Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells / 中国结合医学杂志

OBJECTIVE@#To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action.@*METHODS@#This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively.@*RESULTS@#GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+ and CD45+CD4+ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05).@*CONCLUSION@#GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

E.faecium QH06 alleviates TNBS-induced colonic mucosal injury in rats / 南方医科大学学报

OBJECTIVE@#To investigate the effect of Enterococcus faecium QH06 on TNBS-induced ulcerative colitis (UC) in rats and explore the mechanisms in light of intestinal flora and intestinal immunity.@*METHODS@#Thirty-six male Wistar rats were randomized equally into control group, UC model group, and E.faecium QH06 intervention group. The rats in the latter two groups were subjected to colonic enema with 5% TNBS/ethanol to induce UC, followed by treatment with intragastric administration of distilled water or E.faecium QH06 at the dose of 0.21 g/kg. After 14 days of treatment, the rats were examined for colon pathologies with HE staining. The mRNA and protein expression levels of IL-4, IL-10, IL-12, and IFN-γ in the colon tissues were detected using RT-qPCR and ELISA, and the expression of TLR2 protein was detected with immunohistochemistry and ELISA. Illumina Miseq platform was used for sequencing analysis of the intestinal flora of the rats with bioinformatics analysis. The correlations of the parameters of the intestinal flora with the expression levels of TLR2 and cytokines were analyzed.@*RESULTS@#The rats with TNBS- induced UC showed obvious weight loss (P < 0.01) and severe colon tissue injury with high pathological scores (P < 0.01). The protein expression levels of IFN-γ, IL-12, and TLR2 (P < 0.01) and the mRNA expression levels of IFN-γ, IL-12 and IL-10 (P < 0.05) were significantly increased in the colon tissues of the rats with UC. Illumina Miseq sequence analysis showed that in UC rats, the Shannon index (P < 0.05) ACE (P < 0.01)and Chao (P < 0.05) index for the diversity of intestinal flora both decreased with a significantly increased abundance of Enterobacteriaceae (P < 0.01) and a lowered abundance of Burkholderiaceae (P < 0.05). Compared with the UC rats, the rats treated with E. faecium QH06 showed obvious body weight gain (P < 0.05), lessened colon injuries, lowered pathological score of the colon tissue (P < 0.05), decreased protein expressions of IFN- γ, IL- 12, and TLR2 and mRNA expressions of IFN- γ and IL-12 (P < 0.01 or 0.05), and increased protein expressions of IL- 4 (P < 0.05). The Shannon index ACE (P < 0.05) and Chao (P < 0.05) index of intestinal microflora were significantly increased, the abundance of Enterobacteriaceae was lowered and that of Burkholderiaceae and Rikenellaceae was increased in E.faecium QH06- treated rats (P < 0.01 or 0.05). Correlation analysis showed that IFN-γ was positively correlated with the abundance of Enterobacteriaceae, and IFN-γ was negatively correlated with the abundance of Prevotellaceae, Desulfovibrionaceae, norank_o_Mollicutes_RF39 and Clostridiales_vadinBB60_group; TLR2 was negatively correlated with Clostridiales_vadinBB60_group, norank_o_Mollicutes_RF39 and Prevotellaceae.@*CONCLUSION@#E.faecium QH06 can alleviate TNBS-induced colonic mucosal injury in rats, and its effect is mediated possibly by increasing the abundance of SCFA-producing bacteria such as Prevotellaceae and inhibiting abnormal immune responses mediated by TLR2.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B / 中国结合医学杂志

OBJECTIVE@#To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines.@*METHODS@#Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively.@*RESULTS@#Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS.@*CONCLUSIONS@#Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.@*REGISTRATION NO@#ChiCTR-RCS-13004001.

Ausência de associação do polimorfismo IL4 e características clínicas do acidente vascular encefálico hemorrágico / Absence of association of IL4 polymorphism and clinical characteristics of hemorrhagic stroke / Ausencia de asociación de polimorfismo IL4 y características clínicas del accidente cerebrovascular hemorrágico

Objetivo: investigar a associação entre o polimorfismo do tipo VNTR, do gene IL4, localizado na região do intron 3, em pacientes diagnosticados com acidente vascular encefálico hemorrágico (AVEH) ou aneurisma intracerebral em uma amostra do Distrito Federal. Método: Tratou-se de um estudo observacional, retrospectivo, transversal, com 55 indivíduos, dos quais foram anotadas as características clínicas do prontuário e realizada análise da genotipagem por meio da estratégia de PCR. As frequências genotípicas foram estimadas por contagem direta. O nível de significância adotado foi de 5% e o teste estatístico utilizado foi o Qui-Quadrado. Resultados: Foi verificado que o genótipo mais frequente foi o B1/B2 (50,9%; n=28), seguido pelo genótipo ancestral B1/B1 (27,3%, N=15), sendo que o menos frequente foi o genótipo B2/B2 (21,8%, N=12). Não foi encontrada associação estatística entre as variáveis hipertensão arterial sistêmica, diabetes, tabagismo e etilismo e a presença do polimorfismo no grupo estudado. Conclusão: A presença do polimorfismo IL4 INTRON 3 VNTR teve associação com a variável sexo, demonstrando que na amostra estudada, o AVEH é mais frequente em mulheres do que em homens, divergindo de estudos nos quais indivíduos do sexo masculino são mais propensos a desenvolverem AVE.

Objective: to investigate the association between The IL4 gene VNTR polymorphism, located in the intron 3 region, in patients diagnosed with hemorrhagic stroke (Stroke) or intracerebral aneurysm in a sample from the Federal District. Method: This was an observational, retrospective, cross-sectional study with 55 individuals, from which the clinical characteristics of the medical records were recorded and genotyping analysis was performed using the PCR strategy. Genotypic frequencies were estimated by direct counting. The level of significance adopted was 5% and the statistical test used was Chi-Square. Results: It was verified that the most frequent genotype was B1/B2 (50.9%; n=28), followed by the ancestral genotype B1/B1 (27.3%, N=15), and the least frequent was genotype B2/B2 (21.8%, N=12). No statistical association was found between the variables systemic arterial hypertension, diabetes, smoking and alcohol consumption and the presence of polymorphism in the studied group. Conclusion: The presence of IL4 INTRON 3 VNTR polymorphism was associated with the gender variable, demonstrating that in the sample studied, AVEH is more frequent in women than in men, diverging from studies in which males are more likely to develop a VENa

Objetivo: investigar la asociación entre el polimorfismo VNTR del gen IL4, localizado en la región intrón 3, en pacientes diagnosticados de accidente cerebrovascular hemorrágico (Stroke) o aneurisma intracerebral en una muestra del Distrito Federal. Método: Estudio observacional, retrospectivo, transversal, con 55 individuos, del cual se registraron las características clínicas de las historias clínicas y se realizó un análisis de genotipado mediante la estrategia de PCR. Las frecuencias genotípicas se estimaron mediante conteo directo. El nivel de significancia adoptado fue del 5% y la prueba estadística utilizada fue Chi-Cuadrado. Resultados: Se verificó que el genotipo más frecuente fue B1/B2 (50,9%; n=28), seguido del genotipo ancestral B1/B1 (27,3%, N=15), y el menos frecuente fue el genotipo B2/B2 (21,8%, N=12). No se encontró asociación estadística entre las variables hipertensión arterial sistémica, diabetes, tabaquismo y consumo de alcohol y la presencia de polimorfismo en el grupo estudiado. Conclusión: La presencia del polimorfismo IL4 INTRON 3 VNTR se asoció con la variable género, demostrando que en la muestra estudiada, AVEH es más frecuente en mujeres que en hombres, divergiendo de los estudios en los que los varones tienen más probabilidades de desarrollar una VENa.

Polimorfismo IL1B -511 e associação com suscetibilidade e prognóstico em pacientes com carcinoma papilífero de tireoide / IL1B -511 polymorphism and association with susceptibility and prognosis in patients with papillary thyroid carcinoma / IL1B -511 polimorfismo y asociación con susceptibilidad y pronóstico en pacientes con carcinoma papilar de tiroides

Objetivo: Associar a presença do SNP IL1B -511 (rs16944) à susceptibilidade ao CPT, bem como comparar níveis séricos da citocina antes e sete dias após a Iodoterapia, juntamente com outras características clínicas dos pacientes. Método: Trata-se de um estudo caso-controle, no qual foram obtidas amostras de sangue de 52 indivíduos (26 em cada grupo). A genotipagem foi realizada por meio da estratégia PCR-RFLP. Os níveis séricos de IL-1ß foi medido por meio de kit para ensaio imunoenzimático (ELISA). Testes para médias e estudos de associação foram executados considerando-se um nível de significância de 5%. Resultados: Não houve diferença estatística com relação a distribuição genotípica entre indivíduos caso e controle, e estes grupos não diferiram em relação às dosagens de citocina. Porém, os níveis de citocina aumentaram significativamente após a Iodoterapia, sendo que os portadores do genótipo CC apresentaram maior produção da proteína, mas este aumento não estava correlacionado com a dose de radiofármaco administrada. Conclusão: O polimorfismo IL1B -511 não foi associado à susceptibilidade ao CPT, porém os níveis séricos da citocina elevaram-se com o tratamento da iodoterapia, e esta elevação foi genótipo dependente

Objective: To associate the presence of SNP IL1B -511 (rs16944) with susceptibility to TLC, as well as to compare serum cytokine levels before and seven days after iodotherapy, along with other clinical characteristics of patients. Method: This is a case-control study, in which blood samples were obtained from 52 individuals (26 in each group). Genotyping was performed using the PCR-RFLP strategy. Serum IL-1ß levels were measured using an enzyme immunoassay kit (ELISA). Tests for means and association studies were performed considering a significance level of 5%. Results: There was no statistical difference regarding genotypic distribution between case and control individuals, and these groups did not differ in relation to cytokine dosages. However, cytokine levels increased significantly after iodine therapy, and patients with the CC genotype showed higher protein production, but this increase was not correlated with the administered radiopharmaceutical dose. Conclusion: IL1B-511 polymorphism was not associated with susceptibility to TLC, but serum cytokine levels increased with the treatment of iodotherapy, and this elevation was genotype dependent.

Objetivo: investigar la asociación entre el polimorfismo VNTR del gen IL4, localizado en la región intrón 3, en pacientes diagnosticados de accidente cerebrovascular hemorrágico (Stroke) o aneurisma intracerebral en una muestra del Distrito Federal. Método: Estudio observacional, retrospectivo, transversal, con 55 individuos, del cual se registraron las características clínicas de las historias clínicas y se realizó un análisis de genotipado mediante la estrategia de PCR. Las frecuencias genotípicas se estimaron mediante conteo directo. El nivel de significancia adoptado fue del 5% y la prueba estadística utilizada fue Chi-Cuadrado. Resultados: Se verificó que el genotipo más frecuente fue B1/B2 (50,9%; n=28), seguido del genotipo ancestral B1/B1 (27,3%, N=15), y el menos frecuente fue el genotipo B2/B2 (21,8%, N=12). No se encontró asociación estadística entre las variables hipertensión arterial sistémica, diabetes, tabaquismo y consumo de alcohol y la presencia de polimorfismo en el grupo estudiado. Conclusión: La presencia del polimorfismo IL4 INTRON 3 VNTR se asoció con la variable género, demostrando que en la muestra estudiada, AVEH es más frecuente en mujeres que en hombres, divergiendo de los estudios en los que los varones tienen más probabilidades de desarrollar una VENa.

Study on effect of anemoside B4 in improving COPD rats by regulating IL-12/STAT4 and IL-4/STAT6 signaling pathways / 中国中药杂志

To study the effect of anemoside B4 on rats with chronic obstructive pulmonary disease (COPD).Seventy-two SD male rats were randomly divided into blank group and model group.The method of exposure to cigarette smoke and combined with lipopolysaccharide (LPS) was used to replicate the rat model of COPD.After the model was maintained for 5 weeks,the rats were randomly divided into model group,dexamethasone group (0.81 mg·kg~(-1)) and anemoside B4 low,medium and high (2,4,8 mg·kg~(-1)) dose groups,a group of 12 animals were administered,and then the administration was started.The administration was maintained until the28th day,and the pulmonary function parameters of rats were measured by an animal pulmonary function instrument.After testing the rat lung function parameters,immediately draw rat alveolar lavage fluid (BALF),and use high-throughput protein chip technology to determined the expression levels of inflammatory cytokines in rat BALF.HE staining was used to observe the general pathological changes of rat lung and tracheal tissue.Masson staining was used to observe the collagen deposition in rat lung tissue.Real-time q PCR method was used to determine the mRNA expression level of related genes in rat lung tissue.Western blot method was used to determine the expression levels of related proteins in rat lung tissues.According to the findings,compared with the model group,the dexamethasone group and the anemoside B4 drug groups had different degrees of increase in the lung function parameters of rats (P<0.01,P<0.05),improved the expression level of inflammatory cytokines in the BALF of rats to varying degrees (P<0.01,P<0.05),and improved the pathological structure of rat lung tissue to varying degrees.Relative mRNA expressions of matrix metalloproteinase 2 (MMP-2),matrix metalloproteinase 12 (MMP-12),matrix metalloproteinase inhibitor 1 (TIMP-1),interleukin-6 (IL-6),and transforming growth factor-β1 (TGF-β1) were significantly reduced (P<0.01);whereas relative mRNA expressions of matrix metalloproteinase 9(MMP-9) and matrix metalloproteinase inhibitor 2 (TIMP-2) were increased significantly (P<0.01).The mRNA and protein expression levels of T-box transcription factor (T-bet),interleukin-12 (IL-12) and signal transducer and activator of transcription 4(STAT4) reduced to varying degrees (P<0.01,P<0.05).The mRNA of transcription factor GATA3 (binding protein-3),interleukin-4 (IL-4) and signal transducer and activator of transcription 6 (STAT6) in rat lung tissues and the protein expression levels of IL-4 and STAT6 were increased to varying degrees (P<0.01,P<0.05).In conclusion,anemoside B4 has a certain protective effect on COPD rats caused by cigarette smoke exposure and combined with LPS.The mechanism of action may be related to the regulation of IL-12/STAT4 and IL-4/STAT6 signaling pathways.

Suckling by Schistosoma mansoni-infected mothers restored IgG2a and TGF-β production, but not IL-6 and delayed-type hypersensitivity in IL-12/IL-23-deficient mice

Abstract INTRODUCTION Suckling by schistosomotic mice improves anti-ovalbumin (OA) antibody production, while delayed-type hypersensitivity (DTH) remains unaffected. This property of milk from schistosomotic mice was investigated in IL-12/IL-23-deficient mice (IL-12p40KO). METHODS We compared anti-OA DTH, IgG2a and cytokines in wild-type and IL-12p40KO mice suckled by infected (SIM) or non-infected (CONTROL) mothers. RESULTS SIM mice showed similar intensity and eosinophils in the DTH, which was abolished in IL-12p40KO and IL-12p40KO-SIM mice. In IL-12p40KO-SIM, IgG2a and TGF-β levels were higher, but IL-6 levels were lower. CONCLUSIONS Milk from schistosomotic mothers may evoke IgG2a without eliciting DTH in IL-12/IL-23 deficiencies, by changing TGF-β/IL-6 levels.

Irritable bowel syndrome in celiac disease - relationships to celiac disease antibodies and levels of pro-inflammatory cytokines / Síndrome del intestino irritable en la enfermedad celíaca: relaciones con los anticuerpos contra la enfermedad celíaca y los niveles de citocinas proinflamatorias

ABSTRACT Background: Evidence indicates that low-grade inflammation can alter gastrointestinal motor and sensory function and might contribute to the genesis of symptoms in IBS. Objective: To examine relationships between IBS, disease antibodies and cytokine titers in celiac patients and a control group. Materials and methods: IBS, CD activity and serum levels of IL-6, IL-8 and IL12/23p40 were determined in celiac patients and controls. Results: 123 celiac patients were included, 89% were female. 59% demonstrated disease activity and 32% met IBS criteria. Prevalence of IBS was not different between patients who adhered or did not adhere to GFD as well as between patients with or without positive antibodies. Celiac patients had increased levels of IL-6, IL-8 and IL12/23p40 as compared to controls. Higher levels of cytokines were found in celiac patients with IBS than in those without IBS. No difference in levels of cytokines was found between patients with and without CD positive antibodies. A significant negative correlation between the mental component of QoL and IL-6 and IL12/23p40 levels was found, but not with IL-8. Conclusion: Higher levels of inflammatory cytokines were found in CD patients with IBS than in either those without IBS or controls, indicating that IBS symptoms are associated with an increase in the inflammatory response and a decrease in quality of life of CD patients. These differences in cytokine levels were not related to CD antibodies status suggesting that IBS, in CD, is related to a different inflammatory process than that which is relevant to CD.

RESUMEN Antecedentes: la evidencia indica que la inflamación de bajo grado puede alterar la función motora y sensorial gastrointestinal y puede contribuir a la aparición de síntomas en el SII. Objetivo: Examinar la relación entre SII, anticuerpos contra enfermedades y títulos de citocinas en pacientes celíacos y un grupo de control. Materiales y métodos: se determinaron los síntomas de SII, actividad de CD y niveles séricos de IL-6, IL-8 e IL12 / 23p40 en pacientes celíacos y controles. Resultados: se incluyeron 123 pacientes celíacos, el 89% eran mujeres. El 59% demostró actividad de la enfermedad y el 32% cumplió con los criterios del SII. La prevalencia del SII no fue diferente entre los pacientes que se adhirieron o no se adhirieron a GFD, así como entre los pacientes con o sin anticuerpos positivos. Los pacientes celíacos tenían niveles aumentados de IL-6, IL-8 e IL12 / 23p40 en comparación con los controles. Se encontraron niveles más altos de citocinas en pacientes celíacos con SII que en aquellos sin SII. No se encontraron diferencias en los niveles de citocinas entre pacientes con y sin anticuerpos CD positivos. Se encontró una correlación negativa significativa entre el componente mental de la calidad de vida y los niveles de IL-6 e IL12 / 23p40, pero no con IL-8. Conclusión: Se encontraron niveles más altos de citocinas inflamatorias en pacientes con EC con SII que en aquellos sin SII o controles, lo que indica que los síntomas del SII están asociados con un aumento en la respuesta inflamatoria y una disminución en la calidad de vida de los pacientes con CD. Estas diferencias en los niveles de citocinas no estaban relacionadas con el estado de los anticuerpos contra la CD, lo que sugiere que el SII, en la CD, está relacionado con un proceso inflamatorio diferente al que es relevante para la CD.

Increased platelet distribution width and reduced IL-2 and IL-12 are associated with thrombocytopenia in Plasmodium vivax malaria

BACKGROUND Thrombocytopenia in malaria involves platelet destruction and consumption; however, the cellular response underlying this phenomenon has still not been elucidated. OBJECTIVE To find associations between platelet indices and unbalanced Th1/Th2/Th17 cytokines as a response to thrombocytopenia in Plasmodium vivax infected (Pv-MAL) patients. METHODS Platelet counts and quantification of Th1/Th2/Th17 cytokine levels were compared in 77 patients with uncomplicated P. vivax malaria and 37 healthy donors from the same area (endemic control group - ENCG). FINDINGS Thrombocytopenia was the main manifestation in 55 patients, but was not associated with parasitaemia. The Pv-MAL patients showed increases in the mean platelet volume (MPV), which may be consistent with larger or megaplatelets. Contrary to the findings regarding the endemic control group, MPV and platelet distribution width (PDW) did not show an inverse correlation, due the increase in the heterogeneity of platelet width. In addition, the Pv-MAL patients presented increased IL-1β and reduced IL-12p70 and IL-2 serum concentrations. Furthermore, the reduction of these cytokines was associated with PDW values. MAIN CONCLUSIONS Our data demonstrate that an increase in MPV and the association between reductions of IL-2 and IL-12 and PDW values may be an immune response to thrombocytopenia in uncomplicated P. vivax malaria.

Interleukin-12 over-expression in malignant melanoma B16 cells reduces programmed death-1 expression on T cells in mice with immune reconstitution / 南方医科大学学报

OBJECTIVE@#To investigate whether interleukin-12 (IL-12) over-expression in malignant melanoma B16 cells affects the expression level of programmed death-1 (PD-1) on T cells in mice during immune microenvironment reconstruction.@*METHODS@#B16 cells were transfected with an IL-12 expression lentiviral vector, and IL-12 over-expression in the cells was verified qPCR and ELISA. Plate cloning assay was used to compare the cell proliferation activity between B16 cells and B16/IL-12 cells. The expression of IL-12 protein in B16/IL-12 cells-derived tumor tissue were detected by ELISA. C57BL/6 mice were inoculated with B16 cells or B16/IL-12 cells, and 14 days later the proportion of T cells with high expression of PD-1 in the tumor-draining lymph nodes was detected by flow cytometry. Mouse models of immune reconstitution established by 650 cGy X-ray radiation were inoculated with B16 (B16+RT group) or B16/IL-12 (B16/IL-12+RT group) cells, with the mice without X-ray radiation prior to B16 cell inoculation as controls. Tumor growth in the mice was recorded at different time points, and on day 14, flow cytometry was performed to detect the proportion of T cells with high PD-1 expression in the tumor-draining lymph nodes and in the tumor tissue.@*RESULTS@#B16 cells infected with the IL-12-overexpressing lentiviral vector showed significantly increased mRNA and protein levels of IL-12 ( < 0.001) without obvious changes in cell viability (>0.05). B16/IL-12 cells expressed higher levels of IL-12 than B16 cells ( < 0.01). In the tumor-bearing mouse models, the proportion of CD4 PD-1 T cells was significantly lower in B16/IL-12 group than in B16 group ( < 0.01). In the mice with X-ray radiation-induced immune reconstitution, PD-1 expressions on CD4 T cells ( < 0.05) and CD8+ T cells ( < 0.01) were significantly higher in B16+ RT group than in the control mice and in B16/IL-12+RT group ( < 0.01 or 0.001); the tumors grew more slowly in B16/IL-12+RT group than in B16 + RT group ( < 0.001).@*CONCLUSIONS@#During immune microenvironment reconstruction, overexpression IL-12 in the tumor microenvironment can reduce the percentage of PD-1 T cells and suppress the growth of malignant melanoma in mice.

Increased Expression of Interleukin-12 in Lesional Skin of Atopic Dermatitis Patients with Psoriasiform Features on Histopathology: An Immunohistochemical Study

Preliminary Study on the Characteristic of Plasma Cytokine Profiles in Patients with Idiopathic Multicentric Castleman Diseases / 中国实验血液学杂志

OBJECTIVE@#To investigate the characteristic changes of the plasma cytokine profile in Chinese patients with idiopathic multicentric Castleman diseases (iMCD).@*METHODS@#The plasma samples from 22 patients with confirmed diagnosis of iMCD were collected before treatments; Specimens from 17 patients with newly diagnosed multiple myeloma, 10 non Hodgkin's lymphoma, and 15 healthy donors were used as control. Seventeen kinds of cytokines were measured by cytokine beads array (CBA) and ELISA respectively.@*RESULTS@#Six cytokines were measured by ELISA. The concentrations of IL-2, IL-6, IL-21 and VEGF were significantly higher in the plasma of iMCD patients than those of the healthy donors (P＜0.01) and the level of IL-21 was highest in the iMCD group. There was no significant difference in the levels of IL-1β and IL-4 between the iMCD and healthy donor groups. Thirteen cytokines were measured by CBA assay, besides IL-6 level was confirmed to be higher in iMCD group than that in healthy controls (P＜0.01）, IL-12-p70 and IL-33 levels were also higher in iMCD group than those in control group (P＜0.05）, no significant difference of the rest cytokines was found between iMCD and the control group.@*CONCLUSION@#IL-6 and VEGF has shown to involved in the pathogenesis of iMCD, the results of preliminary study imply the role of IL-2 、IL-21、IL-12-p70 and IL-33 in this rare lymphoproliferative disease. Further studies are needed to elucidate the mechanism of these cytokines, which may shed some light on the identification of novel therapeutic targets against iMCD.

IL-12 Enhances Immune Response by Modulation of Myeloid Derived Suppressor Cells in Tumor Microenvironment / 전남의대학술지

Myeloid derived suppressor cells (MDSCs) are a heterogenous population of immature cells that play a critical role in tumor associated immune suppression. In tumor conditions, the population of MDSCs increases. The main feature of these cells is their ability to suppress the T cell response in antigen specific or nonspecific manners depending on the condition of T cell activation. IL-12 can modulate MDSC in preliminary reports, so we investigated how IL-12 can affect MDSC in a tumor microenvironment. After implanting tumor based cells on syngeneic host, 4T-1/BALB/c or EL4/C57BL6 mice, MDSCs (Gr1+CD11b+) were isolated from splenocytes. Isolated MDSCs were treated with GM-CSF with or without IL-12 and analyzed based on their phenotypes and functions. Treatment of MDSC with IL-12 increased co-stimulatory molecules of CD80, CD86, OX-40L, enhancing the DC phenotype (CD11c) and maturation markers such as p-NF-κB and p-GSK3β. In addition to a change of surface markers, T-cell suppressive function of MDSC after IL-12 treatment was significantly improved compared with the control MDSC. In addition, PD-L1+F4/80+ macrophages, which show aninhibitory effect in phagocytosis, were decreased after IL-12 treatment. The changes of cell surface expression of CD80, CD86, MHC class II were also shown in vivo. Our results showed that the IL-12 can modulate MDSC into APC and recover the macrophage function. These results suggested that IL-12 plays a role in improving the tumor immune microenvironment through MDSC modulation.

A Novel Niosomal Combination of Selenium Coupled with Glucantime against Leishmania tropica

There is no effective treatment modality available against different forms of leishmaniasis. Therefore, the aim of this study was to improve the penetration and efficacy of selenium and glucantime coupled with niosomes and compared them with their simple forms alone on in vitro susceptibility assays. In this study, the niosomal formulations of selenium and in combination with glucantime were prepared. The size and morphology of the niosomal formulations were characterized and the effectivity of the new formulation was also evaluated using in vitro MTT assay, intra-macrophage model, and gene expression profile. From the results obtained, no cytotoxicity effect was observed for niosomal and simple forms of drugs, as alone or in combination. Niosomal formulations of the drugs significantly showed more inhibitory effects (P≤0.001) than the simple drugs when the selectivity index was considered. The gene expression levels of Interleukin (IL-10) significantly decreased, while the level of IL-12 and metacaspase significantly increased (P≤0.001). The results of the present study showed that selenium plus glucantime niosome possess a potent anti-leishmanial effect and enhanced their lethal activity as evidenced by the in vitro experiments.

Antileishmanial Activity of Niosomal Combination Forms of Tioxolone along with Benzoxonium Chloride against Leishmania tropica

In this study, we carried out extensive in vitro studies on various concentrations of tioxolone along with benzoxonium chloride and their niosomal forms against Leishmania tropica. Niosomes were prepared by the hydration method and were evaluated for morphology, size, release study, and encapsulation efficiency. This study measured leishmanicidal activity against promastigote and amastigote, apoptosis and gene expression levels of free solution and niosomal-encapsulated tioxolone along with benzoxonium chloride. Span/Tween 60 niosome had good physical stability and high encapsulation efficiency (more than 97%). The release profile of the entrapped compound showed that a gradual release rate. The combination of niosomal forms on promastigote and amastigote were more effective than glucantime. Also, the niosomal form of this compound was significantly less toxic than glucantime (P≤0.05). The flowcytometric analysis on niosomal form of drugs showed that higher number of early apoptotic event as the principal mode of action (89.13% in 200 μg/ml). Also, the niosomal compound increased the expression level of IL-12 and metacaspase genes and decreased the expression level of the IL-10 gene, which further confirming the immunomodulatory role as the mechanism of action. We observed the synergistic effects of these 2 drugs that induced the apoptotic pathways and also up regulation of an immunomodulatory role against as the main mode of action. Also, niosomal form of this combination was safe and demonstrated strong anti-leishmaniasis effects highlights further therapeutic approaches against anthroponotic cutaneous leishmaniasis in future planning.

Distinct Inflammation Biomarkers in Healthy Individuals and Patients with Schizophrenia: A Reliability Testing of Multiplex Cytokine Immunoassay by Bland-Altman Analysis

OBJECTIVE: Since the inflammatory process has been implicated in the pathophysiology of psychiatric disorder, an important issue emerging is to assess the test-retest reliability of cytokine measurement in healthy individuals and patients with schizophrenia. The objective of the present study was to investigate the test-retest reliability of bead-based multiplex immunoassay technology (BMIT) for cytokine measurement by using a Bland-Altman plot (BAP). METHODS: Twenty healthy individuals and twenty patients with schizophrenia were enrolled, and a 17-plex cytokine assay was used to measure inflammatory biomarkers at baseline and two weeks later. The test-retest reliability was examined by BAP, 95% limits of agreement (LOA), intraclass correlation coefficient (ICC), and coefficient of repeatability (CoR). RESULTS: In the healthy controls, only interleukin (IL)-2, IL-13, IL-10, IL-17, and macrophage inflammatory protein-1β showed excellent ICC. The BAP with 95% LOA determined that 13 cytokines showed acceptable 95% LOA for a 2-week test-retest reliability, and only IL-1β, IL-12 and tumor necrosis factor (TNF)-α had significant test-retest bias. The CoR of cytokines varied significantly, ranging from 1.72 to 218.1. Compared with healthy controls, patients with schizophrenia showed significantly higher levels of IL-5, IL-13, and TNF-α and significantly lower levels of IL-4, IL-12, and interferon-gamma (IFN-γ). Of these six cytokines, IL-12 and TNF-α were considered suboptimal reliability. CONCLUSION: The findings from ICC and CoR implied that the test-retest reliability of BMIT for cytokine measurement were suboptimal. However, the BAP with 95% LOA confirmed that BMIT can reliably distinguish schizophrenia from healthy individuals in cytokine measurement, while significant within-subject variation and between-group overlapping were evident in cytokine expression.

Níveis séricos de citocinas IL-2, IL-10 e IL-12 em pacientes com adenocarcinoma do estômago / Serum cytokine of il-2, il-10 and il-12 levels in patients with stomach adenocarcinoma

ABSTRACT BACKGROUND: Gastric adenocarcinoma is the fourth most common cause of cancer-associated death worldwide. OBJECTIVE: We evaluated the immunological status of patients with gastric cancer before surgery and circulating cytokines as potential diagnostic biomarkers for gastric cancer. METHODS: We included 90 healthy controls and 95 patients with distal Gastric adenocarcinoma in Mazandaran, Sari, Iran. We measured serum IL-2, IL-10 and IL-12 Levels by a sandwich enzyme-linked immunosorbent assay using the IBL international GMBH kit. RESULTS: The serum IL-10 levels in the patients with Gastric adenocarcinoma were significantly higher than those of the healthy controls (P=0.02). There were no significant differences in serum IL-2 and IL-12 levels between patients with gastric cancer and healthy controls. CONCLUSION: Increased levels of IL-10 might be useful as diagnostic biomarkers for Gastric adenocarcinoma; however, this needs to be confirmed with larger number of patients and with control groups other than blood donors, properly age paired. These results suggest that positive expression of IL-10 may be useful as a molecular marker to distinguish stage of gastric cancers which can be more readily controlled.

RESUMO CONTEXTO: O adenocarcinoma gástrico é a quarta causa mais comum de morte relacionada ao câncer em todo o mundo. OBJETIVO: Avaliar o status imunológico dos pacientes com câncer gástrico antes da cirurgia e as citocinas circulantes como potenciais biomarcadores diagnósticos para câncer gástrico. MÉTODOS: Incluímos 90 indivíduos controles saudáveis e 95 pacientes com adenocarcinoma gástrico distal em Mazandaran, Sari, Iran. Os níveis de soro Il-2, IL-10 e Il-12 foram medidos por um ensaio de imunoabsorção enzimática pela técnica de sanduíche usando o kit IBL International GmbH. RESULTADOS: Os níveis séricos IL-10 nos pacientes com adenocarcinoma gástrico foram significativamente superiores aos dos controles saudáveis (P=0,2). Não houve diferenças significativas nos níveis de soro IL-2 e IL-12 entre pacientes com câncer gástrico e controles saudáveis. CONCLUSÃO: Níveis aumentados de IL-10 podem ser úteis como biomarcadores diagnósticos para adenocarcinoma gástrico; no entanto, isso precisa ser confirmado com maior número de pacientes e com grupos de controle que não sejam doadores de sangue, adequadamente emparelhado por idade. Estes resultados sugerem que a expressão positiva do IL-10 pode ser útil como um marcador molecular para distinguir a fase de câncer gástrico que pode ser mais facilmente controlada.

Interleukin-12 as a biomarker of the beneficial effects of food restriction in mice receiving high fat diet or high carbohydrate diet

The impact of food restriction (FR) during 56 days on serum levels of cytokines in mice fed a high-fat diet (HFD) or high-carbohydrate diet (HCD) were evaluated. The amount of food was reduced 50% for HFD-FR and HCD-FR groups compared to mice receiving free access to HFD (HFD group) or HCD (HCD group). We quantified the serum levels of basic fibroblast growth factor, granulocyte-macrophage colony-stimulating factor, inducible protein 10, interferon γ, interleukin 1α (IL-1α), IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, keratinocyte chemoattractant, macrophage inflammatory protein-1α, monocyte chemotactic protein 1, monokine induced by IFN-γ, and tumor necrosis factor α. Only IL-12 levels were lower (P<0.05), for both HFD-FR (HFD-FR vs HFD) and HCD-FR (HCD-FR vs HCD). Therefore, IL-12 levels could be considered a biological marker of the beneficial effects of FR.

A Case of Paradoxical Flare of Pustular Psoriasis after Ustekinumab Therapy / 대한피부과학회지

Biologics are the most advanced treatment for psoriasis. Ustekinumab, one of the biologics for psoriasis, is a human monoclonal antibody that binds to the p40 subunit of interleukin-12 and interleukin-23. A 41-year-old woman with a 17-year history of plaque psoriasis and psoriatic arthritis presented with worsening lesions. The patient had previously been treated with a number of topical and systemic medications and narrow band ultraviolet B. However, none of the treatments consistently controlled her disease. Thus, treatment with ustekinumab 45 mg via subcutaneous injection was initiated. Approximately 7 days after the first treatment, she experienced a flare with generalized pustules in her whole body. The condition was controlled with systemic steroid treatment. The patient was subsequently treated with adalimumab, and improvement in her plaque and pustular lesions was noted. Herein, we report a case of psoriasis that flared up after ustekinumab therapy, which was accompanied by a morphological change from plaque to pustular lesions.