Experimental vaccine induces Th1-driven immune responses and resistance to Neisseria gonorrhoeae infection in a murine model.

Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMVs) plus microencapsulated interleukin-12 (IL-12), and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10-13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6-9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal immunoglobulin G (IgG) and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted interferon-γ (IFNγ), but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as elongation factor-Tu (EF-Tu) and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a nonliving gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate type 1 T helper cell (Th1)-driven immune responses in the genital tract.

Suppression of host adaptive immune responses by Neisseria gonorrhoeae: role of interleukin 10 and type 1 regulatory T cells.

Infection with Neisseria gonorrhoeae triggers an intense inflammatory response characterized by an influx of neutrophils in the genital tract, yet natural gonococcal infection does not induce a state of protective immunity. Our previous studies in a mouse model of N. gonorrhoeae infection demonstrated that transforming growth factor-ß (TGF-ß) is involved in the suppression of adaptive immunity by this organism, but complete inhibition of TGF-ß activity only partially reverses N. gonorrhoeae-mediated suppression of T helper type 1 (Th1) and Th2 responses. In this study, we show that N. gonorrhoeae strongly induced the production of interleukin (IL)-10 and type 1 regulatory T (Tr1) cells. Blockade of IL-10 and Tr1 cell activity enhanced both Th1/Th2-dependent adaptive immune responses and Th17-governed innate responses to N. gonorrhoeae. Treatment of mice with anti-IL-10 antibody during gonococcal challenge led to faster clearance of infection and induced protection against secondary infection, with the generation of circulating and vaginal anti-gonococcal antibodies. Our results suggest that inhibition of IL-10 and Tr1 cells affords a new approach to the treatment of gonorrhea and facilitates the development of specific protective immunity.

Neisseria gonorrhoeae selectively suppresses the development of Th1 and Th2 cells, and enhances Th17 cell responses, through TGF-ß-dependent mechanisms.

Infection with Neisseria gonorrhoeae does not induce specific immunity or immune memory. Our previous studies in a murine model of vaginal gonococcal infection showed that innate immunity governed by Th17 cells was a critical aspect of the immune response elicited by this pathogen. Herein we show that N. gonorrhoeae selectively inhibited Th1 and Th2 cells and enhanced Th17 cell development through the induction of TGF-ß. Whereas Th17 responses depended on gonococcal lipooligosaccharide acting through TLR4, the inhibitory effect of N. gonorrhoeae on Th1/Th2 responses involved gonococcal Opa proteins. In vitro Th17 responses to N. gonorrhoeae could be diverted to Th1/Th2 by blockade of TGF-ß, but not by blockade of IL-17. The results reveal that N. gonorrhoeae suppresses Th1/Th2-mediated adaptive immune response through mechanisms dependent on TGF-ß, and that this effect can be manipulated to promote the development of adaptive immunity.

Interstitial fluid flow and cyclic strain differentially regulate cardiac fibroblast activation via AT1R and TGF-ß1.

Cardiac fibroblasts are exposed to both cyclic strain and interstitial fluid flow in the myocardium. The balance of these stimuli is affected by fibrotic scarring, during which the fibroblasts transition to a myofibroblast phenotype. The present study investigates the mechanisms by which cardiac fibroblasts seeded in three-dimensional (3D) collagen gels differentiate between strain and fluid flow. Neonatal cardiac fibroblast-seeded 3D collagen gels were exposed to interstitial flow and/or cyclic strain and message levels of collagens type I and III, transforming growth factor ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA) were assessed. Flow was found to significantly increase and strain to decrease expression of myofibroblast markers. Corresponding immunofluorescence indicated that flow and strain differentially regulated α-SMA protein expression. The effect of flow was inhibited by exposure to losartan, an angiotensin II type 1 receptor (AT1R) blocker, and by introduction of shRNA constructs limiting AT1R expression. Blocking of TGF-ß also inhibited the myofibroblast transition, suggesting that flow-mediated cell signaling involved both AT1R and TGF-ß1. Reduced smad2 phosphorylation in response to cyclic strain suggested that TGF-ß is part of the mechanism by which cardiac fibroblasts differentiate between strain-induced and flow-induced mechanical stress. Our experiments show that fluid flow and mechanical deformation have distinct effects on cardiac fibroblast phenotype. Our data suggest a mechanism in which fluid flow directly acts on AT1R and causes increased TGF-ß1 expression, whereas cyclic strain reduces activation of smad proteins. These results have relevance to the pathogenesis and treatment of heart failure.

Structure and function relationships in IgA.

Immunoglobulin A (IgA) has a critical role in immune defense particularly at the mucosal surfaces, and is equipped to do so by the unique structural attributes of its heavy chain and by its ability to polymerize. Here, we provide an overview of human IgA structure, describing the distinguishing features of the IgA1 and IgA2 subclasses and mapping the sites of interaction with host receptors important for IgA's functional repertoire. Remarkably, these same interaction sites are targeted by binding proteins and proteases produced by various pathogens as a means to subvert the protective IgA response. As interest in the prospect of therapeutic IgA-based monoclonal antibodies grows, the emerging understanding of the relationship between IgA structure and function will be invaluable for maximizing the potential of these novel reagents.

Characterization of antigen-presenting cells induced by intragastric immunization with recombinant chimeric immunogens constructed from Streptococcus mutans AgI/II and type I or type II heat-labile enterotoxins.

Intragastric (i.g.) immunization with recombinant chimeric proteins constructed from the saliva-binding region (SBR) of Streptococcus mutans surface antigen AgI/II and the A2/B subunits of enterobacterial heat-labile enterotoxins has been successfully used to induce salivary and circulating antibodies against S. mutans that have protective potential against dental caries. To investigate the mode of action of these vaccine constructs, mice were immunized i.g. with chimeric proteins constructed from SBR and cholera toxin (CT) or the type II enterotoxins of Escherichia coli, LT-IIa and LT-IIb. Antigen-presenting cells (APC) in Peyer's patches (PP) and mesenteric lymph nodes (MLN) were characterized by flow cytometry. Compared with immunization with SBR alone, chimeric proteins SBR-LTIIaA2/B and SBR-LTIIbA2/B increased the number of B cells and macrophages in PP and diminished B cell numbers in MLN, whereas SBR-CTA2/B diminished the numbers of B cells and macrophages in PP and MLN. Immunization with all three chimeric proteins led to upregulation of MHC class II molecules and co-stimulatory receptors CD40, CD80, and CD86 especially on dendritic cells in PP and also on APC in MLN. The results provide a molecular basis for the enhanced immune responses induced by chimeric proteins compared with uncoupled antigen, and for differential responses to chimeric proteins based on CT or type II enterotoxins.

The development of community health indicators: a district-wide approach.

INTRODUCTION: In response to high rates of chronic disease, the Capital District Health Authority in Nova Scotia recognized a need to move from a focus on acute care in decision making to one that also values a population health approach guided by community health indicators. METHODS: Stakeholders were surveyed on the choice, knowledge and utility of selected indicators. RESULTS: Respondents reported high scores for changes in their knowledge and attitude regarding community health indicators, and identified priority indicators for action.Decision makers' use of community health indicators was increased by stakeholder involvement, supporting evidence in plain language, and wide dissemination.

Human RFamide-related peptide-1 diminishes cellular and integrated cardiac contractile performance.

Peptides influence cardiac dysfunction; however, peptidergic modulation of contractile performance remains relatively uncharacterized. We identified a novel human peptide that modulates mammalian contractile performance. Members of the FMRFamide-related peptide (FaRP) family contain a C-terminal RFamide but structurally variant N-terminal extension. We report human RFamide-related peptide-1 (hRFRP-1) and rat RFRP-1 rapidly and reversibly decreased shortening and relaxation in isolated mammalian cardiac myocytes in a dose dependent manner. The mammalian FaRP, 26RFa, structurally related to RFRP-1 by only an RFamide did not influence myocyte contractile function. The protein kinase C (PKC) inhibitor bisindolylmaleimide-1 blocked hRFRP-1 activity. Pretreatment with pertussis toxin (PTX) did not diminish hRFRP-1 influence on contractile function. In addition, intravenous injection of hRFRP-1 in mice decreased heart rate, stroke volume, ejection fraction, and cardiac output. Collectively these findings are consistent with the conclusion RFRP-1 is an endogenous signaling molecule that activates PKC and acts through a PTX-insensitive pathway to modulate cardiac contractile function. Taken together these negative chronotropic, inotropic, and lusitropic effects of hRFRP-1 are significant; they suggest direct acute cellular and organ-level responses in mammalian heart. This is the first known study to identify a mammalian FaRP with cardio-depressant effects, opening a new area of research on peptidergic modulation of contractile performance. The high degree of RFRP structure conservation from amphibians to mammals, and similarity to invertebrate cardioinhibitory peptides suggests RFRP-1 is involved in important physiological functions. Elucidation of mechanisms involved in hRFRP-1 synthesis, release, and signaling may aid the development of strategies to prevent or attenuate cardiac dysfunction.

Critical role of Th17 responses in a murine model of Neisseria gonorrhoeae genital infection.

Host immune responses, including the characteristic influx of neutrophils, against Neisseria gonorrhoeae are poorly understood; adaptive immunity is minimal and non-protective. We hypothesize that N. gonorrhoeae selectively elicits Th17-dependent responses, which trigger innate defense mechanisms, including neutrophils and antimicrobial proteins, that it can resist. We found that N. gonorrhoeae induced the production of interleukin-17 (IL-17) in mouse T-cells and Th17-inducing cytokines in mouse and human APCs in vitro. IL-17 was induced in the iliac lymph nodes in vivo in a female mouse model of genital tract gonococcal infection. Antibody blockade of IL-17 or deletion of the major IL-17 receptor (IL-17R) in IL-17RA(KO) mice led to prolonged infection and diminished neutrophil influx. Genital tract tissue from IL-17RA(KO) mice showed reduced production of neutrophil-attractant chemokines in response to culture with N. gonorrhoeae. These results imply a crucial role for IL-17 and Th17 cells in the immune response to N. gonorrhoeae.

Mucosal immunization of mice with recombinant OMP P2 induces antibodies that bind to surface epitopes of multiple strains of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) is a significant cause of otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease. Vaccine research for NTHI has focused on the outer membrane proteins (OMPs) of NTHI. The goal of this study was to evaluate mucosal and systemic immune responses to recombinant OMP P2 (rP2) of NTHI. Enzyme-linked immunosorbent assay (ELISA) demonstrated that both mucosal and systemic routes of immunization resulted in antibodies to rP2. Whole-cell ELISA and flow cytometry indicated that mucosal immunization induced antibodies to epitopes that are on the bacterial surface of the homologous strain as well as several heterologous strains. In contrast, systemic immunization induced antibodies to non-surface exposed epitopes. These data show for the first time that mucosal immunization of mice with rP2 induces antibodies that recognize surface exposed epitopes on multiple strains, indicating that P2 is a candidate for development of a mucosal vaccine for NTHI.

Terminology: nomenclature of mucosa-associated lymphoid tissue.

Stimulation of mucosal immunity has great potential in vaccinology and immunotherapy. However, the mucosal immune system is more complex than the systemic counterpart, both in terms of anatomy (inductive and effector tissues) and effectors (cells and molecules). Therefore, immunologists entering this field need a precise terminology as a crucial means of communication. Abbreviations for mucosal immune-function molecules related to the secretory immunoglobulin A system were defined by the Society for Mucosal Immunolgy Nomenclature Committee in 1997, and are briefly recapitulated in this article. In addition, we recommend and justify standard nomenclature and abbreviations for discrete mucosal immune-cell compartments, belonging to, and beyond, mucosa-associated lymphoid tissue.

Predictors of hospital length of stay and cost in patients with intracerebral hemorrhage.

We used the 2002 Healthcare Cost and Utilization Project Nationwide Inpatient Sample to assess hospital length of stay (LOS) and cost among adults with a principal diagnosis of intracerebral hemorrhage (n = 13,239). Sixty-nine percent of patients were aged > or =65 years, and 31% died during hospitalization. Mean LOS (cost) was 7.7 days (15,256 dollars) (survivors: 9.6 days, 17,442 dollars). Patient, hospital, and payer characteristics accounted for 69.1% of variation in cost per discharge.

Immunomodulation with enterotoxins for the generation of secretory immunity or tolerance: applications for oral infections.

The heat-labile enterotoxins, such as cholera toxin (CT), and the labile toxins types I and II (LT-I and LT-II) of Escherichia coli have been extensively studied for their immunomodulatory properties, which result in the enhancement of immune responses. Despite superficial similarity in structure, in which a toxic A subunit is coupled to a pentameric binding B subunit, different toxins have different immunological properties. Administration of appropriate antigens admixed with or coupled to these toxins by oral, intranasal, or other routes in experimental animals induces mucosal IgA and circulating IgG antibodies that have protective potential against a variety of enteric, respiratory, or genital infections. These include the generation of salivary antibodies that may protect against colonization with mutans streptococci and the development of dental caries. However, exploitation of these adjuvants for human use requires an understanding of their mode of action and the separation of their desirable immunomodulatory properties from their toxicity. Recent findings have revealed that adjuvant action is not critically dependent upon the enzymic activity of the A subunits, and that the isolated B subunits may exert different effects on cells of the immune system than do the intact toxins. Interaction of the toxins with immunocompetent cells is not exclusively dependent upon their conventional ganglioside receptors. Immunomodulatory effects have been observed on dendritic cells, macrophages, CD4(+) and CD8(+) T-cells, and B-cells. Numerous factors-including the precise form of the toxin adjuvant, properties of the antigen, whether and how they are coupled, route of administration, and species of animal model-affect the outcome, whether this is enhanced humoral and cellular immunity, or specific induced tolerance toward the antigen.

Systemic treatment for moderate to severe psoriasis: estimates of failure rates and direct medical costs in a north-eastern US managed care plan.

BACKGROUND: Estimates of US medical costs related to psoriasis treatment are limited and tend to understate the economic burden of moderate to severe psoriasis, which often requires the use of systemic agents, phototherapy or both. OBJECTIVE: To estimate treatment failure rates and direct medical costs associated with the use of systemic agents and phototherapy in US patients with psoriasis. METHODS: Claims records from a large New England-based health insurer were used to obtain patient-level data. Eligible patients with at least one claim listing an ICD-9-CM code for psoriasis (696.0; 696.1) were identified. Patients not receiving systemic treatments (methotrexate, cyclosporine, acitretin) or phototherapy (ultraviolet B with or without tar or petrolatum, psoralen and ultraviolet A [PUVA]) were excluded. Treatment failure was defined as a switch in therapy, augmentation with non-topical therapies, discontinuation following uptitration of dose or discontinuation following hospitalization. Medical costs included those related to pharmacy (over-the-counter medication excluded), institutional services (inpatient and outpatient) and professional services. RESULTS: A total of 2068 patients with moderate to severe psoriasis were included in the analysis. Over a 1-year period, approximately 20% of patients experienced treatment failure. The mean time to failure among patients who switched therapy ranged from 3 to 6 months. Mean annual pharmacy costs in the various treatment groups (categorized according to initial therapy received) ranged from 257 dollars to 1992 dollars per patient. Mean annual costs for institutional and professional services ranged from 156 dollars to 799 dollars and 183 dollars to 481 dollars per patient, respectively. The 99th percentile annual pharmacy and institutional costs exceeded 10,000 dollars and 18,000 dollars, respectively. CONCLUSION: Treatment of moderate to severe psoriasis with traditional systemic agents or phototherapy is associated with a high likelihood of treatment failure and a considerable economic burden.

Large fetal pulmonary arteriovenous fistula: impact on pulmonary development.

A case report of a patient with a large pulmonary arteriovenous fistula and valvar pulmonary stenosis is presented. The fistula was diagnosed prenatally and its effect on in utero cardiovascular growth and development documented. Due to concerns about massive intrapulmonary shunting potentially causing profound cyanosis after delivery, an EXIT (EX-utero Intrapartum Treatment) procedure was used to transfer the infant from placental to extracorporeal membrane oxygenation (ECMO) support. Severe pulmonary microvascular disease resulted in prohibitive pulmonary hypertension despite surgical ligation of the fistula. Prenatal and postnatal hemodynamic assessments of the fistula are presented and are compared to the pathologic findings.

Mechanisms of immune tolerance to food antigens in humans.

Immune responses and the mechanisms of tolerance to the common dietary antigens bovine gamma globulin (BGG), ovalbumin (OVA), and soybean protein were evaluated in normal human volunteers. Humoral and T cell proliferative responses to these antigens were measurable but low, consistent with immune tolerance. There were limited correlations between responses in the systemic and mucosal compartments, and in general the responses to one dietary antigen could not predict the response to another. T cell proliferation to dietary antigens increased significantly by addition of recombinant human interleukin-2 (rhuIL-2). Peripheral blood mononuclear cells stimulated with BGG or OVA expressed IL-2Ralpha chain but not IL-2 mRNA, consistent with T cell anergy. Incubation with exogenous IL-2 alone did not restore T cell proliferation to BGG or OVA. In some individuals T cell proliferation to an unrelated vaccine antigen was suppressed by addition of BGG or OVA, but could be reversed with low doses of rhuIL-2. We conclude that in humans anergy is the major mechanism of tolerance to chronic antigen feeding, and we propose that such anergic, antigen-specific T cells actively contribute to maintenance of homeostasis in the intestine in the face of massive antigen challenge.

Effect of attenuated Salmonella enterica serovar Typhimurium expressing a Streptococcus mutans antigen on secondary responses to the cloned protein.

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route with Salmonella expressing an insoluble protein and to study the ability to augment recall responses by boosting with either Salmonella-expressed protein or purified soluble protein alone. The glucan-binding domain (GLU) of the enzyme glucosyltransferase (GTF), which is an important virulence factor of Streptococcus mutans, was recombinantly expressed in the insoluble phase in S. enterica serovar Typhimurium, and the immunogenicity of this construct was studied in mice. We examined the induction of primary immune responses by insoluble GLU polypeptide delivered in Salmonella at week 1 (groups 1 and 2) and recall responses after a week 15 boost with either Salmonella expressing GLU (group 1) or purified GLU polypeptide (groups 2 and 3). Group 4 served as the control and received phosphate-buffered saline alone by the i.n. route. Significant anti-GLU serum immunoglobulin G (IgG) levels were seen in groups 1, 2, and 3 at week 18 (P < 0.001), i.e., 3 weeks after the booster immunization. Mice in group 2, who received Salmonella followed by GLU, had the highest GLU-specific IgG levels among all groups. The serum IgG levels persisted in all responding groups for at least 7 weeks after the boost (week 22). The IgG2a/IgG1 subclass ratio of serum anti-GLU antibodies in group 1 significantly increased after the boost. These results support the induction of a type 1-like immune response to GLU after primary and booster immunizations with Salmonella expressing GLU. On the other hand, group 2 mice, which received Salmonella expressing GLU as the primary dose and soluble protein as the booster dose, exhibited a shift from a type 1-like to a more type 2-like immune response to GLU following the boost. These results indicate that S. enterica serovar Typhimurium is an excellent delivery vehicle for the insoluble and recombinantly expressed GLU of GTF and that this construct was especially effective in priming the host for a secondary response to soluble GLU polypeptide.

Urethral cytokine and immune responses in Chlamydia trachomatis-infected males.

Penile urethral swabs collected from PCR-confirmed Chlamydia trachomatis-infected, C. trachomatis-uninfected, and non-C. trachomatis-infected, nongonococcal urethritis-infected males were analyzed for cytokine, total immunoglobulin (Ig), and specific antibody levels by enzyme-linked immunosorbent assay. Differential cellular components of the swab transport medium were also enumerated for the same groups. Although low, the levels of C. trachomatis-specific IgA and IgG antibodies and interleukin 8 cytokine were significantly higher in C. trachomatis-infected individuals. There were no significant differences in the levels of seven additional cytokines evaluated.

Intestinal macrophages lack CD14 and CD89 and consequently are down-regulated for LPS- and IgA-mediated activities.

The intestinal mucosa normally displays minimal inflammation despite the close proximity between mucosal macrophages and lumenal bacteria. Macrophages interact with bacteria and their products through CD14, a surface receptor involved in the response to LPS, and CD89, the receptor for IgA (FcalphaR). Here we show that resident macrophages isolated from normal human intestine lack CD14 and CD89. The absence of CD14 and CD89 was not due to the isolation procedure or mucosal cell products, but was evident at the transcriptional level, as the macrophages expressed neither CD14- nor CD89-specific mRNAs, but did express Toll-like receptor 2 and 4 transcripts. Consistent with their CD14(-) phenotype, lamina propria macrophages displayed markedly reduced LPS-induced cytokine production and LPS-enhanced phagocytosis. In addition, IgA-enhanced phagocytosis was sharply reduced in lamina propria macrophages. Thus, the absence of CD14 and CD89 on resident intestinal macrophages, due to down-regulated gene transcription, causes down-modulated LPS- and IgA-mediated functions and probably contributes to the low level of inflammation in normal human intestinal mucosa.

Distinct cytokine regulation by cholera toxin and type II heat-labile toxins involves differential regulation of CD40 ligand on CD4(+) T cells.

Cholera toxin (CT) and the type II heat-labile enterotoxins (HLT) LT-IIa and LT-IIb act as potent systemic and mucosal adjuvants and induce distinct T-helper (Th)-cell cytokine profiles. In the present study, CT and the type II HLT were found to differentially affect cytokine production by anti-CD3-stimulated human peripheral blood mononuclear cells (PBMC), and the cellular mechanisms responsible were investigated. CT suppressed interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-alpha), and IL-12 production by PBMC cultures more than either LT-IIa or LT-IIb. CT but not LT-IIa or LT-IIb reduced the expression of CD4(+) T-cell surface activation markers (CD25 and CD69) and subsequent proliferative responses of anti-CD3-stimulated T cells. CT but not LT-IIa or LT-IIb significantly reduced the expression of CD40 ligand (CD40L) on CD4(+) T cells. In a coculture system, CT-treated CD4(+) T cells induced significantly less TNF-alpha and IL-12 p70 production by both autologous monocytes and monocyte-derived dendritic cells than either LT-IIa- or LT-IIb-treated CD4(+) T cells. These findings demonstrate that CT, LT-IIa, and LT-IIb differentially affect CD40-CD40L interactions between antigen-presenting cells and T cells and help explain the distinct cytokine profiles observed with type I and type II HLT when used as mucosal adjuvants.