Development and implementation of standardized method for detecting immunogenicity of acellular pertussis vaccines in Korea

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.

Enzyme-linked immunosorbent assay for detecting anti-pertussis toxin antibody in mouse

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.

Effect of Sphingosine-1-Phosphate on Intracellular Free Ca2+ in Cat Esophageal Smooth Muscle Cells

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.

The activation of α₂-adrenergic receptor in the spinal cord lowers sepsis-induced mortality

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.

Effect of pertussis toxin pretreated centrally on blood glucose level induced by stress

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.

Effect of D-glucose feeding on mortality induced by sepsis

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.

Transcriptional Analysis of Nod-Like Receptors in a Mouse Model of Experimental Autoimmune Uveitis

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.

Multiple Signaling Pathways Contribute to the Thrombin-induced Secretory Phenotype in Vascular Smooth Muscle Cells

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.

Preliminary study on the immunogenicity of a newly developed GCC Tdap vaccine and its protection efficacy against Bordetella pertussis in a murine intranasal challenge model

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.

Recent Trends of Antigenic Variation in Bordetella pertussis Isolates in Korea

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.

Pertussis Seroprevalence in Korean Adolescents and Adults Using Anti-Pertussis Toxin Immunoglobulin G

This study was conducted to evaluate age-specific seroprevalence of pertussis in Korea and to formulate a strategy to prevent and reduce the incidence of pertussis. Residual serum samples of healthy adolescents and adults 11 yr of age or older were collected between July 2012 and December 2012, and anti-pertussis toxin (PT) IgG titers were measured using a commercial ELISA kit. We compared the mean anti-PT IgG titers and seroprevalence of pertussis of the six age groups: 11-20, 21-30, 31-40, 41-50, 51-60, and > or = 61 yr. A total of 1,192 subjects were enrolled. The mean anti-PT IgG titer and pertussis seroprevalence were 35.53 +/- 62.91 EU/mL and 41.4%, respectively. The mean anti-PT IgG titers and seroprevalence were not significantly different between the age groups. However, the seroprevalence in individuals 51 yr of age or older was significantly higher than in individuals younger than 51 yr (46.5% vs 39.1%, P = 0.017). Based on these results, a new pertussis prevention strategy is necessary for older adults.

Wnt5a stimulates chemotactic migration and chemokine production in human neutrophils

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.

Signaling Pathway of Lysophosphatidic Acid-Induced Contraction in Feline Esophageal Smooth Muscle Cells

Lysolipids such as LPA, S1P and SPC have diverse biological activities including cell proliferation, differentiation, and migration. We investigated signaling pathways of LPA-induced contraction in feline esophageal smooth muscle cells. We used freshly isolated smooth muscle cells and permeabilized cells from cat esophagus to measure the length of cells. Maximal contraction occurred at 10(-6) M and the response peaked at 30s. To identify LPA receptor subtypes in cells, western blot analysis was performed with antibodies to LPA receptor subtypes. LPA1 and LPA3 receptor were detected at 50 kDa and 44 kDa. LPA-induced contraction was almost completely blocked by LPA receptor (1/3) antagonist KI16425. Pertussis toxin (PTX) inhibited the contraction induced by LPA, suggesting that the contraction is mediated by a PTX-sensitive G protein. Phospholipase C (PLC) inhibitors U73122 and neomycin, and protein kinase C (PKC) inhibitor GF109203X also reduced the contraction. The PKC-mediated contraction may be isozyme-specific since only PKCepsilon antibody inhibited the contraction. MEK inhibitor PD98059 and JNK inhibitor SP600125 blocked the contraction. However, there is no synergistic effect of PKC and MAPK on the LPA-induced contraction. In addition, RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y27632 significantly, but not completely, reduced the contraction. The present study demonstrated that LPA-induced contraction seems to be mediated by LPA receptors (1/3), coupled to PTX-sensitive G protein, resulting in activation of PLC, PKC-epsilon pathway, which subsequently mediates activation of ERK and JNK. The data also suggest that RhoA/ROCK are involved in the LPA-induced contraction.

Influence of the Adjuvants and Genetic Background on the Asthma Model Using Recombinant Der f 2 in Mice

Der f 2 is the group 2 major allergen of a house dust mite (Dermatophagoides farinae) and its function has been recently suggested. To determine the optimal condition of sensitization to recombinant Der f 2 (rDer f 2) in murine model of asthma, we compared the effectiveness with different adjuvants in BALB/c and C57BL/6 mice. Mice from both strains sensitized with rDer f 2 by intraperitoneal injection or subcutaneous injection on days 1 and 14. The dosage was 20 microg. Freund's adjuvants with pertussis toxin (FP) or alum alone were used as adjuvants. On days 28, 29, and 30, mice were challenged intranasally with 0.1% rDer f 2. We evaluated airway hyperresponsivenss, eosinophil proportion in lung lavage, airway inflammation, and serum allergen specific antibody responses. Naive mice were used as controls. Airway hyperresponsiveness was increased in C57BL/6 with FP, and BALB/c with alum (PC200: 13.5+/-6.3, 13.2+/-6.7 vs. >50 mg/ml, p<0.05). The eosinophil proportion was increased in all groups; C57BL/6 with FP, BALB/c with FP, C57BL/6 with alum, BALB/c with alum (24.8+/-3.6, 20.3+/-10.3, 11.0+/-6.9, 5.7+/-2.8, vs. 0.0+/-0.0%, p<0.05). The serum allergen specific IgE levels were increased in C57BL/6 with FP or alum (OD: 0.8+/-1.4, 1.1+/-0.8, vs. 0.0+/-0.0). C57BL/6 mice were better responders to rDer f 2 and as for adjuvants, Freund's adjuvant with pertussis toxin was better.

A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 microm in diameter) exhibiting tonic firing and prominent Ih, SKL-NP inhibited Ih and spike firings in a concentration dependent manner (IC50=7.85 microM). SKL-NP-induced inhibition of Ih was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates Ih in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (I h) in DRG neurons of rats.

Immunoassay of Pertussis According to Ages / 소아감염

PURPOSE: We conducted the immunoassay of pertussis according to ages, in order to evaluate protective immunity against pertussis in Korean populations. METHODS: Healthy subjects were enrolled at four university hospitals in Korea. The subjects were grouped as seven age groups (every 10 years). Antibodies against pertussis toxin (PT) in sera were measured by enzyme linked immunosorbent assay (ELISA) kits. Geometric mean concentrations (GMC) of antibodies and the ratios of the subjects with seroprotective antibody levels were determined. The subjects with antibody titers > or =24.0 EU/mL were considered to seroprotective as the manufacturer's protocol. RESULTS: Total 1,605 subjects (age: 2 months-65 years) participated in this study, and their GMC was 56.16+/-50.54 EU/mL. Among seven age groups, age group or =11 year) showed lower levels of antibody against pertussis and lower ratio of the subjects with seroprotective antibody titers than children (age group <11 year).

Development of a time-resolved fluoroimmunoassay for detecting S1 subunit of pertussis toxin and its application / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a time-resolved fluoroimmunoassay (TRFIA) for detection of pertussis toxin (PT) S1 subunit for quality control of human PT vaccine.</p><p><b>METHODS</b>A double antibody sandwich one-step method was used to establish the TRFIA for detecting PT S1 subunit in the vaccine.</p><p><b>RESULTS</b>The sensitivity of c peptide analysis reached 2.5 ng/ml without cross-reactions with other antigens. This assay could be used in detecting S1 subunit in the vaccine.</p><p><b>CONCLUSION</b>The TRFIA for detecting PT S1 subunit is simple, sensitive and rapid for quality control of the PT vaccine.</p>

Differential effects of CXCR4 antagonists on the survival and proliferation of myeloid leukemia cells in vitro

BACKGROUND: Antagonists of CXC chemokine receptor 4 (CXCR4), including AMD3100, induce peripheral mobilization of hematopoietic stem cells and have been approved for clinical use. We explored whether the CXCR4 antagonists affected the survival and proliferation of myeloid leukemia cells in vitro. METHODS: The effects of CXCR4 antagonists AMD3100 and T140 on the survival and proliferation of myeloid leukemia cell lines (U937, HL-60, MO7e, KG1a, and K562) as well as CD34+ cells obtained from patients with AML and CML were analyzed by flow cytometry by using annexin V and a colorimetric cell proliferation assay. RESULTS: AMD3100, but not T140, stimulated the proliferation of leukemia cells in vitro in a dose-dependent manner for up to 5 days (~2-fold increase at a concentration of 10-5 M), which was not abrogated by pretreatment of the cells with pertussis toxin, but was attenuated by RNAi knockdown of CXCR7 transcripts. In contrast, AMD3100 induced a marked decrease in the cell numbers after 5-7 days. AMD3100, but not T140, induced phosphorylation of MAPK p44/p42. AMD3100 increased the number and size of leukemia cell colonies and reduced cell apoptosis during the first 5-7 days of incubation, but the phenomena were reversed during the later period of incubation. CONCLUSION: The effects of CXCR4 antagonists on the proliferation of myeloid leukemia cells are not uniform. AMD3100, but not T140, exerts dual effects, initially enhancing and subsequently inhibiting the survival and proliferation of the cells in vitro.

Native Low-Density Lipoprotein-Dependent Interleukin-8 Production Through Pertussis Toxin-Sensitive G-Protein Coupled Receptors and Hydrogen Peroxide Generation Contributes to Migration of Human Aortic Smooth Muscle Cells

PURPOSE: Stimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration. MATERIALS AND METHODS: nLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H2O2 generation was determined by microscopic analysis using 2',7'-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates. RESULTS: nLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H2O2 generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H2O2. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody. CONCLUSION: PTX-sensitive G-protein coupled receptor-dependent H2O2 generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.

Effect of Pertussis Toxin and Herbimycin A on Proteinase-Activated Receptor 2-Mediated Cyclooxygenase 2 Expression in Helicobacter pylori-Infected Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.