Structural comparison of protiated, H/D-exchanged and deuterated human carbonic anhydrase IX.

Human carbonic anhydrase IX (CA IX) expression is upregulated in hypoxic solid tumours, promoting cell survival and metastasis. This observation has made CA IX a target for the development of CA isoform-selective inhibitors. To enable structural studies of CA IX-inhibitor complexes using X-ray and neutron crystallography, a CA IX surface variant (CA IXSV; the catalytic domain with six surface amino-acid substitutions) has been developed that can be routinely crystallized. Here, the preparation of protiated (H/H), H/D-exchanged (H/D) and deuterated (D/D) CA IXSV for crystallographic studies and their structural comparison are described. Four CA IXSV X-ray crystal structures are compared: two H/H crystal forms, an H/D crystal form and a D/D crystal form. The overall active-site organization in each version is essentially the same, with only minor positional changes in active-site solvent, which may be owing to deuteration and/or resolution differences. Analysis of the crystal contacts and packing reveals different arrangements of CA IXSV compared with previous reports. To our knowledge, this is the first report comparing three different deuterium-labelled crystal structures of the same protein, marking an important step in validating the active-site structure of CA IXSV for neutron protein crystallography.

The influence of macrophages on mesenchymal stromal cell therapy: passive or aggressive agents?

Mesenchymal stromal cells (MSC) have emerged as promising cell therapies for multiple conditions based on demonstrations of their potent immunomodulatory and regenerative capacities in models of inflammatory disease. Understanding the effects of MSC on T cells has dominated the majority of work carried out in this field to date; recently, however, a number of studies have shown that the therapeutic effect of MSC requires the presence of macrophages. It is timely to review the mechanisms and manner by which MSC modulate macrophage populations in order to design more effective MSC therapies and clinical studies. A complex cross-talk exists through which MSC and macrophages communicate, a communication that is not controlled exclusively by MSC. Here, we examine the evidence that suggests that MSC not only respond to inflammatory macrophages and adjust their secretome accordingly, but also that macrophages respond to encounters with MSC, creating a feedback loop which contributes to the immune regulation observed following MSC therapy. Future studies examining the effects of MSC on macrophages should consider the antagonistic role that macrophages play in this exchange.

Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication following allogeneic haematopoietic stem cell transplantation (HSCT), occurring in up to 30-50% of patients who receive human leucocyte antigen (HLA)-matched sibling transplants. Current therapies for steroid refractory aGVHD are limited, with the prognosis of patients suboptimal. Mesenchymal stem or stromal cells (MSC), a heterogeneous cell population present in many tissues, display potent immunomodulatory abilities. Autologous and allogeneic ex-vivo expanded human MSC have been utilized to treat aGVHD with promising results, but the mechanisms of therapeutic action remain unclear. Here a robust humanized mouse model of aGVHD based on delivery of human peripheral blood mononuclear cells (PBMC) to non-obese diabetic (NOD)-severe combined immunodeficient (SCID) interleukin (IL)-2rγ(null) (NSG) mice was developed that allowed the exploration of the role of MSC in cell therapy. MSC therapy resulted in the reduction of liver and gut pathology and significantly increased survival. Protection was dependent upon the timing of MSC therapy, with conventional MSC proving effective only after delayed administration. In contrast, interferon (IFN)-γ-stimulated MSC were effective when delivered with PBMC. The beneficial effect of MSC therapy in this model was not due to the inhibition of donor PBMC chimerism, as CD45(+) and T cells engrafted successfully in this model. MSC therapy did not induce donor T cell anergy, FoxP3(+) T regulatory cells or cause PBMC apoptosis in this model; however, it was associated with the direct inhibition of donor CD4(+) T cell proliferation and reduction of human tumour necrosis factor-α in serum.

Fabrication of CaO-NaO-SiO(2)/TiO (2) scaffolds for surgical applications.

A series of titanium (Ti) based glasses were formulated (0.62 SiO(2)-0.14 Na(2)O-0.24 CaO, with 0.05 mol% TiO(2) substitutions for SiO(2)) to develop glass/ceramic scaffolds for bone augmentation. Glasses were initially characterised using X-ray diffraction (XRD) and particle size analysis, where the starting materials were amorphous with 4.5 µm particles. Hot stage microscopy and high temperature XRD were used to determine the sintering temperature (~700 °C) and any crystalline phases present in this region (Na(2)Ca(3)Si(6)O(16), combeite and quartz). Hardness testing revealed that the Ti-free control (ScC-2.4 GPa) had a significantly lower hardness than the Ti-containing materials (Sc1 and Sc2 ~6.6 GPa). Optical microscopy determined pore sizes ranging from 544 to 955 µm. X-ray microtomography calculated porosity from 87 to 93 % and surface area measurements ranging from 2.5 to 3.3 SA/mm(3). Cytotoxicity testing (using mesenchymal stem cells) revealed that all materials encouraged cell proliferation, particularly the higher Ti-containing scaffolds over 24-72 h.

Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells.

BACKGROUND: Adult bone marrow-derived mesenchymal stem cells (MSC) possess potent immune modulatory effects which support their possible use as a therapy for immune-mediated disease. MSC induce regulatory T cells (T(reg)) in vitro although the in vivo relevance of this is not clear. OBJECTIVE: This study addressed the hypothesis that adult bone marrow derived-MSC would prevent the pathology associated with allergen-driven airway inflammation, and sought to define the effector mechanism. METHODS: The influence of allogeneic MSC was examined in a model system where T(reg) induction is essential to prevent pathology. This was tested using a combination of a model of ovalbumin-driven inflammation with allogeneic MSC cell therapy. RESULTS: Systemic administration of allogeneic MSC protected the airways from allergen-induced pathology, reducing airway inflammation and allergen-specific IgE. MSC were not globally suppressive but induced CD4(+) FoxP3(+) T cells and modulated cell-mediated responses at a local and systemic level, decreasing IL-4 but increasing IL-10 in bronchial fluid and from allergen re-stimulated splenocytes. Moderate dose cyclophosphamide protocols were used to differentially ablate T(reg) responses; under these conditions the major beneficial effect of MSC therapy was lost, suggesting induction of T(reg) as the key mechanism of action by MSC in this model. In spite of the elimination of T(reg) , a significant reduction in airway eosinophilia persisted in those treated with MSC. CONCLUSION: These data demonstrate that MSC induce T(reg) in vivo and reduce allergen-driven pathology. Multiple T(reg) dependent and independent mechanisms of therapeutic action are employed by MSC.

Zinc and silver glass polyalkenoate cements: an evaluation of their antibacterial nature.

A biofilm is an accumulation of micro-organisms and their extracellular products forming a structured community on a surface. Biofilm formation on medical devices has severe health consequences as bacteria growing in this lifestyle are tolerant to both host defence mechanisms and antibiotic therapies. However, silver and zinc ions inhibit the attachment and proliferation of immature biofilms. The objective of this study is to evaluate whether silver and zinc ions eluted from novel glass polyalkenoate cement (GPC) coatings have the ability to inhibit Methicillin-resistant Staphylococcus aureus (MRSA) in vivo. A silver and zinc-containing GPC coating was synthesised, deposited onto Ti6Al4V discs and placed in a specified amount of analytical water for 1, 7 and 30 days. The resulting elutes were collected and Atomic absorption spectroscopy was used to measure ion release. The elutes were injected into Galleria mellonella larvae infected with MRSA and the antibacterial properties of these elutes were evaluated in vivo. The majority of the zinc and silver ions were released within the first 24 h; this corresponded with the greatest degree of protection observed in infected larvae. Results were compared to a conventional in vitro model where identical elutes were incubated with MRSA on nutrient agar. These results were consistent with those observed in the larval model, demonstrating a reduction in bacterial viability when co-cultured with elutes for 2 h. This work confirms the promise of the Galleria mellonella as a model for the assessment of antimicrobial agents and demonstrates the capacity of novel silver and zinc-containing GPCs to retard the colonisation of MRSA.

Attenuated Bordetella pertussis vaccine strain BPZE1 modulates allergen-induced immunity and prevents allergic pulmonary pathology in a murine model.

BACKGROUND: Virulent Bordetella pertussis, the causative agent of whooping cough, exacerbates allergic airway inflammation in a murine model of ovalbumin (OVA) sensitization. A live genetically attenuated B. pertussis mucosal vaccine, BPZE1, has been developed that evokes full protection against virulent challenge in mice but the effect of this attenuated strain on the development of allergic responses is unknown. OBJECTIVE: To assess the influence of attenuated B. pertussis BPZE1 on OVA priming in a murine model of allergic airway inflammation. METHODS: Mice were challenged with virulent or attenuated strains of B. pertussis, and sensitized to allergen (OVA) at the peak of bacterial carriage. Subsequently, airway pathology, local inflammation and OVA-specific immunity were examined. RESULTS: In contrast to virulent B. pertussis, live BPZE1 did not exacerbate but reduced the airway pathology associated with allergen sensitization. BPZE1 immunization before allergen sensitization did not have an adjuvant effect on allergen specific IgE but resulted in a statistically significant decrease in airway inflammation in tissue and bronchoalveolar lavage fluid. BPZE1 significantly reduced the levels of OVA-driven IL-4, IL-5 and IL-13 but induced a significant increase in IFN-gamma in response to OVA re-stimulation. CONCLUSIONS: These data demonstrate that, unlike virulent strains, the candidate attenuated B. pertussis vaccine BPZE1 does not exacerbate allergen-driven airway pathology. BPZE1 may represent an attractive T-helper type 1 promoting vaccine candidate for eradication of whooping cough that is unlikely to promote atopic disease.

Cell contact, prostaglandin E(2) and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4+CD25(High) forkhead box P3+ regulatory T cells.

Adult human mesenchymal stromal or stem cells (MSC) can differentiate into a variety of cell types and are candidate cellular therapeutics in regenerative medicine. Surprisingly, these cells also display multiple potent immunomodulatory capabilities, including allosuppression, making allogeneic cell therapy a possibility. The exact mechanisms involved in regulatory T cell induction by allogeneic human MSC was examined, using purified CD4+ populations and well-characterized bone marrow-derived adult human MSC. Allogeneic MSC were shown to induce forkhead box P3 (FoxP3)+ and CD25+ mRNA and protein expression in CD4+ T cells. This phenomenon required direct contact between MSC and purified T cells, although cell contact was not required for MSC induction of FoxP3 expression in an unseparated mononuclear cell population. In addition, through use of antagonists and neutralizing antibodies, MSC-derived prostaglandins and transforming growth factor (TGF)-beta1 were shown to have a non-redundant role in the induction of CD4+CD25+FoxP3+ T cells. Purified CD4+CD25+ T cells induced by MSC co-culture expressed TGF-beta1 and were able to suppress alloantigen-driven proliferative responses in mixed lymphocyte reaction. These data clarify the mechanisms of human MSC-mediated allosuppression, supporting a sequential process of regulatory T cell induction involving direct MSC contact with CD4+ cells followed by both prostaglandin E(2) and TGF-beta1 expression. Overall, this study provides a rational basis for ongoing clinical studies involving allogeneic MSC.

Interferon-gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells.

The ability of mesenchymal stem cells (MSC) to suppress alloresponsiveness is poorly understood. Herein, an allogeneic mixed lymphocyte response was used as a model to investigate the mechanisms of MSC-mediated immunomodulation. Human MSC are demonstrated to express the immunosuppressive cytokines hepatocyte growth factor (HGF), interleukin (IL)-10 and transforming growth factor (TGF)-beta1 at concentrations that suppress alloresponses in vitro. MSC also express cyclooxygenase 1 and 2 and produce prostaglandin E2 constitutively. Blocking studies with indomethacin confirmed that prostaglandins contribute to MSC-mediated allosuppression. The proinflammatory cytokine interferon (IFN)-gamma did not ablate MSC inhibition of alloantigen-driven proliferation but up-regulated HGF and TGF-beta1. IFN-gamma also induced expression of indoleamine 2,3, dioxygenase (IDO), involved in tryptophan catabolism. Use of an antagonist, 1-methyl-L-tryptophan, restored alloresponsiveness and confirmed an IDO contribution to IFN-gamma-induced immunomodulation by MSC. Addition of the tryptophan catabolite kynurenine to mixed lymphocyte reactions (MLR), blocked alloproliferation. These findings support a model where IDO exerts its effect through the local accumulation of tryptophan metabolites rather than through tryptophan depletion. Taken together, these data demonstrate that soluble factors, or products derived from MSC, modulate immune responses and suggest that MSC create an immunosuppressive microenvironment capable of modulating alloresponsiveness even in the presence of IFN-gamma.

gammadelta T cells regulate the early inflammatory response to bordetella pertussis infection in the murine respiratory tract.

The role of gammadelta T cells in the regulation of pulmonary inflammation following Bordetella pertussis infection was investigated. Using a well-characterized murine aerosol challenge model, inflammatory events in mice with targeted disruption of the T-cell receptor delta-chain gene (gammadelta TCR-/- mice) were compared with those in wild-type animals. Early following challenge with B. pertussis, gammadelta TCR-/- mice exhibited greater pulmonary inflammation, as measured by intra-alveolar albumin leakage and lesion histomorphometry, yet had lower contemporaneous bacterial lung loads. The larger numbers of neutrophils and macrophages and the greater concentration of the neutrophil marker myeloperoxidase in bronchoalveolar lavage fluid from gammadelta TCR-/- mice at this time suggested that differences in lung injury were mediated through increased leukocyte trafficking into infected alveoli. Furthermore, flow cytometric analysis found the pattern of recruitment of natural killer (NK) and NK receptor+ T cells into airspaces differed between the two mouse types over the same time period. Taken together, these findings suggest a regulatory influence for gammadelta T cells over the early pulmonary inflammatory response to bacterial infection. The absence of gammadelta T cells also influenced the subsequent adaptive immune response to specific bacterial components, as evidenced by a shift from a Th1 to a Th2 type response against the B. pertussis virulence factor filamentous hemagglutinin in gammadelta TCR-/- mice. The findings are relevant to the study of conditions such as neonatal B. pertussis infection and acute respiratory distress syndrome where gammadelta T cell dysfunction has been implicated in the inflammatory process.

Prior Bordetella pertussis infection modulates allergen priming and the severity of airway pathology in a murine model of allergic asthma.

BACKGROUND: It has been proposed that T helper (Th)2-driven immune deviation in early life can be countered by Th1 inducing childhood infections and that such counter-regulation can protect against allergic asthma. OBJECTIVE: To test whether Th1-inducing infection with Bordetella pertussis protects against allergic asthma using well-characterized murine models. METHODS: Groups of mice were sensitized to ovalbumin (OVA) in the presence or absence of B. pertussis, a well-characterized Th1 inducing respiratory infection. Immunological, pathological and physiological parameters were measured to assess the impact of infection on immune deviation and airway function. RESULTS: We demonstrate that OVA sensitization does not affect the development of B. pertussis-specific immune responses dominated by IgG2a and IFN-gamma and does not impair Th1-mediated clearance of airway infection. In contrast, B. pertussis infection at the time of sensitization modulated the response to OVA and significantly reduced total serum and OVA-specific IgE. The pattern of cytokine responses, in particular OVA-specific IL-5 responses in the spleen was also modulated. However, B. pertussis did not cause global suppression as IL-10 and IL-13 levels were enhanced in OVA-stimulated spleen cell cultures and in lavage fluid from infected co-sensitized mice. Histopathological examination revealed that B. pertussis infection prior to OVA sensitization resulted in increased inflammation of bronchiolar walls with accompanying hyperplasia and mucous metaplasia of lining epithelia. These pathological changes were accompanied by increased bronchial hyper-reactivity to methacholine exposure. CONCLUSION: Contrary to the above premise, a Th1 response induced by a common childhood infection does not protect against bronchial hyper-reactivity, but rather exacerbates the allergic asthmatic response, despite modulation of immune mediators.

High-sensitivity PCR detection of parvovirus B19 in plasma.

Parvovirus B19 (B19) is a human pathogen transmitted to susceptible individuals via respiratory secretions and contaminated blood or blood products. B19 levels in pooled plasma of less than 10(4) genome equivalents/ml may not be infectious, while those greater than 10(7)/ml are capable of transmitting infection. A World Health Organization (WHO) B19 DNA international standard has been recently introduced. The purpose of the present work was to develop a PCR-enzyme-linked immunosorbent assay (PCR-ELISA) calibrated against the WHO B19 DNA international standard which could easily and reliably detect B19 DNA levels in plasma above 10(4) IU/ml (6.5 x 10(3) genome equivalents/ml). A B19 PCR-ELISA system was developed which uses a dinitrophenylated oligonucleotide probe to detect immobilized biotinylated amplicons following single-round PCR amplification. The level of B19 DNA (in international units per milliliter) in individual and pooled plasma specimens was evaluated. Proteinase K treatment of plasma was found to be sufficient to quantitatively release B19 DNA. The B19 PCR-ELISA had a sensitivity of detection of 1.6 x 10(3) IU/ml B19 DNA and a dynamic range extending from 8 to 1,000 IU of B19 DNA (equivalent to 1.6 x 10(3) to 2 x 10(5) IU of B19 DNA/ml). Furthermore, the antibody profile of pooled plasma products was determined in terms of B19 immunoglobulin G (IgG) (in international units per milliliter). The B19 IgG level was found to be 64.7 +/- 17.5 IU/ml (mean +/- standard deviation). The B19 PCR-ELISA, which is calibrated against the B19 DNA international standard, may have an application for the rapid screening of plasma minipools for B19 DNA, thereby leading to an improvement in blood product safety.

The rational design of vaccine adjuvants for mucosal and neonatal immunization.

There is an urgent requirement for neonatal vaccines that induce effective and long-lasting immune responses at the mucosal surfaces of the gut and respiratory tract. The delay in their development has been due in part to a lack of understanding of the mucosal and neonatal immune systems. This work reviews recent advances in the understanding of the cells and molecules that mediate immunity, describing the importance of different T helper populations in determining the success of vaccination strategies. These advances have allowed the rational design of novel vaccine adjuvants and delivery systems that can selectively induce immunity at different anatomical sites mediated by distinct T cell populations. Five functional classes of adjuvant are described. These exploit mechanisms which a) create an antigen depot, b) preserve antigen conformation, c) direct antigen to specific immune cells, d) induce mucosal responses and e) induce cytotoxic T cell responses. Comparisons are made between the chemical structures of bacterial toxins and non-toxic derivatives that retain adjuvanticity. The concept of DNA immunization is introduced and the advantages and disadvantages of this novel approach are discussed. The specific problems relating to neonatal immunization are explored with particular reference to the functional immaturity of the neonatal immune system and interference by maternal antibody. Finally, recent work suggesting that there is no intrinsic barrier to designing effective neonatal vaccines deliverable by the mucosal route is discussed.

Baculovirus expression of parvovirus B19 (B19V) NS1: utility in confirming recent infection.

BACKGROUND: The presence of anti-parvovirus B19 (B19V) IgM against viral capsid proteins (VP1 and VP2) has long been used to detect recent infection. The utility of antibodies directed against B19V NS1 protein has received less attention as a serological indicator of recent infection, although anti-B19V NS1 IgG has been associated with persistent infection. OBJECTIVES: To elucidate the role of anti-B19V NS1 antibody detection in recent infection, full-length B19V NS1 was expressed and purified. The resultant antigen was used to develop both Western blot assays and microplate ELISA for the detection of NS1 antibodies. STUDY DESIGN: Serum specimens were obtained from individuals recently infected with B19V (children (n=16), adults (n=40)) and from 17 individuals with no evidence of recent B19V infection. All specimens were screened for anti-B19V NS1 IgG and IgM. RESULTS: It was observed that 68.8% (11/16) of children recently infected with B19V were anti-B19V NS1 IgG seropositive. Furthermore, 27.5% (11/40) anti-B19V VP2 IgM positive specimens also contained anti-B19V NS1 IgM when tested by ELISA, while no reactivity was observed following Western blot analysis, possibly due to the absence of conformational epitopes. CONCLUSIONS: Anti-B19V NS1 IgM detection may have utility in the confirmation of recent infection with B19V.

Impaired gamma interferon responses against parvovirus B19 by recently infected children.

Parvovirus B19 is the causative agent of "fifth disease" of childhood. It has been implicated in a variety of conditions, including unsuccessful pregnancy and rheumatoid arthritis, and is a potential contaminant of blood products. There has been little study of immunity to parvovirus B19, and the exact nature of the protective humoral and cell-mediated immune response is unclear. Immune responses to purified virus capsid proteins, VP1 and VP2, were examined from a cohort of recently infected children and compared with responses from long-term convalescent volunteers. The results demonstrate that antibody reactivity is primarily maintained against conformational epitopes in VP1 and VP2. The unique region of VP1 appears to be a major target for cell-mediated immune responses, particularly in recently infected individuals. We confirm that antibody reactivity against linear epitopes of VP2 is lost shortly after infection but find no evidence of the proposed phenotypic switch in either the subclass of parvovirus B19-specific antibody or the pattern of cytokine production by antigen-specific T cells. The dominant subclass of specific antibody detected from both children and adults was immunoglobulin G1. No evidence was found for interleukin 4 (IL-4) or IL-5 production by isolated lymphocytes from children or adults. In contrast, lymphocytes from convalescent adults produced a typical type 1 response associated with high levels of IL-2 and gamma interferon (IFN-gamma). However, we observed a significant (P<0.001) deficit in the production of IFN-gamma in response to VP1 or VP2 from lymphocytes isolated from children. Taken together, these results imply that future parvovirus B19 vaccines designed for children will require the use of conformationally preserved capsid proteins incorporating Th1 driving adjuvants. Furthermore, these data suggest novel mechanisms whereby parvovirus B19 infection may contribute to rheumatoid arthritis and unsuccessful pregnancy.

Protection against Bordetella pertussis in mice in the absence of detectable circulating antibody: implications for long-term immunity in children.

Most vaccines used for humans work through humoral immunity, yet many appear to be protective even after specific circulating antibody levels have waned to undetectable levels. Furthermore, it has been difficult to define a serologic correlate of protection against a number of infectious diseases, including those caused by Bordetella pertussis. B. pertussis clearance in immunized mice has been shown to correlate with pertussis vaccine efficacy in children. This murine respiratory challenge model was used to demonstrate persistent vaccine-induced protection against B. pertussis in the absence of circulating antibody at the time of challenge. Whole-cell and acellular pertussis vaccines induced persistent memory T and B cells and anamnestic antibody responses after challenge. The findings suggest that immunologic memory is more significant in protection than is the induction of immediate antibody responses and imply that vaccinated children still may be protected against disease following the disappearance of specific serum IgG.

Interferon-gamma mediated immune effector mechanisms against Bordetella pertussis.

The role of IFN-gamma in reducing the intracellular load of Bordetella pertussis in murine macrophages in vitro has been examined. The results demonstrate that exposure to IFN-gamma can reduce bacterial load in viable macrophages and that this is associated with production of nitric oxide (NO). These observations provide a mechanism by which IFN-gamma may mediate its antimicrobial effect and support an important role for activated alveolar macrophages in the elimination of B. pertussis from the respiratory tract. Using intracellular iron chelation, it is shown that intracellular survival of B. pertussis is dependent on iron availability and suggest that iron restriction may be an important mechanism by which IFN-gamma influences bacterial survival within mouse macrophages. It is also shown that IFN-gamma may mediate its effect through NO independent mechanisms and that B. pertussis is sensitive to agents that stimulate the respiratory burst. Finally, it is shown that the concentration of L-tryptophan may be a limiting step in the intracellular survival of B. pertussis and that the induction of tryptophan degrading enzymes may be an additional mechanism through which IFN-gamma exerts its antimicrobial effects against B. pertussis.

A respiratory challenge model for infection with Bordetella pertussis: application in the assessment of pertussis vaccine potency and in defining the mechanism of protective immunity.

The evaluation of vaccines for human use usually requires the development of appropriate animal models and the definition of laboratory correlates of immunity. Traditionally whole cell pertussis vaccines have been controlled by using an active mouse protection test, which measures protection following intracerebral challenge with Bordetella pertussis. However, this test is unsuitable for assessing the potency of the new generation acellular pertussis vaccines. In the present study we demonstrate that a murine respiratory challenge model for infection with B. pertussis is suitable for assessing the potency of acellular and whole cell pertussis vaccines. To allow standardization of different vaccines we have expressed the area under the clearance curve for immunized mice as a ratio of that for non-immunized controls to obtain a potency index. A comparison of estimated vaccine efficacy in children with potency in the murine model results in a highly significant correlation (r = 0.976, p < 0.001). Furthermore, we have used this model to define the protective mechanism of immunity against respiratory infection with B. pertussis and demonstrate a requirement for both specific T and B cells. In accordance with studies in humans, no clear relationship was observed between monotypic serum antibody responses against the putative protective antigens of B. pertussis and protection. In contrast, the most potent protection was observed when the T cell response is polarized to the Th1 subtype.

Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the co-stimulatory molecules B7-1, B7-2 and CD28.

Pertussis toxin (PT) is a major virulence factor of Bordetella pertussis which exerts a range of effects on the immune system, including the enhancement of IgE, IgA and IgG production, delayed-type hypersensitivity reactions, and the induction of experimental autoimmune diseases. However, the mechanism by which PT mediates adjuvanticity remains to be defined. In this investigation we have shown that PT can potentiate antigen-specific T cell proliferation and the secretion of IFN-gamma, IL-2, IL-4 and IL-5 when injected with foreign antigens. A chemically detoxified PT and a genetic mutant with substitutions/deletions in the S-1 and B oligomer components that abrogate enzymatic and binding activity displayed no adjuvant properties. In contrast, a non-toxic S-1 mutant devoid of enzymatic activity but still capable of receptor binding retained its adjuvanticity, augmenting the activation of both Th1 and Th2 subpopulations of T cells. In an attempt to address the mechanism of T cell activation, we found that PT stimulated the production of IFN-gamma and IL-2 by naive T cells and IL-1 by macrophages. Therefore potentiation of distinct T cell subpopulations may have resulted in part from the positive influence of IFN-gamma on the development of Th1 cells and the co-stimulatory role of IL-1 for Th2 cells. Furthermore, PT augmented expression of the co-stimulatory molecules B7-1 and B7-2 on macrophages and B cells, and CD28 on T cells, suggesting that the adjuvant effect may also be associated with facilitation of the second signal required for maximal T cell activation. This study demonstrates that the immunopotentiating properties of PT are largely independent of ADP-ribosyltransferase activity, but are dependent on receptor binding activity and appear to involve enhanced activation of T cells.