Validation of an automated enzyme immunoassay for interleukin-6 for routine clinical use

Serum levels of Interleukin-6 (IL-6), a proinflammatory cytokine, are increased in early stages of inflammatory diseases such as infection and sepsis. Assay systems which permit its measurement within a few hours and as a single measurement have not been reported so far. We therefore evaluated a now commercially available automated method for IL-6 measurement on the Cobas Core(R) immunological analyzer (Roche Diagnostic Systems) which enables single IL-6 measurement within about 1 hour. The automated assay correlates well with an established, manual microtiter plate assay (Biosource GmbH) which uses the same antibodies and reagents (r=0.98). Accuracy of the automated method was established by adding known amounts of IL-6 international reference preparation. Recovery of the international standard was in the range of 92-104%. The automated assay had a precision of singletons below 6% and was linear up to 2800 pg/ml. This automated assay provides a suitable, convenient and time saving method for measurement of IL-6 serum levels in the routine clinical laboratory

Tumor microenvironment in NSCLC suppresses NK cells function

NK cells, which contribute to tumor immunosurveillance, are present in the microenvironment of Non-Small-Cell Lung Carcinoma. However, they display strongly altered phenotype with decreased expression of NKp30, NKp80, DNAM-1, CD16 and ILT2, and impaired cytotoxic functions. The possible mechanisms leading to these defects are discussed

Phenothiazines Inhibit Hepatitis C Virus Entry, Likely by Increasing the Fluidity of Cholesterol-Rich Membranes

Despite recent progress in the development of direct-acting antiviral agents against hepatitis C virus (HCV), more effective therapies are still urgently needed. We and others previously identified three phenothiazine compounds as potent HCV entry inhibitors. In this study, we show that phenothiazines inhibit HCV entry at the step of virus-host cell fusion, by intercalating into cholesterol-rich domains of the target membrane and increasing membrane fluidity. Perturbation of the alignment/packing of cholesterol in lipid membranes likely increases the energy barrier needed for virus-host fusion. A screening assay based on the ability of molecules to selectively increase the fluidity of cholesterol-rich membranes was subsequently developed. One compound that emerged from the library screen, topotecan, is able to very potently inhibit the fusion of liposomes with cell culture-derived HCV (HCVcc). These results yield new insights into HCV infection and provide a platform for the identification of new HCV inhibitors

Adipose tissue transcriptomic signature highlights the pathological relevance of extracellular matrix in human obesity

Analysis of the transcriptomic signature of white adipose tissue in obese human subjects revealed increased interstitial fibrosis and an infiltration of inflammatory cells into the tissue

Unraveling the complex interplay between anti-tumor immune response and autoimmunity mediated by B cells and autoantibodies in the era of anti-checkpoint monoclonal antibody therapies

The intricate relationship between anti-tumor immunity and autoimmunity is a complex yet crucial aspect of cancer biology. Tumor microenvironment often exhibits autoimmune features, a phenomenon that involves natural autoimmunity and the induction of humoral responses against self-antigens during tumorigenesis. This induction is facilitated by the orchestration of anti-tumor immunity, particularly within organized structures like tertiary lymphoid structures (TLS). Paradoxically, a significant number of cancer patients do not manifest autoimmune features during the course of their illness, with rare instances of paraneoplastic syndromes. This discrepancy can be attributed to various immune-mediated locks, including regulatory or suppressive immune cells, anergic autoreactive lymphocytes, or induction of effector cells exhaustion due to chronic stimulation. Overcoming these locks holds the risk to induce autoimmune mechanisms during cancer progression, a phenomenon notably observed with anti-immune checkpoint therapies, in contrast to more conventional treatments like chemotherapy or radiotherapy. Therefore, the challenge arises in managing immune-related adverse events (irAEs) induced by immune checkpoint inhibitors treatment, as decoupling them from the anti-tumor activity poses a significant clinical dilemma. This review summarizes recent advances in understanding the link between B-cell driven anti-tumor responses and autoimmune reactions in cancer patients, and discusses the clinical implications of this relationship

Nine newly identified individuals refine the phenotype associated with MYT1L mutations

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Prognostic impact of vitamin B6 metabolism in lung cancer

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.publishedVersio

Conditional Transgenesis Using Dimerizable Cre (DiCre)

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. We have recently developed a conceptually new approach to regulate Cre recombinase, that we have called Dimerizable Cre or DiCre. It is based on splitting Cre into two inactive moieties and fusing them to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin associated protein), respectively. These latter can be efficiently hetero-dimerized by rapamycin, leading to the reinstatement of Cre activity. We have been able to show, using in vitro approaches, that this ligand-induced dimerization is an efficient way to regulate Cre activity, and presents a low background activity together with a high efficiency of recombination following dimerization. To test the in vivo performance of this system, we have, in the present work, knocked-in DiCre into the Rosa26 locus of mice. To evaluate the performance of the DiCre system, mice have been mated with indicator mice (Z/EG or R26R) and Cre-induced recombination was examined following activation of DiCre by rapamycin during embryonic development or after birth of progenies. No recombination could be observed in the absence of treatment of the animals, indicating a lack of background activity of DiCre in the absence of rapamycin. Postnatal rapamycin treatment (one to five daily injection, 10 mg/kg i.p) induced recombination in a number of different tissues of progenies such as liver, heart, kidney, muscle, etc. On the other hand, recombination was at a very low level following in utero treatment of DiCre×R26R mice. In conclusion, DiCre has indeed the potentiality to be used to establish conditional Cre-deleter mice. An added advantage of this system is that, contrary to other modulatable Cre systems, it offers the possibility of obtaining regulated recombination in a combinatorial manner, i.e. induce recombination at any desired time-point specifically in cells characterized by the simultaneous expression of two different promoters

Consensus guidelines for the detection of immunogenic cell death

none82siApoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.Kepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G; Cirone, Mara; Colombo, Maria I; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G; Faggioni, Alberto; Formenti, Silvia C; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong-Min; Huang, Xing; Illidge, Tim M; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V; Loi, Sherene; Lowenstein, Pedro R; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean-Ehrland; Riganti, Chiara; Rovere-Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T; Yang, Haining; Zong, Wei-Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, LorenzoKepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G; Cirone, Mara; Colombo, Maria I; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G; Faggioni, Alberto; Formenti, Silvia C; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong Min; Huang, Xing; Illidge, Tim M; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V; Loi, Sherene; Lowenstein, Pedro R; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean Ehrland; Riganti, Chiara; Rovere Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T; Yang, Haining; Zong, Wei Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenz

Molecular Epidemiology and Evolutionary Trajectory of Emerging Echovirus 30, Europe

In 2018, an upsurge in echovirus 30 (E30) infections was reported in Europe. We conducted a large-scale epidemiologic and evolutionary study of 1,329 E30 strains collected in 22 countries in Europe during 2016-2018. Most E30 cases affected persons 0-4 years of age (29%) and 25-34 years of age (27%). Sequences were divided into 6 genetic clades (G1-G6). Most (53%) sequences belonged to G1, followed by G6 (23%), G2 (17%), G4 (4%), G3 (0.3%), and G5 (0.2%). Each clade encompassed unique individual recombinant forms; G1 and G4 displayed >= 2 unique recombinant forms. Rapid turnover of new clades and recombinant forms occurred over time. Clades G1 and G6 dominated in 2018, suggesting the E30 upsurge was caused by emergence of 2 distinct clades circulating in Europe. Investigation into the mechanisms behind the rapid turnover of E30 is crucial for clarifying the epidemiology and evolution of these enterovirus infections.Peer reviewe