Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding.

A new metabolite profiling approach combined with an ultrarapid sample preparation procedure was used to study the temporal and spatial dynamics of the wound-induced accumulation of jasmonic acid (JA) and its oxygenated derivatives in Arabidopsis thaliana. In addition to well known jasmonates, including hydroxyjasmonates (HOJAs), jasmonoyl-isoleucine (JA-Ile), and its 12-hydroxy derivative (12-HOJA-Ile), a new wound-induced dicarboxyjasmonate, 12-carboxyjasmonoyl-l-isoleucine (12-HOOCJA-Ile) was discovered. HOJAs and 12-HOOCJA-Ile were enriched in the midveins of wounded leaves, strongly differentiating them from the other jasmonate metabolites studied. The polarity of these oxylipins at physiological pH correlated with their appearance in midveins. When the time points of accumulation of different jasmonates were determined, JA levels were found to increase within 2-5 min of wounding. Remarkably, these changes occurred throughout the plant and were not restricted to wounded leaves. The speed of the stimulus leading to JA accumulation in leaves distal to a wound is at least 3 cm/min. The data give new insights into the spatial and temporal accumulation of jasmonates and have implications in the understanding of long-distance wound signaling in plants.

Detection of the acute phase of abdominal angiostrongyliasis with a parasite-specific IgG enzyme linked immunosorbent assay

Angiostrongylus costaricensis may cause intestinal lesions of varied severity when it accidentally infects man in Central and South America. First-stage larvae have never been detected in stools. Therefore, a parasite-specific IgG ELISA was evaluated for the determination of the acute phase of infection. The specificity and the sensitivity of the immunoassay was shown to be 76.2% and 91.1%, respectively. Eight serum samples taken from patients with histopathological diagnosis, at different time points (3 to 15 months) after surgical treatment, showed a sharp and early decline in antibody reactivity. The titration of anti-A. costaricensis antibodies has proved to be a useful method for the diagnosis of acute abdominal angiostrongyliasis.

Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.

Introduction: Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice. Methods: Male Balb/C mice (n = 80) were injected intravenously with 1-5 mu g recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NF kappa B) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin. Results: TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NF kappa B and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNF alpha, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1 beta and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ. Conclusions: Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms

Attachment among Adoptive Families: Two Emipirical Studies in an International Context

The present dissertation analyzed the construct of attachment at different time points, specifically focusing on two phases of adoptive family life that have so far received little attention from investigators. Study 1 focused on the first months of adoption, and analyzed the development of the attachment relationship to new caregivers. The sample was composed of a small but homogeneous group (n=6) of Korean-born children, adopted by Italian parents. The Parent Attachment Diary (Dozier & Stovall, 1997) was utilized to assess the child's attachment behavior. We assessed these behavior for the first 3 months after placement into adoption. Results showed a double variability of attachment behavior: within subjects during the 3-months, and between subjects, with just half of the children developing a stable pattern of attachment. In order to test the growth trajectories of attachment behavior, Hierarchical Linear Models (Bryk & Raudenbush, 1992) were also applied, but no significant population trend was identified. Study 2 analyzed attachment among adoptees during the sensitive period of adolescence. Data was derived from an international collection (n= 104, from Belgium Italy, and Romania) of semi-structured clinical interviews (with adolescents and with their adoptive parents), as well as from questionnaires. The purpose of this study was to detect the role played by risk and protective factors on the adoptee's behavioral and socio-emotional outcomes. In addition, we tested the possible interactions between the different attachment representations within the adoptive family. Results showed that pre-adoptive risk predicted the adolescent's adjustment; however, parental representations constituted an important moderator of this relationship. Moreover, the adolescent's security of attachment partially mediated the relationship between age at placement and later behavioral problems. In conclusion, the two present attachment studies highlighted the notable rate of change of attachment behavior over time, which showed its underlying plasticity, and thus the possible reparatory value of the adoption practice. Since parents have been proven to play an important role, especially in adolescence, the post-adoption support acquires even more importance in order to help parents promoting a positive and stable relational environment over time. - L'objectif de cette thèse est de décrire la formation des relations d'attachement chez les enfants et les adolescents adoptés, lors de deux phases particulières de la vie de la famille adoptive, qui ont été relativement peu étudiées. L'Étude 1 analyse les premiers mois après l'adoption, avec le but de comprendre si, et comment, une relation d'attachement aux nouveaux parents se développe. L'échantillon est composé d'un petit groupe (n = 6) d'enfants provenant de Corée du Sud, adoptés par des parents Italiens. A l'aide du Parent Attachment Diary (Dozier & Stovall, 1997), des observations des comportements d'attachement de l'enfant ont été recueillies chaque jour au cours des 3 premiers mois après l'arrivée. Les résultats montrent une double variabilité des comportements d'attachement: au niveau inter- et intra-individuel ; au premier de ces niveaux, seuleme la moitié des enfants parvient à développer un pattern stable d'attachement ; au niveau intra-individuel, les trajectoires de développement des comportements d'attachement ont été testées à l'aide de Modèles Linéaires Hiérarchiques (Bryk et Raudenbush, 1992), mais aucune tendance significative n'a pu être révélée. L'Étude 2 vise à analyser l'attachement chez des enfants adoptés dans l'enfance, lors de la période particulièrement sensible de l'adolescence. Les données sont issues d'un base de données internationale (n = 104, Belgique, Italie et Roumanie), composée d' entretiens cliniques semi-structurées (auprès de l'adolescents et des ses parents adoptifs), ainsi que de questionnaires. Les analyses statistiques visent à détecter la présence de facteurs de risque et de protection relativement à l'attachement et aux problèmes de comportement de l'enfant adopté. En outre, la présence d'interactions entre les représentations d'attachement des membres de la famille adoptive est évaluée. Les résultats montrent que les risques associés à la période pré-adoptive prédisent la qualité du bien-être de l'adolescent, mais les représentations parentales constituent un modérateur important de cette relation. En outre, la sécurité de l'attachement du jeune adopté médiatise partiellement la relation entre l'âge au moment du placement et les problèmes de comportement lors de l'adolescence. En conclusion, à l'aide de multiples données relatives à l'attachement, ces deux études soulignent son évolution notable au fil du temps, ce qui sous-tend la présence d'une certaine plasticité, et donc la possible valeur réparatrice de la pratique de l'adoption. Comme les parents semblent jouer un rôle important de ce point de vue, surtout à l'adolescence, cela renforce la notion d'un soutien post-adoption, en vue d'aider les parents à la promotion d'un environnement relationnel favorable et stable. - Il presente lavoro è volto ad analizzare l'attaccamento durante le due fasi della vita della famiglia adottiva che meno sono state indagate dalla letteratura. Lo Studio 1 aveva l'obiettivo di analizzare i primi mesi che seguono il collocamento del bambino, al fine di capire se e come una relazione di attaccamento verso i nuovi genitori si sviluppa. Il campione è composto da un piccolo gruppo (n = 6) di bambini provenienti dalla Corea del Sud e adottati da genitori italiani. Attraverso il Parent Attachment Diary (Stovall e Dozier, 1997) sono stati osservati quotidianamente, e per i primi tre mesi, i comportamenti di attaccamento del bambino. I risultati hanno mostrato una duplice variabilità: a livello intraindividuale (nell'arco dei 3 mesi), ed interindividuale, poiché solo la metà dei bambini ha sviluppato un pattern stabile di attaccamento. Per verificare le traiettorie di sviluppo di tali comportamenti, sono stati applicati i Modelli Lineari Gerarchici (Bryk & Raudenbush, 1992), che però non hanno stimato una tendenza significativa all'interno della popolazione. Obiettivo dello Studio 2 è stato quello di esaminare l'attaccamento nelle famiglie i cui figli adottivi si trovavano nella delicata fase adolescenziale. I dati, provenienti da una raccolta internazionale (n = 104, Belgio, Italia e Romania), erano costituiti da interviste cliniche semi-strutturate (con gli adolescenti e i propri genitori adottivi) e da questionari. Le analisi hanno indagato il ruolo dei fattori di rischio sullo sviluppo socio-emotivo e sugli eventuali problemi comportamentali dei ragazzi. Inoltre, sono state esaminate le possibili interazioni tra le diverse rappresentazioni di attaccamento dei membri della famiglia adottiva. I risultati hanno mostrato che il rischio pre-adottivo predice l'adattamento dell'adolescente, sebbene le rappresentazioni genitoriali costituiscano un importante moderatore di questa relazione. Inoltre, la sicurezza dell'attaccamento dell'adolescente media parzialmente la relazione tra età al momento dell'adozione e problemi comportamentali in adolescenza. In conclusione, attraverso i molteplici dati relativi all'attaccamento, i due studi ne hanno evidenziato il cambiamento nel tempo, a riprova della sua plasticità, e pertanto sottolineano il possibile valore riparativo dell'adozione. Dal momento che i genitori svolgono un ruolo importante, soprattutto in adolescenza, il supporto nel post- adozione diventa centrale per aiutarli a promuovere un ambiente relazionale favorevole e stabile nel tempo.

Postoperative cognitive dysfunction POCD, serum anticholinergic activity and intraoperative cerebral perfusion in geriatric patients

Background: Inadequate intraoperative cerebral perfusion and increased serum anticholinergic activity (SAA) have been suggested as possible causes of postoperative cognitive dysfunction (POCD). Methods: 53 patients aged >65 yrs undergoing elective major surgical procedures under standardized general anaesthesia. Cerebral perfusion was monitored with transcranial Doppler and near-infrared spectroscopy. Mx, an index of cerebral autoregulation was calculated based on the correlation of spontaneous changes inmean arterial blood pressure (MAP) and cerebral blood flow velocity. Cognitive function was measured preoperatively and 7 days postoperatively using the CERAD-Neuropsychological Battery. A postoperative decline >1 z-score in at least 2 cognitive variables was defined as POCD. SAA was measured preoperatively and 7 days postoperatively (data available for 38 patients). CRP was measured at the same time points and 2 days postoperatively. Results: Age was 75_7 yrs (mean_SD). 23 patients (43%) developed POCD. There were no statistical significant differences between patients with POCD and without POCD in age (77_7 vs 73_6 yrs), MAP (74_12 vs 78_11 mmHg), cerebral tissue oxygenation indices (67_6 vs 69_4 %) SAA preoperatively (1.74_1.52 vs 1.74_1.21) and 7 days postoperatively (1.90_1.63 vs 1.84_1.39) and CRP preoperatively (32_72 vs 7_9), 2 days postoperatively (176_129 vs 111_69) and 7days postoperatively (53_43 vs 48_25). Patients with POCD had less efficient autoregulation than patients without POCD (Mx 0.55_0.15 vs 0.45_0.20, p = 0.046). However, the percentage of patients with clearly impaired autoregulation (ie, Mx>0.5) was statistically not different between groups (with POCD: 65%; without POCD: 38%; p = 0.06) but there seems to be a trend. Conclusions: Our data on the association between cerebral perfusion and POCD in elderly patients are inconclusive and more patients need to be investigated. In this small group of patients SAA seems not to be associated with POCD.

Neonatal hyperglycemia inhibits angiogenesis and induces inflammation and neuronal degeneration in the retina.

Recent evidence suggests that transient hyperglycemia in extremely low birth weight infants is strongly associated with the occurrence of retinopathy of prematurity (ROP). We propose a new model of Neonatal Hyperglycemia-induced Retinopathy (NHIR) that mimics many aspects of retinopathy of prematurity. Hyperglycemia was induced in newborn rat pups by injection of streptozocine (STZ) at post natal day one (P1). At various time points, animals were assessed for vascular abnormalities, neuronal cell death and accumulation and activation of microglial cells. We here report that streptozotocin induced a rapid and sustained increase of glycemia from P2/3 to P6 without affecting rat pups gain weight or necessitating insulin treatment. Retinal vascular area was significantly reduced in P6 hyperglycemic animals compared to control animals. Hyperglycemia was associated with (i) CCL2 chemokine induction at P6, (ii) a significant recruitment of inflammatory macrophages and an increase in total number of Iba+ macrophages/microglia cells in the inner nuclear layer (INL), and (iii) excessive apoptosis in the INL. NHIR thereby reproduces several aspects of ischemic retinopathies, including ROP and diabetic retinopathies, and might be a useful model to decipher hyperglycemia-induced cellular and molecular mechanisms in the small rodent.

Novel relationship between Vegf expression and retinal pigmented epithelium permeability following light damage in the mouse retina

Purpose:In the retina, the balance between pro- and anti-angiogenic factors is critical for angiogenesis control but is also involved in cell survival and maintenance. For instance, the anti-angiogenic factor PEDF is neuroprotective for photoreceptors (PRs) in models of retinal degeneration. We previously reported upregulation of VEGF (24h to 48h post lesion) in the light-damage (LD) model. Furthermore, systemic delivery of PEDF, as well as lentiviral gene transfer of an anti-VEGF antibody rescue PRs from cell death. Studies in vitro show that VEGF induces retinal endothelial cells apoptosis via the alteration of the Akt1/p38 MAPK signalling pathway under hypoxic conditions. Thus, in this study, we investigate the effect of high levels of VEGF on retinal pigmented epithelium (RPE) permeability and molecular targets expression after light-induced PR degeneration. Methods:To characterize the action of VEGF in the retina during the course of LD, we exposed adult Balb/c mice to 5'000 lux for 1h, and we collected neural retinas and eye-cups (containing RPE) at different time points after the LD. We analysed protein expression by Elisa and Western blotting. In order to study RPE cell permeability after the LD we stained β-catenin on flat mounted RPE. Results:In the neural retina, preliminary results indicate that high levels of VEGF induce a significant upregulation of VEGF receptor 2, whereas VEGF receptor 1 expression is decreased. Concomitantly with VEGF upregulation, LD increases the Src phosphorylation between 24h to 48h. Furthermore, we observe that β-catenin translocates to the cytoplasm of RPE cells between 24h to 36h after the lesion, indicating an increase on the RPE permeability, which could contribute indirectly to the deleterious effect of VEGF observed during light-induced PR apoptosis. Conclusions:This study further involves VEGF in LD and highlights the prime importance of angiogenic factor balance for PR survival. Our results suggest that PR apoptosis is augmented by RPE cell permeability, which may induce high level of VEGF and could be deleterious. The specific action of RPE permeability on PR survival and the role of Src in the retina are under investigation.

Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.

Expression of two important glucose transporter proteins, GLUT 2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/brain type glucose transporter GLUT 1 (representing the typical glucose transporter in fetal liver parenchyma), was studied immunocytochemically during hepatocarcinogenesis in rats at different time points between 7 and 65 wk after cessation of 7-wk administration of 12 mg/kg of body weight of N-nitrosomorpholine p.o. (stop model). Foci of altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci (MCF) composed of both glycogenotic and glycogen-poor cells were present at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes. Morphometrical evaluation of the focal lesions revealed that the number and volume fraction of GSF increased steadily until Wk 65. MCF were rare at 7 wk, increased slightly in number and size until Wk 37, but showed a pronounced elevation in their number and volume fraction from Wk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreased or partially absent at all time points. Consequently, foci of decreased GLUT 2 expression showed a steady increase in number and volume fraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurred in some MCF from Wk 50 onward. The liver type glucose transporter GLUT 2 was decreased in all adenomas and hepatocellular carcinomas (HCC). In three of seven adenomas and 10 of 12 carcinomas, expression of GLUT 1 was increased compared with normal liver parenchyma. In two cases of adenoid HCC, cells of ductular formations coexpressed GLUT 2 and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, and cystic cholangiomas expressed only GLUT 1. Seven of 12 HCC contained many microvessels intensely stained for GLUT 1, a phenomenon never observed in normal liver. Whenever adenoid tumor formations occurred, GLUT 1-positive microvessels were located in the immediate vicinity of these formations. Only in one HCC were such microvessels found in the absence of adenoid formations. Our studies indicate that a reduction of GLUT 2 expression occurs already in early preneoplastic hepatic foci and is maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a few MCF and in the majority of adenomas and carcinomas.

In vitro characterization of sunitinib eluting beads.

Purpose: To load embolization particles (DC-Beads, Biocompatibles, UK) with an anti-angiogenic agent (sunitinib) and to characterize the in vitro properties of the Beads-drug association.Materials: DC Beads of 100-300µm were loaded using a specially designed 10mg/ml sunitinib solution. Loading profile was studied by spectrophotometry of the supernatant solution at 430nm at different time points. Release experiment was performed using the USP method 4 (flow-through cell). Spectrophotometric determination at 430nm was used to measure drug concentration in the eluting solution.Results: We were able to load >98% of the drug in the DC-Beads in 2 hours. The maximum concentration was 20mg sunitinib/ml DC Beads. Loaded Beads gradually released 59% of the loaded drug in the eluting solution, by an ionic exchange mechanism,over 6 hours.Conclusions: DC Beads could be loaded with the multi tyrosine kinase inhibitor sunitinib using a specially designed solution. High drug payload can be achieved. The loaded DC Beads released the drug in an ionic eluting solution with an interesting release profile.

Not in Education, Employment, or Training status among young Swiss men. Longitudinal associations with mental health and substance use.

PURPOSE: Not in Education, Employment, or Training (NEET) youth are youth disengaged from major social institutions and constitute a worrying concern. However, little is known about this subgroup of vulnerable youth. This study aimed to examine if NEET youth differ from other contemporaries in terms of personality, mental health, and substance use and to provide longitudinal examination of NEET status, testing its stability and prospective pathways with mental health and substance use. METHODS: As part of the Cohort Study on Substance Use Risk Factors, 4,758 young Swiss men in their early 20s answered questions concerning their current professional and educational status, personality, substance use, and symptomatology related to mental health. Descriptive statistics, generalized linear models for cross-sectional comparisons, and cross-lagged panel models for longitudinal associations were computed. RESULTS: NEET youth were 6.1% at baseline and 7.4% at follow-up with 1.4% being NEET at both time points. Comparisons between NEET and non-NEET youth showed significant differences in substance use and depressive symptoms only. Longitudinal associations showed that previous mental health, cannabis use, and daily smoking increased the likelihood of being NEET. Reverse causal paths were nonsignificant. CONCLUSIONS: NEET status seemed to be unlikely and transient among young Swiss men, associated with differences in mental health and substance use but not in personality. Causal paths presented NEET status as a consequence of mental health and substance use rather than a cause. Additionally, this study confirmed that cannabis use and daily smoking are public health problems. Prevention programs need to focus on these vulnerable youth to avoid them being disengaged.

Decreased expression of peroxisome proliferator-activated receptor alpha and liver fatty acid binding protein after partial hepatectomy of rats and mice.

BACKGROUND AIMS: Marked changes in metabolism, including liver steatosis and hypoglycemia, occur after partial hepatectomy. Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a nuclear hormone receptor that is activated by fatty acids and involved in hepatic fatty acid metabolism and regeneration. Liver fatty acid binding protein (LFABP) is an abundant protein in liver cytosol whose expression is regulated by PPAR alpha. It is involved in fatty acid uptake and diffusion and in PPAR alpha signaling. The aim of this study was to investigate the expression of PPAR alpha and LFABP during liver regeneration. METHODS: Male Sprague-Dawley rats and male C57 Bl/6 mice were subjected to 2/3 hepatectomy and LFABP and PPAR alpha mRNA and protein levels were measured at different time points after surgery. The effect of partial hepatectomy was followed during 48 h in rats and 72 h in mice. RESULTS: PPAR alpha mRNA and protein levels were decreased 26 h after hepatectomy of rats. The LFABP mRNA and protein levels paralleled those of PPAR alpha and were also decreased 26 h after hepatectomy. In mice, the mRNA level was decreased after 36 and 72 h after hepatectomy. In this case, LFABP mRNA levels decreased more slowly after partial hepatectomy than in rats. CONCLUSIONS: A marked decrease in PPAR alpha expression may be important for changed gene expression, e.g. LFABP, and metabolic changes, such as hypoglycemia, during liver regeneration.

Participation of cytokines in the necrotic-inflammatory lesions in the heart and skeletal muscles of Calomys callosus infected with Trypanosoma cruzi

Calomys callosus, a sylvatic reservoir of Trypanosoma cruzi, when infected with the Colombian strain (Biodeme Type III, T. cruzi I ) develops necrotic-inflammatory lesions and intense early fibrogenesis in the heart and skeletal muscles, that spontaneously regress. Participation of pro-inflammatory and pro-fibrogenic cytokines, such as tumor necrosis factor-alpha (TNF-alpha), gamma interferon (IFN-gamma) , and tumor growth factor-beta (TGF-beta), in the pathogenesis of the lesions is herein studied. Eighty C. callosus weighing 20 to 30 g were used. Seventy of them were inoculated with the Colombian strain (10(5) blood forms) and 10 were maintained as intact non-infected controls. After infection, C. callosus were sacrificed at different time-points from 15 to 70 days. The heart and skeletal muscle were processed for histopathology and cryopreserved for immunohistochemistry. Early necrotic lesions of parasitized skeletal muscle and myocardium with intense inflammatory lesions were present. Search for the in situ presence of TNF-alpha and IFN-gamma, was performed using rat-IgG anti-mouse antibodies against these cytokines. For the in situ search of TGF-beta, rabbit IgG anti-mouse antibodies were used. Immunolabeling of the cytokines in tissues of infected C. callosus was successful. The cytokines TNF-alpha, IFN-gamma , and TGF-beta were detected in the cytoplasm of macrophages and in the necrotic material from 15 to 45 days post-infection, decreasing their intensity until complete disappearance by the 65th day, which correlated with subsiding histopathological lesions. These findings suggest the participation of these cytokines in the control of parasite multiplication, in the development of an early fibrogenesis and in the regression of fibrotic-inflammatory lesions observed in C. callosus.

Vaccination with a Melan-A peptide selects an oligoclonal T cell population with increased functional avidity and tumor reactivity.

Both the underlying molecular mechanisms and the kinetics of TCR repertoire selection following vaccination against tumor Ags in humans have remained largely unexplored. To gain insight into these questions, we performed a functional and structural longitudinal analysis of the TCR of circulating CD8(+) T cells specific for the HLA-A2-restricted immunodominant epitope from the melanocyte differentiation Ag Melan-A in a melanoma patient who developed a vigorous and sustained Ag-specific T cell response following vaccination with the corresponding synthetic peptide. We observed an increase in functional avidity of Ag recognition and in tumor reactivity in the postimmune Melan-A-specific populations as compared with the preimmune blood sample. Improved Ag recognition correlated with an increase in the t(1/2) of peptide/MHC interaction with the TCR as assessed by kinetic analysis of A2/Melan-A peptide multimer staining decay. Ex vivo analysis of the clonal composition of Melan-A-specific CD8(+) T cells at different time points during vaccination revealed that the response was the result of asynchronous expansion of several distinct T cell clones. Some of these T cell clones were also identified at a metastatic tumor site. Collectively, these data show that tumor peptide-driven immune stimulation leads to the selection of high-avidity T cell clones of increased tumor reactivity that independently evolve within oligoclonal populations.

Thogoto virus infection induces sustained type I interferon responses that depend on RIG-I-like helicase signaling of conventional dendritic cells.

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.

N-Terminal Modifications Improve the Receptor Affinity and Pharmacokinetics of Radiolabeled Peptidic Gastrin-Releasing Peptide Receptor Antagonists: Examples of 68Ga- and 64Cu-Labeled Peptides for PET Imaging.

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS: The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS: The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION: We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.