Supplementary Figures 1-5, Tables 1-2 from Activation of Toll-like Receptor 5 on Breast Cancer Cells by Flagellin Suppresses Cell Proliferation and Tumor Growth

Microprocessors are widely used in space applications. ARM processors fabricated with FDSOI technologies are promising for future space programs. However, radiation-hardening approaches can greatly affect the resource overhead and performance. These hardening techniques need to be evaluated at system level to better understand their effectiveness, especially for modern CMOS technologies. In this paper, two ARM (R) Cortex (R) M0 cores, a reference core and a hardened core were implemented on the same die in a 28-nm FDSOI technology. The reference core was designed with components from the standard cell library, while the other one was implemented with hardened DICE flip-flops. Both of the cores shared the same on-chip TMR-protected SRAM. Heavy ion experiments showed that the error cross section of the core with DICE flip-flops was about 2 times smaller at LET of 40 MeV.cm(2).mg(-1). This suggests that the contribution from logic circuits in the microprocessor cores also need to be considered for better error reduction.

The objective of this study was to compare nucleated cell fractions and mesenchymal stromal cells (MSCs) from adipose tissue to bone marrow processed by a point-of-care device that are available for immediate implantation. A paired comparison using adipose and bone marrow from five horses was done. The number of nucleated cells, viability, total adherent cells on day 6 of culture and colony-forming unit fibroblasts (CFU-Fs) were determined. Gene expression for markers of stemness, adipogenic, chondrogenic, osteogenic lineage, and collagen formation was measured in total RNA isolated from adherent adipose and bone marrow cells. Day 6 adherent adipose-derived MSC was frozen briefly, whereas day 6 adherent bone marrow–derived MSC was passaged two additional times to obtain adequate cell numbers for chondrogenic, osteogenic, and adipogenic cell differentiation assays. The total cell count per gram was significantly greater for bone marrow, whereas total adherent cells per gram and the CFU-F per million nucleated cells on day 6 were significantly greater for the adipose. In undifferentiated adherent cells, relative gene expression for CD34, adipogenic, and chondrogenic markers and collagen II was significantly lower in the adipose-derived cells. Conversely, expression of collagen I was significantly higher in the undifferentiated adipose-derived cells. Cell density and total RNA were higher in differentiated adipogenic and osteogenic cultures of adipose cells and in chondrogenic cultures of bone marrow cells. This cell preparation method provides a stromal vascular fraction with a large proportion of multipotent MSCs. There are differences in the cells obtained from the two sources. This method can provide an adequate number of multipotent cells from adipose tissue for immediate implantation.

AbstractIncreasing evidence showed that Toll-like receptors (TLR), key receptors in innate immunity, play a role in cancer progression and development but activation of different TLRs might exhibit the exact opposite outcome, antitumor or protumor effects. TLR function has been extensively studied in innate immune cells, so we investigated the role of TLR signaling in breast cancer epithelial cells. We found that TLR5 was highly expressed in breast carcinomas and that TLR5 signaling pathway is overly responsive in breast cancer cells. Interestingly, flagellin/TLR5 signaling in breast cancer cells inhibits cell proliferation and an anchorage-independent growth, a hallmark of tumorigenic transformation. In addition, the secretion of soluble factors induced by flagellin contributed to the growth-inhibitory activity in an autocrine fashion. The inhibitory activity was further confirmed in mouse xenografts of human breast cancer cells. These findings indicate that TLR5 activation by flagellin mediates innate immune response to elicit potent antitumor activity in breast cancer cells themselves, which may serve as a novel therapeutic target for human breast cancer therapy. Cancer Res; 71(7); 2466–75. ©2011 AACR.

Abstract Toll-like receptors (TLRs) play key roles in both the innate and adaptive immune systems, particularly in inflammatory responses against pathogen infection. Recent evidence showed that functional TLRs are also expressed on a wide variety of tumor cells; however, their activation exhibits either antitumor or pro-tumor effects. In order to investigate the function of TLR signaling pathways in breast cancers, we analyzed the expression of TLRs and their functional status in breast cancer cells by RT-PCR and NF-κB reporter gene assays in response to synthetic ligands for these receptors. We found that only TLR5 signaling is highly responsive in breast cancer cells. The phosphorylation levels of IκB, ERK, and JNK after flagellin (an agonist of TLR5) stimulation were increased in tumor cells. Activation of TLR5 signaling in tumor cells by flagellin induces the secretion of various cytokines and chemokines including interleukin-6, interleukin-8, and TNF-α. Moreover, flagellin/TLR5 signaling in MCF-7 cells inhibits an anchorage-independent growth, a hallmark of tumorigenic transformation. In addition, the secretion of soluble factors induced by flagellin contributed to the growth-inhibitive activity in an autocrine fashion. The inhibitive activity was further confirmed in mouse xenografts of human breast cancer model. Administration of flagellin inhibits tumor growth and therefore prolongs the survival of tumor-bearing mice. These findings indicate that TLR5 activation by flagellin mediates innate immunity to elicit potent antitumor activity in breast cancers, which may serve as a novel therapeutic target for human breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3819.

Toll-like receptors (TLRs) are the key molecular sensors used by the mammalian innate immune system to detect various types of pathogens. Tlr13 is a novel and uncharacterized member of the mammalian TLR family. Here we report the cloning and characterization of tlr13. Tlr13 is predominantly expressed in the spleen, particularly in dendritic cells and macrophages. Tlr13 appears to activate a MyD88- and TAK1-dependent TLR signaling pathway, inducing the activation of NF-κB. This receptor can also activate type 1 interferon through IRF7. Furthermore, Tlr13 seems to be another intracellular TLR. Remarkably, cells expressing tlr13 fail to respond to known TLR ligands but instead respond specifically to vesicular stomatitis virus. Cells with the knockdown of tlr13 are highly susceptible to vesicular stomatitis virus infection. Thus, these results provide an important insight into the potential role of the novel Toll-like receptor tlr13 in the recognition of viral infection.

The high prevalence and incidence of atopic asthma has highlighted this disease as an important health problem to be addressed. This chronic disorder is characterized by airflow obstruction, bronchial hyperresponsiveness, and inflammation. The role of adaptive immunity in atopic asthma has been extensively studied; however the importance of innate immunity in this disease is beginning to be uncovered. Toll-like receptors are pathogen-recognition receptors ( PRRs) that recognize pathogen-associated molecular patterns ( PAMPs). These receptors have been found to be expressed extensively in the airway. Epidemiological and genetic studies have linked TLR2 and TLR4 with atopic disease. Also, in vivo and in vitro studies of T1,K2, TLR4 and TLR9 with their respective ligands have associated these receptors with the activation or decrease of the adaptive response. This review attempts to discuss the most relevant data that links allergic asthma with TLRs.

Little has been known about Tlr13 (Toll-like receptor 13), a novel member of the Toll-like receptor family. To elucidate the molecular basis of murine Tlr13 gene expression, the activity of the Tlr13 gene promoter was characterized. Reporter gene analysis and electrophoretic mobility shift assays demonstrated that Tlr13 gene transcription was regulated through three cis-acting elements that interacted with the Ets2, Sp1, and PU.1 transcription factors. Furthermore, our work suggests that these transcription factors may cooperate, culminating in maximal transcription of the Tlr13 gene. In contrast, NF-kappa B appeared to act as an inhibitor of Tlr13 transcription. Overexpression of Ets2 caused a strong increase in the transcriptional activity of the Tlr13 promoter; however, overexpression of NF-kappa B p65 dramatically inhibited it. Additionally, interferon-beta is capable of acting Tlr13 transcription, but the activated signaling of lipopolysaccharide/TLR4 and peptidoglycan/TLR2 strongly inhibited the Tlr13 gene promoter. Thus, these findings reveal the mechanism of Tlr13 gene regulation, thereby providing insight into the function of Tlr13 in the immune response to pathogen.

As sensors of invading microorganisms, Toll-like receptors (TLRs) are expressed not only on macrophages and dendritic cells (DCs) but also on epithelial cells. In the TLR family, Tlr11 appears to have the unique feature in that it is expressed primarily on epithelial cells, although it is also expressed on DCs and macrophages. Here, we demonstrate that transcription of the Tlr11 gene is regulated through two cis-acting elements, one Ets-binding site and one interferon regulatory factor (IRF)-binding site. The Ets element interacts with the epithelium-specific transcription factors, ESE-1 and ESE-3, and the IRF motif interacts with IRF-8. Thus, Tlr11 expression on epithelial cells is regulated by the transcription factors that are presumably distinct from transcription factors that regulate the expression of TLRs in innate immune cells such as macrophages and DCs. Our results imply that the distinctive transcription regulatory machinery for TLRs on epithelium may represent a promising new avenue for the development of epithelia-specific therapeutic interventions.