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.

How Could Antibiotics, Probiotics, and Corticoids Modify Microbiota and Its Influence in Cancer Immune Checkpoint Inhibitors: A Review.

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.

Point-of-care lung ultrasound assessment for risk stratification and therapy guiding in COVID-19 patients: a prospective noninterventional study.

BACKGROUND: Lung ultrasound is feasible for assessing lung injury caused by coronavirus disease 2019 (COVID-19). However, the prognostic meaning and time-line changes of lung injury assessed by lung ultrasound in COVID-19 hospitalised patients are unknown. METHODS: Prospective cohort study designed to analyse prognostic value of lung ultrasound in COVID-19 patients by using a quantitative scale (lung ultrasound Zaragoza (LUZ)-score) during the first 72 h after admission. The primary end-point was in-hospital death and/or admission to the intensive care unit. Total length of hospital stay, increase of oxygen flow and escalation of medical treatment during the first 72 h were secondary end-points. RESULTS: 130 patients were included in the final analysis; mean±sd age was 56.7±13.5 years. Median (interquartile range) time from the beginning of symptoms to admission was 6 (4-9) days. Lung injury assessed by LUZ-score did not differ during the first 72 h (21 (16-26) points at admission versus 20 (16-27) points at 72 h; p=0.183). In univariable logistic regression analysis, estimated arterial oxygen tension/inspiratory oxygen fraction ratio (PAFI) (hazard ratio 0.99, 95% CI 0.98-0.99; p=0.027) and LUZ-score >22 points (5.45, 1.42-20.90; p=0.013) were predictors for the primary end-point. CONCLUSIONS: LUZ-score is an easy, simple and fast point-of-care ultrasound tool to identify patients with severe lung injury due to COVID-19, upon admission. Baseline score is predictive of severity along the whole period of hospitalisation. The score facilitates early implementation or intensification of treatment for COVID-19 infection. LUZ-score may be combined with clinical variables (as estimated by PAFI) to further refine risk stratification.

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.

Lipid Nanoparticles Decorated with TNF-Related Aptosis-Inducing Ligand (TRAIL) Are More Cytotoxic than Soluble Recombinant TRAIL in Sarcoma.

Sarcomas are rare and heterogeneous cancers classically associated with a poor outcome. Sarcomas are 1% of the cancer but recent estimations indicate that sarcomas account for 2% of the estimated cancer-related deaths. Traditional treatment with surgery, radiotherapy, and chemotherapy has improved the outcome for some types of sarcomas. However, novel therapeutic strategies to treat sarcomas are necessary. TNF-related apoptosis-inducing ligand (TRAIL) is a death ligand initially described as capable of inducing apoptosis on tumor cell while sparing normal cells. Only few clinical trials have used TRAIL-based treatments in sarcoma, but they show only low or moderate efficacy of TRAIL. Consequently, novel TRAIL formulations with an improved TRAIL bioactivity are necessary. Our group has developed a novel TRAIL formulation based on tethering this death ligand on a lipid nanoparticle surface (LUV-TRAIL) resembling the physiological secretion of TRAIL as a trasmembrane protein inserted into the membrane of exosomes. We have already demonstrated that LUV-TRAIL shows an improved cytotoxic activity when compared to soluble recombinant TRAIL both in hematological malignancies and epithelial-derived cancers. In the present study, we have tested LUV-TRAIL in several human sarcoma tumor cell lines with different sensitivity to soluble recombinant TRAIL, finding that LUV-TRAIL was more efficient than soluble recombinant TRAIL. Moreover, combined treatment of LUV-TRAIL with distinct drugs proved to be especially effective, sensitizing even more resistant cell lines to TRAIL.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells.

PURPOSE: Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. METHODS/PATIENTS: LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. RESULTS: LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. CONCLUSION: The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.

Decreased activation-induced cell death by EBV-transformed B-cells from a patient with autoimmune lymphoproliferative syndrome caused by a novel FASLG mutation.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency characterized by chronic lymphoproliferation, autoimmune manifestations, expansion of double-negative T-cells, and susceptibility to malignancies. Most cases of ALPS are caused by germline or somatic FAS mutations. We report the case of an ALPS patient due to a novel homozygous Fasligand gene mutation (ALPS-FASLG). METHODS: ALPS biomarkers were measured and FASLG mutation was identified. Functional characterization was carried out based on activation-induced cell death (AICD) and cytotoxicity assays. RESULTS: This report describes the cases of a patient who presented a severe form of ALPS-FASLG, and his brother who had died due to complications related to ALPS. Moreover, in another family, we present the first case of lymphoma in a patient with ALPS-FASLG. Functional studies showed defective Fasligand-mediated apoptosis, cytotoxicity, and AICD in T-cell blasts. Otherwise, expression of the FASLG gene and corresponding protein was normal, but the shedding of the Fasligand was impaired in T-cells. Additionally, analyzing Epstein-Barr virus (EBV)-transformed B-cells, our results indicate impaired AICD in ALPS-FASLG patients. CONCLUSION: Patients with autosomal recessive inheritance of ALPS-FASLG have a severe phenotype and a partial defect in AICD in T- and B-cell lines. The Fasligand could play a key role in immune surveillance preventing malignancy.

Identification of the novel HLA-C*01:65:02 allele by next-generation sequencing.

The novel HLA-C*01:65:02 allele was detected during routine HLA typing.

Serum levels of IL-6 and IL-10 on admission correlate with complications in elderly patients with hip fracture.

OBJECTIVES: Ageing may cause a progressive pro-inflammatory environment and alter functionality of different immune-cell populations. The aim of the present study is to examine the influence of certain serum immunological parameters on hospitalization stay and complications in patients who have suffered a hip fracture. PATIENTS AND METHODS: A prospective study was carried out with 87 patients (63 women) presenting with either trochanteric femoral fracture or Garden IV displaced subcapital fracture. The average age was 84.43 ± 9, ranging from 65 to 104 years old. Data regarding different comorbidities were recorded at the time of arrival. The morning after patient's admission peripheral blood samples were obtained and a series of immunological parameters were determined: leukocyte formula, platelets count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), IL-6 and IL-10 levels, T-lymphocytes count, number of cells/mm3 and percentages of CD3, CD4, CD8, CD3-/CD16/56+ (NK cells), and CD3-/CD19+ (B cells). RESULTS: IL-6 serum levels presented a positive and significant correlation with higher levels of CRP (p < 0.001), IL-10 (p = 0.002), and higher percentages of NK CD56+ cells (p = 0.046). IL-6 serum levels at hospitalization presented a positive and significant correlation with a longer hospitalization stay (p = 0.037). Hospitalization increased by 0.231 days for every 1 pg/mL above the IL-6 mean value (40.43 pg/mL). Lower serum IL-10 levels on admission were associated with the appearance of symptomatic urinary tract infection during hospitalization (p = 0.032). Higher number of CD19+ cells/mm3 presented a significant relationship with pneumonia (p = 0.018) and symptomatic urinary tract infection (p = 0.0019). CONCLUSIONS: IL-6 serum levels on admission showed a positive and significant correlation with a longer hospitalization stay in elderly patients presenting with hip fracture. Lower levels of IL-10 in peripheral blood on admission were associated with symptomatic urinary tract infections. A higher number of CD19+ cells/mm³ was significantly associated with pneumonia and symptomatic urinary tract infection. These immunological variables on admission may serve as risk indicators of complications during hospitalization.

Peripheral Blood TCRß Repertoire, IL15, IL2 and Soluble Ligands for NKG2D Activating Receptor Predict Efficacy of Immune Checkpoint Inhibitors in Lung Cancer.

The development of immune checkpoint inhibitors (ICIs) has changed the therapeutic paradigm of lung cancer (LC), becoming the standard of treatment for previously untreated advanced non-small cell lung cancer (NSCLC) without actionable mutations. It has allowed the achievement of durable responses and resulted in significant survival benefits. However, not all patients respond; hence, molecular biomarkers are needed to help us predict which patients will respond. With this objective, a prospective observational study was designed, including a cohort of 55 patients with NSCLC who received ICIs. We studied whether biomarkers such as TCRß and specific cytokines involved in the regulation of T cell activity were related to the immunotherapy response. In the survival analysis, it was found that patients with higher TCRß clonality, lower TCRß evenness, higher TCRß Shannon diversity and lower TCRß convergence had higher overall survival (OS) and progression-free survival (PFS). However, no statistically significant association was observed. Regarding cytokines, those patients with higher levels of IL-2 and IL-15 presented statistically significantly shorter OS and PFS, respectively. In fact, in the multivariable analysis, the high IL-15 level increased the risk of death by three times. Although the sample size was small and more studies are needed to confirm our results, our study reveals promising markers of responses to ICIs.

Impact of antibiotics, corticosteroids, and microbiota on immunotherapy efficacy in patients with non-small cell lung cancer.

Lung cancer is a leading cause of morbidity and mortality globally, with its high mortality rate attributed mainly to non-small cell lung cancer (NSCLC). Although immunotherapy with immune checkpoint inhibitors (ICI) has revolutionized its treatment, patient response is highly variable and lacking predictive markers. We conducted a prospective study on 55 patients with NSCLC undergoing ICI therapy to identify predictive markers of both response and immune-related adverse events (IrAEs) in the airway microbiota. We also analyzed the clinical evolution and overall survival (OS) with respect to treatments that affect the integrity of the microbiota, such as antibiotics and corticosteroids. Our results demonstrated that respiratory microbiota differ significantly in ICI responders: they have higher alpha diversity values and lower abundance of the Firmicutes phylum and the Streptococcus genus. Employing a logistic regression model, the abundance of Gemella was the major predictor of non-ICI response, whereas Lachnoanaerobaculum was the best predictor of a positive response to ICI. The most relevant results were that antibiotic consumption is linked to a lower ICI response, and the use of corticosteroids correlated with poorer overall survival. Whereas previous studies have focused on gut microbiota, our findings highlight the importance of the respiratory microbiota in predicting the treatment response. Future research should explore microbiota modulation strategies to enhance immunotherapy outcomes. Understanding the impact of antibiotics, corticosteroids, and microbiota on NSCLC immunotherapy will help personalize treatment and improve patient outcomes.

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

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.

Biomarkers of Immunotherapy Response in Patients with Non-Small-Cell Lung Cancer: Microbiota Composition, Short-Chain Fatty Acids, and Intestinal 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 gut ecosystems were rich and diverse, and the intestinal barrier's integrity was preserved. These results add new knowledge on the composition of microbiota and its correlation with barrier permeability and short-chain fatty acids and suggest that more studies are required before these potential biomarkers can be incorporated into the clinical management of patients via immune checkpoint inhibitor treatment.

Liposomes decorated with Apo2L/TRAIL overcome chemoresistance of human hematologic tumor cells.

Human Apo2-ligand/TRAIL is a member of the TNF cytokine superfamily capable of inducing apoptosis on tumor cells while sparing normal cells. Besides its antitumor activity, Apo2L/TRAIL is also implicated in immune regulation. Apo2L/TRAIL is stored inside activated T cells in cytoplasmic multivesicular bodies and is physiologically released to the extracellular medium inserted in the internal membrane vesicles, known as exosomes. In this study we have generated artificial lipid vesicles coated with bioactive Apo2L/TRAIL, which resemble natural exosomes, to analyze their apoptosis-inducing ability on cell lines from hematological tumors. We have tethered Apo2L/TRAIL to lipid vesicles by using a novel Ni(2+)-(N-5-amino-1-carboxylpentyl)-iminodiacetic acid, NTA)-containing liposomal system. This lipidic framework (LUVs-Apo2L/TRAIL) greatly improves Apo2L/TRAIL activity, decreasing by around 14-fold the LC50 on the T-cell leukemia Jurkat. This increase in bioactivity correlated with the greater ability of LUVs-Apo2L/TRAIL to induce caspase-3 activation and is probably due to the increase in local concentration of Apo2L/TRAIL, improving its receptor cross-linking efficiency. More important, liposome-bound Apo2L/TRAIL overcame the resistance to soluble recombinant Apo2L/TRAIL exhibited by tumor cell mutants overexpressing Bcl-xL or by a Bax and Bak-defective Jurkat cell mutant (Jurkat-shBak) and are also effective against other hematologic tumor cells. Jurkat-Bcl-xL and Jurkat-shBak cells are resistant to most chemotherapeutic drugs currently used in cancer treatment, and their sensitivity to LUVs-Apo2L/TRAIL could have potential clinical applications.

DR5 Up-Regulation Induced by Dichloroacetate Sensitizes Tumor Cells to Lipid Nanoparticles Decorated with TRAIL.

Cancer resistance to treatments is a challenge that researchers constantly seek to overcome. For instance, TNF-related apoptosis-inducing ligand (TRAIL) is a potential good prospect as an anti-cancer therapy, as it attacks tumor cells but not normal cells. However, treatments based in soluble TRAIL provided incomplete clinical results and diverse formulations have been developed to improve its bioactivity. In previous works, we generated a new TRAIL formulation based in its attachment to the surface of unilamellar nanoliposomes (LUV-TRAIL). This formulation greatly increased apoptosis in a wide selection of tumor cell types, albeit a few of them remained resistant. On the other hand, it has been described that a metabolic shift in cancer cells can also alter its sensitivity to other treatments. In this work, we sought to increase the sensitivity of several tumor cell types resistant to LUV-TRAIL by previous exposure to the metabolic drug dichloroacetate (DCA), which forces oxidative phosphorylation. Results showed that DCA + LUV-TRAIL had a synergistic effect on both lung adenocarcinoma A549, colorectal HT29, and breast cancer MCF7 cells. Despite DCA inducing intracellular changes in a cell-type specific way, the increase in cell death by apoptosis was clearly correlated with an increase in death receptor 5 (DR5) surface expression in all cell lines. Therefore, DCA-induced metabolic shift emerges as a suitable option to overcome TRAIL resistance in cancer cells.

Granzyme-A deficiency attenuates experimental osteoarthritis in mice, but perforin deficiency does not.

OBJECTIVES: This study aims to assess the development of osteoarthritis (OA) in granzyme A- (gzmA) and B- (gzmB) and perforin- (perf) knockout mice. MATERIALS AND METHODS: A total of 75 male and female C57BL/6 (eight to nine-week-old) mice were allocated to: gzmA-deficient (gzmA-/-) (11 females, 8 males), gzmB-deficient (gzmB-/-) (9 females, 8 males), perf-deficient (perf-/-) (10 females, 9 males), and control group (10 females, 10 males). Osteoarthritis was induced in the right knee by instability of the meniscus medial ligament. Sham surgery was practiced in the left knee. Knee samples obtained eight weeks after surgery were stained (Safranin-O) and blindly scored in lateral and medial femur and tibia using the Osteoarthritis Research Society International scale (OARSI) (from Grade 0, cartilage intact to 6, deformation), (five stages from 0, no OA to 4, >50% surface involvement); OARSI score (Grade x Stage); and a semi-quantitative scale from Grade 0 (normal) to 6 (cartilage erosion >80%). RESULTS: Significantly higher values in all scales in the right knees compared to the left knees in male and female mice were observed (p<0.05). Males of all strains showed in the right knee higher values than females on all scales. Deficiency of perforin did not modify OA severity in any sex. The gzmA-/- females presented less degenerative changes than the other groups. CONCLUSION: Our study results show that sex plays an important role in the development of experimental OA in mice. Deficiency of gzmA can protect from the development of OA in female mice.

A Subset of PD-1-Expressing CD56bright NK Cells Identifies Patients with Good Response to Immune Checkpoint Inhibitors in Lung Cancer.

(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.

Unravelling the cellular response to the SARS-COV-2 vaccine in inflammatory bowel disease patients on biologic drugs.

Suboptimal vaccine response is a significant concern in patients with Inflammatory Bowel Disease (IBD) receiving biologic drugs. This single-center observational study involved 754 patients with IBD. In Phase I (October 2020-April 2021), 754 IBD participants who had not previously received the SARS-CoV-2 vaccine, underwent blood extraction to assess the seroprevalence of SARS-CoV-2 infection and IBD-related factors. Phase II (May 2021-October 2021) included a subgroup of 52 IBD participants with confirmed previous SARS-CoV-2 infection, who were studied for humoral and cellular response to the SARS-CoV-2 vaccine. In Phase I, treatment with anti-TNF was associated with lower rates of seroconversion (aOR 0.25 95% CI [0.10-0.61]). In Phase II, a significant increase in post-vaccination IgG levels was observed regardless of biologic treatment. However, patients treated with anti-TNF exhibited significantly lower IgG levels compared to those without IBD therapy (5.32 ± 2.47 vs. 7.99 ± 2.59 U/ml, p = 0.042). Following vaccination, a lymphocyte, monocyte, and NK cell activation pattern was observed, with no significant differences between patients receiving biologic drugs and those without IBD treatment. Despite lower seroprevalence and humoral response to the SARS-CoV-2 vaccine in patients treated with anti-TNF, the cellular response to the vaccine did not differ significantly from that patients without IBD therapy.

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

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.

Liposome-bound APO2L/TRAIL is an effective treatment in a rabbit model of rheumatoid arthritis.

OBJECTIVE: We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen-induced arthritis (AIA). METHODS: We used a novel Ni(2+)-(N-5-amino-1-carboxypentyl)-iminodiacetic acid)-containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed. RESULTS: Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic. CONCLUSION: Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA.