Effect and mechanism of Xihuang Pills on rats with precancerous lesions of breast / 中国中药杂志

The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P<0.05 or P<0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P<0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P<0.05 or P<0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P<0.05 or P<0.01), and the protein expression of Bcl-2 was lowered(P<0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P<0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P<0.01), while an increase in the mRNA expression of caspase-3(P<0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P<0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.

Effect of naringenin on the anti-inflammatory, vascularization, and osteogenesis differentiation of human periodontal ligament stem cells via the stromal cell-derived factor 1/C-X-C motif chemokine receptor 4 signaling axis stimulated by lipopolysaccharide / 华西口腔医学杂志

OBJECTIVES@#This study aimed to investigate how naringenin (Nar) affected the anti-inflammatory, vascula-rization, and osteogenesis differentiation of human periodontal ligament stem cells (hPDLSCs) stimulated by lipopolysaccharide (LPS) and to preliminarily explore the underlying mechanism.@*METHODS@#Cell-counting kit-8 (CCK8), cell scratch test, and Transwell assay were used to investigate the proliferation and migratory capabilities of hPDLSCs. Alkaline phosphatase (ALP) staining, alizarin red staining, lumen-formation assay, enzyme-linked immunosorbent assay, quantitative timed polymerase chain reaction, and Western blot were used to measure the expression of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), vascular endothlial growth factor (VEGF), basic fibroblast growth factor (bFGF), von Willebrand factor (vWF), tumor necrosis factor-α (TNF-α), and interleukin (IL)-6.@*RESULTS@#We observed that 10 μmol/L Nar could attenuate the inflammatory response of hPDLSCs stimulated by 10 μg/mL LPS and promoted their proliferation, migration, and vascularization differentiation. Furthermore, 0.1 μmol/L Nar could effectively restore the osteogenic differentiation of inflammatory hPDLSCs. The effects of Nar's anti-inflammatory and promotion of osteogenic differentiation significantly decreased and inflammatory vascularization differentiation increased after adding AMD3100 (a specific CXCR4 inhibitor).@*CONCLUSIONS@#Nar demonstrated the ability to promote the anti-inflammatory, vascularization, and osteogenic effects of hPDLSCs stimulated by LPS, and the ability was associated with the stromal cell-derived factor/C-X-C motif chemokine receptor 4 signaling axis.

Novo Efeito Cardioprotetor da L-Carnitina em Camundongos Obesos Diabéticos: Regulação da Expressão de Quemerina e CMKLRI no Coração e Tecidos Adiposos / Novel Cardioprotective Effect of L-Carnitine on Obese Diabetic Mice: Regulation of Chemerin and CMKLRI Expression in Heart and Adipose Tissues

Resumo Fundamentos A L-carnitina (LC) tem muitos efeitos benéficos em animais diabéticos e humanos, mas seu efeito regulatório sobre a quemerina como uma citocina inflamatória e seu receptor no estado diabético são desconhecidos. Objetivos O presente estudo teve como objetivo investigar o efeito regulatório da LC na expressão do receptor semelhante ao de quimiocina 1 e quemerina (CMKLRI) em tecidos adiposo e cardíaco de camundongos diabéticos. Métodos Sessenta camundongos NMARI foram divididos em quatro grupos, incluindo controle, diabético, diabético + suplementação com LC e controle + suplementação com LC. O diabetes foi induzido pela alimentação dos animais com dieta hipercalórica por 5 semanas e injeção de estreptozotocina. Os animais foram tratados com 300 mg/kg de LC por 28 dias. Nos dias 7, 14 e 28 após o tratamento, os níveis de mRNA e proteína da quemerina e CMKLRI nos tecidos cardíacos e adiposos de animais foram determinados utilizando análise por qPCR e ELISA. Os índices de resistência à insulina também foram medidos em todos os grupos experimentais. A diferença com p<0,05 foi considerada significativa. Resultados A expressão de quemerina e CMKLRI aumentou nos tecidos cardíaco e adiposo de camundongos diabéticos nos dias 14 e 28 após a indução do diabetes, concomitantemente com a incidência de resistência à insulina e níveis aumentados de quemerina circulante (p<0,05). O tratamento com LC causou uma diminuição significativa na expressão de ambos os genes nos tecidos estudados e redução dos sintomas de resistência à insulina e dos níveis séricos de quemerina (p<0,05). Conclusão Os resultados sugerem que o tratamento com LC pode diminuir a expressão de quemerina e CKLR1 em tecidos cardíacos e adiposos de animais experimentais obesos e diabéticos.

Abstract Background L-carnitine (LC) has many beneficial effects on diabetic animals and humans, but its regulatory effect on chemerin as an inflammatory cytokine, and its receptor in diabetes status is unknown. Objectives The present study aimed to investigate the regulatory effect of LC on the expression of chemerin and chemokine-like receptor I (CMKLRI) in adipose and cardiac tissues of diabetic mice. Methods Sixty NMARI mice were divided into four groups including control, diabetic, diabetic + LC supplementation and control + LC supplementation. Diabetes was induced by feeding the animals a high-calorie diet for 5 weeks and injection of Streptozotocin. The animals were treated with 300 mg/kg LC for 28 days. On days 7, 14, and 28 after treatment, the mRNA and protein levels of chemerin and CMKLRI in the cardiac and adipose tissues of the animals were determined using qPCR analysis and ELISA. Insulin resistance indices were also measured in all experimental groups. Differences with p <0.05 were considered significant. Results Chemerin and CMKLRI expressions levels were increased in cardiac and adipose tissues of diabetic mice on days 14 and 28 after diabetes induction, concurrent with the incidence of insulin resistance and increased levels of circulating chemerin (p<0.05). The treatment with LC caused a significant decrease in the expression of both genes in studied tissues and the reduction of insulin resistance symptoms and serum chemerin levels (p<0.05). Conclusion The results suggest that LC treatment were able to downregulate the expression of chemerin and CKLR1 in cardiac and adipose tissues of obese, diabetic experimental animals.

Screening of Variants in the Transcript Profile of Eutopic Endometrium from Infertile Women with Endometriosis during the Implantation Window / Rastreio de variantes no perfil de tanscritos do endométrio eutópico de mulheres inférteis com endometriose durante a janela de implantação

Abstract Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial

Resumo Objetivo Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação. Métodos Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença. Resultados Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez. Conclusão Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.

O receptor de quimiocina CXCR3/CD183 na tuberculose / The chemokine receptor CXCR3/CD183 in tuberculosis

Introdução: o receptor CXCR3/CD183 juntamente com seu indutor IFNy e seus ligantes CXCL9, CXCL10 e CXCL11 têm sido descritos como de grande importância na resposta imune do perfil T helper 1 (Th1). Este grupo de quimiocinas é expresso no microambiente e permite a migração de células ao sítio da infecção para combater o patógeno. Objetivo: revisar o atual estado da arte sobre o papel do receptor CXCR3/CD183 na tuberculose. Metodologia: o presente estudo inclui a revisão narrativa de 12 artigos que foram selecionados a partir de 74 artigos encontrados nas bases de dados PubMed e Sciencedirect entre primeiro de agosto e 31 de outubro de 2014. Resultados: diferentes abordagens vêm sendo utilizadas para o estudo desse receptor. A utilização de modelos animais como camundongos, coelhos e macacos é a mais comum. Porém, ensaios in vitro com células humanas do sangue periférico e efusão pleural também já foram utilizados para representar, com maior fidelidade, a resposta ao Mycobacterium tuberculosis (Mtb) pelo sistema imune humano. Esses estudos resultaram em importantes achados sobre o papel do receptor CXCR3 na tuberculose (TB), principalmente quanto à expressão em linfócitos e neutrófilos, assim como o padrão de coexpressão de outros receptores. Conclusão: o CXCR3 é o receptor de uma importante citocina (IP-10) induzida pelo IFN-gama, produzida na resposta Th1, eficaz na resposta à tuberculose. Nesse trabalho, resssalta-se que foram encontrados poucos estudos sobre o tema e isso demonstra a necessidade de realização de novas pesquisas, a fim de melhor investigar o papel desse importante receptor na tuberculose.

Introduction: the CXCR3/CD183 receptor along with its IFNy inducer and its ligands: the chemokines named CXCL9, CXCL10 and CXCL11 are of great importance in the Th1 (T helper 1) immune response. This group of chemokine modulates the migration of cells to the site of infection to defend against the pathogen. Objective: to investigate the current state of the art on the role of the receptor CXCR3/CD183 in tuberculosis. Methodology: the present study includes the narrative review of 12 articles that were selected from 74 articles found in the PubMed and Sciencedirect databases between August 1 and October 31, 2014. Results: different approaches have been used for the study of this receptor. The use of animal models such as mice, rabbits and monkeys is more common. However, in vitro assays with human peripheral blood cells and pleural effusion were also used to represent more faithfully the response to Mycobacterium tuberculosis (Mtb) by the human immune system. These studies resulted in significant findings on the role of the CXCR3 receptor in tuberculosis (TB), especially for expression in lymphocytes and neutrophils, as well as the pattern of co-expression of other receptors. Conclusion: CXCR3 is the receptor for an important cytokine (IP-10) induced by IFN-gamma, produced in the Th1 response, effective in responding to tuberculosis. In this work, it is emphasized that cheeses found few studies on the subject and demonstration, the need for conducting research, in order to better investigate the role of this important receptor in tuberculosis.

Avaliação da expressão apical de fatores pró-angiogênicos e citocinas em reposta à infecção endodôntica / Evaluation of apical expression of pro-angiogenic factors and cytokines in response to endodontic infection

A progressão e desenvolvimento de patologias pulpares e periapicais estão intimamente relacionados à presença de microrganismos e seus subprodutos nos sistema de canais radiculares (SCR), que induzem uma resposta de defesa adjacente ao ápice radicular. A angiogênese é apontada como fator essencial na patogênese das alterações periapicais crônicas, estando relacionada ao seu estabelecimento e manutenção, por ser fonte constante de citocinas, quimiocinas e proteases. A angiogênse também está relacionada ao reparo tecidual que segue à resolução das alterações perirradiculares após a realização da terapia endodôntica. Neste estudo, avaliou-se a expressão de fatores pró-angiogêncios e citocinas relacionadas, em amostras coletadas de pacientes (n=20) com dentes portadores de Periodontite Apical Crônica imediatamente após a instrumentação dos SCR e 7 dias após os procedimentos de desinfecção. As amostras foram analisadas por meio de reação em cadeia da polimerase em tempo real (PCR-RT). Verificou-se a expressão gênica de fatores pró-angiogênicos e citocinas Angiopoetina-1 (AGT1), Fator de crescimento endotelial vascular-A (VEGF-A), Fator de crescimento fibroblástico básico (FGF-ß), Proteína quimiotática de monócitos (CCL2/MCP-1), Proteína inflamatória de macrófagos-1ß (CCL4), C-X-C Receptor de quimiocina tipo 4 (CXCR4), C-C Receptor de quimiocina tipo 6 (CCR6), TNF-α, IFN-γ, IL-17, IL-10, IL6, RANK-L e MMP-9. A expressão do mRNA dos mediadores avaliados revelou aumento significativo nos níveis de AGT1, CCL2/MCP-1, CCL4, CCR6, TNF-α, IFN- γ, IL-10, RANK-L e MMP-9 no dia 7 quando comparado com o dia 0 (P <0,05). Para VEGF-A, FGF-ß, CXCR4, IL-17 e IL-6 as expressões de mRNA foram semelhantes em ambos tempos mensurados (P> 0,05). Pode-se concluir que, após desinfecção do SCR, houve aumento nos níveis de expressão de mRNA de importantes mediadores envolvidos nos fenômenos angioproliferativos e osteogênicos.(AU)

The progression and development of pulpal and periapical pathologies are closely related to the presence of microorganisms and their by-products in the infected root canal system (RCS), which induces a defense response adjacent to the root apex. Angiogenesis has been identified as an essential factor in the pathogenesis of chronic periapical alterations, being related to its establishment and maintenance, being a constant source of cytokines, chemokines and proteases. Angiogenesis is also related to the tissue repair that follows the resolution of the periradicular alterations after the implementation of endodontic therapy. In this study, was evaluated the expression of pro-angiogenic factors and correlated cytokines in samples collected from patients (n = 20) on teeth with Chronic Apical Periodontitis immediately after RCS instrumentation and 7 days after disinfection procedures. Samples were analyzed by real-time polymerase chain reaction (RT-PCR). The gene expression of pro-angiogenic factors and cytokines Angiopoetin-1 (AGT1), Vascular endothelial growth factor-A (VEGF-A), Basic fibroblast growth factor (FGF-ß), Monocyte Chemoattractant Protein-1 (CCL2/MCP-1), Macrophage inflammatory protein-1ß (CCL4), C-X-C chemokine receptor motif 4 (CXCR4), C-C chemokine receptor motif 6 (CCR6), TNF-α, IFN-γ, IL-17, IL-10, IL-6, RANK-L and MMP-9. The mRNA expression of the mediators evaluated revealed a significant increase in levels of AGT1, CCL2/MCP-1, CCL4, CCR6, TNF-α, IFN-γ, IL-10, RANK-L and MMP-9 on day 7 when compared to day 0 (P <0.05). As for VEGF-A, FGF-ß, CXCR4, IL-17 and IL-6 their mRNA expressions was similar at both observed times (P> 0.05). In conclusion, after cleaning and shaping procedures of the RCS, there was an increase in mRNA expression levels of important mediators involved in angioproliferative and osteogenic phenomenon.(AU)

Evaluation of chemokines and receptors in gnotobiotic root canal infection by F. nucleatum and E. faecalis

Abstract The present study aims to evaluate the longitudinal effects of induced experimental infections in gnotoxenic animals on the expression of inflammatory chemokines and their receptors in periradicular tissues. The null hypothesis tested was that Enterococcus faecalis and Fusobacterium nucleatum had no effect on CCR5, CCL5, CXCL10, CCL2/MCP-1, CXCR2 and CCR1 expression. Two groups of five animals (n = 5) aged between 8 and 12 weeks were used in this study. The animals were anaesthetized, and coronary access was performed in the first molar on the right and left sides. Microorganisms were inoculated into the left molar, and the right molar was sealed without contamination to function as a control. Animals were sacrificed 7 and 14 days after infection, and periapical tissues were collected. The cytokine mRNA expression levels were assessed using real-time PCR. The chemokine mRNA expression levels demonstrated that the experimental infection was capable of inducing increased chemokine expression on day 7 compared to that on day 14, except for CCR5 and CCL5, which showed no changes. The gnotoxenic animal model proved to be effective and allowed evaluation of the immune response against a known infection. Additionally, this study demonstrates that gene expression of chemokines and their receptors against the experimental infection preferentially prevailed during the initial phase of induction of the periradicular alteration (i.e., on day 7 post-infection).

Role of chemerin/CMKLR1 in the maintenance of early pregnancy / 医学前沿

Chemerin is a cytokine that attracts much attention in the reproductive process. This study aimed to explore the effects of chemerin and its receptor chemokine-like receptor 1 (CMKLR1) on the maintenance of early pregnancy. The expression levels of chemerin and CMKLR1 in the decidua tissues of 20 early normal pregnant women and 20 early spontaneous abortion women were examined by Western blot and real-time polymerase chain reaction analyses. CMKLR1 receptor antagonist (α-NETA) was then intrauterinely injected into normal pregnant mice model to assess its effect on the outcome of pregnancy and the phosphorylation rate of ERK1/2 in decidua tissues.We found that the expression level of chemerin in women who had experienced early spontaneous abortion was lower than in those who had experienced normal early pregnancy (P < 0.01); conversely, CMKLR1 expression was higher in the former than in the latter (P < 0.01). In a pregnant-mouse model, the embryo resorption rate of α-NETA group was higher than that in the negative control group (61.5% vs. 10.8%) (P < 0.001). Compared with the control group, ERK1/2 phosphorylation in decidua tissues decreased in the α-NETA-treated group (P < 0.01). These results suggested that the inhibition of the chemerin/CMKLR1 signaling pathway can lead to the abortion of mouse embryos, and that chemerin/CMKLR1 may play an important role in the maintenance of early pregnancy possibly by regulating ERK1/2 phosphorylation.

Association of Chemokines and Chemokine Receptor Expression with Monocytic-Myeloid-Derived Suppressor Cells during Tumor Progression

Myeloid-derived suppressor cells (MDSCs) are highly immunosuppressive myeloid cells that show increased expression in cancer patients; however, the molecular mechanisms underlying their generation and function are unclear. Whereas granulocytic-MDSCs correlate with poor overall survival in breast cancer (BC), the presence and relevance of monocytic (Mo)-MDSCs are unknown. Here, we report for the first time increased chemokine and chemokine receptor production by Mo-MDSCs in BC patients. A clear population of Mo-MDSCs with the typical cell surface phenotype (human leukocyte antigen-antigen D related [HLA-DR]low/− CD11b+ CD33+ CD14+) increased significantly during disease progression. In addition, the chemokine receptor expression level on Mo-MDSCs in patients with invasive BC was the highest. Furthermore, different chemokine receptor expression patterns were noted in Mo-MDSCs between healthy controls (HC) and BC patients. Additionally, CD4 T cells proliferations were significantly decreased in the invasive BC groups compared with the HC group. However, the ductal carcinoma in situ (DCIS) group had no significantly compared with the HC group. Our data suggest that monitoring chemokine and chemokine receptor production by Mo-MDSCs may represent a novel and simple biomarker for assessing disease progression in BC patients.

Neuron-derived CCL2 contributes to microglia activation and neurological decline in hepatic encephalopathy

BACKGROUND: CCL2 was up-regulated in neurons and involved in microglia activation and neurological decline in mice suffering from hepatic encephalopathy (HE). However, no data exist concerning the effect of neuron-derived CCL2 on microglia activation in vitro. METHODS: The rats were pretreated with CCL2 receptor inhibitors (INCB or C021, 1 mg/kg/day i.p.) for 3 days prior to thioacetamide (TAA) administration (300 mg/kg/day i.p.) for inducing HE model. At 8 h following the last injection (and every 4 h after), the grade of encephalopathy was assessed. Blood and whole brains were collected at coma for measuring CCL2 and Iba1 expression. In vitro, primary neurons were stimulated with TNF-α, and then the medium were collected for addition to microglia cultures with or without INCB or C021 pretreatment. The effect of the medium on microglia proliferation and activation was evaluated after 24 h. RESULTS: CCL2 expression and microglia activation were elevated in the cerebral cortex of rats received TAA alone. CCL2 receptors inhibition improved neurological score and reduced cortical microglia activation. In vitro, TNF-α treatment induced CCL2 release by neurons. Medium from TNF-α stimulated neurons caused microglia proliferation and M1 markers expression, including iNOS, COX2, IL-6 and IL-1ß, which could be suppressed by INCB or C021 pretreatment. The medium could also facilitate p65 nuclear translocation and IκBα phosphorylation, and NF-κB inhibition reduced the increased IL-6 and IL-1ß expression induced by the medium. CONCLUSION: Neuron-derived CCL2 contributed to microglia activation and neurological decline in HE. Blocking CCL2 or inhibiting microglia excessive activation may be potential strategies for HE.

Chemokine Receptors Expression in MSCs: Comparative Analysis in Different Sources and Passages

MSC-based therapy is providing a cure for degenerative diseases with unmet medical need and usually iliac crest bone marrow (ICBM) are being applied in clinics. Alternative sources, including adipose tissue and reamer/irrigator/ aspirator hold great potential for isolating MCSs. Here, we compared original MSCs features of adipose tissue (Ad-MSCs) and bone marrow of long-bone (RIA-MSCs) or iliac crest, and the expression of chemokine receptors (including CXCR4, CX3CR1, CXCR6, CXCR2, CCR1 and CCR7) in these three sources, which are important in the context of homing. We further investigated the role of SDF-1/CXCR4 axis as a key player in motility of different population of MSCs using Transwell migration assay. All cells exhibited typical MSCs characteristics. However, different MSCs sources expressed different levels of chemokine receptors. Generally, the expression of these chemokine receptors was decreased with increasing passage (P) number from 2 to 3. Interestingly, it was observed that the CXCR4 expression and migration capacity in Ad-MSCs is significantly higher than ICBM and RIA-MSCs in P2. Although our data showed that CXCR4 had highest expression in P2 Ad-MSCs, but it dramatically declined following sub-culturing in the P3. Hence, to improve homing of MSCs by means of chemokine/their receptors axis, the source of isolation and passage number should be considered for clinical applications.

Serosal Cavities Contain Two Populations of Innate-like integrin α4highCD4+ T Cells, Integrin α4β1+α6β1+α4β7− and α4β1+α6β1−α4β7+ Cells

We previously reported peritoneal innate-like integrin α4 (CD49d)highCD4+ T cells that provided help for B-1a cells. Here we analyzed the expression of various integrin chains on the peritoneal and pleural integrin α4highCD4+ T cells and investigated the functional heterogeneity of the subpopulations based on the integrin expression. Pleural cavity contained a lower ratio of integrin α4highCD4+ T cells to integrin α4lowCD4+ T cells than peritoneal cavity, but the pleural integrin α4highCD4+ T cells have the same characteristics of the peritoneal integrin α4highCD4+ T cells. Most of integrin α4highCD4+ T cells were integrin β1highβ7−, but a minor population of integrin α4highCD4+ T cells was integrin β1+β7+. Interestingly, the integrin α4highβ1highβ7− CD4+ T cells expressed high levels of integrin α4β1 and α6β1, whereas integrin α4highβ1+β7+ CD4+ T cells expressed high levels of integrin α4β1 and α4β7, suggesting an alternative expression of integrin α6β1 or α4β7 in combination with α4β1 in respective major and minor populations of integrin α4highCD4+ T cells. The minor population, integrin α4highβ1+β7+ CD4+ T cells, were different from the integrin α4highβ1highβ7− CD4+ T cells in that they secreted a smaller amount of Th1 cytokines upon stimulation and expressed lower levels of Th1-related chemokine receptors CCR5 and CXCR3 than the integrin α4highβ1 highβ7− CD4+ T cells. In summary, the innate-like integrin α4highCD4+ T cells could be divided into 2 populations, integrin α4β1+α6β1+α4β7− and α4β1+α6β1−α4β7+ cells. The functional significance of serosal integrin α4β7+ CD4+ T cells needed to be investigated especially in view of mucosal immunity.

Hematopoietic progenitor cell mobilization for autologous transplantation - a literature review

ABSTRACT The use of high-dose chemotherapy with autologous support of hematopoietic progenitor cells is an effective strategy to treat various hematologic neoplasms, such as non-Hodgkin lymphomas and multiple myeloma. Mobilized peripheral blood progenitor cells are the main source of support for autologous transplants, and collection of an adequate number of hematopoietic progenitor cells is a critical step in the autologous transplant procedure. Traditional strategies, based on the use of growth factors with or without chemotherapy, have limitations even when remobilizations are performed. Granulocyte colony-stimulating factor is the most widely used agent for progenitor cell mobilization. The association of plerixafor, a C-X-C Chemokine receptor type 4 (CXCR4) inhibitor, to granulocyte colony stimulating factor generates rapid mobilization of hematopoietic progenitor cells. A literature review was performed of randomized studies comparing different mobilization schemes in the treatment of multiple myeloma and lymphomas to analyze their limitations and effectiveness in hematopoietic progenitor cell mobilization for autologous transplant. This analysis showed that the addition of plerixafor to granulocyte colony stimulating factor is well tolerated and results in a greater proportion of patients with non-Hodgkin lymphomas or multiple myeloma reaching optimal CD34+ cell collections with a smaller number of apheresis compared the use of granulocyte colony stimulating factor alone.

Inducing Effect of Modified Cytokine Cocktail on Dendritic Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the inducing effect of 'modified' cytokine cocktail on the dendritic cell maturation and migration capability.</p><p><b>METHODS</b>PBMNC were isolated from human peripheral blood stem cell (PBSC) by using density gradient centrifugation, the immature DC (imDC) were induced by using GM-CSF and IL-4 in vitro. Total A549 RNA was transfected into imDC by using electroporation, which was stimulated to matuation by the "gold standard" cytokine cocktail and "modified" cytokine cocktail, respectively. The expression of DC surface markers (CD11c, HLA-DR, CD80, CD83 and CD86) and chemokine receptor (CCR5, CCR7 and CXCR4) were detected by flow cytometry; the mRNA expression levels of DC chemokine receptor (CCR2, CCR5, CCR7, CXCR3 and CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12) were detected by RT-PCR.</p><p><b>RESULTS</b>As compared with "gold standard cytokine cocktail", the "modified" cytokine cocktail-induced DC expressed higher levels of surface markers (CD11c, HLA-DR, CD80, CD83 and CD86), chemokine receptors (CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12).</p><p><b>CONCLUSION</b>The "modified" cytokine cocktail can more effectively induce the DC maturation, enhace the migratory capability of DC and more generate the immunostimulatory DC, when compared with the "gold standard" cytokine cocktail effect.</p>

Differential Chemokine Signature between Human Preadipocytes and Adipocytes

Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity.

Sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.

Activation and Recruitment of Regulatory T Cells via Chemokine Receptor Activation in Trichinella spiralis-Infected Mice

As most infections by the helminth parasite elicit the recruitment of CD4+CD25+Foxp3+ T (T(reg)) cells, many scientists have suggested that these cells could be used for the treatment of immune-mediated inflammation and associated diseases. In order to investigate the distribution and alteration of activated T(reg) cells, we compared the expression levels of T(reg) cell activation markers in the ileum and gastrocnemius tissues 1, 2, and 4 weeks after infection. The number of T(reg) cells was monitored using GFP-coded Foxp3 transgenic mice. In mice at 1 week after Trichinella spiralis infection, the number of activated T(reg) cells was higher than in the control group. In mice at 2 weeks after infection, there was a significant increase in the number of cells expressing Foxp3 and CTLA-4 when compared to the control group and mice at 1 week after infection. At 4 weeks after infection, T. spiralis was easily identifiable in nurse cells in mouse muscles. In the intestine, the expression of Gzmb and Klrg1 decreased over time and that of Capg remained unchanged for the first and second week, then decreased in the 4th week. However, in the muscles, the expression of most chemokine genes was increased due to T. spiralis infection, in particular the expression levels of Gzmb, OX40, and CTLA-4 increased until week 4. In addition, increased gene expression of all chemokine receptors in muscle, CXCR3, CCR4, CCR5, CCR9, and CCR10, was observed up until the 4th week. In conclusion, various chemokine receptors showed increased expressions combined with recruitment of T(reg) cells in the muscle tissue.

Receptor expression and responsiveness of human peripheral blood mononuclear cells to a human cytomegalovirus encoded CC chemokine

Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitrohas the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitroinvestigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.

Avaliação das quimiocinas e da expressão de seus receptores em células mononucleares de pacientes portadores da doença de Chagas submetidas à infecção in vitro com Trypanosoma cruzi e tratamento com benzonidazol / Evaluation of chemokines and their receptors expression in patient mononuclear patients of Chagas disease undergoing in vitro infection with Trypanosoma cruzi and treatment with benznidazole

Atualmente o Brasil apresenta 3 milhões de indivíduos portadores da cardiomiopatia chagásica. Porém, tratamento etiológico com o fármaco Benzonidazol (BZ) na fase crônica da doença ainda não está elucidado. Acredita-se que a recomendação do BZ nessa fase, pode prevenir ou retardar a evolução clínica da cardiomiopatia na Doença Chagas (DC). Assim o objetivo do estudo é avaliar a produção de quimiocinas e expressão de seus receptores em Células mononucleares do sangue periférico - PBMC (de portadores crônicos da doença de Chagas) submetidas in vitro ao tratamento com BZ, após a infecção com T.cruzi. Foram selecionados 11 pacientes na fase crônica da doença. Amostras de sangue desses pacientes foram coletadas para obtenção de PBMC, em que foram cultivadas em placas de cultivo na concentração de 106 células/ml por poço. Após a adesão das células aderentes (principalmente macrófagos), as células não aderentes (principalmente linfócitos) foram removidas e as formas tripomastigotas foram adicionadas ao cultivo para infecção das células aderentes. Subsequente a incubação, as células não aderentes foram adicionadas novamente ao cultivo juntamente com o fármaco Bz (1µg/mL), ficando um co-cultivo de células aderentes infectadas com T.cruzi, células não aderentes e o BZ (C+T+BZ). As placas de cultura foram incubadas por períodos de 24h e 5 dias. Para uma análise fidedigna da ação do BZ nas células aderentes e não aderentes foi necessário a criação dos controles: células (C), células e tripomastigotas (C+T) e células e o BZ (C+BZ). Após o cultivo, foram coletados os sobrenadantes das culturas, para avaliação da produção de quimiocinas (CCL2, CXL9, CXL10, CCL5 e CXCL8) por CBA (Cytometric Bead Array). Posteriormente foi realizada a imunofenotipagem...