The effect of exercise on innate mucosal immunity.

METHODS: The authors conducted a prospective observational study comparing salivary lactoferrin and lysozyme concentration over 5 months (chronic changes) in elite rowers (n=17, mean age 24.3+/-4.0 years) with sedentary individuals (controls) (n=18, mean age=27.2+/-7.1 years) and a graded exercise test to exhaustion (acute changes) with a cohort of elite rowers (n=11, mean age 24.7+/-4.1). RESULTS: Magnitudes of differences and changes were interpreted as a standardised (Cohen's) effect size (ES). Lactoferrin concentration in the observational study was approximately 60% lower in rowers than control subjects at baseline (7.9+/-1.2 microg/ml mean+/-SEM, 19.4+/-5.6 microg/ml, p=0.05, ES=0.68, 'moderate') and at the midpoint of the season (6.4+/-1.4 microg/ml mean +/- SEM, 21.5+/-4.2 microg/ml, p=0.001, ES=0.89, 'moderate'). The concentration of lactoferrin at the end of the study was not statistically significant (p=0.1) between the groups. There was no significant difference between rowers and control subjects in lysozyme concentration during the study. There was a 50% increase in the concentration of lactoferrin (p=0.05, ES=1.04, 'moderate') and a 55% increase in lysozyme (p=0.01, ES=3.0, 'very large') from pre-exercise to exhaustion in the graded exercise session. CONCLUSION: Lower concentrations of these proteins may be indicative of an impairment of innate protection of the upper respiratory tract. Increased salivary lactoferrin and lysozyme concentration following exhaustive exercise may be due to a transient activation response that increases protection in the immediate postexercise period.

Pseudomonas aeruginosa: the potential to immunise against infection.

Pseudomonas aeruginosa remains a serious pathogen for specific cohorts of patients where chronic infection is a poor prognostic indicator, such as those with cystic fibrosis, burn wounds or those who are immunocompromised. Significant disease burden is associated with a diverse spectrum of both nosocomial and community-acquired infections. To date, vaccines against P. aeruginosa have shown limited and often conflicting efficacy data, especially against heterologous strains, which are increasingly identified as co-colonisers of biofilms. While few studies have gone beyond Phase II clinical trials, a particular concern is the ability of P. aeruginosa to evade the immune system while provoking an immune response that contributes to the destructive nature of infection. Therefore, vaccine development needs to focus on preventing attachment and colonisation, as well as preventing conversion to a mucoid phenotype that is characteristic of the chronic condition that promotes pathology.

CD8+ T cells have an essential role in pulmonary clearance of nontypeable Haemophilus influenzae following mucosal immunization.

A rodent respiratory experimental model has proved useful for investigating the immune mechanisms responsible for clearance of bacteria from the lungs. Immunohistochemical studies in immune and nonimmune rats have identified the cellular kinetics of response to bacterial pulmonary infection for CD8+, CD4+, and gammadelta+ T cells; B cells; and the expression of major histocompatibility complex class II (MHC-II). During the course of bacterial clearance, there was no apparent proliferation or extravasation of lymphocytes, nor was there increased expression of MHC-II in nonimmune animals despite an influx of polymorphonuclear leukocytes, whereas in immunized animals there was an early influx of CD8+ and gammadelta+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes, and finally an increased number of CD4+ T cells. Depletion of CD8+ T cells confirmed their vital contribution in the preprimed immune response to pulmonary infection by significantly decreasing the animals' ability to clear bacteria following challenge.

Vaccines and mucosal immunisation.

The earliest attempts to protect humans against infectious diseases and toxins were by administering foreign substances to mucosal membranes, predominantly by the oral route. In the late 1880s, significant attention was given to the concept of 'local' immunisation, and the discipline of mucosal immunology was born in the early 1900s. However, despite the early enthusiasm, progress has been slow, with few mucosal vaccines being efficacious. The complexities of mucosal immune regulation and the lack of appropriate antigen delivery systems which can access mucosal inductive sites, have remained substantial obstacles. Recent studies demonstrating compartmentalisation of the common mucosal immune system create further challenges for the development of organ-specific vaccines. In the 21st century, our knowledge of mucosal immunoregulatory mechanisms, coupled with new technology for antigen delivery and immunomodulation will provide the necessary know-how to see the development and widespread use of mucosal vaccines for both preventative and therapeutic use.

Programmed inflammatory processes induced by mucosal immunisation.

Inflammation is essential to repair tissue damaged by physical, microbial or allergic mechanisms. Inappropriately zealous responses lead to destructive pathology or chronic disease cycles, whereas ideal outcomes are associated with complete and rapid restoration of tissue structure and function. The establishment of a rodent model investigating the different immune responses to non-typeable Haemophilus influenzae infection in both the lung and the ear indicate an ability to clear bacteria and reduce inflammation following mucosal immunisation. Lung histochemistry, upregulaion of macrophages and polymorphonuclear neutrophils, recruitment of gammadelta(+) and CD8(+) T cells, cytokine levels and depletion studies all support the hypothesis that mucosal immunisation facilitates control of the immune response resulting in enhanced bacterial clearance and programming of inflammation which limits damage and promotes the rapid restoration of structural normality.

Mucosal immunity in the lung and upper airway.

The mucosal surfaces of the lungs and upper airways are common sites for infection. Extensive studies of the mechanisms associated with immune responses in the respiratory tract have found that understanding the system is challenging and involves many complex interactions to prevent and eliminate infection. Immune protection against diseases transmitted through the respiratory tract requires an understanding of the important aspects associated with beneficial, detrimental or ineffective immune responses. Two critical aspects of an immune response against a pathogen are that of the inductive stage, either induced by vaccination or primary infection, and the effector stage, the ability to recognise, respond to and eliminate the infection without detriment to the host. An immunisation strategy must not only have a measure of the induced antigen specific response, but this response must also be protective.

Viral co-infection does not reduce the efficacy of vaccination against non-typeable Haemophilus influenzae middle ear infection in a rat model.

The mucosal vaccination of rodents with killed non-typeable Haemophilus influenzae (NTHi) has been previously shown to enhance live NTHi clearance following middle ear challenge. This study assessed the efficacy of mucosal anti-NTHi vaccination during a concomitant viral infection of the respiratory tract. Animals were mucosally immunised with killed NTHi by intra-Peyer's patch primary inoculation and lung (intratracheal) boost. At the time of both immunisations rats were also infected intra-nasally with Sendai virus. Concomitant Sendai virus infection did not influence the efficacy of anti-NTHi vaccination mediated clearance of NTHi from the middle ear. This would suggest that immunisation strategies to prevent bacterial middle ear infection would be effective despite the presence of concomitant viral agents.

Catalase immunization from Pseudomonas aeruginosa enhances bacterial clearance in the rat lung.

Pseudomonas aeruginosa is a common cause of infection in immunocompromised patients and is the major contributor to morbidity in individuals with cystic fibrosis (CF). The antibiotic resistance shown by this pathogen and morbidity in patients with chronic infection has encouraged investigations into the development of a vaccine. This study reports the purification of a 60 kDa protein, isolated from a mucoid strain of P. aeruginosa, identified by amino acid sequence analysis as the catalase protein (KatA). A rat model of acute P. aeruginosa respiratory infection was used to investigate the immunogenicity of KatA and determine the potential of mucosal immunization with KatA to protect against infection. Immunization regimens compared a single intra-Peyer's patch (IPP) immunization with an IPP primary inoculation followed by an intratracheal boost to the lungs. Mucosal immunization with KatA resulted in significant pulmonary clearance of both homologous (p<0.001) and heterologous (p<0.05) strains of P. aeruginosa. Both immunization regimens enhanced bacterial clearance, increased the rate of recruitment of phagocytes to the bronchoalveoli and induced KatA-specific antibody. However, the regimen that included a boost induced a more effective immune response that also resulted in better clearance of P. aeruginosa from the lungs. Mucosal immunization induced KatA- specific antibodies in the serum and the bronchoalveolar lavage, and KatA-specific lymphocyte proliferation in vitro in cells isolated from the mesenteric lymph nodes of immunized rats. The data presented suggests that KatA has the potential to afford a protective immune response against pulmonary infection by P. aeruginosa

Killed whole bacterial cells, a mucosal delivery system for the induction of immunity in the respiratory tract and middle ear: an overview.

Infectious diseases remain a leading cause of morbidity and mortality worldwide with mucosal membranes being the most frequent portals of entry of pathogenic micro-organisms. This has prompted studies aimed at the development of vaccination protocols that would lead to an increased protection of mucosae through an understanding of the common mucosal immune system as an immune communication network between mucosal sites. Recent studies have suggested that preferential sub-networks exist within the system and these studies have exploited the gut-associated lymphoid tissue (GALT)-lung sub-network in the development of oral vaccine strategies for infections of the respiratory tract and middle ear. Mucosal immunization with whole formalin killed Pseudomonas aeruginosa (Pa), Branhamella catarrhalis, nontypable Haemophilus influenzae (NTHi) or Streptococcus pneumoniae (Spn) results in enhanced homologous bacterial clearance from the lung of immune animals challenged with live bacteria. These studies have been extended to the middle ear where similar results have been observed for NTHi and Spn. Mechanisms responsible for inducing enhanced bacterial clearance from the airways include opsonising antibody, antigen specific CD4+ T helper cells, cytokine responses and recruitment of activated polymophonuclear neutrophils. The mechanisms induced by immunization which stimulates the immune system to rapidly mobilise both innate and specific immune responses during infection are the subject of ongoing research.

Characterization of the gene encoding a 26-kilodalton protein (OMP26) from nontypeable Haemophilus influenzae and immune responses to the recombinant protein.

A 26-kDa protein (OMP26) isolated and purified from nontypeable Haemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzae Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.

Enhancement of pulmonary clearance of Moraxella (Branhamella) catarrhalis following immunization with outer membrane protein CD in a mouse model.

Moraxella (Branhamella) catarrhalis is an important human respiratory tract pathogen. Outer membrane protein (OMP) CD is highly conserved among strains and has characteristics that indicate it may be an effective vaccine antigen. This study investigated the effect of immunization with OMP CD on pulmonary clearance following intratracheal challenge of mice with M. catarrhalis. Two routes of immunization were studied: mucosal immunization (intra-Peyer's patch followed by intratracheal boost) and intramuscular immunization with OMP CD. Both resulted in enhanced pulmonary clearance of M. catarrhalis compared with sham-immunized controls. Immunization with OMP CD induced specific antibodies in serum and bronchoalveolar lavage fluid and induced a specific lymphocyte proliferative response in T cells from mesenteric lymph nodes from mice mucosally immunized with OMP CD. On the basis of these results, OMP CD should undergo continued testing to determine whether it will induce a protective immune response in humans.

Characteristics of the immunological response in the clearance of non-typeable Haemophilus influenzae from the lung.

Clearance of non-typeable Haemophilus influenzae (NTHi) from the respiratory tract was investigated, over time, in immune and non-immune rats. A triphasic pattern characterized the clearance of bacteria from the lungs. Mucosal immunization enhanced bacterial clearance from the lungs in each of the three phases compared with clearance from non-immunized animals. Total clearance of bacteria was observed from lung tissue by 12 h in immune animals and 24 h in non-immune animals. Polymorphonuclear leucocytes not only arrived earlier and initially in greater numbers, but disappeared earlier in immune animals (peaking at 8 h post-challenge), compared with non-immune animals (peaking at 12h post-challenge). Systemically derived and locally produced NTHi-specific IgA and IgG correlated with enhanced bacterial clearance during the secondary phase. This model demonstrates that immunized animals up-regulate and resolve inflammatory responses to pulmonary infection more rapidly than the non-immunized controls.

Nontypeable Haemophilus influenzae: pathogenesis and prevention.

In this paper, we describe the ability of nontypeable Haemophilus influenzae (NTHi) to coexist with the human host and the devastating results associated with disruption of the delicate state of balanced pathogenesis, resulting in both acute and chronic respiratory tract infections. It has been seen that the strains of NTHi causing disease show a marked genetic and phenotypic diversity but that changes in the lipooligosaccharide (LOS) and protein size and antigenicity in chronically infected individuals indicate that individual strains of NTHi can remain and adapt themselves to avoid expulsion from their infective niche. The lack of reliance of NTHi on a single mechanism of attachment and its ability to interact with the host with rapid responses to its environment confirmed the success of this organism as both a colonizer and a pathogen. In vitro experiments on cell and organ cultures, combined with otitis media and pulmonary models in chinchillas, rats, and mice, have allowed investigations into individual interactions between NTHi and the mammalian host. The host-organism interaction appears to be a two-way process, with NTHi using cell surface structures to directly interact with the mammalian host and using secreted proteins and LOS to change the mammalian host in order to pave the way for colonization and invasion. Many experiments have also noted that immune system evasion through antigenic variation, secretion of enzymes and epithelial cell invasion allowed NTHi to survive for longer periods despite a specific immune response being mounted to infection. Several outer membrane proteins and LOS derivatives are discussed in relation to their efficacy in preventing pulmonary infections and otitis media in animals. General host responses with respect to age, genetic makeup, and vaccine delivery routes are considered, and a mucosal vaccine strategy is suggested.

Potential of a novel protein, OMP26, from nontypeable Haemophilus influenzae to enhance pulmonary clearance in a rat model.

A major outer membrane protein band of approximately 25 to 27 kDa is commonly observed in strains of Haemophilus influenzae. This study has investigated the potential of a 26-kDa protein (OMP26) from nontypeable H. influenzae (NTHI) as a vaccine candidate. OMP26 was used to immunize rats via intestinal Peyer's patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain. Significant levels of anti-OMP26 were found in the serum and bronchoalveolar lavage from immunized rats, and isotypes of immunoglobulin G (IgG) were also measured in serum. Analysis of IgG isotypes present in serum following OMP26-immunization suggest that predominantly a T-helper 1-type response was induced. The OMP26 protein was amino-terminally sequenced and found to have no homology with the P5 of H. influenzae type b P5 or the fimbrin protein of NTHI, both can migrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis at similar molecular masses but OMP26 has 100% homology with a segment of the H. influenzae Rd genome. The results of this study suggest that OMP26 may be a suitable vaccine candidate against NTHI infection and warrants continued investigation and characterization.

Kinetics of inflammatory cytokines in the clearance of non-typeable Haemophilus influenzae from the lung.

Levels of the pro-inflammatory cytokines TNF-alpha and IFN-gamma were measured from the time of infection to the time of complete clearance of non-typeable Haemophilus influenzae (NTHi) from the lung in immune and non-immune rats. Mucosal immunization facilitated production of significant levels of TNF-alpha as early as 30 min post-pulmonary challenge with NTHi in immune animals. Following the peak at 2 h, rapid decline of TNF-alpha levels occurred from the alveolar spaces. Levels of TNF-alpha in non-immunized animals increased at a slower rate, peaked at a lower concentration and were slower to decline. The significantly larger number of macrophages seen in the immune animals at 1 h after bacterial challenge could partially account for the higher levels of TNF-alpha. Interferon-gamma was not detected in immune or non-immune rats at any time point before NTHi clearance after pulmonary challenge. Study of the kinetics of TNF-alpha release demonstrates that immunized animals control the release of pro-inflammatory cytokines more effectively than non-immunized animals for enhanced clearance of bacterial infection from the lungs.

Outer-membrane antigen expression by Moraxella (Branhamella) catarrhalis influences pulmonary clearance.

Moraxella (Branhamella) catarrhalis is a common respiratory tract pathogen in man. The bacterium shows a strong tendency to form aggregates in vitro. A variant strain of M. catarrhalis that showed a reduced tendency to form aggregates was selected by successive in-vitro passage in broth culture from which aggregates had settled. The non-clumping variant strain showed alteration in expression of outer-membrane antigens, including the HMW-OMP, an outer-membrane protein of c. 200 kDa, outer-membrane protein CD and lipo-oligosaccharide. A mouse model for pulmonary challenge with M. catarrhalis revealed significant differences in the rate of clearance of the isogenic variant strains from the lung. The parent strain caused enhanced recruitment of neutrophils to the lung and more rapid clearance of bacteria from the lungs in comparison to the non-clumping variant. It is concluded that alteration of expression of surface molecules by M. catarrhalis has a significant impact in an in-vivo model of pulmonary clearance.

Modulation of antigen-specific T and B cell responses influence bacterial clearance of non-typeable Haemophilus influenzae from the lung in a rat model.

This study investigates antigen-specific B and T cell responses following mucosal immunization with the major outer membrane protein, P2, from non-typeable Haemophilus influenzae (NTHi) and the role of these responses in bacterial clearance following pulmonary challenge. Modification of the immunization preparation by the inclusion of sodium dodecyl sulphate (SDS) with the adjuvant, incomplete Freund's, differentially affects the T and B cell responses to the P2 antigen. Rats received an intra-Peyer's patch-immunization with P2 with or without the inclusion of 1% (w/v) SDS, were boosted via an intratracheal administration of P2 alone on day 14, and challenged with live NTHi in the lungs on day 21. There were significant differences in the rate of bacterial clearance between the different P2-immunized groups and the non-immune group. The inclusion of SDS with P2 resulted in enhanced bacterial clearance. This clearance corresponded to an enhancement of P2-specific lymphocyte proliferation by CD4+ T helper cells but a decrease (reduced approximately 75%) in anti-P2 IgG and IgA in both serum and bronchoalveolar lavage washings. P2-specific IgM levels were not altered. IgG subclass analysis indicated that the inclusion of SDS had caused a significant reduction in IgG2a and an increase in IgG1. The data indicates that the magnitude of antibody levels to P2 may not be as important as T cell responses in enhancing clearance of NTHi in the lung, in vivo, and that immunization targeting enhancement of antigen-specific T cells may be important to inducing effective immunity to NTHi.

Enhanced respiratory clearance of nontypeable Haemophilus influenzae following mucosal immunization with P6 in a rat model.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of infection of the respiratory tract in children and adults. The search for an effective vaccine against this pathogen has focused on components of the outer membrane, and peptidoglycan-associated lipoprotein P6 is among the proposed candidates. This study investigated the immunogenicity of P6 in a rat respiratory model. P6 was purified from two strains of NTHi, one capsule-deficient strain and an H. influenzae type b strain, and assessed for clearance of both homologous and heterologous bacterial strains following mucosal immunization. A protective immune response was determined by enhancement of pulmonary clearance of live bacteria and an increased rate of recruitment of phagocytic cells to the lungs. This was most effective when Peyer's patch immunization was accompanied by an intratracheal (IT) boost. However, the rate of bacterial clearance varied between strains, which suggests some differences in anti-P6 immunological defenses recognizing the expression of the highly conserved P6 lipoprotein on the bacterial surface in some strains. P6-specific antibodies in both serum and bronchoalveolar lavage fluid were cross-reactive and did not differ significantly in strain specificity, demonstrating that difference in clearance was unlikely due to differences in P6-specific antibody levels. Serum homologous and heterologous P6-antibody was bactericidal against NTHi even when enhanced clearance had not been observed. Peyer's patch immunization induced P6-specific CD4+ T-helper cell proliferation in lymphocytes isolated from the mesenteric lymph nodes. An IT boost increased the level of P6-specific antibodies in serum and bronchoalveolar lavage fluid, and P6-specific mesenteric node lymphocyte proliferation. Cells from rats immunized with P6 demonstrated proliferation following stimulation with P6 from nonhomologous strains; however, there was some variation in proliferative responses to P6 from different strains in lymphocytes isolated from animals immunized with killed bacteria. The increase in P6-specific antibodies and T-helper cell responses following an IT boost correlated with an increased rate of recruitment of phagocytic cells and enhanced bacterial clearance of both homologous and heterologous bacteria in the lungs. The data suggests that P6 has the potential to afford protection against pulmonary infection by NTHi following the induction of effective antigen-specific B- and T-cell responses in mucosal tissues.

Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae.

Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell-mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.