PURPOSE: Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice. MATERIALS AND METHODS: Bordetella pertussis Tohama phase I was cultured for 24–30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250–1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP). RESULTS: Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively. CONCLUSION: The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.
Animals ; Antibodies ; Bordetella pertussis ; Chromatography ; Diagnosis ; Enzyme-Linked Immunosorbent Assay* ; Horseradish Peroxidase ; Methods ; Mice* ; Models, Animal ; Pertussis Toxin ; Streptavidin ; Vaccines ; Whooping Cough

PURPOSE: There is no standard method for confirming the immunogenicity of acellular pertussis vaccines. We tried to develop a local standard method for evaluating the immunogenicity of the three-component of acellular pertussis vaccines which was developed by a Korean local company. MATERIALS AND METHODS: The developed pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin) were evaluated by in-house enzyme-linked immunosorbent assay (ELISA) using 189 negative sera, 25 positive sera, and 73 paired sera (pre- and post-Tdap [tetanus, diphtheria, and acellular pertussis] vaccinated sera). ELISA units were calculated by the reference line method, compared with World Health Organization reference sera, and the cut-off value was calculated using negative sera. RESULTS: When compared to National Institute for Biological Standards and Control control antigen (NIBSC) control antigens, the developed pertussis toxin (PT) and filamentous haemagglutinin (FHA) antigens were 203.48 and 61.60 IU/µg, respectively. Each in-house ELISA was established by validating the coefficients of variation % (PT, 11.53%; FHA, 8.60%; pertactin [PRN], 9.86%) obtained from the results of inter- and intra-assay variation. Also, the cut-off values of PT, FHA, and PRN were 11.65, 38.95, and 5.66 EU/mL, respectively. The distributions of antibody levels in paired showed that 93.15% (68/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were higher than a 100% increase after vaccination. Additionally, the values of 89.04% (65/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were below each cut-off point. CONCLUSION: We established an in-house ELISA method using self-developed antigens, and these immunoassays have provided a way to standardize measuring the immunogenicity of newly developed vaccines, through single- and dual-serology.
Diphtheria ; Enzyme-Linked Immunosorbent Assay ; Hemagglutinins ; Immunoassay ; Immunoglobulin G ; Korea* ; Methods* ; Pertussis Toxin ; Pertussis Vaccine ; Vaccination ; Vaccines* ; Whooping Cough* ; World Health Organization

A comprehensive collection of proteins senses local changes in intracellular Ca²⁺ concentrations ([Ca²⁺](i) and transduces these signals into responses to agonists. In the present study, we examined the effect of sphingosine-1-phosphate (S1P) on modulation of intracellular Ca²⁺ concentrations in cat esophageal smooth muscle cells. To measure [Ca²⁺](i) levels in cat esophageal smooth muscle cells, we used a fluorescence microscopy with the Fura-2 loading method. S1P produced a concentration-dependent increase in [Ca²⁺](i) in the cells. Pretreatment with EGTA, an extracellular Ca²⁺ chelator, decreased the S1P-induced increase in [Ca²⁺](i), and an L-type Ca²⁺-channel blocker, nimodipine, decreased the effect of S1P. This indicates that Ca²⁺ influx may be required for muscle contraction by S1P. When stimulated with thapsigargin, an intracellular calcium chelator, or 2-Aminoethoxydiphenyl borate (2-APB), an InsP₃ receptor blocker, the S1P-evoked increase in [Ca²⁺](i) was significantly decreased. Treatment with pertussis toxin (PTX), an inhibitor of G(i)-protein, suppressed the increase in [Ca²⁺](i) evoked by S1P. These results suggest that the S1P-induced increase in [Ca²⁺](i) in cat esophageal smooth muscle cells occurs upon the activation of phospholipase C and subsequent release of Ca²⁺ from the InsP₃-sensitive Ca²⁺ pool in the sarcoplasmic reticulum. These results suggest that S1P utilized extracellular Ca²⁺ via the L type Ca²⁺ channel, which was dependent on activation of the S1P₄ receptor coupled to PTX-sensitive G(i) protein, via phospholipase C-mediated Ca²⁺ release from the InsP₃-sensitive Ca²⁺ pool in cat esophageal smooth muscle cells.
Animals ; Calcium ; Cats* ; Egtazic Acid ; Fura-2 ; Methods ; Microscopy, Fluorescence ; Muscle Contraction ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Nimodipine ; Pertussis Toxin ; Phospholipases ; Sarcoplasmic Reticulum ; Thapsigargin ; Type C Phospholipases

The effect of clonidine administered intrathecally (i.t.) on the mortality and the blood glucose level induced by sepsis was examined in mice. To produce sepsis, the mixture of D-galactosamine (GaLN; 0.6 g/10 ml)/lipopolysaccharide (LPS; 27 µg/27 µl) was treated intraperitoneally (i.p.). The i.t. pretreatment with clonidine (5 µg/5 µl) increased the blood glucose level and attenuated mortality induced by sepsis in a dose-dependent manner. The i.t. post-treatment with clonidine up to 3 h caused an elevation of the blood glucose level and protected sepsis-induced mortality, whereas clonidine post-treated at 6, 9, or 12 h did not affect. The pre-treatment with oral D-glucose for 30 min prior to i.t. post-treatment (6 h) with clonidine did not rescue sepsis-induced mortality. In addition, i.t. pretreatment with pertussis toxin (PTX) reduced clonidine-induced protection against mortality and clonidine-induced hyperglycemia, suggesting that protective effect against sepsis-induced mortality seems to be mediated via activating PTX-sensitive G-proteins in the spinal cord. Moreover, pretreatment with clonidine attenuated the plasma tumor necrosis factor α (TNF-α) induced by sepsis. Clonidine administered i.t. or i.p. increased p-AMPKα1 and p-AMPKα2, but decreased p-Tyk2 and p-mTOR levels in both control and sepsis groups, suggesting that the up-regulations of p-AMPKα1 and p-AMPKα2, or down-regulations of p-mTOR and p-Tyk2 may play critical roles for the protective effect of clonidine against sepsis-induced mortality.
Animals ; Blood Glucose ; Clonidine ; Glucose ; GTP-Binding Proteins ; Hyperglycemia ; Mice ; Mortality* ; Pertussis Toxin ; Plasma ; Sepsis ; Spinal Cord* ; Tumor Necrosis Factor-alpha

Sepsis is the life-threatening response to infection which can lead to tissue damage, organ failure, and death. In the current study, the effect of orally administered D-glucose on the mortality and the blood glucose level induced by D-Galactosamine (GaLN)/lipopolysaccharide (LPS)-induced sepsis was examined in ICR mice. After various amounts of D-glucose (from 1 to 8 g/kg) were orally fed, sepsis was induced by injecting intraperitoneally (i.p.) the mixture of GaLN /LPS. Oral pre-treatment with D-glucose dose-dependently increased the blood glucose level and caused a reduction of sepsis-induced mortality. The oral post-treatment with D-glucose (8 g/kg) up to 3 h caused an elevation of the blood glucose level and protected the mortality observed in sepsis model. However, D-glucose post-treated at 6, 9, or 12 h after sepsis induction did not affect the mortality and the blood glucose level induced by sepsis. Furthermore, the intrathecal (i.t.) pretreatment once with pertussis toxin (PTX; 0.1 microg/5 ml) for 6 days caused a reduction of D-glucose-induced protection of mortality and hyperglycemia. Furthermore, once the hypoglycemic state is continued up to 6 h after sepsis initiated, sepsis-induced mortality could not be reversed by D-glucose fed orally. Based on these findings, it is assumed that the hypoglycemic duration between 3 and 6 h after the sepsis induction may be a critical time of period for the survival. D-glucose-induced protective effect against sepsis-induced mortality appears to be mediated via activating PTX-sensitive G-proteins in the spinal cord. Finally, the production of hyperglycemic state may be critical for the survival against the sepsis-induced mortality.
Animals ; Blood Glucose ; Glucose* ; GTP-Binding Proteins ; Hyperglycemia ; Mice ; Mice, Inbred ICR ; Mortality* ; Pertussis Toxin ; Sepsis* ; Spinal Cord

In the present study, we examined the effect of pertussis toxin (PTX) administered centrally in a variety of stress-induced blood glucose level. Mice were exposed to stress after the pretreatment of PTX (0.05 or 0.1 µg) i.c.v. or i.t. once for 6 days. Blood glucose level was measured at 0, 30, 60 and 120 min after stress stimulation. The blood glucose level was increased in all stress groups. The blood glucose level reached at maximum level after 30 min of stress stimulation and returned to a normal level after 2 h of stress stimulation in restraint stress, physical, and emotional stress groups. The blood glucose level induced by cold-water swimming stress was gradually increased up to 1 h and returned to the normal level. The intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with PTX, a Gi inhibitor, alone produced a hypoglycemia and almost abolished the elevation of the blood level induced by stress stimulation. The central pretreatment with PTX caused a reduction of plasma insulin level, whereas plasma corticosterone level was further up-regulated in all stress models. Our results suggest that the hyperglycemia produced by physical stress, emotional stress, restraint stress, and the cold-water swimming stress appear to be mediated by activation of centrally located PTX-sensitive G proteins. The reduction of blood glucose level by PTX appears to due to the reduction of plasma insulin level. The reduction of blood glucose level by PTX was accompanied by the reduction of plasma insulin level. Plasma corticosterone level up-regulation by PTX in stress models may be due to a blood glucose homeostatic mechanism.
Animals ; Blood Glucose* ; Corticosterone ; GTP-Binding Proteins ; Hyperglycemia ; Hypoglycemia ; Insulin ; Mice ; Pertussis Toxin* ; Plasma ; Stress, Psychological ; Swimming ; Up-Regulation ; Whooping Cough*

PURPOSE: Active reduced dose tetanus-diphtheria-acellular pertussis (Tdap) vaccination for adolescents and adults is necessary because waning immunity after primary diphtheria-tetanus-pertussis vaccination is related to the recent emergence of pertussis. This study was conducted to compare the immunogenicity and protection efficacy against Bordetella pertussis between a new GCC Tdap vaccine and a commercially available Tdap vaccine in a murine model. MATERIALS AND METHODS: BALB/c mice were immunized with two doses of diphtheria-tetanus-acellular pertussis (DTaP) vaccine for priming and a subsequent Tdap booster vaccination. According to the type of booster vaccine, mice were divided into four groups: commercially available Tdap vaccine in group 1 and GCC Tdap vaccines of different combinations of pertussis antigens in groups 2 to 4. Humoral and cell-mediated immune responses and protection efficacy using a murine intranasal challenge model after booster vaccination were compared among the four groups. RESULTS: Every group showed significant increases in antibody titers against pertussis antigens such as pertussis toxin, filamentous hemagglutinin, and pertactin after booster vaccination. Spleen cells showed both Th1 and Th2 cell-mediated immune responses stimulated by pertussis antigens in all groups without any significant difference. In the intranasal B. pertussis infection model, bacteria were eradicated in all groups five days after challenge infection. CONCLUSION: This preliminary study did not show significantly different immunogenicity or protection efficacy of the new GCC Tdap vaccines compared to the commercially available Tdap vaccine, although a more extensive study is necessary to assess the differing efficacies of the new GCC Tdap vaccines.
Adolescent ; Adult ; Animals ; Bacteria ; Bordetella pertussis* ; Hemagglutinins ; Humans ; Mice ; Pertussis Toxin ; Republic of Korea ; Spleen ; Vaccination ; Vaccines ; Whooping Cough

PURPOSE: To evaluate the transcription pattern of Nod-like receptors (NLRs), the intracellular sensors, to detect danger signals in murine eyes with experimental autoimmune uveitis (EAU). METHODS: EAU was induced in B6 (C57BL/6) mice by subcutaneous injection of human interphotoreceptor retinoid binding protein and intraperitoneal injection of pertussis toxin. At 1, 2, and 3 weeks post-immunization, the eyeballs were extracted and subjected to histological and molecular assays using real-time reverse transcription polymerase chain reaction. RESULTS: The levels of nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain 1 (NLRP1), NLRP3, nucleotide-binding oligomerization domain-containing protein 1 (NOD1), and NOD2 transcripts were increased at 2 weeks and gradually reduced thereafter. Notably, NLRP3 showed the highest expression in the eyes with EAU. Similarly, the transcript level of pro-inflammatory cytokine, interleukin-1beta, increased and reached a peak at 2 weeks post-immunization. The retinal structure was severely damaged by inflammation at 3 weeks post-immunization. CONCLUSIONS: Among NLRs, NLRP3 may induce inflammation in eyes after EAU immunization.
Animals ; Carrier Proteins ; Humans ; Immunization ; Inflammation ; Injections, Intraperitoneal ; Injections, Subcutaneous ; Interleukin-1beta ; Leucine ; Mice* ; Pertussis Toxin ; Polymerase Chain Reaction ; Retinaldehyde ; Reverse Transcription ; Uveitis*

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors/PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.
Humans ; Muscle, Smooth, Vascular* ; Myocytes, Smooth Muscle ; Pertussis Toxin ; Phenotype* ; Phosphorylation ; Protein Kinase C ; Proto-Oncogene Proteins c-raf ; Thrombin

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.
*Antigenic Variation ; Antigens/*genetics/immunology/metabolism ; Bacterial Proteins/genetics/metabolism ; Bordetella pertussis/*genetics/isolation & purification/*metabolism ; Genes, Bacterial ; Genotype ; Humans ; Pertussis Toxin/genetics/metabolism ; Promoter Regions, Genetic ; Republic of Korea ; Sequence Analysis, DNA ; Whooping Cough/immunology/*microbiology/pathology