Suppressors of Cytokine Signaling (SOCS)1 and SOCS3 Proteins Are Mediators of Interleukin-10 Modulation of Inflammatory Responses Induced by Chlamydia muridarum and Its Major Outer Membrane Protein (MOMP) in Mouse J774 Macrophages.

The immunopathology of chlamydial diseases is exacerbated by a broad-spectrum of inflammatory mediators, which we reported are inhibited by IL-10 in macrophages. However, the chlamydial protein moiety that induces the inflammatory mediators and the mechanisms by which IL-10 inhibits them are unknown. We hypothesized that Chlamydia major outer membrane protein (MOMP) mediates its disease pathogenesis, and the suppressor of cytokine signaling (SOCS)1 and SOCS3 proteins are mediators of the IL-10 inhibitory actions. Our hypothesis was tested by exposing mouse J774 macrophages to chlamydial stimulants (live Chlamydia muridarum and MOMP) with and without IL-10. MOMP significantly induced several inflammatory mediators (IL-6, IL-12p40, CCL5, CXCL10), which were dose-dependently inhibited by IL-10. Chlamydial stimulants induced the mRNA gene transcripts and protein expression of SOCS1 and SOCS3, with more SOCS3 expression. Notably, IL-10 reciprocally regulated their expression by reducing SOCS1 and increasing SOCS3. Specific inhibitions of MAPK pathways revealed that p38, JNK, and MEK1/2 are required for inducing inflammatory mediators as well as SOCS1 and SOCS3. Chlamydial stimulants triggered an M1 pro-inflammatory phenotype evidently by an enhanced nos2 (M1 marker) expression, which was skewed by IL-10 towards a more M2 anti-inflammatory phenotype by the increased expression of mrc1 and arg1 (M2 markers) and the reduced SOCS1/SOCS3 ratios. Neutralization of endogenously produced IL-10 augmented the secretion of inflammatory mediators, reduced SOCS3 expression, and skewed the chlamydial M1 to an M2 phenotype. Inhibition of proteasome degradation increased TNF but decreased IL-10, CCL5, and CXCL10 secretion by suppressing SOCS1 and SOCS3 expressions and dysregulating their STAT1 and STAT3 transcription factors. Our data show that SOCS1 and SOCS3 are regulators of IL-10 inhibitory actions, and underscore SOCS proteins as therapeutic targets for IL-10 control of inflammation for Chlamydia and other bacterial inflammatory diseases.

Cu(II) Complexes with FomA Protein Fragments of Fusobacterium Nucleatum Increase Oxidative Stress and Malondialdehyde Level.

An explanation of carcinogenesis processes may certainly contribute to the prevention and development of novel methods for cancer treatment. In this paper, we considered the probable relationship between the presence of Fusobacterium nucleatum in the colon and its possible influence on the development of colorectal cancer. For this purpose, intracellular and/or extracellular generation of reactive oxygen species (ROS) by mouse colon carcinoma cells (CT26) was stimulated by two fragments of FomA adhesin from F. nucleatum and their complexes with copper(II): Cu(II)-Ac-KGHGNG-NH2 (1Cu) and Cu(II)-Ac-PTVHNE-NH2 (2Cu). Incubation of the cells with copper complexes was followed with ICP-MS technique. The overall generation of ROS was shown by means of fluorescence spectroscopy with two proper probes, whereas identification of ROS was achieved by the spin trapping technique and electron paramagnetic resonance measurements. As a result, an abundant production of the hydroxyl radicals, both inside and outside the cells, was observed upon the stimulation of the CT26 cells with the copper complexes. Clearly both compounds induced strong oxidation stress which triggered a radicals' cascade that finally resulted in the pronounced lipid peroxidation. The latter was evidenced with the measured level of malondialdehyde, a biomarker of the peroxidation process. By applying N-acetylcysteine antioxidant to the studied system, the free radical mechanism of the lipid peroxidation process was confirmed. Hypothetically this mechanism can lead to colon cell damage and further cancerogenesis processes.

Group A streptococcal M-protein specific antibodies and T-cells drive the pathology observed in the rat autoimmune valvulitis model.

Acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are autoimmune mediated diseases triggered by group A streptococcal (GAS) infections. Molecular mimicry between GAS M-proteins and host tissue proteins has been proposed as the mechanism that initiates autoreactive immune responses in ARF/RHD. However, the individual role of antibodies and T-cells specific for GAS M-proteins in the pathogenesis of autoimmune carditis remains under-explored. The current study investigated the role of antibodies and T-cells in the development of carditis in the Lewis rat autoimmune valvultis (RAV) model by transferring serum and/or splenic T-cells from rats previously injected with GAS recombinant M5 protein. Here we report that serum antibodies alone and serum plus in vitro expanded rM5-specific T-cells from hyperimmune rats were capable of transferring carditis to naïve syngeneic animals. Moreover, the rats that received combined serum and T-cells developed more severe carditis. Recipient rats developed mitral valvulitis and myocarditis and showed prolongation of P-R intervals in electrocardiography. GAS M5 protein-specific IgG reactivity and T-cell recall response were also demonstrated in recipient rats indicating long-term persistence of antibodies and T-cells following transfer. The results suggest that both anti-GAS M5 antibodies and T-cells have differential propensity to induce autoimmune mediated carditis in syngeneic rats following transfer. The results highlight that antibodies and effector T-cells generated by GAS M protein injection can also independently home into cardiac tissue to cross-react with tissue proteins causing autoimmune mediated immunopathology.

Nontoxic outer membrane vesicles efficiently increase the efficacy of an influenza vaccine in mice and ferrets.

In this study, we developed a further-modified outer membrane vesicle (fmOMV) from the ΔmsbB/ΔpagP mutant of Escherichia coli transformed with the plasmid, pLpxF, in order to use it as an adjuvant for pandemic H1N1 (pH1N1) influenza vaccine. We evaluated the efficacy of the pH1N1 influenza vaccine containing the fmOMV in animal models as compared to the commercial adjuvants, alum or AddaVaxTM. The fmOMV-adjuvanted pH1N1 influenza vaccine induced a significant increase in the humoral immunity; however, this effect was less than that of the AddaVaxTM. The fmOMV-adjuvanted vaccine displayed pronounced an enhanced protective efficacy with increased T cell immune response and reduced the viral load in the lungs of the infected mice after challenging them with a lethal dose of the homologous virus. Moreover, it resulted in a significantly higher cross-protection against heterologous virus challenge than that of the pH1N1 vaccine with alum or with no adjuvants. In ferrets, the fmOMV-adjuvanted vaccine elicited a superior antibody response based on the HI titer and efficiently protected the animals from the lethal viral challenges. Taken together, the nontoxic fmOMV could be a promising adjuvant for inducing robust T cell priming into the pH1N1 vaccine and might be broadly applicable to the development of preventive measures against influenza virus infection.

Retinoic acid pre-treatment down regulates V. cholerae outer membrane vesicles induced acute inflammation and enhances mucosal immunity.

Bacterial outer membrane vesicles have been extensively investigated and considered as a next generation vaccine. Recently, we have demonstrated that the cholera pentavalent outer membrane vesicles (CPMVs) immunogen induced adaptive immunity and had a strong protective efficacy against the circulating V. cholerae strains in a mouse model. In this present study, we are mainly focusing on reducing outer membrane vesicle (OMV) -mediated toxicity without altering its antigenic property. Therefore, we have selected All-trans Retinoic Acid (ATRA), active metabolites of vitamin A, which have both anti-inflammatory and mucosal adjuvant properties. Pre-treatment of ATRA significantly reduced CPMVs induced TLR2 mediated pro-inflammatory responses in vitro and in vivo. Furthermore, we also found ATRA pre-treatment significantly induced mucosal immune response and protective efficacy after two doses of oral immunization with CPMVs (75µg). This study can help to reduce OMV based vaccine toxicity and induce better protective immunity where children and men suffered from malnutrition mainly in developing countries.

Brucella abortus-activated microglia induce neuronal death through primary phagocytosis.

Inflammation has long been implicated as a contributor to pathogenesis in neurobrucellosis. Many of the associated neurocognitive symptoms of neurobrucellosis may be the result of neuronal dysfunction resulting from the inflammatory response induced by Brucella abortus infection in the central nervous system. In this manuscript, we describe an immune mechanism for inflammatory activation of microglia that leads to neuronal death upon B. abortus infection. B. abortus was unable to infect or harm primary cultures of mouse neurons. However, when neurons were co-cultured with microglia and infected with B. abortus significant neuronal loss occurred. This phenomenon was dependent on TLR2 activation by Brucella lipoproteins. Neuronal death was not due to apoptosis, but it was dependent on the microglial release of nitric oxide (NO). B. abortus infection stimulated microglial proliferation, phagocytic activity and engulfment of neurons. NO secreted by B. abortus-activated microglia induced neuronal exposure of the "eat-me" signal phosphatidylserine (PS). Blocking of PS-binding to protein milk fat globule epidermal growth factor-8 (MFG-E8) or microglial vitronectin receptor-MFG-E8 interaction was sufficient to prevent neuronal loss by inhibiting microglial phagocytosis without affecting their activation. Taken together, our results indicate that B. abortus is not directly toxic to neurons; rather, these cells become distressed and are killed by phagocytosis in the inflammatory surroundings generated by infected microglia. Neuronal loss induced by B. abortus-activated microglia may explain, in part, the neurological deficits observed during neurobrucellosis.

Escherichia coli Braun Lipoprotein (BLP) exhibits endotoxemia - like pathology in Swiss albino mice.

The endotoxin lipopolysaccharide (LPS) promotes sepsis, but bacterial peptides also promote inflammation leading to sepsis. We found, intraperitoneal administration of live or heat inactivated E. coli JE5505 lacking the abundant outer membrane protein, Braun lipoprotein (BLP), was less toxic than E. coli DH5α possessing BLP in Swiss albino mice. Injection of BLP free of LPS purified from E. coli DH5α induced massive infiltration of leukocytes in lungs and liver. BLP activated human polymorphonuclear cells (PMNs) ex vivo to adhere to denatured collagen in serum and polymyxin B independent fashion, a property distinct from LPS. Both LPS and BLP stimulated the synthesis of platelet activating factor (PAF), a potent lipid mediator, in human PMNs. In mouse macrophage cell line, RAW264.7, while both BLP and LPS similarly upregulated TNF-α and IL-1ß mRNA; BLP was more potent in inducing cyclooxygenase-2 (COX-2) mRNA and protein expression. Peritoneal macrophages from TLR2-/- mice significantly reduced the production of TNF-α in response to BLP in contrast to macrophages from wild type mice. We conclude, BLP acting through TLR2, is a potent inducer of inflammation with a response profile both common and distinct from LPS. Hence, BLP mediated pathway may also be considered as an effective target against sepsis.

Phase 1 testing of detoxified LPS/group B meningococcal outer membrane protein vaccine with and without synthetic CPG 7909 adjuvant for the prevention and treatment of sepsis.

BACKGROUND: Gram-negative bacteria (GNB) are a leading cause of nosocomial infection and sepsis. Increasing multi-antibiotic resistance has left clinicians with fewer therapeutic options. Antibodies to GNB lipopolysaccharide (LPS, or endotoxin) have reduced morbidity and mortality as a result of infection and are not subject to the resistance mechanisms deployed by bacteria against antibiotics. In this phase 1 study, we administered a vaccine that elicits antibodies against a highly conserved portion of LPS with and without a CpG oligodeoxynucleotide (ODN) TLR9 agonist as adjuvant. METHODS: A vaccine composed of the detoxified LPS (dLPS) from E. coli O111:B4 (J5 mutant) non-covalently complexed to group B meningococcal outer membrane protein (OMP). Twenty healthy adult subjects received three doses at 0, 29 and 59 days of antigen (10 µg dLPS) with or without CPG 7909 (250 or 500 µg). Subjects were evaluated for local and systemic adverse effects and laboratory findings. Anti-J5 LPS IgG and IgM antibody levels were measured by electrochemiluminesence. Due to premature study termination, not all subjects received all three doses. RESULTS: All vaccine formulations were well-tolerated with no local or systemic events of greater than moderate severity. The vaccine alone group achieved a ≥ 4-fold "responder" response in IgG and IgM antibody in only one of 6 subjects. In contrast, the vaccine plus CPG 7909 groups appeared to have earlier and more sustained (to 180 days) responses, greater mean-fold increases, and a higher proportion of "responders" achieving ≥ 4-fold increases over baseline. CONCLUSIONS: Although the study was halted before all enrolled subjects received all three doses, the J5dLPS/OMP vaccine, with or without CpG adjuvant, was safe and well-tolerated. The inclusion of CpG increased the number of subjects with a ≥ 4-fold antibody response, evident even after the second of three planned doses. A vaccine comprising J5dLPS/OMP antigen with CpG adjuvant merits further investigation. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov Identifier: NCT01164514.

Random mutagenesis identifies a C-terminal region of YopD important for Yersinia type III secretion function.

A common virulence mechanism among bacterial pathogens is the use of specialized secretion systems that deliver virulence proteins through a translocation channel inserted in the host cell membrane. During Yersinia infection, the host recognizes the type III secretion system mounting a pro-inflammatory response. However, soon after they are translocated, the effectors efficiently counteract that response. In this study we sought to identify YopD residues responsible for type III secretion system function. Through random mutagenesis, we identified eight Y. pseudotuberculosis yopD mutants with single amino acid changes affecting various type III secretion functions. Three severely defective mutants had substitutions in residues encompassing a 35 amino acid region (residues 168-203) located between the transmembrane domain and the C-terminal putative coiled-coil region of YopD. These mutations did not affect regulation of the low calcium response or YopB-YopD interaction but markedly inhibited MAPK and NFκB. [corrected] activation. When some of these mutations were introduced into the native yopD gene, defects in effector translocation and pore formation were also observed. We conclude that this newly identified region is important for YopD translocon function. The role of this domain in vivo remains elusive, as amino acid substitutions in that region did not significantly affect virulence of Y. pseudotuberculosis in orogastrically-infected mice.

Dual toxic-peptide-coding Staphylococcus aureus RNA under antisense regulation targets host cells and bacterial rivals unequally.

Produced from the pathogenicity islands of Staphylococcus aureus clinical isolates, stable SprG1 RNA encodes two peptides from a single internal reading frame. These two peptides accumulate at the membrane, and inducing their expression triggers S. aureus death. Replacement of the two initiation codons by termination signals reverses this toxicity. During growth, cis-antisense RNA SprF1 is expressed, preventing mortality by reducing SprG1 RNA and peptide levels. The peptides are secreted extracellularly, where they lyse human host erythrocytes, a process performed more efficiently by the longer peptide. The two peptides also inactivate Gram-negative and -positive bacteria, with the shorter peptide more effective against S. aureus rivals. Two peptides are secreted from an individual RNA containing two functional initiation codons. Thus, we present an unconventional type I toxin-antitoxin system expressed from a human pathogen producing two hemolytic and antibacterial peptides from a dual-coding RNA, negatively regulated by a dual-acting antisense RNA.

Ras regulates alveolar macrophage formation of CXC chemokines and neutrophil activation in streptococcal M1 protein-induced lung injury.

Streptococcal toxic shock syndrome (STSS) is associated with a high mortality rate. The M1 serotype of Streptococcus pyogenes is most frequently associated with STSS. Herein, we examined the role of Ras signaling in M1 protein-induced lung injury. Male C57BL/6 mice received the Ras inhibitor (farnesylthiosalicylic acid, FTS) prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Administration of FTS reduced M1 protein-induced neutrophil recruitment, edema formation and tissue damage in the lung. M1 protein challenge increased Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Ras activity decreased M1 protein-induced expression of Mac-1 on neutrophils and secretion of CXC chemokines in the lung. Moreover, FTS abolished M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. Ras inhibition decreased chemokine-mediated neutrophil migration in vitro. Taken together, our novel findings indicate that Ras signaling is a potent regulator of CXC chemokine formation and neutrophil infiltration in the lung. Thus, inhibition of Ras activity might be a useful way to antagonize streptococcal M1 protein-triggered acute lung injury.

Simulated systemic recurrent Mycoplasma infection in rats induces recurrent sickness responses without residual impairment in spatial learning and memory.

In spite of their prevalence and importance, recurrent acute infections seldom have been investigated in the laboratory. We set out to measure fever and sickness behaviour in simulated recurrent Mycoplasma infection; Mycoplasma is a common clinical cause of recurrent acute infection. Male Sprague-Dawley rats had radiotransponders implanted to measure abdominal temperature and cage activity. After recovery, rats received three intraperitoneal (I.P.) injections, 10 days apart, of either fibroblast-stimulating lipopeptide-1 (FLS-1), a pyrogenic moiety of Mycoplasma salivarium, at a dose of 500 µg.kg(-1) in 1 ml.kg(-1) phosphate-buffered saline (PBS), or vehicle (PBS, 1 ml.kg(-1)). Body mass and food intake were measured daily. For measurement of learning and memory, training in a Morris Water Maze commenced 10 days after the last of the three successive injections and continued daily for 4 days. Spatial memory was assessed on the following day. Hippocampal tissue of rats was collected on the day of the last exposure to the maze. Recurrent FSL-1 administration induced recurrent fevers (~1°C) for about 9h, recurrent lethargy (~40-60%) for 1 day, recurrent anorexia (~16-30%) for 1 day, and recurrent reductions in the rate of mass gain (~112%) for 1 day, but did not induce persistent stunting. Recurrent FSL-1 administration did not result in tolerance to fever, lethargy or anorexia. There was no residual histological damage to the hippocampus and no residual detrimental effect in learning or memory in rats. Though we cannot extrapolate our results directly to humans, clinical recurrent acute Mycoplasma infection may not impose a high risk of stunting or impaired spatial learning and memory.

Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells.

Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K. kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K. kingae isolate were characterized. Among several major proteins, K. kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K. kingae infection. This study is the first report of OMV production by K. kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells.

Molecular and virulence characteristics of an outer membrane-associated RTX exoprotein in Pasteurella pneumotropica.

BACKGROUND: Pasteurella pneumotropica is a ubiquitous bacterium that is frequently isolated from laboratory rodents and causes various clinical symptoms in immunodeficient animals. Currently two RTX toxins, PnxIA and PnxIIA, which are similar to hemolysin-like high-molecular-weight exoproteins are known in this species. In this study, we identified and analyzed a further RTX toxin named PnxIIIA and the corresponding type I secretion system. RESULTS: The RTX exoprotein, PnxIIIA, contains only a few copies of the RTX repeat-like sequence and 3 large repeat sequences that are partially similar to the outer membrane protein found in several prokaryotes. Recombinant PnxIIIA protein (rPnxIIIA) was cytotoxic toward J774A.1 mouse macrophage cells, whereas cytotoxicity was attenuated by the addition of anti-CD11a monoclonal antibody. rPnxIIIA could bind to extracellular matrices (ECMs) and cause hemagglutination of sheep erythrocytes. Binding was dependent on the 3 large repeat sequences in PnxIIIA. Protein interaction analyses indicated that PnxIIIA is mainly localized in the outer membrane of P. pneumotropica ATCC 35149 in a self-assembled oligomeric form. PnxIIIA is less cytotoxic to J774A.1 cells than PnxIA and PnxIIA. CONCLUSIONS: The results implicate that PnxIIIA is located on the cell surface and participates in adhesion to ECMs and enhanced hemagglutination in the rodent pathogen P. pneumotropica.

Real-time impedance analysis of host cell response to meningococcal infection.

Measuring cell proliferation and cell death during bacterial infection involves performing end-point assays that represent the response at a single time point. A new technology from Roche Applied Science and ACEA Biosciences allows continuous monitoring of cells in real-time using specialized cell culture microplates containing micro-electrodes. The xCELLigence system enables continuous measurement and quantification of cell adhesion, proliferation, spreading, cell death and detachment, thus creating a picture of cell function during bacterial infection. Furthermore, lag and log phases can be determined to estimate optimal times to infect cells. In this study we used this system to provide valuable insights into cell function in response to several virulence factors of the meningitis causing pathogen Neisseria meningitidis, including the lipopolysaccharide (LPS), the polysaccharide capsule and the outer membrane protein Opc. We observed that prolonged time of infection with pathogenic Neisseria strains led to morphological changes including cell rounding and loss of cell-cell contact, thus resulting in changed electrical impedance as monitored in real-time. Furthermore, cell function in response to 14 strains of apathogenic Neisseria spp. (N. lactamica and N. mucosa) was analyzed. In contrast, infection with apathogenic N. lactamica isolates did not change electrical impedance monitored for 48 h. Together our data show that this system can be used as a rapid monitoring tool for cellular function in response to bacterial infection and combines high data acquisition rates with ease of handling.

Streptococcal M1 protein-induced lung injury is independent of platelets in mice.

Streptococcus pyogenes of the M1 serotype is frequently associated with severe streptococcal infections. M1 protein challenge can cause widespread microthrombosis, suggesting a role of platelets in streptococcal sepsis. Herein, we hypothesized that platelets may play a role in M1 protein-induced lung inflammation and injury. M1 protein was injected intravenously in C57Bl/6 mice. For platelet and neutrophil depletion, an anti-GP1bα antibody and an anti-Gr-1 antibody, respectively, were administered before M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for analysis of neutrophil infiltration, edema, and macrophage inflammatory protein 2 (MIP-2) formation. Blood was collected for analysis of membrane-activated complex 1 (Mac-1) and CD40 ligand (CD40L) expression on neutrophils and platelets as well as soluble CD40L in plasma. M1 protein caused significant pulmonary damage characterized by neutrophil infiltration, increased formation of edema and MIP-2 in the lung, and enhanced Mac-1 expression on neutrophils. However, M1 protein challenge had no effect on platelet surface expression of CD40L or soluble CD40L levels in plasma. Interestingly, platelet depletion had no influence on M1 protein-induced neutrophil recruitment, MIP-2 production, and tissue damage in the lung or Mac-1 expression on neutrophils. Moreover, we observed that M1 protein could bind to neutrophils but not to platelets. On the other hand, neutrophil depletion abolished M1 protein-induced edema formation and tissue damage in the lung. Our data suggest that neutrophils but not platelets are involved in the pathophysiology of M1 protein-provoked pulmonary damage. Thus, neutrophils may constitute a key target in infections caused by S. pyogenes of the M1 serotype.

B7-H3 augments the inflammatory response and is associated with human sepsis.

B7-H3, a new member of the B7 superfamily, acts as both a T cell costimulator and coinhibitor, and thus plays a key role in the regulation of T cell-mediated immune responses. However, it is unclear whether B7-H3 is involved in the innate immune monocyte/macrophage-mediated inflammatory response. In this paper, we show that, although B7-H3 alone failed to stimulate proinflammatory cytokine release from murine macrophages, it strongly augmented both LPS- and bacterial lipoprotein-induced NF-kappaB activation and inflammatory response. This occurred in both a TLR4- and TLR2-dependent manner. Blockage of B7-H3 in vivo attenuated LPS-induced proinflammatory cytokine release and endotoxic shock-related lethality. Furthermore, we found that patients diagnosed with sepsis, in contrast to healthy individuals, exhibited significant levels of raised plasma soluble B7-H3 (sB7-H3) and that this level correlated with the clinical outcome and levels of plasma TNF-alpha and IL-6. In addition, a putative receptor for B7-H3 was detected on monocytes and peritoneal macrophages from septic patients but not on monocytes from healthy donors. Stimulation of human monocytes with LPS and inflammatory cytokines led to a substantial release of sB7-H3. Taken together, our data indicate that significantly elevated plasma sB7-H3 in septic patients may predict a poor outcome. Furthermore, we demonstrate that B7-H3 functions as a costimulator of innate immunity by augmenting proinflammatory cytokine release from bacterial cell wall product-stimulated monocytes/macrophages and may contribute positively to the development of sepsis.

Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting.

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a potential virulence factor that induces epithelial cell death, but its pathologic effects on the immune system have yet to be determined. The present study investigated the pathologic events occurring in dendritic cells (DCs) exposed to a cytotoxic concentration of AbOmpA. AbOmpA induced early-onset apoptosis and delayed-onset necrosis in DCs. AbOmpA targeted the mitochondria and induced the production of reactive oxygen species (ROS). ROS were directly responsible for both apoptosis and necrosis of AbOmpA-treated DCs. These results demonstrate that the AbOmpA secreted from A. baumannii induces DC death, which may impair T cell biology to induce adaptive immune responses against A. baumannii.

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions.

OmpA-like protein Loa22 from Leptospira interrogans serovar Lai is cytotoxic to cultured rat renal cells and promotes inflammatory responses.

Leptospirosis renal disease is one of the common clinical manifestations of leptospirosis, including acute renal failure and tubulointerstitial nephritis. Outer membrane protein A-like protein Loa22 is a lipoprotein from Leptospira interrogans and has been suggested to be a corresponding virulence factor. However, the role of Loa22 in leptospiral nephropathy is not yet understood. In the present study, we constructed a vector and artificially expressed Loa22 in Escherichia coli BL21(DE)pLysS cells. After extensive purification, along with a GST tag protein control, Loa22 protein was used to test the cytotoxicity in cultured rat proximal tubule cells (NRK52E) and examine its effects on the induction of inflammatory responses. Using morphological examination, 2,3-bis(2-methoxy-4- nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoium hydrixide absorbance, lactate dehydrogenase assays and an analysis of apoptosis via flow cytometry, it was found that Loa22 protein mediates a direct cytotoxic effect on NRK52E cells in a dose-dependent manner. Using real-time PCR, western blotting and immunofluorescence, it was found that Loa22 protein upregulates the expression of toll-like receptor 2 (TLR2), induces nitric oxide synthase and promotes the production of nitric oxide (NO) and monocyte chemoattractant protein-1 (MCP-1) by NRK52E cells. Additionally, using a TLR2 blocking antibody, it was found that enhanced NO and MCP-1 production by NRK52E cells after Loa22 stimulation requires the activation of TLR2. Collectively, our data suggested that Loa22 is a critical virulence factor of L. interrogans and is involved in the leptospiral nephropathy through mediating direct cytotoxicity and enhancing inflammatory responses.