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BACKGROUND: Transfusion-associated circulatory overload (TACO) is a leading cause of transfusion-related morbidity and mortality. TACO follows a two-hit pathophysiology, where comorbidities like cardiac or renal failure act as the first hit followed by blood transfusion as a second hit. Observational studies suggest that plasma transfusion is more likely to cause TACO than other blood products. We conducted a randomized animal study to gather evidence that plasma transfusion can induce TACO. MATERIAL AND METHODS: As a first hit a large myocardial infarction was created in male Wistar rats. Then animals were randomized to receive 4 units of solvent/ detergent-treated pooled plasma (SDP), fresh frozen plasma (FFP), a colloid control (albumin 5%) or a crystalloid fluid control (Ringer’s lactate) (n=10 per group). The primary outcome was the difference between pre- and post-transfusion left-ventricular end diastolic pressure (ΔLVEDP). Secondary outcomes were markers for acute lung injury; lung wet/dry weight ratio, PaO(2)/FiO(2) ratio and pulmonary histological assessment. RESULTS: Pre-transfusion characteristics were similar between groups. ΔLVEDP increased significantly after transfusion with SDP (7.7 mmHg; 4.5–10.5) and albumin (13.0 mmHg; 6.5–15.2), but not after FFP (7.9 mmHg, 1.1; 11.3) compared to infusion with Ringer’s lactate (0.6 mmHg; 0.4–2.2), p=0.007, p=0.0005 and p=0.14 respectively. There were no significant differences in ΔLVEDP between groups receiving SDP, FFP or albumin. There was no increase in acute lung injury in any group compared to other groups. DISCUSSION: Circulatory overload, measured as ΔLVEDP, was induced after transfusion of SDP or albumin, but not after infusion of Ringer’s lactate. These results show that the effect of plasma transfusion on ΔLVEDP differs from fluid overload induced by crystalloid infusion. Colloid osmotic pressure may be an important component in the development of TACO and should be a target for future research.

Background and Objectives: Transfusion-associated circulatory overload (TACO) is a pulmonary transfusion complication and a leading cause of transfusion-related morbidity and mortality. Volume overload and rising hydrostatic pressure as a consequence of transfusion are seen as the central pathway leading to TACO. A possible preventative measure for TACO could be the use of low-volume blood products like volume-reduced lyophilized plasma. We hypothesize that volume-reduced lyophilized plasma decreases circulatory overload leading to a reduced pulmonary capillary pressure and can therefore be an effective strategy to prevent TACO. Materials and Methods: A validated two-hit animal model in rats with heart failure was used. Animals were randomized to receive 4 units of either solvent-detergent pooled plasma (SDP) as control, standard volume lyophilized plasma (LP-S) or hyperoncotic volume-reduced lyophilized plasma (LP-VR). The primary outcome was the difference between pre-transfusion and post-transfusion left ventricular end-diastolic pressure (ΔLVEDP). Secondary outcomes included markers for acute lung injury. Results: LVEDP increased in all randomization groups following transfusion. The greatest elevation was seen in the group receiving LP-VR (+11.9 mmHg [5.9–15.6]), but there were no significant differences when compared to groups receiving either LP-S (+6.3 mmHg [2.9–13.4], p = 0.29) or SDP (+7.7 mmHg [4.5–10.5], p = 0.55). There were no significant differences in markers for acute lung injury, such as pulmonary wet/dry weight ratios, lung histopathology scores or PaO2/FiO2 ratio between the three groups. Conclusion: Transfusion with hyperoncotic volume-reduced plasma did not attenuate circulatory overload compared to standard volume plasma and was therefore not an effective preventative strategy for TACO in this rat model.

The lancet / Digital health 4(1), e18-e26 (2022). doi:10.1016/S2589-7500(21)00211-9, Published by The Lancet, London

Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration ‘hotspot’ regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photo-convertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4thIg-like dimerization domain are not involved, indicating that intracellular signalling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

Hypoxia-inducible factor (HIF)1α is a transcription factor involved in cellular metabolism and regulation of immune cell effector functions. Here, we studied the role of HIF1α in myeloid cells during pneumonia caused by the major causative pathogen, Streptococcus pneumoniae (Spneu). Mice deficient for HIF1α in myeloid cells (LysMcreHif1αfl/fl) were generated to study the in vitro responsiveness of bone marrow-derived macrophages (BMDMs) and alveolar macrophages (AMs) to the Gram-positive bacterial wall component lipoteichoic acid (LTA) and heat-killed Spneu, and the in vivo host response after infection with Spneu via the airways. Both BMDMs and AMs released more lactate upon stimulation with LTA or Spneu, indicative of enhanced glycolysis; HIF1α-deficiency in these cells was associated with diminished lactate release. In BMDMs, HIF1α-deficiency resulted in reduced secretion of tumor necrosis factor (TNF)α and interleukin (IL)-6 upon activation with Spneu but not LTA, while HIF1α-deficient AMs secreted less TNFα and IL-6 in response to LTA, and TNFα after Spneu stimulation. However, no difference was found in the host response of LysMcreHif1αfl/fl mice after Spneu infection as compared to controls. Similar in vivo findings were obtained in neutrophil (Mrp8creHif1αfl/fl) HIF1α-deficient mice. These data suggest that myeloid HIF1α is dispensable for the host defense during pneumococcal pneumonia.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPβ or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan–Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPβ and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPβ as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPβ and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBβ and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression.

Background and Objectives: Transfusion-associated circulatory overload (TACO) is the primary cause of transfusion-related mortality. Speed and volume of transfusion are major risk factors. The aim of this study was to investigate the interaction of red blood cell (RBC) transfusion speed and volume on the development of TACO. Materials and Methods: A validated model for TACO in anaemic Lewis rats with an acute myocardial infarction was used. The effect on pulmonary hydrostatic pressure of one, two or four units of packed RBCs transfused in either 30 or 60 min was evaluated (3.3–26.6 ml·kg −1·hr −1). Pulmonary capillary pressure was measured as left ventricular end-diastolic pressure (LVEDP). Cardiac stress biomarkers atrial natriuretic-peptide (ANP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were measured 1-h post-transfusion. Results: Thirty animals were included (n = 5 per group). Transfusion of RBCs increased LVEDP in a volume-dependent manner (ΔLVEDP [mmHg]: −0.95, +0.50, +6.26, p < 0.001). Fast transfusion increased overall ΔLVEDP by +3.5 mmHg and up to +11.8 mmHg in the four units' group (p = 0.016). Doubling transfusion speed increased ΔLVEDP more than doubling volume in the larger volume groups. No difference in ANP or NT-proBNP were seen in high transfusion volume or groups. Conclusion: Transfusion volume dose-dependently increased LVEDP, with speed of transfusion rapidly elevating LVEDP at higher transfusion volumes. ANP and NT-proBNP were not impacted by transfusion volume or speed in this model. TACO is seen as purely volume overload, however, this study emphasizes that limiting transfusion speed, as a modifiable risk factor, might aid in preventing TACO.

Peritonitis and abdominal sepsis remain major health problems and challenge for clinicians. Bruton's tyrosine kinase (Btk) is a versatile signaling protein involved in the regulation of B cell development and function, as well as innate host defense. In the current study, we aimed to explore the role of Btk in the host response during peritonitis and sepsis in mice induced by a gradually growing pathogenic strain of Escherichia coli bacteria. We found that Btk deficiency ameliorated antibacterial host defense during the late stage of E. coli-induced peritonitis. Btk was not required for cytokine and chemokine release in response to either E. coli or lipopolysaccharide and did not impact organ damage evoked by E. coli. Btk deficiency also did not alter neutrophil influx to the primary site of infection. However, the absence of Btk modestly enhanced phagocytosis of E. coli by neutrophils. These results indicate that Btk-mediated signaling is superfluous for inflammatory responses and remarkably detrimental for antibacterial defense during E. coli-induced peritonitis.

Lipid accumulation is associated with various forms of acute renal injury; however, the causative factors and pathways underpinning this lipid accumulation have not been thoroughly investigated. In this study, we performed lipidomic profiling of renal tissue following ischaemia–reperfusion injury (IRI). We identified a significant accumulation of cholesterol and specific phospholipids and sphingolipids in kidneys 24 h after IRI. In light of these findings, we hypothesised that pathways involved in lipid metabolism may also be altered. Through the analysis of published microarray data, generated from sham and ischaemic kidneys, we identified nephron‐specific metabolic pathways affected by IRI and validated these findings in ischaemic renal tissue. In silico analysis revealed the downregulation of several energy and lipid metabolism pathways, including mitochondrial fatty acid beta‐oxidation (FAO), peroxisomal lipid metabolism, fatty acid (FA) metabolism, and glycolysis. The pentose phosphate pathway (PPP), which is fuelled by glycolysis, was the only metabolic pathway that was upregulated 24 h following IRI. In this study, we describe the effect of renal IRI on metabolic pathways and how this contributes to lipid accumulation. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Rationale: Bronchial thermoplasty (BT) is a bronchoscopic treatment for severe asthma targeting airway smooth muscle (ASM). Observational studies have shown ASM mass reduction after BT, but appropriate control groups are lacking. Furthermore, as treatment response is variable, identifying optimal candidates for BT treatment is important. Objectives: First, to assess the effect of BT on ASM mass, and second, to identify patient characteristics that correlate with BT response. Methods: Patients with severe asthma (n = 40) were randomized to immediate (n = 20) or delayed (n = 20) BT treatment. Before randomization, clinical, functional, blood, and airway biopsy data were collected. In the delayed control group, reassessment, including biopsies, was performed after 6 months of standard clinical care, followed by BT. In both groups, post-BT data including biopsies were obtained after 6 months. ASM mass (% positive desmin or a-smooth muscle actin area in the total biopsy) was calculated with automated digital analysis software. Associations between baseline characteristics and Asthma Control Questionnaire and Asthma Quality of Life Questionnaire (AQLQ) improvement were explored. Measurements and Main Results: Median ASM mass decreased by .50% in the immediate BT group (n = 17) versus no change in the delayed control group (n = 19) (P = 0.0004). In the immediate group, Asthma Control Questionnaire scores improved with 20.79 (interquartile range [IQR], 21.61 to 0.02) compared with 0.09 (IQR, 20.25 to 1.17) in the delayed group (P = 0.006). AQLQ scores improved with 0.83 (IQR, 20.15 to 1.69) versus 20.02 (IQR, 20.77 to 0.75) (P = 0.04). Treatment response in the total group (n = 35) was positively associated with serum IgE and eosinophils but not with baseline ASM mass. Conclusions: ASM mass significantly decreases after BT when compared with a randomized non–BT-treated control group. Treatment response was associated with serum IgE and eosinophil levels but not with ASM mass.

Introduction: Placental site trophoblastic tumor (PSTT) is a rare subtype of gestational trophoblastic disease. Association of PSTT and nephrotic syndrome is exceedingly rare and has been described in 8 cases thus far. In all cases hysterectomy was performed within months after onset of symptoms, leading to immediate remission of nephrotic syndrome, except for one patient who died of complications of PSTT. Case: We describe the history of a woman in which PSTT was discovered years after onset of nephrotic syndrome. Kidney biopsy revealed lupus-like mesangiocapillary nephritis and over time the patient developed additional symptoms mimicking systemic lupus erythematosus (SLE). Discussion: We provide an overview of the literature on this clinical entity and elaborate on its pathophysiology. In addition, we reflect on the phenomenon of anchoring bias, that led physicians to assume the patient had SLE without questioning this diagnosis in the light of the unexplained finding of increased tumor markers.

Over the past 2 decades, scoring systems for multiple glomerular diseases have emerged, as have consortia of pathologists and nephrologists for the study of glomerular diseases, including correlation of pathologic findings with clinical features and outcomes. However, one important limitation faced by members of these consortia and other renal pathologists and nephrologists in both investigative work and routine practice remains a lack of uniformity and precision in clearly defining the morphologic lesions on which the scoring systems are based. In response to this issue, the Renal Pathology Society organized a working group to identify the most frequently identified glomerular lesions observed by light microscopy and electron microscopy, review the literature to capture the published definitions most often used for each, and determine consensus terms and definitions for each lesion in a series of online and in-person meetings. The defined lesions or abnormal findings are not specific for any individual disease or subset of diseases, but rather can be applied across the full spectrum of glomerular diseases and within the context of the different scoring systems used for evaluating and reporting these diseases. In addition to facilitating glomerular disease research, standardized terms and definitions should help harmonize reporting of medical kidney diseases worldwide and lead to more-precise diagnoses and improved patient care.

Diabetic nephropathy (DN) is a major complication of diabetes and is associated with high risk for cardiovascular mortality, which is partially related to elevated platelet activity. Platelets are also active players in inflammation and fibrosis. In this study, we examine the effect of ticagrelor-induced platelet inhibition on the development of DN. DN was induced by unilateral nephrectomy followed by streptozotocin injections for 5 days. Mice received ticagrelor (300 mg/kg) or vehicle every other day, for 16 weeks. Experimental groups: non-diabetic control, diabetic control, non-diabetic ticagrelor, and diabetic ticagrelor. Ticagrelor treatment in diabetic mice lowered urinary albumin excretion, it prevented diabetes-induced mesangial matrix expansion, podocyte effacement, and glomerular endothelial cell injury, which includes loss of endothelial fenestrations, ICAM-1 expression, and PECAM expression. In addition, ticagrelor treatment prevented collagen IV deposition and macrophage infiltration in the tubulointerstitium and these diabetic mice showed lower systemic and tubular inflammation and tubular apoptosis. This tubular protection is likely to be a result of protection to the glomerular endothelium by ticagrelor, which reduces albuminuria and albumin toxicity to the tubules and reduced tubular and interstitial inflammation and fibrosis. In conclusion, ticagrelor-induced platelet inhibition protects against renal injury in diabetic mice, likely by protecting the glomerular endothelial cells.

BackgroundThe microbiome has emerged as an important player in the pathophysiology of a whole spectrum of diseases that affect the critically ill. We hypothesized that differences in microbiota composition across vendors can influence murine models of pulmonary lipopolysaccharide (LPS) inflammation and Gram-negative pneumonia.MethodsA multi-vendor approach was used with genetically similar mice derived from three different vendors (Janvier, Envigo, Charles River). This model was employed to study the effect on the host response to a pulmonary LPS challenge (1 μgKlebsiella pneumoniaeLPS, intranasal), as well as experimentalK. pneumoniaeinfection (ATCC43816,1 × 104CFU, intranasal).ResultsGut microbiota analysis revealed profound intervendor differences in bacterial composition as shown by beta diversity and at various taxonomic levels. Tumor necrosis factor (TNF)-α and interleukin (IL)-6 release in lung and bronchoalveolar lavage fluid (BALF) were determined 6 and 24 h after intranasal treatment with LPS. No differences were found between the groups, with the exception for Envigo, showing a higher level of TNFα in lung and BALF at 6 h compared to Janvier and Charles River. In another set of experiments, mice from different vendors were subjected to a clinically relevant model of Gram-negative pneumonia (K. pneumoniae). At 12 and 36 h post-infection, no intervendor differences were found in bacterial dissemination, or TNFα and IL-6 levels in the lungs. In line, markers for organ failure did not differ between groups.ConclusionsAlthough there was a marked variation in the gut microbiota composition of mice from different vendors, the hypothesized impact on our models of pulmonary inflammation and severe pneumonia was limited. This is of significance for experimental settings, showing that differences in gut microbiota do not have to lead to differences in outcome.

Background Ischaemia-reperfusion (IR) injury is an important determinant of delayed graft function (DGF) affecting allograft function. Mitochondrial DNA (mtDNA) is released upon cell death and platelet activation into the extracellular environment and has been suggested to be a biomarker in several diseases. Whether extracellular mtDNA accumulates in plasma and/or urine upon renal IR and predisposes DGF is unknown. Methods C57BL/6J wild-type mice were subjected to renal IR. In addition, an observational case–control study was set up enrolling 43 patients who underwent kidney transplantation. One day post-IR in mice and a few days following renal transplantation in human, blood and urine were collected. Patients were stratified into DGF and non-DGF groups. Results mtDNA-encoded genes accumulate in urine and plasma in both mice subjected to renal IR injury and in humans following renal transplantation. In human renal transplant recipients, cold ischaemia time and renal function correlate with urinary mtDNA levels. Urinary mtDNA levels but not urinary nuclear DNA levels were significantly higher in the DGF group compared with the non-DGF group. Multiple receiver operating characteristic curves revealed significant diagnostic performance for mtDNA-encoded genes cytochrome c oxidase III (COXIII); nicotinamide adenine dinucleotide hydrogen subunit 1 (NADH-deh); mitochondrially encoded, mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 2 (MT-ND2) with an area under the curve of, respectively, 0.71 [P = 0.03; 95% confidence interval (CI) 0.54–0.89], 0.75 (P = 0.01; 95% CI 0.58–0.91) and 0.74 (P = 0.02; 95% CI 0.58–0.89). Conclusions These data suggest that renal ischaemia time determines the level of mtDNA accumulation in urine, which associates with renal allograft function and the diagnosis of DGF following renal transplantation.

Background Liver kinase B1 (LKB1) has been studied extensively as a tumor suppressor gene (Stk11) in the context of cancer. We hypothesized that myeloid LKB1 plays a role in innate immunity during pneumonia. Methods Mice deficient for LKB1 in myeloid cells (LysM-cre × Stk11fl/fl) or neutrophils (Mrp8-cre × Stk11fl/fl) were infected with Klebsiella pneumoniae via the airways. LysM-cre × Stk11fl/fl mice were also intranasally challenged with lipopolysaccharide (LPS). Results Mice with myeloid LKB1 deficiency, but not those with neutrophil LKB1 deficiency, had increased bacterial loads in lungs 6–40 hours after infection, compared with control mice, pointing to a role for LKB1 in macrophages. Myeloid LKB1 deficiency was associated with reduced cytokine release into the airways on local LPS instillation. The number of classic (SiglecFhighCD11bneg) alveolar macrophages (AMs) was reduced by approximately 50% in the lungs of myeloid LKB1–deficient mice, which was not caused by increased cell death or reduced proliferation. Instead, these mice had AMs with a “nonclassic” (SiglecFlowCD11bpos) phenotype. AMs did not up-regulate glycolysis in response to LPS, irrespective of LKB1 presence. Conclusion Myeloid LKB1 is important for local host defense during Klebsiella pneumonia by maintaining adequate AM numbers in the lung.

Tet methylcytosine dioxygenase 2 (Tet2) mediates demethylation of DNA. We here sought to determine the expression and function of Tet2 in macrophages upon exposure to lipopolysaccharide (LPS), and in the host response to LPS induced lung and peritoneal inflammation, and during Escherichia (E.) coli induced peritonitis. LPS induced Tet2 expression in mouse macrophages and human monocytes in vitro, as well as in human alveolar macrophages after bronchial instillation in vivo. Bone marrow-derived macrophages from myeloid Tet2 deficient (Tet2fl/flLysMCre) mice displayed enhanced production of IL-1β, IL-6 and CXCL1 upon stimulation with several Toll-like receptor agonists; similar results were obtained with LPS stimulated alveolar and peritoneal macrophages. Histone deacetylation was involved in the effect of Tet2 on IL-6 production, whilst methylation at the Il6 promoter was not altered by Tet2 deficiency. Tet2fl/flLysMCre mice showed higher IL-6 and TNF levels in bronchoalveolar and peritoneal lavage fluid after intranasal and intraperitoneal LPS administration, respectively, whilst other inflammatory responses were unaltered. E. coli induced stronger production of IL-1β and IL-6 by Tet2 deficient peritoneal macrophages but not in peritoneal lavage fluid of Tet2fl/flLysMCre mice after in vivo intraperitoneal infection. Tet2fl/flLysMCre mice displayed enhanced bacterial growth during E. coli peritonitis, which was associated with a reduced capacity of Tet2fl/flLysMCre peritoneal macrophages to inhibit the growth of E. coli in vitro. Collectively, these data suggest that Tet2 is involved in the regulation of macrophage functions triggered by LPS and during E. coli infection.

Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disease (LSD) in which sphingomyelin accumulates due to deficient acid sphingomyelinase. In the chronic visceral subtype, organ manifestations are generally limited to the spleen, liver, and lungs. We report a male patient with the chronic visceral subtype who developed proteinuria and renal insufficiency at the age of 49. In renal tissue, foam cells were observed in the glomeruli as well as sphingomyelin accumulation within podocytes, mesangial cells, endothelial cells, and tubular epithelial cells. Although macrophages are the primary storage cells in both ASMD and Gaucher disease, comparison to the histopathological findings in Gaucher and Fabry disease revealed a diffuse storage pattern in multiple renal cell types, closer resembling the pattern found in Fabry disease.

Background Hemorrhagic shock is associated with acute kidney injury and increased mortality. Targeting the endothelial angiopoietin/Tie2 system, which regulates endothelial permeability, previously reduced hemorrhagic shock-induced vascular leakage. We hypothesized that as a consequence of vascular leakage, renal perfusion and function is impaired and that activating Tie2 restores renal perfusion and function. Methods Rats underwent 1 h of hemorrhagic shock and were treated with either vasculotide or PBS as control, followed by fluid resuscitation for 4 h. Microcirculatory perfusion was measured in the renal cortex and cremaster muscle using contrast echography and intravital microscopy, respectively. Changes in the angiopoietin/Tie2 system and renal injury markers were measured in plasma and on protein and mRNA level in renal tissue. Renal edema formation was determined by wet/dry weight ratios and renal structure by histological analysis. Results Hemorrhagic shock significantly decreased renal perfusion (240 ± 138 to 51 ± 40, p p p = 0.08, cremaster perfusion 7 ± 2 perfused vessels, p p p p = 0.05), of which angiopoietin-2 elevation was associated with renal edema formation (r = 0.81, p p p p p > 0.9 at all time points) or reduce renal injury (NGAL p = 0.26, KIM-1 p = 0.78, creatinine p > 0.9, renal edema p = 0.08), but temporarily improved cremaster perfusion at 3 h following start of fluid resuscitation compared to untreated rats (resuscitation + 3 h: 11 ± 3 vs 8 ± 3 perfused vessels, p Conclusion Hemorrhagic shock-induced renal impairment cannot be restored by standard fluid resuscitation, nor by activation of Tie2. Future treatment strategies should focus on reducing angiopoietin-2 levels or on activating Tie2 via an alternative strategy.

Bruton’s tyrosine kinase (Btk) is a cytoplasmic kinase expressed in B cells and myeloid cells. It is essential for B cell development and natural antibody-mediated host defense against bacteria in humans and mice, but little is known about the role of Btk in innate host defensein vivo. Previous studies have indicated that lack of (natural) antibodies is paramount for impaired host defense againstStreptococcus (S.) pneumoniaein patients and mice with a deficiency in functional Btk. In the present study, we re-examined the role of Btk in B cells and myeloid cells during pneumococcal pneumonia and sepsis in mice. The antibacterial defense of Btk-/-mice was severely impaired during pneumococcal pneumosepsis and restoration of natural antibody production in Btk-/-mice by transgenic expression of Btk specifically in B cells did not suffice to protect against infection. Btk-/-mice with reinforced Btk expression in MhcII+cells, including B cells, dendritic cells and macrophages, showed improved antibacterial defense as compared to Btk-/-mice. Bacterial outgrowth in Lysmcre-Btkfl/Y mice was unaltered despite a reduced capacity of Btk-deficient alveolar macrophages to respond to pneumococci. Mrp8cre-Btkfl/Y mice with a neutrophil specific paucity in Btk expression, however, demonstrated impaired antibacterial defense. Neutrophils of Mrp8cre-Btkfl/Y mice displayed reduced release of granule content after pulmonary installation of lipoteichoic acid, a gram-positive bacterial cell wall component relevant for pneumococci. Moreover, Btk deficient neutrophils showed impaired degranulation and phagocytosis upon incubation with pneumococciex vivo. Taken together, the results of our study indicate that besides regulating B cell-mediated immunity, Btk is critical for regulation of myeloid cell-mediated, and particularly neutrophil-mediated, innate host defense againstS. pneumoniae in vivo.

BACKGROUND: With current immunosuppressive regimens, BK polyomavirus-associated nephropathy (BKPyVAN) is still a matter of concern. Stratification of patients at risk for allograft loss is of uttermost importance to guide treatment choice and assess prognosis. In 2018, the Banff working group proposed a classification scheme for the prognosis of BKPyVAN, but external application on independent cohorts is yet to be performed. We investigated how the 2018 Banff classification would perform in a multicenter cohort comprising 50 cases of biopsy-proven BKPyVAN compared to previously published classification systems. METHODS: We analyzed consecutive BKPyVAN cases from two Dutch university hospitals between 2002 and 2013, retrieved clinical data, and scored all biopsies according to the Banff 2018 classification, and as a comparison, 4 previously proposed BKPyVAN classification systems. We used estimated glomerular filtration rate trajectories and death-censored graft survival as primary endpoints. RESULTS: The 2018 Banff classification did not associate with estimated glomerular filtration rate decline or graft failure and performed only slightly better than the 4 previously proposed classifiers. Anti-human leukocyte antigen donor-specific antibodies (DSAs), especially in combination with ongoing biopsy-proven BKPyVAN on follow-up, did correlate with graft function and survival. Patients who were DSA+/BKPyVAN+ on follow-up had more inflammation at the baseline biopsy, which by itself was not associated with graft outcomes. CONCLUSIONS: Neither the 2018 Banff BKPyVAN classification nor previously published stratification systems could be applied to our multicenter patient cohort. Our data suggest that there might be a prognostic value for follow-up biopsies and DSA measurements to improve risk stratification after BKPyVAN, although prospective multicenter efforts with protocol measurements are needed to confirm this.

Background Platelet dysfunction importantly contributes to trauma-induced coagulopathy (TIC). Our aim was to examine the impact of transfusing platelets (PLTs) in a 2:1 PLT-to-red blood cell (RBC) ratio versus the standard 1:1 ratio on transfusion requirements, correction of TIC, and organ damage in a rat multiple trauma transfusion model. Methods Mechanically ventilated male Sprague Dawley rats were traumatized by crush injury to the small intestine and liver and a fracture of the femur, followed by exsanguination until a mean arterial pressure (MAP) of 40 mmHg. Animals were randomly assigned to receive resuscitation in a high PLT dose (PLT to plasma to RBC in a ratio of 2:1:1) or a standard PLT dose (ratio of 1:1:1) until a MAP of 60 mmHg was reached (n = 8 per group). Blood samples were taken for biochemical and thromboelastometry (ROTEM) assessment. Organs were harvested for histopathology.Outcome measures were transfusion requirements needed to reach a pretargeted MAP, as well as ROTEM correction and organ failure. Results Trauma resulted in coagulopathy as assessed by deranged ROTEM results. Mortality rate was 19%, with all deaths occurring in the standard dose group. The severity of hypovolemic shock as assessed by lactate and base excess was not different in both groups. The volume of transfusion needed to reach the MAP target was lower in the high PLT dose group compared to the standard dose, albeit not statistically significant (p = 0.054). Transfusion with a high PLT dose resulted in significant stronger clot firmness compared to the standard dose at all time points following trauma, while platelet counts were similar. Organ failure as assessed by biochemical analysis and histopathology was not different between groups, nor were there any thromboembolic events recorded. Conclusions Resuscitation with a high (2:1) PLT-to-RBC ratio was more effective compared to standard (1:1) PLT-to-RBC ratio in treating TIC, with a trend towards reduced transfusion volumes. Also, high PLT dose did not aggravate organ damage. Transfusion strategies using higher PLT dose regiments might be a feasible treatment option in hemorrhaging trauma patients for the correction of TIC.

Background Complement factor C5 can either aggravate or attenuate the T-helper type 2 (TH2) immune response and airway hyperresponsiveness (AHR) in murine models of allergic asthma. The effect of C5 during the effector phase of allergen-induced asthma is ill-defined. Objectives We aimed to determine the effect of C5 blockade during the effector phase on the pulmonary TH2 response and AHR in a house dust mite (HDM) driven murine asthma model. Methods BALB/c mice were sensitized and challenged repeatedly with HDM via the airways to induce allergic lung inflammation. Sensitized mice received twice weekly injections with a blocking anti-C5 or control antibody 24 h before the first challenge. Results HDM challenge in sensitized mice resulted in elevated C5a levels in bronchoalveolar lavage fluid. Anti-C5 administered to sensitized mice prior to the first HDM challenge prevented this rise in C5a, but did not influence the influx of eosinophils or neutrophils. While anti-C5 did not impact the recruitment of CD4 T cells upon HDM challenge, it reduced the proportion of TH2 cells recruited to the airways, attenuated IL-4 release by regional lymph nodes restimulated with HDM ex vivo and mitigated the plasma IgE response. Anti-C5 did not affect innate lymphoid cell (ILC) proliferation or group 2 ILC (ILC2) differentiation. Anti-C5 attenuated HDM induced AHR in the absence of an effect on lung histopathology, mucus production or vascular leak. Conclusions Generation of C5a during the effector phase of HDM induced allergic lung inflammation contributes to TH2 cell differentiation and AHR without impacting ILC2 cells. Electronic supplementary material The online version of this article (10.1186/s12931-019-1136-5) contains supplementary material, which is available to authorized users.

Humans with dysfunctional Bruton's tyrosine kinase (Btk) are highly susceptible to bacterial infections. Compelling evidence indicates that Btk is essential for B cell-mediated immunity, whereas its role in myeloid cell-mediated immunity against infections is controversial. In this study, we determined the contribution of Btk in B cells and neutrophils to host defense against the extracellular bacterial pathogen Klebsiella pneumoniae, a common cause of pulmonary infections and sepsis. Btk-/- mice were highly susceptible to Klebsiella infection, which was not reversed by Btk re-expression in B cells and restoration of natural antibody levels. Neutrophil-specific Btk deficiency impaired host defense against Klebsiella to a similar extent as complete Btk deficiency. Neutrophil-specific Btk deficiency abolished extracellular reactive oxygen species production in response to Klebsiella. These data indicate that expression of Btk in neutrophils is crucial, while in B cells, it is dispensable for in vivo host defense against K. pneumoniae.

CCAAT/enhancer-binding protein delta (C/EBPδ) is a transcription factor involved in apoptosis and proliferation, which is downregulated in pancreatic ductal adenocarcinoma (PDAC) cells. Loss of nuclear C/EBPδ in PDAC cells is associated with decreased patient survival and pro-tumorigenic properties in vitro. Interestingly however, next to C/EBPδ expression in tumor cells, C/EBPδ is also expressed by cells constituting the tumor microenvironment and by cells comprising the organs and parenchyma. However, the functional relevance of systemic C/EBPδ in carcinogenesis remains elusive. Here, we consequently assessed the potential importance of C/EBPδ in somatic tissues by utilizing an orthotopic pancreatic cancer model. In doing so, we show that genetic ablation of C/EBPδ does not significantly affect primary tumor growth but has a strong impact on metastases, wildtype mice developed metastases at multiple sites, whilst this was not the case in C/EBPδ-/- mice. In line with reduced metastasis formation in C/EBPδ-/- mice, C/EBPδ-deficiency also limited tumor cell dissemination in a specific extravasation model. Tumor cell extravasation was dependent on the platelet-activating factor receptor (PAFR) as a PAFR antagonist inhibited tumor cell extravasation in wildtype mice but not in C/EBPδ-/- mice. Overall, we show that systemic C/EBPδ facilitates pancreatic cancer metastasis, and we suggest this is due to C/EBPδ-PAFR-dependent tumor cell extravasation.

Background and Aims Thrombotic microangiopathies (TMAs) manifest themselves in arteries, arterioles and glomeruli. Nephropathologists need to differentiate TMAs from mimickers like hypertensive nephropathy and vasculitis which can be problematic due to interobserver disagreement and poorly defined diagnostic criteria over a wide spectrum of morphological changes with partial overlap. As a first step towards a machine learning analysis of TMAs, we developed a computer vision model for segmenting arteries, arterioles and glomeruli in TMA and mimickers. Method We manually segmented n=939 arteries, n=6,023 arterioles, n=4,507 glomeruli on whole slide images (WSIs) of 34 renal biopsies and their HE, PAS, trichrome and Jones sections (19 TMA, 11 hypertensive nephropathy, 4 vasculitis with preglomerular involvement). As a segmentation model we used DeepLab V3, pretrained on 61,734 segmented glomeruli from 768 WSIs. 58 randomly chosen WSIs served as the intrainstitutional holdout testing set after training of the model on the remaining slides. Automatic segmentation accuracies were reported as Cohen’s kappa, intersection over union (IoU) and Matthews correlation coefficient (MCC) against the nephropathologist’s segmentation as ground truth. Results Over all classes (artery, arteriole, glomerulus) Cohen’s kappa was 0.86. IoU was 0.716 for artery, 0.491 for arteriole and 0.829 for glomerulus. MCC was 0.837 for artery, 0.664 for arteriole and 0.907 for glomerulus. Conclusion We achieved good automatic segmentation of arteries, arterioles and glomeruli, even with severe pathological distortion on routine histopathological slides. We will further improve this segmentation technology in order to enable the bulk analysis of these descisive tissue compartments in large clinicopathological repositories of native kidney biopsies with TMA using supervised and unsupervised machine learning algorithms.

From rat studies, human case reports and cohort studies, bisphosphonates seem to impair renal function. However, when critically reviewing the literature, zoledronate and pamidronate are more frequently involved in renal deterioration than other bisphosphonates. When bisphosphonate nephropathy occurs, zoledronate more frequently induces tubular toxicity whereas pamidronate typically induces focal segmental glomerulosclerosis. Thus, although bisphosphonates are highly effective in preventing complications for patients with osseous metastases and are highly effective in preventing fractures for patients with osteoporosis, renal function should be monitored closely after initiation of these drugs.

Tenascin C (TNC) is an extracellular matrix protein with immunomodulatory properties that plays a major role during tissue injury and repair. TNC levels are increased in patients with pneumonia and pneumosepsis, and they are associated with worse outcomes. Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium that is a major causative pathogen in nosocomial pneumonia and a rising cause of community-acquired pneumonia. To study the role of TNC during MRSA-induced pneumonia, TNC sufficient (TNC+/+) and TNC-deficient (TNC-/-) mice were infected with MRSA via the airways and euthanized after 6, 24, and 48 h for analysis. Pulmonary transcription of TNC peaked at 6 h, while immunohistochemistry revealed higher protein levels at later time points. Although TNC deficiency was not associated with changes in bacterial clearance, TNC-/- mice showed increased levels of TNF-α and IL-6 in bronchoalveolar lavage fluid during the acute phase of infection when compared with TNC+/+ mice. In addition, TNC-/- mice showed more severe pulmonary pathology at 6, but not at 24 or 48 h, after infection. Together, these data suggest that TNC plays a moderate protective role against tissue pathology during the acute inflammatory phase, but not during the bacterial clearance phase, of MRSA-induced pneumonia. These results argue against an important role of TNC on disease outcome during MRSA-induced pneumonia. IMPORTANCE Recently, the immunomodulatory properties of TNC have drawn substantial interest. However, to date most studies made use of sterile models of inflammation. In this study, we examine the pathobiology of MRSA-induced pneumonia in a model of TNC-sufficient and TNC-deficient mice. We have studied the immune response and tissue pathology both during the initial insult and also during the resolution phase. We demonstrate that MRSA-induced pneumonia upregulates pulmonary TNC expression at the mRNA and protein levels. However, the immunomodulatory role of TNC during bacterial pneumonia is distinct from models of sterile inflammation, indicating that the function of TNC is context dependent. Contrary to previous descriptions of TNC as a proinflammatory mediator, TNC-deficient mice seem to suffer from enhanced tissue pathology during the acute phase of infection. Nonetheless, besides its role during the acute phase response, TNC does not seem to play a major role in disease outcome during MRSA-induced pneumonia., peer-reviewed