Differential Induction of Complement Fragment C5a and Inflammatory Cytokines during Intramammary Infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1β (IL-1β), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Compartmentalization of Immune Responses during Staphylococcus aureus Cranial Bone Flap Infection

Decompressive craniectomy is often required after head trauma, stroke, or cranial bleeding to control subsequent brain swelling and prevent death. The infection rate after cranial bone flap replacement ranges from 0.8% to 15%, with an alarming frequency caused by methicillin-resistant Staphylococcus aureus, which is problematic because of recalcitrance to antibiotic therapy. Herein we report the establishment of a novel mouse model of S. aureus cranial bone flap infection that mimics several aspects of human disease. Bacteria colonized bone flaps for up to 4 months after infection, as revealed by scanning electron microscopy and quantitative culture, demonstrating the chronicity of the model. Analysis of a human cranial bone flap with confirmed S. aureus infection by scanning electron microscopy revealed similar structural attributes as the mouse model, demonstrating that it closely parallels structural facets of human disease. Inflammatory indices were most pronounced within the subcutaneous galeal compartment compared with the underlying brain parenchyma. Specifically, neutrophil influx and chemokine expression (CXCL2 and CCL5) were markedly elevated in the galea, which demonstrated substantial edema on magnetic resonance images, whereas the underlying brain parenchyma exhibited minimal involvement. Evaluation of immune mechanisms required for bacterial containment and inflammation revealed critical roles for MyD88-dependent signaling and neutrophils. This novel mouse model of cranial bone flap infection can be used to identify key immunologic and therapeutic mechanisms relevant to persistent bone flap infection in humans.Decompressive craniectomy is performed after head trauma, stroke, or cranial bleeding, where a portion of the skull is removed to control subsequent brain swelling and prevent death. After removal, the bone flap is often cryopreserved until replacement; however, this increases the likelihood of destroying its blood supply, which substantially augments risk of infection.¹ The prevalence of infection after craniotomy ranges from 0.8% to 15%, with an alarming frequency caused by methicillin-resistant Staphylococcus aureus (MRSA), a major community and nosocomial gram-positive pathogen.² This high infection rate subjects patients to at least two additional surgical procedures since it is not possible to clear the infected bone in situ because of its recalcitrance to antibiotic therapy.³ In the first procedure, the infected skull flap is removed, and after a variable period of antibiotic therapy ranging from 6 weeks to >12 months, a second procedure is performed to place an expensive custom alloplastic flap composed of either acrylic resins, titanium mesh, or hydroxyapatite.^3,4 In approximately 13% of patients, prolonged absence of the skull flap can lead to syndrome of the trephined, a series of adverse effects that can include headache, seizures, mood swings, and behavioral disturbances.^5–7 Treatment of trephine syndrome consists of replacement of the original bone flap or synthetic device^8,9; however, this cannot be performed until there is convincing evidence that any residual infection associated with the original bone/artificial flap has been eliminated.Currently, cranial bone flap infections cannot be prevented or effectively treated without removal of the infected flap, and little information is available about the immune or microbial attributes that contribute to disease chronicity. These are important issues because a better appreciation of key pathogenic factors may reveal new targets to prevent and/or treat bone flap infections. Here we report a novel mouse model of S. aureus cranial bone flap infection that accurately mimics several facets of human disease in response to a relatively low infectious inoculum. This model has revealed that distinct immune responses are elicited within the subcutaneous space and brain parenchyma even though both bone flap surfaces communicate with these compartments and harbor similar numbers of bacteria. This information will help to facilitate the future design of novel therapeutic targets to prevent and/or treat bacterial cranial bone flap infections.     

Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection

Staphylococcus aureus and Escherichia coli are among the most prevalent species of gram-positive and gram-negative bacteria, respectively, that induce clinical mastitis. The innate immune system comprises the immediate host defense mechanisms to protect against infection and contributes to the initial detection of and proinflammatory response to infectious pathogens. The objective of the present study was to characterize the different innate immune responses to experimental intramammary infection with E. coli and S. aureus during clinical mastitis. The cytokine response and changes in the levels of soluble CD14 (sCD14) and lipopolysaccharide-binding protein (LBP), two proteins that contribute to host recognition of bacterial cell wall products, were studied. Intramammary infection with either E. coli or S. aureus elicited systemic changes, including decreased milk output, a febrile response, and induction of the acute-phase synthesis of LBP. Infection with either bacterium resulted in increased levels of interleukin 1beta (IL-1beta), gamma interferon, IL-12, sCD14, and LBP in milk. High levels of the complement cleavage product C5a and the anti-inflammatory cytokine IL-10 were detected at several time points following E. coli infection, whereas S. aureus infection elicited a slight but detectable increase in these mediators at a single time point. Increases in IL-8 and tumor necrosis factor alpha were observed only in quarters infected with E. coli. Together, these data demonstrate the variability of the host innate immune response to E. coli and S. aureus and suggest that the limited cytokine response to S. aureus may contribute to the well-known ability of the bacterium to establish chronic intramammary infection.     

BDCA1-Positive Dendritic Cells (DCs) Represent a Unique Human Myeloid DC Subset That Induces Innate and Adaptive Immune Responses to Staphylococcus aureus Infection

Staphylococcus aureus bloodstream infection (bacteremia) is a major cause of morbidity and mortality and places substantial cost burdens on health care systems. The role of peripheral blood dendritic cells (PBDCs) in the immune responses against S. aureus infection has not been well characterized. In this study, we demonstrated that BDCA1⁺ myeloid DCs (mDCs) represent a unique PBDC subset that can induce immune responses against S. aureus infection. BDCA1⁺ mDCs could engulf S. aureus and strongly upregulated the expression of costimulatory molecules and production of proinflammatory cytokines. Furthermore, BDCA1⁺ mDCs expressed high levels of major histocompatibility complex (MHC) class I and II molecules in response to S. aureus and greatly promoted proliferation and gamma interferon (IFN-γ) production in CD4 and CD8 T cells. Moreover, BDCA1⁺ mDCs expressed higher levels of Toll-like receptor 2 (TLR-2) and scavenger receptor A (SR-A) than those on CD16⁺ and BDCA3⁺ mDCs, and these two receptors were both required for the recognition of S. aureus and the subsequent activation of BDCA1⁺ mDCs. Finally, BDCA1⁺ mDC-mediated immune responses against S. aureus were dependent on MyD88 signaling pathways. These results demonstrate that human BDCA1⁺ mDCs represent a unique subset of mDCs that can respond to S. aureus to undergo maturation and activation and to induce Th1 and Tc1 immune responses.     

Coagulases as Determinants of Protective Immune Responses against Staphylococcus aureus

During infection, Staphylococcus aureus secretes two coagulases (Coa and von Willebrand factor binding protein [vWbp]), which, following an association with host prothrombin and fibrinogen, form fibrin clots and enable the establishment of staphylococcal disease. Within the genomes of different S. aureus isolates, coagulase gene sequences are variable, and this has been exploited for a classification of types. We show here that antibodies directed against the variable prothrombin binding portion of coagulases confer type-specific immunity through the neutralization of S. aureus clotting activity and protection from staphylococcal disease in mice. By combining variable portions of coagulases from North American isolates into hybrid Coa and vWbp proteins, a subunit vaccine that provided protection against challenge with different coagulase-type S. aureus strains in mice was derived.     

Enhancement of Escherichia coli and Staphylococcus aureus antibiotic susceptibility using sesquiterpenoids

The present work examines the potential of sesquiterpenoids to sensitize Escherichia coli and Staphylococcus aureus, and modulate their susceptibility to the standard antibiotics ciprofloxacin, erythromycin, gentamicin and vancomycin. It was tested samples of three sesquiterpenoids: guaiazulene, nerolidol (racemic mixture of the cis and trans isomers) and germacrene D enriched natural extract. Experiments were conducted aiming to assess the antimicrobial effects of the antibiotic-sesquiterpenoid combination on bacterial growth inhibition, by the disc diffusion assay and the minimum inhibitory concentration (MIC) assessment, the bactericidal effects, the post-antibiotic effect (PAE) and the effect on membrane permeability. The data related with the antimicrobial activity evidenced, through the disc diffusion assay, an antibiotic S. aureus antimicrobial activity enhancement by sesquiterpenoids presence. The MIC value for E. coli decreased significantly by sesquiterpenoids combination with ciprofloxacin, erythromycin and gentamicin, and for S. aureus, with all four selected antibiotics. This combination also increased the PAE, with the exception of guaiazulene, which seemed to quench antibiotic antimicrobial action. A moderate correlation between antimicrobial action and impairment of cell membrane function was detected for germacrene D enriched extract, and nerolidol, as single treatments and in combination with antibiotic, while a poor correlation was obtained for guaiazulene. This study provides basis for the evaluation of sesquiterpenoids as alternative or possible synergistic compounds for current antimicrobial chemotherapeutics, showing the practical utility of natural derived products to increase the susceptibility of E. coli and S. aureus.     

Enterotoxigenic Escherichia coli Elicits Immune Responses to Multiple Surface Proteins

Enterotoxigenic Escherichia coli (ETEC) causes considerable morbidity and mortality due to diarrheal illness in developing countries, particularly in young children. Despite the global importance of these heterogeneous pathogens, a broadly protective vaccine is not yet available. While much is known regarding the immunology of well-characterized virulence proteins, in particular the heat-labile toxin (LT) and colonization factors (CFs), to date, evaluation of the immune response to other antigens has been limited. However, the availability of genomic DNA sequences for ETEC strains coupled with proteomics technology affords opportunities to examine novel uncharacterized antigens that might also serve as targets for vaccine development. Analysis of whole or fractionated bacterial proteomes with convalescent-phase sera can potentially accelerate identification of secreted or surface-expressed targets that are recognized during the course of infection. Here we report results of an immunoproteomics approach to antigen discovery with ETEC strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407. Immunoblotting of proteins separated by two-dimensional electrophoresis (2DE) with sera from mice infected with strain {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 or with convalescent human sera obtained following natural ETEC infections demonstrated multiple immunoreactive molecules in culture supernatant, outer membrane, and outer membrane vesicle preparations, suggesting that many antigens are recognized during the course of infection. Proteins identified by this approach included established virulence determinants, more recently identified putative virulence factors, as well as novel secreted and outer membrane proteins. Together, these studies suggest that existing and emerging proteomics technologies can provide a useful complement to ongoing approaches to ETEC vaccine development.Infectious diarrhea substantially impacts human health in the developing world, where hundreds of millions of infections occur each year. Several pathogens, rotavirus, Shigella, Vibrio cholerae, and enterotoxigenic Escherichia coli (ETEC), each contribute significantly to this disease burden and collectively result in an estimated 2 million deaths due to diarrheal illness annually (52). Therefore, ETEC remains a high priority for vaccine development.Enterotoxigenic E. coli strains constitute a phenotypically and genetically diverse pathotype that have in common the production of enterotoxin heat-labile toxin (LT) and/or heat-stable toxin (ST). In the classic paradigm for ETEC pathogenesis, organisms must colonize the small intestine via fimbrial colonization factor antigens (CFAs) for effective toxin delivery and subsequent diarrhea (18). Since the early identification of colonization factors (CFs) as important virulence determinants (15), these structures have been a central focus of ETEC vaccine development, and significant inroads have been made into the identification of a broad array of CFs (22, 43), with over 25 antigens identified thus far. ETEC vaccines currently in development are designed to target the most prevalent CFs (56). Moreover, recent elegant structural characterization of the colonization factor antigen I (CFA/I) pilus has provided additional molecular details of pilus tip adhesin molecules that might be exploited (33) as more highly conserved vaccine targets.However, the remarkable plasticity of E. coli genomes (45) and studies demonstrating that many ETEC strains do not produce an identifiable CF (40, 54) suggest that additional antigens would likely need to be considered to produce a broadly protective vaccine. While much is known about the immunology of the CFs and LT following infection (44, 46, 63), very little is known about the nature of immune responses to ETEC in general, and there is no information regarding immunogenicity of more recently discovered putative virulence factors.Furthermore, large-scale epidemiologic studies have suggested that additional plasmid or chromosomally encoded factors contribute to the development of an effective protective immune response attributable to prior natural infections with ETEC (55). However, the identity of other antigens that might be involved in the development of protective immune responses to ETEC remains largely unexplored.The advent of high-throughput sequencing of multiple genomes and advances in proteomics permit avenues for discovery of novel antigens which might be useful in ETEC vaccine development. Two complete ETEC genomes, ETEC {"type":"entrez-nucleotide","attrs":{"text":"H10407","term_id":"875229","term_text":"H10407"}}H10407 and E24377A (45), and one draft genome sequence, B7A (45), as well as several plasmid sequences (21) are now publicly available. While it is anticipated that dozens if not hundreds of ETEC genome sequences will ultimately be made available, these existing genomes permit some initial antigen discovery and validation efforts that were not previously possible.Recent studies of mice have demonstrated that mice exposed to ETEC are protected from subsequent intestinal colonization (47). Therefore, these studies were undertaken to characterize the nature of protective immune responses afforded by prior exposures to ETEC in this model and to validate immune responses to selected antigens using sera from patients naturally infected with ETEC.     

Mouse Strain-Dependent Differences in Susceptibility to Neisseria gonorrhoeae Infection and Induction of Innate Immune Responses

Acute gonorrhea in women is characterized by a mucopurulent exudate that contains polymorphonuclear leukocytes (PMNs) with intracellular gonococci. Asymptomatic infections are also common. Information on the innate response to Neisseria gonorrhoeae in women is limited to studies with cultured cells, isolated immune cells, and analyses of cervicovaginal fluids. 17β-Estradiol-treated BALB/c mice can be experimentally infected with N. gonorrhoeae, and a vaginal PMN influx occurs in 50 to 80% of mice. Here, we compared the colonization loads and proinflammatory responses of BALB/c, C57BL/6 and C3H/HeN mice to N. gonorrhoeae. BALB/c and C57BL/6 mice were colonized at similar levels following inoculation with 10⁶ CFU of N. gonorrhoeae. BALB/c, but not C57BL/6, mice exhibited a marked vaginal PMN influx. Tumor necrosis factor alpha, interleukin-6, macrophage inflammatory protein 2 (MIP-2), and keratinocyte-derived chemokine were elevated in vaginal secretions from infected BALB/c mice, but not in those from C57BL/6 mice. MIP-2 levels positively correlated with a vaginal PMN influx. In contrast to BALB/c and C57BL/6 mice, C3H/HeN mice were resistant to infection, and there was no evidence of an inflammatory response. We conclude that N. gonorrhoeae causes a productive infection in BALB/c mice that is characterized by the induction of proinflammatory cytokines and chemokines and the recruitment of PMNs. Infection of C57BL/6 mice, in contrast, is more similar to asymptomatic infection. C3H/HeN mice are inherently resistant to N. gonorrhoeae infection, and this resistance is not due to an overwhelming inflammatory response to infection. Host genetic factors can therefore impact susceptibility and the immune response to N. gonorrhoeae.Uncomplicated gonorrhea is most commonly an infection of the urethra in men and the cervix. The female urethra may also be infected, and rectal and pharyngeal infection can occur in either sex. The hallmark of symptomatic gonococcal infection is the presence of a purulent exudate containing numerous polymorphonuclear leukocytes (PMNs), many of which contain intracellular gonococci. Asymptomatic infections are also common, particularly in females. Epithelial cells that line the genital mucosal surface are the first line of defense against this human-specific pathogen, and Neisseria gonorrhoeae produces a robust proinflammatory cytokine and chemokine response when incubated with cultured human vaginal, endocervical, ectocervical (12, 33), urethral (17), endometrial (3), and fallopian tube (31) tissue culture cells. Similarly, studies using the complex fallopian tube organ culture model suggest that N. gonorrhoeae induces the proinflammatory cytokines interleukin-1α (IL-1α) and tumor necrosis factor alpha (TNF-α) (29). Signaling through cellular receptors on epithelial cells results in the activation and recruitment of phagocytic cells, including PMNs and macrophages. Primary macrophages and peripheral blood mononuclear cells also elicit a proinflammatory response when incubated with N. gonorrhoeae (30, 34, 39). These innate immune cells further contribute to the array of proinflammatory cytokines and antimicrobial factors.Due to the multiple cell types that contribute to the host innate response to infection, it is important that whole model systems be utilized to measure the impact of N. gonorrhoeae infection on the host immune response. Experimental urethral colonization in male volunteers with N. gonorrhoeae evokes a strong innate response that is characterized by the production of proinflammatory cytokines (37, 38). Similar studies with female subjects are not feasible due to the risk of complications of gonococcal infection in women. Therefore, features of the innate response to N. gonorrhoeae in the female genital tract are predicted solely from tissue culture systems and the analysis of clinical samples. It is unclear whether women elicit a cytokine response to gonococcal infection. The reason why some infections are asymptomatic is also not known. Hedges et al. (18, 19) were unable to detect local proinflammatory cytokines in cervicovaginal secretions from infected women and detected a low anti-gonococcal antibody response. Based on these observations, it was proposed that N. gonorrhoeae fails to induce host inflammatory responses or is actively immunosuppressive. This finding is in marked contrast with the robust induction of proinflammatory cytokines observed from in vitro cell lines that constitute the female genital tract. The absence of various other cell types in tissue culture cell models could influence the cytokine response to infection; alternatively, the timing of sample collection from infected subjects may also influence the data. Therefore, a systematic analysis of cytokine induction over the course of infection in a female animal model is needed.The 17β-estradiol-treated mouse model is the only small-animal model available for studying the immune response to N. gonorrhoeae genital tract infection. While the mechanism by which estradiol promotes long-term colonization in female mice is not known, it is likely that promotion of an estrus-like state is beneficial for the gonococcus based on the fact that untreated mice can be transiently colonized with N. gonorrhoeae provided they are inoculated in the proestrus stage of the reproductive cycle (7, 46). The 17β-estradiol-treated mouse model has been a useful system for studying many aspects of gonococcal infection, including gonococcal evasion of PMN killing (43, 49) and antimicrobial peptides (23, 48), antigenic variation in vivo (41), and interactions between N. gonorrhoeae and commensal flora (32). This model is based on the use of BALB/c mice. Approximately 50 to 80% of infected BALB/c mice that are treated with a slow-release estradiol pellet exhibit a significant vaginal PMN response following inoculation with N. gonorrhoeae strain FA1090 based on examination of stained vaginal smears (21, 22, 43), and PMNs and macrophages are also found in vaginal and cervical tissue samples from infected mice (44). Gonococci are localized within vaginal and cervical tissue, and similar to that which occurs in humans, an insignificant and transient humoral response to N. gonorrhoeae occurs which was not protective against reinfection with the same strain (44). A recent modification of the model utilizes water-soluble estradiol to reduce the length of time that mice are exposed to nonphysiological concentrations of estradiol. A vaginal PMN influx also occurs during infection of mice treated with water-soluble estradiol, and as with pelleted mice, infection persists despite the presence of PMNs (44).One advantage of using inbred mouse strains for studies of infectious diseases is that environmental and genetic components can be controlled. Interestingly, the susceptibility to infectious agents can often vary with the genetic background of the mouse. One example in the area of sexually transmitted infections is that genetically controlled differences in the development of infertility in inbred mouse strains following inoculation with chlamydia have been reported, with pregnancy rates following infection of C3H/HeN mice being significantly lower than those of C57BL/6 mice (10). Darville and colleagues (8) found similar results, and their data suggested that an earlier and more severe acute inflammatory response in the C57BL/6 strain may lead to earlier eradication of the infection, thus protecting the upper tract from disease. Numerous examples of vulnerability have been found for other infectious agents, including Leishmania major, Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Plasmodium chabaudi, Legionella pneumophila, and Mycobacterium tuberculosis (reviewed by Kramnik and Boyartchuk [27]).In this study, we sought to characterize in greater detail the cytokine and inflammatory response to genital tract infection with N. gonorrhoeae in 17β-estradiol-treated BALB/c mice and to determine if susceptibility to colonization and the host inflammatory response to infection vary between inbred mouse strains. Our data demonstrate that BALB/c, C57BL/6, and C3H/HeN mouse strains differ widely in their response to infection. While both BALB/c and C57BL/6 strains support colonization with the gonococcus, only the BALB/c strain appears to mount an inflammatory response. In contrast, the C3H/HeN strain appears to be resistant to colonization with the gonococcus. These data demonstrate significant divergence among inbred mouse strains in terms of susceptibility and inflammatory response to gonococcal infection, and they suggest that future studies can be designed to correlate genetic markers with the host response.     

GSL-Enriched Membrane Microdomains in Innate Immune Responses

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host–pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.     

Influence of Innate Immune Responses on Autoimmunity

Objective: To explore the therapeutic alliance effects of adenovirus vector-mediated gene transfer of ICOSIg and CTLA4Ig fusion protein on experimental autoimmune myocarditis (EAM).

Methods: Expression vector pAdeno-CTLA4Ig and pAdeno-ICOSIg was constructed and transfected into HEK293 cells. Adenovirus expresses CTLA4Ig and ICOSIg was produced. Ad-CMV-GFP was used as controls. EAM was induced in Lewis rats by injection of procine cardiac myosin. All the immunized rats were divided into four groups. Group A (n = 15) received adenovirus containing CTLA4Ig and ICOSIg from day 14–28; group B (n = 15), group C (n = 15) and group D (n = 15) received adenovirus containing CTLA4Ig, ICOSIg and GFP, respectively. Group E (n = 10) was normal controls never received immunization. On day 28, all the rats were killed after echocardiography examination. Histopathological examination was used to observe inflammation in the myocardium. Western blot was used to detect CTLA4, ICOS, ICOSL and competitive RT-PCR for B7-1, B7-2 expression. T lymphocyte proliferation assay was performed and ELISPOT was used to detect the Th1 and Th2 production.

Results: Alliance application of CTLA4Ig and ICOSIg exerts therapeutic effects on EAM. After a treatment duration of 14 days, cardiac function and myocardial inflammation improved significantly compared to group D. Expression of CTLA-4, ICOS and ICOSL, B7-1 was statistically decreased in group A, B and C compared with group D. T-cell proliferation was inhibited by costimulatory blockade in a dose-dependent style. ICOSIg blockade significantly augments IL-4 and IL-10 production while diminished IFN-γ production.

Conclusions: Blockade of costimulatory pathway with alliance therapy of CTLA4Ig and ICOSIg alleviated autoimmune damage in EAM and improved cardiac function. The mechanisms may be downregulation of costimulatory molecules and anti-inflammation.     

Malnutrition Alters the Innate Immune Response and Increases Early Visceralization following Leishmania donovani Infection

Malnutrition is a risk factor for the development of visceral leishmaniasis. However, the immunological basis for this susceptibility is unknown. We have developed a mouse model to study the effect of malnutrition on innate immunity and early visceralization following Leishmania donovani infection. Three deficient diets were studied, including 6, 3, or 1% protein; these diets were also deficient in iron, zinc, and calories. The control diet contained 17% protein, was zinc and iron sufficient, and was provided ab libitum. Three days after infection with L. donovani promastigotes, the total extradermal (lymph nodes, liver, and spleen) and skin parasite burdens were equivalent in the malnourished (3% protein) and control mice, but in the malnourished group, a greater percentage (39.8 and 4.0%, respectively; P = 0.009) of the extradermal parasite burden was contained in the spleen and liver. The comparable levels of parasites in the footpads in the two diet groups and the higher lymph node parasite burdens in the well-nourished mice indicated that the higher visceral parasite burdens in the malnourished mice were not due to a deficit in local parasite killing but to a failure of lymph node barrier function. Lymph node cells from the malnourished, infected mice produced increased levels of prostaglandin E(2) (PGE(2)) and decreased levels of interleukin-10. Inducible nitric oxide synthase activity was significantly lower in the spleen and liver of the malnourished mice. Thus, malnutrition causes a failure of lymph node barrier function after L. donovani infection, which may be related to excessive production of PGE(2) and decreased levels of IL-10 and nitric oxide.     

Biofilm Matrix Exoproteins Induce a Protective Immune Response against Staphylococcus aureus Biofilm Infection

The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and protein-based biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.     

Differential induction of complement fragment C5a and inflammatory cytokines during intramammary infections with Escherichia coli and Staphylococcus aureus

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.     

Divergent Signal Transduction Responses to Infection with Attaching and Effacing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined. We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells. In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic E. coli (EPEC) strain CVD206. The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ([158 ± 21] % of control). Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling espB mutant of EPEC. E. coli JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206. In contrast, JM101(pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15). These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC. Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens.     

Kinetics of phagocytosis of Staphylococcus aureus and Escherichia coli by human granulocytes.

Although phagocytosis of micro-organisms by granulocytes is one of the most important defence mechanisms against infection, little is known about the kinetics of this process. The present study showed that the rate of ingestion of Staphylococcus aureus and Escherichia coli depends on the concentrations of the granulocytes and bacteria. Phagocytosis of bacteria at a bacteria-to-cell ratio in the range between 100:1 and 1:10 showed an exponential course during the first 30 min. At a bacteria-to-cell ratio of 1:1, application of a correction for the outgrowth of extracellular bacteria gave an exponential course of ingestion over the first 90-min period. Since it was found that the phagocytosis of bacteria by granulocytes at various bacteria-to-cell ratios can be described with Michaelis-Menten kinetics, we studied the kinetics of phagocytosis on the basis of the initial rate for the first 30-min period. The rate of phagocytosis and the maximal degree of ingestion of bacteria by granulocytes proved to be related to the concentration of serum used in the assay. The minimal serum concentration required for maximal ingestion was 2.5% for Staphylococcus aureus and 5% for Escherichia coli. When bacteria were pre-opsonized, the duration of pre-opsonization proved to be limiting for the rate of phagocytosis in dependence on the serum concentration. The effect of temperature on the phagocytosis of micro-organisms proved to be two-fold. First, at temperatures between 4 and 33 degrees a decrease in the functioning of the cells leads to a decrease in the rate of phagocytosis. Above 42 degrees, the temperature affects mainly the opsonization of the micro-organisms and has only a slight influence on the ingestion process. From the data obtained in this study, maximal rates of 6.3 X 10(6) Staphylococcus aureus/5 X 10(6) granulocytes/min and of 7.1 X 10(6) Escherichia coli/5 X 10(6) granulocytes/min were calculated for phagocytosis at a bacteria-to-cell ratio of 100:1 at 37 degrees, i.e. on average about one bacterium per granulocyte per min. The maximum calculated number of bacteria ingested by one granulocyte lies between 40 and 50.