Lymphopenia is associated with broad host response aberrations in community-acquired pneumonia.

OBJECTIVES: Lymphopenia at hospital admission occurs in over one-third of patients with community-acquired pneumonia (CAP), yet its clinical relevance and pathophysiological implications remain underexplored. We evaluated outcomes and immune features of patients with lymphopenic CAP (L-CAP), a previously described immunophenotype characterized by admission lymphocyte count <0.724 × 109 cells/L. METHODS: Observational study in 149 patients admitted to a general ward for CAP. We measured 34 plasma biomarkers reflective of inflammation, endothelial cell responses, coagulation, and immune checkpoints. We characterized lymphocyte phenotypes in 29 patients using spectral flow cytometry. RESULTS: L-CAP occurred in 45 patients (30.2%) and was associated with prolonged time-to-clinical-stability (median 5 versus 3 days), also when we accounted for competing events for reaching clinical stability and adjusted for baseline covariates (subdistribution hazard ratio 0.63; 95% confidence interval 0.45-0.88). L-CAP patients demonstrated a proportional depletion of CD4 T follicular helper cells, CD4 T effector memory cells, naïve CD8 T cells and IgG+ B cells. Plasma biomarker analyses indicated increased activation of the cytokine network and the vascular endothelium in L-CAP. CONCLUSIONS: L-CAP patients have a protracted clinical recovery course and a more broadly dysregulated host response. These findings highlight the prognostic and pathophysiological relevance of admission lymphopenia in patients with CAP.

The shifting lipidomic landscape of blood monocytes and neutrophils during pneumonia.

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

One-year effectiveness of high-load compared with low-load strengthening exercise on self-reported function in patients with hypermobile shoulders: a secondary analysis from a randomised controlled trial.

OBJECTIVES: To investigate the long-term effectiveness of high-load versus low-load strengthening exercise on self-reported function in patients with hypermobility spectrum disorder (HSD) and shoulder symptoms. METHODS: A secondary analysis of a superiority, parallel-group, randomised trial (balanced block randomisation 1:1, electronic concealment) including adult patients (n=100) from primary care with HSD and shoulder pain and/or instability ≥3 months. Patients received 16 weeks of shoulder exercises (three sessions/week): HEAVY (n=50, full-range, high-load, supervised twice/week) or LIGHT (n=50, neutral/mid-range, low-load, supervised three times in total). The 1-year between-group difference in change in self-reported function was measured using the Western Ontario Shoulder Instability Index (WOSI, scale 0-2100, 0=best). Secondary outcomes were self-reported measures including changes in shoulder-related symptoms, function, emotions and lifestyle, quality of life, patient-perceived effect, treatment utility and adverse events. A blinded analyst conducted the analyses using linear mixed model repeated measurements analysis. RESULTS: One-year data were available in 86 out of 100 participants (79% women, mean age 37.8 years) (LIGHT 84%, HEAVY 88%). The mean WOSI score between-group difference favoured HEAVY (-92.9, 95% CI -257.4 to 71.5, p=0.268) but was not statistically significant. The secondary outcomes were mostly inconclusive, but patients in HEAVY had larger improvement in the WOSI emotions subdomain (-36.3; 95% CI -65.4 to -7.3, p=0.014). Patient-perceived effect favoured HEAVY anchored in WOSI-emotions (55% vs 31%, p=0.027) and WOSI-lifestyle (50% vs 29%, p=0.042). CONCLUSION: High-load shoulder strengthening exercise was not superior to low-load strengthening exercise in improving self-reported function at 1 year. High-load strengthening exercise may be more effective in improving patient emotions about shoulder pain and function, but more robust data are needed to support these findings. TRIAL REGISTRATION NUMBER: NCT03869307.

High-dimensional phenotyping of the peripheral immune response in community-acquired pneumonia.

Background: Community-acquired pneumonia (CAP) represents a major health burden worldwide. Dysregulation of the immune response plays an important role in adverse outcomes in patients with CAP. Methods: We analyzed peripheral blood mononuclear cells by 36-color spectral flow cytometry in adult patients hospitalized for CAP (n=40), matched control subjects (n=31), and patients hospitalized for COVID-19 (n=35). Results: We identified 86 immune cell metaclusters, 19 of which (22.1%) were differentially abundant in patients with CAP versus matched controls. The most notable differences involved classical monocyte metaclusters, which were more abundant in CAP and displayed phenotypic alterations reminiscent of immunosuppression, increased susceptibility to apoptosis, and enhanced expression of chemokine receptors. Expression profiles on classical monocytes, driven by CCR7 and CXCR5, divided patients with CAP into two clusters with a distinct inflammatory response and disease course. The peripheral immune response in patients with CAP was highly similar to that in patients with COVID-19, but increased CCR7 expression on classical monocytes was only present in CAP. Conclusion: CAP is associated with profound cellular changes in blood that mainly relate to classical monocytes and largely overlap with the immune response detected in COVID-19.

Inflammatory and glycolytic programs underpin a primed blood neutrophil state in patients with pneumonia.

Neutrophils are potent immune cells with key antimicrobial functions. Previous in vitro work has shown that neutrophil effector functions are mainly fueled by intracellular glycolysis. Little is known about the state of neutrophils still in the circulation in patients during infection. Here, we combined flow cytometry, stimulation assays, transcriptomics, and metabolomics to investigate the link between inflammatory and metabolic pathways in blood neutrophils of patients with community-acquired pneumonia. Patients' neutrophils, relative to neutrophils from age- and sex- matched controls, showed increased degranulation upon ex vivo stimulation, and portrayed distinct upregulation of inflammatory transcriptional programs. This neutrophil phenotype was accompanied by a high-energy state with increased intracellular ATP content, and transcriptomic and metabolic upregulation of glycolysis and glycogenolysis. One month after hospital admission, these metabolic and transcriptomic changes were largely normalized. These data elucidate the molecular programs that underpin a balanced, yet primed state of blood neutrophils during pneumonia.

Towards the Validation of Executive Functioning Assessments: A Clinical Study.

Neuropsychological assessment needs a more profound grounding in psychometric theory. Specifically, psychometrically reliable and valid tools are required, both in patient care and in scientific research. The present study examined convergent and discriminant validity of some of the most popular indicators of executive functioning (EF). A sample of 96 neurological inpatients (aged 18-68 years) completed a battery of standardized cognitive tests (Raven's matrices, vocabulary test, Wisconsin Card Sorting Test, verbal fluency test, figural fluency test). Convergent validity of indicators of intelligence (Raven's matrices, vocabulary test) and of indicators of EF (Wisconsin Card Sorting Test, verbal fluency test, figural fluency) were calculated. Discriminant validity of indicators of EF against indicators of intelligence was also calculated. Convergent validity of indicators of intelligence (Raven's matrices, vocabulary test) was good (rxtyt = 0.727; R2 = 0.53). Convergent validity of fluency indicators of EF against executive cognition as indicated by performance on the Wisconsin Card Sorting Test was poor (0.087 ≤ rxtyt ≤ 0.304; 0.008 ≤ R2 ≤ 0.092). Discriminant validity of indicators of EF against indicators of intelligence was good (0.106 ≤ rxtyt ≤ 0.548; 0.011 ≤ R2 ≤ 0.300). Our conclusions from these data are clear-cut: apparently dissimilar indicators of intelligence converge on general intellectual ability. Apparently dissimilar indicators of EF (mental fluency, executive cognition) do not converge on general executive ability. Executive abilities, although non-unitary, can be reasonably well distinguished from intellectual ability. The present data contribute to the hitherto meager evidence base regarding the validity of popular indicators of EF.

Rectal microbiota are coupled with altered cytokine production capacity following community-acquired pneumonia hospitalization.

Human studies describing the immunomodulatory role of the intestinal microbiota in systemic infections are lacking. Here, we sought to relate microbiota profiles from 115 patients with community-acquired pneumonia (CAP), both on hospital admission and following discharge, to concurrent circulating monocyte and neutrophil function. Rectal microbiota composition did not explain variation in cytokine responses in acute CAP (median 0%, IQR 0.0%-1.9%), but did one month following hospitalization (median 4.1%, IQR 0.0%-6.6%, p = 0.0035). Gene expression analysis of monocytes showed that undisrupted microbiota profiles following hospitalization were associated with upregulated interferon, interleukin-10, and G-protein-coupled-receptor-ligand-binding pathways. While CAP is characterized by profoundly distorted gut microbiota, the effects of these disruptions on cytokine responses and transcriptional profiles during acute infection were absent or modest. However, rectal microbiota were related to altered cytokine responses one month following CAP hospitalization, which may provide insights into potential mechanisms contributing to the high risk of recurrent infections following hospitalization.

Intracellular pyruvate levels positively correlate with cytokine production capacity in tolerant monocytes from patients with pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. OBJECTIVE: To obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. METHODS: Monocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-10. RESULTS: 50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1ß and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. CONCLUSION: These results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.

The host response in different aetiologies of community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) can be caused by a variety of pathogens, of which Streptococcus pneumoniae, Influenza and currently SARS-CoV-2 are the most common. We sought to identify shared and pathogen-specific host response features by directly comparing different aetiologies of CAP. METHODS: We measured 72 plasma biomarkers in a cohort of 265 patients hospitalized for CAP, all sampled within 48 hours of admission, and 28 age-and sex matched non-infectious controls. We stratified the biomarkers into several pathophysiological domains- antiviral response, vascular response and function, coagulation, systemic inflammation, and immune checkpoint markers. We directly compared CAP caused by SARS-CoV-2 (COVID-19, n=39), Streptococcus pneumoniae (CAP-strep, n=27), Influenza (CAP-flu, n=22) and other or unknown pathogens (CAP-other, n=177). We adjusted the comparisons for age, sex and disease severity scores. FINDINGS: Biomarkers reflective of a stronger cell-mediated antiviral response clearly separated COVID-19 from other CAPs (most notably granzyme B). Biomarkers reflecting activation and function of the vasculature showed endothelial barrier integrity was least affected in COVID-19, while glycocalyx degradation and angiogenesis were enhanced relative to other CAPs. Notably, markers of coagulation activation, including D-dimer, were not different between the CAP groups. Ferritin was most increased in COVID-19, while other systemic inflammation biomarkers such as IL-6 and procalcitonin were highest in CAP-strep. Immune checkpoint markers showed distinctive patterns in viral and non-viral CAP, with highly elevated levels of Galectin-9 in COVID-19. INTERPRETATION: Our investigation provides insight into shared and distinct pathophysiological mechanisms in different aetiologies of CAP, which may help guide new pathogen-specific therapeutic strategies. FUNDING: This study was financially supported by the Dutch Research Council, the European Commission and the Netherlands Organization for Health Research and Development.

Association of Hyperferritinemia With Distinct Host Response Aberrations in Patients With Community-Acquired Pneumonia.

BACKGROUND: Strongly elevated ferritin levels have been proposed to reflect systemic hyperinflammation in patients admitted to the intensive care unit. Knowledge of the incidence and pathophysiological implications of hyperferritinemia in patients with acute infection admitted to a non-intensive care setting is limited. METHODS: We determined the association between hyperferritinemia, defined by 2 cutoff values (500 and 250 ng/mL), and aberrations in key host response mechanisms among patients with community-acquired pneumonia (CAP) on admission to a general hospital ward (clinicaltrials.gov NCT02928367; trialregister.nl NTR6163). RESULTS: Plasma ferritin levels were higher in patients with CAP (n = 174; median [interquartile ranges], 259.5 [123.1-518.3] ng/mL) than in age- and sex-matched controls without infection (n = 50; 102.8 [53.5-185.7] ng/mL); P < .001); they were ≥500 ng/mL in 46 patients (26%) and ≥250 ng/mL in 90 (52%). Measurements of 26 biomarkers reflective of distinct pathophysiological domains showed that hyperferritinemia was associated with enhanced systemic inflammation, neutrophil activation, cytokine release, endothelial cell activation and dysfunction, and activation of the coagulation system. Results were robust across different cutoff values. CONCLUSIONS: Hyperferritinemia identifies patients with CAP with a broad deregulation of various host response mechanisms implicated in the pathogenesis of sepsis. This could inform future therapeutic strategies targeting subgroups within the CAP population.

Bacterial and Viral Respiratory Tract Microbiota and Host Characteristics in Adults With Lower Respiratory Tract Infections: A Case-Control Study.

BACKGROUND: Viruses and bacteria from the nasopharynx are capable of causing community-acquired pneumonia (CAP), which can be difficult to diagnose. We aimed to investigate whether shifts in the composition of these nasopharyngeal microbial communities can be used as diagnostic biomarkers for CAP in adults. METHODS: We collected nasopharyngeal swabs from adult CAP patients and controls without infection in a prospective multicenter case-control study design. We generated bacterial and viral profiles using 16S ribosomal RNA gene sequencing and multiplex polymerase chain reaction (PCR), respectively. Bacterial, viral, and clinical data were subsequently used as inputs for extremely randomized trees classification models aiming to distinguish subjects with CAP from healthy controls. RESULTS: We enrolled 117 cases and 48 control subjects. Cases displayed significant beta diversity differences in nasopharyngeal microbiota (P = .016, R2 = .01) compared to healthy controls. Our extremely randomized trees classification models accurately discriminated CAP caused by bacteria (area under the curve [AUC] .83), viruses (AUC .95) or mixed origin (AUC .81) from healthy control subjects. We validated this approach using a dataset of nasopharyngeal samples from 140 influenza patients and 38 controls, which yielded highly accurate (AUC .93) separation between cases and controls. CONCLUSIONS: Relative proportions of different bacteria and viruses in the nasopharynx can be leveraged to diagnose CAP and identify etiologic agent(s) in adult patients. Such data can inform the development of a microbiota-based diagnostic panel used to identify CAP patients and causative agents from nasopharyngeal samples, potentially improving diagnostic specificity, efficiency, and antimicrobial stewardship practices.

What is the spatial-temporal behavior of a low, medium and high adsorptive compound in two contrasting natural sediments in OECD 218/219 test systems?

Artificial sediment used in studies according to OECD 218/219 (Sediment Water Chironomid Toxicity Test Using Spiked Sediment/Water) does not necessarily mirror the characteristics of natural sediments. To investigate the influence of sediment characteristics on the spatial-temporal behaviors of bixafen (KfOM = 2244 mL/g), fluopyram (KfOM = 162 mL/g) and N,N-dimethylsulfamide (KfOM ≈ 0 mL/g), experiments according to OECD 218/219 with two contrasting natural sediments were conducted. The silt loam sediment provided a high content of organic matter (OM) (13.1%), while the OM (0.45%) of the sandy sediment was low. Diffusion into (OECD 219) or out (OECD 218) of the sediment was dependent on the extent of adsorption, which is linked to the model compounds ́ adsorption affinities and the sediments ́ OM. Consequently, N,N-dimethylsulfamide showed unhindered mobility in each experimental set up, while the high adsorption affinities of fluopyram and bixafen limited the diffusion in the respective sediments. Therefore, in experiments with the silt loam sediment, both compounds revealed a limited mobility and either accumulated in the top 5 mm of the sediment (OECD 219) or remained homogenously distributed over the sediment depth (OECD 218). A greater mobility was observed within the sandy sediment.The influence of OM as found in a study using artificial sediment could be confirmed. Moreover, the applicability of a TOXSWA model was reassured to predict the measured concentrations at different sediment depths. TOXSWA is used in the regulatory exposure assessment to simulate the behavior of pesticides in surface waters. Calibration of three driving input parameters by inverse modelling (diffusion-, adsorption coefficient and OM) revealed no potential for improvement. The core sampling technique used and the model may contribute to a more realistic determination of concentration to which the Chironomid larvae are exposed to. This applies to water sediment test systems where the test organisms do not evenly inhabit the sediment.

Rectal bacteriome and virome signatures and clinical outcomes in community-acquired pneumonia: An exploratory study.

Background Bacterial intestinal communities interact with the immune system and may contribute to protection against community-acquired pneumonia (CAP). Intestinal viruses are closely integrated with these bacterial communities, yet the composition and clinical significance of these communities in CAP patients are unknown. The aims of this exploratory study were to characterise the composition of the rectal bacteriome and virome at hospital admission for CAP, and to determine if microbiota signatures correlate with clinical outcomes. Methods We performed a prospective observational cohort study in CAP patients, admitted to a university or community hospital in the Netherlands between October 2016 and July 2018, and controls. Rectal bacteriome and virome composition were characterised using 16S ribosomal RNA gene sequencing and virus discovery next-generation sequencing, respectively. Unsupervised multi-omics factor analysis was used to assess the co-variation of bacterial and viral communities, which served as primary predictor. The clinical outcomes of interest were the time to clinical stability and the length of hospital stay. Findings 64 patients and 38 controls were analysed. Rectal bacterial alpha (p = 0•0015) and beta diversity (r2 =0•023, p = 0•004) of CAP patients differed from controls. Bacterial and viral microbiota signatures correlated with the time to clinical stability (hazard ratio 0•43, 95% confidence interval 0•20-0•93, p = 0•032) and the length of hospital stay (hazard ratio 0•37, 95% confidence interval 0•17-0•81, p = 0•012), although only the latter remained significant following p-value adjustment for examining multiple candidate cut-points (p = 0•12 and p = 0•046, respectively). Interpretation This exploratory study provides preliminary evidence that intestinal bacteriome and virome signatures could be linked with clinical outcomes in CAP. Such exploratory data, when validated in independent cohorts, could inform the development of a microbiota-based diagnostic panel used to predict clinical outcomes in CAP. Funding Netherlands Organization for Scientific Research and Netherlands Organization for Health Research and Development.

An epigenetic and transcriptomic signature of immune tolerance in human monocytes through multi-omics integration.

BACKGROUND: The plasticity of monocytes enables them to exert multiple roles during an immune response, including promoting immune tolerance. How monocytes alter their functions to convey immune tolerance in the context of lower respiratory tract infections in humans is not well understood. Here, we sought to identify epigenetic and transcriptomic features of cytokine production capacity in circulating monocytes during community-acquired pneumonia (CAP). METHODS: Circulating CD14+ monocytes were obtained from the blood of CAP patients included in a longitudinal, observational cohort study, on hospitalization (acute stage, n=75), and from the same patients after a 1-month follow-up (recovery stage, n=56). Age and sex-matched non-infectious participants were included as controls (n=41). Ex vivo cytokine production after lipopolysaccharide (LPS) exposure was assessed by multiplex assay. Transcriptomes of circulating monocytes were generated by RNA-sequencing, and DNA methylation levels in the same monocytes were measured by reduced representation bisulfite sequencing. Data were integrated by fitting projection-to-latent-structure models, and signatures derived by partial least squares discrimination. RESULTS: Monocytes captured during the acute stage exhibited impaired TNF, IL-1ß, IL-6, and IL-10 production after ex vivo stimulation with LPS, relative to controls. IL-6 production was not resolved in recovery monocytes. Multivariate analysis of RNA-sequencing data identified 2938 significantly altered RNA transcripts in acute-stage monocytes (fold expression ≤-1.5 or ≥1.5; adjusted p ≤ 0.01), relative to controls. Comparing DNA methylation levels in circulating monocytes of CAP patients to controls revealed minimal differences, specifically in DNAse hypersensitive sites (HS) of acute-stage monocytes. Data integration identified a cholesterol biosynthesis gene signature and DNAse HS axis of IL-1ß and IL-10 production (R2 =0.51). CONCLUSIONS: Circulating monocytes obtained from CAP patients during the acute stage exhibited impaired cytokine production capacities, indicative of reprogramming to a state of immune tolerance, which was not fully resolved after 1 month. Our split-sample study showed that 51% of the immune tolerance phenotype can be explained, at least in part, by coordinated shifts in cholesterol biosynthesis gene expression and DNAse HS methylation levels. A multi-scale model identified an epigenetic and transcriptomic signature of immune tolerance in monocytes, with implications for future interventions in immunosuppression. TRIAL REGISTRATION: NCT number NCT02928367.

What is the actual exposure of organic compounds on Chironomus riparius? - A novel methodology enabling the depth-related analysis in sediment microcosms.

A novel active sampling method enabled determination of sediment depth profiles revealing the spatial distribution of model compounds N,N-dimethylsulfamide, fluopyram and bixafen (low, medium, high adsorption affinity) in sediment microcosms according to OECD Test 218/219 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment/Spiked Water). After the overlying water was removed, plastic tubes were inserted into the sediment and the microcosms were frozen. For depth-related analysis, each "sediment core" was mounted in a cutting device and sawed into three 5-mm-slices, respectively (top, middle, bottom). Each slice was centrifuged for sediment and pore water separation. By various sampling dates within 28 days, we could follow the behavior of model compounds depending on sorption affinities and display specific distribution patterns within the sediment. N,N-dimethylsulfamide showing no sediment adsorption, migrated unhindered in (OECD 219) and out (OECD 218) of the sediment via pore water, resulting in homogenous distributions in both test designs. Fluopyram with moderate adsorption affinity revealed a concentration gradient with declining amounts from top to bottom layer (OECD 219) and higher amounts in the middle and bottom layer as compared to the top layer (OECD 218). Bixafen providing a strong adsorption affinity accumulated in the top layer in OECD 219, while no concentration gradients became visible in OECD 218. For establishing a Toxic Substances in Surface Waters (TOXSWA) model, we compared our measurements with simulated results revealing good agreements. The presented methodology is a useful tool to determine more realistic sediment and pore water concentrations, which the Chironomid larvae are exposed to.

Corrigendum: Concurrent Immune Suppression and Hyperinflammation in Patients With Community-Acquired Pneumonia.

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Concurrent Immune Suppression and Hyperinflammation in Patients With Community-Acquired Pneumonia.

Background: The nature and timing of the host immune response during infections remain uncertain and most knowledge is derived from critically ill sepsis patients. We aimed to test the hypothesis that community-acquired pneumonia (CAP) is associated with concurrent immune suppression and systemic inflammation. Methods: Blood was collected from 79 CAP patients within 24 h after hospitalization and 1 month after discharge; 42 age- and sex-matched subjects without acute infection served as controls. Blood leukocytes were stimulated with lipopolysaccharide (LPS) or Klebsiella pneumoniae, and cytokines were measured in supernatants. Fifteen plasma biomarkers reflective of key host response pathways were compared between CAP patients with the strongest immune suppression (lowest 25% blood leukocyte tumor necrosis factor (TNF)-α production in response to LPS) and those with the least immune suppression (highest 25% of LPS-induced TNF-α production). Results: Blood leukocytes of CAP patients (relative to control subjects) showed a reduced capacity to release TNF-α, interleukin (IL)-1ß, IL-6 and IL-10 upon stimulation with LPS or K. pneumoniae, with a concurrently enhanced ability to release the anti-inflammatory mediator IL-1 receptor antagonist, irrespective of the presence of sepsis (18.9% of cases). Low (relative to high) TNF-α producers displayed higher plasma levels of biomarkers reflecting systemic inflammation, neutrophil degranulation, endothelial cell activation, a disturbed vascular barrier function and coagulation activation. Conclusion: CAP replicates a common feature of immune suppression in sepsis. The coexistence of immune suppression and hyperinflammation in CAP argues against the theory of two distinct phases during the host response to sepsis.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Optimizing the design of a reproduction toxicity test with the pond snail Lymnaea stagnalis.

This paper presents the results from two ring-tests addressing the feasibility, robustness and reproducibility of a reproduction toxicity test with the freshwater gastropod Lymnaea stagnalis (RENILYS strain). Sixteen laboratories (from inexperienced to expert laboratories in mollusc testing) from nine countries participated in these ring-tests. Survival and reproduction were evaluated in L. stagnalis exposed to cadmium, tributyltin, prochloraz and trenbolone according to an OECD draft Test Guideline. In total, 49 datasets were analysed to assess the practicability of the proposed experimental protocol, and to estimate the between-laboratory reproducibility of toxicity endpoint values. The statistical analysis of count data (number of clutches or eggs per individual-day) leading to ECx estimation was specifically developed and automated through a free web-interface. Based on a complementary statistical analysis, the optimal test duration was established and the most sensitive and cost-effective reproduction toxicity endpoint was identified, to be used as the core endpoint. This validation process and the resulting optimized protocol were used to consolidate the OECD Test Guideline for the evaluation of reproductive effects of chemicals in L. stagnalis.

Manipulation of tumor metabolism for therapeutic approaches: ovarian cancer-derived cell lines as a model system.

BACKGROUND: Malignant transformation of cells is often accompanied by up-regulation of glycolysis-related enzymes and transporters, as well as a distortion of mitochondrial respiration. As a consequence, most malignant tumors utilize high amounts of glucose and produce and accumulate high concentrations of lactate, even in the presence of oxygen. This phenomenon has been termed 'Warburg Effect'. Here, we aimed at resolving the interrelation between tumor metabolism, reactive oxygen species, double strand DNA breaks and radio-resistance in ovarian cancer-derived cells. METHODS: As a model system two ovarian cancer-derived cell lines, OC316 and IGROV-1, and its corresponding xenografts were used. First, the metabolic properties of the xenografts were tested to ensure that initial in vitro data might later be transferred to in vivo data. In parallel, three inhibitors of tumor cell metabolism, 2-deoxy-D-glucose, an inhibitor of glycolysis, oxamate, a pyruvate analogue and inhibitor of lactate dehydrogenase, and rotenone, a specific inhibitor of mitochondrial electron complex I, were tested for their effect on the metabolism and radio-sensitivity of the respective ovarian cancer-derived cell lines. RESULTS: We found that all three inhibitors tested led to significant changes in the tumor cell energy metabolism at non-cytotoxic concentrations. Furthermore, we found that inhibition of tumor glycolysis by 2-deoxy-D-glucose in combination with rotenone decreased the radio-resistance at a clinically relevant radiation dose. This apparent radio-sensitizing effect appears to be based on an increased level of double strand DNA breaks 1 h and 24 h after gamma irradiation. Both cancer-derived cell lines maintained their metabolic properties, as well as their protein expression profiles and levels of reactive oxygen species in xenografts, thus providing a suitable model system for further in vivo investigations. CONCLUSION: A combination of metabolic inhibitors and reactive oxygen species-generating therapies, such as irradiation, may effectively enhance the therapeutic response in particularly metabolically highly active (ovarian) tumors.