Linking Morphology, Toxicokinetic, and Toxicodynamic Traits of Aquatic Invertebrates to Pyrethroid Sensitivity.

Pyrethroid insecticides are known to be highly toxic to most aquatic nontarget organisms, but little is known about the mechanisms causing some species to be highly sensitive while others are hardly affected by the pyrethroids. The aim of the present study was to measure the sensitivity (EC50-values) of 10 aquatic invertebrates toward a 24 h pulse of the pyrethroid cypermethrin and subsequently test if the difference in sensitivity could be explained by measured morphological and physiological traits and modeled toxicokinetic (TK) and toxicodynamic (TD) parameters. Large differences were observed for the measured uptake and elimination kinetics, with bioconcentration factors (BCFs) ranging from 53 to 2337 at the end of the exposure. Similarly, large differences were observed for the TDs, and EC50-values after 168 h varied 120-fold. Modeling the whole organism cypermethrin concentrations indicated compartmentation into a sorbed fraction and two internal fractions: a bioavailable and non-bioavailable internal fraction. Strong correlations between surface/volume area and the TK parameters (sorption and uptake rate constants and the resulting BCF) were found, but none of the TK parameters correlated with sensitivity. The only parameter consistently correlating with sensitivity across all species was the killing rate constant of the GUTS-RED-SD model (the reduced general unified threshold models of survival assuming stochastic death), indicating that sensitivity toward cypermethrin is more related to the TD parameters than to TK parameters.

Toxicometabolomics as a tool for next generation environmental risk assessment.

Traditionally applied methodology in environmental risk assessment (ERA) has fallen out of step with technological advancements and regulatory requirements, challenging effectiveness and accuracy of the assessments. Extensive efforts have been focused towards a transition to a more data-driven and mechanistically-based next generation risk assessment. Metabolomics can produce detailed and comprehensive molecular insight into affected biochemical processes. Combining metabolomics with environmental toxicology can help to understand the mechanisms and/or modes of action underlying toxicity of environmental pollutants and inform adverse outcome pathways, as well as facilitate identification of biomarkers to quantify effects and/or exposure. This Technical Report describes the activities and work performed within the frame of the European Food Risk Assessment Fellowship Programme (EU-FORA), implemented at the section 'Environmental Chemistry and Toxicology' at the Department of Environmental Science at Aarhus University in Denmark with synergies to an ongoing H2020 RIA project 'EndocRine Guideline Optimisation' (ERGO). In accordance with the 'training by doing' principles of the EU-FORA, the fellowship project combined the exploration of the status of scientific discussion on methodology in ERA through literature study with hands-on training, using the metabolomics analysis pipeline established at Aarhus University. For the hands-on training, an amphibian metamorphosis assay (OECD test no.231) was used as a proof-of-concept toxicometabolomics study case. Both a targeted biomarker - and an untargeted metabolomics approach was applied.

A Network of Serum Proteins Predict the Need for Systemic Immunomodulatory Therapy at Diagnosis in Noninfectious Uveitis.

Purpose: Early identification of patients with noninfectious uveitis requiring steroid-sparing immunomodulatory therapy (IMT) is currently lacking in objective molecular biomarkers. We evaluated the proteomic signature of patients at the onset of disease and associated proteomic clusters with the need for IMT during the course of the disease. Design: Multicenter cohort study. Participants: Two hundred thirty treatment-free patients with active noninfectious uveitis. Methods: We used aptamer-based proteomics (n = 1305 proteins) and a bioinformatic pipeline as a molecular stratification tool to define the serum protein network of a Dutch discovery cohort (n = 78) of patients and healthy control participants and independently validated our results in another Dutch cohort (n = 111) and a United States cohort (n = 67). Multivariate Cox analysis was used to assess the relationship between the protein network and IMT use. Main Outcome Measures: Serum protein levels and use of IMT. Results: Network-based analyses revealed a tightly coexpressed serum cluster (n = 85 proteins) whose concentration was consistently low in healthy control participants (n = 26), but varied among patients with noninfectious uveitis (n = 52). Patients with high levels of the serum cluster at disease onset showed a significantly increased need for IMT during follow-up, independent of anatomic location of uveitis (hazard ratio, 3.42; 95% confidence interval, 1.22-9.5; P = 0.019). The enrichment of neutrophil-associated proteins in the protein cluster led to our finding that the neutrophil count could serve as a clinical proxy for this proteomic signature (correlation: r = 0.57, P = 0.006). In an independent Dutch cohort (n = 111), we confirmed that patients with relatively high neutrophil count at diagnosis (> 5.2 × 109/L) had a significantly increased chance of requiring IMT during follow-up (hazard ratio, 3.2; 95% confidence interval, 1.5-6.8; P = 0.002). We validated these findings in a third cohort of 67 United States patients. Conclusions: A serum protein signature correlating with neutrophil levels was highly predictive for IMT use in noninfectious uveitis. We developed a routinely available tool that may serve as a novel objective biomarker to aid in clinical decision-making for noninfectious uveitis.

Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation.

Cytomegalovirus (CMV) and its therapy continue to contribute to morbidity and mortality in hemopoietic stem cell transplantation (HSCT). Many studies have demonstrated the feasibility of in vitro generation of CMV-specific T cells for adoptive immunotherapy of CMV. Few clinical trials have been performed showing the safety and efficacy of this approach in vivo. In this study, donor-derived, CMV-specific T cells were generated for 12 adult HSCT patients by stimulation with dendritic cells transduced with an adenoviral vector encoding the CMV-pp65 protein. Patients received a prophylactic infusion of T cells after day 28 after HSCT. There were no infusion related adverse events. CMV DNAemia was detected in 4 patients after infusion but was of low level. No patient required CMV-specific pharmacotherapy. Immune reconstitution to CMV was demonstrated by enzyme linked immunospot assay in all recipients with rapid increases in predominantly CMV-pp65 directed immunity in 5. Rates of graft-versus-host disease, infection, and death were not increased compared with expected. These results add to the growing evidence of the safety and efficacy of immunotherapy of CMV in HSCT, supporting its more widespread use. This study was registered at www.anzctr.org.au as #ACTRN12605000213640.

Activation of invariant NKT cells ameliorates experimental ocular autoimmunity by a mechanism involving innate IFN-gamma production and dampening of the adaptive Th1 and Th17 responses.

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.

Clinical-scale elutriation as a means of enriching antigen-presenting cells and manipulating alloreactivity.

BACKGROUND AIMS: Clinical-scale elutriation using the Elutra(c) has been shown to enrich monocytes reliably for immunotherapy protocols. Until now, a detailed assessment of the four (F1-F4) non-monocyte fractions derived from this process has not been performed. METHODS: Using fluorescence-activated cell sorting (FACS), we performed phenotypic analyses to investigate the possible enrichment of T, B, natural killer (NK) and dendritic cells (DC) or their subsets in one or more Elutra fractions. RESULTS: Blood DC were enriched up to 10-fold in some fractions (F3 and F4) compared with the pre-elutriation apheresis product. This increased the number of DC that could be isolated from a given cell number by immunomagnetic separation. It was also found that CD62L(-) effector memory CD4(+) T cells were enriched in later fractions. In four of five cases tested, cells from F3 demonstrated decreased alloreactive proliferation in a mixed lymphocyte reaction compared with cells from the apheresis product. B cells were enriched in F1 compared with the apheresis product. CONCLUSIONS: In addition to providing enrichment of monocytes for the generation of DC, the Elutra enriches cell subsets that may be incorporated into and enhance existing immunotherapy and stem cell transplantation protocols.

STAT-3-independent production of IL-17 by mouse innate-like αß T cells controls ocular infection.

Appropriate regulation of IL-17 production in the host can mean the difference between effective control of pathogens and uncontrolled inflammation that causes tissue damage. Investigation of conventional CD4+ T cells (Th17 cells) has yielded invaluable insights into IL-17 function and its regulation. More recently, we and others reported production of IL-17 from innate αß+ T cell populations, which was shown to occur primarily via IL-23R signaling through the transcription factor STAT-3. In our current study, we identify promyelocytic leukemia zinc finger (PLZF)-expressing iNKT, CD4-/CD8+, and CD4-/CD8- (DN) αß+T cells, which produce IL-17 in response to TCR and IL-1 receptor ligation independently of STAT-3 signaling. Notably, this noncanonical pathway of IL-17 production may be important in mucosal defense and is by itself sufficient to control pathogenic Staphylococcus aureus infection at the ocular surface.

Brain gene expression, metabolism, and bioenergetics: interrelationships in murine models of cerebral and noncerebral malaria.

Malaria infection can cause cerebral symptoms without parasite invasion of brain tissue. We examined the relationships between brain biochemistry, bioenergetics, and gene expression in murine models of cerebral (Plasmodium berghei ANKA) and noncerebral (P. berghei K173) malaria using multinuclear NMR spectroscopy, neuropharmacological approaches, and real-time RT-PCR. In cerebral malaria caused by P. berghei ANKA infection, we found biochemical changes consistent with increased glutamatergic activity and decreased flux through the Krebs cycle, followed by increased production of the hypoxia markers lactate and alanine. This was accompanied by compromised brain bioenergetics. There were few significant changes in expression of mRNA for metabolic enzymes or transporters or in the rate of transport of glutamate or glucose. However, in keeping with a role for endogenous cytokines in malaria cerebral pathology, there was significant up-regulation of mRNAs for TNF-alpha, interferon-gamma, and lymphotoxin. These changes are consistent with a state of cytopathic hypoxia. By contrast, in P. berghei K173 infection the brain showed increased metabolic rate, with no deleterious effect on bioenergetics. This was accompanied by mild up-regulation of expression of metabolic enzymes. These changes are consistent with benign hypermetabolism whose cause remains a subject of speculation.

Increased expression of indoleamine 2,3-dioxygenase in murine malaria infection is predominantly localised to the vascular endothelium.

Products of the kynurenine pathway of tryptophan metabolism have been implicated in the pathogenesis of murine and human cerebral malaria. Indoleamine 2,3-dioxygenase is the first and rate-limiting enzyme in this pathway and we have developed an immunohistochemical method for its detection in tissues from normal and malaria-infected mice. Mice were infected with Plasmodium berghei ANKA, a murine model of cerebral malaria, or P. berghei K173, a non-cerebral malaria model. Vascular endothelial cells were the primary sites of indoleamine 2,3-dioxygenase expression in both types of malaria infection and this response was systemic, with positive staining of vascular endothelium in all tissues examined. No indoleamine 2,3-dioxygenase expression was detected in uninfected or interferon-gamma-/- mice. Corroborative data were obtained using quantitative reverse transcription PCR for indoleamine 2,3-dioxygenase mRNA. These results suggest that interferon-gamma-dependent indoleamine 2,3-dioxygenase expression is part of a normal systemic host response to the parasite, perhaps performing some tissue protective functions that may become deranged under some circumstances and contribute to the pathogenesis of cerebral malaria. On the other hand, constitutive indoleamine 2,3-dioxygenase expression in the epididymis and the placenta was detected in both C57Bl/6 wild-type and interferon-gamma-/- mice, suggesting a distinct regulatory mechanism for its induction in these normal physiological situations. Although increased indoleamine 2,3-dioxygenase production during murine malaria infection may not by itself cause cerebral pathology, metabolites of the kynurenine pathway may combine with other features of cerebral malaria, such as breakdown of the blood-brain barrier, to influence CNS function and contribute to the symptoms and pathology observed.

Cerebral malaria: gamma-interferon redux.

There are two theories that seek to explain the pathogenesis of cerebral malaria, the mechanical obstruction hypothesis and the immunopathology hypothesis. Evidence consistent with both ideas has accumulated from studies of the human disease and experimental models. Thus, some combination of these concepts seems necessary to explain the very complex pattern of changes seen in cerebral malaria. The interactions between malaria parasites, erythrocytes, the cerebral microvascular endothelium, brain parenchymal cells, platelets and microparticles need to be considered. One factor that seems able to knit together much of this complexity is the cytokine interferon-gamma (IFN-γ). In this review we consider findings from the clinical disease, in vitro models and the murine counterpart of human cerebral malaria in order to evaluate the roles played by IFN-γ in the pathogenesis of this often fatal and debilitating condition.

Th1 and Th17 cells: adversaries and collaborators.

Autoreactive effector CD4+ T cells have been associated with the pathogenesis of autoimmune disorders. Early studies implicated the interferon (IFN)-gamma-producing T helper (Th)1 subset of CD4+ cells as the causal agents in the pathogenesis of autoimmunity. However, further studies have suggested a more complex story. In models thought to be driven by Th1 cells, mice lacking the hallmark Th1 cytokine IFN-gamma were not protected but tended to have enhanced susceptibility to disease. Identification of the IL-17-producing CD4+ effector cell lineage (Th17) has helped shed light on this issue. Th17 effector cells are induced in parallel to Th1, and, like Th1, polarized Th17 cells have the capacity to cause inflammation and autoimmune disease. This, together with the finding that deficiency of the Th17-related cytokine IL-23 but not the Th1-related cytokine IL-12 causes resistance, led to the notion that Th17 cells are the chief contributors to autoimmune tissue inflammation. Nevertheless, mice lacking IL-17 are not protected from disease and display elevated numbers of IFN-gamma-producing CD4+ T cells, and, in some cases, lack of IFN-gamma does confer resistance. Recent studies report overlapping as well as differential roles of these cells in tissue inflammation, which suggests the existence of a more complex relationship between these two effector T-cell subsets than has hitherto been suspected. This review will attempt to bring together current information regarding interaction, balance, and collaborative potential between the Th1 and Th17 effector lineages.

Cutting edge: NKT cells constitutively express IL-23 receptor and RORgammat and rapidly produce IL-17 upon receptor ligation in an IL-6-independent fashion.

Th17 cells require IL-6 and TGFbeta for lineage commitment and IL-23 for maintenance. Unexpectedly, naive IL-6(-/-) splenocytes stimulated with anti-CD3 and IL-23 produced normal amounts of IL-17 during the first 24 h of culture. These rapid IL-6-independent IL-17 producers were identified as predominantly DX5(+) TCRbeta(+) NKT cells, and a comparable response could be found using the invariant NKT-specific ligand alpha-galactosylceramide. Human NKT cells also produced IL-17. NKT cells constitutively expressed IL-23R and RORgammat. Ligation of either TCR or IL-23R triggered IL-17 production and both together had a synergistic effect, suggesting independent but convergent pathways. IL-17 production was not restricted to a particular subset of NKT cells but they were NK1.1 negative. Importantly, in vivo administration of alpha-galactosylceramide triggered a rapid IL-17 response in the spleen. These data suggest an important biological role for innate IL-17 production by NKT cells that is rapid and precedes the adaptive IL-17 response.

Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.

BACKGROUND: TNF-related lymphotoxin alpha (LTalpha) is essential for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The pathway involved has been attributed to TNFR2. Here we show a second arm of LTalpha-signaling essential for ECM development through LTbeta-R, receptor of LTalpha1beta2 heterotrimer. METHODOLOGY/PRINCIPAL FINDINGS: LTbetaR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTalphabeta deficient mice. Resistance of LTalphabeta or LTbetaR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin(+) CD8(+) T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTbetaR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM. CONCLUSIONS/SIGNIFICANCE: LTbetaR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTbetaR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.

Early cytokine production is associated with protection from murine cerebral malaria.

Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise mechanisms leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-gamma) at 24 h p.i. and with increased splenic and hepatic mRNAs for a range of cytokines (IFN-gamma, interleukin-10 [IL-10], and IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase. In contrast, P. berghei ANKA infection was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN-gamma production at 3 to 4 days p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a burst of IFN-gamma at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated with early production of IFN-gamma. Early IFN-gamma production was present in NK-cell-depleted, gammadelta-cell-depleted, and Jalpha281(-/-) (NKT-cell-deficient) mice but absent from beta2-microglobulin mice that had been infected with P. berghei K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN-gamma and CD8(+) T cells in P. berghei ANKA infection allows the development of cerebral immunopathology.

A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation.

By ligating CD80/CD86 (B7) molecules, the synthetic immunomodulatory reagent CTLA4-Ig (soluble synthetic CTLA4 fusion protein) induces expression of the enzyme indoleamine 2,3-dioxygenase (IDO) in some dendritic cells (DCs), which acquire potent T cell regulatory functions as a consequence. Here we show that this response occurred exclusively in a population of splenic DCs co-expressing the marker CD19. B7 ligation induced activation of the transcription factor signal transducer and activator of transcription (STAT1) in sorted CD19+, but not CD19(NEG), DCs. STAT1 activation occurred even when DCs lacked receptors for type II IFN (IFNgamma); however, STAT1 activation and IDO up-regulation were not observed when DCs lacked receptors for type I IFN (IFNalphabeta). Thus, IFNalpha, but not IFNgamma, signaling was essential for STAT1 activation and IDO up-regulation in CD19+ DCs following B7 ligation. Consistent with these findings, B7 ligation also induced sorted CD19+, but not CD19(NEG), DCs to express IFNalpha. Moreover, recombinant IFNalpha induced CD19+, but not CD19(NEG), DCs to mediate IDO-dependent T cell suppression, showing that IFNalpha signaling could substitute for upstream signals from B7. These data reveal that a minor population of splenic DCs expressing the CD19 marker is uniquely responsive to B7 ligation, and that IFNalpha-mediated STAT1 activation is an essential intermediary signaling pathway that promotes IDO induction in these DCs. Thus, CD19+ DCs may be a target for regulatory T cells expressing surface CTLA4, and may suppress T cell responses via induction of IDO.

Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase.

Murine dendritic cells (DCs) expressing indoleamine 2,3 dioxygenase (IDO) catabolize tryptophan and can suppress T cell responses elicited in vivo. Here, we identify specific subsets of splenic (CD11c+) dendritic cells competent to mediate IDO-dependent T cell suppression following CTLA4-mediated ligation of B7 molecules. IDO-competent DC subsets acquired potent and dominant T cell suppressive properties as a consequence of IDO up-regulation, as they blocked the ability of T cells to respond to other stimulatory DCs in the same cultures. Soluble CTLA4 (CTLA4-Ig) and cloned CTLA4+ regulatory T cells (Tr1D1) up-regulated IDO selectively in DC subsets co-expressing B220 or CD8alpha. The ability of Tr1D1 T cells to suppress CD8+ T cell responses was completely dependent on their ability to induce tryptophan catabolism in DCs. Selective IDO up-regulation in DCs did not inhibit T cell activation, but prevented T cell clonal expansion due to rapid death of activated T cells. T cell responses were restored by genetic or pharmacologic inhibition of IDO enzyme activity, or by adding excess tryptophan. DCs from interferon gamma (IFNgamma)-receptor-deficient mice were effective in promoting IDO-dependent T cell suppression following CTLA4-Ig exposure in vivo, indicating that IFNgamma signaling was not necessary for IDO up-regulation in this model. These findings suggest that IDO-competent DCs provide a regulatory bridge, mediated by CTLA4-B7 engagement, between certain regulatory T cell subsets and naive responder T cells.