Near-Infrared Spectroscopy-Derived Dynamic Cerebral Autoregulation in Experimental Human Endotoxemia-An Exploratory Study.

Cerebral perfusion may be altered in sepsis patients. However, there are conflicting findings on cerebral autoregulation (CA) in healthy participants undergoing the experimental endotoxemia protocol, a proxy for systemic inflammation in sepsis. In the current study, a newly developed near-infrared spectroscopy (NIRS)-based CA index is investigated in an endotoxemia study population, together with an index of focal cerebral oxygenation. Methods: Continuous-wave NIRS data were obtained from 11 healthy participants receiving a continuous infusion of bacterial endotoxin for 3 h (ClinicalTrials.gov NCT02922673) under extensive physiological monitoring. Oxygenated-deoxygenated hemoglobin phase differences in the (very)low frequency (VLF/LF) bands and the Tissue Saturation Index (TSI) were calculated at baseline, during systemic inflammation, and at the end of the experiment 7 h after the initiation of endotoxin administration. Results: The median (inter-quartile range) LF phase difference was 16.2° (3.0-52.6°) at baseline and decreased to 3.9° (2.0-8.8°) at systemic inflammation (p = 0.03). The LF phase difference increased from systemic inflammation to 27.6° (12.7-67.5°) at the end of the experiment (p = 0.005). No significant changes in VLF phase difference were observed. The TSI (mean ± SD) increased from 63.7 ± 3.4% at baseline to 66.5 ± 2.8% during systemic inflammation (p = 0.03) and remained higher at the end of the experiment (67.1 ± 4.2%, p = 0.04). Further analysis did not reveal a major influence of changes in several covariates such as blood pressure, heart rate, PaCO2, and temperature, although some degree of interaction could not be excluded. Discussion: A reversible decrease in NIRS-derived cerebral autoregulation phase difference was seen after endotoxin infusion, with a small, sustained increase in TSI. These findings suggest that endotoxin administration in healthy participants reversibly impairs CA, accompanied by sustained microvascular vasodilation.

Kinetics of Neutrophil Subsets in Acute, Subacute, and Chronic Inflammation.

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease.

Human in vivo neuroimaging to detect reprogramming of the cerebral immune response following repeated systemic inflammation.

Despite increasing evidence that immune training within the brain may affect the clinical course of neuropsychiatric diseases, data on cerebral immune tolerance are scarce. This study in healthy volunteers examined the trajectory of the immune response systemically and within the brain following repeated lipopolysaccharide (LPS) challenges. Five young males underwent experimental human endotoxemia (intravenous administration of 2 ng/kg LPS) twice with a 7-day interval. The systemic immune response was assessed by measuring plasma cytokine levels. Four positron emission tomography (PET) examinations, using the translocator protein (TSPO) ligand 18F-DPA-714, were performed in each participant, to assess brain immune cell activation prior to and 5 hours after both LPS challenges. The first LPS challenge caused a profound systemic inflammatory response and resulted in a 53% [95%CI 36-71%] increase in global cerebral 18F-DPA-714 binding (p < 0.0001). Six days after the first challenge, 18F-DPA-714 binding had returned to baseline levels (p = 0.399). While the second LPS challenge resulted in a less pronounced systemic inflammatory response (i.e. 77 ± 14% decrease in IL-6 compared to the first challenge), cerebral inflammation was not attenuated, but decreased below baseline, illustrated by a diffuse reduction of cerebral 18F-DPA-714 binding (-38% [95%CI -47 to -28%], p < 0.0001). Our findings constitute evidence for in vivo immunological reprogramming in the brain following a second inflammatory insult in healthy volunteers, which could represent a neuroprotective mechanism. These results pave the way for further studies on immunotolerance in the brain in patients with systemic inflammation-induced cerebral dysfunction.

Endotoxemia-Induced Release of Pro-inflammatory Mediators Are Associated With Increased Glomerular Filtration Rate in Humans in vivo.

Introduction: Sepsis is the most prevalent cause of Acute Kidney Injury (AKI). Conversely, in some septic patients the glomerular filtration rate (GFR) is augmented. The role of the inflammatory response and blood pressure to induce this increased GFR is unknown. Herein, we relate inflammatory mediators and blood pressure to the iohexol clearance-derived "true" GFR and kidney injury markers during systemic inflammation in healthy volunteers. Methods: Twelve healthy subjects underwent experimental endotoxemia (i.v. administration of 2 ng/kg Escherichia coli-derived lipopolysaccharide, LPS). As a gold-standard to determine the GFR, iohexol plasma clearance (GFRiohexol) was calculated during a 6-h period on the day before (baseline) as well as 2 and 24 h after LPS administration. Intra-arterial blood pressure was recorded continuously using a radial artery catheter. Circulating inflammatory mediators and urinary excretion of kidney injury markers were serially measured. Results: Experimental endotoxemia profoundly increased plasma concentrations of inflammatory mediators, including [mean ± SD or median [IQR] peak values (pg/mL) of tumor necrosis factor (TNF)-α: 92 ± 40, interleukin (IL)-6: 1,246 ± 605, IL-8: 374 ± 120, IL-10: 222 ± 119, IL-1 receptor antagonist (RA): 8,955 ± 2,429, macrophage chemoattractant protein (MCP)-1: 2,885 [2,706 - 3,765], vascular adhesion molecule (VCAM)-1: 296,105 ± 34,822, intercellular adhesion molecule (ICAM)-1: 25,0170 ± 41,764]. Mean arterial pressure decreased with 13 ± 11 mmHg (p < 0.0001). No significant increase in the urinary excretion of tubular injury markers was observed following LPS administration. GFRiohexol increased from 97 ± 6 at baseline to 118 ± 10 mL/min/1.73m2 (p < 0.0001) post-LPS administration and returned to baseline levels at 24 h post-LPS (99 ± 9 mL/min/1.73m2). Peak plasma concentrations of IL-6 (R 2 = 0.66, p = 0.001) and IL-8 (R 2 = 0.51, p = 0.009), MCP-1 (R 2 = 0.38, p = 0.03) and VCAM-1 levels (R 2 = 0.37, p = 0.04) correlated with the increase in GFRiohexol, whereas a trend was observed for TNF-α (R 2 = 0.33, p = 0.0509) and IL-1RA (R 2 = 0.28, p = 0.08). None of the kidney injury markers or changes in blood pressure were associated with GFRiohexol. In multiple linear regression analysis, both peak IL-6 (p = 0.002) and IL-8 (p = 0.01) concentrations remained significantly correlated with GFRiohexol, without collinearity. Discussion: Concentrations of pro-inflammatory cytokines, but not blood pressure, are correlated with the endotoxemia-induced increase in GFR in healthy volunteers. These findings may indicate that inflammatory mediators orchestrate the augmented GFR observed in a subgroup of sepsis patients.

The effect of BCG vaccination on alveolar macrophages obtained from induced sputum from healthy volunteers.

The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) is able to boost innate immune responses through a process called 'trained immunity'. It is hypothesized that BCG-induced trained immunity contributes to protection against Mycobacterium tuberculosis infection. Since alveolar macrophages are the first cell type to encounter M. tuberculosis upon infection, we aimed to investigate the immunomodulatory effects of BCG vaccination on alveolar macrophages. Searching for a less-invasive method than bronchoalveolar lavage, we optimized the isolation of alveolar macrophages from induced sputum of healthy volunteers. Viable alveolar macrophages could be successfully isolated from induced sputum and showed signs of activation already upon retrieval. Further flow cytometric analyses revealed that at baseline, higher expression levels of activation markers were observed on the alveolar macrophages of smokers compared to non-smokers. In addition, BCG vaccination resulted in decreased expression of the activation markers CD11b and HLA-DR on alveolar macrophages. Future studies should evaluate the functional consequences of this reduced activation of alveolar macrophages after BCG vaccination.

Monocytic HLA-DR expression kinetics in septic shock patients with different pathogens, sites of infection and adverse outcomes.

BACKGROUND: Decreased monocytic (m)HLA-DR expression is the most studied biomarker of sepsis-induced immunosuppression. To date, little is known about the relationship between sepsis characteristics, such as the site of infection, causative pathogen, or severity of disease, and mHLA-DR expression kinetics. METHODS: We evaluated mHLA-DR expression kinetics in 241 septic shock patients with different primary sites of infection and pathogens. Furthermore, we used unsupervised clustering analysis to identify mHLA-DR trajectories and evaluated their association with outcome parameters. RESULTS: No differences in mHLA-DR expression kinetics were found between groups of patients with different sites of infection (abdominal vs. respiratory, p = 0.13; abdominal vs. urinary tract, p = 0.53) and between pathogen categories (Gram-positive vs. Gram-negative, p = 0.54; Gram-positive vs. negative cultures, p = 0.84). The mHLA-DR expression kinetics differed between survivors and non-survivors (p < 0.001), with an increase over time in survivors only. Furthermore, we identified three mHLA-DR trajectories ('early improvers', 'delayed or non-improvers' and 'decliners'). The probability for adverse outcome (secondary infection or death) was higher in the delayed or non-improvers and decliners vs. the early improvers (delayed or non-improvers log-rank p = 0.03, adjusted hazard ratio 2.0 [95% CI 1.0-4.0], p = 0.057 and decliners log-rank p = 0.01, adjusted hazard ratio 2.8 [95% CI 1.1-7.1], p = 0.03). CONCLUSION: Sites of primary infection or causative pathogens are not associated with mHLA-DR expression kinetics in septic shock patients. However, patients showing delayed or no improvement in or a declining mHLA-DR expression have a higher risk for adverse outcome compared with patients exhibiting a swift increase in mHLA-DR expression. Our study signifies that changes in mHLA-DR expression over time, and not absolute values or static measurements, are of clinical importance in septic shock patients.

Proenkephalin Compared to Conventional Methods to Assess Kidney Function in Critically Ill Sepsis Patients.

BACKGROUND: The assessment of renal function in clinical practice remains challenging. Using creatinine to assess the glomerular filtration rate (GFR) is notoriously inaccurate, and determination of the true GFR, e.g., using inulin or iohexol, is laborious and not feasible in daily practice. Proenkephalin (PENK) is a novel candidate biomarker for kidney function that is filtrated in the glomerulus, has shown to represent steady-state GFR in patients with different severities of renal insufficiency. In this pilot study in non-steady-state critically ill patients, we compared plasma PENK concentrations with creatinine-based GFR assessments and validated both against the "true GFR" measured using a gold standard method: iohexol plasma clearance. METHODS: Twenty-three critically ill patients with septic shock were included. Kidney function was determined using the Modification of Diet in Renal Disease formula (eGFRMDRD), Endogenous Creatinine Clearance (GFRECC), and iohexol plasma clearance (GFRiohexol) during a 6-h window. Plasma PENK concentrations were measured using the penKid immunoassay. RESULTS: The eGFRMDRD and GFRECC correlated with the GFRiohexol (R = 0.82, P < 0.0001 and R = 0.82, P < 0.0001 respectively); however, bias and variability were considerable: the eGFRMDRD overestimated the true GFR with 31 ±â€Š35% (95% limits of agreement: -37% to 100%) and the GFRECC with 37 ±â€Š49% (95% limits of agreement: -59% to 133%). Plasma PENK concentrations showed a very strong inverse correlation with the GFRiohexol (R = 0.90, P < 0.0001) which tended to be better compared with the correlation of eGFRMDRD (P = 0.06) and GFRECC (P = 0.08) with the GFRiohexol. CONCLUSIONS: In this pilot study in non-steady-state critically ill sepsis patients, GFR appears to be more accurately reflected by plasma PENK concentrations compared to conventional creatinine-based methods. Therefore, PENK holds promise as an accurate and feasible biomarker to determine kidney function during non-steady-state conditions in the critically ill.

Frontline Science: Endotoxin-induced immunotolerance is associated with loss of monocyte metabolic plasticity and reduction of oxidative burst.

Secondary infections are a major complication of sepsis and associated with a compromised immune state, called sepsis-induced immunoparalysis. Molecular mechanisms causing immunoparalysis remain unclear; however, changes in cellular metabolism of leukocytes have been linked to immunoparalysis. We investigated the relation of metabolic changes to antimicrobial monocyte functions in endotoxin-induced immunotolerance, as a model for sepsis-induced immunoparalysis. In this study, immunotolerance was induced in healthy males by intravenous endotoxin (2 ng/kg, derived from Escherichia coli O:113) administration. Before and after induction of immunotolerance, circulating CD14+ monocytes were isolated and assessed for antimicrobial functions, including cytokine production, oxidative burst, and microbial (Candida albicans) killing capacity, as well metabolic responses to ex vivo stimulation. Next, the effects of altered cellular metabolism on monocyte functions were validated in vitro. Ex vivo lipopolysaccharide stimulation induced an extensive rewiring of metabolism in naive monocytes. In contrast, endotoxin-induced immunotolerant monocytes showed no metabolic plasticity, as they were unable to adapt their metabolism or mount cytokine and oxidative responses. Validation experiments showed that modulation of metabolic pathways, affected by immunotolerance, influenced monocyte cytokine production, oxidative burst, and microbial (C. albicans) killing in naive monocytes. Collectively, these data demonstrate that immunotolerant monocytes are characterized by a loss of metabolic plasticity and these metabolic defects impact antimicrobial monocyte immune functions. Further, these findings support that the changed cellular metabolism of immunotolerant monocytes might reveal novel therapeutic targets to reverse sepsis-induced immunoparalysis.

Comparison of different lots of endotoxin and evaluation of in vivo potency over time in the experimental human endotoxemia model.

The experimental human endotoxemia model is used to study the systemic inflammatory response in vivo. The previously used lot of endotoxin, which was used for over a decade, is no longer approved for human use and a new Good Manufacturing Practices-grade batch has become available. We compared the inflammatory response induced by either bolus or continuous administration of either the previously used lot #1188844 or new lots of endotoxin (#94332B1 and #94332B4). Compared with lot #1188844, bolus administration of lot #94332B1 induced a more pronounced systemic inflammatory response including higher plasma levels of pro-inflammatory cytokines and more pronounced clinical signs of inflammation. In contrast, continuous infusion of lot #94332B4 resulted in a slightly less pronounced inflammatory response compared with lot #1188844. Furthermore, we evaluated whether lot #1188844 displayed in vivo potency loss by reviewing inflammatory parameters obtained from 17 endotoxemia studies performed in our centre between 2007 and 2016. Despite inter-study variability in endotoxemia-induced effects on temperature, heart rate, symptoms, and leukocyte counts, the magnitude of these effects did not decrease over time. In conclusion, although all lots of endotoxin induce a pronounced inflammatory response, the magnitude differs between lots. We observed no potency loss of endotoxin over time.

Experimental human endotoxemia as a model of systemic inflammation.

Systemic inflammation plays a pivotal role in a multitude of conditions, including sepsis, trauma, major surgery and burns. However, comprehensive analysis of the pathophysiology underlying this systemic inflammatory response is greatly complicated by variations in the immune response observed in critically ill patients, which is a result of inter-individual differences in comorbidity, comedication, source of infection, causative pathogen, and onset of the inflammatory response. During experimental human endotoxemia, human subjects are challenged with purified endotoxin (lipopolysaccharide) intravenously which induces a short-lived, well-tolerated and controlled systemic inflammatory response, similar to that observed during sepsis. The human endotoxemia model can be conducted in a highly standardized and reproducible manner, using a carefully selected homogenous study population. As such, the experimental human endotoxemia model does not share the aforementioned clinical limitations and enables us to investigate both the mechanisms of systemic inflammation, as well as to evaluate novel (pharmacological) interventions in humans in vivo. The present review provides a detailed overview of the various designs, organ-specific changes, and strengths and limitations of the experimental human endotoxemia model, with the main focus on its use as a translational model for sepsis research.

New frontiers in precision medicine for sepsis-induced immunoparalysis.

INTRODUCTION: In the last decade, the sepsis research field has shifted focus from targeting hyperinflammation to reversing sepsis-induced immunoparalysis. Sepsis-induced immunoparalysis is very heterogeneous: the magnitude and the nature of the underlying immune defects differ considerably between patients, but also within individuals over time. Therefore, a 'one-treatment-fits-all' strategy for sepsis-induced immunoparalysis is bound to fail, and an individualized 'precision medicine' approach is required. Such a strategy is nevertheless hampered by the unsuitability of the currently available markers to identify the many immune defects that can manifest in individual patients. Areas covered: We describe the currently available markers for sepsis-induced immunoparalysis and limitations pertaining to their use. Furthermore, future prospects and caveats are discussed, focusing on 'omics' approaches: genomics, transcriptomics, epigenomics, and metabolomics. Finally, we present a contemporary overview of adjuvant immunostimulatory therapies. Expert opinion: The integration of multiple omics techniques offers a systems biology approach which can yield biomarker profiles that accurately and comprehensively gauge the extent and nature of sepsis-induced immunoparalysis. We expect this development to be instrumental in facilitating precision medicine for sepsis-induced immunoparalysis, consisting of the application of targeted immunostimulatory therapies and follow-up measurements to monitor the response to treatment and to titrate or adjust medication.

Treatment With Acetylsalicylic Acid Reverses Endotoxin Tolerance in Humans In Vivo: A Randomized Placebo-Controlled Study.

OBJECTIVE: To investigate immunostimulatory effects of acetylsalicylic acid during experimental human endotoxemia and in sepsis patients. DESIGN: Double-blind, randomized, placebo-controlled study in healthy volunteers and ex vivo stimulation experiments using monocytes of septic patients. SETTING: Intensive care research unit of an university hospital. SUBJECTS: Thirty healthy male volunteers and four sepsis patients. INTERVENTIONS: Healthy volunteers were challenged IV with endotoxin twice, at a 1-week interval, with each challenge consisting of a bolus of 1 ng/kg followed by continuous administration of 1 ng/kg/hr during 3 hours. Volunteers were randomized to acetylsalicylic acid prophylaxis (80 mg acetylsalicylic acid daily for a 14-d period, starting 7 d before the first endotoxin challenge), acetylsalicylic acid treatment (80 mg acetylsalicylic acid daily for the 7-d period in-between both endotoxin challenges), or the control group (receiving placebo). Furthermore, monocytes of sepsis patients were incubated with acetylsalicylic acid preexposed platelets and were subsequently stimulated with endotoxin. MEASUREMENTS AND MAIN RESULTS: Acetylsalicylic acid prophylaxis enhanced plasma tumor necrosis factor-α concentrations upon the first endotoxin challenge by 50% compared with the control group (p = 0.02) but did not modulate cytokine responses during the second endotoxin challenge. In contrast, acetylsalicylic acid treatment resulted in enhanced plasma levels of tumor necrosis factor-α (+53%; p = 0.02), interleukin-6 (+91%; p = 0.03), and interleukin-8 (+42%; p = 0.02) upon the second challenge, whereas plasma levels of the key antiinflammatory cytokine interleukin-10 were attenuated (-40%; p = 0.003). This proinflammatory phenotype in the acetylsalicylic acid treatment group was accompanied by a decrease in urinary prostaglandin E metabolite levels (-27% ± 7%; p = 0.01). Ex vivo exposure of platelets to acetylsalicylic acid increased production of tumor necrosis factor-α (+66%) and decreased production of interleukin-10 (-23%) by monocytes of sepsis patients. CONCLUSIONS: Treatment, but not prophylaxis, with low-dose acetylsalicylic acid, partially reverses endotoxin tolerance in humans in vivo by shifting response toward a proinflammatory phenotype. This acetylsalicylic acid-induced proinflammatory shift was also observed in septic monocytes, signifying that patients suffering from sepsis-induced immunoparalysis might benefit from initiating acetylsalicylic acid treatment.

Increased Plasma Levels of Danger-Associated Molecular Patterns Are Associated With Immune Suppression and Postoperative Infections in Patients Undergoing Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

Introduction: Danger-associated molecular patterns (DAMPs) can elicit immune responses and may subsequently induce an immune-suppressed state. Previous work showed that increased plasma levels of DAMPs are associated with immune suppression and increased susceptibility toward infections in trauma patients. Like trauma, major surgical procedures, such as cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC), are also thought to cause profound DAMP release. Furthermore, the incidence of postoperative infections in these patients, ranging from 10 to 36%, is very high compared to that observed in patients undergoing other major surgical procedures. We hypothesized that the double hit of surgical trauma (CRS) in combination with HIPEC causes excessive DAMP release, which in turn contributes to the development of immune suppression. To investigate this, we assessed DAMP release in patients undergoing CRS-HIPEC, and investigated its relationship with immune suppression and postoperative infections. Methods: In 20 patients undergoing CRS-HIPEC, blood was obtained at five time points: just before surgery (baseline), after CRS, after HIPEC, at ICU admission, and 1 day after surgery. Circulating levels of DAMPs [heat shock protein (HSP)70, high mobility group box (HMGB)1, S100A12, S100A8/S100A9, nuclear (n)DNA, mitochondrial (mt)DNA, lactate dehydrogenase (LDH), a marker of unscheduled cell death], and cytokines [tumor necrosis factor (TNF)α, IL-6, IL-8, IL-10, macrophage inflammatory protein (MIP)-1α, MIP-1ß, and MCP-1] were measured. The extent of immune suppression was determined by measuring HLA-DR gene expression and ex vivo leukocytic cytokine production capacity. Results: Plasma levels of DAMPs (maximum fold increases of HSP70: 2.1 [1.5-2.8], HMGB1: 5.9 [3.2-9.8], S100A8/S100A9: 3.6 [1.8-5.6], S100A12: 2.6 [1.8-4.3], nDNA 3.9 [1.0-10.8], LDH 1.7 [1.2-2.5]), and all measured cytokines increased profoundly following CRS-HIPEC. Evidence of immune suppression was already apparent during the procedure, illustrated by a decrease of HLA-DR expression compared with baseline (0.5-fold [0.3-0.9]) and diminished ex vivo pro-inflammatory cytokine production capacity. The increase in HMGB1 levels correlated with the decrease in HLA-DR expression (r = -0.46, p = 0.04), and peak HMGB1 concentrations were significantly higher in the five patients who went on to develop a postoperative infection (p = 0.04). Conclusion: CRS-HIPEC is associated with profound DAMP release and immune suppression, and plasma HMGB1 levels are related with the occurrence of postoperative infections in these patients.

Release of Danger-Associated Molecular Patterns following Chemotherapy Does Not Induce Immunoparalysis in Leukemia Patients.

Chemotherapy may result in the release of danger-associated molecular patterns (DAMPs), which can cause immunoparalysis (deactivation of the immune system). We investigated DAMPs following chemotherapy and their relationship with markers of immunoparalysis in leukemia patients. In 6 patients with acute myeloid leukemia or myelodysplastic syndrome and 12 healthy subjects, DAMPs, cytokines, and markers of immunoparalysis were determined before and during the first week after chemotherapy initiation. In the patients, plasma levels of nuclear DNA (a marker of general DAMP release) were profoundly increased before chemotherapy and further increased 4-6 h afterwards, while the specific DAMP mitochondrial DNA showed only a trend towards increase. Circulating cytokine levels did not change following chemotherapy. Leukocyte cytokine production capacity and HLA-DR expression were similar in patients and healthy controls until day 4 when leukocytes were found to be virtually absent. In conclusion, in the early phase following chemotherapy in leukemia patients, increased DAMP release does not induce immunoparalysis.