Propylthiouracil (PTU)-induced antineutrophil cytoplasmic antibody (ANCA)-related vasculitis and nephritis were recently reported in about 30 patients with hyperthyroidism. The objective of this study was to clarify the prevalence of ANCA and the relationship between ANCA and thyroid antibodies in children with Graves' disease. Titers of myeloperoxidase (MPO)-ANCA in sera of 51 patients with childhood onset Graves' disease (16 before treatment, 25 and 10 treated with PTU and methimazole, respectively) were measured by enzyme-linked immunosolvent assay. Antithyroglobulin antibodies (TGAbs) and antithyroperoxidase antibodies (TPOAbs) were also measured by RIA in 25 PTU-treated patients. No patients had clinical manifestations of vasculitis and nephritis. MPO-ANCA was positive in 6.7% of patients before treatment and in 64.0% of those treated with PTU and in none of those treated with methimazole. MPO-ANCA had a significantly positive correlation with TGAbs (P < 0.05) and no significant correlation with TPOAbs. These findings show the high prevalence of the MPO-ANCA positivity in PTU-treated childhood onset Graves' disease, suggesting that PTU may not be preferred as the first line for the treatment of children with Graves' disease. The significant correlation between MPO-ANCA and TGAbs indicates that the severity of Graves' disease may be a factor responsible for the MPO-ANCA positivity. The cross-reactivity between MPO-ANCA and TPOAbs may not play a role in the high prevalence of MPO-ANCA in the patients exposed to PTU.
Abstract Our ability to improve treatments for blood-related cancers such as multiple myeloma (MM) is currently hindered by limitations in lab procedures for studying cell signaling mechanisms in drug resistance. Conventional assays for studying signal transduction (gel shift assays) require large quantities of cell sample (> 100,000 cells per condition) that often cannot be acquired safely and reliably from primary bone marrow aspirations of different patients. Furthermore, in vitro assays often do not adequately represent key aspects of the complex bone marrow microenvironment, including spatial organization of stromal cell types. The objective of this study was to develop a new functional assay using microfluidics technology to examine signal transduction events, specifically in improved in vitro microenvironments, for non-adherent cells such as those associated with hematologic cancers, and for low cell number applications (∼1000 cells per condition) including patient samples. Successful development of this assay would provide an alternative to gel shift assays and streamline experiments. We developed microscale cell culture chambers that used a surface tension-based passive pumping method for fluidic delivery, obviating the need for external pumps and tubing commonly used in microfluidic systems, thus reducing dead volume and cell loss. To validate the platform, non-adherent RPMI8226 cells (human MM cell line) were cultured, treated with cytokines/drugs, fixed, and immunostained to determine cytoplasmic versus nuclear localization of NF-κB subunit RelA as a measure of canonical NF-κB activation. Fluorescent images of RelA were analyzed using custom image analysis for calculating ratio of nuclear to cytoplasmic signals (intensity ratio, IR) at single cell resolution. IR values of cells in a population were calculated to determine overall nuclear translocation and NF-κB activation. Results showed fluorescence immunostaining coupled with custom image analysis was able to detect dose-dependent TNF-α activation of NF-κB, and dose-dependent inhibition of NF-κB activity by bortezomib treatment. Ongoing work involves applying the platform to study effects of various drug treatments on NF-κB signaling in primary patient MM cells in monoculture and in coculture with bone marrow stromal cells. Our results demonstrate the platform is a functional, enabling tool for studying single-cell NF-κB signal transduction with low numbers of non-adherent cells. The platform is extensible to other hematologic cancers, applicable for other transcription factors (e.g., STAT3), amenable to coculture studies, and has potential to enable novel experiments with limited primary samples that may have significant impact in translational research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3197. doi:1538-7445.AM2012-3197
<div>Abstract<p>We recently showed that a subset of human T acute lymphoblastic leukemia (T-ALL) cell lines expresses low basal levels of p50, a nuclear factor-κB (NF-κB)/Rel family member, resulting in their capacity to activate the atypical p65:cRel complex rather than the classic p50:p65 dimer. Here, we show that the transcription factor TAL1 (also known as SCL) binds to the promoter of the NFKB1 gene that encodes p50 and represses its transcription to set up this unique response in T-ALL cells. When TAL1 expression is reduced in CEM T leukemia cells, basal NFKB1 expression is increased, and the levels of p65:cRel complex and transcription of its target gene, such as intercellular adhesion molecule-1 (ICAM-1), are reduced in response to etoposide treatment. Moreover, a significant negative correlation between NFKB1 and TAL1 or LMO1 was found in primary human TAL1/LMO1 double-positive T-ALL samples previously described by Ferrando et al. Thus, TAL1 modulates NFKB1 expression and an NF-κB-dependent transcriptional program in a subset of human T-cell leukemia cells. (Cancer Res 2006; 66(12): 6008-13)</p></div>
We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.
To analyze the utility of insulin-like growth factor binding protein-3 (IGFBP-3) radioimmunoassay for diagnosis of growth hormone deficiency (GHD) we measured IGFBP-3 in sera from normal children, short children and patients with GHD. The sensitivity (true positive ratio) of IGFBP-3 for complete GHD (cGHD) was 93%, while the specificity (true negative ratio) for normal short children (NS) was 88%. In contrast, the sensitivity of IGFBP-3 for partial GHD (pGHD) was only 43%. The poor discrimination between patients with pGHD and NS may be the result of their relatively similar GH level, as compared to cGHD, or due to the limitations of GH stimulation tests. The specificity of IGFBP-3 for NS was excellent in children of all ages: less than 10 years old (87%) and older than 10 (88%). However, sensitivity for GHD was good for children less than 10 years old (84%) but poor for children older than 10 (64%). IGFBP-3 may be less sensitive for diagnosing GHD in older children because IGFBP-3 levels may also increase during puberty due to mechanisms independent of the GH-IGF-I axis.
Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β–activated kinase-1 (TAK1) phosphorylation of NF-κB–activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB–mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.
Abstract The nuclear factor-κB (NF-κB)/Rel transcription factors are recognized as critical apoptosis regulators. We reported previously that NF-κB contributes to chemoresistance of CEM human T leukemic cells in part through its ability to induce p21waf1/cip1. Here, we provide evidence that sequential NF-κB-activating signals induce heightened NF-κB DNA binding and p21waf1/cip1 induction in CEM and additional T leukemic cell lines. This response arises from exceedingly low basal expression of the p105/p50 NF-κB subunit encoded by the NFKB1 gene in these cell lines. An initial NF-κB activation event enhances the recruitment of p65 and ELF1 to the NFKB1 promoter, leading to p65- and ELF1-dependent synthesis of p105/p50, which promotes an exchange of NF-κB complexes to p50-containing complexes with an increased DNA-binding activity to certain NF-κB target elements. Subsequent stimulation of these cells with an anticancer agent, etoposide, results in augmented NF-κB-dependent p21waf1/cip1 induction and increased chemoresistance of the leukemia cells. Thus, we propose that low basal NFKB1 expression coupled with sequential NF-κB activation events can promote increased chemoresistance in certain T leukemic cells. (Mol Cancer Res 2006;4(2):101–12)
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