ABSTRACT
Background: Salmonella is a significant pathogens of foodborne illness. The widespread use of antibiotics in clinical practice and animal husbandry has resulted in increasing drug resistance of Salmonella. In this study, we examined the serotype distribution and drug resistance of Salmonella in pediatric patients with diarrhea in Chenzhou City to provide a basis for the scientific control and rational use of antibiotics in clinical practice in relation to Salmonellosis. Methods: Stool Salmonella spp. were collected from patients younger than 18 years of age who met the definition for foodborne illness at two sentinel hospitals from 2017 through 2022 tested Salmonella, and a descriptive analysis of the epidemiologic characteristics. Salmonella strains isolated from the stool underwent serology and drug-sensitivity tests. The following 14 antibiotics were used for the drug-sensitivity tests: ampicillin (AMP), ampicillin/sulbactam (AMS), cefazolin (CFZ), cefoxitin, cefotaxime, ceftazidime, imipenem (IPM), tetracycline (TET), nalidixic acid, ciprofloxacin, chloramphenicol (CHL), gentamicin, trimethoprim/sulfamethoxazole (SXT), and azithromycin. Results: Samples from 1,263 pediatric with diarrhea, and Salmonella was detected in 221 (17.5%) of these patients. Positive test results were principally observed in the second and third quarters of each year, accounting for 21.1% and 19.6% of the cases, respectively. The infection rates of infants aged less than 12 months and toddlers aged 1-3 years with diarrhea were the highest at 21.3% and 17.8%, respectively. The 221 Salmonella strains were divided into 32 serotypes, of which Salmonella Typhimurium (S. Typhimurium) was the dominant strain (79.2%). The resistance rates to TET (86.9%), AMP (75.6%), AMS (58.4%), CFZ (55.7%), CHL (54.3%), and SXT (45.2%) predominated, and the differences in the drug-resistance rates to 1st-, 2nd-, and 3rd-generation cephalosporins were high (2.3-55.7%). Only 0.9% of the strains were resistant to IPM. The multidrug resistance (MDR) rate was 76.5% (169/221), and 48.9% (108/221) of the strains were resistant to five or more classes of antibiotics, of which the most common drug-resistance profile was AMP-AMS-TET-CHL-CFZ-SXT, accounting for 10.9% of Salmonella strains (24/221). Conclusions: Foodborne salmonellosis tended to occur during the summer and autumn in children, and infants and toddlers were more likely to develop salmonellosis than children in the other age groups. The dominant Salmonella serotype was S. Typhimurium. The drug-resistance rate of the tested strains was high, and the MDR problem was severe. We recommend that in the treatment of salmonellosis, antibiotics be selected rationally based on the drug-resistance status of local Salmonella resistance situation to ensure safety and efficacy.
ABSTRACT
In the present study, we aimed to explore the effect and underlying mechanism of metformin on lipopolysaccharide (LPS)-induced acute kidney injury (AKI). A total of 24 BALB/C mice were randomly divided into four groups: control group, LPS group and metformin group (50 or 100 mg/kg). The histological changes and cell apoptosis in kidney tissues were detected by hematoxylin-eosin staining and terminal-deoxynucleotidyl transferase-mediated nick end labeling assay, respectively. Enzyme-linked immunosorbent assay was applied to determine serum levels of blood urea nitrogen (BUN), kidney injury molecule-1 (Kim-1), creatinine (Cre), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß). Western blotting analysis were carried out to confirm the expressions of monocyte chemotactic protein-inducible protein 1 (MCPIP1), silent information regulator sirtuin 1 (SIRT1), and NF-κB p65 (acetyl K310). Compared with the control group, the mice in LPS group had glomerular capillary dilatation, renal interstitial edema, tubular cell damage and apoptosis. The serum levels of BUN, KIM-1, Cre, TNF-α, and IL-1ß in LPS group were significantly higher than those in control group. Moreover, LPS also elevated the expressions of MCPIP1 and NF-κB p65 (acetyl K310) but decreased the expression of SIRT1 in kidney tissues. However, metformin distinctly decreased LPS-induced renal dysfunction, the serum levels of BUN, KIM-1, Cre, TNF-α, and IL-1ß. In addition, metformin markedly increased the expressions of MCPIP1 and SIRT1 but decreased the expression of NF-κB p65 (acetyl K310) in kidney tissues. Metformin prevented LPS-induced AKI by up-regulating the MCPIP1/SIRT1 signaling pathway and subsequently inhibiting NF-κB-mediated inflammation response.
ABSTRACT
Background: Most previous studies on Clostridium difficile infection (CDI) mainly focused on adults with underlying diseases or critical illnesses. However, the number of CDI cases in children has also significantly increased, especially the growth of community-acquired CDI, which has attracted attention. This study was conducted to examine the toxin gene characteristics and the risk factors associated with community-acquired CDI (CA-CDI) in children with diarrhea. Methods: Children with diarrhea before admission or within 48 hours of hospitalization were included in the study. Stool samples were collected from children with community-acquired diarrhea who were treated at the Children's Hospital of the First People's Hospital of Chenzhou, China from June of 2021 to June of 2022. Fluorescence real-time polymerase chain reaction was utilized to detect Clostridioides difficile (CD) toxins A (tcdA) and B (tcdB) genes as well as binary toxin gene A (cdtA) and B (cdtB) in the specimens cultured for CD. Each child with CA-CDI was matched with four control children of the same sex, age, and place of residence. Necessary clinical data were extracted from the hospital's electronic medical record system. Then, a multivariate conditional logistic regression analysis was applied to identify potential risk factors for CA-CDI. Results: Sixteen (8.3%) of the 193 stool specimens who tested positive for CD were selected for the case group, and their matching 64 control patients were in the study cohort. The breakdown of the CD genotypes of the 16 positive cases were follows: 14 (tcdA+ and tcdB+) (7.25%) and 2 (tcdA+ and tcdB-) (1.04%). The cdtA and cdtB binary toxin genes were negative in all. The results of multivariate conditional logistic regression analysis identified antibiotic use within the previous month [odds ratio (OR) =5.13; 95% confidence interval (CI): 1.65-15.91] and non-breastfeeding (OR =4.89; 95% CI: 1.11-21.53) as independent risk factors for CDI in pediatric patients experiencing community-acquired diarrhea. Conclusions: Children who had been treated with antibiotics and not breastfed were more susceptible to CDI. Therefore, in order to prevent and to control the spread of CD infection, being prudent to the aforementioned high-risk factors is strongly advocated in clinical practice.
ABSTRACT
Renal cell carcinoma (RCC) with poor prognosis and high incidence rate is a common malignant disease. Current therapies could bring little benefit for the patients with advanced-stage RCC. PDIA2 is an isomerase responsible for protein folding and its role in cancer including RCC is under investigation. In this study, we found that PDIA2 was expressed much higher in RCC tissues than the control but the methylation level of PDIA2 promoter was lower based on the TCGA data. Patients with higher PDIA2 expression exerted worse survival. In clinical specimen, PDIA2 expression was correlated to patients clinical factors such as TNM stage (I/II vs III/IV, p = 0.025) and tumor size (≤ 7 cm vs > 7 cm, p = 0.004). Moreover, K-M analysis showed that PDIA2 was associated with patients survival in RCC. PDIA2 was expressed much higher in cancer cells A498 than 786-O than that in 293 T cells. After PDIA2 was knocked down, cell proliferation, migration and invasion was potently inhibited. But cell apoptotic rate increased reversely. Furthermore, the efficacy of Sunitinib on RCC cells was strengthened after PDIA2 knockdown. In addition, knockdown of PDIA2 gene leaded to downregulation of levels of JNK1/2, phosphorylated JNK1/2, c-JUN, and Stat3. But this inhibition was partially released when JNK1/2 was overexpressed. In consistent, cell proliferation was also partially recovered. In summary, PDIA2 plays important role in progression of RCC and JNK signaling pathway might be regulated by PDIA2. This study suggests PDIA2 as a candidate target for therapy of RCC (AU)
Subject(s)
Humans , MAP Kinase Signaling System/genetics , Carcinoma, Renal Cell/pathology , Carcinoma, Renal Cell/genetics , Kidney Neoplasms/pathology , Kidney Neoplasms/genetics , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , PrognosisABSTRACT
Renal cell carcinoma (RCC) with poor prognosis and high incidence rate is a common malignant disease. Current therapies could bring little benefit for the patients with advanced-stage RCC. PDIA2 is an isomerase responsible for protein folding and its role in cancer including RCC is under investigation. In this study, we found that PDIA2 was expressed much higher in RCC tissues than the control but the methylation level of PDIA2 promoter was lower based on the TCGA data. Patients with higher PDIA2 expression exerted worse survival. In clinical specimen, PDIA2 expression was correlated to patients' clinical factors such as TNM stage (I/II vs III/IV, p = 0.025) and tumor size (≤ 7 cm vs > 7 cm, p = 0.004). Moreover, K-M analysis showed that PDIA2 was associated with patients' survival in RCC. PDIA2 was expressed much higher in cancer cells A498 than 786-O than that in 293 T cells. After PDIA2 was knocked down, cell proliferation, migration and invasion was potently inhibited. But cell apoptotic rate increased reversely. Furthermore, the efficacy of Sunitinib on RCC cells was strengthened after PDIA2 knockdown. In addition, knockdown of PDIA2 gene leaded to downregulation of levels of JNK1/2, phosphorylated JNK1/2, c-JUN, and Stat3. But this inhibition was partially released when JNK1/2 was overexpressed. In consistent, cell proliferation was also partially recovered. In summary, PDIA2 plays important role in progression of RCC and JNK signaling pathway might be regulated by PDIA2. This study suggests PDIA2 as a candidate target for therapy of RCC.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Humans , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Kidney Neoplasms/pathology , MAP Kinase Signaling System , PrognosisABSTRACT
Effectively interfering energy metabolism in tumor cells and simultaneously activating the in vivo immune system to perform immune attacks are meaningful for tumor treatment. However, precisely targeted therapy is still a huge challenge. Herein, a mitochondrial-targeting phototheranostic system, FE-T nanoparticles (FE-T NPs) are developed to damage mitochondria in tumor cells and change the tumor immunosuppressive microenvironment. FE-T NPs are engineered by encapsulating the near-infrared (NIR) absorbed photosensitizer IR-FE-TPP within amphiphilic copolymer DSPE-SS-PEG-COOH for high-performing with simultaneous mitochondrial-targeting, near-infrared II (NIR-II) fluorescence imaging, and synchronous photothermal therapy (PTT) /photodynamic therapy (PDT) /immune therapy (IMT). In tumor treatment, the disulfide in the copolymer can be cleaved by excess intracellular glutathione (GSH) to release IR-FE-TPP and accumulate in mitochondria. After 808 nm irradiation, the mitochondrial localization of FE-T NPs generated reactive oxygen species (ROS), and hyperthermia, leading to mitochondrial dysfunction, photoinductive apoptosis, and immunogenic cell death (ICD). Notably, in situ enhanced PDT/PTT in vivo via mitochondrial-targeting with FE-T NPs boosts highly efficient ICD toward excellent antitumor immune response. FE-T NPs provide an effective mitochondrial-targeting phototheranostic nanoplatform for imaging-guided tumor therapy.
Subject(s)
Nanoparticles , Neoplasms , Photochemotherapy , Humans , Combined Modality Therapy , Photosensitizing Agents , Neoplasms/diagnostic imaging , Neoplasms/therapy , Polymers , Mitochondria , Photochemotherapy/methods , Cell Line, Tumor , Phototherapy/methods , Tumor MicroenvironmentABSTRACT
Lung adenocarcinoma (LUAD) is the most common type of lung cancer and the leading cause of cancer incidence and mortality worldwide. Despite the improvement of traditional and immunological therapies, the clinical outcome of LUAD is still far from satisfactory. Patients given the same treatment regimen had different responses and clinical outcomes due to the heterogeneity of LUAD. How to identify the targets based on heterogeneity analysis is crucial for treatment strategies. Recently, the single-cell RNA-sequencing (scRNA-seq) technology has been used to investigate the tumor microenvironment (TME) based on cell-specific changes and shows prominently valuable for biomarker prediction. In this study, we systematically analyzed a meta-dataset from the multiple LUAD scRNA-seq datasets in LUAD, identified 15 main types of cells and 57 cell subgroups, and revealed a series of potential biomarkers in M2b, exhausted CD8+T, endothelial cells, fibroblast, and metabolic patterns in TME, which further validated with immunofluorescence in clinical cohorts of LUAD. In the prognosis analysis, M0 macrophage and T cell activation were shown correlated to a better prognosis (p<0.05). Briefly, our study provided insights into the heterogeneity of LUAD and assisted in novel therapeutic strategies for clinical outcome improvement.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Humans , Prognosis , Endothelial Cells , Single-Cell Gene Expression Analysis , Adenocarcinoma of Lung/genetics , Lung Neoplasms/genetics , Tumor Microenvironment/geneticsABSTRACT
The diversity of cyclic peptides was expanded by elaborating Mitsunobu macrocyclization, tethering various hydroxy acid building blocks with different Nε-amine substituents. This new strategy was then applied in synthesizing peptidomimetic estrogen receptor modulator (PERM) analogs on the solid support. The PERM analogs exhibited increased serum peptidase stability, cell penetration, and estrogen receptor α binding affinity. Studying diversity-oriented methods for preparing azacyclopeptides provides a new tool for macrocycle construction and further structural information for optimizing ERα modulators for ER positive breast cancers.
Subject(s)
Breast Neoplasms , Peptidomimetics , Breast Neoplasms/metabolism , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/metabolism , Female , Humans , Peptides, Cyclic , Protein BindingABSTRACT
Objective: To observe the morphological changes of corneal endothelial cells in healthy Chinese children and youngsters and analyze the sensitive and specificity of the endothelial assessments. Methods: 14,670 Chinese healthy volunteers enrolled were examined by specular microscopy, and the endothelial descriptive indexes: the central corneal thickness (CCT), endothelial cell density (ECD), coefficient of variation in average cell size (CV), the percentage of regular hexagonal cells (hexagonality, HEX), cell size of minimal cell (S min), cell size of maximal cell (S max), average cell size (S avg), and size of standard deviation of cell area (S sd) as well as sex and age were analyzed. Results: The average age of this study is 17.36 ± 7.58 (4-30) years. There is no sex predominance: 7,260 male (49.5%) and 7,410 female (50.5%). The mean CCT, ECD, CV, HEX, S min/max, S avg, and S sd are 529.94 ± 31.53 (437-644) µm, 3,051.28 ± 375.49 (2,031-4,074) cells/mm2, 28.34 ± 4.36 (18-40) %, 61.21 ± 10.29 (17-89) %, (147.79 ± 21.94 to 678.29 ± 120.96) µm2, 332.74 ± 44.62 µm2, and 95.02 ± 23.17 µm2, respectively. The CCTs keep consistency. The ECD decreased rate is 1.02%/year. The curve of ECD and hexagonality expresses the same linear tender. The CCT and endothelial evaluation indexes have no sex predominant (p > 0.05); the quantitative indicators: CCT, ECD, and HEX are significant negative associated with age (p = 0.001 or p < 0.001); the variability indexes: the CV, S min, S max, S avg, and S sd are positive correlation (p < 0.001). The coefficients of CCT, HE, and S min are -0.35, -0.59, and 1.17, respectively. Conclusions: The ECD decrease rate is 1.02%/year of the normal Chinese Han childhood to the earlier adulthood. The ages 4 to 12, 13 to 20, and 21 to 30 can be named as the childhood, puberty and adulthood from endothelial biologic identity. The HEX is the sensitivity marks for the polymorphisms while the S min is the specificity indicator CVs upon the Topcon Noncon Specular microscopy results.
Subject(s)
Endothelial Cells , Microscopy , Adolescent , Adult , Cell Count , Child , Child, Preschool , China , Female , Humans , Male , Prospective Studies , Young AdultABSTRACT
Esculetin, a natural derivative from the traditional and widely-used Chinese medicinal herb Cortex Fraxini, has a variety of pharmacological effects, especially in anti-inflammation. However, it is not clear whether esculetin has a therapeutic effect on sepsis. This study aimed to investigate the anti-inflammatory and protective effects of esculetin on early sepsis. The results showed that the lung injury was significantly relieved with the treatment of esculetin, accompanied with the restrained production of inflammatory factors including IL-1ß, IL-6, TNF-α, CCL2 and iNOS during the early phase of E.coli-induced sepsis. Of note, activation of NF-κB and STAT1/STAT3 signals, the main upstream signals of many inflammatory factors, were attenuated by esculetin in both lung tissues from septic mice and LPS-stimulated macrophage. These findings suggested that the protection of esculetin against early sepsis should be related to its anti-inflammatory effect, which was at least partly due to its inhibition on NF-κB and STAT1/STAT3 signaling pathway in macrophage. Thus, esculetin could serve as a potential therapeutic agent by rebalancing innate immune response in macrophage for the treatment of early sepsis.
Subject(s)
NF-kappa B , Sepsis , Signal Transduction/drug effects , Umbelliferones/pharmacology , Animals , Inflammation/drug therapy , Lipopolysaccharides , Mice , NF-kappa B/antagonists & inhibitors , STAT1 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/antagonists & inhibitors , Sepsis/drug therapyABSTRACT
The abnormal expression of protein tyrosine phosphatase nonreceptor type 6 (PTPN6) has been proved to be associated with the progression of colorectal cancer. However, its role in chemosensitivity and related molecular mechanism have not been clarified. It has been reported that PTPN6 was down-regulated in colorectal cancer cells compared with the normal colorectal cells. To evaluate the effects of PTPN6 on the proliferation and survival of colorectal cancer cells, PTPN6 was overexpressed in colorectal cancer cells in the present study. We found that cell proliferation and viability were both decreased after overexpression of PTPN6. The IC50 of 5-Fu against colorectal cells was also declined in PTPN6 transfected cells. And further, we verified that PTPN6 could down-regulate the expression of P-gp and MRP-1. Moreover, SP1 was the target protein of PTPN6 predicated by ChIPBase software and confirmed through Co-immunoprecipitation assay and it was negatively regulated by PTPN6. To further verify the effect of SP1 on chemoresistance, SP1 was overexpressed. SP1 overexpression enhanced the drug-resistance to 5-Fu and abrogated the effects of PTPN6 upregulation on 5-Fu resistance. All the above changes were associated with the down-regulation of proteins related to MAPK signalling pathway, such as phosphorylation of extracellular regulated protein kinases (ERK) and p38. In summary, PTPN6 promoted chemosensitivity of colorectal cancer cells by targeting SP1 and inhibiting the activation of MAPK signalling pathway. SIGNIFICANCE OF THE STUDY: It has been demonstrated that the abnormal expression of PTPN6 was related to the progression of colorectal cancer. However, the chemosensitivity of PTPN6 and its molecular mechanisms were still unclear. Here, we identified that PTPN6 was down-regulated in colorectal cancer cells. Moreover, PTPN6 overexpression not only reduced cell proliferation and viability, but decreased the resistance of colorectal cells to 5-Fu. In our research, we found that the SP1 was the target protein of PTPN6 and it was negatively regulated by PTPN6. In addition, SP1 could increase the resistance of colorectal cells to 5-Fu. Molecular mechanism studies have shown that PTPN6 promoted the chemosensitivity of colorectal cancer cells by inhibiting the activation of MAPK signalling pathway.
Subject(s)
Colorectal Neoplasms/metabolism , Drug Resistance, Neoplasm/drug effects , Fluorouracil/pharmacology , MAP Kinase Signaling System/drug effects , Neoplasm Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Sp1 Transcription Factor/metabolism , Caco-2 Cells , Colorectal Neoplasms/drug therapy , HCT116 Cells , HT29 Cells , HumansABSTRACT
Schistosomiasis has been a fatal obstinate disease that threatens global human health, resulting in the granulomatous inflammation and liver fibrosis. Objective:The aim of this study was to evaluate the therapeutic effects and mechanisms of hydroxyasiaticoside combined with praziquantel in the treatment of schistosomiasis-induced liver fibrosis. Methods:Mice were randomly distributed into four experimental groups: normal control group, model group, praziquantel group, praziquantel + hydroxyasiaticoside group. Except for the normal control group, they were infected with Schistosomia cercariae through the abdominal skin to induce liver fibrosis. In the intervention group, mice were administered with the respective drugs by gavage after 8 weeks of infection. At the end of the treatment, mice were sacrificed to collect blood for the determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels. Moreover, the liver was excised, weighed, and liver indices were calculated. Histopathological examination was performed to assess liver morphology. Besides, the expression of collagen type I and III in liver was determined; the mRNA expression levels of IL-6 and TNF-α in liver tissues were measured using Real-time PCR while ELISA and western blotting were performed on liver tissue homogenate to determine the protein expression of IL-6 and TNF-α. Results:The combination of praziquantel and hydroxyasiaticoside lowered the pathological scores of schistosomiasis-induced hepatic fibrosis, the liver indice, serum AST and ALT levels, improved liver morphology, downregulated the expression levels of hepatic type I and III collagen, inhibited the mRNA expression levels of pro-inflammatory factors (IL-6 and TNF-α) in the liver of mice relative to the praziquantel alone. Conclusion:The combination of hydroxyasiaticoside and praziquantel is a potential therapeutic option for schistosomiasis-induced hepatic fibrosis. Notably, this combination noticeably suppresses the protein and mRNA expression levels of pro-inflammatory factors (TNF-α and IL-6) in the liver.
ABSTRACT
The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells, the low cultivated rate and complicated operation. To solve these problems, researchers made many studies on culture process of human normal primary epithelial cell. In this paper, we mainly introduce some methods used in separation and purification of human normal epithelial cells, such as tissue separation method, enzyme digestion separation method, mechanical brushing method, red blood cell lysis method, percoll layered medium density gradient separation method. We also review some methods used in the culture and subculture, including serum-free medium combined with low mass fraction serum culture method, mouse tail collagen coating method, and glass culture bottle combined with plastic culture dish culture method. The biological characteristics of human normal epithelial cells, the methods of immunocytochemical staining, trypan blue exclusion are described. Moreover, the factors affecting the aseptic operation, the conditions of the extracellular environment, the conditions of the extracellular environment during culture, the number of differential adhesion, and the selection and dosage of additives are summarized.
Subject(s)
Cell Culture Techniques , Cell Separation/methods , Epithelial Cells/cytology , Animals , Cells, Cultured , Collagen , Culture Media , Culture Media, Serum-Free , Humans , MiceABSTRACT
Both systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) diseases are related to the genetic and environmental factors, causing damage to the skin. The mutations of keratin 1 gene (KRT1) were reported to associate with skin diseases. The single-nucleotide polymorphism (SNP, rs14024) and the indel polymorphism (cds-indel, rs267607656), consisting mostly of the common haplotypes and could be used for genotyping of KRT1. We used the PCR with sequence specific primers (PCR-SSP) to determine the genotype of KRT1 in 164 SLE, 99 SSc patients, and 418 healthy controls. The results showed that the mutant with G at SNP rs14024 was associated with the high risk to SLE (p = 6.48×10-5) and SSc (p = 8.75×10-5), while the deletion allele at rs267607656 was associated with the low risk to SSc (p = 4.89×10-4) comparing to the normal controls. Haplogenotype, Del-/MU+ was associated with high susceptibility to SLE (OR = 1.87, p = 0.001) and SSc (OR = 2.29, p = 2.34×10-4). In contrast, the Haplogenotype Del+/MU- was associated with resistance to SLE (OR = 0.35, p = 6.24×10-5) and SSc (OR = 0.34, p = 0.001). This study demonstrates that the variations in KRT1 and the specific polymorphism of KRT1 in this Chinese Han population are associated with autoimmune diseases SLE and SSc. Typing KRT1 might be helpful to identify SLE and SSc patients.
Subject(s)
Asian People/genetics , Keratin-1/genetics , Lupus Erythematosus, Systemic/genetics , Polymorphism, Single Nucleotide , Scleroderma, Systemic/genetics , Adult , Base Sequence , Female , Haplotypes , Humans , MaleABSTRACT
OBJECTIVE: To compare the percentages of γδT cells in the lymphocytes and CD3âº; T cells isolated from the livers, lungs, spleen and mesenteric lymph nodes of C57BL/6 mice and the phonotype and function of γδT cells in different tissues or organs. METHODS: Lymphocytes were isolated from livers, lungs, spleen and mesenteric lymph nodes of normal C57BL/6 mice, respectively. Then, percentages of γδT cells in lymphocytes and CD3âº; T cells, and phenotypic characteristics of γδT cells were examined by flow cytometry. Moreover, IFN-γ, IL-4, IL-9 and IL-17 secreted by γδT cells were detected by means of intracellular cytokine staining after stimulation with PMA plus ionomycin. RESULTS: The percentage of γδT in lymphocyte cells of the livers was significantly higher than that of the lungs, spleen and mesenteric lymph nodes (P<0.05), and the percentage in CD3âº; T cells of the mesenteric lymph nodes was the lowest (P<0.05). CD4â»; CD8â»; γδT cells were the main subpopulation in these tissues and organs and there was also a small proportion of CD8âº; γδT cells. The proportion of CD4âº; γδT cells in the mesenteric lymph nodes was significantly higher than that in the others (P<0.05), and a group of CD4âº; CD8âº; γδT cells existed obviously. The percentage of IL-17âº; cells in γδT cells was significantly higher than IFN-γâº; and IL-4âº; cells, and almost no IL-9 γδT cells were found. The ability of secreting cytokines of γδT cells in the lungs was the strongest and the percentage of IL-17âº; γδT was (26.6 ± 12.1) %. In addition, the proportion of IFN-γâºâ»; γδT cells in the livers and lungs were (1.36 ± 0.37)% and (1.6 ± 0.7)%, respectively. CONCLUSION: The proportion, phenotype and function of γδT cells had significant difference in the livers, lungs, spleen, mesenteric lymph nodes of C57BL/6 mice.
Subject(s)
Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocyte Subsets/metabolism , Animals , Cytokines/biosynthesis , Female , Flow Cytometry , Mice , Mice, Inbred C57BL , Organ Specificity/immunology , T-Lymphocyte Subsets/immunologyABSTRACT
Schistosomiasis japonica is a severe tropical disease caused by the parasitic worm Schistosoma japonicum. Among the most serious pathological effects of S. japonicum infection are hepatic lesions (cirrhosis and fibrosis) and portal hypertension. Interleukin-17 (IL-17) is a pro-inflammatory cytokine involved in the pathogenesis of many inflammatory and infectious conditions, including schistosomiasis. We infected C57BL/6 mice with S. japonicum and isolated lymphocytes from the liver to identify cell subsets with high IL-17 expression and release using flow cytometry and ELISA. Expression and release of IL-17 was significantly higher in hepatic lymphocytes from infected mice compared with control mice in response to both non-specific stimulation with anti-CD3 monoclonal antibody plus/anti-CD28 monoclonal antibody and PMA plus ionomycin. We then compared IL-17 expression in three hepatic T-cell subsets, T helper, natural killer T and γδT cells, to determine the major source of IL-17 during infection. Interleukin-17 was induced in all three subsets by PMA + ionomycin, but γδT lymphocytes exhibited the largest increase in expression. We then established a mouse model to further investigate the role of IL-17 in granulomatous and fibrosing inflammation against parasite eggs. Reducing IL-17 activity using anti-IL-17A antibodies decreased infiltration of inflammatory cells and collagen deposition in the livers of infected C57BL/6 mice. The serum levels of soluble egg antigen (IL)-specific IgGs were enhanced by anti-IL-17A monoclonal antibody blockade, suggesting that IL-17 normally serves to suppress this humoral response. These findings suggest that γδT cells are the most IL-17-producing cells and that IL-17 contributes to granulomatous inflammatory and fibrosing reactions in S. japonicum-infected C57BL/6 mouse liver.
Subject(s)
Inflammation Mediators/metabolism , Interleukin-17/metabolism , Liver/immunology , Schistosoma japonicum/immunology , Schistosomiasis japonica/immunology , T-Lymphocyte Subsets/immunology , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Protozoan/blood , Cells, Cultured , Collagen Type III/metabolism , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Granuloma/drug therapy , Granuloma/immunology , Granuloma/parasitology , Ionomycin/pharmacology , Liver/drug effects , Liver/parasitology , Liver/pathology , Liver Cirrhosis/drug therapy , Liver Cirrhosis/immunology , Liver Cirrhosis/parasitology , Mice , Mice, Inbred C57BL , Natural Killer T-Cells/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Schistosoma japonicum/pathogenicity , Schistosomiasis japonica/drug therapy , Schistosomiasis japonica/parasitology , Schistosomiasis japonica/pathology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/parasitology , Th17 Cells/immunology , Up-RegulationABSTRACT
Schistosome infection could cause significant liver damage in animal; Th2 cells play an important role in the progress of this disease. In our study, C57BL/6 mice were infected by Schistosoma japonicum and lymphocytes were isolated from the liver to detect some characteristics of interleukin-5 (IL-5)-producing T cells by different methods. The results revealed that S. japonicum infection could induce a large amount of IL-5 in mouse liver T cells by the means of fluorescent bead immunoassay and RT-PCR. Although, mouse liver contained many T cell subsets, such as Th cells, Tc cells, NKT cells, and γδ T cells. Fluorescence activated cell sorting results indicated that Th cells were the main source of IL-5 in the T cell population after phorbol 12-myristate 13-acetate and ionomycin stimulation. Moreover, the percentage of IL-5-producing Th cells continued to increase from 4 to 8 weeks after S. japonicum infection, which differed from the changes of IFN-γ(+) Th1 cells, IL-4(+) Th2 cells, and IL-17A(+) Th17 cells during S. japonicum infection. Additionally, cytokines co-expression results demonstrated that 36.2 % of IL-5(+) Th cells could express IL-4, and 10 % of it could produce IFN-γ or IL-17A. Collectively, these findings implied that IL-5-producing Th cells posses some properties which differ from other cytokines secreting Th cells.
Subject(s)
Interleukin-5/biosynthesis , Liver/immunology , Schistosoma japonicum/pathogenicity , Schistosomiasis japonica/immunology , T-Lymphocytes/immunology , Animals , Interferon-gamma/biosynthesis , Interleukin-17/metabolism , Interleukin-4/biosynthesis , Liver/parasitology , Mice , Mice, Inbred C57BL , Schistosoma japonicum/immunology , Schistosomiasis japonica/parasitology , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Th1 Cells/cytology , Th1 Cells/immunology , Th1 Cells/metabolism , Th2 Cells/cytology , Th2 Cells/immunology , Th2 Cells/metabolismABSTRACT
OBJECTIVE: To observe the immune response of Th17 cells in mesenteric lymph node (MLN) of C57BL/6 mice infected by Schistosoma japonicum. METHODS: Twenty C57BL/6 mice were randomly divided into infected group and control group each with ten mice. The mice in infected group were infected each with 40 +/- 5 S. japonicum cercariae. Five to six weeks later, MLN lymphocytes were separated and stimulated for 4 h by anti-CD3 (1 microg/ml) and anti-CD28 (1 microg/ml) before examination of IL-17 and retinoic acid receptor-related orphan receptor gammat (ROR-gammat) mRNA by reverse transcription PCR. The level of IL-17 and IFN-gamma was detected by ELISA after culturing with supernatant for 72h. MLN lymphocytes were stimulated for 5h by 10 ng/ml phorbol myristoyl acetate (PMA) and 1 microg/ml ionomycin. The intracellular cytokines were stained and the content of Th17 and other cytokines was examined by flow cytometry. RESULTS: The level of IFN-gamma [(214.3 +/- 62.6) pg/ml] and IL-17 [(176.8 +/- 62.1) pg/ml] in the supernatant of cultured MLN cells from the infected mice was significantly higher than that of normal mice [(467 +/- 13.9) and 0 pg/ml) (P < 0.05). The expression level of IL-17 and ROR-gammat mRNA was also considerably higher than that of normal mice. IL-17+ IL-4+, IL-17+ IFN-gamma+, IL-17+ IL-5+ and IL-17+ IL-9 cells accounted for 0.06%, 0.02%, 0.02%, and 0.01% of the mesenteric lymph node CD4+ T cells of the infected mice, respectively. However, IL-17+ IL-10+ and IL-17+ Foxp3+ cells were undetected. CONCLUSION: The MLN of S. japonicum-infected C57BL/6 mice can induce the production of Th17 cells, and these cells can secrete IL-4, less IFN-gamma, IL-5 and IL-9, but not IL-10, and can not express Foxp3 in the infected mice.
Subject(s)
Lymph Nodes/parasitology , Schistosoma japonicum/immunology , Schistosomiasis japonica/immunology , Th17 Cells/immunology , Animals , Female , Mesenteric Lymphadenitis/parasitology , Mice , Mice, Inbred C57BLABSTRACT
To validate the protective efficacy against schistosomiasis by immunization with cells from juvenile Schistosoma japonicum in a murine model and to analyze possible factors related to protection, in this study, two independent repeated vaccination trials were performed. After three subcutaneous vaccinations, in trial one, in the absence of adjuvant, primary juvenile worm cells (pJCs) from S. japonicum induced remarkable average reductions in worm burden (54.3%), liver eggs per gram (LEPG) load (59.8%) as well as egg granulomas size (66.5%) compared to PBS control group (P<0.01), which were significantly higher than those elicited by fractions of juvenile worm cells (JCFs) or fractions of juvenile worms (JWFs) (P<0.05). Non-cell components of worms (WNCs) showed no significant protection. In trial two, compared to PBS control group, significant protective effect was also observed for cultured juvenile worm cells (cJCs) from S. japonicum with 58.4% worm reduction and 68.1% LEPG reduction (P<0.01). However, cultured adult worms cells (cACs) showed significantly higher worm burden (P<0.05) and egg burden (P<0.01) when compared to cJCs. Immunological analysis of trial two revealed that cJCs engendered a Th1-biased mixed Th1/Th2 type of immune response while cACs elicited a Th2-type response. Our data indicated that immunization with both primary and cultured cells from S. japonicum juvenile worms provided high immunoprotection, for which the physical character of immunogens, stage-specific parasite and the type of immune response induced might be responsible, suggesting that vaccination with whole cells from S. japonicum larvae is a promising approach to produce protective immunity against schistosomiasis.