Cleaning solutions for orthodontic clear aligners: a microbiological and colorimetric analysis.

Insulinoma-associated 1 (INSM1) is neuroendocrine (NE) transcription factor which temporal and spatial expression pattern is restricted to regions undergoing NE differentiation during embryogenesis. INSM1 is however re-expressed at high levels in small-cell lung cancer (SCLC) together with an array of other NE markers. The NE signature of SCLC is a central diagnostic tool for the disease but recent data pinpoints that this distinct signature might play a role in SCLC pathogenesis. The functional role of INSM1 in SCLC was here investigated by shRNA-mediated silencing of INSM1 in a panel of SCLC cell lines. Upon silencing of INSM1, significant reduction in cell viability was observed as measured by MTT assay. This reduction in cell viability was concomitant with decreased cell proliferation and increased apoptosis as measured by BrdU incorporation and cleaved caspase 3/7 levels, respectively. Accordingly with phenotypic growth changes it was demonstrated that INSM1 silencing caused reduced phosphorylation of members of the growth- and survival-promoting PI3K/Akt, MAPK and JAK-STAT signaling pathways and reduced levels of the inhibitors of apoptosis proteins (IAPs) members c-IAP1, XIAP and survivin. Importantly, it was shown that INSM1 is an upstream regulator of signatures previously shown to play a role in SCLC tumorigenesis, including achate-scute homolog 1 (ASCL1) and Sonic Hedgehog (SHh) pathway. Upon silencing of INSM1, a decrease in mRNA and protein levels of ASCL1 and downstream targets aldehyde dehydrogenase 1 (ALDH1) and delta-like ligand 3 (DLL3) was observed. Furthermore, mRNA levels of the SHh positive mediators Gli2, Gli3 and Smoothened was downregulated while the SHh inhibitor Patched was upregulated upon INSM1 silencing. Futhermore, overexpression of the Notch1 intracellular domain in SCLC cells resulted in a significant reduction in INSM1 expression and cell viability. This suggest that a master inhibitor of the NE signature, Notch1 signaling, could be an inhibitor of INSM1-driven tumorigenesis. In conclusion, INSM1 is a potential key driver of growth- and survival-promoting pathways in SCLC and warrants further investigation of INSM1 as a therapeutic target. Citation Format: Camilla L. Christensen, Takeshi Shimamura, Esra A. Akbay, Signe R. Michaelsen, Hans S. Poulsen, Kwok-kin Wong. Insulinoma-associated 1 is a key transcriptional regulator of growth and survival-promoting pathways in small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3126. doi:10.1158/1538-7445.AM2013-3126

Cerebral vascular deposition of the amyloid-β (Aβ) peptide, a condition known as cerebral amyloid angiopathy (CAA), is associated with intracerebral hemorrhaging and contributes to disease progression in Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID). Familial mutations at positions 22 and 23 within the Aβ peptide lead to early onset and severe CAA pathology. Here, we evaluate the effects of fibrillar Aβ peptides on the viability of primary-cultured human cerebral smooth muscle (HCSM) cells, which are the major site of amyloid deposition in cerebral blood vessel walls. Comparisons are made of the familial E22Q (Dutch) mutant of Aβ40 with wild-type Aβ40 and Aβ42. In agreement with previous studies, we find that there is a significant reduction in cell viability when Aβ40-Dutch or Aβ42-WT peptides are added to HCSM cell cultures as monomeric Aβ, whereas Aβ40-WT is relatively nontoxic. The binding of Aβ fibrils derived from sporadic CAA or familial Dutch-type CAA brain tissue to the membrane surface of HCSM cells does not result in a significant loss of cell viability. In contrast, when Aβ40-WT monomers and sporadic CAA fibrils are coincubated in HCSM cell cultures, there is a significant reduction in HCSM cell viability that is accompanied by an increase in cell surface fibril formation. Lastly, intrathecal administration of Aβ40-Dutch fibrillar seeds promotes fibrillar amyloid accumulation in the smooth muscle of meningeal vessels in the rTg-D transgenic rat model of CAA. Together, the present findings suggest that fibrillar Aβ seeds propagate the expansion of new amyloid fibrils on cerebral vascular smooth muscle, leading to membrane disruption and cell death.

Children with Group 3 medulloblastoma (G3 MB) have a very poor prognosis, and many do not survive beyond 5 years after diagnosis. A factor that may contribute to this is the lack of available targeted therapy. Expression of protein lin‐28 homolog B (LIN28B), a regulator of developmental timing, is upregulated in several cancers, including G3 MB, and is associated with worse survival in this disease. Here, we investigate the role of the LIN28B pathway in G3 MB and demonstrate that the LIN28B–lethal‐7 (let‐7; a microRNA that is a tumor suppressor)–lymphokine‐activated killer T‐cell‐originated protein kinase (PBK; also known as PDZ‐binding kinase) axis promotes G3 MB proliferation. LIN28B knockdown in G3‐MB‐patient‐derived cell lines leads to a significant reduction in cell viability and proliferation in vitro and in prolonged survival of mice with orthotopic tumors. The LIN28 inhibitor N‐methyl‐N‐[3‐(3‐methyl‐1,2,4‐triazolo[4,3‐b]pyridazin‐6‐yl)phenyl]acetamide (1632) significantly reduces G3 MB cell growth and demonstrates efficacy in reducing tumor growth in mouse xenograft models. Inhibiting PBK using HI‐TOPK‐032 also results in a significant reduction in G3 MB cell viability and proliferation. Together, these results highlight a critical role for the LIN28B–let‐7–PBK pathway in G3 MB and provide preliminary preclinical results for drugs targeting this pathway.

We investigated the possible anticancer mechanisms of Pteris vittata [PV] n-hexane extract on MCF-7 [breast cancer cell line]. Cultured cell lines were treated with various concentrations of this extract ± Baf-A1 [autophagic inhibitor]. Cells’ viability, apoptotic markers [caspase-7, Bax, and Bcl-2], autophagic markers [light chain 3 [LC-3] and P62/SQSTM1]], and the tumor suppressor P53 and its mRNA were checked by their corresponding methods. Treated cell lines showed significant concentration and time-dependent reductions in cell viability in response to PV-n-hexane extract and also exhibited a concomitant induction of apoptosis [increased chromatin condensation, nuclear fragmentation, and pro-apoptotic Bax, and cleaved caspase-7 levels while decreased Bcl-2 levels] and autophagy [increased autophagosomes vacuoles, and LC3B II levels while decreased P62/SQSTM1 levels]. Moreover, PV-n-hexane extract-treated cells showed significant increases in the P53 and its mRNA levels. The addition of Baf-A1 reversed the PV-n-hexane extract autophagic effects and increased apoptotic cell percentage with a much increase in the cleaved caspase-7 and P53 protein and its mRNA levels. We concluded that the PV-n-hexane extract exhibits cytotoxic effects on the MCF-7 cell line with significant reductions in cell viability and concomitant autophagy and apoptosis induction. Inhibition of autophagy in the PV-treated MCF-7 cells enhances apoptosis via a p35-dependent pathway.

Purpose: The aim of this study was to access the safety profiles of 2 fusion proteins with anti-vascular endothelial growth factor action (ziv-aflibercept and aflibercept) on retinal pigment epithelium cells and Muller-Glia cells in culture by assessing cell viability post drug exposure. Methods: Primary human retinal pigment epithelium cells (pRPE) and Muller-Glia cells (Mio-M1) were exposed to the clinical standardized concentrations of ziv-aflibercept (25 mg/mL) and aflibercept (40 mg/mL). Progressively higher concentrations of NaCl (300, 500, 1,000, 1,500, 2,000, 5,000, and 10,000 mosm/kg) were also applied to cells to assess the possibility of potentiating hyperosmotic cytotoxity effect. The study was applied to measure pRPE and Mio-M1 viability by a tetrazolium dye-reduction assay (XTT). Results: Cell viability of both pRPE and Mio-M1 presented no significant changes after exposure of ziv-aflibercept and aflibercept. Progressive NaCl concentrations did not significantly alter cell viability. The exposure to the negative control of 75 µL/mL of dimethyl sulfoxide showed significant reduction in cell viability. Conclusions: At clinical doses, neither ziv-aflibercept nor aflibercept caused any significant reduction in cell viability in vitro. Furthermore, injection solutions of NaCl with higher osmolality caused no significant reduction in cell viability.

CRLF2 Rearrangement Status in Ph-like ALL Predicts Intrinsic Glucocorticoid Resistance In Vitro that is Reversible with Targeted MAPK and PI3K Pathway Inhibition

Anandamide (AEA) and 2‐arachidonoylglycerol (2‐AG) are compounds naturally produced in humans and animals that interact with the cannabinoid system. These compounds activate specific cannabinoid receptors on cells to alter the neurotransmitter release from presynaptic neuron. There are studies which claim that endocannabinoids are selective towards tumor cells without affecting the functioning of normal cells, mainly by regulating different cell‐death pathways and downstream signaling molecules. The purpose of this study is to evaluate the antiproliferative activity of AEA and 2‐AG in U87‐MG glioblastoma cells. A cell viability assay utilizing 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) was performed to evaluate the antiproliferative effects. The results demonstrated that AEA at concentrations 10 and 30 μM decreased cell viability as compared to control (P≤ 0.001). 2‐arachidonoylglycerol at concentrations 10, 30 and 100 μM decreased cell viability as compared to control (P≤ 0.001). Interestingly, a combination of AEA and 2‐AG at concentrations 10 and 30 μM, respectively reduced cell viability as compared to their individual effects suggesting a synergistic approach. Moreover, a significant reduction in cell viability was observed with a combination of OL‐135, a reversible inhibitor of fatty acid amide hydrolase (FAAH) and AEA as compared to AEA alone. Similarly, a significant reduction in cell viability was observed with a combination of JZL‐184, an irreversible inhibitor for monoacylglycerol lipase (MAGL) and 2‐AG as compared to 2‐AG alone. These results suggest that by inhibiting the enzymes FAAH and MAGL which are involved in the degradation of AEA and 2‐AG, respectively a further reduction in cell viability could be observed, suggesting a strategy to inhibit endocannabinoid degrading enzymes and use of endocannabinoids as potential anti‐cancer agents. Further assays will be performed to determine the mechanism of anti‐cancer activity demonstrated by these compounds.

Many epithelial cancers have been shown to overexpress the enzyme cyclooxygenase-2 (COX-2), an enzyme responsible for metabolizing anandamide (AEA) to prostamides. AEA has demonstrated cytotoxicity in COX-2 overexpressing cancers via its metabolism to novel J-series prostamides, namely 15d-PMJ2. Fatty acid amide hydrolase (FAAH) degrades AEA into arachidonic acid and ethanolamine (EA), limiting the cytotoxic capability of AEA. Cell lines with high FAAH expression have demonstrated resistance to AEA. By understanding the metabolic characteristics of FAAH, we can design AEA analogs which circumvent FAAH breakdown. To examine the effects of altering polarity, steric bulk, and functional groups on AEA-mediated cytotoxicity, we investigated known AEA derivatives which possess these properties. Arvanil, Arachidonoyl Diethanolamine (ADA), Arachidonoyl Serinol (AS), and R1-methanandamide (m-AEA) add steric bulk to the molecule via aromatic rings, extra EA arms and additional alcohol/methyl functional groups respectively. Arachidonoyl glycine (NAGly) substitutes the terminal EA alcohol with a carboxylic acid increasing polarity. Arachidonoyl-2’-chloroethylamine (AC) substitutes the terminal EA alcohol with a highly soluble chlorine. Furthermore, it is known that Arvanil, ADA, AS, and m-AEA resist degradation of the molecule by FAAH and NAGly is a known substrate of COX-2. Therefore our goal was to determine which structural modifications improve AEA-mediated cytotoxicity. To determine this, JWF2 tumorigenic keratinocytes were exposed to differing concentrations of the AEA analogs for 24 hours and cell viability was measured by conducting MTS assays. Arvanil demonstrated a 90% reduction in cell viability, NAGly demonstrated a 70% reduction in cell viability, and m-AEA demonstrated a 100% reduction in cell viability at 20 µM, the optimal dosage of AEA. Due to the fact that ADA, AS, and AC did not show a significant reduction in cell viability these molecules were not further examined. MTS assays were conducted in other epithelial cancer cell lines with varying expressions of COX-2 and FAAH. Each cell line was exposed to varying concentrations of either NAGly, Arvanil, or both for 24 hours. NAGly demonstrated a 40% reduction in cell viability in HT-29 colon cancer cells (low COX-2, high FAAH). Arvanil demonstrated a 60% reduction in A431 tumorigenic keratinocytes. NAGly demonstrated a 60% reduction in cell viability and Arvanil demonstrated a 100% reduction in cell viability in patient-derived primary melanoma. These findings suggest that modulation and substitute to the core structure of AEA will result in decreased susceptibility to FAAH degradation and enhanced antineoplastic activity. Citation Format: Andrew Morris, Eman Soliman, Rukiyah Van Dross, Colin Burns. Structural modification of the chemotherapeutic anandamide: Designing anti-cancer agents and investigating their COX-2 metabolic products [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2193. doi:10.1158/1538-7445.AM2017-2193

[Purpose] This study aims to evaluate the possible antitumor effect of vitamin D. Vitamin D is a group of lipophilic compounds comprising of vitamin D2 and vitamin D3. Vitamin D3 has been shown to play an important role in humans. It has been previously reported that the onset risk of malignant melanoma and leukemia can be decreased by maintaining high vitamin D3 levels in the body. In addition, the cytotoxic effect of vitamin D on breast cancer cells has also been shown. In this study, the relationship between the cytotoxicity of vitamin D3 and vitamin D3 receptor (VDR) expression on melanoma and leukemia cells was examined. [Method] B16F10 melanoma (B16F10) and P388 leukemia (P388) cells were used in this study. Western blot analysis was performed to determine VDR expression in each cell type. To evaluate cytotoxicity, B16F10 or P388 cells were seeded at 1×105 cells/mL, and calcitriol as vitamin D3 (range 9 ng/mL-90 μg/mL) was added at once. After two days, cytotoxicity was evaluated by colorimetric analysis using Cell Counting Kit-8. Additionally, the cytotoxicity of P388 cells was determined following the combined treatment of vitamin D3 and retinoic acid. [Results and Discussion] Western blot analysis revealed that both B16F10 and P388 cell types, expressed VDR with a higher expression in P388 cells. In P388 cells, there was a significant reduction in cell viability following vitamin D3 treatment, which decreased to 10% at 90 μg/mL. In contrast, the viability of B16F10 cells following vitamin D3 treatment only slightly decreased. Interestingly, these results suggest that the cytotoxicity of vitamin D3 correlates with VDR expression. VDR reportedly forms a heterodimer with the retinoid X receptor. However, the presence of retinoic acid did not alter vitamin D3 cytotoxicity. Therefore, it was considered that cytotoxicity induced by vitamin D3 was not connected with the formation of the VDR-retinoid X receptor heterodimer. In conclusion, there was a higher expression of VDR in P388 cells compared with B16F10 cells. This higher expression correlated with a higher level of vitamin D3 cytotoxicity, suggests an antitumor effect of vitamin D3. This study proposes the possibility of using vitamin D3 as a novel treatment for leukemia and other cancers. Citation Format: Takaya Hoshi, Yuta Abe, Ikumi Sugiyama, Yasuyuki Sadzuka. The connection of vitamin D3 induced cytotoxicity with the vitamin D3 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 367.

IntroductionGold nanoparticles (GNPs) have drawn immense attention in cancer diagnosis and therapy during the past few years. Nanoparticles preferably leak and accumulate inside the tumour due to many reasons including high vascular density, increased vessel permeability, and defective lymphatic draining of tumours via a process called the enhanced permeability and retention (EPR) effect. Very small particles (<30 nm), although can enter the tumour tissue by the EPR effect, they also may leave it again by passive diffusion. This size-dependent cellular uptake thus limits the use of very small nanoparticles in cancer therapy. This study was conducted to improve the retention of small nanoparticles and overcome its size limitation by linking it to a noncoding DNA fragment.Material and methodsA noncoding DNA fragment was amplified by PCR using a 5’ thiol-labelled forward primer and an unmodified 3’ reverse primer. After amplification, the PCR product was purified then conjugate to citrate stabilized10 nm GNPs. MCF-7 cells were treated with GNP-DNA conjugate using metafectene as a transfection agent for cytotoxicity studies. The GNP-DNA conjugate chemosensitization of MCF-7 cells to doxorubicin (Adriamycin ) was also tested.Results and discussionsTreatment of MCF-7 cells with GNP-DNA conjugate resulted in a significant reduction in cell viability when compared to both control and unmodified GNP groups. The cell toxicity tests revealed that the conjugation of GNP with DNA lead to a 69.4-fold decrease in the IC 50 of GNP in MCF-7 cells. Furthermore, GNP-DNA conjugate showed a synergistic effect with doxorubicin leading to a significant reduction in cell viability at concentrations as low as 70 µM GNP-DNA and 1.6 µM doxorubicin.ConclusionThe results of this study shed light on the marked effect of the modification of small GNP with DNA fragment on the effect of GNP in cancer cells and hence enables studying the effects of GNP size on cellular chemosensitivity without being restricted by GNP size-related differences in uptake efficiency.

Increasing evidence suggests that unknown collagen remodeling mechanisms in the sclera underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantify collagen remodeling at the tissue- and lamella-level. Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; gluing the eye to a washer using (iii) 50 μL and (iv) 200 μL of cyanoacrylate adhesive; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in our new bioreactor. Two scleral shells of normal juvenile tree shrews were fluorescently labeled using a collagen specific protein and cultured in our bioreactor. Using two-photon microscopy, grid patterns were photobleached into and across multiple scleral lamellae. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns. No significant reduction in cell viability was observed under conditions (i) and (v). Compared to condition (i), cell viability was significantly reduced starting at day 0 (condition (ii)) and day 3 (conditions (iii, iv, vi)). Tissue-level strain and intralamellar shear angel increased significantly during the culture period. Some scleral lamellae elongated while others shortened. Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are sensitive to tissue manipulations and tissue gluing. However, Ham's F-12 Nutrient Mixture has a protective effect on cell viability and can offset the cytotoxic effect of cyanoacrylate adhesive. This is the first study to quantify collagen micro-deformations over a prolonged period in organ culture providing a new methodology to study scleral remodeling in myopia.

This study introduces advanced nanoparticle-based drug delivery systems (NDDS) designed for targeted colorectal cancer treatment. We developed and characterized three distinct formulations: Bevacizumab-loaded chitosan nanoparticles (BEV-CHI-NP), polymeric micelles (BEV-PM), and BEV-conjugated exosomes enriched with AS1411 and N1-methyladenosine (AP-BEV + M1A-EXO). Each formulation exhibited optimized physicochemical properties, with particle sizes between 150 and 250nm and surface charges ranging from + 14.4 to + 43mV, ensuring stability and targeted delivery. The AP-BEV + M1A-EXO formulation demonstrated targeted delivery to VEGF, a protein commonly overexpressed in colorectal cancer cells, as indicated by localized staining. This suggests a more precise delivery of the therapeutic agent to VEGF-enriched regions. In contrast, the BEV-CHI-NP formulation exhibited a broader pattern of tumor suppression, evidenced by reduced overall staining intensity. The BEV-PM group showed moderate effects, with a relatively uniform protein expression across tumor tissues. In vivo studies indicated that the AP-BEV + M1A-EXO formulation achieved a notable reduction in tumor volume (~ 65.4%) and decreased levels of tumor biomarkers, including CEA and CA 19-9, compared to conventional BEV-API treatment. In vitro experiments using human colon tumor organoids (HCTOs) further supported these findings, showing a significant reduction in cell viability following exposure to AP-BEV + M1A-EXO. These results suggest that combining aptamer specificity with exosome-based delivery systems could enhance the precision and effectiveness of colorectal cancer therapies, representing a potential advancement in treatment strategies. In vivo experiments further revealed that the AP-BEV + M1A-EXO formulation outperformed conventional BEV-API treatment, achieving a four-fold increase in tumor suppression. This formulation resulted in a 65.4% reduction in tumor volume and a significant decrease in tumor biomarkers, including CEA and CA 19-9. In vitro studies also demonstrated a significant reduction in cell viability in human colon tumor organoids exposed to AP-BEV + M1A-EXO. These findings highlight the potential of combining aptamer specificity with exosome-based delivery systems to enhance the precision and efficacy of colorectal cancer therapies, marking a promising step forward in cancer treatment innovation.

BackgroundThe role of the complement system and its membrane-bound regulatory proteins (mCRPs) in the pathogenesis of cancer is still a debatable issue. The current study aimed to evaluate the role of the complement regulatory protein, the decay-accelerating factor (CD55), in the pathogenesis of acute leukemia.MethodsCD55 gene expression was assessed in the peripheral blood of 34 patients with acute myeloid leukemia (AML), 26 patients with acute lymphoblastic leukemia (ALL), and 30 healthy controls by qRT-PCR. Also, CD55 gene knockdown was performed in HSB-2 (ALL cell line) using customized short hairpin RNA (shRNA). Flowcytometric analysis was done to ensure successful transfection, and MTT assay was performed to evaluate the cell viability post-transfection and silencing of CD55.ResultsThere was a significant downregulation of CD55 in acute leukemia patients compared to the healthy controls (p < 0.001) with RQ values of AML, and ALL patients were 0.2499 ± 0.07427 and 0.2581 ± 0.09467, respectively. The MTT assay showed a significantly reduced viability of HSB-2 cells following posttranscriptional silencing of CD55 (p < 0.001) by 78.6% as compared to the non-transfected or mock-transfected cells. In the presence of human serum, there was a significant reduction in cell viability by 66.3% as compared to non-transfected controls (p = 0.01). Regarding the cells co-transfected with CD55 and CD46 silencing plasmids, cell viability was significantly decreased by 70.6% compared to non-transfected cells.ConclusionCD55 was significantly downregulated in acute leukemia. However, its in vitro silencing showed significant reduction in cell viability, giving it a dual opposing role in cancer.

Multiplex Drug Screening Identifies a Novel Therapeutic Backbone for the Treatment of Acute Myeloid Leukaemia

Simple SummaryThe transformation process of chronic lymphocytic leukemia into an aggressive lymphoma, called Richter syndrome (RS), is incompletely understood, and therapeutic options are limited. Here, we report CARD9 to be expressed in a subset of RS tissue specimen and in the first and only available RS cell line, U-RT1. In U-RT1, CARD9 attaches to BCL10 and MALT1, and knockdown of CARD9 leads to a significant reduction in cell viability. We hypothesized that CARD9 plays an oncogenic role in RS through the activation of NF-κB signaling. Our findings may help to extend the current knowledge about the pathogenesis of RS and promote the development of targeted therapies for this aggressive disease.Richter syndrome (RS) is defined as the transformation of chronic lymphocytic leukemia (CLL) into an aggressive lymphoma, mostly diffuse large B-cell lymphoma (DLBCL). Despite intensive therapy, patients with RS have an unfavorable clinical outcome. The detailed pathobiology of Richter transformation still needs to be elucidated. Here, we report high mRNA and protein levels of CARD9 in the RS cell line U-RT1. Co-immunoprecipitation revealed the assembly of a CBM complex using CARD9 instead of CARD11. CARD9 is known to be an activator of NF-кB signaling in myeloid cells. U-RT1 Western blot analyses showed phosphorylation of IκB as well as IKK, indicating a constitutively active canonical NF-кB pathway. This was further supported by the significant reduction in cell viability and CYLD cleavage products after CARD9 siRNA knockdown. We also showed immunostaining for CARD9 in 53% of cases analyzed in a series of RS tissue specimens, whereas other lymphomas rarely show CARD9 expression. This is the first report on ectopic expression and function of CARD9 in an aggressive B-cell lymphoma. Our findings suggest that CARD9 may contribute to the pathogenesis of RS.