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

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.

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.

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.

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.

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

Hypoxia-inducible factor- (HIF-) 1α has been implicated in the ability of cells to adapt to alterations in oxygen levels. Bacterial stimuli can induce HIF1α in immune cells, including those of myeloid origin. We here determined the role of myeloid cell HIF1α in the host response during pneumonia and sepsis caused by the common human pathogen Klebsiella pneumoniae. To this end, we generated mice deficient for HIF1α in myeloid cells (LysM-cre × Hif1αfl/fl) or neutrophils (Mrp8-cre × Hif1αfl/fl) and infected these with Klebsiella pneumoniae via the airways. Myeloid, but not neutrophil, HIF1α-deficient mice had increased bacterial loads in the lungs and distant organs after infection as compared to control mice, pointing at a role for HIF1α in macrophages. Myeloid HIF1α-deficient mice did not show increased bacterial growth after intravenous infection, suggesting that their phenotype during pneumonia was mediated by lung macrophages. Alveolar and lung interstitial macrophages from LysM-cre × Hif1αfl/fl mice produced lower amounts of the immune enhancing cytokine tumor necrosis factor upon stimulation with Klebsiella, while their capacity to phagocytose or to produce reactive oxygen species was unaltered. Alveolar macrophages did not upregulate glycolysis in response to lipopolysaccharide, irrespective of HIF1α presence. These data suggest a role for HIF1α expressed in lung macrophages in protective innate immunity during pneumonia caused by a common bacterial pathogen.

The Banff antibody mediated rejection (ABMR) classification is vulnerable to misinterpretation, but the reasons are unclear. To better understand this vulnerability, we evaluated how ABMR is diagnosed in practice. To do this, the Banff Antibody‐Mediated Injury Workgroup electronically surveyed an international cohort of nephrologists/surgeons (n=133) and renal pathologists (n=99). Most providers (97%) responded that they use the Banff ABMR classification at least sometimes, but DSA information is often not readily available. Only 41.1%(55/133) of nephrologists/surgeons and 19.2%(19/99) of pathologists reported that they always have DSA results when the biopsy is available. Additionally, only 19.6%(26/133) of nephrologists/surgeons responded that non‐HLA antibody or molecular transcripts are obtained when ABMR histologic features are present but DSA is undetected. Several respondents agreed that histologic features concerning for ABMR in the absence of DSA and/or C4d are not well accounted for in the current classification [31.3% (31/99) pathologists and 37.6%(50/133) nephrologist/surgeons]. The Banff ABMR classification appears widely accepted, but efforts to improve the accessibility of DSA information for the multidisciplinary care team are needed. Further clarity is also needed in Banff ABMR nomenclature to account for the spectrum of ABMR and for histologic features suspicious for ABMR when DSA is absent. Refereed/Peer-reviewed

BackgroundDiabetic nephropathy (DN) is a major complication of diabetes and the main cause of end-stage renal disease. Extracellular vesicles (EVs) are small cell-derived vesicles that can alter disease progression by microRNA (miRNA) transfer.MethodsIn this study, we aimed to characterize the cellular origin and miRNA content of EVs in plasma samples of type 2 diabetes patients at various stages of DN. Type 2 diabetes patients were classified in three groups: normoalbuminuria, microalbuminuria and macroalbuminuria. The concentration and cellular origin of plasma EVs were measured by flow cytometry. A total of 752 EV miRNAs were profiled in 18 subjects and differentially expressed miRNAs were validated.ResultsDiabetic patients with microalbuminuria and/or macroalbuminuria showed elevated concentrations of total EVs and EVs from endothelial cells, platelets, leucocytes and erythrocytes compared with diabetic controls. miR-99a-5p was upregulated in macroalbuminuric patients compared with normoalbuminuric and microalbuminuric patients. Transfection of miR-99a-5p in cultured human podocytes downregulated mammalian target of rapamycin (mTOR) protein expression and downregulated the podocyte injury marker vimentin.ConclusionsType 2 diabetes patients with microalbuminuria and macroalbuminuria display differential EV profiles. miR-99a-5p expression is elevated in EVs from macroalbuminuria and mTOR is its validated mRNA target.

Background In systemic amyloidosis, the kidney is frequently affected and renal involvement has a major impact on survival. Renal involvement is clinically characterized by decreased estimated glomerular filtration rate (eGFR) and proteinuria. The two most common renal amyloidosis types are light chain-related amyloidosis (AL) and serum amyloid A (AA) amyloidosis. Standardized histopathological scoring of amyloid deposits is crucial to assess disease progression. Therefore, we aimed to validate the proposed scoring system from Rubinstein et al. (Novel pathologic scoring tools predict end-stage kidney disease in light chain (AL) amyloidosis. Amyloid 2017; 24: 205–211) in an independent patient cohort. Methods We attempt to reproduce the scoring system, consisting of an amyloid score (AS) and a composite scarring injury score (CSIS), in a multicentre AL and AA case series. Additionally, we analysed all renal amyloidosis kidney biopsies performed in the Netherlands between 1993 and 2012. Results Similar to the original study, AS and CSIS correlated to eGFR (r = −0.45, P = 0.0061 and r = −0.60, P Conclusions In our AL case series and the original study, AS and CSIS were correlated to eGFR but not to proteinuria, and AS correlated with renal outcome. Overall, we regard this scoring system as competent for standardized histopathological assessment of amyloid deposits burden and thereby disease advancement in renal biopsies.

Introduction In hemorrhaging trauma patients, the endothelium is activated, resulting in excessive endothelial synthesis of von Willebrand Factor (vWF), which may enhance micro-thrombi formation, resulting in obstruction of the microcirculation and endothelial injury, aggravating bleeding, as well as contributing to organ failure. Under normal conditions, vWF is cleaved by the metalloprotease ADAMTS-13. After trauma, ADAMTS-13 levels are reduced. Objectives To assess whether recombinant human ADAMTS-13 inhibits endothelial injury and organ failure in a rat trauma-transfusion model. Methods Blood products were prepared from syngeneic rat blood according to blood bank standards. Polytrauma was induced in rats by crush injury to the intestines and liver and by fracture of the femur. The rats were hemorrhaged until a mean arterial pressure (MAP) of 40 mmHg was reached. Rats were randomized to receive transfusion of RBCs, FFPs, and platelets in a 1:1:1 ratio to achieve a MAP of 70 mmHg, with or without the addition of ADAMTS-13 (50 μg/kg). Blood samples were assessed for biochemistry and rotational thromboelastometry (ROTEM). Syndecan-1 and VE-cadherin levels were measured as a reflection of endothelial integrity. The amount of leakage of dextran-FITC from the vascular system to the parenchyma in lungs was quantified. To assess inflammation, IL-6 and IL-8 levels were determined. Organ damage was assessed by histopathology. Results All rats were severely shocked, with no significant differences in shock parameters between groups. Rats treated with ADAMTS-13 showed signs of a more effective shock reversal (higher blood pressure, lower lactate levels) compared to controls. Also, ROTEM parameters of clot formation in rats receiving ADAMTS-13 improved compared to controls, which was mainly platelet-dependent. Syndecan-1 levels relative to baseline trended to be lower in ADAMTS-13 treated rats compared to controls (107 vs 149%, p = 0.08). ADAMTS-13 reduced albuminuria (1.7 vs 4.4 g/L, p < 0.01) and organ-specific inflammation (pulmonary IL-6 243 vs 369 pg/mL, p = 0.08; splenic IL-6 253 vs 307, p = 0.03) compared to controls, but did not improve histopathological scores. Conclusions The use of ADAMTS-13 in a rat trauma-transfusion model improves parameters of shock, platelet-driven coagulation, endothelial damage, and organ inflammation. These results suggest that ADAMTS-13 is important in mediating outcome of trauma. Whether ADAMTS-13 can be used as a therapeutic adjunct to treat bleeding trauma patients remains to be determined.

Summary: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets multiple organs and causes severe coagulopathy. Histopathological organ changes might not only be attributable to a direct virus-induced effect, but also the immune response. The aims of this study were to assess the duration of viral presence, identify the extent of inflammatory response, and investigate the underlying cause of coagulopathy. Methods: This prospective autopsy cohort study was done at Amsterdam University Medical Centers (UMC), the Netherlands. With informed consent from relatives, full body autopsy was done on 21 patients with COVID-19 for whom autopsy was requested between March 9 and May 18, 2020. In addition to histopathological evaluation of organ damage, the presence of SARS-CoV-2 nucleocapsid protein and the composition of the immune infiltrate and thrombi were assessed, and all were linked to disease course. Findings: Our cohort (n=21) included 16 (76%) men, and median age was 68 years (range 41–78). Median disease course (time from onset of symptoms to death) was 22 days (range 5–44 days). In 11 patients tested for SARS-CoV-2 tropism, SARS-CoV-2 infected cells were present in multiple organs, most abundantly in the lungs, but presence in the lungs became sporadic with increased disease course. Other SARS-CoV-2-positive organs included the upper respiratory tract, heart, kidneys, and gastrointestinal tract. In histological analyses of organs (sampled from nine to 21 patients per organ), an extensive inflammatory response was present in the lungs, heart, liver, kidneys, and brain. In the brain, extensive inflammation was seen in the olfactory bulbs and medulla oblongata. Thrombi and neutrophilic plugs were present in the lungs, heart, kidneys, liver, spleen, and brain and were most frequently observed late in the disease course (15 patients with thrombi, median disease course 22 days [5–44]; ten patients with neutrophilic plugs, 21 days [5–44]). Neutrophilic plugs were observed in two forms: solely composed of neutrophils with neutrophil extracellular traps (NETs), or as aggregates of NETs and platelets.. Interpretation: In patients with lethal COVID-19, an extensive systemic inflammatory response was present, with a continued presence of neutrophils and NETs. However, SARS-CoV-2-infected cells were only sporadically present at late stages of COVID-19. This suggests a maladaptive immune response and substantiates the evidence for immunomodulation as a target in the treatment of severe COVID-19. Funding: Amsterdam UMC Corona Research Fund.

CCAAT/enhancer-binding protein &delta, (C/EBP&delta, ) is a transcription factor involved in growth arrest and differentiation, which has consequently been suggested to harbor tumor suppressive activities. However, C/EBP&delta, over-expression correlates with poor prognosis in glioblastoma and promotes genomic instability in cervical cancer, hinting at an oncogenic role of C/EBP&delta, in these contexts. Here, we explore the role of C/EBP&delta, in pancreatic cancer. We determined C/EBP&delta, expression in biopsies from pancreatic cancer patients using public gene-expression datasets and in-house tissue microarrays. We found that C/EBP&delta, is highly expressed in healthy pancreatic ductal cells but lost in pancreatic ductal adenocarcinoma. Furthermore, loss of C/EBP&delta, correlated with increased lymph node involvement and shorter overall survival in pancreatic ductal adenocarcinoma patients. In accordance with this, in vitro experiments showed reduced clonogenic capacity and proliferation of pancreatic ductal adenocarcinoma cells following C/EBP&delta, re-expression, concurrent with decreased sphere formation capacity in soft agar assays. We thus report a previously unrecognized but important tumor suppressor role of C/EBP&delta, in pancreatic ductal adenocarcinoma. This is of particular interest since only few tumor suppressors have been identified in the context of pancreatic cancer. Moreover, our findings suggest that restoration of C/EBP&delta, activity could hold therapeutic value in pancreatic ductal adenocarcinoma, although the latter claim needs to be substantiated in future studies.

Human hematopoiesis is critically dependent on the transcription factor GATA2. Patients with GATA2 deficiency typically present with myelodysplastic syndrome, reduced numbers of monocytes, NK cells and B cells, and/or opportunistic infections. Here, we present two families that harbor distinct GATA2 mutations with highly variable onset and course of disease. We discuss the use of allogeneic hematopoietic cell transplantation in these patients, especially as treatment for pulmonary alveolar proteinosis.

Background Septic patients are often anemic, requiring red blood cell (RBC) transfusions. However, RBC transfusions are associated with organ injury. The mechanisms of RBC-induced organ injury are unknown, but increased clearance of donor RBCs from the circulation with trapping in the organs could play a role. We hypothesized that washing of RBCs prior to transfusion may reduce clearance and trapping of donor cells and thereby reduce organ injury. Methods Sprague-Dawley rats were inoculated intratracheally with 10 colony-forming units (CFU) of Streptococcus pneumoniae or vehicle as a control and transfused with either a washed or standard (non-washed) biotinylated RBC transfusion from syngeneic rats. Controls received saline. Blood samples were taken directly after transfusion and at 24 h to calculate the 24 h post transfusion recovery (PTR). After sacrifice, flow cytometry was used to detect donor RBCs in organs and blood. The organs were histologically scored by a pathologist and CFUs in the lung and blood were counted. Results The 24h-PTR was similar between healthy and pneumoseptic rats after a standard transfusion. In healthy rats, a washed transfusion resulted in a higher PTR and less accumulation of donor RBCs in the organs compared with a standard transfusion. However, during pneumonia, this effect of washing was not seen. Transfusion did not further augment lung injury induced by pneumonia, but washing decreased bacterial outgrowth in the lungs associated with reduced lung injury. Conclusion In healthy recipients, washing increased 24h-PTR of donor RBCs and decreased trapping in organs. In pneumoseptic rats, washing reduced bacterial outgrowth and lung injury, but did not improve PTR.

Thrombocytopenia predicts mortality in melioidosis patients and during experimental melioidosis, platelets play a protective role in both innate immunity and vascular integrity., Background Infection with the gram-negative bacillus Burkholderia pseudomallei (melioidosis) is an important cause of pneumosepsis in Southeast Asia and has a mortality of up to 40%. We aimed to assess the role of platelets in the host response against B. pseudomallei infection. Methods Association between platelet counts and mortality was determined in 1160 patients with culture-proven melioidosis. Mice treated with (low- or high-dose) platelet-depleting antibody were inoculated intranasally with B. pseudomallei and killed. Additional studies using functional glycoprotein Ibα–deficient mice were conducted. Results Thrombocytopenia was present in 31% of patients at admission and predicted mortality in melioidosis patients even after adjustment for confounders. In our murine-melioidosis model, platelet counts decreased, and mice treated with a platelet-depleting antibody showed enhanced mortality and higher bacterial loads compared to mice with normal platelet counts. Low platelet counts had a modest impact on early-pulmonary neutrophil influx. Reminiscent of their role in hemostasis, platelet depletion impaired vascular integrity, resulting in early lung bleeding. Glycoprotein Ibα–deficient mice had reduced platelet counts during B. pseudomallei infection together with an impaired local host defense in the lung. Conclusions Thrombocytopenia predicts mortality in melioidosis patients and, during experimental melioidosis, platelets play a protective role in both innate immunity and vascular integrity.

Acute kidney injury (AKI), a major public health problem associated with high mortality and increased risk of progression towards end-stage renal disease, is characterized by the activation of intra-renal haemostatic and inflammatory processes. Platelets, which are present in high numbers in the circulation and can rapidly release a broad spectrum of bioactive mediators, are important acute modulators of inflammation and haemostasis, as they are the first cells to arrive at sites of acute injury, where they interact with endothelial cells and leukocytes. Diminished control of platelet reactivity by endothelial cells and/or an increased release of platelet-activating mediators can lead to uncontrolled platelet activation in AKI. As increased platelet sequestration and increased expression levels of the markers P-selectin, thromboxane A2, CC-chemokine ligand 5 and platelet factor 4 on platelets have been reported in kidneys following AKI, platelet activation likely plays a part in AKI pathology. Results from animal models and some clinical studies highlight the potential of antiplatelet therapies in the preservation of renal function in the context of AKI, but as current strategies also affect other cell types and non-platelet-derived mediators, additional studies are required to further elucidate the extent of platelet contribution to the pathology of AKI and to determine the best therapeutic approach by which to specifically target related pathogenic pathways.

High-molecular-weight kininogen (HK), together with factor XI, factor XII and prekallikrein, is part of the contact system that has proinflammatory, prothrombotic, and vasoactive properties. We hypothesized that HK plays a role in the host response during pneumonia-derived sepsis. To this end mice were depleted of kininogen (KNG) to plasma HK levels of 28% of normal by repeated treatment with a specific antisense oligonucleotide (KNG ASO) for 3 wk before infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways. Whereas plasma HK levels increased during infection in mice treated with a scrambled control ASO (Ctrl ASO), HK level in the KNG ASO-treated group remained reduced to 25–30% of that in the corresponding Ctrl ASO group both before and after infection. KNG depletion did not influence bacterial growth in lungs or dissemination to distant body sites. KNG depletion was associated with lower lung CXC chemokine and myeloperoxidase levels but did not impact neutrophil influx, lung pathology, activation of the vascular endothelium, activation of the coagulation system, or the extent of distant organ injury. These results were corroborated by studies in mice with a genetic deficiency of KNG, which were indistinguishable from wild-type mice during Klebsiella-induced sepsis. Both KNG depletion and KNG deficiency were associated with strongly reduced plasma prekallikrein levels, indicating the carrier function of HK for this zymogen. This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia-derived sepsis.

Systemic inflammatory response syndrome (SIRS) is characterized by the activation of the innate immune system resulting in stimulation of inflammatory responses, coagulation, and platelet activation that may contribute to complication such as the development of acute kidney injury (AKI). AKI importantly worsens the outcome of SIRS, implying the existence of a detrimental cross talk via systemic messages. Mitochondria are a source of damage-associated molecular patterns (DAMPs) and are thought to form a molecular link between tissue injury and stimulation of innate immunity. The role of mitochondrial DNA (mtDNA) in the cross talk between the onset of SIRS and subsequent development of AKI is unknown. Hence, we performed a case control study in critically ill patients with SIRS diagnosed with or without AKI, in which we determined mtDNA levels in plasma and urine, and correlated these to markers of renal impairment, inflammation, coagulation, and platelet activation. In addition, we exposed mice, primary renal tubular epithelial cells (TECs), and platelets to mtDNA or purified mitochondrial ligands, and measured their response to elucidate underlying pathophysiological mechanisms. Our data reveal that increased systemic mtDNA levels in SIRS patients do not correlate with systemic inflammation and renal disease activity. Moreover, AKI does not have an additional effect on circulating mtDNA levels. In contrast, we found that urinary mtDNA levels correlate with an elevated albumin creatinine ratio (ACR) as well as with increased urinary markers of inflammation, coagulation, and platelet activation. Both renal TECs and platelets respond to mtDNA and mtDNA ligands, leading to increased expression of, respectively, inflammatory cytokines and P-selectin. Moreover, activation of platelets results in mtDNA release. Together, these data suggest that circulating mtDNA is probably not important in the detrimental cross talk between SIRS and AKI, whereas renal mtDNA accumulation may be related to intrarenal inflammation, coagulation processes, and renal dysfunction in the pathophysiology of SIRS.

Lupus nephritis (LN) often results in progressive renal dysfunction. The inactive rhomboid 2 (iRhom2) is a newly identified key regulator of A disintegrin and metalloprotease 17 (ADAM17), whose substrates, such as TNF-α and heparin-binding EGF (HB-EGF), have been implicated in the pathogenesis of chronic kidney diseases. Here, we demonstrate that deficiency of iRhom2 protects the lupus-prone Fcgr2b-/- mice from developing severe kidney damage without altering anti-double-stranded DNA (anti-dsDNA) Ab production by simultaneously blocking HB-EGF/EGFR and TNF-α signaling in the kidney tissues. Unbiased transcriptome profiling of kidneys and kidney macrophages revealed that TNF-α and HB-EGF/EGFR signaling pathways are highly upregulated in Fcgr2b-/- mice, alterations that were diminished in the absence of iRhom2. Pharmacological blockade of either TNF-α or EGFR signaling protected Fcgr2b-/- mice from severe renal damage. Finally, kidneys from LN patients showed increased iRhom2 and HB-EGF expression, with interstitial HB-EGF expression significantly associated with chronicity indices. Our data suggest that activation of iRhom2/ADAM17-dependent TNF-α and EGFR signaling plays a crucial role in mediating irreversible kidney damage in LN, thereby uncovering a target for selective and simultaneous dual inhibition of 2 major pathological pathways in the effector arm of the disease.

Platelet collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal via an immunoreceptor tyrosine-based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI-/- mice with the common human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI-/- mice showed increased bacterial growth in lungs, and GPVI-/- mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet-leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet-leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of Klebsiella GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI-/- and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.

Background It is now well established that there are two types of appendicitis: simple (nonperforating) and complex (perforating). This study evaluates differences in the composition of the immune cellular infiltrate in children with simple and complex appendicitis. Materials and methods A total of 47 consecutive children undergoing appendectomy for acute appendicitis between January 2011 and December 2012 were included. Intraoperative criteria were used to identify patients with either simple or complex appendicitis and were confirmed histopathologically. Immune histochemical techniques were used to identify immune cell markers in the appendiceal specimens. Digital imaging analysis was performed using Image J. Results In the specimens of patients with complex appendicitis, significantly more myeloperoxidase positive cells (neutrophils) (8.7% versus 1.2%, P

The pathophysiology of renal ischemia/reperfusion (I/R) injury is characterized by excessive activation of inflammation and coagulation processes followed by abnormal renal tissue repair, resulting in renal injury and function loss. Platelets are important actors in these processes, however to what extent platelets contribute to the pathophysiology of renal I/R injury still needs to be elucidated. In the current study, we treated wild-type mice with a platelet depleting antibody, which caused thrombocytopenia. We then investigated the role of platelets during the pathophysiology of renal I/R by subjecting control wild-type mice with normal platelet counts and thrombocytopenic wild-type mice to renal I/R injury. Our results showed that in the early phase of renal I/R injury, thrombocytopenia 24 hours after ischemia reperfusion does not influence renal injury, neutrophil infiltration and accumulation of inflammatory chemokines (e.g. keratinocyte chemoattractant, monocyte chemoattractant protein 1, tumor necrosis factor alpha). In the recovery and regeneration phase of I/R injury, respectively 5 and 10 days post-ischemia, thrombocytopenia did also not affect the accumulation of intra-renal neutrophils and macrophages, renal injury, and renal fibrosis. Together, these results imply that lowering platelet counts do not impact the pathogenesis of I/R injury in mice.

BACKGROUND Transfusion‐associated circulatory overload (TACO) is the predominant complication of transfusion resulting in death. The pathophysiology is poorly understood, but inability to manage volume is associated with TACO, and observational data suggest it is different from simple cardiac overload due to fluids. We developed a two‐hit TACO animal model to assess the role of volume incompliance (“first‐hit”) and studied whether volume overload (“second‐hit”) by red blood cell (RBC) transfusion is different compared to fluids (Ringer's lactate [RL]). MATERIALS AND METHODS Male adult Lewis rats were stratified into a control group (no intervention) or a first hit: either myocardial infarction (MI) or acute kidney injury (AKI). Animals were randomized to a second hit of either RBC transfusion or an equal volume of RL. A clinically relevant difference was defined as an increase in left ventricular end‐diastolic pressure (ΔLVEDP) of +4.0 mm Hg between the RBC and RL groups. RESULTS In control animals (without first hit) LVEDP was not different between infusion groups (Δ + 1.6 mm Hg). LVEDP increased significantly more after RBCs compared to RL in animals with MI (Δ7.4 mm Hg) and AKI (Δ + 5.4 mm Hg), respectively. Volume‐incompliant rats matched clinical TACO criteria in 92% of transfused versus 25% of RL‐infused animals, with a greater increase in heart rate and significantly higher blood pressure. CONCLUSION To our knowledge, this is the first animal model for TACO, showing that a combination of volume incompliance and transfusion is essential for development of circulatory overload. This model allows for further testing of mechanistic factors as well as therapeutic approaches.

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity., peer-reviewed

Volatile organic compounds in breath can reflect host and pathogen metabolism and might be used to diagnose pneumonia. We hypothesized that rats with Streptococcus pneumoniae ( SP) or Pseudomonas aeruginosa ( PA) pneumonia can be discriminated from uninfected controls by thermal desorption-gas chromatography-mass-spectrometry (TD-GC-MS) and selected ion flow tube-mass spectrometry (SIFT-MS) of exhaled breath. Male adult rats ( n = 50) received an intratracheal inoculation of 1) 200 µl saline, or 2) 1 × 107 colony-forming units of SP or 3) 1 × 107 CFU of PA. Twenty-four hours later the rats were anaesthetized, tracheotomized, and mechanically ventilated. Exhaled breath was analyzed via TD-GC-MS and SIFT-MS. Area under the receiver operating characteristic curves (AUROCCs) and correct classification rate (CCRs) were calculated after leave-one-out cross-validation of sparse partial least squares-discriminant analysis. Analysis of GC-MS data showed an AUROCC (95% confidence interval) of 0.85 (0.73–0.96) and CCR of 94.6% for infected versus noninfected animals, AUROCC of 0.98 (0.94–1) and CCR of 99.9% for SP versus PA, 0.92 (0.83–1.00), CCR of 98.1% for SP versus controls and 0.97 (0.92–1.00), and CCR of 99.9% for PA versus controls. For these comparisons the SIFT-MS data showed AUROCCs of 0.54, 0.89, 0.63, and 0.79, respectively. Exhaled breath analysis discriminated between respiratory infection and no infection but with even better accuracy between specific pathogens. Future clinical studies should not only focus on the presence of respiratory infection but also on the discrimination between specific pathogens.

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BACKGROUND: The development of deep neural networks is facilitating more advanced digital analysis of histopathologic images. We trained a convolutional neural network for multiclass segmentation of digitized kidney tissue sections stained with periodic acid-Schiff (PAS). METHODS: We trained the network using multiclass annotations from 40 whole-slide images of stained kidney transplant biopsies and applied it to four independent data sets. We assessed multiclass segmentation performance by calculating Dice coefficients for ten tissue classes on ten transplant biopsies from the Radboud University Medical Center in Nijmegen, The Netherlands, and on ten transplant biopsies from an external center for validation. We also fully segmented 15 nephrectomy samples and calculated the network's glomerular detection rates and compared network-based measures with visually scored histologic components (Banff classification) in 82 kidney transplant biopsies. RESULTS: The weighted mean Dice coefficients of all classes were 0.80 and 0.84 in ten kidney transplant biopsies from the Radboud center and the external center, respectively. The best segmented class was "glomeruli" in both data sets (Dice coefficients, 0.95 and 0.94, respectively), followed by "tubuli combined" and "interstitium." The network detected 92.7% of all glomeruli in nephrectomy samples, with 10.4% false positives. In whole transplant biopsies, the mean intraclass correlation coefficient for glomerular counting performed by pathologists versus the network was 0.94. We found significant correlations between visually scored histologic components and network-based measures. CONCLUSIONS: This study presents the first convolutional neural network for multiclass segmentation of PAS-stained nephrectomy samples and transplant biopsies. Our network may have utility for quantitative studies involving kidney histopathology across centers and provide opportunities for deep learning applications in routine diagnostics.

The causative agents of Lyme borreliosis, spirochetes belonging to the Borrelia burgdorferi sensu lato group, have developed several ways to protect themselves against killing by the host complement system. In addition, it has been shown that serum sensitive isolates are (partially) protected by the Ixodes Tick Salivary Lectin Pathway Inhibitor (TSLPI) protein; a salivary gland protein that inhibits the function of Mannose Binding Lectin (MBL). MBL is a C-type lectin that recognizes oligosaccharides on pathogens and activates the complement system via the lectin pathway. MBL deficiency has been linked to a more severe course of several infectious diseases and humans with detectable antibodies against B. burgdorferi are significantly more often MBL deficient compared to humans without antibodies against B. burgdorferi. Here we set out to investigate the role of MBL in the immune response against B. burgdorferi in more detail. We demonstrate that B. burgdorferi N40 needle-infected C57BL/6 MBL deficient mice harbored significantly higher B. burgdorferi numbers in skin tissue during the early course of infection. In line with these findings they also developed higher anti-B. burgdorferi IgG serum antibodies compared to WT controls. In contrast, B. burgdorferi loads in distant tissue such as heart, joints or bladder at later time points were similar for both mouse strains. These in vivo findings were corroborated using a B. burgdorferi N40-infected I. scapularis infestation model. We showed that MBL is capable of binding B. burgdorferi through its carbohydrate recognition domains, but in vitro complement killing assays, peritoneal macrophage and whole blood stimulations, phagocytosis assays and an in vivo migration experiment did not reveal the mechanism by which MBL facilitates early clearance of B. burgdorferi. To conclude, we show a protective role of MBL in the early stages of B. burgdorferi infection, yet the underlying mechanism warrants further investigation.

Ischemia reperfusion (I/R) injury triggers the activation of coagulation and inflammation processes involved in the pathophysiology of acute kidney injury (AKI). Coagulation proteases upregulated upon renal I/R injury activate protease activated receptors (PARs), which form an important molecular link between inflammation and coagulation. PAR4 is the major thrombin receptor on mouse platelets, and the only PAR that is expressed on both human and murine platelets. In addition, PAR4 is expressed on other cells including podocytes. We here sought to determine the contribution of PAR4 in the host response to renal I/R injury. Hence, we subjected PAR4 knockout and wild-type mice to renal I/R injury. PAR4 knockout mice exhibited an increased tolerance to renal tubular necrosis and showed a decreased neutrophil influx in response to renal I/R, independent from platelet PAR4. On the other hand, PAR4 deficiency resulted in albumin cast formation in peritubular capillaries and showed a tendency towards albuminuria. Transmission Electron Microscopy revealed an increase in podocyte foot process effacement. Our findings suggest that PAR4 contributes to renal injury likely through facilitating neutrophil migration, independent from platelet PAR4. In addition, PAR4 fulfils an important function in the maintenance of podocyte integrity following renal I/R insult. Subsequently, loss of PAR4 results in albuminuria.

Critically ill patients are at a constant risk of direct (e.g., by pneumonia) or indirect lung injury (e.g., by sepsis). Excessive alveolar fibrin deposition is a prominent feature of lung injury, undermining pulmonary integrity and function. We examined the effect of local administration of recombinant human tissue factor pathway inhibitor (rh-TFPI), a natural anticoagulant, in two well-established models of lung injury in rats. Rats received intratracheal instillation of Pseudomonas aeruginosa, causing direct lung injury, or they received an intravenous injection of Escherichia coli lipopolysaccharide (LPS), causing indirect lung injury. Rats were randomized to local treatment with rh-TFPI or placebo through repeated nebulization. Challenge with P. aeruginosa or LPS was associated with increased coagulation and decreased fibrinolysis in bronchoalveolar lavage fluid (BALF) and plasma. Rh-TFPI levels in BALF increased after nebulization, whereas plasma rh-TFPI levels remained low and systemic TFPI activity was not affected. Nebulization of rh-TFPI attenuated pulmonary and systemic coagulation in both models, without affecting fibrinolysis. Nebulization of rh-TFPI modestly reduced the inflammatory response and bacterial growth of P. aeruginosa in the alveolar compartment. Local treatment with rh-TFPI does not alter systemic TFPI activity; however, it attenuates both pulmonary and systemic coagulopathy. Furthermore, nebulized rh-TFPI modestly reduces the pulmonary inflammatory response and allows increased bacterial clearance in rats with direct lung injury caused by P. aeruginosa

SummaryBacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.Supplementary Material to this article is available online at www.thrombosis-online.com.

Supplemental Digital Content is available in the text, Background: Melioidosis, caused by the gram-negative bacterium Burkholderia pseudomallei, is a common cause of community-acquired sepsis in Southeast Asia and Northern Australia. The NLRP3 inflammasome and its downstream product interleukin-1 beta (IL-1β) have been proposed to play crucial roles in melioidosis. In this study, we characterized the role of IL-1β more closely and we assessed its therapeutic potential. Methods: mRNA expression of inflammasome components was determined in isolated leukocytes of 32 healthy controls and 34 patients with sepsis caused by B pseudomallei. Wild-type (WT), NLRP3-deficient (Nlrp3−/−), and Asc−/− mice were infected with B pseudomallei. In additional experiments, infected WT mice were treated with an anti-IL-1β antibody. After 24, 48, and 72 hours (h) mice were sacrificed and organs were harvested. Furthermore, survival studies were performed. Results: Patients with melioidosis exhibited lower mRNA levels of caspase-1, NLRP3, and ASC. Bacterial dissemination and organ damage were increased in B pseudomallei-infected Nlrp3−/− and Asc−/− mice, together with a reduced pulmonary cell influx. Anti-IL-1β treatment of B pseudomallei challenged mice resulted in strongly reduced bacterial counts, organ damage, and pulmonary granulocyte influx together with reduced mortality. Postponement of anti-IL-1β treatment for 24 h postinfection still protected mice during melioidosis. Conclusion: Expression of caspase-1, NLRP3, and ASC is altered in melioidosis patients. In mice, both NLRP3 and ASC contribute to the host defense against melioidosis. Anti-IL-1β treatment protects mice against B pseudomallei infection and might be a novel treatment strategy in melioidosis.

Platelet-dense (δ) granules are important for platelet function. Platelets contribute to host defense and vascular integrity during pneumonia and sepsis, and δ granule products, including adenosine diphosphate (ADP), can influence inflammatory responses. We therefore aimed to study the role of platelet δ granules in the host response during sepsis. Hermansky-Pudlak syndrome (Hps)3coa mice (with reduced δ granule content), mice treated with the platelet ADP receptor inhibitor clopidogrel, and appropriate control mice were infected with the human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia and sepsis. In order to override potential redundancy in platelet functions, we also studied Hps3coa and control mice with moderate antibody-induced thrombocytopenia (10%) prior to infection. We found that sepsis-induced thrombocytopenia tended to be less severe in Hps3coa mice, and was significantly ameliorated in Hps3coa mice with low pre-infection platelet counts. Bacterial growth was similar in Hps3coa and control mice in the presence of normal platelet counts prior to infection, but lower in the lungs of Hps3coa mice with low pre-infection platelet counts. Hps3coa mice had unaltered lung pathology and distant organ injury during pneumosepsis, irrespective of pre-infection platelet counts; lung bleeding did not differ between Hps3coa and control mice. Clopidogrel did not influence any host response parameter. These data suggest that platelet δ granules can play a detrimental role in pneumosepsis by aggravating thrombocytopenia and impairing local antibacterial defense, but that these unfavorable effects only become apparent in the presence of low platelet counts.

Obesity has become a worldwide health crisis and is associated with a plethora of comorbidities. The multi-organ effects of obesity have been linked to ectopic lipid accumulation. Thus, there is an urgent need to tackle the obesity crisis by developing effective lipid-lowering therapies. 2-hydroxypropyl-β-Cyclodextrin (2HP-β-CD) has been previously shown to reduce lysosomal cholesterol accumulation in a murine model of Niemann Pick Type C (NPC) disease. Using a murine model of Western diet-induced obesity (DIO), we report the effects of 2HP-β-CD in counteracting weight gain, expansion of adipose tissue mass and ectopic lipid accumulation. Interestingly, DIO caused intracellular storage of neutral lipids in hepatic tissues and of phospholipids in kidneys, both of which were prevented by 2HP-β-CD. Importantly, this report brings attention to the nephrotoxic effects of 2HP-β-CD: renal tubular damage, inflammation and fibrosis. These effects may be overlooked, as they are best appreciated upon assessment of renal histology.

Diabetic nephropathy (DN) is a microvascular complication of diabetes mellitus that results in both tubular and glomerular injury. Low-grade inflammation and oxidative stress are two mechanisms known to drive the progression of DN. Nucleotide-binding leucine-rich repeat containing family member X1 (NLRX1) is an innate immune receptor, uniquely located in mitochondria, that has been found to regulate inflammatory responses and to dampen renal oxidative stress by regulating oxidative phosphorylation. For this reason, we investigated the role of NLRX1 in the development of DN in a Type 1 Diabetes mouse model. We analyzed the effect of NLRX1 deficiency on diabetes development and the accompanied renal damage, inflammation, and fibrosis. We found that multiple low doses of streptozotocin induced body weight loss, polydipsia, hyperglycemia, glycosuria, and a mild DN phenotype in wildtype and NLRX1-deficient mice, without significant differences between these mouse strains. Despite increased NLRX1 expression in diabetic wildtype mice, NLRX1 deficiency did not affect the diabetic phenotype induced by streptozotocin treatment, as reflected by similar levels of polyuria, microalbuminuria, and increased renal markers of oxidative stress and inflammation in wildtype and NLRX1-deficient mice. The present findings show that NLRX1 does not mediate the development of streptozotocin-induced diabetes and diabetic-induced nephropathy in mice after multiple low doses of streptozotocin. This data implies that, while NLRX1 can be triggered by cellular stress, its regulatory and functional effects may be dependent on the specific physiological conditions. In the case of DN, NLRX1 may be neither helpful nor harmful, but rather a marker of metabolic stress.

The Gram-negative intracellular pathogen Burkholderia pseudomallei is the causative agent of melioidosis, an important cause of sepsis in Southeast Asia. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is essential for an appropriate immune response during pathogen invasion. In patients with melioidosis, TLR5 is the most abundantly expressed TLR, and a hypofunctional TLR5 variant has been associated with improved survival. Here, we studied the functional role of TLR5 and its ligand flagellin in experimental melioidosis. First, we observed differential TLR5 expression in the pulmonary and hepatic compartments upon infection with B. pseudomallei. Next, we found that B. pseudomallei-challenged TLR5-deficient (Tlr5(−/−)) mice were more susceptible to infection than wild-type (WT) mice, as demonstrated by higher systemic bacterial loads, increased organ injury, and impaired survival. Lung bacterial loads were not different between the two groups. The phenotype was flagellin independent; no difference in in vivo virulence was observed for the flagellin-lacking mutant MM36 compared to the wild-type B. pseudomallei strain 1026b. Tlr5(−/−) mice showed a similar impaired antibacterial defense when infected with MM36 or 1026b. Ex vivo experiments showed that TLR5-deficient macrophages display markedly impaired phagocytosis of B. pseudomallei. In conclusion, these data suggest that TLR5 deficiency has a detrimental flagellin-independent effect on the host response against pulmonary B. pseudomallei infection.

Diabetes is associated with endothelial dysfunction and a hypercoagulable state of the blood. There is increasing evidence that the hypercoagulability in diabetes patients can be causally linked to microvascular complications, such as diabetic nephropathy.

Hyperglycemia has classically been identified as the principal factor in the pathogenesis and progression of diabetic nephropathy. Hyperglycemia induces renal damage directly but also through hemodynamic alterations. Because it has been noted that not all hyperglycemic diabetes patients will develop diabetic nephropathy, other factors contributing to the pathogenesis of the disease, e.g., genetic predisposition, hypertension, dyslipidemia, and proteinuria, have been identified. This chapter describes in brief the prevailing pathogenetic factors involved in the development and progression of diabetic nephropathy and serves as an overview of the chapters of Section II in which a detailed description of each individual component in the pathogenesis of the disease is discussed. A comprehensive understanding of each underlying mechanisms and downstream pathways as well as cross talks between these mechanisms is mandatory to development of novel therapeutic options.

High-molecular-weight kininogen is an important substrate of the kallikrein-kinin system. Activation of this system has been associated with aggravation of hallmark features in asthma. We aimed to determine the role of kininogen in enhanced pause (Penh) measurements and lung inflammation in a house dust mite (HDM)-induced murine asthma model. Normal wild-type mice and mice with a genetic deficiency of kininogen were subjected to repeated HDM exposure (sensitization on days 0, 1, and 2; challenge on days 14, 15, 18, and 19) via the airways to induce allergic lung inflammation. Alternatively, kininogen was depleted after HDM sensitization by twice-weekly injections of a specific antisense oligonucleotide (kininogen ASO) starting at day 3. In kininogen-deficient mice HDM induced in Penh was completely prevented. Remarkably, kininogen deficiency did not modify HDM-induced eosinophil/neutrophil influx, T helper 2 responses, mucus production, or lung pathology. kininogen ASO treatment started after HDM sensitization reduced plasma kininogen levels by 75% and reproduced the phenotype of kininogen deficiency: kininogen ASO administration prevented the HDM-induced increase in Penh without influencing leukocyte influx, Th2 responses, mucus production, or lung pathology. This study suggests that kininogen could contribute to HDM-induced rise in Penh independently of allergic lung inflammation. Further research is warranted to confirm these data using invasive measurements of airway responsiveness.