterologous infections, including non-typeable strains, at the nasopharynx, and prompted efficient killing of heterologous strains, suggesting that Δpep27 immunization provides a wide range of cross-protection. Furthermore, Δpep27 immunization significantly increased both the survival rate and the level of IgG 3months post-immunization, demonstrating long-lasting immunity. Thus, Δpep27 could serve as a highly feasible mucosal vaccine once it is further developed into a non-transformable strain.

Prodigiosin was isolated from marine bacteria Hahella chejuensis which has been recently discovered from Marado, Cheju Island, Republic of Korea. Immunosuppressive properties have been reported for prodigiosin members such as undecylprodigiosin, metacycloprodigiosin, prodigiosin and its synthetic analogue PNU156804 (PNU). However, the effect of this agent on macrophage function has not been characterized in detail. In the present study, we examined the effects of prodigiosin on the production of inflammatory cytokines and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine macrophage. When thioglycollate-elicited macrophages pre-exposed to prodigiosin (1-100 ng/ml) were stimulated with LPS, pretreatment with prodigiosin resulted in the inhibition of NO production and inducible nitric oxide synthase (iNOS) protein and mRNA expression in a concentration-dependent manner. In contrast, the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 was not altered. Inhibition of iNOS protein expression appears to be at the transcriptional level, since prodigiosin decreased LPS-induced NF-kappaB activity through preventing the degradation of IkBalpha, with significant inhibition achieved following pretreatment with prodigiosin. However, prodigiosin did not exert any effect on AP-1 activity. Prodigiosin blocked phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK), but not that of extracellular signal-regulated kinase 1/2 (ERK 1/2). These results indicate that the inhibition of these signaling molecules expression was correlated with the reduced production of NO in macrophages. Taken together, the present data suggest that prodigiosin reduces NO production and iNOS expression by inhibiting LPS-triggered p38 MAPK and JNK phosphorylation and NF-kappaB activation, thereby implicating a mechanism by which prodigiosin may exert its immunosuppressive effects.

Streptococci cause a variety of diseases, such as dental caries, pharyngitis, meningitis, pneumonia, bacteremia, endocarditis, erysipelas, and necrotizing fasciitis. The natural niche of this genus of bacteria ranges from the mouth and nasopharynx to the skin, indicating that the bacteria will inevitably be subjected to environmental changes during invasion into the host, where it is exposed to the host immune system. Thus, the Streptococcus-host interaction determines whether bacteria are cleared by the host's defenses or whether they survive after invasion to cause serious diseases. If this interaction was to be deciphered, it could aid in the development of novel preventive and therapeutic agents. Streptococcus species possess many virulent factors, such as peroxidases and heat-shock proteins (HSPs), which play key roles in protecting the bacteria from hostile host environments. This review will discuss insights into the mechanism(s) by which streptococci adapt to host environments. Additionally, we will address how streptococcal infections trigger host stress responses; however, the mechanism by which bacterial components modulate host stress responses remains largely unknown.

More than 50% of sepsis cases are caused by Streptococcus pneumoniae, and hospital mortality related to sepsis comprises 52% of all hospital deaths. Therefore, sepsis is a medical emergency, and any treatment against the agent that produces it, is welcome.The role of Panax ginseng C.A. Meyer (Araliaceae) aqueous extract in bacterial infection in vivo is not well understood. Here, the protective effect of Korean red ginseng (KRG) extract against pneumococcal infection and sepsis was elucidated.In this study, mice were administrated KRG (25, 50, 100 mg/kg) for 15 days, and then infected with a lethal S. pneumoniae strain. Survival rate, body weight, and colonization were determined.The RAW 264.7 macrophage cells were infected with S. pneumoniae and cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Inflammation was examined using an enzyme-linked immunosorbent assay (ELISA) and hematoxylin and eosin (HE) staining while gene expression was determined using western blotting.KRG-pre-treated mice (100 mg/kg of KRG) had significantly higher survival rates and body weights than those of the non-treated controls; KRG-pre-treated mice had lower bacterial number and morbidity than those of the non-treated controls. 100 mg/kg of KRG administration decreased cytokine levels including tumor necrosis factor (TNF)-α (897 and 623 pg/ml, control and KRG groups, respectively, P0.05) and interleukin (IL)-1β (175 and 127 pg/ml, control and KRG groups, respectively, P = 0.051), nitric oxide level (149 and 81 nM, control and KRG groups, respectively, P0.05), and neutrophil infiltration 48 h post-infection, in vivo. In pneumococcal infection, KRG pre-treatment downregulated toll-like receptor (TLR) 4 and TNF-ɑ expressions in RAW 264.7 macrophage cells and increased cell survival by activating phosphoinositide 3-kinase (PI3K)/AKT signaling.Taken together, 100 mg/kg of KRG appeared to protect host cells from lethal pneumococcal sepsis by inhibiting inflammation as well as by enhancing bacterial clearance thereby reinforcing cell survival against pneumococcal infection.

Activating signal cointegrator 2 (ASC-2) is a transcriptional coactivator of many nuclear receptors (NRs) and other transcription factors and contains two NR-interacting LXXLL motifs (NR boxes). In the pancreas, ASC-2 is expressed only in the endocrine cells of the islets of Langerhans, but not in the exocrine cells. Thus, we examined the potential role of ASC-2 in insulin secretion from pancreatic beta-cells. Overexpressed ASC-2 increased glucose-elicited insulin secretion, whereas insulin secretion was decreased in islets from ASC-2+/- mice. DN1 and DN2 are two dominant-negative fragments of ASC-2 that contain NR boxes 1 and 2, respectively, and block the interactions of cognate NRs with the endogenous ASC-2. Primary rat islets ectopically expressing DN1 or DN2 exhibited decreased insulin secretion. Furthermore, relative to the wild type, ASC-2+/- mice showed reduced islet mass and number, which correlated with increased apoptosis and decreased proliferation of ASC-2+/- islets. These results suggest that ASC-2 regulates insulin secretion and beta-cell survival and that the regulatory role of ASC-2 in insulin secretion appears to involve, at least in part, its interaction with NRs via its two NR boxes.

The abuse of methamphetamine (MA) is an increasingly growing problem globally and produces serious side effects. In the present study, the immunomodulating effects of MA were examined on the immune system after MA (5 mg/kg body weight) was administered daily orally for 14 d. The immune system was evaluated by the antibody response to sheep red blood cells (SRBC; plaque assay and serum immunoglobulin [Ig] G), natural killer (NK) activity, lymphocyte subpopulations in the spleen and thymus, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. Body weight, spleen weight, and thymus weight generally decreased in MA-treated mice. MA treatment induced an increase in the percentage of CD4(+) cells with simultaneous decrease in the percentages of CD8(+) and double-positive CD4(+)CD8(+) in thymus. MA inhibited the IgM plaque-forming cell number, and lowered the level of IgG, the proliferation of mitogen-stimulated B and T cells, and the growth of granulocyte-macrophage colony-forming units (CFU-GM). Exposure to MA also decreased interleukin-2 production by splenocytes. In contrast, splenic NK activity in exposed mice was significantly enhanced. Taken together, data indicate that the immune system was suppressed by oral MA exposure.

The in vivo immunomodulatory function of the activity of murine natural killer (NK) cells induced by high mannuronic acid-containing alginate (HMA) was examined. HMA was injected i.p at doses of 25 and 100 mg/kg. The NK activity was 3 times higher with 100 mg/kg HMA than the baseline. In addition, in vitro studies of splenocytes cultured with HMA for 20 h showed a significant increase in NK activity at E:T ratio of 100:1; a 160% and 210% increase at 10 and 100 microg/mL, respectively. There was a six fold increase in interferon-gamma production in a postculture of splenocytes with 100 microg/mL HMA. HMA had no suppressive effects on the lymphocyte function in the presence or absence of mitogens. This suggests that HMA is useful in cancer immunotherapy.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.

Spread of Streptococcus pneumoniae from the nasopharynx to other host tissues would require the organism to adapt to a variety of environmental conditions. Since heat shock proteins are induced by environmental stresses, we investigated the effect of heat shock on ClpL and ClpP synthesis and the effect of clpL and clpP mutations on the expression of key pneumococcal virulence genes. Pulse labeling with [ 35 S]methionine and chase experiments as well as immunoblot analysis demonstrated that ClpL, DnaK, and GroEL were stable. Purified recombinant ClpL refolded urea-denatured rhodanese in a dose-dependent manner, demonstrating ClpL's chaperone activity. Although growth of the clpL mutant was not affected at 30 or 37°C, growth of the clpP mutant was severely affected at these temperatures. However, both clpL and clpP mutants were sensitive to 43°C. Although it was further induced by heat shock, the level of expression of ClpL in the clpP mutant was high at 30°C, suggesting that ClpP represses expression of ClpL. Furthermore, the clpP mutation significantly attenuated the virulence of S. pneumoniae in a murine intraperitoneal infection model, whereas the clpL mutation did not. Interestingly, immunoblot and real-time reverse transcription-PCR analysis demonstrated that pneumolysin and pneumococcal surface antigen A were induced by heat shock in wild-type S. pneumoniae. Other virulence genes were also affected by heat shock and clpL and clpP mutations. Virulence gene expression seems to be modulated not only by heat shock but also by the ClpL and ClpP proteases.

Multidrug resistance has been a major problem in cancer chemotherapy. In this study, in vitro and in vivo modulations of MDR by ginsenoside Rg(3), a red ginseng saponin, were investigated. In flow cytometric analysis using rhodamine 123 as an artificial substrate, Rg(3) promoted accumulation of rhodamine 123 in drug-resistant KBV20C cells in a dose-dependent manner, but it had no effect on parental KB cells. Additionally Rg(3) inhibited [3H]vinblastine efflux and reversed MDR to doxorubicin, COL, VCR, and VP-16 in KBV20C cells. Reverse transcriptase-polymerase chain reaction and immuno-blot analysis after exposure of KBV20C cells to Rg(3) showed that inhibition of drug efflux by Rg(3) was due to neither repression of MDR1 gene expression nor Pgp level. Photo-affinity labeling study with [3H]azidopine, however, revealed that Rg(3) competed with [3H]azidopine for binding to the Pgp demonstrating that Rg(3) competed with anticancer drug for binding to Pgp thereby blocking drug efflux. Furthermore, Rg(3) increased life span in mice implanted with DOX-resistant murine leukemia P388 cells in vivo and inhibited body weight increase significantly.

Simazine, a triazine herbicide, was investigated for its in vivo immunomodulatory properties. Male C57Bl/6 mice were treated with vehicle or 300 or 600 mg/kg body weight (bw) simazine daily orally for 4 wk. The immune system was evaluated by the antibody response to sheep red blood cells (SRBC; plaque assay and serum immunoglobulin G), natural killer (NK) and macro-phage activities, lymphocyte subpopulations in the spleen and thymus, and concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated lymphocyte proliferation using splenocytes. Body weight and spleen and thymus weight decreased generally in simazine-treated mice, while the weight of adrenal glands was higher than in the control. Simazine treatment (600 mg/kg) induced an increase in the percentage of CD4(+) cells in spleen and CD8 + in thymus. Simazine inhibited the IgM plaque-forming cell numbers and lowered the level of IgG and the proliferation of mitogen-stimulated B cells and T cells. In addition, splenic NK and peritoneal macrophage activities in exposed mice were significantly decreased. Exposure to simazine also decreased cytokine production by macrophages, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha). Taken together, data indicate that the immune system was suppressed by oral simazine exposure.

Capsular polysaccharide type 2 (PS) from Streptococcus pnemoniae induced the secretory and cellular macrophage response. However, the exact mechanism by which PS regulates the macrophage functions remains unclear. In this study, we examined signal molecules which may participate in PS-elicited responses by macrophages. Our data demonstrated that tumoricidal activation of macrophages induced by PS was inhibited by either protein kinase C (PKC) inhibitor, H7 or protein tyrosine kinase (PTK) inhibitor, genistein. In addition, these inhibitors blocked the production of TNF-α and NO in PS-stimulated macrophages. Furthermore, PS-induced cell activation is possibly mediated by Toll-like receptor 2. These data suggest that PKC and PTK are involved in the activation of macrophages with PS.

Senescence, which is irreversible cell cycle arrest, is induced by various types of DNA damage, including genotoxic stress. Senescent cells show dysregulation of tumor suppressor genes and other regulators of cellular proliferation. Activating transcription factor 3 (ATF3) plays a pleiotropic role in biological processes through genotoxic stress. In this study, we examined the effects of acrylamide (ACR), a genotoxic carcinogen, on cellular senescence and the molecular mechanisms of ATF3 function in macrophages. Treatment of macrophages with ACR at low concentrations (

Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae , including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae , as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (Δ adhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H 2 O 2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the Δ adhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the Δ adhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence.

Background Activating transcription factor-3 (ATF3) is known as a suppressor of cytokine production after exposure to lipopolysaccharide or during gram-negative bacterial infection. However, the mechanism by which ATF3 regulates innate immunity against gram-positive bacterial infection, particularly Streptococcus pneumoniae, remains unknown. Methods The wild-type and ATF3 knock-out (KO) mice were infected intranasally (i.n) or intraperitoneally with S. pneumoniae, and bacterial colonization or survival rate was determined. Pneumococcal pneumonia was induced by i.n infection, and ATF3 level was determined by Western blot. ATF3 KO cells or ATF3 siRNA transfection were used to determine expression of ATF3 downstream genes. Enzyme-linked immunosorbent assay was used to examine cytokines levels. Results ATF3 was highly expressed in various cell lines in vitro and in many organs in vivo. Pneumolysin was a novel inducer of ATF3. Pneumococcal infection induced ATF3, which subsequently stimulated production of cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and interferon [IFN]-γ). ATF3-mediated cytokine induction protected the host from pneumococcal infection. In the pneumonia infection model, the bacterial clearance of wild-type mice was more efficient than those of ATF3 KO mice. Conclusions Taken together, we can conclude that ATF3 regulates innate immunity positively upon pneumococcus infection by enhancing TNF-α, IL-1β, and IFN-γ expression and modulating bacterial clearance.

Aflatoxin B 1 (AFB 1 ) is a potent hepatocarcinogen which is thought to exhibit an impairment of specific and non-specific immunity. Macrophages are responsible for non-specific immunity in host defense against tumors and microorganisms, and release a number of cytotoxic compounds, including nitric oxide (NO). We investigate whether the effect of AFB 1 on signal transduction is related to the decrease of NO production in murine peritoneal macrophages. When macrophages were stimulated with lipopolysaccharide (LPS) after AFB 1 -pretreatment, AFB1 decreased the NO production. The percentage of NO production in AFB 1 -pretreated macrophages was inversely increased by the addition of cholera toxin, phorbol 12-myrisate 13-acetate (PMA) and ionomycin. This suggests that AFB 1 affects the function of signaling constituents, including guanine nucleotide-binding protein (G protein), protein kinase C (PKC) and the calcium ion. AFB 1 -pretreatment significantly decreased PKC activity and tyrosine phosphorylation after LPS-stimulation. Taken together, these data propose that the inhibition of LPS-stimulated NO production by AFB 1 is related to the suppression of kinase-mediated intracellular signal transduction in murine peritoneal macrophages.

In this study we examined the potential for the synergistic augmentation of the antitumor activity of inflammatory mouse peritoneal macrophages by stimulation with protein A combined with IFN-gamma. The moderate augmentative effect induced by preincubation with protein A was demonstrated to be concentration-dependent, whereas IFN-gamma had a very low activating effect. Following preincubation with both protein A and IFN-gamma, a marked enhancement of macrophage activity was noted. In addition, based on the utilization of neutralizing antibody to TNF-alpha or the inhibition of NO production, TNF-alpha and NO were proven to be involved as mediators during the activation of tumoricidal macrophages by protein A in combination with IFN-gamma. We also demonstrated that supernatants from macrophages treated with protein A plus IFN-gamma contained both TNF-alpha and NO at markedly increased levels. Thus, tumor cell lysis in the combined system was mediated via TNF-alpha or NO. These results demonstrate the synergistic effects on mouse peritoneal macrophage function of protein A in combination with IFN-gamma and suggest that combinations of such agents may serve as the basis for future in vivo immunotherapy.

We examined the immunosuppressive effects of aflatoxin B1 (AFB1), a toxic compound produced by the Aspergillus flavus, on murine peritoneal macrophages after in vitro pre-exposure. When thioglycollate-elicited macrophages pre-exposed to AFB1 were stimulated with lipopolysaccharide (LPS), antitumor activity induced by LPS was suppressed by 10 and 50 μM AFB1. In addition, the production of reactive intermediates including nitric oxide (NO), superoxide anion and hydrogen peroxide which have been known to be implicated in macrophage-mediated cytotoxicity, was decreased by AFB1 pretreatment in a dose-dependent manner. We also determined whether the macrophage-mediated cytokine production was altered by AFB1 in vitro pretreatment. AFB1 markedly inhibited TNF-α, interleukin-1 (IL-1) and IL-6 production by LPS-stimulated macrophages. Taken together, these data indicate that AFB1 inhibits the killing ability of murine macrophages, decreases various secretory molecules in those cells and the macrophages would be one of many systems affected by AFB1.

Aflatoxin B, (AFB1), a potent hepatocarcinogen, has been known to impair non-specific and specific immune responses. Nitric oxide (NO), hydrogen peroxide (H2O2), superoxide anion (O2-) and tumor necrosis factor-alpha (TNF-alpha) produced by macrophages play an important role in host defense against tumors and microorganisms. In the present studies, we investigated the involvement of those products in the reduced antitumor activities by AFB1. When macrophages are stimulated with LPS after AFB1-pretreatment, the cytolytic activities decrease in a dose-dependent manner. The addition of N(G)-monomethyl arginine (NMMA), anti-TNF-alpha antibodies, catalase and peroxidase decreases antitumor activities further. In contrast, superoxide dismutase (SOD) does not change the antitumor activities. NO and TNF-alpha production was reduced by the addition of NMMA and anti-TNF-alpha antibodies, respectively. Taken together, these data indicate that the reduced antitumor activities in murine peritoneal macrophages are mediated by the suppressed production of NO, TNF-alpha and H2O2 by AFB1 pretreatment, suggesting that the inhibitory effect of AFB1 on those materials may provide the tumors with readily growing condition in vivo.

Atherosclerosis is a chronic inflammatory disease, which is associated with increased expression of adhesion molecules and monocyte recruitment into the arterial wall. This study evaluated whether hexane extracts from the edible part (DB-H1) or bark region (DB-H2) of Dioscorea. batatas Decne have anti-atherosclerotic properties in vivo and in vitro experiments. We also identified bioactive components in the hexane extracts. Thirty-six apolipoprotein E (ApoE(-/-) ) mice and 12 control (C57BL/6J) mice were given a Western-type diet for 11 or 21 wk. To examine the effects of yam extracts on lesion development, ApoE(-/-) mice were orally administered DB-H1 or DB-H2 for the duration of the study (200 mg/kg b.w./day, 3 times per wk). Both DB-H1 and DB-H2 significantly reduced the total atherosclerotic lesion area in the aortic root. In addition, plasma concentrations of total cholesterol, oxidized-low-density lipoprotein, and c-reactive protein were decreased by administration of DB-H1 and DB-H2. Consistent with the in vivo observations, DB-H1 and DB-H2 inhibited tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule-1 expression and adhesion of THP-1 monocytes to TNF-α-activated vascular smooth muscle cells. It was also found that treatment with DB-H1 or DB-H2 resulted in the inhibition nitric oxide (NO) and reactive oxygen species production and iNOS expression in macrophages. Thus, DB-H1 and DB-H2 seem to influence atherosclerosis by affecting the production of inflammatory mediators in vivo. Our results suggest that yam extracts have the potential to be used in the prevention of atherosclerosis.