Improvement of Asia-Pacific colorectal screening score and evaluation of its use combined with fecal immunochemical test.

BACKGROUND: The Asia-Pacific Colorectal Screening (APCS) score is effective to screen high-risk groups of advanced colorectal neoplasia (ACN) patients but needs revising and can be combined with the fecal immunochemical test (FIT). This paper aimed to improve the APCS score and evaluate its use with the FIT in stratifying the risk of ACN. METHODS: This prospective and multicenter study enrolled 955 and 1201 asymptomatic Chinese participants to form the derivation and validation set, respectively. Participants received the risk factor questionnaire, colonoscopy and FIT. Multiple logistic regression was applied, and C-statistic, sensitivity and negative predictive values (NPVs) were used to compare the screening efficiency. RESULTS: A modified model was developed incorporating age, body mass index (BMI), family history, diabetes, smoking and drinking as risk factors, stratifying subjects into average risk (AR) or high risk (HR). In the validation set, the HR tier group had a 3.4-fold (95% CI 1.8-6.4) increased risk for ACN. The C-statistic for the modified score was 0.69 ± 0.04, and 0.67 ± 0.04 for the original score. The sensitivity of the modified APCS score combined with FIT for screening ACN high-risk cohorts was 76.7% compared with 36.7% of FIT alone and 70.0% of the modified APCS score alone. The NPVs of the modified score combined with FIT for ACN were 98.0% compared with 97.0% of FIT alone and 97.9% of the modified APCS score alone. CONCLUSIONS: The modified score and its use with the FIT are efficient in selecting the HR group from a Chinese asymptomatic population.

Signal Transducer and Activator of Transcription 3 for the Differentiation of Hepatocellular Carcinoma from Cirrhosis.

BACKGROUND: Overexpression and constitutive activation of signal transducer and activator of transcription (STAT) 3 have been suggested in the tumorigenesis of many human cancers, including multiple carcinomas, melanoma, and lymphoma. The diagnosis of hepatocellular carcinoma (HCC) in lobectomy specimens is usually straightforward, but distinguishing cirrhosis from well-differentiated HCC can be challenging in core biopsies. Our aims were to investigate the expression level of STAT3 and phosphorylated STAT3 (pSTAT3) in HCC and cirrhosis, and the application of STAT3 in the differential diagnosis of HCC and cirrhosis. METHODS: Sixty cases were divided into three groups: patients with HCC only (Group 1), HCC and cirrhosis (Group 2), and cirrhosis only (Group 3). Formalin-fixed and paraffin-embedded tissue sections were stained immunohistochemically for STAT3, pSTAT3, and CD163. The values obtained from the tissue sections of each group were compared in statistical analysis. RESULTS: STAT3 showed a high level in HCC and was a significant marker for differentiating HCC from cirrhosis (P < 0.0001). The odds ratio between HCC and cirrhosis increased 34.4 times when the intensity of STAT3 increased by 1 level. Spearman's correlation and Chi-square tests also demonstrated that expression level of STAT3 did not correlate with age, gender, or the presence of a cirrhotic background. CONCLUSIONS: STAT3 staining differs significantly in HCC and cirrhosis. The findings reinforce the role of STAT3 in the tumorigenesis of HCC and provide a useful marker to differentiate HCC from cirrhosis in challenging liver biopsies.

The Neuronal Kinesin UNC-104/KIF1A Is a Key Regulator of Synaptic Aging and Insulin Signaling-Regulated Memory.

Aging is the greatest risk factor for a number of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. Furthermore, normal aging is associated with a decline in sensory, motor, and cognitive functions. Emerging evidence suggests that synapse alterations, rather than neuronal cell death, are the causes of neuronal dysfunctions in normal aging and in early stages of neurodegenerative diseases. However, little is known about the mechanisms underlying age-related synaptic decline. Here, we uncover a surprising role of the anterograde molecular motor UNC-104/KIF1A as a key regulator of neural circuit deterioration in aging C. elegans. Through analyses of synapse protein localization, synaptic transmission, and animal behaviors, we find that reduced function of UNC-104 accelerates motor circuit dysfunction with age, whereas upregulation of UNC-104 significantly improves motor function at advanced ages and also mildly extends lifespan. In addition, UNC-104-overexpressing animals outperform wild-type controls in associative learning and memory tests. Further genetic analyses suggest that UNC-104 functions downstream of the DAF-2-signaling pathway and is regulated by the FOXO transcription factor DAF-16, which contributes to the effects of DAF-2 in neuronal aging. Together, our cellular, electrophysiological, and behavioral analyses highlight the importance of axonal transport in the maintenance of synaptic structural integrity and function during aging and raise the possibility of targeting kinesins to slow age-related neural circuit dysfunction.

Activated p38 MAPK in Peripheral Blood Monocytes of Steroid Resistant Asthmatics.

Steroid resistance is a significant problem in management of chronic inflammatory diseases, including asthma. Accessible biomarkers are needed to identify steroid resistant patients to optimize their treatment. This study examined corticosteroid resistance in severe asthma. 24 asthmatics with forced expiratory volume in one second of less then 80% predicted were classified as steroid resistant or steroid sensitive based on changes in their lung function following a week of treatment with oral prednisone. Heparinised blood was collected from patients prior to oral prednisone administration. Phosphorylated mitogen activated kinases (MAPK) (extracellular regulated kinase (ERK), p38 and jun kinase (JNK)) were analyzed in whole blood samples using flow cytometry. Activation of phospho-p38 MAPK and phospho-mitogen- and stress-activated protein kinase 1 (MSK1) in asthmatics' peripheral blood mononuclear cells (PBMC) were confirmed by Western blot. Dexamethasone suppression of the LPS-induced IL-8 mRNA production by steroid resistant asthmatics PBMC in the presence of p38 and ERK inhibitors was evaluated by real time PCR. Flow cytometry analysis identified significantly stronger p38 phosphorylation in CD14+ monocytes from steroid resistant than steroid sensitive asthmatics (p = 0.014), whereas no difference was found in phosphorylation of ERK or JNK in CD14+ cells from these two groups of asthmatics. No difference in phosphorylated p38, ERK, JNK was detected in CD4+, CD8+ T cells, B cells and NK cells from steroid resistant vs. steroid sensitive asthmatics. P38 MAPK pathway activation was confirmed by Western blot, as significantly higher phospho-p38 and phospho-MSK1 levels were detected in the PBMC lysates from steroid resistant asthmatics. P38 inhibitor significantly enhanced DEX suppression of LPS-induced IL-8 mRNA by PBMC of steroid resistant asthmatics. This is the first report demonstrating selective p38 MAPK pathway activation in blood monocytes of steroid resistant asthmatics, suggesting that p38 and MSK1 phosphorylation can serve as blood biomarkers of steroid resistance.

Parkinson's disease genes VPS35 and EIF4G1 interact genetically and converge on α-synuclein.

Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and α-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to α-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future interrogation.

Inhibition of histone deacetylase 2 expression by elevated glucocorticoid receptor beta in steroid-resistant asthma.

RATIONALE: Cross-talk between glucocorticoid receptors and histone deacetylases (HDACs) under steroid-insensitive conditions has not been explored. OBJECTIVES: To evaluate expression and interaction of HDACs with glucocorticoid receptor isoforms in bronchoalveolar lavage and peripheral blood mononuclear cells from steroid-resistant versus steroid-sensitive patients with asthma. METHODS: Expression of HDACs 1 through 11 was measured by real-time polymerase chain reaction in primary cells and in the DO11.10 cell line, designed to overexpress glucocorticoid receptor ß. Glucocorticoid receptor ß expression was inhibited in bronchoalveolar lavage cells by small interfering RNA. Human HDAC2 promoter fragments were cloned into a luciferase reporter vector, and transiently transfected with glucocorticoid receptor α- and ß-encoding plasmids into the cells. Luciferase activity was then assayed in response to glucocorticoids. MEASUREMENTS AND MAIN RESULTS: Levels of HDAC2 mRNA, but not other histone deacetylases, were significantly decreased in bronchoalveolar lavage cells but not in peripheral blood mononuclear cells from steroid-resistant patients with asthma. Overexpression of glucocorticoid receptor ß in DO11.10 cells selectively reduced HDAC2 mRNA and protein levels. Silencing of glucocorticoid receptor ß in bronchoalveolar lavage cells from patients with asthma significantly increased HDAC2 mRNA. Luciferase activity assays with HDAC2 promoter reporter constructs identified two glucocorticoid-inducible regions in the HDAC2 promoter. Promoter activity was increased more than fourfold in dexamethasone-treated cells cotransfected with glucocorticoid receptor α. Cotransfection of glucocorticoid receptor ß abolished this effect in a dose-dependent manner. CONCLUSIONS: Glucocorticoid receptor ß controls expression of histone deacetylase 2 by inhibiting glucocorticoid response elements in its promoter. This highlights a novel mechanism by which glucocorticoid receptor ß promotes steroid insensitivity (Li et al.: J Allergy Clin Immunol 2009;123:S146; and Li et al.: J Allergy Clin Immunol 2010;125:AB104).

Roles of trinucleotide-repeat RNA in neurological disease and degeneration.

A large number of human diseases are caused by expansion of repeat sequences - typically trinucleotide repeats - within the respective disease genes. The abnormally expanded sequence can lead to a variety of effects on the gene: sometimes the gene is silenced, but in many cases the expanded repeat sequences confer toxicity to the mRNA and, in the case of polyglutamine diseases, to the encoded protein. This article highlights mechanisms by which the mRNAs with abnormally expanded repeats can confer toxicity leading to neuronal dysfunction and loss. Greater understanding of these mechanisms will provide the foundation for therapeutic advances for this set of human disorders.

IFN-gamma reverses IL-2- and IL-4-mediated T-cell steroid resistance.

Corticosteroids are the most common therapeutic approach for control of tissue inflammation. Combination IL-2/IL-4 is known to induce T-cell steroid resistance. This can be reversed with IFN-gamma; however, the mechanism by which this occurs is unknown. In the current study, we found that treatment of peripheral blood mononuclear cells with combination IL-2/IL-4 for 48 hours, but not with IL-2 or IL-4 alone, abrogated dexamethasone (DEX)-induced glucocorticoid receptor (GCR)-alpha nuclear translocation in both CD4(+) and CD8(+) T cells. The presence of IL-4 significantly down-regulated IFN-gamma production by IL-2-stimulated cells. Importantly, addition of IFN-gamma to the IL-2/IL-4 combination restored GCRalpha nuclear translocation in response to DEX. Furthermore, DEX-induced mitogen-activated protein kinase (MAPK) phosphatase-1 induction, used as a readout for corticosteroid-induced transactivation, was significantly greater (P < 0.05) in media and IL-2/IL-4/IFN-gamma-treated conditions compared with IL-2/IL-4-treated cells. The combination of IL-2/IL-4 induced p38 MAPK activation in CD3(+) cells (30.5 +/- 5.7% cells expressed phospho-p38 MAPK versus no phospho-p38 MAPK expression after media treatment). The presence of the p38 MAPK inhibitor, SB203580, or IFN-gamma inhibited p38 MAPK phosphorylation and enhanced GCRalpha nuclear translocation in response to DEX. These data indicate that combination IL-2/IL-4 inhibits GCRalpha nuclear translocation in human T cells, and this effect is reversed by IFN-gamma via inhibition of p38 MAPK activation.

RNA toxicity is a component of ataxin-3 degeneration in Drosophila.

Polyglutamine (polyQ) diseases are a class of dominantly inherited neurodegenerative disorders caused by the expansion of a CAG repeat encoding glutamine within the coding region of the respective genes. The molecular and cellular pathways underlying polyQ-induced neurodegeneration are the focus of much research, and it is widely considered that toxic activities of the protein, resulting from the abnormally long polyQ tract, cause pathogenesis. Here we provide evidence for a pathogenic role of the CAG repeat RNA in polyQ toxicity using Drosophila. In a Drosophila screen for modifiers of polyQ degeneration induced by the spinocerebellar ataxia type 3 (SCA3) protein ataxin-3, we isolated an upregulation allele of muscleblind (mbl), a gene implicated in the RNA toxicity of CUG expansion diseases. Further analysis indicated that there may be a toxic role of the RNA in polyQ-induced degeneration. We tested the role of the RNA by altering the CAG repeat sequence to an interrupted CAACAG repeat within the polyQ-encoding region; this dramatically mitigated toxicity. In addition, expression of an untranslated CAG repeat of pathogenic length conferred neuronal degeneration. These studies reveal a role for the RNA in polyQ toxicity, highlighting common components in RNA-based and polyQ-protein-based trinucleotide repeat expansion diseases.

Suppression of polyglutamine toxicity by the yeast Sup35 prion domain in Drosophila.

The propensity of proteins to form beta-sheet-rich amyloid fibrils is related to a variety of biological phenomena, including a number of human neurodegenerative diseases and prions. A subset of amyloidogenic proteins forms amyloid fibrils through glutamine/asparagine (Q/N)-rich domains, such as pathogenic polyglutamine (poly(Q)) proteins involved in neurodegenerative disease, as well as yeast prions. In the former, the propensity of an expanded poly(Q) tract to abnormally fold confers toxicity on the respective protein, leading to neuronal dysfunction. In the latter, Q/N-rich prion domains mediate protein aggregation important for epigenetic regulation. Here, we investigated the relationship between the pathogenic ataxin-3 protein of the human disease spinocerebellar ataxia type 3 (SCA3) and the yeast prion Sup35, using Drosophila as a model system. We found that the capacity of the Sup35 prion domain to mediate protein aggregation is conserved in Drosophila. Although select yeast prions enhance poly(Q) toxicity in yeast, the Sup35N prion domain suppressed poly(Q) toxicity in the fly. Suppression required the oligopeptide repeat of the Sup35N prion domain, which is critical for prion properties in yeast. These results suggest a trans effect of prion domains on pathogenic poly(Q) disease proteins in a multicellular environment and raise the possibility that Drosophila may allow studies of prion mechanisms.

[Inhibition of proliferation, adhesion and invasion ability of human lung carcinoma cell A549 by tumor necrosis factor-alpha converting enzyme (TACE)].

We constructed prokaryotic expression vectors for different domains of TACE gene and expressed the fusion proteins, so as to explore their effects on the proliferation, adhesion and invasion potential of tumor cells in vitro. The total RNA was isolated from THP1 cell. TACE cDNA was amplified by RT-PCR and subcloned into pMD18-T vector to construct pMD-18T-TACE vector. The different cDNA fragment of TACE were amplified from plasmid pMD-18T-TACE and then cloned into pET-28a( + ) to construct expression vector pET28a( + )- 300, pET28a( + )-T800, and pET28a( + )-T1300, which respectively transformed into E. coli BL21 (DE3). The expression of His-tagged fusion proteins were induced with IPTG and purified through BBST NTA resin. The proliferation ability was examined by MTT assay. The adhesive and invasive ability were examined by plated adhesion model and Transwell assay. The protein pET28a( + )-T300 and pET28a( + )-T1300 can reduce the proliferation, adhesion and invasion ability of human lung carcinoma cell A549 in vitro, but otherwise the protein pET28a( + )-T800 had not shown the inhibitive function. The fusion protein of disintegrin domain of TACE have the similar biological function to other disintegrins, which can be used for further research on function of TACE in inflammation and tumor.

ATF2 impairs glucocorticoid receptor-mediated transactivation in human CD8+ T cells.

Chronic inflammatory diseases often have residual CD8(+) T-cell infiltration despite treatment with systemic corticosteroids, which suggests divergent steroid responses between CD4(+) and CD8(+) cells. To examine steroid sensitivity, dexamethasone (DEX)-induced histone H4 lysine 5 (K5) acetylation and glucocorticoid receptor alpha (GCR alpha) translocation were evaluated. DEX treatment for 6 hours significantly induced histone H4 K5 acetylation in normal CD4(+) cells (P = .001) but not in CD8(+) cells. DEX responses were functionally impaired in CD8(+) compared with CD4(+) cells when using mitogen-activated protein kinase phosphatase (1 hour; P = .02) and interleukin 10 mRNA (24 hours; P = .004) induction as a readout of steroid-induced transactivation. Normal DEX-induced GCR alpha nuclear translocation and no significant difference in GCR alpha and GCR beta mRNA expression were observed in both T-cell types. In addition, no significant difference in SRC-1, p300, or TIP60 expression was found. However, activating transcription factor-2 (ATF2) expression was significantly lower in CD8(+) compared with CD4(+) cells (P = .009). Importantly, inhibition of ATF2 expression by small interfering RNA in CD4(+) cells resulted in inhibition of DEX-induced transactivation in CD4(+) cells. The data indicate refractory steroid-induced transactivation but similar steroid-induced transrepression of CD8(+) cells compared with CD4(+) cells caused by decreased levels of the histone acetyltransferase ATF2.

Divergent expression and function of glucocorticoid receptor beta in human monocytes and T cells.

Glucocorticoid (GC) insensitivity is a significant problem in the treatment of immune-mediated diseases. The current study examined whether T cells and monocytes differed in their response to GC and the potential molecular basis for their variation in response to steroids. Functional studies revealed that dexamethasone (DEX) inhibited phorbol 12-myristate 13-acetate/ionomycin-induced tumor necrosis factor alpha and interleukin-6 production to a significantly lesser extent in monocytes than T cells. In parallel, a significantly longer period of time was required for DEX to induce the steroid-responsive gene, mitogen-activated protein kinase phosphatase-1 (MKP-1), in human monocytes as compared with T cells. It is interesting that such differences were not observed between murine T cells and monocytes. GC receptor beta (GCRbeta) is a splicing variant of the classic GCR, GCRalpha, which functions as a dominant-negative inhibitor of GCRalpha in humans, not mice (as mice do not express GCRbeta mRNA as a result of a difference in the murine GCR 9b exon sequence). It was found that human monocytes had a significantly higher level of GCRbeta than T cells. Furthermore, GCRbeta was found in the cytoplasm and nucleus of monocytes, and GCRbeta was localized to the nucleus of T cells. This raised the possibility that GCRbeta in the cytoplasm could affect GCRalpha cellular shuttling in response to DEX. Indeed, we found that DEX-induced nuclear translocation of GCRalpha was decreased in monocytes as compared with T cells. Specific RNA silencing of GCRbeta in human monocytes resulted in enhanced steroid-induced GCRalpha transactivation and transrepression. Our data suggest that GCRbeta contributes to variation in the GC responses of monocytes versus T cells.

Increased glucocorticoid receptor beta alters steroid response in glucocorticoid-insensitive asthma.

RATIONALE: Glucocorticoids (GCs) are highly effective in the treatment of asthma. However, some individuals have GC-insensitive asthma. OBJECTIVES: To evaluate the functional response to steroids of bronchoalveolar lavage (BAL) cells from sites of airway inflammation from patients with GC-insensitive versus GC-sensitive asthma. As well, to attempt to define the functional role of glucocorticoid receptor (GCR)beta (a splicing variant, and dominant negative inhibitor of, the classic GCRalpha) in controlling GCRalpha nuclear translocation and transactivation at a molecular level. METHODS AND MEASUREMENTS: Fiberoptic bronchoscopy with collection of BAL fluid was performed on seven patients with GC-sensitive asthma and eight patients with GC-insensitive asthma. GCRalpha cellular shuttling in response to 10(-6) M dexamethasone treatment and GCRbeta expression were analyzed in BAL cells by immunofluorescence staining. The effects of overexpression and silencing of GCRbeta mRNA on GCRalpha function were assessed. MAIN RESULTS: Significantly reduced nuclear translocation of GCRalpha in response to steroids was found in BAL cells from patients with GC-insensitive asthma. BAL macrophages from patients with GC-insensitive asthma had significantly increased levels of cytoplasmic and nuclear GCRbeta. It was demonstrated that GCRalpha nuclear translocation and its transactivation properties were proportionately reduced by level of viral transduction of the GCRbeta gene into the DO-11.10 cell line. RNA silencing of GCRbeta mRNA in human BAL macrophages from patients with GC-insensitive asthma resulted in enhanced dexamethasone-induced GCRalpha transactivation. CONCLUSIONS: GC insensitivity is associated with loss of GCRalpha nuclear translocation in BAL cells and elevated GCRbeta, which may inhibit GCRalpha transactivation in response to steroids.

Divergent expression and function of glucocorticoid receptor ß in human monocytes and T cells.

Glucocorticoid (GC) insensitivity is a significant problem in the treatment of immune-mediated diseases. The current study examined whether T cells and monocytes differed in their response to GC and the potential molecular basis for their variation in response to steroids. Functional studies revealed that dexamethasone (DEX) inhibited phorbol 12-myristate 13-acetate/ionomycin-induced tumor necrosis factor α and interleukin-6 production to a significantly lesser extent in monocytes than T cells. In parallel, a significantly longer period of time was required for DEX to induce the steroid-responsive gene, mitogen-activated protein kinase phosphatase-1 (MKP-1), in human monocytes as compared with T cells. It is interesting that such differences were not observed between murine T cells and monocytes. GC receptor ß (GCRß) is a splicing variant of the classic GCR, GCRα, which functions as a dominant-negative inhibitor of GCRα in humans, not mice (as mice do not express GCRß mRNA as a result of a difference in the murine GCR 9b exon sequence). It was found that human monocytes had a significantly higher level of GCRß than T cells. Furthermore, GCRß was found in the cytoplasm and nucleus of monocytes, and GCRß was localized to the nucleus of T cells. This raised the possibility that GCRß in the cytoplasm could affect GCRα cellular shuttling in response to DEX. Indeed, we found that DEX-induced nuclear translocation of GCRα was decreased in monocytes as compared with T cells. Specific RNA silencing of GCRß in human monocytes resulted in enhanced steroid-induced GCRα transactivation and transrepression. Our data suggest that GCRß contributes to variation in the GC responses of monocytes versus T cells.

[Screening of TACE peptide inhibitors from a phage display random 15-peptide library by recombinant TACE ecotodomain].

Tumour necrosis factor-alpha converting enzyme (TACE) is the major protease responsible for processing proTNF from membrane-anchored precursor into secreted TNF-alpha. It was validated that TACE is involved in many diseases such as arthritis, multiple sclerosis and Alzheimers, therefore it represents a novel and significant target for therapeutic intervention in a variety of inflammatory and neuroimmunological diseases. To obtain the recombinant TACE ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coded for catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RT-PCR, the expression plasmid was constructed by inserting T800/T1300 into plasmid pET-28a/pET-28c and transformed into E. coli BL21 (DE3). SDS-PAGE and Western blotting analysis revealed that T800/T1300 was highly expressed in the form of inclusion body being induced by IPTG. After Ni2+ -NTA resin affinity chromatography, the purity of the recombinant T800/T1300 protein was more than 90%. T800 and T1300 protein were used in the screening of TACE-binding peptides from the phage display random 15-peptide library. After four rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence-TRWLVYFSRPYLVAT was found and synthesized. The synthetic peptide was shown to bind to TACE and inhibit the TNF-alpha release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3%. FACS analysis revealed that the peptide mediated the accumulation of TNF-alpha on LPS-stimulated PBMC surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE and the deduced motif might be applied to molecular design of anti-inflammation drugs.

Superantigen-induced corticosteroid resistance of human T cells occurs through activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK-ERK) pathway.

BACKGROUND: Microbial superantigens induce human T-cell resistance to corticosteroids. OBJECTIVE: Understanding the molecular pathways resulting in corticosteroid-resistant T cells is important because this condition can complicate the treatment of inflammation. METHODS: The response of human PBMCs to steroids was assessed by using proliferation assays after stimulation with superantigens or anti-CD3 in the presence of various kinase inhibitors. Glucocorticoid receptor alpha (GCRalpha) localization was defined on the basis of intracellular staining. Protein phosphorylation was measured by means of Western blotting. RESULTS: In the current study we found that PBMCs stimulated with superantigen, but not anti-CD3, induced corticosteroid-resistant T cells. However, the purified T cells stimulated either with staphylococcal enterotoxin B (SEB) or anti-CD3 are susceptible to corticosteroid inhibition. These results imply that signals on antigen-presenting cells might act in concert with the T-cell receptor to cause steroid resistance. Blockade of CD40-CD40 ligand interaction had no effect on superantigen-induced corticosteroid resistance. However, CD28 costimulation with T-cell receptor activation induced corticosteroid resistance of human T cells in a dose-dependent manner. Superantigen stimulation, compared with anti-CD3 stimulation, was found to induce a more rapid and sustained phosphorylation of mitogen-activated extracellular signal-regulated kinase (ERK). Treatment with PD98059 and UO126 (specific mitogen-activated protein kinase kinase [MEK]/ERK inhibitors), but not a p38 inhibitor or a c-Jun N-terminal kinase inhibitor, restored the response to steroids, as indicated by proliferation assays. Furthermore, purified ERK1 and ERK2 were able to phosphorylate recombinant human GCRalpha directly in an in vitro kinase assay. Of note, superantigen-induced corticosteroid resistance was associated with abrogation of GCRalpha nuclear translocation. This effect could be reversed by treatment with MEK/ERK pathway inhibitors. CONCLUSIONS: These data are compatible with the hypothesis that superantigen-induced corticosteroid resistance involves the Raf-MEK-ERK1/ERK2 pathway of T-cell receptor signaling, which leads to GCRalpha phosphorylation and inhibition of dexamethasone-induced GCRalpha nuclear translocation.

CD56+ cells induce steroid resistance in B cells exposed to IL-15.

Interleukin-2 can induce steroid resistance in T cells. IL-15 shares biological activities with IL-2, as both cytokines use IL-2Rgamma for signal transduction. We therefore sought to determine whether IL-15 contributes to induction of PBMC corticosteroid resistance. Surprisingly, we found that incubation of unfractionated PBMC with IL-15 for 48 h resulted in the inhibition of glucocorticoid receptor (GCR) nuclear translocation in response to dexamethasone (DEX) treatment in CD19-positive B cells significantly greater than CD19-negative non-B cells (p < 0.01). However, pure B cells incubated with IL-15 responded normally with nuclear translocation of GCR in response to steroids, but failed to translocate GCR when they were grown in the presence of CD19(-) cells. Coculture of B cells with CD3(+) (T cells), CD14(+) (monocytes), or CD56(+) (NK and NKT cells) in the presence of IL-15 revealed that only CD56(+) cells contributed to the steroid insensitivity of B cells. IL-15 stimulation significantly increased production of IL-4 by CD56(+) cells (p < 0.02). Treatment of purified B cells with combination IL-15/IL-4 resulted in abrogation of glucocorticoid receptor nuclear translocation and the inability of DEX to suppress cytokine production by B cells. In the presence of IL-4-neutralizing Ab, when B cells were cocultured with CD56(+) cells and IL-15, the B cells were found to be steroid sensitive, i.e., DEX induced GCR nuclear translocation. This study demonstrates that B cells develop steroid resistance in the presence of CD56(+) cells after IL-15 stimulation. Furthermore, IL-15 and IL-4 have the capacity to induce B cell insensitivity to steroids.