Individual outcome prediction models for patients with COVID-19 based on their first day of admission to the intensive care unit.

BACKGROUND: Currently, good prognosis and management of critically ill patients with COVID-19 are crucial for developing disease management guidelines and providing a viable healthcare system. We aimed to propose individual outcome prediction models based on binary logistic regression (BLR) and artificial neural network (ANN) analyses of data collected in the first 24 h of intensive care unit (ICU) admission for patients with COVID-19 infection. We also analysed different variables for ICU patients who survived and those who died. METHODS: Data from 326 critically ill patients with COVID-19 were collected. Data were captured on laboratory variables, demographics, comorbidities, symptoms and hospital stay related information. These data were compared with patient outcomes (survivor and non-survivor patients). BLR was assessed using the Wald Forward Stepwise method, and the ANN model was constructed using multilayer perceptron architecture. RESULTS: The area under the receiver operating characteristic curve of the ANN model was significantly larger than the BLR model (0.917 vs 0.810; p < 0.001) for predicting individual outcomes. In addition, ANN model presented similar negative predictive value than the BLR model (95.9% vs 94.8%). Variables such as age, pH, potassium ion, partial pressure of oxygen, and chloride were present in both models and they were significant predictors of death in COVID-19 patients. CONCLUSIONS: Our study could provide helpful information for other hospitals to develop their own individual outcome prediction models based, mainly, on laboratory variables. Furthermore, it offers valuable information on which variables could predict a fatal outcome for ICU patients with COVID-19.

Inhibition of RANK signaling in breast cancer induces an anti-tumor immune response orchestrated by CD8+ T cells.

Most breast cancers exhibit low immune infiltration and are unresponsive to immunotherapy. We hypothesized that inhibition of the receptor activator of nuclear factor-κB (RANK) signaling pathway may enhance immune activation. Here we report that loss of RANK signaling in mouse tumor cells increases leukocytes, lymphocytes, and CD8+ T cells, and reduces macrophage and neutrophil infiltration. CD8+ T cells mediate the attenuated tumor phenotype observed upon RANK loss, whereas neutrophils, supported by RANK-expressing tumor cells, induce immunosuppression. RANKL inhibition increases the anti-tumor effect of immunotherapies in breast cancer through a tumor cell mediated effect. Comparably, pre-operative single-agent denosumab in premenopausal early-stage breast cancer patients from the Phase-II D-BEYOND clinical trial (NCT01864798) is well tolerated, inhibits RANK pathway and increases tumor infiltrating lymphocytes and CD8+ T cells. Higher RANK signaling activation in tumors and serum RANKL levels at baseline predict these immune-modulatory effects. No changes in tumor cell proliferation (primary endpoint) or other secondary endpoints are observed. Overall, our preclinical and clinical findings reveal that tumor cells exploit RANK pathway as a mechanism to evade immune surveillance and support the use of RANK pathway inhibitors to prime luminal breast cancer for immunotherapy.

PD-L1/PD-1 crosstalk in colorectal cancer: are we targeting the right cells?

BACKGROUND: The analysis of tumour-infiltrating immune cells within patients' tumour samples in colorectal cancer (CRC) has become an independent predictor of patient survival. The tumour microenvironment and the immune checkpoints, such as PD-L1/PD-1, are relevant to the prognoses and also appear to be relevant for further CRC therapies. METHODS: We analysed the presence and features of the infiltrated monocyte/macrophage and lymphocyte populations in both tumour and peritumour samples from patients with CRC (n = 15). RESULTS: We detected a large number of CD14+ monocytes/macrophages with an alternative phenotype (CD64+CD163+) and CD4+ lymphocytes that infiltrated the tumour, but not the peritumour area. The monocytes/macrophages expressed PD-L1, whereas the lymphocytes were PD-1+; however, we did not find high PD-L1 levels in the tumour cells. Coculture of circulating naïve human monocytes/macrophages and lymphocytes with tumour cells from patients with proficient mismatch repair CRC induced both an alternative phenotype with higher expression of PD-L1 in CD14+ cells and the T-cell exhaustion phenomenon. The addition of an α-PD-1 antibody restored lymphocyte proliferation. CONCLUSION: These results emphasise the interesting nature of immune checkpoint shifting therapies, which have potential clinical applications in the context of colorectal cancer.

Obstructive Sleep Apnea Monocytes Exhibit High Levels of Vascular Endothelial Growth Factor Secretion, Augmenting Tumor Progression.

Obstructive sleep apnea (OSA) is a syndrome characterized by repeated pauses in breathing induced by a partial or complete collapse of the upper airways during sleep. Intermittent hypoxia (IH), a hallmark characteristic of OSA, has been proposed to be a major determinant of cancer development, and patients with OSA are at a higher risk of tumors. Both OSA and healthy monocytes have been found to show enhanced HIF1α expression under IH. Moreover, these cells under IH polarize toward a tumor-promoting phenotype in a HIF1α-dependent manner and influence tumor growth via vascular endothelial growth factor (VEGF). Monocytes from patients with OSA increased the tumor-induced microenvironment and exhibited an impaired cytotoxicity in a 3D tumor in vitro model as a result of the increased HIF1α secretion. Adequate oxygen restoration both in vivo (under continuous positive airway pressure treatment, CPAP) and in vitro leads the monocytes to revert the tumor-promoting phenotype, demonstrating the plasticity of the innate immune system and the oxygen recovery relevance in this context.

Generation and Characterization of Antibodies against Opioid Receptors from Zebrafish.

The opioid system is well conserved among species and plays a critical role in pain and addiction systems. The use of zebrafish as an experimental model to study development and genetics is extraordinary and has been proven to be relevant for the study of different diseases. The main drawback to its use for the analysis of different pathologies is the lack of protein tools. Antibodies that work in other models are not suitable for zebrafish due to the low degree of homology that exists among the opioid receptor protein sequences in different species. Here we report the successful generation and characterization of antibodies against the mu, delta 1 and delta 2 opioid receptors in zebrafish. The antibodies obtained, which are specific for each receptor due to the use of the C-terminus as antigens, work for Western blotting and immunohistochemistry. In addition, the antibodies against mu and delta 1 opioid receptors, but not those against delta 2, are able to immunoprecipitate the corresponding receptor from zebrafish lysates. The development of opioid receptor antibodies is an asset to the further study of the endogenous opioid system in zebrafish.

PD-L1 Overexpression During Endotoxin Tolerance Impairs the Adaptive Immune Response in Septic Patients via HIF1α.

Sepsis, among other pathologies, is an endotoxin tolerance (ET)-related disease. On admission, we classified 48 patients with sepsis into 3 subgroups according to the ex vivo response to lipopolysaccharide. This response correlates with the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the ET degree. Moreover, the ET-related classification determines the outcome of these patients. Programmed cell death-ligand 1 (PD-L1) expression on septic monocytes is also linked with ET status. In addition to the regulation of cytokine production, one of the hallmarks of ET that significantly affects patients with sepsis is T-cell proliferation impairment or a poor switch to the adaptive response. PD-L1/programmed cell death-1 (PD-1) blocking and knockdown assays on tolerant monocytes from both patients with sepsis and the in vitro model reverted the impaired adaptive response. Mechanistically, the transcription factor hypoxia-inducible factor-1α (HIF1α) has been translocated into the nucleus and drives PD-L1 expression during ET in human monocytes. This fact, together with patient classification according to the ex vivo lipopolysaccharide response, opens an interesting field of study and potential personalized clinical applications, not only for sepsis but also for all ET-associated pathologies.

Hypoxia-induced PD-L1/PD-1 crosstalk impairs T-cell function in sleep apnoea.

Obstructive sleep apnoea (OSA) is associated with higher cancer incidence, tumour aggressiveness and cancer mortality, as well as greater severity of infections, which have been attributed to an immune deregulation. We studied the expression of programmed cell death (PD)-1 receptor and its ligand (PD-L1) on immune cells from patients with OSA, and its consequences on immune-suppressing activity. We report that PD-L1 was overexpressed on monocytes and PD-1 was overexpressed on CD8+ T-cells in a severity-dependent manner. PD-L1 and PD-1 overexpression were induced in both the human in vitro and murine models of intermittent hypoxia, as well as by hypoxia-inducible factor-1α transfection. PD-L1/PD-1 crosstalk suppressed T-cell proliferation and activation of autologous T-lymphocytes and impaired the cytotoxic activity of CD8+ T-cells. In addition, monocytes from patients with OSA exhibited high levels of retinoic acid related orphan receptor, which might explain the differentiation of myeloid-derived suppressor cells. Intermittent hypoxia upregulated the PD-L1/PD-1 crosstalk in patients with OSA, resulting in a reduction in CD8+ T-cell activation and cytotoxicity, providing biological plausibility to the increased incidence and aggressiveness of cancer and the higher risk of infections described in these patients.

A System Dynamics Model to Predict the Human Monocyte Response to Endotoxins.

System dynamics is a powerful tool that allows modeling of complex and highly networked systems such as those found in the human immune system. We have developed a model that reproduces how the exposure of human monocytes to lipopolysaccharides (LPSs) induces an inflammatory state characterized by high production of tumor necrosis factor alpha (TNFα), which is rapidly modulated to enter into a tolerant state, known as endotoxin tolerance (ET). The model contains two subsystems with a total of six states, seven flows, two auxiliary variables, and 14 parameters that interact through six differential and nine algebraic equations. The parameters were estimated and optimized to obtain a model that fits the experimental data obtained from human monocytes treated with various LPS doses. In contrast to publications on other animal models, stimulation of human monocytes with super-low-dose LPSs did not alter the response to a second LPSs challenge, neither inducing ET, nor enhancing the inflammatory response. Moreover, the model confirms the low production of TNFα and increased levels of C-C motif ligand 2 when monocytes exhibit a tolerant state similar to that of patients with sepsis. At present, the model can help us better understand the ET response and might offer new insights on sepsis diagnostics and prognosis by examining the monocyte response to endotoxins in patients with sepsis.

Monocytes inhibit NK activity via TGF-ß in patients with obstructive sleep apnoea.

Obstructive sleep apnoea (OSA) is associated with cancer incidence and mortality. The contribution of the immune system appears to be crucial; however, the potential role of monocytes and natural killer (NK) cells remains unclear.Quantitative reverse transcriptase PCR, flow cytometry and in vitro assays were used to analyse the phenotype and immune response activity in 92 patients with OSA (60 recently diagnosed untreated patients and 32 patients after 6 months of treatment with continuous positive airway pressure (CPAP)) and 29 healthy volunteers (HV).We determined that monocytes in patients with OSA exhibit an immunosuppressive phenotype, including surface expression of glycoprotein-A repetitions predominant protein (GARP) and transforming growth factor-ß (TGF-ß), in contrast to those from the HV and CPAP groups. High levels of TGF-ß were detected in OSA sera. TGF-ß release by GARP+ monocytes impaired NK cytotoxicity and maturation. This altered phenotype correlated with the hypoxic severity clinical score (CT90). Reoxygenation eventually restored the altered phenotypes and cytotoxicity.This study demonstrates that GARP+ monocytes from untreated patients with OSA have an NK-suppressing role through their release of TGF-ß. Our findings show that monocyte plasticity immunomodulates NK activity in this pathology, suggesting a potential role in cancer incidence.

Circulating Monocytes Exhibit an Endotoxin Tolerance Status after Acute Ischemic Stroke: Mitochondrial DNA as a Putative Explanation for Poststroke Infections.

Patients with acute ischemic stroke (AIS) suffer from infections associated with mortality. The relevance of the innate immune system, and monocytes in particular, has emerged as an important factor in the evolution of these infections. The study enrolled 14 patients with AIS, without previous treatment, and 10 healthy controls. In the present study, we show that monocytes from patients with AIS exhibit a refractory state or endotoxin tolerance. The patients were unable to orchestrate an inflammatory response against LPS and expressed three factors reported to control the evolution of human monocytes into a refractory state: IL-1R-associated kinase-M, NFkB2/p100, and hypoxia-inducible factor-1α. The levels of circulating mitochondrial DNA (mtDNA) in patients with AIS correlated with impaired inflammatory response of isolated monocytes. Interestingly, the patients could be classified into two groups: those who were infected and those who were not, according to circulating mtDNA levels. This finding was validated in an independent cohort of 23 patients with AIS. Additionally, monocytes from healthy controls, cultured in the presence of both sera from patients and mtDNA, reproduced a refractory state after endotoxin challenge. This effect was negated by either a TLR9 antagonist or DNase treatment. The present data further extend our understanding of endotoxin tolerance implications in AIS. A putative role of mtDNA as a new biomarker of stroke-associated infections, and thus a clinical target for preventing poststroke infection, has also been identified.

Friedelane-type triterpenoids as selective anti-inflammatory agents by regulation of differential signaling pathways in LPS-stimulated macrophages.

A series of 31 pentacyclic triterpenoids isolated from the root barks of Celastrus vulcanicola and Maytenus jelskii were tested for cytotoxicity and inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophages. Compounds 18 (C18) and 25 (C25) exhibited significant inhibition of LPS-induced NO release at 50 and 25µM concentrations, respectively, and decreased mRNAs of pro-inflammatory cytokines. At the molecular level, C18 neither inhibited LPS-mediated phosphorylation of mitogen activated protein kinases (MAPKs) nor nuclear translocation of nuclear factor kappa beta (NFκB). Instead, C18 enhanced and prolonged nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and increased the expression of its target genes including hemeoxigenase 1 (HO1). C25 efficiently inhibited LPS-mediated phosphorylation of JNK, p38 and ERK, without affecting NFκB or Nrf2 signaling pathways. Both compounds reduced LPS-mediated processing of caspase-1 and the cleavage of interleukin 1ß (IL1ß) proform, reflecting their ability to target the inflammasome. C25 also counteracted LPS effects on iNOS expression and pro-inflammatory cytokines mRNA levels in Bv-2 microglial cells. The anti-inflammatory effect of both compounds was also assessed in human macrophages. Our results suggest that triterpenoids C18 and C25 possess anti-inflammatory effects, which may be therapeutically relevant for diseases linked to inflammation.

p21 mediates macrophage reprogramming through regulation of p50-p50 NF-κB and IFN-ß.

M1 and M2 macrophage phenotypes, which mediate proinflammatory and antiinflammatory functions, respectively, represent the extremes of immunoregulatory plasticity in the macrophage population. This plasticity can also result in intermediate macrophage states that support a balance between these opposing functions. In sepsis, M1 macrophages can compensate for hyperinflammation by acquiring an M2-like immunosuppressed status that increases the risk of secondary infection and death. The M1 to M2 macrophage reprogramming that develops during LPS tolerance resembles the pathological antiinflammatory response to sepsis. Here, we determined that p21 regulates macrophage reprogramming by shifting the balance between active p65-p50 and inhibitory p50-p50 NF-κB pathways. p21 deficiency reduced the DNA-binding affinity of the p50-p50 homodimer in LPS-primed and -rechallenged macrophages, impairing their ability to attenuate IFN-ß production and acquire an M2-like hyporesponsive status. High p21 levels in sepsis patients correlated with low IFN-ß expression, and p21 knockdown in human monocytes corroborated its role in IFN-ß regulation. The data demonstrate that p21 adjusts the equilibrium between p65-p50 and p50-p50 NF-κB pathways to mediate macrophage plasticity in LPS tolerance. Identifying p21-related pathways involved in monocyte reprogramming may lead to potential targets for sepsis treatment.

Actin polymerisation after FCγR stimulation of human fibroblasts is BCL10 independent.

B-cell lymphoma 10 (BCL10) is not essential for actin polymerisation after FcγR stimulation in human fibroblasts.

Ibrutinib as an antitumor immunomodulator in patients with refractory chronic lymphocytic leukemia.

Ibrutinib has emerged as a promising therapy for patients with chronic lymphocytic leukemia (CLL) who are nonresponsive to standard therapies. The refractory state of monocytes and T-cell exhaustion in patients with CLL could explain the morbidity and mortality reported in these patients. We studied the effect of ibrutinib on the immune response of four relapsed patients with CLL during the first treatment cycle. We observed the ability to recover the standard response against bacterial stimulus in CD14+ cells, improving levels of phospho-Erk1/2 and antigen presentation. Meanwhile, ibrutinib drove Th1-selective pressure in T lymphocytes, thus, reducing the PD-1 and PDL-1 expression. Our data suggest the impact of BTK inhibition along with immunomodulation on the innate immune response and a switch to the specific adaptive immune response, which might help to decrease infectious complications. The potential effect of ibrutinib on CLL patient outcomes is worthy of further study, because infections could be reduced with the use of ibrutinib.

CC-122 immunomodulatory effects in refractory patients with diffuse large B-cell lymphoma.

In the three patients included in a phase I clinical trial (NCT01421524), we report the immunomodulatory effects and efficacy of CC-122, a novel pleiotropic pathway modifier compound originally developed for broad diffuse large B-cell lymphoma (DLBCL). The chemical structure of CC-122 includes the glutarimide moiety that is known to modulate the immune response. The immunomodulatory agents including lenalidomide represent a promising therapeutic strategy targeting tumors in B-cell lymphoid malignancies. We observed that CC-122 might regulate the NK phenotype and its activity due to the reduced accumulation of myeloid-derived suppressor cell and eventually decrease the Tregs subsets. Finally, the activation of T cells through co-stimulatory molecule (CD28) was detected as a delayed CC-122 effect. In this context, CC-122 arises as an alternative option for DLBCL patients refractory to the traditional chemotherapeutic agents.

Galactomannan Downregulates the Inflammation Responses in Human Macrophages via NFκB2/p100.

We show that galactomannan, a polysaccharide consisting of a mannose backbone with galactose side groups present on the cell wall of several fungi, induces a reprogramming of the inflammatory response in human macrophages through dectin-1 receptor. The nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NFκB2)/p100 was overexpressed after galactomannan challenge. Knocking down NFκB2/p100 using small interfering RNA (siRNA) indicated that NFκB2/p100 expression is a crucial factor in the progression of the galactomannan-induced refractoriness. The data presented in this study could be used as a modulator of inflammatory response in clinical situations where refractory state is required.

Human monocytes undergo functional re-programming during sepsis mediated by hypoxia-inducible factor-1α.

Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis.

Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC) into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical practice.