Proinflammatory polarization strongly reduces human macrophage in vitro phagocytosis of tumor cells in response to CD47 blockade.

Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.

Comparison of culture media reveals that non-essential amino acids strongly affect the phenotype of human monocyte-derived macrophages.

Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte-derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte-derived macrophage phenotype. Monocyte-derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT-qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFα, IL-10, SIRPα, LILRB1 and Siglec-10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non-essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non-essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte-derived macrophages cultured in vitro.

Metabolism of 25-Hydroxy-Vitamin D in Human Macrophages Is Highly Dependent on Macrophage Polarization.

Macrophages synthesize active vitamin D (1,25-dihydroxy-vitamin D) and express the vitamin D receptor in the nucleus; however, vitamin D metabolism in relation to macrophage polarization and function is not well understood. We studied monocyte-derived macrophages (MDMs) from human buffy coats polarized into M0, M1 (LPS + IFNγ), M2a (IL4 + IL13) and M2c (IL10) macrophage subtypes stimulated with 25-hydroxy-vitamin D (1000 and 10,000 nanomolar). We measured vitamin D metabolites (25-hydroxy-vitamin D, 1,25-dihydroxy-vitamin D, 24,25-dihydroxy-vitamin D and 3-epi-25-hydroxy-vitamin D) in cell media with liquid chromatography-mass spectrometry-mass spectrometry. The mRNA expression (CYP27B1, CYP24A1 and CYP24A1-SV) was measured with qPCR. We found that reparative MDMs (M2a) had significantly more 1,25-dihydroxy-vitamin D compared to the other MDMs (M0, M1 and M2c). All MDMs were able to produce 3-epi-25-hydroxy-vitamin D, but this pathway was almost completely attenuated in inflammatory M1 MDMs. All MDM subtypes degraded vitamin D through the 24-hydroxylase pathway, although M1 MDMs mainly expressed an inactive splice variant of CYP24A1, coding the degrading enzyme. In conclusion, this study shows that vitamin D metabolism is highly dependent on macrophage polarization and that the C3-epimerase pathway for vitamin D is active in macrophages.

A New Serum Macrophage Checkpoint Biomarker for Innate Immunotherapy: Soluble Signal-Regulatory Protein Alpha (sSIRPα).

Background and Aims: The macrophage "don't eat me" pathway CD47/SIRPα is a target for promising new immunotherapy. We hypothesized that a soluble variant of SIRPα is present in the blood and may function as a biomarker. Methods: Monocyte derived macrophages (MDMs) from human buffy-coats were stimulated into macrophage subtypes by LPS and IFN-γ (M1), IL-4 and IL-13 (M2a), IL-10 (M2c) and investigated using flow cytometry. Soluble SIRPα (sSIRPα) was measured in cell cultures and serum by Western blotting and an optimized ELISA. Serum samples were obtained from 120 healthy individuals and from 8 individuals challenged by an LPS injection. Results: All macrophage phenotypes expressed SIRPα by flowcytometry, and sSIRPα was present in all culture supernatants including unstimulated cells. M1 macrophages expressed the lowest level of SIRPαand released the highest level of sSIRPα (p < 0.05). In vivo, the serum level of sSIRPα increased significantly (p < 0.0001) after an LPS challenge in humans. The median concentration in healthy individuals was 28.7 µg/L (19.8−41.1, 95% reference interval), and 20.5 µg/L in an IFCC certified serum reference material. The protein was stable in serum for prolonged storage and repeated freeze/thawing. Conclusions: We demonstrate that sSIRPα is produced constitutively and the concentration increases upon macrophage activation both in vitro and in vivo. It is present in human serum where it may function as a biomarker for the activity of tumor-associated macrophages (TAMs), and for monitoring the effect of immunotherapy.

Profiling Bispebjerg Acute Cohort: Database Formation, Acute Contact Characteristics of a Metropolitan Hospital, and Comparisons to Urban and Rural Hospitals in Denmark.

Purpose: To present a metropolitan cohort, Bispebjerg acute cohort (BAC), and compare patient characteristics and outcomes with patients from urban and rural hospitals in Denmark. Patients and Methods: We linked data from seven Danish nationwide registries and included all acute contacts to non-psychiatric hospitals in the years 2016-2018. Acute hospital contacts to Bispebjerg and Frederiksberg Hospital constituted BAC, representing a solely metropolitan/urban catchment area. Patient characteristics and outcomes were compared to the rest of Denmark in an urban cohort (UrC) and a rural cohort (RuC), stratified by visit and hospitalization contact types. Results: We identified 4,063,420 acute hospital contacts in Denmark and BAC constituted 8.4% (n=343,200) of them. BAC had a higher proportion of visits (65.1%) compared with UrC (52.1%) and RuC (45.3%). Patients in BAC more often lived alone (visits: BAC: 34.8%, UrC: 30.6%, RuC: 29.2%; hospitalizations: BAC: 50.8%, UrC: 36.7%, RuC: 37.2%) and had temporary CPR number (visits: BAC: 4.4%, UrC: 1.9%, RuC: 1.6%; hospitalizations: BAC: 1.5%, UrC: 0.9%, RuC: 0.8%). Visit patients in BAC were younger (BAC: 36, UrC: 42, RuC: 45 years, median), more often students (BAC: 18.0%, UrC: 14.0%, RuC: 12.5%), and had more contacts due to infectious diseases (BAC: 19.8%, UrC: 14.1%, RuC: 6.2%) but less due to injuries (BAC: 40.0%, UrC: 43.8%, RuC: 60.7%). Hospitalized patients in BAC had higher median age (BAC: 64, UrC: 61, RuC: 64 years) and fewer were in employment than in UrC (BAC: 26.1%, UrC: 32.1%, RuC: 28.1%). BAC Hospitalizations had a lower death rate within 30 days than in RuC (BAC: 3.0% [2.9-3.1%], UrC: 3.1% [3.0-3.1%], RuC: 3.4% [3.3-3.4%]), but a higher readmission-rate (BAC: 20.5% [20.3-20.8%], UrC: 17.3% [17.2-17.4%], RuC: 17.5% [17.5-17.6%]). Conclusion: Significant differences between BAC, urban, and rural cohorts may be explained by differences in healthcare structure and sociodemographics of the catchment areas.

Soluble PD-1 (sPD-1) is expressed in human macrophages.

The PD-1/PD-L1 axis plays a crucial role in regulating the anti-tumour immune response. A soluble PD-1 protein (sPD-1) has previously been observed, which could block the binding of PD-L1 to PD-1. Tumour associated macrophages are abundant in tumours, and evidence suggest they express PD-1. However, whether they also express sPD-1 remains unclear. The objective of this study was to investigate expression of sPD-1 in two in vitro models of human macrophages: THP-1 cells and monocyte-derived macrophages (MDM). Cells were polarised with either LPS + IFN-γ or IL-4 + IL-13 or left unpolarised. PD-1 and sPD-1 mRNAs were measured using droplet digital PCR, sPD-1 protein by electrochemiluminescence immunoassay and PD-1 by flow cytometry. sPD-1 mRNA was induced in both THP-1 cells and MDM after polarisation with LPS + IFN-γ, while IL-4 + IL-13 induced sPD-1 mRNA in MDM only. sPD-1 protein was measurable in culture supernatants. These findings show that macrophages can be induced to express sPD-1.

The Danish Anaesthesia Database.

AIM OF DATABASE: The aim of the Danish Anaesthesia Database (DAD) is the nationwide collection of data on all patients undergoing anesthesia. Collected data are used for quality assurance, quality development, and serve as a basis for research projects. STUDY POPULATION: The DAD was founded in 2004 as a part of Danish Clinical Registries (Regionernes Kliniske Kvalitetsudviklings Program [RKKP]). Patients undergoing general anesthesia, regional anesthesia with or without combined general anesthesia as well as patients under sedation are registered. Data are retrieved from public and private anesthesia clinics, single-centers as well as multihospital corporations across Denmark. In 2014 a total of 278,679 unique entries representing a national coverage of ~70% were recorded, data completeness is steadily increasing. MAIN VARIABLE: Records are aggregated for determining 13 defined quality indicators and eleven defined complications all covering the anesthetic process from the preoperative assessment through anesthesia and surgery until the end of the postoperative recovery period. DESCRIPTIVE DATA: Registered variables include patients' individual social security number (assigned to all Danes) and both direct patient-related lifestyle factors enabling a quantification of patients' comorbidity as well as variables that are strictly related to the type, duration, and safety of the anesthesia. Data and specific data combinations can be extracted within each department in order to monitor patient treatment. In addition, an annual DAD report is a benchmark for departments nationwide. CONCLUSION: The DAD is covering the anesthetic process for the majority of patients undergoing anesthesia in Denmark. Data in the DAD are increasingly used for both quality and research projects.

Frequency of early warning score assessment and clinical deterioration in hospitalized patients: A randomized trial.

AIM: To explore whether early warning score (EWS) measurements at 8h intervals is associated with better outcomes than 12h intervals. We hypothesized that the proportion of patients that deteriorated to a higher EWS at 24h after hospital admission would be lower with 8h interval than with 12h interval. METHOD: This was a pragmatic, ward-level randomized, non-blinded, controlled trial at an urban University hospital. During two six weeks periods acutely admitted surgical and medical patients, with an initial EWS of 0 or 1, were monitored either every 8th hour or every 12th hour. The primary outcome was clinical deterioration 24h post-admission, estimated by the proportion of patients with an EWS≥2 at 24h after the initial EWS on admission. RESULTS: Of 3185 patients screened for eligibility, 1346 patients were included to the trial. Forty-nine percent were allocated to the 8h group and 51% to the 12h group; of these, 23% and 20% had an elevated EWS≥2 at 24h, respectively (p=0.456), OR 1.17 (0.78-1.76); 3.4% and 2.2%, respectively had an EWS≥5 (p=0.391), and one patient in each group had an EWS≥7 at 24h (p=1.0). Multiple logistic regression analysis showed no significant interactions for the primary outcome and the predefined variables: age, gender, ward type, and inclusion period, with an adjusted OR 1.20 (0.79-1.82). There were no significant differences in regard to the secondary outcomes: cardiac arrests, ICU admissions, review by medical emergency team (MET), length of hospital stay, or elevated EWS at 48 h. Thirty-day mortality was 1.1% vs. 1.8% (p=0.357) in the 8h group and the 12h-group, respectively (OR=0.60 (0.23-1.50), p=0.279). CONCLUSION: We found no significant reduction in the proportion of clinical deterioration with monitoring frequencies of 3 vs. 2 times daily among patients acutely admitted to a surgical or medical ward and an initial EWS of 0-1.

Serious adverse events in a hospital using early warning score - what went wrong?

AIM: To evaluate the performance of a new early warning score (EWS) system by reviewing all serious adverse events in our hospital over a 6-month time period. METHOD: All incidents of unexpected death (UD), cardiac arrest (CA) and unanticipated intensive care unit admission(UICU) of adult patients on general wards were reviewed to see if the escalation protocol that is part of the EWS system was followed in the 24h preceding the event, and if not where in the chain of events failure occurred. RESULTS: We found 77 UICU and 67 cases of the combined outcome (CO) of CA and UD. At least two full sets of EWS were recorded in 87, 94 and 75% of UICU, CA and UD. Patients were monitored according to the escalation protocol in 13, 31 and 13% of UICU, CA and UD. Nurses escalated care and contacted physicians in 64% and 60% of events of UICU and the corresponding proportions for CO were 58% and 55%. On call physicians provided adequate care in 49% of cases of UICU and 29% of cases of the CO. Senior staff was involved according to protocol in 53% and 36% of cases of UICU and CO, respectively. CONCLUSION: Poor compliance with the escalation protocol was commonly found when serious adverse events occurred but level of care provided by physicians was also a problem in a hospital with implemented early warning system. This information may prove useful in improving performance of EWS systems.

Abnormal vital signs are strong predictors for intensive care unit admission and in-hospital mortality in adults triaged in the emergency department - a prospective cohort study.

BACKGROUND: Assessment and treatment of the acutely ill patient have improved by introducing systematic assessment and accelerated protocols for specific patient groups. Triage systems are widely used, but few studies have investigated the ability of the triage systems in predicting outcome in the unselected acute population. The aim of this study was to quantify the association between the main component of the Hillerød Acute Process Triage (HAPT) system and the outcome measures; Admission to Intensive Care Unit (ICU) and in-hospital mortality, and to identify the vital signs, scored and categorized at admission, that are most strongly associated with the outcome measures. METHODS: The HAPT system is a minor modification of the Swedish Adaptive Process Triage (ADAPT) and ranks patients into five level colour-coded triage categories. Each patient is assigned a triage category for the two main descriptors; vital signs, T(vitals), and presenting complaint, T(complaint). The more urgent of the two determines the final triage category, T(final). We retrieved 6279 unique adult patients admitted through the Emergency Department (ED) from the Acute Admission Database. We performed regression analysis to evaluate the association between the covariates and the outcome measures. RESULTS: The covariates, T(vitals), T(complaint) and T(final) were all significantly associated with ICU admission and in-hospital mortality, the odds increasing with the urgency of the triage category. The vital signs best predicting in-hospital mortality were saturation of peripheral oxygen (SpO(2)), respiratory rate (RR), systolic blood pressure (BP) and Glasgow Coma Score (GCS). Not only the type, but also the number of abnormal vital signs, were predictive for adverse outcome. The presenting complaints associated with the highest in-hospital mortality were 'dyspnoea' (11.5%) and 'altered level of consciousness' (10.6%). More than half of the patients had a T(complaint) more urgent than T(vitals), the opposite was true in just 6% of the patients. CONCLUSION: The HAPT system is valid in terms of predicting in-hospital mortality and ICU admission in the adult acute population. Abnormal vital signs are strongly associated with adverse outcome, while including the presenting complaint in the triage model may result in over-triage.

The formation and design of the 'Acute Admission Database'- a database including a prospective, observational cohort of 6279 patients triaged in the emergency department in a larger Danish hospital.

BACKGROUND: Management and care of the acutely ill patient has improved over the last years due to introduction of systematic assessment and accelerated treatment protocols. We have, however, sparse knowledge of the association between patient status at admission to hospital and patient outcome. A likely explanation is the difficulty in retrieving all relevant information from one database. The objective of this article was 1) to describe the formation and design of the 'Acute Admission Database', and 2) to characterize the cohort included. METHODS: All adult patients triaged at the Emergency Department at Hillerød Hospital and admitted either to the observationary unit or to a general ward in-hospital were prospectively included during a period of 22 weeks. The triage system used was a Danish adaptation of the Swedish triage system, ADAPT. Data from 3 different data sources was merged using a unique identifier, the Central Personal Registry number; 1) Data from patient admission; time and date, vital signs, presenting complaint and triage category, 2) Blood sample results taken at admission, including a venous acid-base status, and 3) Outcome measures, e.g. length of stay, admission to Intensive Care Unit, and mortality within 7 and 28 days after admission. RESULTS: In primary triage, patients were categorized as red (4.4%), orange (25.2%), yellow (38.7%) and green (31.7%). Abnormal vital signs were present at admission in 25% of the patients, most often temperature (10.5%), saturation of peripheral oxygen (9.2%), Glasgow Coma Score (6.6%) and respiratory rate (4.8%). A venous acid-base status was obtained in 43% of all patients. The majority (78%) had a pH within the normal range (7.35-7.45), 15% had acidosis (pH < 7.35) and 7% had alkalosis (pH > 7.45). Median length of stay was 2 days (range 1-123). The proportion of patients admitted to Intensive Care Unit was 1.6% (95% CI 1.2-2.0), 1.8% (95% CI 1.5-2.2) died within 7 days, and 4.2% (95% CI 3.7-4.7) died within 28 days after admission. CONCLUSIONS: Despite challenges of data registration, we succeeded in creating a database of adequate size and data quality. Future studies will focus on the association between patient status at admission and patient outcome, e.g. admission to Intensive Care Unit or in-hospital mortality.

[Measurement of intraabdominal pressure and abdominal compartment syndrome in surgical and intensive care units]. / Måling af intraabdominalt tryk og abdominalt kompartmentsyndrom på kirurgiske og intensiv terapi-afdelinger.

INTRODUCTION: Over the past number of years, there has been an increasing interest in the pathophysiological phenomena intraabdominal hypertension (IAH) and abdominal compartment syndrome (ACS). Foreign studies have shown considerably national differences in the interpretation of the two phenomena. The purpose of this questionnaire study was to survey which group of patients is examined for IAH and ACS, how the examination is carried out and how the two conditions are treated in surgical and intensive care units in Denmark. MATERIALS AND METHODS: A questionnaire was sent to all relevant surgical and intensive care units in Denmark. RESULTS: The response rate was 81%. 74% intensive care units and 31% surgical wards measured IAP. 100% used the intravesical method. International guidelines were followed in 11% of surgical wards and 18% of the intensive care units regarding how often IAP should be measured and 44%/ 32% regarding at what IAP level surgical intervention should be considered. 78% of the wards and 79% of the intensive care units that measured IAP had celiotomy as a treatment modality. There were major differences between both surgical wards and intensive care units regarding which group of patients should have IAP measured. CONCLUSION: Considering the differences in clinical practice and the discrepancy to international guidelines interdisciplinary national guideline might contribute to a more uniform evaluation and treatment of patients with IAH or ACS.

[Capacity in Danish intensive care units. A national survey of capacity, cancellations and transfers of critically ill patients]. / Kapaciteten på danske intensivafdelinger. En national survey af kapacitet, aflysninger og overflytninger af kritisk syge patienter.

INTRODUCTION: A shortage of intensive care beds and fully-booked intensive care units has a range of undesirable consequences for patients and personnel, eg. transfer to other intensive care units, cancellation of operations, tighter visitation criteria and an increase in the work-load. The problem is illustrated in a national survey. MATERIALS AND METHODS: The survey was undertaken in 3 parts and comprised all 50 adult intensive care units in Denmark. Part 1 was a questionnaire encompassing demographic data, the number of open intensive care beds and how often under or over capacity was experienced in the department. Parts 2 and 3 consisted of a daily registry of the capacity and occupancy rate in the intensive care departments for two weeks along with a contemporary registry of the number of admittances, transfers and cancellations of operations. RESULTS: In Denmark only 2% of all somatic beds are intensive care beds. Under capacity, defined as a 100% occupancy rate, was experienced weekly or monthly in 80% of all intensive care units in Denmark. Occupancy rate was high, a medium of 78%, highest in level III intensive care units with an 88% occupancy rate. The numbers for transfers were equivalent to 800-1000 patient transfers per year. The number of cancelled operations was equivalent to 2000 per year. CONCLUSION: This survey documents that there is a problem with the capacity in Danish intensive care units. Establishing more intensive care beds in selected departments, ensuring personnel for the beds already established and establishing intermediate care beds could relieve the shortage of beds.

[Capacity problems in Danish intensive care units?]. / Er der kapacitetsproblemer på danske intensivafdelinger?

There are documented capacity problems in Danish ICUs. The indications for intensive care have increased in the last decade without any increase in the number of ICU beds. The result is massive pressure on many ICUs and many negative consequences in relation to healthcare, healthcare economics and patient comfort. Possible solutions: 1) an increase in the number of ICU beds, 2) re-organization of Danish ICUs into larger units and 3) creation of "step-down"-units. Intensive care is a costly area in the healthcare system, where there must be distinct guidelines for visitation and use of expensive medicine and advanced technology.