A Transient Inflammatory Response Induced by Lipopolysaccharide Infusion Lowers Markers of Endogenous Cholesterol and Bile Acid Synthesis in Healthy Normocholesterolemic Young Men.

Inflammation is associated with changes in plasma lipids, lipoproteins, and cholesterol efflux capacity (CEC). It is unknown if the changes in lipids and lipoproteins during inflammation are related to changes in cholesterol absorption, synthesis, and bile acid synthesis. We, therefore, examined the effects of acute lipopolysaccharide (LPS)-induced transient systemic inflammation on lipids, lipoproteins, CEC, and markers of cholesterol metabolism. We also evaluated whether markers for cholesterol metabolism at baseline predict the intensity of the inflammatory response. Eight healthy young subjects received LPS infusion, and blood was sampled for the following 24 h. In addition to lipids, lipoproteins, and CEC, we also measured markers for cholesterol absorption and synthesis, bile acid synthesis, and inflammation. Compared with baseline, plasma total cholesterol, low-density lipoprotein cholesterol, and CEC decreased, while triglycerides increased in the 24 h following LPS infusion. TC-standardized levels of cholesterol synthesis markers (lathosterol, lanosterol, and desmosterol) and a bile acid synthesis marker (7α-OH-cholesterol) also decreased, with no changes in cholesterol absorption markers (campesterol, sitosterol, and cholestanol). Baseline TC-standardized levels of desmosterol and 7α-OH-cholesterol were positively correlated with concentrations of various inflammatory markers. Changes in TC-standardized desmosterol and 7α-OH-cholesterol were negatively correlated with concentrations of inflammatory markers. LPS infusion reduced endogenous cholesterol synthesis and bile acid synthesis in healthy young men.

The effect of drone transport on the stability of biochemical, coagulation and hematological parameters in healthy individuals.

OBJECTIVES: Transport of blood tubes is mainly by car or pneumatic transport. The transportation of blood tubes by drones is a novel approach for rapid transportation of blood tubes over long distances. However, limited data on the stability of biochemical, coagulation and hematological parameters is available after transport of blood tubes by drone. METHODS: To investigate the effect of drone transport on the stability of blood parameters, four test flights were performed. Blood was drawn from 20 healthy individuals and 39 of the most frequently measured blood parameters were compared between 4 groups; immediate measurement (control), late measurement, transport by car and transport by drone. Total allowable error (TAE) of the EFLM Biological Variation Database was used to determine the clinical relevance of significant differences. RESULTS: The majority of blood parameters were not affected by drone transport. Eight of the measured parameters showed significant differences between all the groups; glucose, phosphate, potassium, chloride, hemoglobin, platelet count, activated partial thromboplastin time (APTT) and lactate dehydrogenase (LD). A clinically relevant increase for LD after transport and a decrease for glucose values in time and after transport compared with the control group was shown. CONCLUSIONS: Transportation of blood tubes from healthy individuals by drones has a limited clinically relevant effect. From the 39 investigated blood parameters only LD and glucose showed a clinically relevant effect.

Caspase-11 contributes to pulmonary host defense against Klebsiella pneumoniae and local activation of coagulation.

Klebsiella (K.) pneumoniae is a common cause of gram-negative pneumonia and sepsis. Caspase-11 is an intracellular receptor for lipopolysaccharide and regulates pyroptosis, a specific form of inflammatory cell death, which aids in host defense against intracellular gram-negative bacteria. Recently, caspase-11 has also been implicated in blood coagulation. Previously, we found that local fibrin formation contributes to protective immunity against Klebsiella infection of the lung. The aim of the present study was to determine the role of caspase-11 in host defense during K. pneumoniae-evoked pneumonia and sepsis. Therefore, we infected wild-type and caspase-11-deficient (Casp11-/-) mice with a low-dose K. pneumoniae via the airways to induce a gradually evolving pneumosepsis. Casp11-/- mice displayed increased bacterial numbers in the lung 12 h and 48 h after inoculation. Analysis of pulmonary IL-1α, IL-1ß, and TNF levels showed reduced IL-1α levels in bronchoalveolar lavage fluid and increased TNF levels in the lung of Casp11-/- mice at 48 h after inoculation. Lung γH2AX staining (marker for cell death), lung pathology and neutrophil influx in the lung, as well as bacterial dissemination and organ damage, however, were not altered in Casp11-/- mice after Klebsiella infection. Strikingly, analysis of cross-linked fibrin and D-dimer (markers for coagulation) revealed significantly less fibrin formation in the lungs of Casp11-/- mice at either time point after Klebsiella infection. These data reveal that caspase-11 contributes to protective immunity against K. pneumoniae possibly by activation of blood coagulation in the lung.

Role of tissue factor in the procoagulant and antibacterial effects of human adipose-derived mesenchymal stem cells during pneumosepsis in mice.

BACKGROUND: Adult mesenchymal stem cells (MSCs) improve the host response during experimental sepsis in animals. MSCs from various sources express a procoagulant activity that has been linked to the expression of tissue factor. This study sought to determine the role of tissue factor associated with adipose-derived MSCs (ASCs) in their procoagulant and antibacterial effects during pneumonia-derived sepsis. METHODS: Mice were infused intravenously with ASCs or vehicle after infection with the common human pathogen Klebsiella pneumoniae via the airways. RESULTS: Infusion of freshly cultured or cryopreserved ASCs induced the expression of many genes associated with tissue factor signaling and coagulation activation in the lungs. Freshly cultured and cryopreserved ASCs, as well as ASC lysates, exerted procoagulant activity in vitro as determined by a fibrin generation assay, which was almost completely inhibited by an anti-tissue factor antibody. Infusion of cryopreserved ASCs was associated with a rise in plasma thrombin-antithrombin complexes (indicative of coagulation activation) and formation of multiple thrombi in the lungs 4 h post-infusion. Preincubation of ASCs with anti-tissue factor antibody prior to infusion prevented the rise in plasma thrombin-antithrombin complex concentrations but did not influence thrombus formation in the lungs. ASCs reduced bacterial loads in the lungs and liver at 48 h after infection, which was not influenced by preincubation with anti-tissue factor antibody. At this late time point, microthrombi in the lungs were not detected anymore. CONCLUSION: These data indicate that ASC-associated tissue factor is responsible for systemic activation of coagulation after infusion of ASCs but not for the formation of microthrombi in the lungs or antibacterial effects.

Leukocyte transcriptional signatures dependent on LPS dosage in human endotoxemia.

The host immune response is characterized by a complex interplay of signal-specific cellular transcriptional responses. The magnitude of the immune response is dependent on the strength of the external stimulus. Knowledge on leukocyte transcriptional responses altered in response to different stimulus dosages in man is lacking. Here, we sought to identify leukocyte transcriptional signatures dependent on LPS dose in humans. Healthy human volunteers were administered 1 ng/kg (n = 7), 2 ng/kg (n = 6), or 4 ng/kg (n = 7) LPS intravenously. Blood was collected before (pre-LPS) and 4 h after LPS administration. Total RNA was analyzed by microarrays and generalized linear models. Pathway analysis was performed by using Ingenuity pathway analysis. Leukocyte transcriptomes altered per LPS dosage were predominantly shared, with 47% common signatures relative to pre-LPS. A univariate linear model identified a set of 3736 genes that exhibited a dependency on differing LPS dosages. Neutrophil, monocyte, and lymphocyte counts explained 38.9% of the variance in the LPS dose-dependent gene set. A multivariate linear model including leukocyte composition delineated a set of 295 genes with a dependency on LPS dose. Evaluation of the 295 gene signature in patients with sepsis due to abdominal infections showed significant correlations. Promoter regions of the LPS dose gene set were enriched for YY1, EGR1, ELK1, GABPA, KLF4, and REL transcription factor binding sites. Intravenous injection of 1, 2, or 4 ng/kg LPS was accompanied by both shared and distinct leukocyte transcriptional alterations. These data may assist in assessing the severity of the insult in patients with abdominal sepsis.

Human Adipose-Derived Mesenchymal Stem Cells Modify Lung Immunity and Improve Antibacterial Defense in Pneumosepsis Caused by Klebsiella pneumoniae.

Adult mesenchymal stem cells exert immunomodulatory effects that might improve the host response during sepsis. Knowledge on the effect of adipose-derived mesenchymal stem cells (ASCs) in sepsis is limited. Klebsiella (K.) pneumoniae is a common cause of gram-negative pneumonia and sepsis. This study sought to determine the effect of human ASCs on the host response during pneumosepsis in mice. Mice were infected with K. pneumoniae via the airways to induce a gradually evolving infection in the lung culminating pneumosepsis. One or 6 hours after infection, mice were infused intravenously with ASCs or vehicle, and euthanized after 16 hours or 48 hours, respectively. The effects of freshly cultured and cryopreserved ASCs were compared, the latter formulation being more clinically relevant. Intravenously administered ASCs were visualized in lung tissue by immunostaining at 1 and 3 hours, but not at 15 hours after infusion. Although early after infection, ASCs did not or only modestly influence bacterial loads, they reduced bacterial burdens in lungs and distant organs at 48 hours. ASCs reduced the lung levels of pro-inflammatory cytokines and attenuated lung pathology, but did not influence distant organ injury. ASCs strongly modified the lung transcriptome in uninfected mice and especially mice with pneumosepsis. Cryopreserved and cultured ASCs induced largely similar effects on the lung transcriptome. These data indicate that human ASCs induce profound immune modulatory effects in the lungs, resulting in reduced bacterial burdens and lung inflammation during pneumosepsis caused by a common human pathogen, suggesting that ASCs may be an adjunctive therapeutic in this condition. Stem Cells Translational Medicine 2019;8:785&796.

Intravenous Infusion of Human Adipose Mesenchymal Stem Cells Modifies the Host Response to Lipopolysaccharide in Humans: A Randomized, Single-Blind, Parallel Group, Placebo Controlled Trial.

In experimental models, mesenchymal stem cells (MSCs) can modulate various immune responses implicated in the pathogenesis of sepsis. Intravenous injection of lipopolysaccharide (LPS) into healthy subjects represents a model with relevance for the host response to sepsis. To explore the use of MSCs in sepsis, we determined their effect on the response to intravenous LPS in a randomized study in 32 healthy subjects with four treatment arms: placebo or allogeneic adipose MSCs (ASCs) intravenously at either 0.25 × 106 , 1 × 106 , or 4 × 106 cells/kg; all subjects received LPS intravenously (2 ng/kg) one hour after the end of ASC infusion (Trial Register number 2014-002537-63, clinicaltrials.gov identifier NCT02328612). Infusion of ASCs was well tolerated. The high ASC dose increased the febrile response, exerted mixed pro-inflammatory (enhanced interleukin-8 and nucleosome release) and anti-inflammatory effects (increased interleukin-10 and transforming growth factor-ß release), and enhanced coagulation activation and reduced the fibrinolytic response. Blood leukocyte transcriptome analyses showed a biphasic effect of ASCs on the LPS response: at 2 hours post LPS, ASC-infused subjects displayed higher expression of genes involved in innate immune pathways, whereas at 4 hours post LPS these subjects had lower expression of innate immune pathway genes. Infusion of ASCs did not modify the "ex vivo" responsiveness of whole blood to various bacterial agonists. These results indicate that intravenous infusion of allogeneic ASCs (4 × 106 cells/kg) has a variety of proinflammatory, anti-inflammatory, and procoagulant effects during human endotoxemia. Further studies are needed to assess the safety and efficacy of ASCs in sepsis patients. Stem Cells 2018;36:1778-1788.

Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Assay for Diagnosis of Cutaneous and Visceral Leishmaniasis.

A novel pan-Leishmania loop-mediated isothermal amplification (LAMP) assay for the diagnosis of cutaneous and visceral leishmaniasis (CL and VL) that can be used in near-patient settings was developed. Primers were designed based on the 18S ribosomal DNA (rDNA) and the conserved region of minicircle kinetoplast DNA (kDNA), selected on the basis of high copy number. LAMP assays were evaluated for CL diagnosis in a prospective cohort trial of 105 patients in southwest Colombia. Lesion swab samples from CL suspects were collected and were tested using the LAMP assay, and the results were compared to those of a composite reference of microscopy and/or culture in order to calculate diagnostic accuracy. LAMP assays were tested on samples (including whole blood, peripheral blood mononuclear cells, and buffy coat) from 50 suspected VL patients from Ethiopia. Diagnostic accuracy was calculated against a reference standard of microscopy of splenic or bone marrow aspirates. To calculate analytical specificity, 100 clinical samples and isolates from fever-causing pathogens, including malaria parasites, arboviruses, and bacteria, were tested. We found that the LAMP assay had a sensitivity of 95% (95% confidence interval [CI], 87.2% to 98.5%) and a specificity of 86% (95% CI, 67.3% to 95.9%) for the diagnosis of CL. With VL suspects, the sensitivity of the LAMP assay was 92% (95% CI, 74.9% to 99.1%) and its specificity was 100% (95% CI, 85.8% to 100%) in whole blood. For CL, the LAMP assay is a sensitive tool for diagnosis and requires less equipment, time, and expertise than alternative CL diagnostics. For VL, the LAMP assay using a minimally invasive sample is more sensitive than the gold standard. Analytical specificity was 100%.