Expression and purification methods for the production of recombinant human complement component C2.

Human complement component C2 is a critical factor of the classical complement pathway. Here we provide a method for the production of recombinant human C2 (rhC2) protein for research purposes. The human complement component C2 (hC2) is cloned from a human cDNA library by polymerase chain reaction and inserted in a mammalian expression vector (Martini et al., BMC Immunol 11:43, 2010). Transient transfection is utilized to express hC2 in a mammalian cell line, and the expressed C2 is harvested from the conditioned media. rhC2 is purified from the conditioned media by sequential steps of cation exchange and affinity column chromatography. The purified hC2 is characterized for protein purity, stability, and enzymatic activity. The recombinant hC2 activity is tested in a complement activation ELISA assay that measures classical, alternative, and lectin complement pathway activity in C2-depleted serum.

Recombinant human complement component C2 produced in a human cell line restores the classical complement pathway activity in-vitro: an alternative treatment for C2 deficiency diseases.

BACKGROUND: Complement C2 deficiency is the most common genetically determined complete complement deficiency and is associated with a number of diseases. Most prominent are the associations with recurrent serious infections in young children and the development of systemic lupus erythematosus (SLE) in adults. The links with these diseases reflect the important role complement C2 plays in both innate immunity and immune tolerance. Infusions with normal fresh frozen plasma for the treatment of associated disease have demonstrated therapeutic effects but so far protein replacement therapy has not been evaluated. RESULTS: Human complement C2 was cloned and expressed in a mammalian cell line. The purity of recombinant human C2 (rhC2) was greater than 95% and it was characterized for stability and activity. It was sensitive to C1s cleavage and restored classical complement pathway activity in C2-deficient serum both in a complement activation ELISA and a hemolytic assay. Furthermore, rhC2 could increase C3 fragment deposition on the human pathogen Streptococcus pneumoniae in C2-deficient serum to levels equal to those with normal serum. CONCLUSIONS: Taken together these data suggest that recombinant human C2 can restore classical complement pathway activity and may serve as a potential therapeutic for recurring bacterial infections or SLE in C2-deficient patients.

Characterization of regulatory T cells in urban newborns.

BACKGROUND: In the United States, asthma prevalence is particularly high among urban children. Although the underlying immune mechanism contributing to asthma has not been identified, having impaired T regulatory (Treg) cells at birth may be a determining factor in urban children. The objective of this study was to compare Treg phenotype and function in cord blood (CB) of newborns to those in peripheral blood (PB) of a subset of participating mothers. METHODS: Treg numbers, expression, and suppressive function were quantified in subjects recruited prenatally from neighborhoods where >/= 20% of families have incomes below the poverty line. Proportion of Treg cells and expression of naïve (CD45RA) or activated (CD45RO, CD69, and HLA-DR) markers in CD4+T cells was measured by flow cytometry. Treg suppressive capacity was determined by quantifying PHA-stimulated lymphocyte proliferation in mononuclear cell samples with and without CD25 depletion. RESULTS: In an urban cohort of 119 newborns and 82 mothers, we found that newborns had similar number of cells expressing FOXP3 as compared to the mothers but had reduced numbers of CD4+CD25+bright cells that predominantly expressed the naïve (CD45RA) rather than the activated/memory (CD45RO) phenotype found in the mothers. Additionally, the newborns had reduced mononuclear cell TGF-beta production, and reduced Treg suppression of PHA-stimulated lymphocyte proliferation compared to the mothers. CONCLUSION: U.S. urban newborns have Treg cells that express FOXP3, albeit with an immature phenotype and function as compared to the mothers. Longitudinal follow-up is needed to delineate Treg cell maturation and subsequent risk for atopic diseases in this urban birth cohort.

IFN-gamma regulated chemokine production determines the outcome of Staphylococcus aureus infection.

Immunomodulatory therapy represents an attractive approach in treating multidrug-resistant infections. Developing this therapy necessitates a lucid understanding of host defense mechanisms. Neutrophils represent the first line of systemic defense during Staphylococcus aureus infections. However, recent research suggests that survival of S. aureus inside neutrophils may actually contribute to pathogenesis, indicating that neutrophil trafficking to the infection site must be tightly regulated to ensure efficient microbial clearance. We demonstrate that neutrophil-regulating T cells are activated during S. aureus infection and produce cytokines that control the local neutrophil response. S. aureus capsular polysaccharide activates T cell production of IFN-gamma in a novel MHC class II-dependent mechanism. During S. aureus surgical wound infection, the presence of IFN-gamma at the infection site depends upon alphabetaTCR+ cells and functions to regulate CXC chemokine production and neutrophil recruitment in vivo. We note that the reduced neutrophil response seen in IFN-gamma-/- mice during S. aureus infection is associated with reduced tissue bacterial burden. CXC chemokine administration to the infection site resulted in an increased survival of viable S. aureus inside neutrophils isolated from the wound. These data demonstrate that T cell-derived IFN-gamma generates a neutrophil-rich environment that can potentiate S. aureus pathogenesis by facilitating bacterial survival within the neutrophil. These findings suggest avenues for novel immunomodulatory approaches to control S. aureus infections.

Functional Th1 cells are required for surgical adhesion formation in a murine model.

Tissue trauma in the peritoneal and pelvic cavities following surgery or bacterial infection results in adhesions that are a debilitating cause of intestinal obstruction, chronic pelvic pain, and infertility in women. We recently demonstrated that CD4(+) alphabeta T cells are essential for development of this process. Using a murine model of experimental adhesion formation, we now demonstrate that adhesion formation is characterized by the selective recruitment of Tim-3(+), CCR5(+), CXCR3(+), IFN-gamma(+) cells, indicating the presence of a Th1 phenotype. We further demonstrate that adhesion formation is critically dependent on the function of Th1 cells because mice genetically deficient for IFN-gamma, T-bet, or treated with Abs to the Th1-selective chemoattractant IL-16 show significantly less adhesion formation than wild-type mice. In addition, disrupting the interaction of the Th1-specific regulatory molecule Tim-3, with its ligand, significantly exacerbates adhesion formation. This enhanced response is associated with increases in the level of neutrophil-attracting chemokines KC and MIP-2, known to play a role in adhesiogenesis. These data demonstrate that the CD4(+) T cells orchestrating adhesion formation are of the Th1 phenotype and delineate the central role of T-bet, Tim-3, IFN-gamma, and IL-16 in mediating this pathogenic tissue response.

The Bacteroides fragilis transcriptome response to oxygen and H2O2: the role of OxyR and its effect on survival and virulence.

The intestinal anaerobic symbiont, Bacteroides fragilis, is highly aerotolerant and resistant to H(2)O(2). Analysis of the transcriptome showed that expression of 45% of the genome was significantly affected by oxidative stress. The gene expression patterns suggested that exposure to oxidative stress induced an acute response to rapidly minimize the immediate effects of reactive oxygen species, then upon extended exposure a broad metabolic response was induced. This metabolic response induced genes encoding enzymes that can supply reducing power for detoxification and restore energy-generating capacity. An integral aspect of the metabolic response was downregulation of genes related to translation and biosynthesis which correlated with decreased growth and entry into a stationary phase-like growth state. Examination of oxyR mutants showed that they were impaired for the acute response and they induced the expanded metabolic response with only minimal exposure to stress. The oxyR mutants were more sensitive to oxidants in vitro and in vivo they were attenuated in an intra-abdominal abscess infection model. Aerotolerance and resistance to oxidative stress are physiological adaptations of B. fragilis to its environment that enhance survival in extra-intestinal sites and promote opportunistic infections.

Thioredoxin reductase is essential for thiol/disulfide redox control and oxidative stress survival of the anaerobe Bacteroides fragilis.

Results of this study showed that the anaerobic, opportunistic pathogen Bacteroides fragilis lacks the glutathione/glutaredoxin redox system and possesses an extensive number of putative thioredoxin (Trx) orthologs. Analysis of the genome sequence revealed six Trx orthologs and an absence of genes required for synthesis of glutathione and glutaredoxins. In addition, it was shown that the thioredoxin reductase (TrxB)/Trx system is the major or sole redox system for thiol/disulfide cellular homeostasis in this anaerobic bacterium. Expression of the B. fragilis trxB gene was induced following treatment with diamide or H(2)O(2) or exposure to oxygen. This inducible trxB expression was OxyR independent. Northern blot hybridization analysis showed that the trxB mRNA was cotranscribed with lolA as a bicistronic transcript or was present as a monocistronic transcript that was also highly induced under the same conditions. The role of LolA, a prokaryotic periplasmic lipoprotein-specific molecular chaperone in the thiol/disulfide redox system, is unknown. A trxB deletion mutant was more sensitive to the effects of diamide and oxygen than the parent strain. In addition, the trxB mutant was unable to grow in culture media without addition of a reductant. Furthermore, the trxB mutant was not able to induce intraabdominal abscess formation in a mouse model, whereas the parent strain was. Taken together, these data strongly suggest that TrxB/Trx is the major, if not the sole, thiol/disulfide redox system in this anaerobe required for survival and abscess formation in a peritoneal cavity infection model.

A defined O-antigen polysaccharide mutant of Francisella tularensis live vaccine strain has attenuated virulence while retaining its protective capacity.

Francisella tularensis, the causative agent of tularemia, has been designated a CDC category A select agent because of its low infective dose (<10 CFU), its ready transmission by aerosol, and its ability to produce severe morbidity and high mortality. The identification and characterization of this organism's virulence determinants will facilitate the development of a safe and effective vaccine. We report that inactivation of the wbtA-encoded dehydratase of the O-antigen polysaccharide (O-PS) locus of the still-unlicensed live vaccine strain of F. tularensis (LVS) results in a mutant (the LVS wbtA mutant) with remarkably attenuated virulence. Western blot analysis and immune electron microscopy studies associate this loss of virulence with a complete lack of surface O-PS expression. A likely mechanism for attenuation is shown to be the transformation from serum resistance in the wild-type strain to serum sensitivity in the mutant. Despite this significant attenuation in virulence, the LVS wbtA mutant remains immunogenic and confers protective immunity on mice against challenge with an otherwise lethal dose of either F. tularensis LVS or a fully virulent clinical isolate of F. tularensis type B. Recognition and characterization of the pivotal role of O-PS in the virulence of this intracellular bacterial pathogen may have broad implications for the creation of a safe and efficacious vaccine.

Cord blood cytokines and acute lower respiratory illnesses in the first year of life.

OBJECTIVES: Little is known about the relation between cytokine profile at birth and acute lower respiratory illnesses in the first year of life. The purpose of this work was to examine the relation between cytokine secretions by cord blood mononuclear cells and acute lower respiratory illness in a birth cohort of 297 children. METHODS: Cord blood mononuclear cells were isolated, and secretion of interferon-gamma, interleukin-13, interleukin-10, and tumor necrosis factor-alpha at baseline and in response to allergens (Blatella germanica 2 and Dermatophagoides farinae 1) and mitogen (phytohemagglutinin) were quantified using enzyme-linked immunosorbent assay. Acute lower respiratory illness was defined as a parental report of a diagnosis of bronchiolitis, pneumonia, bronchitis, and/or croup by a health care professional in the first year of life. Differences in the levels of cord blood cytokines between children with and without acute lower respiratory illness were examined using 2-sample Wilcoxon tests. Logistic regression models were used to examine the relation between various categories of cord blood cytokines and acute lower respiratory illness. RESULTS: Median levels of interferon-gamma secreted by cord blood mononuclear cells in response to Blatella germanica 2 and Dermatophagoides farinae 1 were higher among children without acute lower respiratory illness as compared with children with acute lower respiratory illness. After adjustment for other covariates, the odds of acute lower respiratory illness was reduced among children in the top category (at or more than the median of detectable values) of interferon-gamma level, significantly so in response to Blatella germanica 2. CONCLUSIONS: In a cohort of children from the general population, we found that upregulated interferon-gamma secretion at birth is associated with reduced risk of acute lower respiratory illness in the first year of life.

A bacterial carbohydrate links innate and adaptive responses through Toll-like receptor 2.

Commensalism is critical to a healthy Th1/Th2 cell balance. Polysaccharide A (PSA), which is produced by the intestinal commensal Bacteroides fragilis, activates CD4+ T cells, resulting in a Th1 response correcting the Th2 cell skew of germ-free mice. We identify Toll-like receptors as crucial to the convergence of innate and adaptive responses stimulated by PSA. Optimization of the Th1 cytokine interferon-gamma in PSA-stimulated dendritic cell-CD4+ T cell co-cultures depends on both Toll-like receptor (TLR) 2 and antigen presentation. Synergy between the innate and adaptive responses was also shown when TLR2-/- mice exhibited impaired intraabdominal abscess formation in response to B. fragilis. Commensal bacteria, using molecules like PSA, potentially modulate the Th1/Th2 cell balance and the response to infection by coordinating both the innate and adaptive pathways.

Hyaluronic acid binding peptides prevent experimental staphylococcal wound infection.

Staphylococcus aureus is a major cause of surgical wound infections. The development of mechanisms of antimicrobial resistance by this and other bacterial pathogens has prompted the search for new approaches to treat infectious diseases. Hyaluronic acid binding peptides have been shown to modulate cellular trafficking during host responses and were assessed for their ability to treat and possibly prevent experimental surgical wound infections caused by S. aureus. Treatment with these peptides was highly efficacious in reducing the number of S. aureus cells at the wound site and ameliorated the inflammatory host response associated with these infections. These data suggest a novel approach for the treatment and prophylaxis of staphylococcal wound infections in the clinical setting.

Mode of delivery and cord blood cytokines: a birth cohort study.

BACKGROUND: The mechanisms for the association between birth by cesarean section and atopy and asthma are largely unknown. OBJECTIVE: To examine whether cesarean section results in neonatal secretion of cytokines that are associated with increased risk of atopy and/or asthma in childhood. To examine whether the association between mode of delivery and neonatal immune responses is explained by exposure to the maternal gut flora (a marker of the vaginal flora). METHODS: CBMCs were isolated from 37 neonates at delivery, and secretion of IL-13, IFN-gamma, and IL-10 (at baseline and after stimulation with antigens [dust mite and cat dander allergens, phytohemagglutinin, and lipopolysaccharide]) was quantified by ELISA. Total and specific microbes were quantified in maternal stool. The relation between mode of delivery and cord blood cytokines was examined by linear regression. The relation between maternal stool microbes and cord blood cytokines was examined by Spearman's correlation coefficients. RESULTS: Cesarean section was associated with increased levels of IL-13 and IFN-gamma. In multivariate analyses, cesarean section was associated with an increment of 79.4 pg/ml in secretion of IL-13 by CBMCs after stimulation with dust mite allergen (P < 0.001). Among children born by vaginal delivery, gram-positive anaerobes and total anaerobes in maternal stool were positively correlated with levels of IL-10, and gram-negative aerobic bacteria in maternal stool were negatively correlated with levels of IL-13 and IFN-gamma. CONCLUSION: Cesarean section is associated with increased levels of IL-13 and IFN-gamma, perhaps because of lack of labor and/or reduced exposure to specific microbes (e.g., gram-positive anaerobes) at birth.

CD4+ T cells and CXC chemokines modulate the pathogenesis of Staphylococcus aureus wound infections.

T cells are critical for the formation of intraabdominal abscesses by Staphylococcus aureus. We hypothesized that T cells modulate the development of experimental staphylococcal infections by controlling polymorphonuclear leukocyte (PMN) trafficking. In models of staphylococcal s.c. abscess formation, hindpaw infection, and surgical wound infection, S. aureus multiplied in the tissues of WT C57BL/6J mice and elicited a marked inflammatory response. CD4(+) alphabeta T cells homed to the surgical wound infection site of WT animals. In contrast, significantly fewer S. aureus were recovered from the tissues of mice deficient in alphabeta T cells, and the inflammatory response was considerably diminished compared with that of WT animals. Alphabeta T cell receptor (-/-) mice had significantly lower concentrations of PMN-specific CXC chemokines in wound tissue than did WT mice. The severity of the wound infection was enhanced by administration of a CXC chemokine and abrogated by antibodies that blocked the CXC receptor. An acapsular mutant was less virulent than the parental S. aureus strain in both the s.c. abscess and the surgical wound infection models in WT mice. These data reveal an important and underappreciated role for CD4(+) alphabeta T cells in S. aureus infections in controlling local CXC chemokine production, neutrophil recruitment to the site of infection, and subsequent bacterial replication.

Modulation of surgical fibrosis by microbial zwitterionic polysaccharides.

Bacterial carbohydrates have long been considered T cell-independent antigens that primarily induce humoral immune responses. Recently, it has been demonstrated that bacterial capsules that possess a zwitterionic charge motif can activate CD4(+) T cells after processing and presentation by antigen-presenting cells. Here we show that these zwitterionic polysaccharides can prevent T helper 1-mediated fibrosis by signaling for the release of IL-10 from CD4(+) T cells in vivo. IL-10 production by these T cells and their ability to prevent fibrosis is controlled by the inducible costimulator (ICOS)-ICOS ligand pathway. These data demonstrate that the interaction of the zwitterionic polysaccharides with T cells results in modulation of surgical fibrosis in vivo and suggest a previously undescribed approach to "harnessing" T cell function to prevent inflammatory tissue disorders in humans.

An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.

The mammalian gastrointestinal tract harbors a complex ecosystem consisting of countless bacteria in homeostasis with the host immune system. Shaped by evolution, this partnership has potential for symbiotic benefit. However, the identities of bacterial molecules mediating symbiosis remain undefined. Here we show that, during colonization of animals with the ubiquitous gut microorganism Bacteroides fragilis, a bacterial polysaccharide (PSA) directs the cellular and physical maturation of the developing immune system. Comparison with germ-free animals reveals that the immunomodulatory activities of PSA during B. fragilis colonization include correcting systemic T cell deficiencies and T(H)1/T(H)2 imbalances and directing lymphoid organogenesis. A PSA mutant of B. fragilis does not restore these immunologic functions. PSA presented by intestinal dendritic cells activates CD4+ T cells and elicits appropriate cytokine production. These findings provide a molecular basis for host-bacterial symbiosis and reveal the archetypal molecule of commensal bacteria that mediates development of the host immune system.

Effect of B7-2 and CD40 signals from activated antigen-presenting cells on the ability of zwitterionic polysaccharides to induce T-Cell stimulation.

Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria elicit potent CD4+-T-cell responses in vivo and in vitro. We demonstrated that HLA-DR on professional antigen-presenting cells (APCs) is required for ZPS-induced T-cell proliferation in vitro (15). Recently, it was shown that ZPSs are processed to low-molecular-weight carbohydrates by a nitric oxide-mediated mechanism in endosomes and locate in the major histocompatibility complex class II pathway (5, 15). The effect of the ZPS-mediated expression of HLA-DR and costimulatory molecules on the APC and T-cell engagement and subsequent T-cell activation has not been elucidated. Herein, we report that ZPS-mediated induction of HLA-DR-surface expression and T-cell proliferation are maximally enhanced after incubation of APCs for 8 h with ZPS. Treatment of APCs with bafilomycin A inhibits the up-regulation of ZPS-mediated HLA-DR surface expression and leads to inhibition of T-cell proliferation. Monoclonal antibodies (MAbs) to the costimulatory molecules B7-2 and CD40L specifically block ZPS-mediated T-cell activation, while a MAb to B7-1 does not. Surface expression of B7-2 and B7-1 but not of CD40 is maximally enhanced at 8 to 16 h of treatment of APCs with ZPS. The results demonstrate that the cellular immune response to ZPS depends on the translocation of HLA-DR to the cell surface and requires costimulation via B7-2 and CD40 on activated APCs. The implication is that activation of ZPS-specific T cells requires an orchestrated arrangement of both presenting and costimulatory molecules to form an immunological synapse.

Cytokines, allergy, and asthma.

PURPOSE OF REVIEW: This review examines recent articles on the relationship of cytokines to allergy and asthma with particular emphasis on immune mechanisms involved in disease development in early life. RECENT FINDINGS: It was previously proposed that reduced microbial exposure in early life is responsible for a shift of the Th1/Th2 balance in the immune system towards the proallergenic Th2 response. This Th1/Th2 imbalance results in the clinical expression of allergy and/or asthma. In recent years, accumulating data from mice and humans have identified Th2 cytokines [interleukin (IL)-4, IL-13, and IL-5] as major contributors to allergy and asthma. Interestingly, the Th1 cytokine interferon-gamma has recently been shown to act concurrently with Th2 cytokines in maintaining the chronic inflammatory response in allergic diseases, particularly in asthmatic airways. Most recently, evidence suggests that suppression of T-regulatory cells may contribute to the underlying immune mechanisms involved in allergy and asthma. SUMMARY: An enhanced Th2 immune response and the elaboration of cytokines such as IL-4, IL-13, and IL-5 contribute to the induction of allergy and asthma. Interferon-gamma, a Th1 cytokine, acts in conjunction with Th2 (IL-4, IL-13, and IL-5) in maintaining chronic allergic inflammation. The mechanisms leading to an enhanced Th2 response are still controversial. Th2-dominated immune responses may result from immune suppression of T-regulatory cells as well as Th1 cells. Understanding early-life immune mechanisms responsible for atopic diseases, specifically how cytokines of T-regulatory cells act to balance the Th1 and Th2 immune response, continues to be a fruitful area of research.

Polysaccharide processing and presentation by the MHCII pathway.

The adaptive immune system functions through the combined action of antigen-presenting cells (APCs) and T cells. Specifically, class I major histocompatibility complex antigen presentation to CD8(+) T cells is limited to proteosome-generated peptides from intracellular pathogens while the class II (MHCII) endocytic pathway presents only proteolytic peptides from extracellular pathogens to CD4(+) T cells. Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria can activate CD4(+) T cells. Here we show that ZPSs are processed to low molecular weight carbohydrates by a nitric oxide-mediated mechanism and presented to T cells through the MHCII endocytic pathway. Furthermore, these carbohydrates bind to MHCII inside APCs for presentation to T cells. Our observations begin to elucidate the mechanisms by which some carbohydrates induce important immunologic responses through T cell activation, suggesting a fundamental shift in the MHCII presentation paradigm.

Regulation of postsurgical fibrosis by the programmed death-1 inhibitory pathway.

Surgical adhesions are a common and often severe complication of abdominal or pelvic injury that cause pelvic pain, bowel obstruction, and infertility in women. Current treatments are of limited effectiveness because little is known about the cellular and subcellular processes underlying adhesiogenesis. Recently, we showed that Th1 alpha beta CD4(+) T cells mediate the pathogenesis of adhesion formation in a rodent model of this disease process. In this study, we demonstrate that in mice these T cells home directly to the site of surgically induced adhesions and control local chemokine production in a manner dependent on the CD28 T cell costimulatory pathway. Conversely, the inhibitory programmed death-1 pathway plays a central role in limiting adhesiogenesis, as programmed death-1 blockade was associated with increased T cell infiltration, chemokine production, and a concomitant exacerbation of disease. Our results reveal for the first time that the development of postsurgical fibrosis is under the tight control of positive and negative T cell costimulation, and suggest that targeting these pathways may provide promising therapies for the prevention of adhesion formation.

Zwitterionic polysaccharides stimulate T cells with no preferential V beta usage and promote anergy, resulting in protection against experimental abscess formation.

Zwitterionic polysaccharides (Zps) from pathogenic bacteria, such as Bacteroides fragilis, are virulence factors responsible for abscess formation associated with intra-abdominal sepsis. The underlying cellular mechanism for abscess formation requires T cell activation. Conversely, abscess formation can be prevented by prophylactic s.c. injection of purified Zps alone, a process also dependent on T cells. Hence, the modulatory role of T cells in abscess formation was investigated. We show that Zps interact directly with T cells with fast association/dissociation kinetics. Vbeta repertoire analysis using RT-PCR demonstrates that Zps have broad Vbeta usage. Zps-specific hybridomas responded to a variety of other Zps, but not to a nonzwitterionic polysaccharide, indicating cross-reactivity between different Zps. Furthermore, Zps-reactive T cell hybridomas could effectively transfer protection against abscess formation. Analysis of the proliferative capacity of T cells recovered from Zps-treated animals revealed that these T cells are anergic to subsequent stimulation by the different Zps or to alloantigens in an MLR. This anergic response was relieved by addition of IL-2. Taken together, the data show that this class of polysaccharides interacts directly with T cells in a nonbiased manner to elicit an IL-2-dependent anergic response that confers protection against abscess formation.