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ObjectiveTo investigate the expression of lysophosphatidic acid （LPA） in patients with liver cancer， as well as its influence on malignant biological behavior of liver cancer and related regulatory mechanism. MethodsFrom January 2016 to December 2022， 26 patients with liver cancer， 28 patients with liver cirrhosis， and 28 individuals undergoing physical examination were enrolled. ELISIA was used to measure the content of LPA in plasma and peritoneal effusion of the patients with liver cancer or liver cirrhosis accompanied by peritoneal effusion， and the content of LPA was measured in plasma of the normal population at the same time， so as to clarify the difference in the expression of LPA in different populations， such as the patients with liver cancer and those with liver cirrhosis. MTT cell proliferation assay and cell migration assay were used to observe the influence of LPA and its inhibitor pertussis toxin （PTX） on the proliferation， migration， and invasion of SMMC7721 cells. In order to investigate the effect of LPA on the expression of RhoA and its upstream and downstream molecules FAK and P53 after binding to its receptor， qPCR and Western blot were used to observe the effect of LPA on the mRNA and protein expression levels of P53， FAK， and RhoA in SMMC7721 cells. A one-way analysis of variance was used for comparison of the means of continuous data between multiple groups， and the SNK-q test was used for comparison between two groups. ResultsCompared with the patients with liver cirrhosis， the patients with liver cancer had a significantly higher concentration of LPA in plasma （4.99±0.55 μmol/L vs 2.63±0.43 μmol/L， P<0.05） and peritoneal effusion （5.19±0.63 μmol/L vs 2.91±0.46 μmol/L， P<0.05）， and the patients with liver cancer also had a significantly higher level of plasma LPA than the normal population （4.99±0.55 μmol/L vs 1.61±0.39 μmol/L， P<0.05）. The cell proliferation assay showed that LPA significantly promoted the proliferation of SMMC7721 cells， and cell proliferation rate increased with the increase in dose and time； in particular， the middle-and high-dose groups had a significantly higher proliferation rate than the control group （P<0.05）. PTX inhibited the proliferative capacity of SMMC7721 cells in a time-dependent manner， and there was a significant difference between the groups （P<0.05）. The proliferation rate of the 72-hour high-dose LPA group was 3.6 times that of the control group， while the proliferation rate of the PTX group was 0.6 times that of the control group； the proliferation rate of the 72-hour high-dose LPA+PTX group was 1.2 times that of the control group. In addition， LPA increased the migration ability of hepatoma cells， while PTX inhibited their migration， in a time-dependent manner， and there was a significant difference between the groups （P<0.05）. The migration rate of the 72-hour high-dose LPA group was 3.09 times that of the control group， while the migration rate of the PTX group was 0.4 times that of the control group； the migration rate of the 72-hour high-dose LPA+PTX group was 0.99 times that of the control group. qPCR and Western blot showed that there were significant reductions in the mRNA and protein expression levels of P53 in SMMC7721 cells after LPA treatment， while there were significant increases in the mRNA and protein expression levels of FAK and RhoA； there was a significant difference between the LPA group and the control group （P<0.05）. ConclusionThere is an abnormal increase in the expression of LPA in patients with liver cancer， and LPA can promote the proliferation of liver cancer cells and increase their migration ability. At the same time， LPA changes the expression levels of P53， FAK， and RhoA， which may be associated with the promotion of tumor development and progression by LPA.
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Objective:To investigate the levels and influencing factors of serum pertussis toxin (PT)-IgG antibody in children with pertussis.Methods:The clinical data including age, course of disease and vaccination status of children with laboratory-confirmed pertussis and tested for PT-IgG antibody in Shenzhen Children′s Hospital from July 2015 to December 2018 were collected. Venous blood samples were obtained to detect PT-IgG antibody levels. Nasopharyngeal swabs were taken for polymerase chain reaction (PCR) test to detect Bordetella pertussis nucleic acid and culture of Bordetella pertussis. Mann-Whitney U test was used for comparison between two groups.Kruskal-Wallis test was used for comparison among multiple groups. Multiple linear regression was used to analyze the influencing factors of PT-IgG antibody levels. Results:A total of 871 children aged 4(2, 7) months were included, among whom, 592(68.0%) cases were under six months and 754 (86.6%) cases were under one year old. The course of disease was 15 (11, 20) days. Among 871 cases, 864 (99.2%) cases were PCR test and (or) culture positive, including 696 cases positive only for PCR test, 35 cases positive only for culture and 133 cases positive for both PCR test and culture. There were 452 (51.9%) children who were not vaccinated and 346 (39.7%) children vaccinated with at least one dose. In terms of age, the PT-IgG amtibody levels of children aged 0 to two months, three to five months, six months to two years and ≥three years were 0.7 (0, 8.2) IU/mL, 2.3 (0, 23.0) IU/mL, 24.6 (0, 112.3) IU/mL and 24.9 (0, 114.7) IU/mL, respectively. The PT-IgG antibody levels of children after onset of symptoms at 0 to two weeks, more than two to four weeks, more than four to eight weeks and more than eight weeks were 0(0, 7.9) IU/mL, 8.7(0, 56.0) IU/mL, 26.6(5.1, 82.9) IU/mL and 68.0(15.3, 118.8) IU/mL, respectively. The differences were both statistically significant ( H=88.346 and 94.076, respectively, both P<0.01). The PT-IgG antibody levels in children who were unvaccinated and vaccinated with at least one dose were 0.9 (0, 12.7) IU/mL and 14.6(0, 86.3) IU/mL, respectively. The difference was statistically significant ( Z=-8.520, P<0.01) PT-IgG≥80 IU/mL accounted for 16%(139/871) in the whole range of age, 34.3%(12/35) in children ≥three years old. There were 13 patients aged ≥three years old with a disease course >two weeks, among whom, six patients had PT-IgG≥80 IU/mL. Age, course of disease and vaccination status were independent influencing factors of PT-IgG levels ( β=0.108, 0.189 and 0.250, respectively, all P<0.01). Conclusions:The levels of PT-IgG antibody in children with pertussis are influenced by age, course of disease and vaccination status. The single serum PT-IgG of 80 IU/mL as cut-off value in the diagnosis of pertussis may lead to a increase of missed diagnosis. Therefore, it is necessary to further explore the standards suitable for children in China.
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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.
Subject(s)
Diphtheria , Enzyme-Linked Immunosorbent Assay , Hemagglutinins , Immunoassay , Immunoglobulin G , Korea , Methods , Pertussis Toxin , Pertussis Vaccine , Vaccination , Vaccines , Whooping Cough , World Health OrganizationABSTRACT
Objective@#To establish a functional antibody detection method for acellular pertussis vaccines in order to conveniently and effectively evaluate the production consistency and potency of acellular pertussis vaccine bulks and final products.@*Methods@#Chinese hamster ovary (CHO) cell clustering assay was optimized and used to measure titers of neutralizing antibodies against pertussis toxin in mouse immune serum samples.@*Results@#Vaccine samples were determined to be immunized intraperitoneally with 1/5 the human dose to ten female NIH mice (20-24 g, 5-week-old). Four weeks after immunization, blood samples were collected to isolate serum. Serially diluted serum samples were used to neutralize 0.1 IU/ml of pertussis toxin national reference product for 2 hours. Results of clustering were determined after 48 hours of incubation in pre-cultured CHO cell wells. The geometric mean of the serum dilution of the final unclustered wells was the neutralizing antibody titer of vaccine sample. There were significant differences in the titers of neutralizing antibodies elicited by acellular pertussis vaccines prepared with different manufacturing processes. Vaccine samples succeed or failed the modified intracerebral challenge assay (MICA) were easily distinguishable by neutralizing antibodies.@*Conclusion@#The method of detecting neutralizing antibodies to pertussis toxin greatly reduces the amount of animals used in research. CHO cell clustering assay that has better repeatability and precision can be used for monitoring and initial evaluation of the consistency and potency of the bulks and final products of pertussis vaccines prepared with different manufacturing processes.
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Pertussis toxin (PT) is one of the major virulence factors of Borrelia pertussis and also an essential component of pertussis vaccines. Before using it in vaccine production, detoxification is necessa-ry because of its biological activity. This paper summarized the structure and detoxification process of PT as well as the safety and immunogenicity of pertussis vaccines prepared with detoxified PT.
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Objective To investigate the effects of different concentrations and adsorption methods of aluminum hydroxide adjuvant produced by different manufacturers on the immunogenicity of the diphtheria-tetanus-acellular pertussis and inactivated poliovirus combined vaccine ( DTaP-sIPV) . Methods Five anti-gens of DTaP were adsorbed onto different concentrations (0. 42 mg/ml, 0. 47 mg/ml and 0. 52 mg/ml) of aluminum hydroxide from different manufacturers through sequential and separate adsorption. Adsorbability, anti-pertussis toxin ( PT)/filamentous hemagglutinin ( FHA)/pertactin ( PRN)/diphtheria toxoid ( DT)/tet-anus toxoid ( TT) antibodies and the potency of vaccines were detected. Results The adsorbability of alu-minum hydroxide adjuvant slightly decreased with the reduction of concentration. No significant difference in potency and antibody level was observed between sequential and separate adsorption. Moreover, no signifi-cant difference in antibody level was observed between vaccines prepared with aluminum hydroxide adjuvant produced by General Chemical Corp and our institute. Conclusion Aluminum hydroxide adjuvant produced by our institute at the concentration of 0. 52 mg/ml and separate adsorption method are suitable for prepara-tion of DTaP-sIPV.
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Objective This cross sectional study was designed to estimate the intensity and duration of DTP by analyzing serum anti-Bordetella pertussis toxin (PT) IgG antibodies among children in Shanghai. The results may provide scientific evidence for developing vaccination protocol and assessing vaccination effect. Methods Blood samples were obtained from 372 children (0-18 years of age) in Shanghai. The serum level of anti-PT IgG was determined by commercial ELISA kits. Anti-PT IgG level ≥30 IU/mL was defined as positive if no pertussis vaccine was vaccinated in the past year, which indicates recent contact with Bordetella pertussis. Anti-PT IgG level ≥100 IU/mL suggests an acute infection. Results The mean concentration of anti-PT IgG antibody was 16.21 IU/mL in the 372 subjects. Serum anti-PT IgG was positive (≥30 IU/mL) in 42 (11.29％) children. The highest positive rate was found in the age group of ＜3 years old (18.60％), followed by the age group of 10-12 years old (11.43％), and the lowest positive rate in the age group of 3-5 years old (7.46％). The mean level of anti-PT IgG antibody was 21.82 IU/mL in the age group of ＜3 years old, 21.16 IU/mL in the age group of 10-12 years old, and the lowest (9.74 IU/mL) in the age group of 6-9 years old. Conclusions Atypical B. pertussis infection is prevalent among younger children in Shanghai. Booster dose of pertussis vaccine may be useful in reducing the incidence of pertussis in children.
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Introducción. La tos convulsa es una enfermedad altamente contagiosa causada por Bordetella pertussis. Tiene una alta tasa de morbilidad y mortalidad, especialmente, en los lactantes menores de seis meses de edad. En la Argentina, la incidencia y la mortalidad se han encontrado en aumento en las últimas 3 décadas. Objetivo. Determinar anticuerpos contra Bordetella pertussis en las mujeres embarazadas en el tercer trimestre de la gestación y en el recién nacido, medidos en la sangre del cordón. Métodos. Se disenó un estudio observacional, transversal. El estudio se inició en 2011 cuando la vacunación contra pertussis en la embarazada no estaba incluida en el Calendario Nacional de Vacunación y era opcional. Los anticuerpos se midieron en las madres en el tercer trimestre del embarazo y en la sangre del cordón umbilical al nacer. Las determinaciones de anticuerpos se realizaron con el kit de ELISA humano para IgG toxina pertussis ABCAMR. Se utilizó la prueba de chi² para comparar la prevalencia. Resultados. Se incluyó a 111 madres y a sus bebés, 35 hijos de madres no vacunadas (antes de la implementación de la vacuna en embarazadas) y 76 hijos de madres vacunadas. Los bebés de madres vacunadas presentaron anticuerpos IgG positivos en el 92% (70/76), mientras que los bebés de madres no vacunadas fueron negativos para anticuerpos IgG en el 100% (35/35) con una p < 0,001. Conclusión. En la población de vacunadas del estudio, se observó que sus hijos presentaron anticuerpos IgG positivos en el 92%. Este estudio apoya la necesidad de la inmunización materna contra Bordetella pertussis para proteger al recién nacido.
Introduction. Pertussis is a highly contagious disease caused by Bordetella pertussis. It poses a high morbidity and mortality rate, especially among infants younger than 6 months old. In Argentina, pertussis incidence and mortality have increased over the past three decades. Objective. To establish Bordetella pertussis antibody titers among pregnant women in their third trimester and among newborn infants, as measured in cord blood. Methods. This was an observational, crosssectional study. The study started in 2011; at that time, pertussis vaccination was not mandatory for pregnant women as per the national immunization schedule, only optional. Maternal antibodies were measured in the last trimester of pregnancy for women and in cord blood for newborn infants. Antibody titers were determined using Abcam's anti-Bordetella pertussis toxin (PT) IgG in vitro ELISA kit. The X2 test was used to compare prevalence rates. Results. The study included 111 mother-newborn infant dyads; 35 infants from unvaccinated mothers (before the introduction of the vaccine) and 76 from vaccinated mothers. Positive IgG antibodies were found in 92% (70/76) of infants born from vaccinated mothers whereas 100% (35/35) of infants born from unvaccinated mothers had negative results for antibodies; p < 0.001. Conclusion. In the vaccinated population of this study, 92% of infants had positive IgG antibodies. This study supports the need for maternal immunization against Bordetella pertussis to provide protection to newborn infants.
Subject(s)
Humans , Male , Female , Infant , Pregnancy Complications, Infectious/prevention & control , Pregnancy Complications, Infectious/blood , Pregnancy Complications, Infectious/epidemiology , Bordetella pertussis/immunology , Whooping Cough/prevention & control , Whooping Cough/blood , Whooping Cough/epidemiology , Antibodies, Bacterial/blood , Argentina , Pregnancy , Seroepidemiologic Studies , Cross-Sectional Studies , Hospitals, UniversityABSTRACT
Objective To investigate the levels of antibody to pertussis toxin (PT) IgG in newborns in Shunyi Women and Children's Hospital of Beijing Children's Hospital in 2016.Methods A total of 419 newborns were enrolled in this study.Umbilical cord blood sample was collected from each subject and detected by enzyme-linked immunosorbent assay to measure the concentration of PT-IgG.Besides,all newborns were followed up to January 31,2017.Chi-square test was used for statistical analysis.Results The detectable rate of umbilical cord blood samples for PT-IgG accounted for 30.1％ (126/419).The median antibody level was ＜ 5 U/ml,and the 90th and the 95th percentile were 14.3 and 24.0 U/ml,respectively.No cases of pertussis occurred at the end of follow-up.Conclusions The newborns born in Shunyi Women and Children's Hospital of Beijing Children's Hospital are generally lack of protective PT antibody.
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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.
Subject(s)
Animals , Mice , Blood Glucose , Corticosterone , GTP-Binding Proteins , Hyperglycemia , Hypoglycemia , Insulin , Pertussis Toxin , Plasma , Stress, Psychological , Swimming , Up-Regulation , Whooping CoughABSTRACT
Background Researches indicated that etiology and epidemiology of pertussis toxin (PTX)dependent experimental autoimmune uveoretinitis(EAU)model are very different with human uveoretinitis owing to the influence of PTX on immune.Our previous study has established lipopolysaccharide (LPS),an endotoxin,which instesad of PTX,mediated EAU model.However,the exact roles of LPS and PTX in EAU still remained unclear.Objective This study was to investigate the roles of LPS and PTX in EAU model.Methods Twenty SPF C57BL/6(H-2b) mice were assigned to 0 d-PTX-EAU group,7 d-PTX-EAU group,0 d-LPS-EAU group and 7 d-LPS-EAU group using random number table method.The mice were immunized with interphotoreceptor retinoid-binding protein 1-20(IRBP 1-20) emulsified in complete Freund adjuvant (CFA),and concurrently with or on day 7 postimmunization,LPS or PTX was injected in the footpad or intraperitoneally respectively.Delayed-type hypersensitivity (DTH) of the mice was evaluated by measuring the ear thickness 48 hours after IRBP was injected into the ear pinna,and lymphocyte proliferation was assessed by tritiated thymidine uptake.Retinal histopathological examination was performed and scored based on criteria of Caspi.The use and care of experimental animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Serious infiltration of inflammatory cells,disorder of entire retinal structure and retinal folds were seen in the mice of the 0 d-PTX-EAU group and 7 d-LPS-EAU group on 21 days after injection of PTX or 14 days after injection of LPS,and severe vitritis and a few granuloma-like lesions were found in the 0 d-PTX-EAU group.However,only mild vasodilatation or less retinal folds were found in the 7 d-PTX-EAU group and 0 d-LPS-EAU group.The pathological scores in the mice of the 0 d-PTX-EAU group and 7 d-LPS-EAU group were higher than those of the 7 d-PTX-EAU group and 0 d-LPS-EAU group (all at P ＜ 0.05).The ear thickness was (62.600 ± 3.362) μm,(60.000±2.345) μm,(30.400± 1.817) μm and (32.800 ± 1.643) μm in the 0 d-PTX-EAU group,7 d-PTX-EAU group,0 d-LPS-EAU group and 7 d-LPS-EAU group,showing a significantly difference among the 4 groups (Fgroup =259.751,P=0.000),and the ear thicknesses of 0 d-PTX-EAU group and 7 d-PTX-EAU group were significantly higher than those of the 0 d-LPS-EAU group and 7 d-LPS-EAU group (all at P＜0.05).The lymphocyte proliferation was strongly enhanced in PTX-EAU groups,and the radiation count per minute (cpm) was (16 150.000±799.218)/min and (16 120.000±729.383)/min in the 0 d-PTX EAU group and 7 d-PTX EAU group,and (8 348.000±258.979)/min and (8 540.000±81.548)/min in the 0 d-LPS EAU group and 7 d-LPS EAU group respectively,with a significant difference among the PTX-EAU groups and LPS-EAU groups (Fgroup =316.978,P=0.000).Conclusions LPS and PTX play different roles during the EAU formation.LPS may be involved in the breakdown of blood-retina barriers (BRB).
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Objective To study the sero-epidemiology of pertussis immunity and related factors among the community-based populations in Tianjin.Methods Antibodies to pertussis toxin immunoglobulin and IgG (PT-IgG) levels among the community-based populations were examined,using ELISA over the three consecutive years.Subjects that were older than 4 years of age,with concentration of anti-PT-IgG higher than 40 IU/ml were recognized as having recent Bordetella (B.) pertussis infection.Results Of the 1 825 study subjects,the average positive rate of anti-PT-IgG was 10.96％.The highest rate appeared as 24.37％-13.61％ among the 0-3 year-olds (P＜0.001).The positive rate was 8.84％,and the estimated incidence of recent infection became 10 852 per 100 000 among those age 4-83 year-olds.Peak of the estimated incidence rate of recent infection was 18 986 per 100 000 in the age group of 51-83 year-olds (P=0.001),but increasing linearly along with the increase of age (r=0.976,P＜0.001).There were statistically significant differences seen in the antibody positive rate of 3-year period of study (P=0.001),and appeared linear correlation with the reported annual incidence (r =0.992,P＜ 0.001).There were statistically significant differences in the antibody positive rates of the 3 monitoring areas (P=0.034),also showing a linear correlation with the reported annual incidence (r=0.996,P＜0.001).Conclusion Results from this study indicated that B.pertussis infection had been common,particularly in adults living in the communities in Tianjin,calling for the current pertussis immunization strategy to be improved in order to control the pertussis reemerge in China.
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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.
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Humans , Antigenic Variation , Antigens/genetics , Bacterial Proteins/genetics , Bordetella pertussis/genetics , Genes, Bacterial , Genotype , Pertussis Toxin/genetics , Promoter Regions, Genetic , Republic of Korea , Sequence Analysis, DNA , Whooping Cough/immunologyABSTRACT
Objective To investigate the influence of pertussis toxin(PTX)on G protein-coupled estrogen receptor(GPER)-mediated activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt)signaling activated by 17 β-estradiol(17β-E2)in endometrial carcinoma cells.Methods Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells.Changes of levels of GPER,ERα and ERβ protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations(0,0.1,0.5,1.0 μg/ml),and then co-stimulated with with 1 × 10-6 mol/L 17β-E2 respectively at different time (Ishikawa 30 minutes,HEC-1A 15 minutes).Results(1)Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell.(2)After co-treated with PTX at different concentrations(0,O.1,0.5,1.0 μg/ml)and 10-6 mol/L 17β-E2,in Ishikawa cell,the ratio of pAkt/Akt was 0.74 ±0.54,0.34 ±0.06,0.18 ±0.03,0.07 ±0.15,the gray values of GPER was 0.872 ± 0.490,0.395 ± 0.054,0.145 ± 0.014,0.034 ± 0.008,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which was most obviously when the concentration was 1.0 μg/ml(F =63.729,P =0.0001;F =160.284,P =0.0001);ERα and ERβ protein had no significant change among different groups(P ＞0.05).In HEC-1A cell,the ratio of pAkt/Akt was 0.73 ±0.09,0.26 ±0.14,0.11 ±0.03,0,the Gray values of GPER is 0.927 ±0.134,0.485 ± 0.022,0.194 ± 0.004,0,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which were also completely inhibited when the concentration was 1 μg/ml(F =1039.321,P =0.0001;F =109.646,P =0.0001),ERα protein had no significant differences(P ＞ 0.05)among different groups.ERβ was negatively expressed.Conclusion The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.
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Objective To discuss the levels of inhibitory protein Ga-2 (Gαi2) in hippocampus of brain and the effects of Gαi2 on neuronal intracellular Ca2+ level in cerebral ischemia reperfusion injury model of rats.Methods Ninety SD rats were randomly (random number) assigned to sham group (n =30),ischemic-reperfusion (IR) group (n =30),Pertussis toxin (PT) group (n =30).The blood flow of right common carotid artery of rat was blocked for 90 min to make ischemia reperfusion model.The levels of Gαi2 in hippocampus was assayed by immunohistochemistry and Western blotting after ischemia reperfusion for 6,12,24 h in each group.The average fluorescence values of intracellular Ca2+ levels in hippocampus of rats in three groups were detected by using Flow CytoMeter (FCM).Neuronal cell apoptosis was measured by TUNEL.Results After restoration of middle cerebral artery blood flow for different lengths of time,the levels of hippocampus Gαi2 and Ca2+ levels in IR group were significantly higher than those in Sham group (P ＜0.01).The hippocampus Ca2+ levels in PT group were higher than those in IR group (P ＜ 0.01).The apoptotic rates of neurons in PT group were lower than those in IR group (P ＜0.05).Conclusions The level of Gαi2 in cerebral ischemia reperfusion injury model was increased.Gαi2 might reduce the calcium ion concentration of neurons after cerebral ischemia and rcduce the neuronal cell apoptosis in this model.Gαi2 might play a role in protecting neuron from cerebral ischemia reperfusion injury.
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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.
Subject(s)
Humans , Cell Movement/physiology , Cells, Cultured , Hydrogen Peroxide/metabolism , Interleukin-8/biosynthesis , Lipoproteins, LDL/pharmacology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Pertussis Toxin/pharmacology , Phosphorylation/drug effects , Reactive Oxygen Species/metabolism , Receptors, G-Protein-Coupled/physiology , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Objectives To analyze the characteristics of antigenic genes of clinical Bordetella pertussis strains recently isolated by analyzing the sequence of pertussis toxin S1 subunit(ptxS1) , pertactin (Prn) , fimbriae 2 (Fim2) and fimbriae 3 (Fim3 ) genes of four clinical isolates. Methods The 4 clinical isolates were collected in 2002 in Shijiazhuang of Hebei province. Four strains were isolated from pertussis patient's nasopharyngeal aspirate. ptxS1, Prn, Fim2 and Fim3 genes of these strains were amplified and sequenced. The sequences of those genes were compared with those of the isolates in GenBank and the isoaltes used in the production of pertussis vaccine in China. Results The results of the gene sequencing showed the four clinical isolates belonged to ptxS1 A type, which were different from those in vaccine strains. In addition, three Prn and three Fim'3 variants were observed in the four clinical isolates. Sequence analysis showed that the nucleotide sequence and deduced amino acid sequence of those strains had more than 99% identity with those in vaccine strains. The phylogenetic trees of those genes also showed these strains had a higher level of similarity with other Bordetella pertussis strains. Conclusion The four clinical isolates are different from vaccine strains in four antigenic genes, which laid a foundation for further studies on pertussis epidemiology,quality control and development of pertussis vaccine in China.
ABSTRACT
Serum amyloid A (SAA) induced CCL2 production via a pertussis toxin (PTX)-insensitive pathway in human umbilical vein endothelial cells (HUVECs). SAA induced the activation of three MAPKs (ERK, p38 MAPK, and JNK), which were completely inhibited by knock-down of formyl peptide receptor 2 (FPR2). Inhibition of p38 MAPK and JNK by their specific inhibitors (SB203580 and SP600125), or inhibition by a dominant negative mutant of p38 MAPK dramatically decreased SAA-induced CCL2 production. Inactivation of Gi protein(s) by PTX inhibited the activation of SAA-induced ERK, but not p38 MAPK or JNK. The results indicate that SAA stimulates FPR2-mediated activation of p38 MAPK and JNK, which are independent of a PTX-sensitive G-protein and are essential for SAA-induced CCL2 production.
ABSTRACT
BACKGROUND: Bone marrow (BM) mesenchymal stem cells (MSCs) can be expanded over 20~30 cell doublings in vitro even in the absence of any growth factors. However, the mechanisms that govern MSC proliferation are not well understood. METHODS: We investigated the role of signaling of the pertussis toxin (PTX)-sensitive G protein-coupled receptor in the proliferation of BM MSCs. RESULTS: PTX inhibited the proliferation of human BM MSCs and murine BM stromal MS-5 cells in a dose-dependent manner. Among the chemokines produced by the BM stromal cells, stromal cell-derived factor-1 (SDF-1) enhanced the proliferation of BM MSCs, while MIP-1alpha, MCP-3 or RANTES did not. PTX also inhibited the proliferation of some fibroblasts, such as MRC-5 and NIH-3T3, but did not affect the proliferation of HeLa and HSF cells. HSF cells did not express CXCR4 mRNA, but did produce SDF-1. In contrast, HeLa cells expressed CXCR4 strongly on the cell surface, but did not produce SDF-1. BM MSCs, MS-5, MRC-5, and NIH-3T3 cells all expressed CXCR4 minimally on the cell surface. These cells, however, had abundant CXCR4 protein in their cytoplasm, which was demonstrated by flow cytometric analysis performed after permeabilization of the cells. In addition, an ELISA performed on the culture supernatants of the cells revealed that these cells constitutively produce and secrete SDF-1. CONCLUSION: These results indicate that the signaling through the PTX-sensitive G protein-coupled receptor, which is induced by autocrine factors, plays an important role in the proliferation of BM MSCs and in some fibroblasts, and that SDF-1 is the most probable candidate for the autocrine growth factor.
Subject(s)
Humans , Bone Marrow , Cell Proliferation , Chemokine CCL3 , Chemokine CCL5 , Chemokines , Cytoplasm , Enzyme-Linked Immunosorbent Assay , Fibroblasts , HeLa Cells , Intercellular Signaling Peptides and Proteins , Mesenchymal Stem Cells , NIH 3T3 Cells , Pertussis Toxin , RNA, Messenger , Stromal Cells , Whooping CoughABSTRACT
PURPOSE: We conducted the study to evaluate the immunogenicity and safety of three component DTaP vaccine (Infanrix(R)) in a group of Korean healthy infants on a three-dose primary vaccination. And we compared the immunogenicity of this DTaP vaccine with two component DTaP vaccine which has been widely used in Korea. METHODS: We enrolled one hundred fifty one healthy infants aged 8-9 weeks. These infants were vaccinated at age 2, 4 and 6 months of age with three component DTaP vaccine. Solicited adverse events were actively monitored for 72 hours following each vaccination, and all adverse events after each vaccination were observed for three weeks. Anti-diphtheria toxoid Ab., anti-tetanus toxoid Ab., anti-pertussis toxin Ab., anti-filamentous hemagglutinin Ab., and anti-pertactin Ab. were measured using ELISA for assessing immunogenicity of study vaccine in 60 infants. Immunogenicity analysis of two component DTaP vaccine was performed with same methods in 14 infants as control. RESULTS: The seroconversion rates of anti-diphtheria toxoid Ab, anti-tetanus toxoid Ab. anti- filamentous hemagglutinin Ab. were 100% in both group. Seroconversion rate of anti-pertactin Ab in study group was 100%, but the rate in control group was 50%. However, geometric mean concentration of anti-pertussis toxin Ab. was higher in control group. Mild local and systemic reactions were observed within three days after vaccination, and no serious adverse events related study vaccine were happened during study period. CONCLUSIONS: Our study results suggest that three component DTaP vaccine (Infanrix(R)) is a well- tolerable and high immunogenic vaccine, especially anti-Pertactin Ab. of the study vaccine is very immunogenic. It can be available as routine DTaP vaccination in our infants.