Targeting DNA damage response components induces enhanced STING-dependent type-I IFN response in ATM deficient cancer cells and drives dendritic cell activation.

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example, ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However, additional antitumor benefits from targeting the DDR pathways, which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal, inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here, we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However, STING deficiency in tumor cells does not prevent DC activation, suggesting that transactivation of the STING pathway occurs within DCs. Furthermore, depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells, indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary, in this study, we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation, which is enhanced in ATM-deficient cells.

Seroprevalencia de infección frente a SARS-CoV-2 en trabajadores de la salud en un hospital monográfico pediátrico en Madrid (España) / Seroprevalence of antibodies against SARS-CoV-2 among health care workers in a pediatric monographic hospital in Madrid (Spain)

Introducción: A 30 de abril de 2020, se habían notificado 203.715 infecciones SARS-CoV-2 en España, 54.486 en Madrid, y el 21,4% eran trabajadores de la salud. El objetivo del estudio es determinar la prevalencia serológica de infección SARS-CoV-2 en trabajadores de un hospital monográfico pediátrico. Método: Del 13 al 30 de abril, 1.523 trabajadores fueron convocados a realizar un test serológico (All Test®) frente a SARS-CoV-2 y respondieron un cuestionario con información demográfica, clínico-epidemiológica y de exposición a COVID-19. Resultados: Mil doscientos noventa y dos (84,8%) fueron estudiados. La prevalencia serológica (IgM y/o IgG+) a SARS-CoV-2 fue del 17,2% (222/1.292) y del 15,5% (201/1.292) considerando IgG positiva. La edad media fue 44±13 años, el 73% eran mujeres. El 33,8% (75/222) fueron asintomáticos. Tenían rRT-PCR positiva previa 81. El 14% (32/222) contacto familiar. Conclusión: La prevalencia serológica SARS-CoV-2 en los trabajadores de un hospital pediátrico fue mayor que en la población general. Muchos pasaron una infección inadvertida.(AU)

Introduction: As of 30 April 2020, 203.715 SARS-CoV-2 infections had been reported in Spain, 54.486 in Madrid, 21.4% were health care workers. Our objective is to determine seroprevalence of COVID-19 among workers in a monographic pediatric hospital. Methods: Between April13th and 30th, 1.523 health workers were recruited to be tested for SARS-CoV-2 serology screening (All Test®) and they answered a questionnaire with demographic, epidemiological and clinical information and previous exposure to COVID-19. Findings: One thousand two hundred ninety two (84.8%) were tested. Positive serology (IgM and/or IgG) to SARS-CoV-2 was found in 17.2% (222/1.292), in 15.5% (201/1.292) if only IgG was considered. Median age was 44±13 years, 73% were female. The 33.8% (75/222) were asymptomatic. Eighty one had a previous positive rRT-PCR. The 14% (32/222) referred a family contact. Conclusion: Serology prevalence for SARS-CoV-2 in workers of a pediatric hospital was higher than in general population. Many of them had an unnoticed infection.(AU)

Seroprevalence of antibodies against SARS-CoV-2 among health care workers in a pediatric monographic hospital in Madrid (Spain).

INTRODUCTION: As of 30 April 2020, 203.715 SARS-CoV-2 infections had been reported in Spain, 54.486 in Madrid, 21.4% were health care workers. Our objective is to determine seroprevalence of COVID-19 among workers in a monographic pediatric hospital. METHODS: Between April13th and 30th, 1.523 health workers were recruited to be tested for SARS-CoV-2 serology screening (All Test®) and they answered a questionnaire with demographic, epidemiological and clinical information and previous exposure to COVID-19. FINDINGS: One thousand two hundred ninety two (84.8%) were tested. Positive serology (IgM and/or IgG) to SARS-CoV-2 was found in 17.2% (222/1.292), in 15.5% (201/1.292) if only IgG was considered. Median age was 44±13 years, 73% were female. The 33.8% (75/222) were asymptomatic. Eighty one had a previous positive rRT-PCR. The 14% (32/222) referred a family contact. CONCLUSION: Serology prevalence for SARS-CoV-2 in workers of a pediatric hospital was higher than in general population. Many of them had an unnoticed infection.

Seroprevalence of antibodies against SARS-CoV-2 among health care workers in a pediatric monographic hospital in Madrid (Spain). / Seroprevalencia de infección frente a SARS-CoV-2 en trabajadores de la salud en un hospital monográfico pediátrico en Madrid (España).

INTRODUCTION: As of 30 April 2020, 203.715 SARS-CoV-2 infections had been reported in Spain, 54.486 in Madrid, 21.4% were health care workers. Our objective is to determine seroprevalence of COVID-19 among workers in a monographic pediatric hospital. METHODS: Between April13th and 30th, 1.523 health workers were recruited to be tested for SARS-CoV-2 serology screening (All Test®) and they answered a questionnaire with demographic, epidemiological and clinical information and previous exposure to COVID-19. FINDINGS: One thousand two hundred ninety two (84.8%) were tested. Positive serology (IgM and/or IgG) to SARS-CoV-2 was found in 17.2% (222/1.292), in 15.5% (201/1.292) if only IgG was considered. Median age was 44±13 years, 73% were female. The 33.8% (75/222) were asymptomatic. Eighty one had a previous positive rRT-PCR. The 14% (32/222) referred a family contact. CONCLUSION: Serology prevalence for SARS-CoV-2 in workers of a pediatric hospital was higher than in general population. Many of them had an unnoticed infection.

A cell-engineered system to assess tumor cell sensitivity to CD8+ T cell-mediated cytotoxicity.

In vitro assays that evaluate CD8+ T cell-mediated cytotoxicity are important to aid in the development of novel therapeutic approaches to enhance anti-tumor immune responses. Here, we describe a novel cytotoxicity co-culture assay that circumvents the problem of highly variable allogeneic responses and obviates the constraints of HLA-restriction between effector and target cells. We show that this assay can be easily applied to a panel of tumor cell lines to provide additional insights into intrinsic drivers of sensitivity/resistance to T cell-mediated killing, and to evaluate the impact of targeted therapies on both tumor and T cell compartments.

Falso positivo a morfina en orina por reactividad cruzada con alimemazina / False positive result to morphine in urine due to cross-reactivity to alimemazine

La alimemazina (Variargil(R)) es un antihistamínico anti-H1 reversible inespecífico que atraviesa la barrera hematoencefálica. Actúa como anticolinérgico. Su forma de presentación es en gotas en suspensión oral. Su uso clínico está extendido a rinitis alérgica estacional, angioedema y urticaria leve a partir de los 2 años de edad, conjuntivitis alérgica e insomnio de conciliación en la infancia. Presenta una farmacocinética muy variable, somnolencia excesiva conocida como «heavy hangover» y efecto de rebote tras su retirada. No está autorizado su uso en menores de 2 años. Presentamos el caso de un paciente de 19 meses que acude a Urgencias por referir somnolencia y falta de respuesta a estímulos tras la administración de alimemazina por dificultad para conciliar el sueño

Alimemazine (Variargil(R)) is a non-specific reversible anti-H1 antihistamine that crosses the blood-brain barrier. It acts as anticholinergic drug. It is marketed in drop form (oral suspension). It is used to relieve seasonal allergic rhinitis, angioedema, mild urticaria, allergic conjunctivitis, and for difficulty in falling asleep in children. It has a very variable pharmacokinetic, excessive "heavy hangover drowsiness" and rebound effect after withdrawal. It is not authorised for children under 2 years of age. The case is presented of a patient seen in the emergency room due to drowsiness and lack of response to stimuli after administration of alimemazine due to difficulties in falling asleep

Suppression of IRAK1 or IRAK4 Catalytic Activity, but Not Type 1 IFN Signaling, Prevents Lupus Nephritis in Mice Expressing a Ubiquitin Binding-Defective Mutant of ABIN1.

Polymorphisms in the TNIP1 gene encoding A20-binding inhibitor of NF-κB1 (ABIN1) predispose to lupus and other autoimmune diseases in at least eight human populations. We found previously that knock-in mice expressing a ubiquitin-binding-defective mutant of ABIN1 (ABIN1[D485N]) develop autoimmunity as they age and succumb to a disease resembling lupus nephritis in humans. In this article, we report that Flt3-derived dendritic cells from these mice overproduced type 1 IFNs upon stimulation with ligands that activate TLR7 or TLR9. However, crossing ABIN1[D485N] mice to IFNAR1-knockout mice that do not express the α-subunit of the type 1 IFNR did not prevent splenomegaly, the appearance of high serum levels of autoantibodies and other Igs, or liver inflammation and only reduced kidney inflammation modestly. In contrast, crossing ABIN1[D485N] mice to knock-in mice expressing catalytically inactive mutants of IRAK1 or IRAK4 prevented splenomegaly, autoimmunity, and liver and kidney inflammation. Our results support the notion that IRAK1 and/or IRAK4 are attractive targets for the development of drugs to prevent, and perhaps treat, lupus nephritis and other autoinflammatory diseases caused by the decreased ability of ABIN1 or other proteins to restrict the strength of MyD88 signaling.

Suppression of interferon ß gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members.

PLK (Polo-like kinase) inhibitors, such as BI-2536, have been reported to suppress IFNB (encoding IFNß, interferon ß) gene transcription induced by ligands that activate TLR3 (Toll-like receptor 3) and TLR4. In the present study, we found that BI-2536 is likely to exert this effect by preventing the interaction of the transcription factors IRF3 (interferon-regulatory factor 3) and c-Jun with the IFNB promoter, but without affecting the TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1}-catalysed phosphorylation of IRF3 at Ser³96, the dimerization and nuclear translocation of IRF3 or the phosphorylation of c-Jun and ATF2 (activating transcription factor 2). Although BI-2536 inhibits few other kinases tested, it interacts with BET (bromodomain and extra-terminal) family members and displaces them from acetylated lysine residues on histones. We found that BET inhibitors that do not inhibit PLKs phenocopied the effect of BI-2536 on IFNB gene transcription. Similarly, BET inhibitors blocked the interaction of IRF5 with the IFNB promoter and the secretion of IFNß induced by TLR7 or TLR9 ligands in the human plasmacytoid dendritic cell line GEN2.2, but without affecting the nuclear translocation of IRF5. We found that the BET family member BRD4 (bromodomain-containing protein 4) was associated with the IFNB promoter and that this interaction was enhanced by TLR3- or TLR4-ligation and prevented by BI-2536 and other BET inhibitors. Our results establish that BET family members are essential for TLR-stimulated IFNB gene transcription by permitting transcription factors to interact with the IFNB promoter. They also show that the interaction of the IFNB promoter with BRD4 is regulated by TLR ligation and that BI-2536 is likely to suppress IFNB gene transcription by targeting BET family members.

Protein kinase IKKß-catalyzed phosphorylation of IRF5 at Ser462 induces its dimerization and nuclear translocation in myeloid cells.

The siRNA knockdown of IFN Regulatory Factor 5 (IRF5) in the human plasmacytoid dendritic cell line Gen2.2 prevented IFNß production induced by compound CL097, a ligand for Toll-like receptor 7 (TLR7). CL097 also stimulated the phosphorylation of IRF5 at Ser462 and stimulated the nuclear translocation of wild-type IRF5, but not the IRF5[Ser462Ala] mutant. The CL097-stimulated phosphorylation of IRF5 at Ser462 and its nuclear translocation was prevented by the pharmacological inhibition of protein kinase IKKß or the siRNA knockdown of IKKß or its "upstream" activator, the protein kinase TAK1. Similar results were obtained in a murine macrophage cell line stimulated with the TLR7 agonist compound R848 or the nucleotide oligomerization domain 1 (NOD1) agonist KF-1B. IKKß phosphorylated IRF5 at Ser462 in vitro and induced the dimerization of wild-type IRF5 but not the IRF5[S462A] mutant. These findings demonstrate that IKKß activates two "master" transcription factors of the innate immune system, IRF5 and NF-κB.

Cot/tpl2-MKK1/2-Erk1/2 controls mTORC1-mediated mRNA translation in Toll-like receptor-activated macrophages.

Cot/tpl2 is the only MAP3K that activates MKK1/2-Erk1/2 in Toll-like receptor-activated macrophages. Here we show that Cot/tpl2 regulates RSK, S6 ribosomal protein, and 4E-BP phosphorylation after stimulation of bone marrow-derived macrophages with lipopolysaccharide (LPS), poly I:C, or zymosan. The dissociation of the 4E-BP-eIF4E complex, a key event in the cap-dependent mRNA translation initiation, is dramatically reduced in LPS-stimulated Cot/tpl2-knockout (KO) macrophages versus LPS-stimulated wild-type (Wt) macrophages. Accordingly, after LPS activation, increased cap-dependent translation is observed in Wt macrophages but not in Cot/tpl2 KO macrophages. In agreement with these data, Cot/tpl2 increases the polysomal recruitment of the 5´ TOP eEF1α and eEF2 mRNAs, as well as of inflammatory mediator gene-encoding mRNAs, such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and KC in LPS-stimulated macrophages. In addition, Cot/tpl2 deficiency also reduces total TNFα, IL-6, and KC mRNA expression in LPS-stimulated macrophages, which is concomitant with a decrease in their mRNA half-lives. Macrophages require rapid fine control of translation to provide an accurate and not self-damaging response to host infection, and our data show that Cot/tpl2 controls inflammatory mediator gene-encoding mRNA translation in Toll-like receptor-activated macrophages.

Cot/tpl2 activity is required for TLR-induced activation of the Akt p70 S6k pathway in macrophages: Implications for NO synthase 2 expression.

LPS stimulation activates IKK and different MAP kinase pathways, as well as the PI3K-Akt-mTOR-p70 S6k pathway, a negative regulator of these MyD88-dependent intracellular signals. Here, we show that Cot/tpl2, a MAP3K responsible for the activation of the MKK1-Erk1/2, controls P-Ser473 Akt and P-Thr389 p70 S6k phosphorylation in LPS-stimulated macrophages. Analysis of the intracellular signalling in Cot/tpl2 KO macrophages versus WT macrophages reveals lower IκBα recovery and higher phosphorylation of JNK and p38α after 1 h of LPS stimulation. Moreover, Cot/tpl2 deficiency increases LPS-induced NO synthase 2 (NOS2) expression in macrophages. Inhibition of the PI3K pathway abolishes the differences in IκBα and NOS2 expression between Cot/tpl2 KO and WT macrophages following LPS administration. Furthermore, in zymosan- and polyI:C-stimulated macrophages, Cot/tpl2 mediates P-Ser473 Akt phosphorylation, increases IκBα levels and decreases NOS2 expression. In conclusion, these data reveal a novel role for the Cot/tpl2 pathway in mediating TLR activation of the Akt-mTOR-p70 S6k pathway, allowing Cot/tpl2 to fine-control the activation state of other signalling pathways.

COX2 expression and Erk1/Erk2 activity mediate Cot-induced cell migration.

The MAPKKK8 Cot/tpl-2, identified as an oncogene (Cot-T), participates in the intracellular signaling activated by members of the TLR and TNFalpha receptor superfamilies. Here we demonstrate that Cot promotes cell migration by regulating different steps involved in this process, such as cell adhesion and metalloproteinase activity. Indeed, Cot also regulates the cytoskeleton and Cot-T overexpression provokes the polarization of microtubules and the loss of stress fibers. Moreover, and in accordance with the increased Rac-GTP levels observed, Cot-T overexpressing cells develop more lamellipodia than control cells. Conversely, depletion of endogenous Cot increases the formation of stress fibers which is correlated with the high levels of Rho-GTP observed in these cells. In addition, the increase in COX2 expression and the activation of Erk1/2 regulated by Cot are essential for the induction of cell migration. Together, these data provide evidence of a new role for both proto-oncogenic and oncogenic Cot.