OBJECTIVE: To investigate the enhancement of macrophage chemotaxis in patients with knee osteoarthritis (KOA) and its correlation with the disease severity. METHODS: Eighty patients with KOA admitted from July 2019 to June 2022 were enrolled as the observation group and divided into 29 cases of moderate group, 30 cases of severe group and 21 cases of extremely severe group. At the same time, 30 healthy subjects were included as the control group. The gene expressions of NF-κB, CXC chemokine receptor 7 (CXCR7) and CXC chemokine ligand 12 (CXCL12) in macrophages of each group were analyzed. Visual analogue scale(VAS) was used to evaluate the degree of joint pain. Joint function was evaluated by knee Joint Society Scoring system(KSS). Finally, data analysis was carried out. RESULTS: The expression levels of NF-κB, CXCR7 and CXCL12 in moderate group, severe group and extreme recombination group were higher than those in control group. The VAS, the expression of NF-κB, CXCR7 and CXCL12 in the severe group and the extreme recombination group were higher than those in the moderate group, whereas KSS was lower than that in the moderate group. The VAS, expression levels of NF-κB, CXCR7 and CXCL12 in the extremely severe group were higher than those in the severe group, and KSS was lower than that in the severe group (all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with VAS score, but negatively correlated with KSS(all P<0.01). The expression levels of NF-κB, CXCR7 and CXCL12 in macrophages were positively correlated with the severity of disease. After excluding the influence of traditional factors (gender, age and disease duration), multiple linear regression analysis further showed that the expression levels of NF-κB, CXCR7 and CXCL12 were still positively correlated with the severity of disease(all P<0.01). CONCLUSION The chemotaxis of macrophages in patients with KOA increased with the aggravation of the disease, and was related to the degree of pain and function impairment.
Humans ; Osteoarthritis, Knee/genetics* ; Chemotaxis/genetics* ; NF-kappa B/metabolism* ; Macrophages/metabolism* ; Receptors, CXCR/metabolism* ; Patient Acuity

OBJECTIVETo construct a knockout fliY gene mutant strain of Campylobacter jejuni for determining the role of FliY protein in flagellar movement related to bacterial motility, chemotaxis and colonization.

METHODSThe plasmid pBluescript-II-SK was used to construct the suicide plasmid; according to homologous exchange principle, the suicide plasmid was utilized to generate fliY gene knockout mutant(fliY) in Campylobacter jejuni strain NCTC11168. The fliY mutant strain was identified by PCR, sequencing and Western blotting. The chemotactic and colonizing abilities of fliY mutant were determined by colony migration test and bacterial chemotactic test in vitro, and colonization test in jejunum of mice.

RESULTSThe fliY(-)mutant strain showed a growth curve in medium similar to that of wild-type strain. PCR, sequencing and Western blotting assay confirmed that the fliY gene in fliY(-)mutant was deleted. Compared to the wild-type strain, the colonies of fliY-mutant on semisolid plate were much smaller (P <0.05), the chemotactic ability of fliY mutant towards sodium deoxycholate and bovine bile was significantly attenuated (P <0.05), and the number of fliY mutant (CFU) in jejunal tissue specimens of the infected mice was significantly decreased (P<0.05).

CONCLUSIONThe function of C.jejuni fliY gene refers to controlling flagellar movement, which is involved in bacterial chemotaxis and colonization.

Animals ; Bacterial Proteins ; genetics ; Campylobacter jejuni ; genetics ; pathogenicity ; Chemotaxis ; genetics ; Gene Knockout Techniques ; Jejunum ; microbiology ; Membrane Proteins ; genetics ; Mice ; Mice, Inbred BALB C

Animals ; Chemotaxis ; physiology ; Dictyostelium ; genetics ; metabolism ; physiology ; Genes, Protozoan ; genetics ; physiology ; Genes, ras ; genetics ; physiology ; Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Protozoan Proteins ; genetics ; metabolism ; Signal Transduction ; Transcription Factors ; genetics ; metabolism

Animals integrate various environmental stimuli within the nervous system to generate proper behavioral responses. However, the underlying neural circuits and molecular mechanisms are largely unknown. The insulin-like signaling pathway is known to regulate dauer formation, fat metabolism, and longevity in Caenorhabditis elegans (C. Elegans). Here, we show that this highly conserved signaling pathway also functions in the integrative response to an olfactory diacetyl and a gustatory Cu(2+) stimuli. Worms of wild-type N2 Bristol displayed a strong avoidance to the Cu(2+) barrier in the migration pathway to the attractive diacetyl. Mutants of daf-2 (insulin receptor), daf-18 (PTEN lipid phosphatase), pdk-1 (phosphoinositide-dependent kinase), akt-1/-2 (Akt/PKB kinase) and sgk-1 (serum- and glucocorticoid-inducible kinase) show severe defects in the elusion from the Cu(2+). Mutations in DAF-16, a forkhead-type transcriptional factor, suppress the integrative defects of daf-2 and akt-1/-2 mutants. We further report that neither cGMP nor TGFβ pathways, two other dauer formation regulators, likely plays a role in the integrative learning. These results suggest that the insulin-like signaling pathway constitutes an essential component for sensory integration and decision-making behavior plasticity.
Animals ; Caenorhabditis elegans ; genetics ; physiology ; Caenorhabditis elegans Proteins ; genetics ; physiology ; Chemotaxis ; genetics ; physiology ; Copper ; physiology ; Cyclic GMP ; genetics ; physiology ; Diacetyl ; metabolism ; Insulin ; metabolism ; Longevity ; Signal Transduction ; Smell ; genetics ; physiology ; Taste ; genetics ; physiology ; Transforming Growth Factor beta ; genetics ; physiology

OBJECTIVETo clone the gene encoding methyl-accepting chemotaxis signal transduction protein (MCSTP) of Helicobacter hepaticus and analyze the gene structures using bioinformatics methods.

METHODSWith the specific primer of Helicobacter hepaticus MCSTP c1977, MCSTP gene was amplified by PCR from the genomic DNA of Helicobacter hepaticus and ligated to the prokaryotic expression vector pET22b(+). After sequencing, the sequence homology and structural feature of MCSTP gene were analyzed by bioinformatics method.

RESULTSA 99% similarity was identified between MCSTP gene cloned and its counterpart in standard Helicobacter hepaticus strain ATCC51449 genome DNA published by GenBank, with only a replacement of A by T at 1160 bp. A low homology was found in the MCSTP genes between Helicobacter hepaticus, Campylobacter jejuni and Helicobacter pylori by bioinformatics analysis, suggesting the specificity of MCSTP gene in Helicobacter hepaticus among the microbes.

CONCLUSIONThe prokaryotic expression plasmid pET22b(+)/MCSTP is constructed successfully, and the bioinformatics analysis provided evidences and clues for further study of the biological functions and pathogenic mechanism of MCSTP.

Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Computational Biology ; methods ; Genetic Vectors ; genetics ; Helicobacter hepaticus ; genetics ; isolation & purification ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Methyl-Accepting Chemotaxis Proteins ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction

OBJECTIVETo construct the murine CCL21 eukaryotic expression plasmid, and to investigate the chemotactic function of its products.

METHODSMurine CCL21 cDNA was amplified by RT-PCR from murine total RNA, and was inserted into eukaryotic expression plasmid pcDNA3.1 after confirmation of sequencing. The recombinant CCL21 plasmid was transferred into mouse forestomach carcinoma (MFC) cells and the chemotactic function of expressed products was detected by chemotaxis assay.

RESULTGene sequencing, gel electrophoresis of PCR products and restrictive digestion proved the successful construction of CCL21, and its expression was confirmed by Western Blot. The transfected tumor cells had a significant chemotactic function to DC.

CONCLUSIONThe recombinant murine CCL21 eukaryotic expression plasmid has been successfully constructed, and its expression products in tumor cells have a marked chemotactic function to DC.

Animals ; Base Sequence ; Chemokine CCL21 ; biosynthesis ; genetics ; Chemotaxis, Leukocyte ; Cloning, Molecular ; DNA, Complementary ; genetics ; Dendritic Cells ; drug effects ; immunology ; Genetic Vectors ; genetics ; Mice ; Mice, Inbred C57BL ; Molecular Sequence Data ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Stomach Neoplasms ; metabolism ; pathology ; Transfection ; Tumor Cells, Cultured

OBJECTIVETo investigate the effects of Annexin II (AnxA2) gene silencing by siRNA on proliferation and invasive potential of lymphoma cell line Jurkat cells.

METHODSA synthesized siRNA duplex targeting to AnxA2 was transfected into Jurkat cells. Transfection efficiency was analyzed by real-time PCR and flow cytometry. MTT assay for cell proliferation and transwell plates for invasive potential were performed.

RESULTSCompared with the negative controls, the cell proliferation inhibitory rate of the AnxA2 siRNA transfected Jurkat cells was significantly increased at 24 h, 48 h and 72 h [(17.4 +/- 2.3)%, (22.4 +/- 3.8)%, (37.6 +/- 1.5)% vs (-1.3 +/- 5.1)%, (-5.5 +/- 4.4)%, (-10.8 +/- 5.5)%, respectively, P<0.05]. The cell invasive potential of the transfected Jurkat cells was inhibited remarkably at 48 h (11.3 +/- 4.2 vs 54.3 +/- 8.7, P<0.01).

CONCLUSIONAnxA2 gene silenced by siRNA can inhibit the proliferation and the invasive potential of Jurkat cells remarkably.

Annexin A2 ; genetics ; metabolism ; Cell Proliferation ; Chemotaxis ; genetics ; Gene Silencing ; Humans ; Jurkat Cells ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the role of CD147 in the proliferation, activation and chemotaxis of Jurkat cell induced by cyclophilin A (CyPA).

METHODSCD147 mRNA and protein level siRNA transfected Jurkat cells were identified by RT-PCR and Western blot respectively. Jurkat cell, Jurkat-vector cell and Jurkat-CD147 siRNA cells were treated with different concentrations of CyPA (0.01 nmol/L, 0.1 nmol/L, 1 nmol/L, 10 nmol/L) or PBS for 24 h and 48 h. Proliferation level was detected by MTT assay. CD25 was measured by flow cytometry. Transwell chamber was used to detect the chemotaxis. The effect of CyPA on the adhesive potential of Jurkat cell was studied by cell-matrix adhesion assay.

RESULTSCD147 mRNA and protein level siRNA transfected cells were decreased significantly than that of control cells. CyPA stimulated the proliferation of Jurkat cell in a dose-dependent manner, its effect peaked at 10 nmol/L CyPA. Blockage of CD147 expression decreased the proliferation level of Jurkat cell induced by CyPA. CyPA increased the activation rate of Jurkat cell, and blockage CD147 expression decreased the activation rate of the cell induced by CypA. CyPA showed a chemotactic activity on Jurkat cell, the chemotaxis index being 2.32, and the chemotactic ability was decreased after inhibition of CD147 expression. CyPA had no effect on adhesion of Jurkat cell to extracellular matrix.

CONCLUSIONCD147 plays a role in the proliferation, activation and chemotaxis of Jurkat cell induced by CyPA.

Basigin ; genetics ; Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Chemotaxis ; drug effects ; Cyclophilin A ; pharmacology ; Humans ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Jurkat Cells ; RNA, Small Interfering ; genetics ; Transfection

UNLABELLEDOBJECTIVE To explore the effects of human macrophage inflammatory protein-1 beta (hMIP-1beta) modification on the in vivo tumorigenicity and vaccine efficacy of tumor cells.

METHODSMurine colorectal adenocarcinoma CT26 cells were transfected with a recombinant adenovirus carring the hMIP-1beta gene (AdhMIP-1beta). The efficacy of gene transfection was tested by X-gal staining. The hMIP-1beta level in the supernatant of hMIP-1beta gene-modified CT26 cells was assayed by ELISA, and the chemotactic activity for CD4+ T cells, CD8+ T cells, NK cells and immature dendritic cells (imDCs) were assayed by a transwell chamber. The changes of growth characteristics and in vivo tumorigenicity of hMIP-1beta gene-modified CT26 cells were also assessed. BALB/c mice were immunized with hMIP-1beta gene-modified CT26 tumor vaccine and the antitumor effect was evaluated.

RESULTShMIP-1beta gene could be transfected into CT26 cells by AdhMIP-1beta with an efficiency over 95%. The level of hMIP-1beta in the culture supernatant of hMIP-1beta gene-modified CT26 cells was 980 pg/ml and the supernatant displayed ramarkable chemotactic activity to CD4+ T cells, CD8+ T cells, NK cells and imDCs compared with LacZ gene-modified CT26 cells and control. When the hMIP-1beta gene-modifited CT26 cells were subcutaneously inoculated in BALB/c mice, the tumorigencity was delayed and suppressed, and overt necrosis and lymphocyte infiltration were observed in the tumor tissue, but not in those inoculated with LacZ gene-modified CT26 cells or parental CT26 cells. The mice immunized with hMIP-1beta gene-modified CT26 tumor vaccine could induce tumor specific CTL activity and nonspecific NK activity, and exhibited resistance to later challenge with wild-type CT26 cells.

CONCLUSIONhMIP-1beta gene-modified CT26 cells exhibit decreased tumorigenicity, and hMIP-1beta gene-modified tumor vaccine may induce a powerful specific and nonspecific antitumor response. The data suggested that hMIP-1beta gene-modified tumor vaccine may play a potent role in prevention of metastasis and recurrence of malignant tumors.

Adenocarcinoma ; metabolism ; pathology ; Adenoviridae ; genetics ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; Cell Line, Tumor ; Chemokine CCL4 ; genetics ; metabolism ; Chemotaxis, Leukocyte ; Colonic Neoplasms ; metabolism ; pathology ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Female ; Genetic Vectors ; Humans ; Killer Cells, Natural ; immunology ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden

Phytosphingosine-1-phosphate (PhS1P) was found to stimulate an intracellular calcium increase via phospholipase C but not pertussis toxin (PTX)- sensitive G-proteins in L2071 mouse fibroblasts. PhS1P also activated ERK and p38 kinase, and these activations by PhS1P were inhibited by PTX. Moreover, PhS1P stimulated the chemotactic migration of L2071 cells via PTX-sensitive Gi protein(s). In addition, the PhS1P-induced chemotactic migration of L2071 cells was also dramatically inhibited by LY294002 and SB203580 (inhibitors of phosphoinositide 3-kinase and p38 kinase, respectively). L2071 cells are known to express four S1P receptors, i.e., S1P1, S1P2, S1P3, and S1P4, and pretreatment with an S1P1 and S1P3 antagonist (VPC 23019) did not affect on PhS1P-induced chemotaxis. This study demonstrates that PhS1P stimulates at least two different signaling cascades, one is a PTX-insensitive but phospholipase C dependent intracellular calcium increase, and the other is a PTX-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and p38 kinase.
1-Phosphatidylinositol 3-Kinase/metabolism ; Animals ; Calcium Signaling/drug effects ; Chemotaxis/*drug effects ; Estrenes/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Fibroblasts/*cytology/*drug effects ; GTP-Binding Proteins/*metabolism ; Gene Expression Regulation/drug effects ; Humans ; Mice ; Pertussis Toxin/*pharmacology ; Phosphorylation/drug effects ; Pyrrolidinones/pharmacology ; RNA, Messenger/genetics/metabolism ; Receptors, Lysosphingolipid/genetics/metabolism ; Sphingosine/*analogs & derivatives/pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism