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1.
Arch Razi Inst ; 74(1): 59-67, 2019 03.
Article in English | MEDLINE | ID: mdl-31013007

ABSTRACT

Whole-cell pertussis vaccine (wP) has been imperative and highly effective in preventing childhood deaths due to pertussis. Pertussis toxin is one of the virulence factors of Bordetella pertussis in all available pertussis vaccines. wP production in Razi Vaccine and Serum Research Institute is according to bioreactor culture of B. pertussis strains in B2 medium. The aim of this study was to evaluate B. pertussis strain 509 PT production in B2 and Thalen-IJssel (THIJS) media by Chinese Hamster Ovary (CHO) cell and enzyme-linked immunosorbent assay methods (ELISA). In the current study, B. pertussis strain 509 was cultured in B2 and THIJS media. Six samples were taken during the log growth phase within 2-3 h intervals (triplicate). The growth rate was calculated using opacity and the quantification of cell-associated and released PT measured by ELISA and CHO cell assays. THIJS medium was significantly showed an increase in the bacterial growth rate. During the first 29 h, bacterial concentrations in B2 and THIJS culture medium were 19 and 29 IOU, respectively. In THIJS medium, greater amount of pertussistoxin production was cell-associated. In B2 medium, maximum cell-associated toxin by ELISA and CHO cell assays were in the ODs of 1.1 and 0.9 and for THIJS medium in the ODs of 1.6 and 1.1, respectively. B. pertussis strain 509 in THIJS medium produced higher cell mass and cell-associated pertussis toxin than that of B2. It can be used for the production of whole-cell vaccine with higher pertussis toxin and accordingly using lower biomass per dose leading to the reduction of vaccine toxicity.


Subject(s)
Bordetella pertussis/physiology , Pertussis Toxin/physiology , Pertussis Vaccine/chemistry , Animals , CHO Cells , Cricetulus , Culture Media , Enzyme-Linked Immunosorbent Assay
2.
Expert Rev Vaccines ; 13(10): 1191-204, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25183193

ABSTRACT

Pertussis toxin (PT) is one of the major virulence factors of Bordetella pertussis and the primary component of all pertussis vaccines available to date. Because of its various noxious effects the toxin needs to be detoxified. In all currently available vaccines, detoxification is achieved by treatment with high quantity of chemical agents such as formaldehyde, glutaraldehyde or hydrogen peroxide. Although effective in detoxification, this chemical treatment alters dramatically the immunological properties of the toxin. In contrast, PT genetically detoxified through the substitution of two residues necessary for its enzymatic activity maintains all functional and immunological properties. This review describes in detail the characteristics of this PT-9K/129G mutant and shows that it is non-toxic and a superior immunogen compared with chemically detoxified PT. Importantly, data from an efficacy trial show that the PT-9K/129G-based vaccine induces earlier and longer-lasting protection, further supporting the hypothesis that PT-9K/129G represents an ideal candidate for future pertussis vaccine formulations.


Subject(s)
Pertussis Toxin/genetics , Pertussis Toxin/immunology , Pertussis Vaccine/genetics , Pertussis Vaccine/immunology , Animals , Bordetella pertussis/enzymology , Bordetella pertussis/genetics , Bordetella pertussis/immunology , Humans , Models, Molecular , Pertussis Toxin/chemistry , Pertussis Toxin/physiology , Pertussis Vaccine/chemistry , Pertussis Vaccine/metabolism
3.
J Immunol ; 186(7): 4347-53, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21368227

ABSTRACT

Sphingosylphosphorylcholine (SPC) is a component of high-density lipoprotein particles. We investigated the functional role of SPC in HUVECs. SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein-dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. Inhibition of p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation, but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3-, NF-κB-, and AP-1-mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Our results suggest that SPC may contribute to atherosclerosis; therefore, SPC and its unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.


Subject(s)
Chemokine CCL2/biosynthesis , Endothelium, Vascular/immunology , Endothelium, Vascular/metabolism , Phosphorylcholine/analogs & derivatives , Sphingosine/analogs & derivatives , Umbilical Veins/immunology , Umbilical Veins/metabolism , Cells, Cultured , Endothelium, Vascular/cytology , Humans , Membrane Lipids/physiology , Pertussis Toxin/physiology , Phosphorylcholine/pharmacology , Signal Transduction/immunology , Sphingosine/pharmacology , Umbilical Veins/cytology
4.
J Immunol ; 186(6): 3294-8, 2011 Mar 15.
Article in English | MEDLINE | ID: mdl-21325623

ABSTRACT

Mast cells (MCs) exert a significant pathologic influence on disease severity in C57BL/6 (B6) strain-dependent experimental allergic encephalomyelitis (EAE), a model of primary progressive multiple sclerosis (MS). However, relapsing-remitting MS, which is modeled in SJL mice, is the more prevalent form. Given genetically determined heterogeneity in numbers and responsiveness of MCs from various strains of mice, we asked whether these cells also influence this more clinically relevant MS model using SJL-Kit(W/W-v) mice. Similar to the commercially available WBB6F(1)-Kit(W/W-v) mice, SJL-Kit(W/W-v) mice are MC-deficient, anemic, and neutropenic and have normal T cell compartments. They exhibit significantly reduced disease severity, but retain the relapsing-remitting course, a phenotype reversed by selective MC reconstitution. These data confirm that MC influence is not confined to an isolated model of EAE and reveal a new system to study the effects of MC heterogeneity on relapsing-remitting EAE and other SJL strain-specific diseases.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Mast Cells/immunology , Mast Cells/pathology , Multiple Sclerosis, Relapsing-Remitting/immunology , Multiple Sclerosis, Relapsing-Remitting/pathology , Severity of Illness Index , Animals , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Cells, Cultured , Crosses, Genetic , Disease Models, Animal , Encephalomyelitis, Autoimmune, Experimental/genetics , Immunophenotyping , Incidence , Mast Cells/transplantation , Mice , Mice, Inbred C57BL , Mice, Knockout , Multiple Sclerosis, Relapsing-Remitting/genetics , Pertussis Toxin/physiology , Proto-Oncogene Proteins c-kit/genetics , Species Specificity
5.
J Immunol ; 182(9): 5730-9, 2009 May 01.
Article in English | MEDLINE | ID: mdl-19380820

ABSTRACT

Pertussis toxin (PTx) has been shown to exert a variety of effects on immune cells independent of its ability to ADP-ribosylate G proteins. Of these effects, the binding subunit of PTx (PTxB) has been shown to block signaling via the chemokine receptor CCR5, but the mechanism involved in this process is unknown. Here, we show that PTxB causes desensitization of a related chemokine receptor, CXCR4, and explore the mechanism by which this occurs. CXCR4 is the receptor for the chemokine stromal cell-derived factor 1alpha (SDF-1alpha) and elicits a number of biological effects, including stimulation of T cell migration. PTxB treatment causes a decrease in CXCR4 surface expression, inhibits G protein-associated signaling, and blocks SDF-1alpha-mediated chemotaxis. We show that PTxB mediates these effects by activating the TCR signaling network, as the effects are dependent on TCR and ZAP70 expression. Additionally, the activation of the TCR with anti-CD3 mAb elicits a similar set of effects on CXCR4 activity, supporting the idea that TCR signaling leads to cross-desensitization of CXCR4. The inhibition of CXCR4 by PTxB is rapid and transient; however, the catalytic activity of PTx prevents CXCR4 signaling in the long term. Thus, the effects of PTx holotoxin on CXCR4 signaling can be divided into two phases: short term by the B subunit, and long term by the catalytic subunit. These data suggest that TCR crosstalk with CXCR4 is likely a normal cellular process that leads to cross-desensitization, which is exploited by the B subunit of PTx.


Subject(s)
Down-Regulation/immunology , Pertussis Toxin/physiology , Receptor Cross-Talk/immunology , Receptors, Antigen, T-Cell/physiology , Receptors, CXCR4/antagonists & inhibitors , Signal Transduction/immunology , Animals , Bordetella pertussis/immunology , Bordetella pertussis/pathogenicity , CHO Cells , Catalytic Domain/immunology , Cells, Cultured , Cricetinae , Cricetulus , Desensitization, Immunologic , Humans , Jurkat Cells , Receptors, Antigen, T-Cell/deficiency , Receptors, Antigen, T-Cell/genetics , Receptors, CXCR4/physiology
6.
Immunology ; 128(1 Suppl): e487-96, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19191907

ABSTRACT

We have recently reported that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in human neutrophils. Here, we report that a similar regulatory system is also functioning in human monocytes, but not lymphocytes. Calpain was constitutively active in resting human monocytes, but not lymphocytes. Mitogen-activated protein kinases, including extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI3K)/Akt and p21-activated kinase (PAK, an effector molecule of Rac) were rapidly (within 1 min) activated in monocytes, but not lymphocytes, upon exposure to calpain inhibitors (PD150606 and N-acetyl-Leu-Leu-Nle-CHO), but not PD145305 (the inactive analogue of PD150606). Following activation of these signalling pathways, monocytes displayed active migration within 5 min after exposure to calpain inhibitors, and active migration was sustained for more than 45 min. The micropipette method revealed that calpain inhibition-mediated monocyte migration was chemotaxis, not random migration. The studies with pharmacological inhibitors suggest that calpain inhibition-mediated monocyte migration is mediated by activation of ERK, p38, JNK, PI3K/Akt and Rac. NSC23766 (Rac inhibitor) and pertussis toxin (PTX) suppressed calpain inhibitor-induced phosphorylation of distinct signalling molecules (PAK, ERK, p38, JNK and Akt) as well as cell migration, suggesting that the PTX-sensitive G protein and Rac axis may be a possible key target of calpain inhibitors. These findings suggest that constitutively active calpain negatively regulates activation of the distinct signalling pathways and cell migration in resting monocytes, but not lymphocytes.


Subject(s)
Calpain/physiology , Chemotaxis/immunology , Monocytes/physiology , Acrylates/pharmacology , Aminoquinolines/pharmacology , Calpain/antagonists & inhibitors , Humans , Lymphocytes/drug effects , Lymphocytes/physiology , Monocytes/drug effects , Neutrophils/drug effects , Neutrophils/physiology , Pertussis Toxin/physiology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Protein Kinases/physiology , Pyrimidines/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , rac GTP-Binding Proteins/antagonists & inhibitors , rac GTP-Binding Proteins/physiology
7.
Cytokine ; 40(1): 35-43, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17881243

ABSTRACT

Experimental autoimmune encephalomyelitis (EAE) is commonly regarded as an animal model of the human disease multiple sclerosis (MS). Pertussis toxin (PTX) is routinely used for EAE induction in mice. Besides opening the blood-brain barrier, it acts as an adjuvant causing strong expansion of antigen-specific cells after coinjection with neuroantigens in IFA. Using an IL-17 ELISPOT assay we developed previously, we investigated the capability of PTX to induce proteolipid protein peptide 139-151(PLPp)-specific Th-17 cells in the immune periphery and in the thymus after coinjection with PLPp/IFA. PTX was found to induce peripheral PLPp-specific Th-17 cells in the draining lymph node and in the spleen, but not in the thymus. Our study indicates a new mechanism by which microbial agents can initiate or maintain autoimmune reactions and supports the growing role in particular for Th-17 cells in organ-specific autoimmune diseases like multiple sclerosis or EAE.


Subject(s)
Interleukin-17/biosynthesis , Myelin Proteolipid Protein/immunology , Peptide Fragments/immunology , Pertussis Toxin/physiology , T-Lymphocyte Subsets/immunology , Animals , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Isoflavones/immunology , Lymphocyte Count , Mice , Mice, Inbred Strains , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , beta-Glucans/immunology
8.
J Immunol ; 178(10): 6123-9, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17475838

ABSTRACT

Compelling evidence has now demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to autoimmune pathogenesis, whereas CD4+CD25+ T regulatory cells (Treg) play a major role in suppression of autoimmunity. Differentiation of proinflammatory Th17 and immunosuppressive Treg from naive CD4 cells is reciprocally related and contingent upon the cytokine environment. We and others have reported that in vivo administration of pertussis toxin (PTx) reduces the number and function of mouse Treg. In this study, we have shown that supernatants from PTx-treated mouse splenic cells, which contained IL-6 and other proinflammatory cytokines, but not PTx itself, overcame the inhibition of proliferation seen in cocultures of Treg and CD4+CD25- T effector cells. This stimulatory effect could be mimicked by individual inflammatory cytokines such as IL-1beta, IL-6, and TNF-alpha. The combination of these cytokines synergistically stimulated the proliferation of CD4+CD25- T effector cells despite the presence of Treg with a concomitant reduction in the percentage of FoxP3+ cells and generation of IL-17-expressing cells. PTx generated Th17 cells, while inhibiting the differentiation of FoxP+ cells, from naive CD4 cells when cocultured with bone marrow-derived dendritic cells from wild-type mice, but not from IL-6-/- mice. In vivo treatment with PTx induced IL-17-secreting cells in wild-type mice, but not in IL-6-/- mice. Thus, in addition to inhibiting the development of Treg, the immunoadjuvant activity of PTx can be attributable to the generation of IL-6-dependent IL-17-producing CD4 cells.


Subject(s)
CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Interleukin-17/biosynthesis , Interleukin-6/biosynthesis , Pertussis Toxin/physiology , Animals , CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation , Cell-Free System/immunology , Cells, Cultured , Coculture Techniques , Female , Gene Expression Regulation/immunology , Immunosuppression Therapy , Interleukin-17/metabolism , Interleukin-6/deficiency , Interleukin-6/genetics , Interleukin-6/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism
9.
J Immunol ; 176(12): 7412-20, 2006 Jun 15.
Article in English | MEDLINE | ID: mdl-16751386

ABSTRACT

The membrane-anchored form of CX3CL1 has been proposed as a novel adhesion protein for leukocytes. This functional property of CX3CL1 is mediated through CX3CR1, a chemokine receptor expressed predominantly on circulating white blood cells. Thus far, it is still uncertain at what stage of the trafficking process CX3CR1 becomes importantly involved and how the CX3CR1-dependent adhesion of leukocytes is regulated during inflammation. The objective of this study was to examine the functional effects of chemokine stimulation on CX3CR1-mediated adhesion of human monocytes. Consistent with previous reports, our data indicate that the activity of CX3CR1 on resting monocytes is sufficient to mediate cell adhesion to CX3CL1. However, the basal, nonstimulated adhesion activity is low, and we hypothesized that like the integrins, CX3CR1 may require a preceding activation step to trigger firm leukocyte adhesion. Compatible with this hypothesis, stimulation of monocytes with MCP-1 significantly increased their adhesion to immobilized CX3CL1, under both static and physiological flow conditions. The increase of the adhesion activity was mediated through CCR2-dependent signaling and obligatory activation of the p38 MAPK pathway. Stimulation with MCP-1 also induced a rapid increase of CX3CR1 protein on the cell surface. Inhibition of the p38 MAPK pathway prevented this increase of CX3CR1 surface expression and blunted the effect of MCP-1 on cell adhesion, indicating a causal link between receptor surface density and adhesion activity. Together, our data suggest that a chemokine signal is required for firm CX3CR1-dependent adhesion and demonstrate that CCR2 is an important regulator of CX3CL1-dependent leukocyte adhesion.


Subject(s)
Chemokine CCL2/physiology , Chemokines, CX3C/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Monocytes/enzymology , Monocytes/immunology , Receptors, Chemokine/biosynthesis , p38 Mitogen-Activated Protein Kinases/physiology , Animals , Antigen Presentation , CX3C Chemokine Receptor 1 , Cell Adhesion/drug effects , Cell Adhesion/immunology , Cell Line, Tumor , Chemokine CCL2/antagonists & inhibitors , Chemokine CX3CL1 , Chemokines, CX3C/antagonists & inhibitors , Chemokines, CX3C/physiology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/physiology , Mice , Monocytes/metabolism , Pertussis Toxin/physiology , Protein Kinase Inhibitors/pharmacology , Receptors, CCR2 , Receptors, Chemokine/deficiency , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Receptors, Chemokine/physiology , Signal Transduction/genetics , Signal Transduction/immunology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
10.
Curr Opin Microbiol ; 9(2): 207-17, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16529981

ABSTRACT

Type IV secretion systems (T4SSs) are membrane-associated transporter complexes used by various bacteria to deliver substrate molecules to a wide range of target cells. T4SSs are involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in DNA uptake from or release into the extracellular milieu, in toxin secretion and in the injection of virulence factors into eukaryotic host target cells by several mammalian pathogens. Rapid progress has been made towards defining the structures and functions of T4SSs, identifying the translocated effector molecules and elucidating the mechanisms by which the effectors subvert eukaryotic cellular processes during infection. These findings have had an important impact on our understanding of how these pathogens manipulate host cell functions to trigger bacterial uptake, facilitate intracellular growth and suppress defence mechanisms, thus facilitating bacterial colonization and disease development.


Subject(s)
Bacteria/chemistry , Bacteria/pathogenicity , Bacterial Proteins/physiology , Animals , Antigens, Bacterial/metabolism , Antigens, Bacterial/physiology , Bacteria/growth & development , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Toxins/metabolism , Bartonella henselae/chemistry , Bartonella henselae/pathogenicity , Biological Transport , Bordetella pertussis/enzymology , Bordetella pertussis/pathogenicity , DNA, Bacterial/metabolism , Eukaryotic Cells/metabolism , Eukaryotic Cells/microbiology , Helicobacter pylori/chemistry , Helicobacter pylori/metabolism , Helicobacter pylori/pathogenicity , Humans , Membrane Proteins/metabolism , Pertussis Toxin/chemistry , Pertussis Toxin/metabolism , Pertussis Toxin/physiology , Phagosomes/metabolism , Phagosomes/microbiology , Rhizobium/chemistry , Rhizobium/metabolism , Rhizobium/pathogenicity , Virulence , Virulence Factors/metabolism
11.
J Immunol ; 175(8): 5115-25, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-16210615

ABSTRACT

Developing thymocytes undergo maturation while migrating through the thymus and ultimately emigrate from the organ to populate peripheral lymphoid tissues. The process of thymic emigration is controlled in part via receptor-ligand interactions between the chemokine stromal-derived factor (SDF)-1, and its cognate receptor CXCR4, and sphingosine 1-phosphate (S1P) and its receptor S1PR. The precise mechanism by which S1P/S1PR and CXCR4/SDF-1 contribute to thymic emigration remains unclear. We proposed that S1P-dependent and -independent mechanisms might coexist and involve both S1P-induced chemoattraction and SDF-1-mediated chemorepulsion or fugetaxis of mature thymocytes. We examined thymocyte emigration in thymi from CXCR4-deficient C57BL/6 embryos in a modified assay, which allows the collection of CD62L(high) and CD69(low) recent thymic emigrants. We demonstrated that single-positive (SP) CD4 thymocytes, with the characteristics of recent thymic emigrants, failed to move away from CXCR4-deficient fetal thymus in vitro. We found that the defect in SP CD4 cell emigration that occurred in the absence of CXCR4 signaling was only partially overcome by the addition of the extrathymic chemoattractant S1P and was not associated with abnormalities in thymocyte maturation and proliferative capacity or integrin expression. Blockade of the CXCR4 receptor in normal thymocytes by AMD3100 led to the retention of mature T cells in the thymus in vitro and in vivo. The addition of extrathymic SDF-1 inhibited emigration of wild-type SP cells out of the thymus by nullifying the chemokine gradient. SDF-1 was also shown to elicit a CXCR4-dependent chemorepellent response from fetal SP thymocytes. These novel findings support the thesis that the CXCR4-mediated chemorepellent activity of intrathymic SDF-1 contributes to SP thymocyte egress from the fetal thymus.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Chemotaxis, Leukocyte/immunology , Receptors, CXCR4/physiology , Signal Transduction/physiology , Thymus Gland/cytology , Thymus Gland/immunology , Animals , Benzylamines , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Cell Proliferation , Chemokine CXCL12 , Chemokines, CXC/physiology , Chemotaxis, Leukocyte/drug effects , Cyclams , Fetus , Heterocyclic Compounds/pharmacology , Immunophenotyping , Integrins/biosynthesis , Integrins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Organ Culture Techniques , Pertussis Toxin/physiology , Signal Transduction/immunology , Thymus Gland/metabolism
12.
Expert Rev Vaccines ; 3(1): 89-101, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14761246

ABSTRACT

Current methods for determining the potency and toxicity of pertussis vaccines are outdated and require improvement. The intracerebral challenge test is effective for determining the potency of whole-cell vaccines but is objectionable on animal welfare and technical grounds. The same applies to its modification for assaying acellular pertussis vaccines. Respiratory challenge methods offer an interim solution pending establishment of validated in vitro correlates of protection, for example nitric oxide induction. Their evaluation is being promoted by the World Health Organization through the Pertussis Vaccines Working Group. Current toxicity assays based on weight gain and histamine sensitization of mice are imprecise and need replacement. Limits need to be established for specific toxin content of both acellular and whole-cell vaccines and should be supported by specific assays. More precise methods based on determination of ribosyltransferase activity in tandem with receptor-binding assays are under evaluation. Genome sequence data and the use of gene microarrays to screen responses triggered by vaccine components may also provide leads to improved methods for assessing both toxicity and immunogenicity.


Subject(s)
Pertussis Vaccine/immunology , Pertussis Vaccine/toxicity , Adhesins, Bacterial/physiology , Animals , Bacterial Toxins , Bordetella pertussis/immunology , Bordetella pertussis/pathogenicity , Fimbriae, Bacterial/physiology , Hemagglutinins/physiology , Histamine/immunology , Lipopolysaccharides/metabolism , Pertussis Toxin/physiology , Toxicity Tests , Transglutaminases/physiology , Virulence Factors, Bordetella
13.
Infect Immun ; 71(11): 6358-66, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14573656

ABSTRACT

In this study, we sought to determine whether pertussis toxin (PT), an exotoxin virulence factor produced exclusively by Bordetella pertussis, is important for colonization of the respiratory tract by this pathogen by using a mouse intranasal infection model. By comparing a wild-type Tohama I strain to a mutant strain with an in-frame deletion of the ptx genes encoding PT (deltaPT), we found that the lack of PT confers a significant peak (day 7) colonization defect (1 to 2 log(10) units) over a range of bacterial inoculum doses and that this defect was apparent within 1 to 2 days postinoculation. In mixed-strain infection experiments, the deltaPT strain showed no competitive disadvantage versus the wild-type strain and colonized at higher levels than in the single-strain infection experiments. To test the hypothesis that soluble PT produced by the wild-type strain in mixed infections enhanced respiratory tract colonization by deltaPT, we coadministered purified PT with the deltaPT inoculum and found that colonization was increased to wild-type levels. This effect was not observed when PT was coadministered via a systemic route. Intranasal administration of purified PT up to 14 days prior to inoculation with deltaPT significantly increased bacterial colonization, but PT administration 1 day after bacterial inoculation did not enhance colonization versus a phosphate-buffered saline control. Analysis of bronchoalveolar lavage fluid samples from mice infected with either wild-type or deltaPT strains at early times after infection revealed that neutrophil influx to the lungs 48 h postinfection was significantly greater in response to deltaPT infection, implicating neutrophil chemotaxis as a possible target of PT activity promoting B. pertussis colonization of the respiratory tract.


Subject(s)
Bordetella pertussis/pathogenicity , Pertussis Toxin/physiology , Respiratory System/microbiology , Animals , Chemotaxis, Leukocyte , Female , Immunity, Innate , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Neutrophils/immunology , Neutrophils/physiology , Pertussis Toxin/administration & dosage
14.
J Immunol ; 170(4): 1728-36, 2003 Feb 15.
Article in English | MEDLINE | ID: mdl-12574336

ABSTRACT

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.


Subject(s)
Adjuvants, Immunologic/pharmacology , Dendritic Cells/immunology , Pertussis Toxin/pharmacology , Th1 Cells/immunology , Th1 Cells/metabolism , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/physiology , Animals , Cell Differentiation/immunology , Cell Movement/immunology , Cells, Cultured , Coculture Techniques , Dendritic Cells/cytology , Dendritic Cells/enzymology , Dendritic Cells/transplantation , Eye Proteins/administration & dosage , Eye Proteins/immunology , Female , Injections, Intraperitoneal , Injections, Subcutaneous , Interferon-gamma/biosynthesis , Interleukin-12/blood , Interleukin-12/metabolism , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocyte Culture Test, Mixed , MAP Kinase Signaling System/immunology , Mice , Mice, Inbred A , Mice, Inbred BALB C , Mice, Inbred C57BL , Pertussis Toxin/administration & dosage , Pertussis Toxin/physiology , Retinol-Binding Proteins/administration & dosage , Retinol-Binding Proteins/immunology , Th1 Cells/enzymology , Uveitis/enzymology , Uveitis/etiology , Uveitis/immunology , Uveitis/pathology
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