Dataset Information

Murine CD4 T cells produce a new form of TGF-β as measured by a newly developed TGF-β bioassay.

ABSTRACT:

Background

It is generally assumed that T cells do not produce active TGF-β since active TGF-β as measured in supernatants by ELISA without acidification is usually not detectable. However, it is possible that active TGF-β from T cells may take a special form which is not detectable by ELISA.

Methodology/principal findings

We constructed a TGF-β bioassay which can detect both soluble and membrane-bound forms of TGF-β from T cells. For this bioassay, 293T cells were transduced with (caga)(12) Smad binding element-luciferase along with CD32 (Fc receptor) and CD86. The resulting cells act as artificial antigen presenting cells in the presence of anti-CD3 and produce luciferase in response to biologically active TGF-β. We co-cultured pre-activated murine CD4(+)CD25(-) T cells or CD4(+)CD25(+) T cells with the 293T-caga-Luc-CD32-CD86 reporter cells in the presence of anti-CD3 and IL-2. CD4(+)CD25(-) T cells induced higher luciferase in the reporter cells than CD4(+)CD25(+) T cells. This T cell-produced TGF-β is in a soluble form since T cell culture supernatants contained the TGF-β activity. The TGF-β activity was neutralized with an anti-mouse LAP mAb or an anti-latent TGF-β/pro-TGF-β mAb, but not with anti-active TGF-β Abs. An anti-mouse LAP mAb removed virtually all acid activatable latent TGF-β from the T cell culture supernatant, but not the ability to induce TGF-β signaling in the reporter cells. The induction of TGF-β signaling by T cell culture supernatants was cell type-specific.

Conclusions/significance

A newly developed 293T-caga-Luc-CD32-CD86 reporter cell bioassay demonstrated that murine CD4 T cells produce an unconventional form of TGF-β which can induce TGF-β signaling. This new form of TGF-β contains LAP as a component. Our finding of a new form of T cell-produced TGF-β and the newly developed TGF-β bioassay system will provide a new avenue to investigate T cell function of the immune system.

SUBMITTER: Oida T

PROVIDER: S-EPMC3073999 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BACKGROUND: The development of microarray technology has greatly enhanced our ability to evaluate gene expression. In theory, the expression of all genes in a given organism can be monitored simultaneously. Sequencing of the chicken genome has provided the crucial information for the design of a comprehensive chicken transcriptome microarray. A long oligonucleotide microarray has been manually curated and designed by our group and manufactured using Agilent inkjet technology. This provides a flexible and powerful platform with high sensitivity and specificity for gene expression studies. RESULTS: A chicken 60-mer oligonucleotide microarray consisting of 42,034 features including the entire Marek's disease virus, two avian influenza virus (H5N2 and H5N3), and 150 chicken microRNAs has been designed and tested. In an important validation study, total RNA isolated from four major chicken tissues: cecal tonsil (C), ileum (I), liver (L), and spleen (S) were used for comparative hybridizations. More than 95% of spots had high signal noise ratio (SNR > 10). There were 2886, 2660, 358, 3208, 3355, and 3710 genes differentially expressed between liver and spleen, spleen and cecal tonsil, cecal tonsil and ileum, liver and cecal tonsil, liver and ileum, spleen and ileum (P < 10-7), respectively. There were a number of tissue-selective genes for cecal tonsil, ileum, liver, and spleen identified (95, 71, 535, and 108, respectively; P < 10-7). Another highlight of these data revealed that the antimicrobial peptides GAL1, GAL2, GAL6 and GAL7 were highly expressed in the spleen compared to other tissues tested. CONCLUSION: A chicken 60-mer oligonucleotide 44K microarray was designed and validated in a comprehensive survey of gene expression in diverse tissues. The results of these tissue expression analyses have demonstrated that this microarray has high specificity and sensitivity, and will be a useful tool for chicken functional genomics. Novel data on the expression of putative tissue specific genes and antimicrobial peptides is highlighted as part of this comprehensive microarray validation study. The information for accessing and ordering this 44K chicken array can be found at http://people.tamu.edu/~hjzhou/TAMUAgilent44KArray/

Project description:Implementation outcome measures are essential for monitoring and evaluating the success of implementation efforts. Yet, currently available measures lack conceptual clarity and have largely unknown reliability and validity. This study developed and psychometrically assessed three new measures: the Acceptability of Intervention Measure (AIM), Intervention Appropriateness Measure (IAM), and Feasibility of Intervention Measure (FIM).Thirty-six implementation scientists and 27 mental health professionals assigned 31 items to the constructs and rated their confidence in their assignments. The Wilcoxon one-sample signed rank test was used to assess substantive and discriminant content validity. Exploratory and confirmatory factor analysis (EFA and CFA) and Cronbach alphas were used to assess the validity of the conceptual model. Three hundred twenty-six mental health counselors read one of six randomly assigned vignettes depicting a therapist contemplating adopting an evidence-based practice (EBP). Participants used 15 items to rate the therapist's perceptions of the acceptability, appropriateness, and feasibility of adopting the EBP. CFA and Cronbach alphas were used to refine the scales, assess structural validity, and assess reliability. Analysis of variance (ANOVA) was used to assess known-groups validity. Finally, half of the counselors were randomly assigned to receive the same vignette and the other half the opposite vignette; and all were asked to re-rate acceptability, appropriateness, and feasibility. Pearson correlation coefficients were used to assess test-retest reliability and linear regression to assess sensitivity to change.All but five items exhibited substantive and discriminant content validity. A trimmed CFA with five items per construct exhibited acceptable model fit (CFI = 0.98, RMSEA = 0.08) and high factor loadings (0.79 to 0.94). The alphas for 5-item scales were between 0.87 and 0.89. Scale refinement based on measure-specific CFAs and Cronbach alphas using vignette data produced 4-item scales (?'s from 0.85 to 0.91). A three-factor CFA exhibited acceptable fit (CFI = 0.96, RMSEA = 0.08) and high factor loadings (0.75 to 0.89), indicating structural validity. ANOVA showed significant main effects, indicating known-groups validity. Test-retest reliability coefficients ranged from 0.73 to 0.88. Regression analysis indicated each measure was sensitive to change in both directions.The AIM, IAM, and FIM demonstrate promising psychometric properties. Predictive validity assessment is planned.