Knowledge, attitude, and adherence to malaria control guidelines and the prevalence of Plasmodium species infection in localities across transmission and ecological zones in Cameroon.

Background: Despite a scale up of control interventions over the years, malaria remains a major public health and economic concern in Cameroon, contributing considerably to hospitalization and deaths. The effectiveness of control strategies depends on the extent of adherence by the population to national guidelines. This study assessed the influence of human knowledge, attitudes, and practices related to malaria and its control on the prevalence of malaria parasite infection, with implications for the elimination of the disease. Methodology: This is a cross-sectional community and hospital-based study, covering the five ecological and three malaria transmission zones in Cameroon. A pre-tested semi-structured questionnaire was used to document socio-demographic and clinical parameters as well as knowledge, attitudes, and practices toward malaria control and management. Consenting participants were screened for malaria parasite with rapid diagnostic test (mRDT) of the peripheral blood. Association between qualitative variables was determined using the chi-square test and logistic regression analysis. Results: A total of 3,360 participants were enrolled, 45.0% (1,513) of whom were mRDT positive, with 14.0% (451/3,216) and 29.6% (951/3,216) having asymptomatic parasitaemia and malaria, respectively. Although most participants knew the cause, symptoms, and control strategies, with 53.6% (1,000/1,867) expertly knowledgeable about malaria overall, only 0.1% (2/1,763) individuals were fully adherent to malaria control measures. Conclusion: The risk of malaria in Cameroon remains high, with the population considerably knowledgeable about the disease but poorly adherent to national malaria control guidelines. Concerted and more effective strategies aimed at improving knowledge about malaria and adherences to control interventions are necessary to ultimately eliminate the disease.

Pre-transplant platelet-to- lymphocyte ratio predicts outcome after allogeneic hematopoietic stem cell transplantation.

For many patients with hematological malignancies such as acute leukemia or myelodysplastic syndrome allogeneic hematopoietic stem cell transplantation (allogeneic HSCT) is the only curative treatment option. Despite the curative potential of this treatment many patients experience relapse of their underlying disease or die due to multiple complications e.g. infections. Risk scores could help to assess the individual prognosis and guide patients and treating physicians to choose between different treatment options. Parameters reflecting the inflammatory status, such as neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and platelet-to-lymphocyte ratio (PLR), have been demonstrated to be associated with prognosis and treatment complications in patients with various cancers. In this study, we evaluate pre-HSCT NLR, MLR and PLR as predictive markers in patients undergoing allogeneic HSCT. We demonstrate that a high (> 133) PLR level is associated with better clinical outcome. Patients with high pre-HSCT PLR show a significant better overall survival (p = 0.001), less relapses (p = 0.016), lower non-relapse-mortality (p = 0.022), less transfusions of red blood cells, platelets and fresh frozen plasma (p = 0.000), fewer episodes of fever (p = 0.002), considerably less different antibiotics (p = 0.005), fewer intensive care unit treatment (p = 0.017) and a lower in-hospital mortality (p = 0.024). Pre-HSCT PLR is easy to calculate by daily routine and could help to predict patient outcome after allogeneic HSCT.

An analysis of social dimensions of podoconiosis and leprosy on affected households in endemic health districts of the North West Region of Cameroon.

Background: Podoconiosis and leprosy are Neglected Tropical Diseases associated with low quality of life, social stigma and isolation of affected people and families. Despite the substantial social burden it imposes, podoconiosis has largely been ignored in the global health literature until recently unlike leprosy. This study assessed and compared the quality of life and social impact of podoconiosis with that of leprosy among affected households and neighborhoods in North West Cameroon. Methods: A comparative cross-sectional design was used. Eighty-six households: 43 podoconiosis and 43 leprosy, plus household neighbours were enrolled from July and August 2015 from three health districts. Podoconiosis patients living in households within Batibo and Ndop health districts were sequentially sampled using a list of confirmed podoconioisis cases from previous studies. Leprosy patients living within communities in Mejang Health Area were sequentially sampled using the Mbingo treatment center register. WHO BREF tool was used to assess quality of life. Franklin Stigma Scale was adapted to assess felt and enacted stigma. Mann-Whitney U test was used to compare differences in stigma and QoL. Results: Physical domain showed a significant difference in the distribution in quality of life between groups (p < 0.05, median:70; U:635, r = 0.2). Overall enacted stigma revealed significant differences with p < 0.05 and r = 0.4. Overall stigma from family members (median:17, U:627 and r = 0.3) and neighbours (median:67, U:336 and r = 0.5) showed significant differences with p < 0.05 in the distribution of scores for both diseases. Sex and age showed significant associations with QoL and stigma. Conclusion: This study reveals the quality of life and stigma associated with podoconiosis on affected households to be comparable to that experienced by households with a leprosy patient. There is need for intensified preventive, management and control schemes to fight podoconiosis in Cameroon, just like leprosy.

A Comparative Analysis of Economic Cost of Podoconiosis and Leprosy on Affected Households in the Northwest Region of Cameroon.

Leprosy and podoconiosis (podo) are neglected tropical diseases that cause severe disfigurement and disability, and may lead to catastrophic health expenditure and hinder economic development of affected persons and households. This study compared economic costs of both diseases on affected households with unaffected neighboring households in the Northwest Region (N.W.R.) of Cameroon. A matched comparative cross-sectional design was used enrolling 170 households (43 podo case households, 41 podo control households, 43 leprosy case households, and 43 leprosy control households) from three health districts in the N.W.R. Direct treatment costs for podo averaged 142 United State dollar (USD), compared with zero for leprosy (P < 0.001). This was also reflected in the proportion of annual household income consumed (0.4 versus 0.0, respectively, P < 0.001). Both diseases caused considerable reductions in working days (leprosy 115 versus podo 135 days. P for comparison < 0.001). The average household income was considerably lower in podo-affected households than unaffected households (410 versus 913 USD, P = 0.01), whereas income of leprosy-affected households was comparable to unaffected households (329 versus 399 USD, P = 0.23). Both leprosy and podo cause financial burdens on affected households, but those on podo-affected families are much greater. These burdens occur through direct treatment costs and reduced ability to work. Improved access to public health interventions for podo including prevention, morbidity management and disability prevention are likely to result in economic returns to affected families. In Cameroon, one approach to this would be through subsidized health insurance for these economically vulnerable households.

Diagnostic value of sTREM-1, IL-8, PCT, and CRP in febrile neutropenia after autologous stem cell transplantation.

Infections and infectious complications are the major cause of morbidity and mortality in febrile neutropenic patients after autologous stem cell transplantation. Laboratory biomarkers are helpful for early identification of critically ill patients and optimal therapy management. Several studies in adult non-neutropenic patients proposed sTREM-1 as a superior biomarker for identification of septic patients as well as a predictor for survival in these patients compared with procalcitonin (PCT), C-reactive protein (CRP), or interleukin-8 (IL-8). Here, to assess the utility of PCT, CRP, IL-8, and sTREM-1 in febrile neutropenia, 44 patients presenting with febrile neutropenia after autologous stem cell transplantation were recruited in a single-center prospective pilot study. We analyzed PCT and CRP as well as IL-8 and sTREM-1 levels pre- and post-transplantation at defined time points. In 20 of 44 patients, concentration of sTREM-1 was under the detection level at appearance of febrile neutropenia. Mean levels of PCT, IL-8, and CRP were significantly increased in infections of critically ill patients who by dysfunction or failure of one or more organs/system depend on survival from advanced instruments of monitoring and therapy. However, all tested biomarkers could not distinguish between presence and absence of bloodstream infection. The combination of the biomarkers PCT and IL-8 achieved a high sensitivity of 90% and specificity of 74% for the identification of serious complications in febrile neutropenia, whereas the combination of CRP and PCT or IL-8 achieved a high sensitivity of 100%, but with the addition of a low specificity of 47or 41%. In conclusion, we found that the measurement of sTREM-1 concentration at presentation of febrile neutropenia is not useful to identify bacterial bloodstream infections and critically ill patients. PCT and IL-8 are useful biomarkers for the early identification of critically ill patients, compared to CRP and sTREM-1 in febrile neutropenia. PCT or IL-8 in combination with clinical parameters should be considered in routine measurement to identify critically ill patients as early as possible.

Understanding of research, genetics and genetic research in a rapid ethical assessment in north west Cameroon.

BACKGROUND: There is limited assessment of whether research participants in low-income settings are afforded a full understanding of the meaning of medical research. There may also be particular issues with the understanding of genetic research. We used a rapid ethical assessment methodology to explore perceptions surrounding the meaning of research, genetics and genetic research in north west Cameroon. METHODS: Eleven focus group discussions (including 107 adults) and 72 in-depth interviews were conducted with various stakeholders in two health districts in north west Cameroon between February and April 2012. RESULTS: Most participants appreciated the role of research in generating knowledge and identified a difference between research and healthcare but gave varied explanations as to this difference. Most participants' understanding of genetics was limited to concepts of hereditary, with potential benefits limited to the level of the individual or family. Explanations based on supernatural beliefs were identified as a special issue but participants tended not to identify any other special risks with genetic research. CONCLUSION: We demonstrated a variable level of understanding of research, genetics and genetic research, with implications for those carrying out genetic research in this and other low resource settings. Our study highlights the utility of rapid ethical assessment prior to complex or sensitive research.

[Immunological tumor therapy]. / Immunologische Tumortherapie.

Tumor cells could fundamentally be recognized and eliminated by the immune system but malignant cells are able to escape the immune surveillance system. The idea of immunotherapy of cancer is to activate, modulate and amplify the host immune response or to genetically equip the immune repertoire of patients with anti-tumor specificities and effectors. In recent years, a variety of promising immunotherapy strategies have been developed, such as bispecific, multispecific and immunoregulatory antibodies, gene-modified T lymphocytes and tumor vaccines. Some drugs have already been approved and others are available for patients in clinical trials. This article presents the current anti-tumor immune strategies and their molecular basis. Even though further research is needed in some areas, such as the establishment of biomarkers for targeted therapy, duration of therapeutic activity and compatibility of combined strategies, cancer immunotherapy is likely to be a key component in oncological treatment concepts in the very near future.

Safety and efficacy of Temsirolimus in combination with Bendamustine and Rituximab in relapsed mantle cell and follicular lymphoma.

In this phase I/II study, we explored the combination of Temsirolimus with Bendamustine and Rituximab (BeRT) in patients with r/r follicular lymphoma (FL) or mantle cell lymphoma (MCL). Patients with 1-3 prior therapies received Bendamustine (90 mg/m(2), day 1+2) and Rituximab (375 mg/m(2), day 1) with Temsirolimus in doses from 25 to 75 mg added on day 1, 8, 15 of a 28-day cycle. Fifteen (11 MCL, 4 FL) patients were included in the phase I. Median age was 73 years and median pretreatment number was 2. No formal dose-limiting toxicity was observed. Dominant non-hematological side effects were fatigue in 11 (73%), nausea in 9 (60%), mucositis in 7 (47%) and vomiting in 6 patients (40%). Cough, diarrhea, pyrexia and rash were observed in five patients (33%) each. Grade 3/4 events included leukopenia in 6 (40%), neutropenia in 4 (27%) and thrombocytopenia in 2 patients (13%). An objective response was observed in 14/15 patients (93%), including 5 complete response (33%; all MCL). After a median follow-up of 19 months, 67% of patients are without signs of progression. Temsirolimus can be safely added to BR with promising preliminary activity. Recruitment in phase II is ongoing.

Perceptions of consent, permission structures and approaches to the community: a rapid ethical assessment performed in North West Cameroon.

BACKGROUND: Understanding local contextual factors is important when conducting international collaborative studies in low-income country settings. Rapid ethical assessment (a brief qualitative intervention designed to map the ethical terrain of a research setting prior to recruitment of participants), has been used in a range of research-naïve settings. We used rapid ethical assessment to explore ethical issues and challenges associated with approaching communities and gaining informed consent in North West Cameroon. METHODS: This qualitative study was carried out in two health districts in the North West Region of Cameroon between February and April 2012. Eleven focus group discussions (with a total of 107 participants) were carried out among adult community members, while 72 in-depth interviews included health workers, non-government organisation staff and local community leaders. Data were collected in English and pidgin, translated where necessary into English, transcribed and coded following themes. RESULTS: Many community members had some understanding of informed consent, probably through exposure to agricultural research in the past. Participants described a centralised permission-giving structure in their communities, though there was evidence of some subversion of these structures by the educated young and by women. Several acceptable routes for approaching the communities were outlined, all including the health centre and the Fon (traditional leader). The importance of time spent in sensitizing the community and explaining information was stressed. CONCLUSIONS: Respondents held relatively sophisticated understanding of consent and were able to outline the structures of permission-giving in the community. Although the structures are unique to these communities, the role of certain trusted groups is common to several other communities in Kenya and Ethiopia explored using similar techniques. The information gained through Rapid Ethical Assessment will form an important guide for future studies in North West Cameroon.

Depletion of naive T cells using clinical grade magnetic CD45RA beads: a new approach for GVHD prophylaxis.

Depletion of naive T cells from donor leukapheresis products (LPs) aims at the reduction of alloreactivity, while preserving memory T-cell reactivity (for example, to pathogens). This study established the immunomagnetic depletion procedure under clean room conditions using CD45RA beads and analyzed LPs of six donors for cell composition and functional immune responses. CD45RA depletion resulted in 3.4-4.7 log (median 4.4) reduction of CD45RA(+) T cells, thereby eliminating naive and late effector T cells. B cells were also completely removed, whereas significant proportions of NK cells, monocytes and granulocytes persisted. CD45RA-depleted LPs contained effector and central memory CD4(+) and CD8(+) T cells that showed sustained IFN-γ secretion to CMV, EBV, Aspergillus and Candida Ags. Alloreactivity was measured in MLRs between donors with complete HLA-mismatch. Alloreactive CD8(+) T cells were strongly reduced (median >1-log) upon CD45RA depletion, whereas alloreactive CD4(+) T cells persisted in significant numbers. In conclusion, clinical grade depletion of CD45RA(+) naive T cells from donor LPs is feasible and highly efficient. The depleted products show sustained CD4(+) and CD8(+) T-cell reactivity to pathogens and effectively reduced CD8-mediated alloreactivity. Prophylactic and preemptive infusions after allogeneic SCT may improve T-cell reconstitution and pathogen-specific immunosurveillance, along with lower risk of inducing GVHD.

SDF1ß expression in renal cell carcinoma correlates with grading and infiltration by CD8+ T-cells.

For several tumor entities, a significant correlation between the chemokine stromal cell-derived factor 1 (SDF1) and its receptor C-X-C chemokine receptor type 4 (CXCR4), metastasis and tumor proliferation, as well as prognosis, has been described. In this study, a series of 105 renal cell carcinoma patients were analyzed in terms of expression of SDF1α and SDF1ß and infiltration by CD4+ and CD8+ T-cells and the data correlated with TNM category, grading and survival. While the splice variant SDF1α had no impact on tumor grading, T-cell invasion or overall survival, expression of SDF1ß showed a significant correlation with tumor grading and also suggested a correlation with metastasis, as well as CD8+ T-cell invasion. These results indicate a potential T-cell-mediated antitumor response induced by SDF1ß up-regulation. Therefore targeting the SDF1ß-CXCR4 signaling pathway may be a promising means for new therapeutic strategies in advanced tumor stages.

NitroScape: a model to integrate nitrogen transfers and transformations in rural landscapes.

Modelling nitrogen transfer and transformation at the landscape scale is relevant to estimate the mobility of the reactive forms of nitrogen (N(r)) and the associated threats to the environment. Here we describe the development of a spatially and temporally explicit model to integrate N(r) transfer and transformation at the landscape scale. The model couples four existing models, to simulate atmospheric, farm, agro-ecosystem and hydrological N(r) fluxes and transformations within a landscape. Simulations were carried out on a theoretical landscape consisting of pig-crop farms interspersed with unmanaged ecosystems. Simulation results illustrated the effect of spatial interactions between landscape elements on N(r) fluxes and losses to the environment. More than 10% of the total N(2)O emissions were due to indirect emissions. The nitrogen budgets and transformations of the unmanaged ecosystems varied considerably, depending on their location within the landscape. The model represents a new tool for assessing the effect of changes in landscape structure on N(r) fluxes.

Observation of the stabilizing effect of a laminated ablator on the ablative Rayleigh-Taylor instability.

A laminated ablator is explored as an alternative concept for stabilizing the ablative Rayleigh-Taylor instability which develops in inertial fusion targets. Experiments measuring the growth of the Rayleigh-Taylor instability of laminated planar foils are reported. Consistent with both theory and simulations, a significant reduction of the perturbation growth is experimentally observed for laminated ablators in comparison to what is observed for classical uniform ablators. Such an enhanced hydrodynamic stability opens opportunities for the design of high-gain inertial fusion targets.

In vitro stimulation and expansion of human tumour-reactive CD8+ cytotoxic T lymphocytes by anti-CD3/CD28/CD137 magnetic beads.

Adoptive immunotherapy with tumour-reactive CD8(+) cytotoxic T lymphocytes (CTLs) requires efficient in vitro approaches allowing the expansion of CTLs to large numbers prior infusion. Here, we investigated the antigen-independent activation and the expansion of human T cells in peripheral blood mononuclear cells (PBMCs) and in tumour-reactive CTLs using Dynabeads coated with monoclonal antibodies to CD3 and to the costimulatory molecules CD28 and CD137 (4-1BB). T cells in PBMCs showed an increased expansion rate of 15- to 17-fold during a 2-week culture period using antibody-conjugated beads with interleukin-2 (IL-2) added versus IL-2 alone. No significant difference between CD3/CD28 beads and CD3/CD28/CD137 beads was observed (P = 0.4). In contrast, expansion of tumour-reactive CD8(+) CTLs over 2 weeks was more efficient using CD3/CD28/CD137 beads (14.4-fold ± 1.2) compared with CD3/CD28 beads (10.6-fold ± 0.7) (P = 0.03) and matched well to the control arm using weekly stimulation with tumour cells. Although all modes of in vitro stimulation decreased the expression of central memory markers CD62L and CCR7 on CTLs, bead-activated cultures expressed consistently higher levels than tumour-stimulated cultures. CTLs analysed after bead-induced expansion versus weekly tumour stimulation showed equal IFN-γ production in ELISPOT assay. Furthermore, cytotoxicity assays demonstrated an either unchanged or slightly reduced capability of tumour cell lysis for antigen-independent stimulated CTLs versus those that maintained on weekly tumour stimulation, regardless of which type of beads was used. Our data suggest that the conjugation of anti-CD137 antibodies to conventional CD3/CD28 beads results in a minor but significant increase in the expansion capacity for tumour-reactive CD8(+) CTLs.

A simple model for screening the local impacts of atmospheric ammonia.

The dry deposition of ammonia from the atmosphere to the surface can lead to eutrophication of sensitive ecosystems and acidification of the soil. A large proportion of the ammonia emitted from agricultural sources can be deposited within a few kilometres and, therefore, impacts of ammonia dry deposition often occur near to the source. To assess these impacts, short-range atmospheric dispersion models are often applied to simulate the emission, dispersion and deposition of ammonia. However, these models can be time-consuming to run and often require detailed input data and, therefore, for multiple assessments it is useful to have a method of screening to discard scenarios where impacts are expected to be negligible. The SCAIL model (Simple Calculation of Ammonia Impact Limits) has been developed for this purpose. SCAIL estimates the atmospheric concentration and dry deposition at the nearest edge of a sensitive ecosystem (receptor) downwind of an ammonia source. These estimates are calculated based on simple meteorological data, the emission rate of the source, land cover type and distance to the receptor. Analysis of the model predictions showed that uncertainty in the model input data leads to an uncertainty in concentration and dry deposition estimates of 25-30% and 40-45% respectively. Detailed atmospheric dispersion models will also have similar uncertainties since they use similar types of input data. Comparison of the concentration predictions with previous measurements made around eight farms showed that the model significantly underestimated concentrations although the model performance was similar to existing screening techniques. The measurement dataset was used to calibrate the SCAIL model which subsequently performed better, using independent verification data, than existing models calibrated in a similar way. The benefits of the SCAIL model are already being seen in the UK, where it is used to screen farms for potential impacts on statutory nature conservation areas.

Very rapid clearance after a joint bleed in the canine knee cannot prevent adverse effects on cartilage and synovial tissue.

OBJECTIVE: Joint bleeding leads to joint destruction. In vitro exposure of human and canine cartilage to blood results in long-lasting severe adverse changes in cartilage. An in vivo joint haemorrhage in the canine knee joint demonstrates similar adverse effects although significantly less outspoken. As a possible explanation for this discrepancy, we studied the clearance rate of blood from the canine knee joints. METHODS: Blood was injected into the knee joint of Beagle dogs either 48 h, 24h or 15 min before termination. The amount of red blood cells (RBC) and white blood cells (WBCs) present in the joint cavity was determined. Chondrocyte activity and cartilage matrix integrity as well as cartilage destructive activity of synovial tissue were determined biochemically. Additionally, synovial tissue was analyzed by use of histochemistry. RESULTS: The amount of blood was decreased to <5% within 48 h. Within this time period the cartilage was negatively affected and the synovial tissue showed cartilage destructive activity. Evaluation of the synovial tissue 15 min post-injection revealed countless numbers of intact RBC that were almost completely disappeared after 48 h without significant recruitment of macrophages. CONCLUSIONS: Blood is cleared very rapidly from the canine knee joint, but already has adverse effects on both cartilage and synovial tissue within that short time span. This rapid clearance can play a role in the discrepancy between long-term in vitro and in vivo effects of blood-induced joint damage since more than 10% v/v blood present for at least 48 h is needed to induce long-term adverse effects in vitro.

The programmed death (PD)-1/PD-ligand 1 pathway regulates graft-versus-host-reactive CD8 T cells after liver transplantation.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after solid-organ transplantation, which is mediated by host-reactive donor T cells emigrating from the allograft. We report on two liver transplant recipients who developed an almost complete donor chimerism in peripheral blood and bone marrow-infiltrating T cells during aGVHD. By analyzing these T cells directly ex vivo, we found that they died by apoptosis over time without evidence of rejection by host T cells. The host-versus-donor reactivity was selectively impaired, as anti-third-party and antiviral T cells were still detectable in the host repertoire. These findings support the acquired donor-specific allotolerance concept previously established in animal transplantation studies. We also observed that the resolution of aGVHD was not accompanied by an expansion of circulating immunosuppressive CD4/CD25/FoxP3-positive T cells. In fact, graft-versus-host-reactive T cells were controlled by an alternative negative regulatory pathway, executed by the programmed death (PD)-1 receptor and its ligand PD-L1. We found high PD-1 expression on donor CD4 and CD8 T cells. In addition, blocking PD-L1 on host-derived cells significantly enhanced alloreactivity by CD8 T cells in vitro. We suggest the interference with the PD-1/PD-L1 pathway as a therapeutic strategy to control graft-versus-host-reactive T cells in allograft recipients.

Degenerated and healthy cartilage are equally vulnerable to blood-induced damage.

BACKGROUND: Joint bleeds have a direct adverse effect on joint cartilage, leading to joint deterioration and, ultimately, to disability. OBJECTIVE: To examine the hypothesis that because degenerated cartilage has a limited repair capacity, it is more susceptible than healthy cartilage to blood-induced cartilage damage. METHODS: Healthy, degenerated (preclinical osteoarthritic) and osteoarthritic (clinically defined) human cartilage was exposed to 10% vol/vol whole blood for 2 days, followed by a recovery period of 12 days in the absence of blood. The effect of exposure to blood on cartilage was determined by measuring proteoglycan synthesis rate, release and content, as well as protease (matrix metalloproteinase (MMP)) activity. RESULTS: In general, exposure to blood led to a decrease in proteoglycan synthesis rate, an increase in the release of proteoglycans and in MMP activity, and therefore, ultimately, in a decrease of the proteoglycan content of the tissue. Impaired cartilage was as least as susceptible as healthy cartilage to this blood-induced damage. CONCLUSION: These results demonstrate that degenerated cartilage is not more susceptible than healthy cartilage to blood-induced damage. Even though these are just in vitro findings, it remains of great importance, also, in joints already affected, to prevent joints bleeds, and when they do occur, to treat them adequately.

Challenges in quantifying biosphere-atmosphere exchange of nitrogen species.

Recent research in nitrogen exchange with the atmosphere has separated research communities according to N form. The integrated perspective needed to quantify the net effect of N on greenhouse-gas balance is being addressed by the NitroEurope Integrated Project (NEU). Recent advances have depended on improved methodologies, while ongoing challenges include gas-aerosol interactions, organic nitrogen and N(2) fluxes. The NEU strategy applies a 3-tier Flux Network together with a Manipulation Network of global-change experiments, linked by common protocols to facilitate model application. Substantial progress has been made in modelling N fluxes, especially for N(2)O, NO and bi-directional NH(3) exchange. Landscape analysis represents an emerging challenge to address the spatial interactions between farms, fields, ecosystems, catchments and air dispersion/deposition. European up-scaling of N fluxes is highly uncertain and a key priority is for better data on agricultural practices. Finally, attention is needed to develop N flux verification procedures to assess compliance with international protocols.