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1. Selective detection of active extracellular granzyme A by using a novel fluorescent immunoprobe with application to inflammatory diseases

Author

Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Gobierno de Aragón, Agencia Estatal de Investigación (España), European Research Council, Ministerio de Economía y Competitividad (España), Senán Salinas, Ana [0009-0009-5597-851X], Comas, Laura [0000-0002-3843-1231], Esteban, Patricia [0000-0003-4123-3524], Garzón, Marcela [0000-0001-6778-0636], Santiago, Llipsy [0000-0002-1861-5981], Domingo, María Pilar [0000-0002-6829-8769], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Paño, José Ramón [0000-0002-9600-8116], Vendrell, Marc [0000-0002-5392-9740], Pardo, Julián [0000-0003-0154-0730], Arias, Maykel [0000-0002-9730-2210], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gálvez Buerba, Eva Mª [eva@icb.csic.es], Arias, Maykel [maykelariascabrero@gmail.com], Senán Salinas, Ana, Comas, Laura, Esteban, Patricia, Garzón, Marcela, Cheng, Zhiming, Santiago, Llipsy, Domingo, María Pilar, Ramírez-Labrada, Ariel, Paño, José Ramón, Vendrell, Marc, Pardo, Julián, Arias, Maykel, Gálvez Buerba, Eva Mª, Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Gobierno de Aragón, Agencia Estatal de Investigación (España), European Research Council, Ministerio de Economía y Competitividad (España), Senán Salinas, Ana [0009-0009-5597-851X], Comas, Laura [0000-0002-3843-1231], Esteban, Patricia [0000-0003-4123-3524], Garzón, Marcela [0000-0001-6778-0636], Santiago, Llipsy [0000-0002-1861-5981], Domingo, María Pilar [0000-0002-6829-8769], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Paño, José Ramón [0000-0002-9600-8116], Vendrell, Marc [0000-0002-5392-9740], Pardo, Julián [0000-0003-0154-0730], Arias, Maykel [0000-0002-9730-2210], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gálvez Buerba, Eva Mª [eva@icb.csic.es], Arias, Maykel [maykelariascabrero@gmail.com], Senán Salinas, Ana, Comas, Laura, Esteban, Patricia, Garzón, Marcela, Cheng, Zhiming, Santiago, Llipsy, Domingo, María Pilar, Ramírez-Labrada, Ariel, Paño, José Ramón, Vendrell, Marc, Pardo, Julián, Arias, Maykel, and Gálvez Buerba, Eva Mª

Abstract

Granzymes (Gzms), a family of serine proteases, expressed by immune and nonimmune cells, present perforin-dependent and independent intracellular and extracellular functions. When released in the extracellular space, GzmA, with trypsin-like activity, is involved in the pathophysiology of different inflammatory diseases. However, there are no validated specific systems to detect active forms of extracellular GzmA, making it difficult to assess its biological relevance and potential use as a biomarker. Here, we have developed fluorescence-energy resonance-transfer (FRET)-based peptide probes (FAM-peptide-DABCYL) to specifically detect GzmA activity in tissue samples and biological fluids in both mouse and human samples during inflammatory diseases. An initial probe was developed and incubated with GzmA and different proteases like GzmB and others with similar cleavage specificity as GzmA like GzmK, thrombin, trypsin, kallikrein, or plasmin. After measuring fluorescence, the probe showed very good specificity and sensitivity for human and mouse GzmA when compared to GzmB, its closest homologue GzmK, and with thrombin. The specificity of this probe was further refined by incubating the samples in a coated plate with a GzmA-specific antibody before adding the probe. The results show a high specific detection of soluble GzmA even when compared with other soluble proteases with very similar cleavage specificity like thrombin, GzmK, trypsin, kallikrein, or plasmin, which shows nearly no fluorescence signal. The high specific detection of GzmA was validated, showing that using pure proteins and serum and tissue samples from GzmA-deficient mice presented a significant reduction in the signal compared with WT mice. The utility of this system in humans was confirmed, showing that GzmA activity was significantly higher in serum samples from septic patients in comparison with healthy donors. Our results present a new immunoprobe with utility to detect extracellular GzmA activity

Published
2024

2. Supporting information for Selective detection of active extracellular granzyme A by using a novel fluorescent immunoprobe with application to inflammatory diseases [Dataset]

Author

Senán Salinas, Ana [0009-0009-5597-851X], Comas, Laura [0000-0002-3843-1231], Esteban, Patricia [0000-0003-4123-3524], Garzón, Marcela [0000-0001-6778-0636], Santiago, Llipsy [0000-0002-1861-5981], Domingo, María Pilar [0000-0002-6829-8769], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Paño, José Ramón [0000-0002-9600-8116], Vendrell, Marc [0000-0002-5392-9740], Pardo, Julián [0000-0003-0154-0730], Arias, Maykel [0000-0002-9730-2210], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gálvez Buerba, Eva Mª [eva@icb.csic.es], Arias, Maykel [maykelariascabrero@gmail.com], Senán Salinas, Ana, Comas, Laura, Esteban, Patricia, Garzón, Marcela, Santiago, Llipsy, Domingo, María Pilar, Ramírez-Labrada, Ariel, Paño, José Ramón, Vendrell, Marc, Pardo, Julián, Arias, Maykel, Gálvez Buerba, Eva Mª, Senán Salinas, Ana [0009-0009-5597-851X], Comas, Laura [0000-0002-3843-1231], Esteban, Patricia [0000-0003-4123-3524], Garzón, Marcela [0000-0001-6778-0636], Santiago, Llipsy [0000-0002-1861-5981], Domingo, María Pilar [0000-0002-6829-8769], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Paño, José Ramón [0000-0002-9600-8116], Vendrell, Marc [0000-0002-5392-9740], Pardo, Julián [0000-0003-0154-0730], Arias, Maykel [0000-0002-9730-2210], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gálvez Buerba, Eva Mª [eva@icb.csic.es], Arias, Maykel [maykelariascabrero@gmail.com], Senán Salinas, Ana, Comas, Laura, Esteban, Patricia, Garzón, Marcela, Santiago, Llipsy, Domingo, María Pilar, Ramírez-Labrada, Ariel, Paño, José Ramón, Vendrell, Marc, Pardo, Julián, Arias, Maykel, and Gálvez Buerba, Eva Mª

Abstract

Supplementary table 1. Concentration of trypsin-like proteases presenting similar enzyme activity using a common substrate.-- The substrate Nɑ-CBZ-L-Lysine thiobenzyl ester hydrochloride was incubated with hGzmA (66.7 nM), hGzmK (2.4 nM), mGzmA (4 nm), Kallikrein (3.7 nM), Plasmin (4.3 nM) and Trypsin (0.4 nM) for 30 min as described in methods. The absorbance was measured. The values obtained at each time were subtracted from the substrate signal in the absence of enzymes. Data represent the mean of three independent replicates.

Published
2024

3. Biomarkers of immunotherapy response in patients with non-small-cell lung cancer: microbiota composition, short-chain fatty acids, and intestinal permeability

Author

Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), European Commission, Gobierno de Aragón, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Moratiel-Pellitero, Alba [0009-0000-9486-6793], Zapata, María [0000-0001-6654-8106], Gascón, Marta [0000-0002-6811-6654], Sesma, Andrea [0000-0001-7209-266X], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Martínez Lostao, Luis [0000-0003-3043-147X], Domingo, María Pilar [0000-0002-6829-8769], Esteban, Patricia [0000-0003-4123-3524], Yubero, Alfonso [0000-0003-4995-0325], Barbero-Herranz, Raquel [0000-0001-8845-1137], Moreno Blanco, Ana [0000-0003-3220-1117], Paño, José Ramón [0000-0002-9600-8116], Lastra, Rodrigo [0000-0002-7498-7798], Pardo, Julián [0000-0003-0154-0730], Isla, Dolores [0000-0002-2483-198X], Campo, Rosa del [0000-0003-1147-7923], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Moratiel-Pellitero, Alba, Zapata, María, Gascón, Marta, Sesma, Andrea, Quílez, Elisa, Ramirez-Labrada, Ariel, Martínez Lostao, Luis, Domingo, María Pilar, Esteban, Patricia, Yubero, Alfonso, Barbero-Herranz, Raquel, Moreno Blanco, Ana, Paño, José Ramón, Lastra, Rodrigo, Pardo, Julián, Isla, Dolores, Campo, Rosa del, Gálvez Buerba, Eva Mª, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación (España), European Commission, Gobierno de Aragón, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Moratiel-Pellitero, Alba [0009-0000-9486-6793], Zapata, María [0000-0001-6654-8106], Gascón, Marta [0000-0002-6811-6654], Sesma, Andrea [0000-0001-7209-266X], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Martínez Lostao, Luis [0000-0003-3043-147X], Domingo, María Pilar [0000-0002-6829-8769], Esteban, Patricia [0000-0003-4123-3524], Yubero, Alfonso [0000-0003-4995-0325], Barbero-Herranz, Raquel [0000-0001-8845-1137], Moreno Blanco, Ana [0000-0003-3220-1117], Paño, José Ramón [0000-0002-9600-8116], Lastra, Rodrigo [0000-0002-7498-7798], Pardo, Julián [0000-0003-0154-0730], Isla, Dolores [0000-0002-2483-198X], Campo, Rosa del [0000-0003-1147-7923], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Moratiel-Pellitero, Alba, Zapata, María, Gascón, Marta, Sesma, Andrea, Quílez, Elisa, Ramirez-Labrada, Ariel, Martínez Lostao, Luis, Domingo, María Pilar, Esteban, Patricia, Yubero, Alfonso, Barbero-Herranz, Raquel, Moreno Blanco, Ana, Paño, José Ramón, Lastra, Rodrigo, Pardo, Julián, Isla, Dolores, Campo, Rosa del, and Gálvez Buerba, Eva Mª

Abstract

There is evidence of the influence of the intestinal microbiota on the response to immunotherapy in cancer. In addition, we lack markers of response to treatment and toxicity, which obliges us to continue our search for them. In this work, we recruited patients with non-small-cell lung cancer receiving immunotherapy who contributed a fecal and blood sample. We analyzed the possible relationship between the response to immune checkpoint inhibitors and the occurrence of immune-related adverse events, the composition (16S rDNA amplification) and functionality (abundance of short-chain fatty acids) of the gut microbiota, and intestinal membrane permeability as a human factor. No correlations were detected between analytical markers and clinical evolution, with a marked individuality of the gut microbiota in terms of composition, but homogeneity in its functionality and permeability. Immune checkpoint inhibitors have been proposed as the standard treatment for different stages of non-small-cell lung cancer in multiple indications. Not all patients benefit from these treatments, however, and certain patients develop immune-related adverse events. Although the search for predictors of response to these drugs is a major field of research, these issues have yet to be resolved. It has been postulated that microbiota could play a relevant role in conditioning the response to cancer treatments; however, the human factor of intestinal permeability also needs to be considered as it is closely related to the regulation of host–microbiota interaction. In this article, we analyzed the possible relationship between the response to immune checkpoint inhibitors and the onset of immune-related adverse events, gut microbiota status, and intestinal membrane permeability. In a pioneering step, we also measured short-chain fatty acid content in feces. Although the correlation analyses failed to identify predictive biomarkers, even when all variables were integrated, our patients’ microbial

Published
2024

4. CAR Immunotherapy for the treatment of infectious diseases: a systematic review

Author

Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (España), Instituto de Salud Carlos III, European Commission, Gobierno de Aragón, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Universidad de Zaragoza, Banco Santander, Asociación Española Contra el Cáncer, Morte Romea, Elena [0000-0001-9262-2461], Pesini, Cecilia [0000-0002-8707-2722], Pellejero, Galadriel [0000-0002-2728-5435], Martínez Lostao, Luis [0000-0003-3043-147X], Toyas, Carla [0000-0002-4217-6317], Redrado, Sergio [0000-0002-8404-0012], Dolader, Elena [0009-0007-5333-4214], Arias, Maykel [0000-0002-9730-2210], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Sanz-Pamplona, Rebeca [0000-0002-2187-3527], Pardo, Julián [0000-0003-0154-0730], Paño, José Ramón [0000-0002-9600-8116], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Ramírez-Labrada, Ariel [aramirezlabrada@yahoo.es], Morte Romea, Elena, Pesini, Cecilia, Pellejero, Galadriel, Martínez-Lostao, Luis, Loscos, Silvia, Toyas, Carla, Redrado, Sergio, Dolader, Elena, Arias, Maykel, Gálvez Buerba, Eva Mª, Sanz-Pamplona, Rebeca, Pardo, Julián, Paño, José Ramón, Ramírez-Labrada, Ariel, Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (España), Instituto de Salud Carlos III, European Commission, Gobierno de Aragón, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Universidad de Zaragoza, Banco Santander, Asociación Española Contra el Cáncer, Morte Romea, Elena [0000-0001-9262-2461], Pesini, Cecilia [0000-0002-8707-2722], Pellejero, Galadriel [0000-0002-2728-5435], Martínez Lostao, Luis [0000-0003-3043-147X], Toyas, Carla [0000-0002-4217-6317], Redrado, Sergio [0000-0002-8404-0012], Dolader, Elena [0009-0007-5333-4214], Arias, Maykel [0000-0002-9730-2210], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Sanz-Pamplona, Rebeca [0000-0002-2187-3527], Pardo, Julián [0000-0003-0154-0730], Paño, José Ramón [0000-0002-9600-8116], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Ramírez-Labrada, Ariel [aramirezlabrada@yahoo.es], Morte Romea, Elena, Pesini, Cecilia, Pellejero, Galadriel, Martínez-Lostao, Luis, Loscos, Silvia, Toyas, Carla, Redrado, Sergio, Dolader, Elena, Arias, Maykel, Gálvez Buerba, Eva Mª, Sanz-Pamplona, Rebeca, Pardo, Julián, Paño, José Ramón, and Ramírez-Labrada, Ariel

Abstract

Immunotherapy treatments aim to modulate the host’s immune response to either mitigate it in inflammatory/autoimmune disease or enhance it against infection or cancer. Among different immunotherapies reaching clinical application during the last years, chimeric antigen receptor (CAR) immunotherapy has emerged as an effective treatment for cancer where different CAR T cells have already been approved. Yet their use against infectious diseases is an area still relatively poorly explored, albeit with tremendous potential for research and clinical application. Infectious diseases represent a global health challenge, with the escalating threat of antimicrobial resistance underscoring the need for alternative therapeutic approaches. This review aims to systematically evaluate the current applications of CAR immunotherapy in infectious diseases and discuss its potential for future applications. Notably, CAR cell therapies, initially developed for cancer treatment, are gaining recognition as potential remedies for infectious diseases. The review sheds light on significant progress in CAR T cell therapy directed at viral and opportunistic fungal infections.

Published
2024

5. Immune response in SARS-CoV-2 infection

Author

Morte Romea, Elena [0000-0001-9262-2461], Uranga Murillo, Iratxe [0000-0001-8411-984X], Hidalgo, Sandra [0000-0003-1629-9978], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Miguel, Diego de [0000-0002-8486-8514], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Martínez Lostao, Luis [0000-0003-3043-147X], Pellejero, Galadriel [0000-0002-2728-5435], Pardo, Julián [0000-0003-0154-0730], Paño, José Ramón [0000-0002-9600-8116], Morte Romea, Elena, Uranga Murillo, Iratxe, Hidalgo, Sandra, Ramírez-Labrada, Ariel, Miguel, Diego de, Gálvez Buerba, Eva Mª, Martínez Lostao, Luis, Pellejero, Galadriel, Letona Jiménez, Santiago, Pardo, Julián, Paño, José Ramón, Morte Romea, Elena [0000-0001-9262-2461], Uranga Murillo, Iratxe [0000-0001-8411-984X], Hidalgo, Sandra [0000-0003-1629-9978], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Miguel, Diego de [0000-0002-8486-8514], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Martínez Lostao, Luis [0000-0003-3043-147X], Pellejero, Galadriel [0000-0002-2728-5435], Pardo, Julián [0000-0003-0154-0730], Paño, José Ramón [0000-0002-9600-8116], Morte Romea, Elena, Uranga Murillo, Iratxe, Hidalgo, Sandra, Ramírez-Labrada, Ariel, Miguel, Diego de, Gálvez Buerba, Eva Mª, Martínez Lostao, Luis, Pellejero, Galadriel, Letona Jiménez, Santiago, Pardo, Julián, and Paño, José Ramón

Abstract

We analyzed several parameters of the immune response of COVID-19 patients who required hospital admission, com paring them to healthy donors (HDs) and other patients admitted for respiratory infections not caused by SARS-CoV-2 (non-COV-RTI). We focused on the main cells involved in innate immunity and soluble inflammatory factors that regulate their activity.

Published
2024

6. A subset of PD-1-expressing CD56bright NK cells identifies patients with good response to immune checkpoint inhibitors in lung cancer

Author

European Commission, Gobierno de Aragón, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Instituto de Salud Carlos III, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Bristol Myers Squibb Foundation, Roche, Ministerio de Ciencia, Innovación y Universidades (España), Gascón, Marta [0000-0002-6811-6654], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Lastra, Rodrigo [0000-0002-7498-7798], Martínez Lostao, Luis [0000-0003-3043-147X], Paño, José Ramón [0000-0002-9600-8116], Sesma, Andrea [0000-0001-7209-266X], Zapata, María [0000-0001-6654-8106], Torres-Ramón, Irene [0000-0003-3387-0558], Yubero, Alfonso [0000-0003-4995-0325], Domingo, María Pilar [0000-0002-6829-8769], Esteban, Patricia [0000-0003-4123-3524], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Pardo, Julián [0000-0003-0154-0730], Isla, Dolores [0000-0002-2483-198X], Gascón, Marta, Ramírez-Labrada, Ariel, Lastra, Rodrigo, Martínez-Lostao, Luis, Paño, José Ramón, Sesma, Andrea, Zapata, María, Moratiel, Alba, Quílez, María Elisa, Torres-Ramón, Irene, Yubero, Alfonso, Domingo, María Pilar, Esteban, Patricia, Gálvez Buerba, Eva Mª, Pardo, Julián, Isla, Dolores, European Commission, Gobierno de Aragón, Ministerio de Ciencia e Innovación (España), Agencia Estatal de Investigación (España), Instituto de Salud Carlos III, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Bristol Myers Squibb Foundation, Roche, Ministerio de Ciencia, Innovación y Universidades (España), Gascón, Marta [0000-0002-6811-6654], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Lastra, Rodrigo [0000-0002-7498-7798], Martínez Lostao, Luis [0000-0003-3043-147X], Paño, José Ramón [0000-0002-9600-8116], Sesma, Andrea [0000-0001-7209-266X], Zapata, María [0000-0001-6654-8106], Torres-Ramón, Irene [0000-0003-3387-0558], Yubero, Alfonso [0000-0003-4995-0325], Domingo, María Pilar [0000-0002-6829-8769], Esteban, Patricia [0000-0003-4123-3524], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Pardo, Julián [0000-0003-0154-0730], Isla, Dolores [0000-0002-2483-198X], Gascón, Marta, Ramírez-Labrada, Ariel, Lastra, Rodrigo, Martínez-Lostao, Luis, Paño, José Ramón, Sesma, Andrea, Zapata, María, Moratiel, Alba, Quílez, María Elisa, Torres-Ramón, Irene, Yubero, Alfonso, Domingo, María Pilar, Esteban, Patricia, Gálvez Buerba, Eva Mª, Pardo, Julián, and Isla, Dolores

Abstract

(1) Despite the effectiveness of immune checkpoint inhibitors (ICIs) in lung cancer, there is a lack of knowledge about predictive biomarkers. The objective of our study is to analyze different subsets of T-lymphocytes and natural killer (NK) cells as predictive biomarkers in a cohort of patients with nonsmall cell lung cancer (NSCLC) treated with ICI. (2) This is an observational, prospective study with 55 NSCLC patients treated with ICI. A total of 43 T and NK cell subsets are analyzed in peripheral blood, including the main markers of exhaustion, differentiation, memory, activation, and inhibition. (3) Regarding the descriptive data, Granzyme B+CD4+ Treg lymphocytes stand out (median 17.4%), and within the NK populations, most patients presented cytotoxic NK cells (CD56+CD3−CD16+GranzymeB+; median 94.8%), and about half of them have highly differentiated adaptive-like NK cells (CD56+CD3−CD16+CD57+ (mean 59.8%). A statistically significant difference was observed between the expression of PD1 within the CD56bright NK cell subpopulation (CD56+CD3−CD16−PD-1+) (p = 0.047) and a better OS. (4) Circulating immune cell subpopulations are promising prognostic biomarkers for ICI. Pending on validation with a larger sample, here we provide an analysis of the major circulating T and NK cell subsets involved in cancer immunity, with promising results despite a small sample size.

Published
2023

7. Supplementary material for Integrated analysis of circulating immune cellular and soluble mediators reveals specific COVID19 signatures at hospital admission with utility for prediction of clinical outcomes [Dataset]

Author

European Commission, Gobierno de Aragón, Instituto de Salud Carlos III, Fundación Banco Santander, Universidad de Zaragoza, Agencia Estatal de Investigación (España), Fundación Inocente Inocente, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Asociación Española Contra el Cáncer, Ministerio de Ciencia, Innovación y Universidades (España), ARAID Foundation, Uranga Murillo, Iratxe [0000-0001-8411-984X], Morte Romea, Elena [0000-0001-9262-2461], Hidalgo, Sandra [0000-0003-1629-9978], Pesini, Cecilia [0000-0002-8707-2722], García-Mulero, Sandra [0000-0003-4931-1267], Sierra Monzón, José L. [0000-0002-8796-2717], Santiago, Llipsy [0000-0002-1861-5981], Arias, Maykel [0000-0002-9730-2210], Miguel, Diego de [0000-0002-8486-8514], Encabo-Berzosa, M. Mar [0000-0001-5533-804X], Gracia Tello, Borja [0000-0003-3248-2908], Sanz-Pamplona, Rebeca [0000-0002-2187-3527], Martínez Lostao, Luis [0000-0003-3043-147X], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Paño, José Ramón [0000-0002-9600-8116], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Pardo, Julián [0000-0003-0154-0730], Paño, José Ramón; Ramírez-Labrada, Ariel; Pardo, Julián, Uranga Murillo, Iratxe, Morte Romea, Elena, Hidalgo, Sandra, Pesini, Cecilia, García-Mulero, Sandra, Sierra Monzón, José L., Santiago, Llipsy, Arias, Maykel, Miguel, Diego de, Encabo-Berzosa, M. Mar, Gracia Tello, Borja, Sanz-Pamplona, Rebeca, Martínez-Lostao, Luis, Gálvez Buerba, Eva Mª, Paño, José Ramón, Ramírez-Labrada, Ariel, Pardo, Julián, European Commission, Gobierno de Aragón, Instituto de Salud Carlos III, Fundación Banco Santander, Universidad de Zaragoza, Agencia Estatal de Investigación (España), Fundación Inocente Inocente, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Asociación Española Contra el Cáncer, Ministerio de Ciencia, Innovación y Universidades (España), ARAID Foundation, Uranga Murillo, Iratxe [0000-0001-8411-984X], Morte Romea, Elena [0000-0001-9262-2461], Hidalgo, Sandra [0000-0003-1629-9978], Pesini, Cecilia [0000-0002-8707-2722], García-Mulero, Sandra [0000-0003-4931-1267], Sierra Monzón, José L. [0000-0002-8796-2717], Santiago, Llipsy [0000-0002-1861-5981], Arias, Maykel [0000-0002-9730-2210], Miguel, Diego de [0000-0002-8486-8514], Encabo-Berzosa, M. Mar [0000-0001-5533-804X], Gracia Tello, Borja [0000-0003-3248-2908], Sanz-Pamplona, Rebeca [0000-0002-2187-3527], Martínez Lostao, Luis [0000-0003-3043-147X], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Paño, José Ramón [0000-0002-9600-8116], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Pardo, Julián [0000-0003-0154-0730], Paño, José Ramón; Ramírez-Labrada, Ariel; Pardo, Julián, Uranga Murillo, Iratxe, Morte Romea, Elena, Hidalgo, Sandra, Pesini, Cecilia, García-Mulero, Sandra, Sierra Monzón, José L., Santiago, Llipsy, Arias, Maykel, Miguel, Diego de, Encabo-Berzosa, M. Mar, Gracia Tello, Borja, Sanz-Pamplona, Rebeca, Martínez-Lostao, Luis, Gálvez Buerba, Eva Mª, Paño, José Ramón, Ramírez-Labrada, Ariel, and Pardo, Julián

Abstract

Supplementary materials and methods: sample processing, flow cytometry, high dimensional flow cytometry data analysis, multiplex plasma protein analyses, granzyme activity assay in serum, statistics. Supplementary figure legends (1-5). Supplementary table legends (1-7).

Published
2022

8. Characterisation of circulating immune cells in a cohort of non-small cell lung cancer patients treated with immunotherapy

Author

Gascón, Marta [0000-0002-6811-6654], Cruellas, Mara [0000-0002-6953-9675], Esteban, Patricia [0000-0003-4123-3524], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Lastra, Rodrigo [0000-0002-7498-7798], Martínez Lostao, Luis [0000-0003-3043-147X], Ocáriz, Maitane [0000-0002-7557-7264], Paño, José Ramón [0000-0002-9600-8116], Quílez Bermejo, J. [0000-0003-2808-1036], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Sesma Goñi, Andrea [0000-0001-7209-266X], Torres-Ramón, Irene [0000-0003-3387-0558], Yubero, Alfonso [0000-0003-4995-0325], Zapata, María [0000-0001-6654-8106], Isla, Dolores [0000-0002-2483-198X], Gascón, Marta, Pardo, Julián, Cruellas, Mara, Esteban, Patricia, Gálvez Buerba, Eva Mª, Lastra, Rodrigo, Martínez-Lostao, Luis, Ocáriz, Maitane, Paño, José Ramón, Quílez Bermejo, J., Ramírez-Labrada, Ariel, Sesma Goñi, Andrea, Torres-Ramón, Irene, Yubero, Alfonso, Zapata, María, Isla, Dolores, Gascón, Marta [0000-0002-6811-6654], Cruellas, Mara [0000-0002-6953-9675], Esteban, Patricia [0000-0003-4123-3524], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Lastra, Rodrigo [0000-0002-7498-7798], Martínez Lostao, Luis [0000-0003-3043-147X], Ocáriz, Maitane [0000-0002-7557-7264], Paño, José Ramón [0000-0002-9600-8116], Quílez Bermejo, J. [0000-0003-2808-1036], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Sesma Goñi, Andrea [0000-0001-7209-266X], Torres-Ramón, Irene [0000-0003-3387-0558], Yubero, Alfonso [0000-0003-4995-0325], Zapata, María [0000-0001-6654-8106], Isla, Dolores [0000-0002-2483-198X], Gascón, Marta, Pardo, Julián, Cruellas, Mara, Esteban, Patricia, Gálvez Buerba, Eva Mª, Lastra, Rodrigo, Martínez-Lostao, Luis, Ocáriz, Maitane, Paño, José Ramón, Quílez Bermejo, J., Ramírez-Labrada, Ariel, Sesma Goñi, Andrea, Torres-Ramón, Irene, Yubero, Alfonso, Zapata, María, and Isla, Dolores

Abstract

[Introduction] Immunotherapy in lung cancer has been one of the most relevant therapeutic advances in recent years. Although many studies demonstrate its antitumor activity, there is still a great lack of knowledge about which may be the best prognostic biomarkers. In recent years, several studies suggest that different immune cell populations might be useful biomarkers to predict immunotherapy efficacy. Among them are specific T cell subsets, natural killer (NK) cells or the ratio between cytotoxic CD8+ T and regulatory CD4+ T (Treg) lymphocytes. In this sense, the objective of our study is to provide a detailed analysis of different circulating immune cell subsets within T and NK cell populations in a cohort of patients with non-small cell lung cancer (NSCLC) treated with immune-checkpoint inhibitors (ICI) and analyze their prognostic value., [Methods] Observational, prospective and analytical study of a cohort of 55 patients diagnosed with NSCLC (unresectable stage III and IV) receiving ICI. Demographic, clinical and tumor variables are collected, as well as a peripheral blood sample. A total of 43 cell subpopulations of CD4+ and CD8+ T and NK cells, including the main exhaustion, differentiation, memory, activation and inhibition markers are analyzed in peripheral blood, correlated with treatment response and survival..., [Results] 55 patients were included (70.9% men) with a median age of 65 years. Regarding histology, there were 40% squamous cell carcinomas and 60% adenocarcinomas, 70.9% being stage IV. After minimum of one year of follow-up, 56.4% of patients had progressed with a median overall survival of 19 months (CI: 11.13-26.87). Regarding the descriptive data of the analyzed cell populations, the regulatory Granzyme B+ CD4+ T lymphocytes stand out with a median of 17.4% (SD 24.4%) and memory CD4+ and CD8+ Tz..., [Conclusions] Circulating immune cell subpopulations are among the most promising prognostic biomarkers for ICI. Here we provide a detailed descriptive analysis of the main circulating T and NK cell subsets involved in cancer immunity in a cohort of NSCLC patients. Some of the results suggest dysfunctional T and NK cell responses that might be related with ICI efficacy, which relevance to predict survival and treatment outcome will be shortly available

Published
2022

9. Determination of the concentration of IgG against the spike receptor-binding domain that predicts the viral neutralizing activity of convalescent plasma and serum against SARS-CoV-2

Author

Gobierno de Aragón, Fundación Banco Santander, Universidad de Zaragoza, Instituto de Salud Carlos III, European Commission, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundación Inocente Inocente, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, ARAID Foundation, Santiago, Llipsy [0000-0002-1861-5981], Uranga Murillo, Iratxe [0000-0001-8411-984X], Arias, Maykel [0000-0002-9730-2210], González Ramírez, Andrés Manuel [0000-0002-5838-0857], Macías León, Javier [0000-0001-6815-6720], Moreo, Eduardo [0000-0002-0182-201X], Redrado, Sergio [0000-0002-8404-0012], Taleb, V. [0000-0001-9224-5854], Lira-Navarrete, Erandi [0000-0003-2462-7580], Hurtado-Guerrero, R. [0000-0002-3122-9401], Encabo-Berzosa, M. Mar [0000-0001-5533-804X], Hidalgo, Sandra [0000-0003-1629-9978], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Miguel, Diego de [0000-0002-8486-8514], Benito, Rafael [0000-0001-5134-1006], Fernández Casanovas, Antonio [0000-0002-2557-4890], Serrano Barcos, Laura [0000-0002-9291-9801], Yuste, Cristina [0000-0003-3667-523X], Villanueva-Saz, Sergio [0000-0001-6209-4282], Paño, José Ramón [0000-0002-9600-8116], Pardo, Julián [0000-0003-0154-0730], Santiago, Llipsy, Uranga Murillo, Iratxe, Arias, Maykel, González Ramírez, Andrés Manuel, Macías-León, Javier, Moreo, Eduardo, Redrado, Sergio, García García, Ana, Taleb, V., Lira-Navarrete, Erandi, Hurtado-Guerrero, R., Aguiló, Nacho, Encabo-Berzosa, M. Mar, Hidalgo, Sandra, Gálvez Buerba, Eva Mª, Ramírez-Labrada, Ariel, Miguel, Diego de, Benito, Rafael, Miranda, Patricia, Fernández Casanovas, Antonio, Domingo, José María, Serrano Barcos, Laura, Yuste, Cristina, Villanueva-Saz, Sergio, Paño, José Ramón, Pardo, Julián, Gobierno de Aragón, Fundación Banco Santander, Universidad de Zaragoza, Instituto de Salud Carlos III, European Commission, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Fundación Inocente Inocente, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, ARAID Foundation, Santiago, Llipsy [0000-0002-1861-5981], Uranga Murillo, Iratxe [0000-0001-8411-984X], Arias, Maykel [0000-0002-9730-2210], González Ramírez, Andrés Manuel [0000-0002-5838-0857], Macías León, Javier [0000-0001-6815-6720], Moreo, Eduardo [0000-0002-0182-201X], Redrado, Sergio [0000-0002-8404-0012], Taleb, V. [0000-0001-9224-5854], Lira-Navarrete, Erandi [0000-0003-2462-7580], Hurtado-Guerrero, R. [0000-0002-3122-9401], Encabo-Berzosa, M. Mar [0000-0001-5533-804X], Hidalgo, Sandra [0000-0003-1629-9978], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Miguel, Diego de [0000-0002-8486-8514], Benito, Rafael [0000-0001-5134-1006], Fernández Casanovas, Antonio [0000-0002-2557-4890], Serrano Barcos, Laura [0000-0002-9291-9801], Yuste, Cristina [0000-0003-3667-523X], Villanueva-Saz, Sergio [0000-0001-6209-4282], Paño, José Ramón [0000-0002-9600-8116], Pardo, Julián [0000-0003-0154-0730], Santiago, Llipsy, Uranga Murillo, Iratxe, Arias, Maykel, González Ramírez, Andrés Manuel, Macías-León, Javier, Moreo, Eduardo, Redrado, Sergio, García García, Ana, Taleb, V., Lira-Navarrete, Erandi, Hurtado-Guerrero, R., Aguiló, Nacho, Encabo-Berzosa, M. Mar, Hidalgo, Sandra, Gálvez Buerba, Eva Mª, Ramírez-Labrada, Ariel, Miguel, Diego de, Benito, Rafael, Miranda, Patricia, Fernández Casanovas, Antonio, Domingo, José María, Serrano Barcos, Laura, Yuste, Cristina, Villanueva-Saz, Sergio, Paño, José Ramón, and Pardo, Julián

Abstract

Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Nevertheless, its efficacy in patients with COVID-19 remains uncertain. Thus, the establishment and validation of standardized methods that predict the viral neutralizing (VN) activity of plasma against SARS-CoV-2 is of utmost importance to appraise its therapeutic value. Using an in-house quantitative ELISA test and two independent cohorts with a total of 345 donors, we found that plasma and serum from most convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, with varying concentrations which correlate with previous disease severity and gender. Anti-RBD IgG plasma concentration significantly correlated with the plasma/serum VN activity against SARS-CoV-2 in vitro., Several hundred millions of people have been diagnosed of coronavirus disease 2019 (COVID-19), causing millions of deaths and a high socioeconomic burden. SARS-CoV-2, the causative agent of COVID-19, induces both specific T- and B-cell responses, being antibodies against the virus detected a few days after infection. Passive immunization with hyperimmune plasma from convalescent patients has been proposed as a potentially useful treatment for COVID-19. Using an in-house quantitative ELISA test, we found that plasma from 177 convalescent donors contained IgG antibodies specific to the spike receptor-binding domain (RBD) of SARS-CoV-2, although at very different concentrations which correlated with previous disease severity and gender. Anti-RBD IgG plasma concentrations significantly correlated with the plasma viral neutralizing activity (VN) against SARS-CoV-2 in vitro. Similar results were found using an independent cohort of serum from 168 convalescent health workers. These results validate an in-house RBD IgG ELISA test in a large cohort of COVID-19 convalescent patients and indicate that plasma from all convalescent donors does not contain a high enough amount of anti-SARS-CoV-2-RBD neutralizing IgG to prevent SARS-CoV-2 infection in vitro. The use of quantitative anti-RBD IgG detection systems might help to predict the efficacy of the passive immunization using plasma from patients recovered from SARS-CoV-2.

Published
2021

10. How could antibiotics, probiotics, and corticoids modify microbiota and its influence in cancer immune checkpoint inhibitors: a review

Author

Cruellas, Mara [0000-0002-6953-9675], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gascón, Marta [0000-0002-6811-6654], Isla, Dolores [0000-0002-2483-198X], Lastra, Rodrigo [0000-0002-7498-7798], Martínez Lostao, Luis [0000-0003-3043-147X], Ocáriz, Maitane [0000-0002-7557-7264], Pardo, Julián [0000-0003-0154-0730], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Sesma, Andrea [0000-0001-7209-266X], Torres-Ramón, Irene [0000-0003-3387-0558], Paño, José Ramón [0000-0002-9600-8116], Cruellas, Mara, Yubero, Alfonso, Zapata, María, Gálvez Buerba, Eva Mª, Gascón, Marta, Isla, Dolores, Lastra, Rodrigo, Martínez-Lostao, Luis, Ocáriz, Maitane, Pardo, Julián, Ramírez-Labrada, Ariel, Sesma, Andrea, Torres-Ramón, Irene, Paño, José Ramón, Cruellas, Mara [0000-0002-6953-9675], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gascón, Marta [0000-0002-6811-6654], Isla, Dolores [0000-0002-2483-198X], Lastra, Rodrigo [0000-0002-7498-7798], Martínez Lostao, Luis [0000-0003-3043-147X], Ocáriz, Maitane [0000-0002-7557-7264], Pardo, Julián [0000-0003-0154-0730], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Sesma, Andrea [0000-0001-7209-266X], Torres-Ramón, Irene [0000-0003-3387-0558], Paño, José Ramón [0000-0002-9600-8116], Cruellas, Mara, Yubero, Alfonso, Zapata, María, Gálvez Buerba, Eva Mª, Gascón, Marta, Isla, Dolores, Lastra, Rodrigo, Martínez-Lostao, Luis, Ocáriz, Maitane, Pardo, Julián, Ramírez-Labrada, Ariel, Sesma, Andrea, Torres-Ramón, Irene, and Paño, José Ramón

Abstract

Immunotherapy has become a new paradigm in oncology, improving outcomes for several types of cancer. However, there are some aspects about its management that remain uncertain. One of the key points that needs better understanding is the interaction between immunotherapy and gut microbiome and how modulation of the microbiome might modify the efficacy of immunotherapy. Consequently, the negative impact of systemic antibiotics and corticosteroids on the efficacy of immunotherapy needs to be clarified.

Published
2021

11. Serum active Granzyme A: a new biomarker that contributes to the pathogenesis of peritoneal sepsis

Author

Garzón, Marcela [0000-0001-6778-0636], Sierra Monzón, José L. [0000-0002-8796-2717], Comas, Laura [0000-0002-3843-1231], Uranga Murillo, Iratxe [0000-0001-8411-984X], Morte Romea, Elena [0000-0001-9262-2461], Algarate, Sonia [0000-0002-0036-6630], Ushakova, Tatiana Khaliulina [0000-0002-7930-2129], Luque Gómez, Pilar [0000-0001-7790-409X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Arias, Maykel [0000-0002-9730-2210], Pardo, Julián [0000-0003-0154-0730], Garzón, Marcela, Sierra Monzón, José L., Comas, Laura, Uranga Murillo, Iratxe, Morte Romea, Elena, Algarate, Sonia, Ushakova, Tatiana Khaliulina, Seral, Cristina, Luque Gómez, Pilar, Paño, José Ramón, Gálvez Buerba, Eva Mª, Arias, Maykel, Pardo, Julián, Garzón, Marcela [0000-0001-6778-0636], Sierra Monzón, José L. [0000-0002-8796-2717], Comas, Laura [0000-0002-3843-1231], Uranga Murillo, Iratxe [0000-0001-8411-984X], Morte Romea, Elena [0000-0001-9262-2461], Algarate, Sonia [0000-0002-0036-6630], Ushakova, Tatiana Khaliulina [0000-0002-7930-2129], Luque Gómez, Pilar [0000-0001-7790-409X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Arias, Maykel [0000-0002-9730-2210], Pardo, Julián [0000-0003-0154-0730], Garzón, Marcela, Sierra Monzón, José L., Comas, Laura, Uranga Murillo, Iratxe, Morte Romea, Elena, Algarate, Sonia, Ushakova, Tatiana Khaliulina, Seral, Cristina, Luque Gómez, Pilar, Paño, José Ramón, Gálvez Buerba, Eva Mª, Arias, Maykel, and Pardo, Julián

Abstract

[Background] Peritonitis is one of the most common leading cause of sepsis. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed in NK cells and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role of serum GzmA as a biomarker and therapeutic target in peritoneal sepsis., [Materials/methods] Concentration and enzyme activity of soluble GzmA were sequentially analyzed in serum from healthy donors and patients with peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (Wt) and GzmA-KO mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics and with serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days and the levels of serum proinflammatory cytokines and bacterial load in blood and spleen were analyzed at 6 and 24h from CLP., [Results] We have found high levels of GzmA in serum of patient with peritonitis. Most importantly, we observed that GzmA activity in serum correlates with SOFA score, suggesting that active GzmA could play an important role in sepsis development in peritonitis patients and could be a new biomarker of sepsis severity. In order to analyze the therapeutical potential of soluble GzmA in peritoneal sepsis, we used the CLP mouse model. After peritonitis induction, GzmA-KO mice exhibit increased survival compared with Wt mice, which correlated with reduced levels of proinflammatory cytokines in serum. The analysis of bacterial load in blood and spleen showed no differences between Wt and GzmA-KO mice suggesting that GzmA does not play an important role in bacterial control. Treatment with serpinb6b reduced mortality, which correlated with reduced cytokine serum levels in serum, confirming the therapeutical potential of gzmA to treat peritoneal sepsis., [Conclusions] Our findings confirm that soluble GzmA plays an important role in the pathogenesis of sepsis and could be a new therapeutic target and a biomarker for the treatment of peritoneal sepsis.

Published
2020

12. The multifaceted function of granzymes in sepsis: some facts and a lot to discover

Author

European Commission, Gobierno de Aragón, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Fundación Banco Santander, Universidad de Zaragoza, Ministerio de Ciencia, Innovación y Universidades (España), ARAID Foundation, Garzón, Marcela [0000-0001-6778-0636], Arias, Maykel [0000-0002-9730-2210], Sierra Monzón, José L. [0000-0002-8796-2717], Morte Romea, Elena [0000-0001-9262-2461], Santiago, Llipsy [0000-0002-1861-5981], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Martínez Lostao, Luis [0000-0003-3043-147X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Pardo, Julián [0000-0003-0154-0730], Garzón, Marcela, Arias, Maykel, Sierra Monzón, José L., Morte Romea, Elena, Santiago, Llipsy, Ramírez-Labrada, Ariel, Martínez-Lostao, Luis, Paño, José Ramón, Gálvez Buerba, Eva Mª, Pardo, Julián, European Commission, Gobierno de Aragón, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Fundación Banco Santander, Universidad de Zaragoza, Ministerio de Ciencia, Innovación y Universidades (España), ARAID Foundation, Garzón, Marcela [0000-0001-6778-0636], Arias, Maykel [0000-0002-9730-2210], Sierra Monzón, José L. [0000-0002-8796-2717], Morte Romea, Elena [0000-0001-9262-2461], Santiago, Llipsy [0000-0002-1861-5981], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Martínez Lostao, Luis [0000-0003-3043-147X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Pardo, Julián [0000-0003-0154-0730], Garzón, Marcela, Arias, Maykel, Sierra Monzón, José L., Morte Romea, Elena, Santiago, Llipsy, Ramírez-Labrada, Ariel, Martínez-Lostao, Luis, Paño, José Ramón, Gálvez Buerba, Eva Mª, and Pardo, Julián

Abstract

Sepsis is a serious global health problem. In addition to a high incidence, this syndrome has a high mortality and is responsible for huge health expenditure. The pathophysiology of sepsis is very complex and it is not well-understood yet. However, it is widely accepted that the initial phase of sepsis is characterized by a hyperinflammatory response while the late phase is characterized by immunosuppression and immune anergy, increasing the risk of secondary infections. Granzymes (Gzms) are a family of serine proteases classified according to their cleavage specificity. Traditionally, it was assumed that all Gzms acted as cytotoxic proteases. However, recent evidence suggests that GzmB is the one with the greatest cytotoxic capacity, while the cytotoxicity of others such as GzmA and GzmK is not clear. Recent studies have found that GzmA, GzmB, GzmK, and GzmM act as pro-inflammatory mediators. Specially, solid evidences show that GzmA and GzmK function as extracellular proteases that regulate the inflammatory response irrespectively of its ability to induce cell death. Indeed, studies in animal models indicate that GzmA is involved in the cytokine release syndrome characteristic of sepsis. Moreover, the GZM family also could regulate other biological processes involved in sepsis pathophysiology like the coagulation cascade, platelet function, endothelial barrier permeability, and, in addition, could be involved in the immunosuppressive stage of sepsis. In this review, we provide a comprehensive overview on the contribution of these novel functions of Gzms to sepsis and the new therapeutic opportunities emerging from targeting these proteases for the treatment of this serious health problem.

Published
2020

13. Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis

Author

Julián Pardo, Pilar Luque, Ludovic Couty, Jose Luis Sierra-Monzón, Maykel Arias, Tatiana Khaliulina-Ushakova, José Ramón Paño-Pardo, Eric Camerer, Ariel Ramirez-Labrada, Cristina Seral, Phillip I. Bird, Laura Comas, Iratxe Uranga-Murillo, Marcela Garzón-Tituaña, Sonia Algarate, Elena Tapia, Llipsy Santiago, Eva M. Galvez, Elena Morte-Romea, Ministerio de Ciencia, Innovación y Universidades (España), European Commission, Gobierno de Aragón, ARAID Foundation, Garzón, Marcela, Sierra Monzón, José L., Comas, Laura, Santiago, Llipsy, Khaliulina, Tatiana, Uranga Murillo, Iratxe, Ramírez-Labrada, Ariel, Tapia, Elena, Morte Romea, Elena, Algarate, Sonia, Camerer, Eric, Bird, Phillip I., Luque Gómez, Pilar, Paño, José Ramón, Gálvez Buerba, Eva Mª, Pardo, Julián, Arias, Maykel, Garzón, Marcela [0000-0001-6778-0636], Sierra Monzón, José L. [0000-0002-8796-2717], Comas, Laura [0000-0002-3843-1231], Santiago, Llipsy [0000-0002-1861-5981], Khaliulina, Tatiana [0000-0002-7930-2129], Uranga Murillo, Iratxe [0000-0001-8411-984X], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Tapia, Elena [0000-0002-4275-1406], Morte Romea, Elena [0000-0001-9262-2461], Algarate, Sonia [0000-0002-0036-663], Camerer, Eric [0000-0002-6271-7125], Bird, Phillip I. [0000-0002-6695-606X], Luque Gómez, Pilar [0000-0001-7790-409X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Pardo, Julián [0000-0003-0154-0730], and Arias, Maykel [0000-0002-9730-2210]

Subjects
0301 basic medicine, Male, Medicine (miscellaneous), Granzymes, Pathogenesis, Mice, 0302 clinical medicine, Molecular Targeted Therapy, Precision Medicine, Pharmacology, Toxicology and Pharmaceutics (miscellaneous), Aged, 80 and over, Mice, Knockout, cecal ligation and puncture, Granzyme A, Middle Aged, Killer Cells, Natural, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Cytokines, Tumor necrosis factor alpha, Female, medicine.symptom, Inflammation Mediators, Research Paper, Peritonitis, Spleen, Inflammation, Proinflammatory cytokine, Sepsis, 03 medical and health sciences, medicine, Extracellular, Animals, Humans, Serpins, Aged, business.industry, Interleukin-6, Macrophages, Cecal ligation and puncture, medicine.disease, Mice, Inbred C57BL, Toll-Like Receptor 4, Disease Models, Animal, 030104 developmental biology, Immunology, business
Abstract

7 figures, 1 table., [Aims]: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis., [Methods]: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times., [Results]: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα., [Conclusions]: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology., This work was supported by grant SAF2017-83120-C2-1-R and SAF2014-54763-C2-2-R from the Ministry of Science, Innovation and Universities and FEDER (Group B29_17R, Aragon Government). MG and LS were supported by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. IUM was supported by a PhD fellowship from Aragon Government, MA was supported by a post-doctoral fellowship “Juan de la Cierva-formación” from the Ministry of Science, Innovation and Universities. JP was supported by ARAID Foundation.

Published
2021

14. Integrated analysis of circulating immune cellular and soluble mediators reveals specific COVID19 signatures at hospital admission with utility for prediction of clinical outcomes

Author

José Ramón Paño-Pardo, Sandra García-Mulero, Ariel Ramirez-Labrada, Borja Gracia-Tello, Rebeca Sanz-Pamplona, Luis Martínez-Lostao, Eva M. Galvez, Jose L. Sierra, Diego de Miguel, Cecilia Pesini, Maria del Mar Encabo-Berzosa, Elena Morte, Iratxe Uranga-Murillo, Llipsy Santiago, Julián Pardo, Sandra Hidalgo, Maykel Arias, European Commission, Gobierno de Aragón, Instituto de Salud Carlos III, Fundación Banco Santander, Universidad de Zaragoza, Agencia Estatal de Investigación (España), Fundación Inocente Inocente, Aspanoa, Asociación Carrera de la Mujer Ciudad de Monzón, Asociación Española Contra el Cáncer, Ministerio de Ciencia, Innovación y Universidades (España), ARAID Foundation, Uranga Murillo, Iratxe, Morte Romea, Elena, Hidalgo, Sandra, Pesini, Cecilia, García-Mulero, Sandra, Sierra Monzón, José L., Santiago, Llipsy, Arias, Maykel, Miguel, Diego de, Encabo-Berzosa, M. Mar, Gracia Tello, Borja, Sanz-Pamplona, Rebeca, Martínez Lostao, Luis, Gálvez Buerba, Eva Mª, Paño, José Ramón, Ramírez-Labrada, Ariel, Pardo, Julián, Uranga Murillo, Iratxe [0000-0001-8411-984X], Morte Romea, Elena [0000-0001-9262-2461], Hidalgo, Sandra [0000-0003-1629-9978], Pesini, Cecilia [0000-0002-8707-2722], García-Mulero, Sandra [0000-0003-4931-1267], Sierra Monzón, José L. [0000-0002-8796-2717], Santiago, Llipsy [0000-0002-1861-5981], Arias, Maykel [0000-0002-9730-2210], Miguel, Diego de [0000-0002-8486-8514], Encabo-Berzosa, M. Mar [0000-0001-5533-804X], Gracia Tello, Borja [0000-0003-3248-2908], Sanz-Pamplona, Rebeca [0000-0002-2187-3527], Martínez Lostao, Luis [0000-0003-3043-147X], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Paño, José Ramón [0000-0002-9600-8116], Ramírez-Labrada, Ariel [0000-0002-3888-7036], and Pardo, Julián [0000-0003-0154-0730]

Subjects
Adult, Male, Chemokine, LAG3, COVID19, Medicine (miscellaneous), Inflammation, NK cells, CD8-Positive T-Lymphocytes, CCL8, Severity of Illness Index, Monocytes, Proinflammatory cytokine, Immune system, Immunitat cel·lular, medicine, ULBP, Humans, Prospective Studies, MIC, Pharmacology, Toxicology and Pharmaceutics (miscellaneous), Respiratory Tract Infections, Aged, Aged, 80 and over, biology, business.industry, GzmA, GzmB, CXCL10, COVID-19, Middle Aged, Immune checkpoint, Cellular immunity, Hospitalization, Killer Cells, Natural, Logistic Models, Case-Control Studies, CXCL9, Immunology, biology.protein, Cytokines, Tumor necrosis factor alpha, Female, medicine.symptom, Chemokines, business, Biomarkers, Research Paper
Abstract

6 figures, 6 tables.-- Supplementary material available., Coronavirus disease 2019 (COVID19), caused by SARS-CoV-2, is a complex disease, with a variety of clinical manifestations ranging from asymptomatic infection or mild cold-like symptoms to more severe cases requiring hospitalization and critical care. The most severe presentations seem to be related with a delayed, deregulated immune response leading to exacerbated inflammation and organ damage with close similarities to sepsis., [Methods]: In order to improve the understanding on the relation between host immune response and disease course, we have studied the differences in the cellular (monocytes, CD8+ T and NK cells) and soluble (cytokines, chemokines and immunoregulatory ligands) immune response in blood between Healthy Donors (HD), COVID19 and a group of patients with non-COVID19 respiratory tract infections (NON-COV-RTI). In addition, the immune response profile has been analyzed in COVID19 patients according to disease severity., [Results]: In comparison to HDs and patients with NON-COV-RTI, COVID19 patients show a heterogeneous immune response with the presence of both activated and exhausted CD8+ T and NK cells characterised by the expression of the immune checkpoint LAG3 and the presence of the adaptive NK cell subset. An increased frequency of adaptive NK cells and a reduction of NK cells expressing the activating receptors NKp30 and NKp46 correlated with disease severity. Although both activated and exhausted NK cells expressing LAG3 were increased in moderate/severe cases, unsupervised cell clustering analyses revealed a more complex scenario with single NK cells expressing more than one immune checkpoint (PD1, TIM3 and/or LAG3). A general increased level of inflammatory cytokines and chemokines was found in COVID19 patients, some of which like IL18, IL1RA, IL36B and IL31, IL2, IFNα and TNFα, CXCL10, CCL2 and CCL8 were able to differentiate between COVID19 and NON-COV-RTI and correlated with bad prognosis (IL2, TNFα, IL1RA, CCL2, CXCL10 and CXCL9). Notably, we found that soluble NKG2D ligands from the MIC and ULBPs families were increased in COVID19 compared to NON-COV-RTI and correlated with disease severity., [Conclusions]: Our results provide a detailed comprehensive analysis of the presence of activated and exhausted CD8+T, NK and monocyte cell subsets as well as extracellular inflammatory factors beyond cytokines/chemokines, specifically associated to COVID19. Importantly, multivariate analysis including clinical, demographical and immunological experimental variables have allowed us to reveal specific immune signatures to i) differentiate COVID19 from other infections and ii) predict disease severity and the risk of death., The authors would like to thank the Biobank of the Aragon Health System integrated in the Spanish National Biobanks Network and the Servicios Científico Técnicos de Citometria de Flujo del CIBA for their collaboration. Work in the JP laboratory is funded by the FEDER (Fondo Europeo de Desarrollo Regional, Gobierno de Aragón, Group B29_17R), Health National Institute Carlos III (COV20-00308), Aragón Government (Fondo COVID-19), Fundación Santander-Universidad de Zaragoza (Programa COVID-19), Agencia Estatal de Investigación (SAF2017-83120-C2-1-R; PID2020-113963RBI00), Fundación Inocente, ASPANOA and Carrera de la Mujer de Monzón. EMG is funded by Agencia Estatal de Investigación (SAF2017-83120-C2-1-R and PID2020-113963RB-I00). IUM and SH are supported by a PhD fellowship from Aragon Government, CP by a PhD fellowship from AECC, LS by a PhD fellowship (FPI) from the Ministry of Science, Innovation and Universities. DDM is supported by a postdoctoral fellowship 'Sara Borrell', and MA is supported by a postdoctoral fellowship 'Juan de la Cierva-incorporacion' from the Ministry of Science, Innovation and Universities. EM and BGT are supported by Rio Hortega contract. JP is supported by the ARAID Foundation.

Published
2021

15. Serum active Granzyme A: a new biomarker that contributes to the pathogenesis of peritoneal sepsis

Author

Garzón, Marcela, Sierra Monzón, José L., Comas, Laura, Uranga Murillo, Iratxe, Morte Romea, Elena, Algarate, Sonia, Ushakova, Tatiana Khaliulina, Seral, Cristina, Luque Gómez, Pilar, Paño, José Ramón, Gálvez Buerba, Eva Mª, Arias, Maykel, Pardo, Julián, Garzón, Marcela, Sierra Monzón, José L., Comas, Laura, Uranga Murillo, Iratxe, Morte Romea, Elena, Algarate, Sonia, Ushakova, Tatiana Khaliulina, Luque Gómez, Pilar, Paño, José Ramón, Gálvez Buerba, Eva Mª, Arias, Maykel, Pardo, Julián, Garzón, Marcela [0000-0001-6778-0636], Sierra Monzón, José L. [0000-0002-8796-2717], Comas, Laura [0000-0002-3843-1231], Uranga Murillo, Iratxe [0000-0001-8411-984X], Morte Romea, Elena [0000-0001-9262-2461], Algarate, Sonia [0000-0002-0036-6630], Ushakova, Tatiana Khaliulina [0000-0002-7930-2129], Luque Gómez, Pilar [0000-0001-7790-409X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Arias, Maykel [0000-0002-9730-2210], and Pardo, Julián [0000-0003-0154-0730]

Subjects
Granzyme, natural sciences, Peritoneal sepsis, Biomarkers
Abstract

of the work presented at the 30th ECCMID 2020, European Congress of Clinical Microbiology and Infectious Diseases, organized by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID).-- www.eccmid.org .-- www.escmid.org.-- Accepted abstract 1508., [Background] Peritonitis is one of the most common leading cause of sepsis. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed in NK cells and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role of serum GzmA as a biomarker and therapeutic target in peritoneal sepsis., [Materials/methods] Concentration and enzyme activity of soluble GzmA were sequentially analyzed in serum from healthy donors and patients with peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (Wt) and GzmA-KO mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics and with serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days and the levels of serum proinflammatory cytokines and bacterial load in blood and spleen were analyzed at 6 and 24h from CLP., [Results] We have found high levels of GzmA in serum of patient with peritonitis. Most importantly, we observed that GzmA activity in serum correlates with SOFA score, suggesting that active GzmA could play an important role in sepsis development in peritonitis patients and could be a new biomarker of sepsis severity. In order to analyze the therapeutical potential of soluble GzmA in peritoneal sepsis, we used the CLP mouse model. After peritonitis induction, GzmA-KO mice exhibit increased survival compared with Wt mice, which correlated with reduced levels of proinflammatory cytokines in serum. The analysis of bacterial load in blood and spleen showed no differences between Wt and GzmA-KO mice suggesting that GzmA does not play an important role in bacterial control. Treatment with serpinb6b reduced mortality, which correlated with reduced cytokine serum levels in serum, confirming the therapeutical potential of gzmA to treat peritoneal sepsis., [Conclusions] Our findings confirm that soluble GzmA plays an important role in the pathogenesis of sepsis and could be a new therapeutic target and a biomarker for the treatment of peritoneal sepsis.

Published
2020

16. The multifaceted function of granzymes in sepsis: some facts and a lot to discover

Author

Garzón, Marcela, Arias, Maykel, Sierra Monzón, José L., Morte Romea, Elena, Santiago, Llipsy, Ramírez-Labrada, Ariel, Martínez Lostao, Luis, Paño, José Ramón, Gálvez Buerba, Eva Mª, Pardo, Julián, European Commission, Gobierno de Aragón, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Fundación Banco Santander, Universidad de Zaragoza, Ministerio de Ciencia, Innovación y Universidades (España), ARAID Foundation, Garzón, Marcela [0000-0001-6778-0636], Arias, Maykel [0000-0002-9730-2210], Sierra Monzón, José L. [0000-0002-8796-2717], Morte Romea, Elena [0000-0001-9262-2461], Santiago, Llipsy [0000-0002-1861-5981], Ramírez-Labrada, Ariel [0000-0002-3888-7036], Martínez Lostao, Luis [0000-0003-3043-147X], Paño, José Ramón [0000-0002-9600-8116], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], and Pardo, Julián [0000-0003-0154-0730]

Subjects
inflammatory cytokine, Endothelial (dys)function, Coagulopathy, Sepsis, Granzymes, Immunosuppression
Abstract

1 figure, 1 table Sepsis is a serious global health problem. In addition to a high incidence, this syndrome has a high mortality and is responsible for huge health expenditure. The pathophysiology of sepsis is very complex and it is not well-understood yet. However, it is widely accepted that the initial phase of sepsis is characterized by a hyperinflammatory response while the late phase is characterized by immunosuppression and immune anergy, increasing the risk of secondary infections. Granzymes (Gzms) are a family of serine proteases classified according to their cleavage specificity. Traditionally, it was assumed that all Gzms acted as cytotoxic proteases. However, recent evidence suggests that GzmB is the one with the greatest cytotoxic capacity, while the cytotoxicity of others such as GzmA and GzmK is not clear. Recent studies have found that GzmA, GzmB, GzmK, and GzmM act as pro-inflammatory mediators. Specially, solid evidences show that GzmA and GzmK function as extracellular proteases that regulate the inflammatory response irrespectively of its ability to induce cell death. Indeed, studies in animal models indicate that GzmA is involved in the cytokine release syndrome characteristic of sepsis. Moreover, the GZM family also could regulate other biological processes involved in sepsis pathophysiology like the coagulation cascade, platelet function, endothelial barrier permeability, and, in addition, could be involved in the immunosuppressive stage of sepsis. In this review, we provide a comprehensive overview on the contribution of these novel functions of Gzms to sepsis and the new therapeutic opportunities emerging from targeting these proteases for the treatment of this serious health problem. This work was supported in part by FEDER/Gobierno de Aragón (group B29), Ministerio de Economia y Competitividad [SAF2014-54763-C2-1 and SAF2017-83120-C2-1-R (JP-P), SAF2014-54763-C2-2-R (EG)] and Instituto de Salud Carlos III (PI16-00526, LM-L; PI18/00527, JP-P). Predoctoral grants/contracts from Fundacion Santander/Universidad de Zaragoza (LS and MA), Ministerio de Ciencia, Innovación y Universidades (MG-T). MA has a Juan de la Cierva Contract (Ministerio de Ciencia, Innovación y Universidades) and JS-M a Rio Hortega Contract (Instituto de Salud Carlos III). JP was supported by Fundación Aragon I+D (ARAID).

Published
2020

17. From tumor mutational burden to blood T cell receptor: Looking for the best predictive biomarker in lung cancer treated with immunotherapy

Author

Sesma, A., Pardo, J., Cruellas, M., Gálvez, E.M., Gascón, M., Isla, D., Martínez-Lostao, L., Ocáriz, M., Paño, J.R., Quílez, E., Ramírez, A., Torres-Ramón, I., Yubero, A., Zapata, M., Lastra, R., Pardo, Julián [0000-0003-0154-0730], Cruellas, Mara [0000-0002-6953-9675], Gálvez Buerba, Eva Mª [0000-0001-6928-5516], Gascón, Marta [0000-0002-6811-6654], Martínez Lostao, Luis [0000-0003-3043-147X], Paño, José Ramón [0000-0002-9600-8116], Ramírez-Labrada, Ariel [0000-0002-3888-7036], and Torres-Ramón, Irene [0000-0003-3387-0558]

Subjects
ICIs (immune checkpoint inhibitors), TCR (T cell receptor), Lung cancer, TMB (tumor mutational burden), Neoantigen, Biomarkers, TCRβ (TCRB)
Abstract

2 tables. Immune control inhibitor drugs (anti-PD1/PD-L1/CTLA-4) (ICIs) are showing efficacy in the treatment of lung cancer. Currently the only biomarker with clinical utility for ICIs, such as the expression of PDL1, does not appear to be perfect or effective. Our working group is conducting a pilot study in lung cancer patients receiving ICIs with the aim of analyze the factors that affect the sensitivity of the immunotherapy in lung Cancer. Tumor Mutational Burden (TMB) and the sequencing of the T Cell Receptor (TCR) repertoire in peripheral blood have been postulated as predictive biomarkers of efficacy of immunotherapy. The review focusses on the predictive value of TMB for clinical responses to ICIs and discusses its clinical need after a discussion of the limitations. TCR CDR3 beta analysis and parameters such as clonality and TCR convergence may be good alternatives. For the moment, the combination of biomarkers may be the optimal tool. Despite therapeutic advances, lung cancer (LC) is one of the leading causes of cancer morbidity and mortality worldwide. Recently, the treatment of advanced LC has experienced important changes in survival benefit due to immune checkpoint inhibitors (ICIs). However, overall response rates (ORR) remain low in unselected patients and a large proportion of patients undergo disease progression in the first weeks of treatment. Therefore, there is a need of biomarkers to identify patients who will benefit from ICIs. The programmed cell death ligand 1 (PD-L1) expression has been the first biomarker developed. However, its use as a robust predictive biomarker has been limited due to the variability of techniques used, with different antibodies and thresholds. In this context, tumor mutational burden (TMB) has emerged as an additional powerful biomarker based on the observation of successful response to ICIs in solid tumors with high TMB. TMB can be defined as the total number of nonsynonymous mutations per DNA megabases being a mechanism generating neoantigens conditioning the tumor immunogenicity and response to ICIs. However, the latest data provide conflicting results regarding its role as a biomarker. Moreover, considering the results of the recent data, the use of peripheral blood T cell receptor (TCR) repertoire could be a new predictive biomarker. This review summarises recent findings describing the clinical utility of TMB and TCRβ (TCRB) and concludes that immune, neontigen, and checkpoint targeted variables are required in combination for accurately identifying patients who most likely will benefit of ICIs.

Published
2020

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