In vivo potential of recombinant granulysin against human melanoma

9-kDa granulysin is a protein expressed into the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. It has been shown to exert cytolysis on microbes and tumors. We showed previously that 9-kDa granulysin exerted cell death by apoptosis in vitro on hematological tumor cell lines and also on cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. In addition, we have shown the anti-tumor efficiency of granulysin as a single agent in two in vivo models of human tumor development in athymic mice, the MDA-MB-231 mammary adenocarcinoma and the NCI-H929 multiple myeloma, without signs of overt secondary effects by itself. In this work, we have tested recombinant 9-kDa granulysin in an in vivo and especially aggressive model of melanoma development, xenografted UACC62 cells in athymic mice. Recombinant granulysin was administered once UACC62-derived tumors were detectable and it substantially retarded the in vivo development of this aggressive tumor. We could also detect apoptosis induction and increased NK cell infiltration inside granulysin-treated tumor tissues. These observations are especially interesting given the possibility of treating melanoma by intra-tumor injection

Conjugation of the 9-kDa isoform of Granulysin with liposomes potentiates its cytotoxicity

Nine kDa granulysin (GRNLY) is a human cytolytic protein secreted by cytotoxic T lymphocytes (CTL) and NK cells of the immune system whose demonstrated physiological function is the elimination of bacteria and parasites. In previous studies by our group, the anti-tumor capacity of recombinant granulysin was demonstrated, both in vitro and in vivo. In the present work, we developed lipid nanoparticles whose surfaces can bind recombinant granulysin through the formation of a complex of coordination between the histidine tail of the protein and Ni2+ provided by a chelating lipid in the liposome composition and termed them LUV-GRNLY, for granulysin-bound large unilamellar vesicles. The objective of this formulation is to increase the granulysin concentration at the site of contact with the target cell and to increase the cytotoxicity of the administered dose. The results obtained in this work indicate that recombinant granulysin binds to the surface of the liposome with high efficiency and that its cytotoxicity is significantly increased when it is in association with liposomes. In addition, it has been demonstrated that the main mechanism of death induced by both granulysin and LUV-GRNLY is apoptosis. Jurkat-shBak cells are resistant to GRNLY and also to LUV-GRNLY, showing that LUV-GRNLY uses the mitochondrial apoptotic pathway to induce cell death. On the other hand, we show that LUV-GRNLY induces the expression of the pro-apoptotic members of the Bcl-2 family Bim and especially PUMA, although it also induced the expression of anti-apoptotic Bcl-xL. In conclusion, we demonstrate that binding of GRNLY to the surfaces of liposomes clearly augments its cytotoxic potential, with cell death executed mainly by the mitochondrial apoptotic pathway

Comparative proteomics of exosomes secreted by tumoral jurkat t cells and normal human t cell blasts unravels a potential tumorigenic role for valosin-containing protein

We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion

Multiple approaches at admission based on lung ultrasound and biomarkers improves risk identification in COVID-19 patients

Background: Risk stratification of COVID-19 patients is fundamental to improving prognosis and selecting the right treatment. We hypothesized that a combination of lung ultrasound (LUZ-score), biomarkers (sST2), and clinical models (PANDEMYC score) could be useful to improve risk stratification. Methods: This was a prospective cohort study designed to analyze the prognostic value of lung ultrasound, sST2, and PANDEMYC score in COVID-19 patients. The primary endpoint was in-hospital death and/or admission to the intensive care unit. The total length of hospital stay, increase of oxygen flow, or escalated medical treatment during the first 72 h were secondary endpoints. Results: a total of 144 patients were included; the mean age was 57.5 ± 12.78 years. The median PANDEMYC score was 243 (52), the median LUZ-score was 21 (10), and the median sST2 was 53.1 ng/mL (30.9). Soluble ST2 showed the best predictive capacity for the primary endpoint (AUC = 0.764 (0.658–0.871); p = 0.001), towards the PANDEMYC score (AUC = 0.762 (0.655–0.870); p = 0.001) and LUZ-score (AUC = 0.749 (0.596–0.901); p = 0.002). Taken together, these three tools significantly improved the risk capacity (AUC = 0.840 (0.727–0.953); p = 0.001). Conclusions: The PANDEMYC score, lung ultrasound, and sST2 concentrations upon admission for COVID-19 are independent predictors of intra-hospital death and/or the need for admission to the ICU for mechanical ventilation. The combination of these predictive tools improves the predictive power compared to each one separately. The use of decision trees, based on multivariate models, could be useful in clinical practice. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/)

Real-time intracellular temperature imaging using lanthanide-bearing polymeric micelles

Measurement of thermogenesis in individual cells is a remarkable challenge due to the complexity of the biochemical environment (such as pH and ionic strength) and to the rapid and yet not well-understood heat transfer mechanisms throughout the cell. Here, we present a unique system for intracellular temperature mapping in a fluorescence microscope (uncertainty of 0.2 K) using rationally designed luminescent Ln3+-bearing polymeric micellar probes (Ln = Sm, Eu) incubated in breast cancer MDA-MB468 cells. Two-dimensional (2D) thermal images recorded increasing the temperature of the cells culture medium between 296 and 304 K shows inhomogeneous intracellular temperature progressions up to ∼20 degrees and subcellular gradients of ∼5 degrees between the nucleolus and the rest of the cell, illustrating the thermogenic activity of the different organelles and highlighting the potential of this tool to study intracellular processes.publishe

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

1 figure, 1 tableSepsis 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).Peer reviewe

Structural insights into the coenzyme mediated monomer-dimer transition of the pro-apoptotic apoptosis inducing factor

The apoptosis-inducing factor (AIF) is a mitochondrial-flavoprotein that, after cell death induction, is distributed to the nucleus to mediate chromatinolysis. In mitochondria, AIF is present in a monomer-dimer equilibrium that after reduction by NADH gets displaced toward the dimer. The crystal structure of the human AIF (hAIF):NAD(H)-bound dimer revealed one FAD and, unexpectedly, two NAD(H) molecules per protomer. A 1:2 hAIF:NAD(H) binding stoichiometry was additionally confirmed in solution by using surface plasmon resonance. The here newly discovered NAD(H)-binding site includes residues mutated in human disorders, and accommodation of the coenzyme in it requires restructuring of a hAIF portion within the 509-560 apoptogenic segment. Disruption of interactions at the dimerization surface by production of the hAIF E413A/R422A/R430A mutant resulted in a nondimerizable variant considerably less efficiently stabilizing charge-transfer complexes upon coenzyme reduction than WT hAIF. These data reveal that the coenzyme-mediated monomer-dimer transition of hAIF modulates the conformation of its C-terminal proapoptotic domain, as well as its mechanism as reductase. These observations suggest that both the mitochondrial and apoptotic functions of hAIF are interconnected and coenzyme controlled: a key information in the understanding of the physiological role of AIF in the cellular life and death cycle. © 2014 American Chemical Society.Peer Reviewe