O fator de virulência relacionado ao H. pylori influencia a expressão de quimiocinas CXCL10, CCL17, CCL20, CCL22, e seus receptores por células mononucleares de sangue periféricos de pacientes com úlceras pépticas / The h. pylori-related virulence factor caga influences the expression of chemokines cxcl10, ccl17, ccl20, ccl22, and their receptors by peripheral blood mononuclear cells from peptic ulcer patients

ABSTRACT BACKGROUND: During the Helicobacter pylori (HP) infection, the infiltration of the leukocytes into stomach mucosa is directed by locally produced chemokines that play a decisive role in infection outcome. The CagA is the most potent virulence factor of HP, so that the infection with CagA + strains is associated with more severe complications than infection with CagA - HP. OBJECTIVE: The aim was to determine the expression of chemokines CXCL10, CCL17, CCL20 and CCL22, and their receptors by CagA + HP- and CagA - HP-derived crude extract (HP-CE)-stimulated peripheral blood mononuclear cells (PBMCs) from peptic ulcer (PU) patients. METHODS: The serum and the PBMCs were collected from 20 HP-infected PU patients, 20 HP-infected asymptomatic subjects (HIA) and 20 non-infected healthy subjects (NHS). The PBMCs were cultured in absence of stimulator or with 10 µg CagA + HP crude extract (CagA + CE), 10 µg CagA - HP crude extract (CagA - CE). Chemokines and receptors were measured by ELISA and real time-PCR respectively. RESULTS: In PU patients, the production of chemokines CXCL10, CCL17, CCL20 and CCL22, and the expression of chemokine receptors CXCR3, CCR4 and CCR6 by CagA + CE-induced PBMCs were significantly higher than non-stimulated and CagA - CE stimulated cultures. The CXCL10 production by CagA + CE stimulated PBMCs from HIA subjects was significantly higher than the equal cultures from PU and NHS groups. The CCL17 and the CCL20 production by non-stimulated, CagA + CE stimulated, and CagA - CE stimulated PBMCs from PU subjects were significantly higher than the equal cultures from NHS and HIA groups. The CCL22 production by non-stimulated, CagA + CE stimulated and CagA - CE stimulated PBMCs from NHS group were significantly higher than the equal cultures from HIA and PU groups. The CagA + CE stimulated PBMCs from HIA subjects expressed lower amounts of CCR6 in comparison with CagA + CE stimulated PBMCs from NHS and PU groups. The serum levels CXCL10 and CCL20 in PU and HIA groups were significantly higher than NHS subjects. NHS and HIA groups displayed higher serum levels of CCL22 in comparison with PU patients. CONCLUSION: Results indicated that the CagA status of bacterium influence the expression of chemokines and receptors by HP-CE stimulated PBMCs from PU patients.

RESUMO CONTEXTO: Durante a infecção por Helicobacter pylori (HP), a infiltração dos leucócitos na mucosa estomacal é dirigida por quimiocinas produzidas localmente que desempenham um papel decisivo no resultado da infecção. O CagA é o fator de virulência mais potente do HP, de modo que a infecção com cepas CagA + está associada a complicações mais graves do que a infecção com CagA - HP. OBJETIVO: O objetivo foi determinar a expressão das quimiocinas CXCL10, CCL17, CCL20 e CCL22, e seus receptores por CagA + HP- e CagA - extrato bruto (EB) derivado de HP (HP-EB) de células mononucleares do sangue periférico (CMSP) de pacientes com úlcera péptica (UP). MÉTODOS: O soro e as CMSP foram coletados de 20 pacientes com UP infectados pelo HP, 20 indivíduos assintomáticos infectados pelo HP (AI-HP) e 20 indivíduos saudáveis não infectados pelo HP (NI-HP). As CMSP foram cultivadas na ausência de estimulador ou com extrato bruto CagA + HP de 10 μg (CagA + EB), 10 μg CagA - extrato bruto HP (CagA - EB). Quimiocinas e receptores foram medidos por ELISA e PCR em tempo real, respectivamente. RESULTADOS: Em pacientes com UP a produção de quimiocinas CXCL10, CCL17, CCL20 e CCL22, e a expressão dos receptores de quimiocina CXCR3, CCR4 e CCR6 por CagA + CMSP induzidos pelo EB foram significativamente maiores do que as culturas não estimuladas e CagA - EB estimulados. A produção de CXCL10 por CagA + EB estimulou as CMSP de sujeitos AI-HP em proporção significativamente maior do que as culturas iguais dos grupos UP e NI-HP. A produção de CCL17 e CCL20 por grupos não estimulados, CagA + EB estimulado, e CagA - EB estimulou CMSP de sujeitos com UP e foram significativamente superiores às culturas iguais dos grupos NI-HP e AI-HP. A produção de CCL22 por grupos não estimulados, CagA + EB estimulado e CagA - EB estimulado pelo grupo NI-HP foram significativamente maiores do que as culturas iguais dos grupos AI-HP e PU. O CagA + EB estimulou as CMSP dos sujeitos do AI-HP, expressando menores quantidades de CCR6 em comparação com as CMSP estimuladas pelo CagA + EB de grupos NI-HP e UP. Os níveis sanguíneos de CXCL10 e CCL20 nos grupos UP e AI-HP foram significativamente superiores aos dos sujeitos do NI-HP. Os grupos NI-HP e AI-HP apresentaram níveis sanguíneos mais elevados de CCL22 em comparação com pacientes com UP. CONCLUSÃO: Os resultados indicaram que o estado CagA da bactéria influencia a expressão de quimiocinas e receptores por HP-EB estimulados CMSP de pacientes com UP.

Lymphocyte B and Th17 chemotactic cytokine levels in peri-implant crevicular fluid of patients with healthy, peri-mucositis, and peri-implantitis implants / Niveles de citoquinas quimiotácticas de linfocitos B y Th17 en el fluido crevicular periimplantario de pacientes con implantes sanos, perimucositis y periimplantitis

Peri-implantitis is one of the leading causes of implant failure and loss, and its early diagnosis is not currently feasible due to the low sensitivity of currents methods. In the current exploratory cross-sectional study, we explored the diagnostic potential of lymphocyte B and Th17-chemotactic cytokine levels in peri-implant crevicular fluid (PICF) in 54 patients with healthy, peri-mucositis, or peri-implantitis implants. Peri-implant crevicular fluid was collected, and the levels of the molecules under study were quantified by Luminex assay. The concentrations of CCL-20 MIP-3 alpha, BAFF/BLYS, RANKL and OPG concentration in PICF were analyzed in the context of patient and clinical variables (smoking status, history of periodontitis, periodontal diagnosis, implant survival, suppuration, bleeding on probing, periodontal probing depth, clinical attachment level, mean of implant probing depth, and plaque index). Patients with peri-implantitis, appear to have an overregulation of the RANKL/BAFF-BLyS axis. This phenomenon needs to be investigated in depth in further studies with a larger sample size.

La periimplantitis es una de las principales causas de falla y pérdida del implante, y su diagnóstico temprano no es factible debido a la baja sensibilidad de los métodos actuales. En este estudio transversal exploratorio, se estudió el potencial diagnóstico de los niveles de citocinas quimiotácticas de linfocitos B y Th17 en el líquido crevicular periimplantario (LCPI) en 54 pacientes con implantes sanos, peri-mucositis o periimplantitis. Se recogió líquido crevicular periimplantario y se cuantificaron los niveles de las moléculas estudiadas mediante Luminex assay. Las concentraciones de CCL-20 MIP-3 alfa, BAFF/BLYS, RANKL y la concentración de OPG en LCPI se analizaron en el contexto de las variables clínicas y del paciente (tabaquismo, antecedentes de periodontitis, diagnóstico periodontal, supervivencia del implante, supuración, sangrado al sondaje, profundidad de sondeo periodontal, nivel de inserción clínica, media de la profundidad de sondeo del implante e índice de placa). Los pacientes con periimplantitis parecen tener una sobrerregulación del eje RANKL/BAFF-BLyS. Este fenómeno debe investigarse en profundidad en futuros estudios con un tamaño de muestra mayor.

In vitro chemokine (C-C motif) receptor 6-dependent non-inflammatory chemotaxis during spermatogenesis

BACKGROUND: Chemokine (C-C motif) receptor 6 (CCR6) is present in sperm and plays a significant role in sperm motility and chemotaxis acting in the reproductive tracts. However, the expression and functional significance of CCR6 in testis are still poorly understood, especially in the process of spermatogenesis. METHODS AND RESULTS: CCR6 was expressed in spermatogenic cell lines and its expression was shown in an age-dependent upregulation manner from puberty to adulthood in mouse testis. Immunostaining results confirmed the localization of CCR 6 in testis. Further chemotaxis assays demonstrated that spermatogenic cells GC-1 and -2 exhibited a directional movement toward CCR6-specific ligand such as CCL20 or Sertoli cells in vitro. CONCLUSIONS: The present findings indicate that CCR6 is involved in the chemotaxis of spermatogenic cells in vitro and promotes chemotaxis under non-inflammatory conditions during normal spermatogenesis.

Relationship between CCL20/CCR6/Th17 axis and vascular invasion and metastasis in patients with primary hepatocellular carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the role of CCL20/CCR6/Th17 axis in vascular invasion and metastasis of primary hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expression levels of CCL20 mRNA in the normal human liver cell line L-02, and human hepatocellular carcinoma cell lines Hep3B, Huh7 and HepG2 were quantified by using SYBR green real time PCR. CCL20 secretions from these cell lines were quantified by using ELISA. The chemotactic effect of HCC cell line Hep3B on human peripheral blood mononuclear cells was determined by using transwell chemotaxis assay. Pre-therapy serum levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17, IL-23, IFN-γ, TNF-α and CCL20 in 93 patients with HCC were measured by using 9-plex array and ELISA. All the patients were chronic hepatitis B virus associated HCC, and 51 cases were those with vascular invasion and metastasis (metastasis group) and 42 cases were not (non-metastasis group). CCL20 and CCR6 mRNA expressions in the HCC and tumor-adjacent tissues were determined by using SYBR Green real time PCR in 41 patients, among them, 20 cases were from the group of patients with metastasis and 21 cases were from the group of patients without metastasis. The CCL20 expression was further determined by immunohistochemistry.</p><p><b>RESULTS</b>The HCC cell lines expressed and secreted higher amount of CCL20, which effectively recruited CCR6(+) T cells. Pre-therapy serum levels of CCL20 in 93 HCC patients were (38.2 ± 28.4)pg/ml, significantly increased than those with benign hepatic hemangiomas [(7.8 ± 17.8)pg/ml, P < 0.01]. In addition, the serum levels of CCL20 were positively correlated with the tumor diameters in HCC patients (r = 0.32, P = 0.0018). CCL20 was dominantly expressed in the cytoplasm in HCC cells, and it was also expressed by some infiltrating immune cells. The mRNA expression levels of CCL20 of the tumor tissues were significantly higher than that in the tumor-adjacent tissues (P < 0.05). Multivariate logistic regression analysis showed that serum levels of IL-17 and CCL20 were independent risk factors of metastasis in HCC patients (P < 0.05 for both). CCL20 mRNA showed no statistically significant differences between patients with metastasis and without metastasis in both tumor tissues and tumor-adjacent tissues (P > 0.05 for both). But the patients with metastasis showed significantly higher expressions of CCR6 both in their tumor [5.75 (1.79, 19.13)]and tumor-adjacent tissues [7.99 (4.49, 19.54)] than those with non-metastasis [1.69 (0.76, 2.87) and 3.58 (1.84, 4.32), P < 0.05 for both].</p><p><b>CONCLUSION</b>CCL20/CCR6/Th17 axis may promote vascular invasion and metastasis hepatocellular carcinoma.</p>

Design and expression of an inhibitor for HIV-1 targeting dendritic cell / 生物工程学报

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.

Expression of CCR6 and CCR7 in laryngeal squamous cell carcinoma / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To evaluate the expressions of chemokine receptor 6 (CCR6), chemokine receptor 7 (CCR7) and their ligands (CCL20, CCL19/CCL21) in laryngeal squamous cell carcinoma (LSCC), and then explore their correlation with the clinicopathological features of LSCC.@*METHOD@#Blood samples, fresh specimens of LSCC and paired adjacent tissues were collected. The expressions of CCR6, CCR7 and their ligands CCL20, CCL19/ CCL21 mRNA as well as the protein CCR6, CCR7 were detected by real-time qRT-PCR and IHC respectively. Flow cytometry was also used to investigate CCR6, CCR7 expressed on PBMC.@*RESULT@#The relative expression levels of CCR6, CCR7, CCL19 and CCL21 mRNA in tumor tissue was significantly higher than that of adjacent tissues (P < 0.05), while the relative expression level of CCL20 mRNA in tumor tissue were significantly lower than that of adjacent tissues (P < 0.05). IHC confirmed the expression of protein CCR6 and CCR7 in both tumor tissue and metastatic ILN and the expression levels of protein CCR6, CCR7 were higher in the cases with lymphatic metastasis than that of those without lymphatic metastasis (P < 0.05). FCM showed the percentage of CD4+ CCR6+ T cells of LSCC was significantly higher than that of normal control (P < 0.05), while that of CD4+ CCR7+ T cells was significantly lower (P < 0.05).@*CONCLUSION@#CCR6 and CCR7 are expressed in tumor situ, metastatic LN and PBMC,and might exert a potential role in LSCC development.

Regulation of immune suppressive cytokines by TLR4 activation in colon cancer cells / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the regulation of Toll-like receptors (TLRs) on immune suppressive cytokines in situ colon cancer cells.</p><p><b>METHODS</b>The mRNA and protein expression spectrum of TLRs in HT-29 cells were determined by RT-PCR and Western blot respectively. The cytokines and chemokines levels of supernant of HT-29 stimulated by lipoplysaccharide(LPS) were detected with ELISA.</p><p><b>RESULTS</b>TLR1-9 were expressed in HT-29 cells on mRNA level. After LPS stimulation, TLR4 mRNA and protein expressions were up-regulated in HT-29 cells, and TGF-beta, VEGF, IL-8, CCL20 and IL-6 levels increased significantly(all P<0.01). Except IL-6, up-regulation of the other cytokines was not suppressed by NF-kappa B inhibitor.</p><p><b>CONCLUSION</b>TLRs expressed on colon cancer cells may elevate the immune suppressive cytokines and chemokines, which promote the immune escape of cancer cells.</p>

Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin / 中华外科杂志

<p><b>OBJECTIVE</b>To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line (HaCaT).</p><p><b>METHODS</b>The three pHSER-CCL20-shRNA-GFP vectors (pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control) have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lentiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively.</p><p><b>RESULTS</b>The titers of three lentiviruses were 7.08 x 10(5) transduced units (TU)/ml, 1.88 x 10(5) TU /ml and 2.08 x 10(5) TU/ml, respectively. Two HaCaT cell clones from each lentiviral vectors were obtained after G418 screening for 5 - 8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly.</p><p><b>CONCLUSIONS</b>The four human immortal keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.</p>

Inhibitory Effect of CCL20 on CD4+ CD25+ regulatory T cell development in mouse thymus / 中国实验血液学杂志

The aim of this study was to investigate the roles of chemokine CCL20 in development of CD4(+)CD25(+) thymocytes by means of fetal thymus organ culture. Fetal mouse thymus lobes were removed at the fetus age of 14.5 days and cultured in complete RPMI 1640 with 20% FBS in vitro. Phenotypes of the thymocytes were analyzed by FACS and the number of cells per lobe was counted. The results revealed that from day 14.5 to day 19, the absolute and relative numbers of the CD4(+)CD25(+) thymocytes varied similarly as their development as in vitro culture at 6 days. Data showed that during the 6 days in vitro culture the CD4(+)CD25(+) cell percentage out of CD4(+) cells was 58.29%, 12.14%, 6.08%, 17.78%, 9.06%, 4.04% and the CD4(+)CD25(+) cell percentage out of CD25(+) cells was 3.75%, 10.81%, 17.20%, 51.93%, 61.64%, 80.06%. All these data indicated similar characters to their development in vivo. Moreover, at interference with CCL20, the percentage of CD4(+)CD25(+) T cells in thymocytes significantly decreased at the 3 and 6 days from 3.24+/-0.18 and 3.96+/-0.24 to 1.27+/-0.11 (p<0.001) and 1.76+/-0.22 (p<0.001) respectively. It is concluded that the development of CD4(+)CD25(+) thymocytes is similar both in vitro and in vivo, interfering with CCL20 significantly downregulate the expression of CD4(+)CD25(+) T cells. The above data may help to understand the development of naturally arising CD4(+)CD25(+) regulatory T cells.

Expression of CCL20 and CXCR4 in epidermal condyloma acuminatum lesions / 南方医科大学学报

<p><b>OBJECTIVE</b>To detect CCL20 and CXCR4 expressions in epidermis infected with condyloma acuminatum (CA) and normal epidermis and investigate the effect of their expressions on Langerhans cells in CA epidermis.</p><p><b>METHODS</b>Gene expression of CCL20 and CXCR4 in 3 epidermal CA lesions and in 3 normal epidermis specimens were detected using Affymetrix oligonucleotide microarrays HG-U 133A 2.0, and the protein levels of CCL20 and CXCR4 in these specimens were measured by Western blotting.</p><p><b>RESULTS</b>Microarray analysis revealed markedly down-regulated mRNA expressions of CCL20 and CXCR4 in the 3 epidermal CA lesions as compared with those in the normal specimens. Western blot analysis showed that the protein expressions of CCL20 and CXCR4 in the CA lesions were significantly lower than those in normal epidermis.</p><p><b>CONCLUSION</b>The protein and mRNA expressions of CCL20 and CXCR4 are markedly down-regulated in epidermal CA lesions, which may contribute to decreased number and backflow disturbance of Langerhans cells in these lesions.</p>

The expression of interleukin-17, interferon-gamma, and macrophage inflammatory protein-3 alpha mRNA in patients with psoriasis vulgaris / 华中科技大学学报（医学）（英德文版）

To investigate the role of Interleukin-17 (IL-17), Interferon-gamma (IFN-gamma), and macrophage inflammatory protein-3 alpha (MIP-3alpha) in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semi-quantitatively analyze the mRNA expression of IL-17, IFN-gamma, and MIP-3alpha in 31 psoriatic lesions and 16 normal skin tissues. The results showed that the mRNA of the three cytokines was present in all specimens. And the expression level of IL-17 mRNA in skin lesions was 1.1416 +/- 0.0591, which was significantly higher than that in normal controls (0.8788 +/- 0.0344, P<0.001). The expression levels of IFN-gamma mRNA were 1.1142 +/- 0.0561 and 0. 9050 +/- 0.0263, respectively, with significant difference (P<0.001). And the expression levels of MIP-3alpha mRNA in psoriatic lesions was 1.1397 +/- 0.0521, which was markedly higher than that in normal controls (0.8681 +/- 0.0308, P<0.001). These findings indicate that up-regulated expression of IL-17, IFN-gamma, and MIP-3alpha might be involved in the pathogenesis of psoriasis.

Expression of CC chemokine ligand 20 and CC chemokine receptor 6 mRNA in patients with psoriasis vulgaris / 华中科技大学学报（医学）（英德文版）

In order to explore the possible role of CC chemokine ligand 20 (CCL20) and its receptor CC chemokine receptor 6 (CCR6) in the pathogenesis of psoriasis, the expression levels of mRNA of them in psoriatic lesions were investigated. The skin biopsies were collected from skin lesions in 35 cases of psoriasis vulgaris and 18 normal controls. RT-PCR was used to semi-quantitatively analyze the mRNA expression of CCL20 and CCR6 in the psoriatic lesions and the normal skin tissues. The results showed that the mRNA of CCL20 and CCR6 was present in every specimen. The expression levels of CCL20 mRNA in skin lesions were 1.1397 +/- 0.0521, which were greatly higher than those in normal controls (0.8681 +/- 0.0308) (P<0.001). The expression levels of CCR6 mRNA in skin lesions were 1.1103 +/- 0.0538, significantly higher than in the controls (0.9131 +/- 0.0433, P<0.001). These findings indicate that up-regulated expression of CCL20 and CCR6 mRNA might be related to the pathogenesis of psoriasis.