Performance of the Sofia SARS-CoV-2 Rapid Antigen Test in Symptomatic and Asymptomatic Pediatric Patients.

The sensitivity and specificity of SARS-CoV-2 antigen tests have not been widely assessed in children. We evaluated children presenting to outpatient care with Quidel Sofia SARS-CoV-2 antigen test (Sofia-Ag-RDT) compared against Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV reverse transcriptase-polymerase chain reaction test from November 2020 to April 2021. Sofia-Ag-RDT had the highest sensitivity in symptomatic (82%; 95% confidence interval, 68%-91%) children.

Lack of Detection of Haemophilus influenzae Using Adult Blood Culture Bottles Inoculated at Low Volumes.

BACKGROUND: Based on guidelines from the Infectious Diseases Society of America and the American Society for Microbiology, many pediatric hospitals are implementing weight-based collection guidelines for blood cultures. To simplify the process of culture collection, there has been interest in validating the use of adult blood culture bottles with low volumes, which may allow for a "one size fits all" bottle. METHOD: This study examined 9 clinically relevant organisms (Staphylococcus aureus, Streptococcus pneumonia, Streptococcus agalactiae, Enterococcus faecium, Escherichia coli, Haemophilus influenzae, Pseudomonas aeruginosa, Candida glabrata, and Candida albicans) utilizing the BD BacTec system at various inoculation volumes and dilutions to assess performance, based on time to positivity, of adult blood culture bottles compared with pediatric blood culture bottles. RESULTS: There was a lack of detection of H. influenzae using adult blood culture bottles inoculated at low volumes, whereas pediatric bottles detected H. influenzae regardless of dilution-volume combinations tested. CONCLUSIONS: Exclusive use of adult blood culture bottles may not detect H. influenzae bacteremia in the setting of low-volume inoculum.

MALT1 is a critical mediator of PAR1-driven NF-κB activation and metastasis in multiple tumor types.

Protease-activated receptor 1 (PAR1), a thrombin-responsive G protein-coupled receptor (GPCR), is implicated in promoting metastasis in multiple tumor types, including both sarcomas and carcinomas, but the molecular mechanisms responsible remain largely unknown. We previously discovered that PAR1 stimulation in endothelial cells leads to activation of NF-κB, mediated by a protein complex comprised of CARMA3, Bcl10, and the MALT1 effector protein (CBM complex). Given the strong association between NF-κB and metastasis, we hypothesized that this CBM complex could play a critical role in the PAR1-driven metastatic progression of specific solid tumors. In support of our hypothesis, we demonstrate that PAR1 stimulation results in NF-κB activation in both osteosarcoma and breast cancer, which is suppressed by siRNA-mediated MALT1 knockdown, suggesting that an intact CBM complex is required for the response in both tumor cell types. We identify several metastasis-associated genes that are upregulated in a MALT1-dependent manner after PAR1 stimulation in cancer cells, including those encoding the matrix remodeling protein, MMP9, and the cytokines, IL-1ß and IL-8. Further, exogenous expression of PAR1 in MCF7 breast cancer cells confers highly invasive and metastatic behavior which can be blocked by CRISPR/Cas9-mediated MALT1 knockout. Importantly, we find that PAR1 stimulation induces MALT1 protease activity in both osteosarcoma and breast cancer cells, an activity that is mechanistically linked to NF-κB activation and potentially other responses associated with aggressive phenotype. Several small molecule MALT1 protease inhibitors have recently been described that could therefore represent promising new therapeutics for the prevention and/or treatment of PAR1-driven tumor metastasis.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

Background & Aims: Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor ß (TGFß) family signaling through SMAD4 in colonic epithelial cells. Methods: The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFß1 and bone morphogenetic protein 2. Results: Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFß signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions: TGFß suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

CARMA3 Is a Critical Mediator of G Protein-Coupled Receptor and Receptor Tyrosine Kinase-Driven Solid Tumor Pathogenesis.

The CARMA-Bcl10-MALT1 (CBM) signalosome is an intracellular protein complex composed of a CARMA scaffolding protein, the Bcl10 linker protein, and the MALT1 protease. This complex was first recognized because the genes encoding its components are targeted by mutation and chromosomal translocation in lymphoid malignancy. We now know that the CBM signalosome plays a critical role in normal lymphocyte function by mediating antigen receptor-dependent activation of the pro-inflammatory, pro-survival NF-κB transcription factor, and that deregulation of this signaling complex promotes B-cell lymphomagenesis. More recently, we and others have demonstrated that a CBM signalosome also operates in cells outside of the immune system, including in several solid tumors. While CARMA1 (also referred to as CARD11) is expressed primarily within lymphoid tissues, the related scaffolding protein, CARMA3 (CARD10), is more widely expressed and participates in a CARMA3-containing CBM complex in a variety of cell types. The CARMA3-containing CBM complex operates downstream of specific G protein-coupled receptors (GPCRs) and/or growth factor receptor tyrosine kinases (RTKs). Since inappropriate expression and activation of GPCRs and/or RTKs underlies the pathogenesis of several solid tumors, there is now great interest in elucidating the contribution of CARMA3-mediated cellular signaling in these malignancies. Here, we summarize the key discoveries leading to our current understanding of the role of CARMA3 in solid tumor biology and highlight the current gaps in our knowledge.

Elevated ALCAM shedding in colorectal cancer correlates with poor patient outcome.

Molecular biomarkers of cancer are needed to assist histologic staging in the selection of treatment, outcome risk stratification, and patient prognosis. This is particularly important for patients with early-stage disease. We show that shedding of the extracellular domain of activated leukocyte cell adhesion molecule (ALCAM) is prognostic for outcome in patients with colorectal cancer (CRC). Previous reports on the prognostic value of ALCAM expression in CRC have been contradictory and inconclusive. This study clarifies the prognostic value of ALCAM by visualizing ectodomain shedding using a dual stain that detects both the extracellular and the intracellular domains in formalin-fixed tissue. Using this novel assay, 105 patients with primary CRCs and 12 normal mucosa samples were evaluated. ALCAM shedding, defined as detection of the intracellular domain in the absence of the corresponding extracellular domain, was significantly elevated in patients with CRC and correlated with reduced survival. Conversely, retention of intact ALCAM was associated with improved survival, thereby confirming that ALCAM shedding is associated with poor patient outcome. Importantly, analysis of patients with stage II CRC showed that disease-specific survival is significantly reduced for patients with elevated ALCAM shedding (P = 0.01; HR, 3.0), suggesting that ALCAM shedding can identify patients with early-stage disease at risk of rapid progression.

XIAP monoubiquitylates Groucho/TLE to promote canonical Wnt signaling.

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing ß-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.

Smad4-mediated signaling inhibits intestinal neoplasia by inhibiting expression of ß-catenin.

BACKGROUND & AIMS: Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of ß-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor ß and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of ß-catenin messenger RNA (mRNA) and Wnt signaling. METHODS: We used microarray analysis to associate levels of Smad4 and ß-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APC(Δ1638/+)) and (APC(Δ1638/+)) × (K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection. RESULTS: In human CRC samples, reduced levels of Smad4 correlated with increased levels of ß-catenin mRNA. In Smad4-depleted cell lines, levels of ß-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the ß-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of ß-catenin mRNA. In mice with heterozygous disruption of Apc(APC(Δ1638/+)), Smad4-deficient intestinal adenomas had increased levels of ß-catenin mRNA and expression of Wnt target genes compared with adenomas from APC(Δ1638/+) mice that expressed Smad4. CONCLUSIONS: Transcription of ß-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor ß, BMP, and Wnt signaling pathways in progression of CRC.