Phagocytic cell death leads to enhanced release of pro-inflammatory S100A12 in familial Mediterranean fever.

BACKGROUND: Familial Mediterranean fever (FMF) is a prototypical autoinflammatory syndrome associated with phagocytic cell activation. Pyrin mutations are the genetic basis of this disease, and its expression has been shown in monocytes, granulocytes, dendritic cells, and synovial fibroblasts. Pyrin functions as a cytosolic pattern recognition receptor and forms a distinct pyrin inflammasome. The phagocyte-specific protein S100A12 is predominantly expressed in granulocytes and belongs to the group of damage associated molecular patterns (DAMP). S100A12 can be detected at massively elevated levels in the serum of FMF patients, even in clinically inactive disease. Whether this is crucial for FMF pathogenesis is as yet unknown, and we therefore investigated the mechanisms of S100A12 release from granulocytes of FMF patients presenting clinically inactive. RESULTS: We demonstrate that FMF neutrophils from patients in clinical inactive disease possess an intrinsic activity leading to cell death even in exogenously unstimulated neutrophils. Cell death resembles NETosis and is dependent on ROS and pore forming protein gasdermin D (GSDMD), as inhibitors for both are capable of completely block cell death and S100A12 release. When pyrin-activator TcdA (Clostridium difficile toxin A) is used to stimulate, neutrophilic cell death and S100A12 release are significantly enhanced in neutrophils from FMF patients compared to neutrophils from HC. CONCLUSIONS: We are able to demonstrate that activation threshold of neutrophils from inactive FMF patients is decreased, most likely by pre-activated pyrin. FMF neutrophils present with intrinsically higher ROS production, when cultured ex vivo. This higher baseline ROS activity leads to increased GSDMD cleavage and subsequent release of, e.g., S100A12, and to increased cell death with features of NETosis and pyroptosis. We show for the first time that cell death pathways in neutrophils of inactive FMF patients are easily triggered and lead to ROS- and GSDMD-dependent activation mechanisms and possibly pathology. This could be therapeutically addressed by blocking ROS or GSDMD cleavage to decrease inflammatory outbreaks when becoming highly active.

Promising results of a new treatment in patients with bowel obstruction in colorectal surgery.

INTRODUCTION: Bowel obstruction increases risk of emergency surgery and leads to suboptimal physical and nutritional condition. Preventing emergency surgery and prehabilitation might improve outcomes. This pilot study aimed to examine the effect of a multimodal obstruction protocol for bowel obstruction patients on the risk of emergency surgery and postoperative morbidity and mortality. MATERIALS AND METHODS: All bowel obstruction patients treated according to the obstruction protocol in the period 2013-2017 were included in this uncontrolled observational cohort study. Benign and malignant causes of bowel obstruction were included. The protocol consisted of: 1. specific dietary adjustments to reduce prestenotic dilatation, 2. oral laxatives and 3. prehabilitation. Emergency surgery and postoperative morbidity and mortality rates were compared to known rates from the literature. RESULTS: Sixty-one patients were included: 44 (72%) were treated for colorectal cancer and 17 (28%) for Crohn's disease or diverticulitis. Four patients (7%) underwent emergency surgery. Primary anastomosis was constructed in 49 out of 57 elective patients (86%). Severe complications (Clavien-Dindo ≥ III) occurred in four patients (7%). No bowel perforation, anastomotic leakages or 30-day mortality was observed. These rates were much lower than rates reported in the literature after surgery for colorectal cancer (3% bowel perforation, 8% anastomotic leakage, 4% 30-day mortality, 15% severe complications) and benign disease (30-day mortality 17%, severe complications 7%). CONCLUSION: Using the obstruction protocol in patients with bowel obstruction reduced emergency surgery and postoperative morbidity and mortality in this pilot study. This protocol seems to be a viable and efficient alternative to emergency surgery.

Global nitrogen budgets in cereals: A 50-year assessment for maize, rice, and wheat production systems.

Industrially produced N-fertilizer is essential to the production of cereals that supports current and projected human populations. We constructed a top-down global N budget for maize, rice, and wheat for a 50-year period (1961 to 2010). Cereals harvested a total of 1551 Tg of N, of which 48% was supplied through fertilizer-N and 4% came from net soil depletion. An estimated 48% (737 Tg) of crop N, equal to 29, 38, and 25 kg ha(-1) yr(-1) for maize, rice, and wheat, respectively, is contributed by sources other than fertilizer- or soil-N. Non-symbiotic N2 fixation appears to be the major source of this N, which is 370 Tg or 24% of total N in the crop, corresponding to 13, 22, and 13 kg ha(-1) yr(-1) for maize, rice, and wheat, respectively. Manure (217 Tg or 14%) and atmospheric deposition (96 Tg or 6%) are the other sources of N. Crop residues and seed contribute marginally. Our scaling-down approach to estimate the contribution of non-symbiotic N2 fixation is robust because it focuses on global quantities of N in sources and sinks that are easier to estimate, in contrast to estimating N losses per se, because losses are highly soil-, climate-, and crop-specific.

[Translational research in pediatric rheumatology. Current research approaches to the innate immune system]. / Translationale Forschung in der pädiatrischen Rheumatologie. Aktuelle Forschungsansätze zum angeborenen Immunsystem.

Translational research aims at closely linking basic research and clinical observations so that important mechanistic insights identified in one field should trigger progress in the other. Particularly in the field of pediatric rheumatology this approach has significantly improved the understanding and therapy of several diseases in recent years. One focus of our research in this respect is on the structure, release mechanisms and function of damage associated molecular patterns (DAMP), particularly S100 proteins. Due to their huge potential as inflammation biomarkers for more specific diagnostics these proteins are of particular clinical interest. Overactivated cells of the innate immune system play a crucial role in the development of rheumatic diseases. Innate mechanisms, such as the generation of neutrophil extracellular traps (NETosis) were linked to the pathogenesis of inflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis. Furthermore, it became increasingly more evident that various excessive sterile inflammatory mechanisms and reactions significantly contribute to an activation of adaptive immune responses and thus to the development of autoimmunity. Studying such potentially DAMP-dependent pathways at the interface between innate and adaptive immunity can provide a better understanding of autoinflammatory conditions in pediatric rheumatology and to identify novel targets for optimization of therapy.

Approximating Phosphorus Leaching from Agricultural Organic Soils by Soil Testing.

Phosphorus applied to soils in excess of crop requirement could create situations favorable to P enrichment in subsurface flow that contributes to eutrophication of surface water. This pathway of P loss can be more severe in muck (i.e., organic) soils where agricultural production is intensive. This study evaluated the suitability of various environmental and agronomic soil P tests initially designed for mineral soils to predict dissolved reactive P (DRP) in subsurface flow from organic soils. Intact soil columns were collected from 44 muck soils in Ontario to provide a wide range of soil test P levels. A lysimeter leaching study was conducted by evenly adding water in an amount equivalent to 5 mm of rainfall. The leachate DRP concentration was linearly related to soil water-extractable P and CaCl-extractable P with values of 0.90 and 0.93, respectively, and to Bray-1 P and FeO-impregnated filter paper extractable P in a split-line model with a change point. Mehlich-3 P and Olsen P, a method recommended for agronomic P calibration in Ontario, were not related to leachate DRP concentration. All P sorption index (PSI) based degree of P saturation (DPS) values were closely related to leachate DRP in split-line models, with the DPS indices expressed as Bray-1 P/PSI and FeO-P/PSI having the highest correlation with leachate DRP concentration. Because it is desirable from practical and economic standpoints that the environmental risk assessment shares the same soil test with agronomic P calibration, the two PSI-based DPS indices as presented can be considered as environmental risk indicators of DRP subsurface loss from organic soils.

Recurrence location after resection of colorectal liver metastases influences prognosis.

AIM: To assess the impact of first recurrence location on survival following surgery of colorectal liver metastases. METHODS: A total of 265 consecutive patients with colorectal liver metastases undergoing liver surgery (2000-2011) were categorized according to first site of tumor recurrence. Time to recurrence (TTR) and overall survival (OS) were determined. Uni- and multivariate analysis were performed to identify factors associated with TTR and OS. RESULTS: Median TTR was 1.16 years following liver resection, and 0.56 years following radiofrequency ablation (RFA). Intrahepatic recurrence following liver resection resulted in a significantly shorter median TTR compared to extrahepatic recurrence. Intrapulmonary recurrence was associated with superior survival compared to other recurrence locations. Such patterns were not observed in the RFA-treated group. Multivariate analysis identified the type of surgical treatment and extra-hepatic first-site recurrence (other than lung) as independent predictors for OS. Pre-operative chemotherapy and simultaneous intrahepatic and extrahepatic recurrence were independent predictors for both TTR and OS. CONCLUSIONS: Patients with intrahepatic recurrence following liver resection have a significantly shorter TTR and OS when compared to patients developing extrahepatic recurrence. Pulmonary recurrence following resection is associated with longer survival. Simultaneous intra- and extrahepatic recurrence is an independent prognostic factor for TTR and OS.

Nitrate leaching in californian rice fields: a field- and regional-scale assessment.

Irrigated croplands can be a major source of nitrate-N (NO-N) in groundwater due to leaching. In California, where high NO-N levels have been found in some areas of the Central Valley aquifer, the contribution from rice systems has not been determined. Nitrate leaching from rice systems was evaluated from soil cores (0-2 m), from the fate of N fertilizer in replicated microplots, and from about 145 regional groundwater wells. Soil NO-N concentrations were ≤3.3 mg kg (usually <1 mg kg) below the root zone (below 33 cm depth). In pore-water samples, NO-N was observed only below the root zone during the first 2 wk after the onset of flooding in either the growing season or the winter fallow period and was always ≤8.4 mg L. Fertilizer N accounted for 0 to 11.8% of NO-N in pore-water samples below the root zone. One year after application, based on an analysis of soil core samples, on average 2.5% of fertilizer N was recovered as N below the root zone (33-100 cm), possibly due to leaching in permeable soils or via preferential flow through cracks in heavy clay soils. Based on a regional assessment, groundwater samples from wells that are located in proximity to rice fields all had measured median NO-N and NO-N levels below 1 mg L. These results indicate that NO-N leaching from the majority of California rice systems poses little risk to groundwater under current crop management practices.

Radiological heterogeneity in response to chemotherapy is associated with poor survival in patients with colorectal liver metastases.

BACKGROUND: In patients with colorectal liver metastases (CLM) there is limited knowledge about the occurrence of radiological heterogeneity in response to chemotherapy. METHODS: A retrospective analysis was performed in the CAIRO and CAIRO II studies on the incidence of intermetastatic heterogeneity in patients with CLM and its association with survival. Mixed response (MR) was defined as >30% difference in individual lesion response, with all lesions showing a similar behaviour; true mixed response (TMR) as two lesions showing progression versus response; homogeneous response (HR) as similar behaviour of all lesions. Patients were classified according to the Response Evaluation Criteria in Solid Tumours (RECIST) categories (partial response (PR), stable disease (SD), progressive disease (PD), complete response (CR)) and then subdivided into MR and TMR in order to compare survival. RESULTS: In the CAIRO and CAIRO II studies, 140 and 150 patients with liver-only disease were identified. 73/290 (25.2%) patients showed MR, and 25/290 (8.6%) patients TMR, and 192/290 (66.2%) patients HR. Overall survival (OS) at 1-4 years was significantly higher for the homogeneous partial responders category compared to other response categories. Median OS was 22.0 months for the entire population. In the partial response category, patients with MR showed significant poorer survival compared to patients with HR (median OS 23.7 versus 36.0 months, respectively, p=0.019). Multivariate analysis identified four independent predictors for OS: serum lactate dehydrogenase (LDH) level (p=0.002), number of first-line chemotherapy cycles (p=0.001), resection of primary tumour (p=0.001) and response category (p=0.012). CONCLUSION: Radiological heterogeneity is present in approximately 35% of patients with CLM. Partial responders according to the RECIST criteria, show a significant poorer survival if classified as heterogeneous partial responder compared to homogeneous partial responders.

Patient tailored resection planning in patients undergoing liver surgery for colorectal liver metastases; how and why should you do it?

BACKGROUND: A systematic preoperative evaluation to determine the individual resection strategy in patients with colorectal liver metastases (CRLM) was assessed as to its clinical value. PATIENTS AND METHODS: From 2009 to 2011, 75 patients with CRLM who were scheduled for surgery were prospectively included and received an additional preoperative systematic evaluation in the presence of a hepatobiliary radiologist and the hepatobiliary surgeon scheduled to perform the surgery. The following items were assessed in a standardized manner: lesion detection and characterization, presence of extrahepatic disease, vascular anatomy, and resection strategy. Intraoperative findings and histopathological results were prospectively recorded. RESULTS: Five out of 75 patients were not considered to be eligible for surgery due to additional findings, such as additional metastases or extrahepatic disease. Sensitivity and specificity for detection of individual CRLM were 80.9% (95% CI 75.7-86.1%) and 69.1% (95% CI 59.1-79.1%), respectively. Radical resections were performed in 87.1%. There was one futile laparotomy (1.4%). CONCLUSION: In patients with colorectal liver metastases, standardized preoperative work-up, with subsequent planning of an individualized resection in a jointed meeting of a hepatobiliary radiologist and the surgeon who will perform the operation, leads to a high level of radical resections and a low number of futile laparotomies.

Focal nodular hyperplasia: hepatobiliary enhancement patterns on gadoxetic-acid contrast-enhanced MRI.

OBJECTIVES: To assess the range of hepatobiliary enhancement patterns of focal nodular hyperplasia (FNH) after gadoxetic-acid injection, and to correlate these patterns to specific histological features. MATERIALS AND METHODS: FNH lesions, imaged with Gadoxetic-acid-enhanced MRI, with either typical imaging findings on T1, T2 and dynamic-enhanced sequences or histologically proven, were evaluated for hepatobiliary enhancement patterns and categorized as homogeneously hyperintense, inhomogeneously hyperintense, iso-intense, or hypo-intense-with-ring. Available histological specimens of FNHs (surgical resection or histological biopsy), were re-evaluated to correlate histological features with observed enhancement patterns. RESULTS: 26 FNHs in 20 patients were included; histology was available in six lesions (four resections, two biopsies). The following distribution of enhancement patterns was observed: 10/26 homogeneously hyperintense, 4/26 inhomogeneously hyperintense, 5/26 iso-intense, 6/26 hypointense-with-ring, and 1/26 hypointense, but without enhancing ring. The following histological features associated with gadoxetic-acid uptake were identified: number and type of bile-ducts (pre-existent bile-ducts, proliferation, and metaplasia), extent of fibrosis, the presence of inflammation and extent of vascular proliferation. CONCLUSION: FNH lesions can be categorized into different hepatobiliary enhancement patterns on Gadoxetic-acid-enhanced MRI, which appear to be associated with histological differences in number and type of bile-ducts, and varying the presence of fibrous tissue, inflammation, and vascularization.

MR liver imaging with Gd-EOB-DTPA: a delay time of 10 minutes is sufficient for lesion characterisation.

OBJECTIVES: To assess whether, in patients with normal liver function, a hepatobiliary delay time of 10 min after Gd-EOB-DTPA injection is sufficient for lesion characterisation. METHODS: In 42 consecutive patients with suspected focal liver lesions, dynamic MRI was performed after intravenous Gd-EOB-DTPA, followed by hepatobiliary phases at 5, 10 and 20 min. The following items were assessed at each hepatobiliary phase: parenchymal enhancement, contrast agent excretion in bile ducts, lesion enhancement characteristics (hypo-, iso-, or hyperintensity, rim enhancement, central non-enhancement), and contrast- and signal-to-noise ratios, separately for hypo- and hyperintense lesions. RESULTS: Following enhancement, parenchymal signal intensity increased significantly up to 10 min (86.3%, P < 0.001), and subsequently stabilised (86.5% after 20 min, P = 0.223). Biliary contrast agent excretion was first observed in 2, 32 and 5 patients after 5, 10 and 20 min respectively. Hepatobiliary lesion enhancement characteristics observed after 5 min persisted during later hepatobiliary phases. CNR and SNR ratios increased significantly (P < 0.05) up to 10 min after enhancement without further increase at 20 min, in hypo- and hyperintense lesions. CONCLUSIONS: If lesion characterisation is the primary reason for performing MRI, a hepatobiliary delay time of 10 min after Gd-EOB-DTPA injection is sufficient in patients with normal liver function. KEY POINTS : • Magnetic resonance imaging is now a first line of investigation of the liver. • Optimal CNR and SNR are achieved 10 min after Gd-EOB-DTPA injection. • Typical enhancement characteristics are observed early and do not change. • Ten-minute hepatobiliary delay is sufficient for characterisation of focal liver lesions.

Accuracy of multislice liver CT and MRI for preoperative assessment of colorectal liver metastases after neoadjuvant chemotherapy.

INTRODUCTION: To determine the best imaging modality for preoperative detection, characterization and measurement of colorectal liver metastases (CRLM) after neoadjuvant chemotherapy (NAC). METHODS: A total of 79 lesions in 15 patients with CRLM were included. Following NAC, all patients received multislice liver CT (MSCT) and magnetic resonance imaging (MRI) that were scored by two observers for lesion number, type, diameter (mm) and segmental location. Intraoperative findings, histopathology and follow-up imaging were used as reference standard for surgically treated patients; non-surgical candidates underwent follow-up imaging. RESULTS: Lesion detection rate was similar for MSCT and MRI (76 and 80%, respectively, p = 0.648). Lesion characterization was significantly superior (p = 0.021) at MRI (89%, κ 0.747, p = 0.001) compared to MSCT (77%, κ 0.235, p = 0.005). Interobserver variability for diameter measurement was not significant at MRI (p = 0.909 [95% CI -1.245 to 1.395]), but significant at MSCT (p = 0.028 [95% CI -3.349 to -2.007]). Differences in diameter measurement were independent of observer (p = 0.131), and no statistical effect from imaging modality on diameter measurement was observed (p = 0.095). CONCLUSION: MRI is superior to MSCT in preoperative characterization and measurement of CRLM after NAC. Lesion detection rates for both modalities are comparable.

Quantifying the impact of regular cutting on vegetative buffer efficacy for nitrogen-15 sequestration.

This study used the stable 15N isotope to quantitatively examine the effects of cutting on vegetative buffer uptake of NO3(-)-N based on the theory that regular cutting would increase N demand and sequestration by encouraging new plant growth. During the summer of 2002, 10 buffer plots were established within a flood-irrigated pasture. In 2003, 15N-labeled KNO3 was applied to the pasture area at a rate of 5 kg N ha(-1) and 99.7 atom % 15N. One-half of the buffer plots were trimmed monthly. In the buffers, the cutting effect was not significant in the first few weeks following 15N application, with both the cut and uncut buffers sequestering 15N. Over the irrigation season, however, cut buffers sequestered 2.3 times the 15N of uncut buffers, corresponding to an increase in aboveground biomass following cutting. Cutting and removing vegetation allowed the standing biomass to take advantage of soil 15N as it was released by microbial mineralization. In contrast, the uncut buffers showed very little change in 15N sequestration or biomass, suggesting senescence and a corresponding decrease in N demand. Overall, cutting significantly improved 15N attenuation from both surface and subsurface water. However, the effect was temporally related, and only became significant 21 to 42 d after 15N application. The dominant influence on runoff water quality from irrigated pasture remains irrigation rate, as reducing the rate by 75% relative to the typical rate resulted in a 50% decrease in total runoff losses and a sevenfold decrease in 15N concentration.

Using nitrogen-15 to quantify vegetative buffer effectiveness for sequestering nitrogen in runoff.

Previous studies have observed higher levels of soluble nutrients leaving vegetative buffers than entering them, suggesting that the buffers themselves are acting as a source rather than a sink by releasing previously stored nutrients. This study used 98 atom % (15)N-labeled KNO(3) at a rate of 5 kg ha(-1) to quantify buffer efficiency for sequestering new inputs of NO(-)(3)-N in an extensively grazed irrigated pasture system. Buffer treatments consisted of an 8-m buffer, a 16-m buffer, and a nonbuffered control. Regardless of the form of runoff N (NO(-)(3), NH(+)(4), or dissolved organic nitrogen [DON]), more (15)N was lost from the nonbuffered treatments than from the buffered treatments. The majority of the N attenuation was by vegetative uptake. Over the course of the study, the 8-m buffer decreased NO(-)(3)-(15)N load by 28% and the 16-m buffer decreased load by 42%. For NH(+)(4)-(15)N, the decrease was 34 and 48%, and for DON-(15)N, the decrease was 21 and 9%. Although the buffers were effective overall, the majority of the buffer impact occurred in the first four weeks after (15)N application, with the buffered plots attenuating nearly twice as much (15)N as the nonbuffered plots. For the remainder of the study, buffer effect was not as marked; there was a steady release of (15)N, particularly NO(-)(3)- and DON-(15)N, from the buffers into the runoff. This suggests that for buffers to be sustainable for N sequestration there is a need to manage buffer vegetation to maximize N demand and retention.

What is the Revised Fear Survey Schedule for Children measuring?

The Fear Survey Schedule for Children-Revised (FSSC-R) is a widely used self-report questionnaire that purports to measure the number of fears and the overall level of fearfulness in children. A number of studies have shown that the ten most common childhood fears can be found on the Danger and Death subscale of the FSSC-R, with upwards of 50% of children endorsing such fears. However, some researchers (e.g., H. McCathie & S.H. Spence, 1991; Behaviour Research and Therapy, 29, 495-502) have questioned the validity of these findings, suggesting that these items do not reflect actual childhood fears that children have or experience on a daily or regular basis. Rather, they suggest that children are responding to these fear items as if they were actually occurring to them in the here and now. The current study examined the occurrence of five Danger and Death fears from the FSSC-R (i.e., "Not being able to breathe", "Being hit by a car or truck", "Falling from high places", "Bombing attacks or being invaded", and "Fire or getting burned") in a sample of normal school children aged eight to 12 years (N=102). More specifically, we used three different methods to asses these fears: (1). prevalence as determined by the standard FSSC-R procedure, (2). prevalence as determined by a fear list procedure, and (3). actual occurrence or prevalence of these fears in the past week, as determined by a diary method. Results indicated that while these fears ranked high when using the standard FSSC-R procedure, they were considerably less common when using the fear list procedure, and had a low probability of actual occurrence on a daily basis, as well as possessing a short duration and low intensity. Implications for the assessment of fears and the use of self-report measures like the FSSC-R are briefly discussed.

Effects of granulocyte colony-stimulating factor (G-CSF) treatment on granulocyte function and receptor expression in patients with ventilator-dependent pneumonia.

Considerable experimental evidence in animals suggests that treatment with G-CSF may have a beneficial effect in the management of severe infections in non-neutropenic hosts. This beneficial effect is attributed to an enhancement of granulopoiesis and neutrophil function, the latter possibly involving up-regulation of receptors on neutrophils that are involved in antibody-mediated cytotoxicity and killing of microorganisms. We compared neutrophil function and phenotype in blood and bronchoalveolar lavage fluid (BALF) of 10 patients with severe ventilator-dependent pneumonia, at baseline and following initiation of G-CSF treatment as adjunct to standard therapy. G-CSF treatment was associated with three-fold increased blood neutrophil counts at day 3 of treatment compared with baseline counts. Mean serum G-CSF concentration increased from 313 to 2007 pg/ml. After correction for lavage dilution effects, BALF G-CSF levels did not differ significantly from baseline, nor did neutrophil receptor expression (FcgammaRI, FcgammaRII, FcgammaRIII, CR3, and L-selectin) or indicators of neutrophil function such as respiratory burst activity, phagocytosis and killing of Candida albicans in BALF or blood. The mortality in this group of patients was 30% and compared favourably to the APACHE II-derived predicted mortality of 60%. We conclude that the possible therapeutic benefit of G-CSF administration in the early phase of severe bacterial pneumonia is not readily explained by its effect on baseline indicators of neutrophil function or receptor expression.

Does nitrogen nutrition restrict the CO2 response of fertile grassland lacking legumes?

The extent of the response of plant growth to atmospheric CO2 enrichment depends on the availability of resources other than CO2. An important growth-limiting resource under field conditions is nitrogen (N). N may, therefore, influence the CO2 response of plants. The effect of elevated CO2 (60 Pa) partial pressure (pCO2) on the N nutrition of field-grown Lolium perenne swards, cultivated alone or in association with Trifolium repens, was investigated using free air carbon dioxide enrichment (FACE) technology over 3 years. The established grassland ecosystems were treated with two N fertilization levels and were defoliated at two frequencies. Under elevated pCO2, the above-ground plant material of the L. perenne monoculture showed a consistent and significant decline in N concentration which, in general, led to a lower total annual N yield. Despite the decline in the critical N concentration (minimum N concentration required for non-N-limited biomass production) under elevated pCO2, the index of N nutrition (ratio of actual N concentration and critical N concentration) was lower under elevated pCO2 than under ambient pCO2 in frequently defoliated L. perenne monocultures. Thus, we suggest that reduced N yield under elevated pCO2 was evoked indirectly by a reduction of plant-available N. For L. perenne grown in association with T. repens and exposed to elevated pCO2, there was an increase in the contribution of symbiotically fixed N to the total N yield of the grass. This can be explained by an increased apparent transfer of N from the associated N2-fixing legume species to the non-fixing grass. The total annual N yield of the mixed grass/legume swards increased under elevated pCO2. All the additional N yielded was due to symbiotically fixed N. Through the presence of an N2-fixing plant species more symbiotically fixed N was introduced into the system and consequently helped to overcome N limitation under elevated pCO2.