Change in defense mechanisms and coping patterns during the course of 2-year-long psychotherapy and psychoanalysis for recurrent depression: a pilot study of a randomized controlled trial.

Very little research has been conducted so far to study the potential mechanisms of change in long-term active psychological treatments of recurrent depression. The present pilot randomized controlled trial aimed to determine the feasibility of studying the change process occurring in patients during the course of 2-year-long dynamic psychotherapy, psychoanalysis, and cognitive therapy, as compared with clinical management. In total, eight outpatients presenting with recurrent depression, two patients per treatment arm, were included. All patients were randomly assigned to one of the four treatment conditions. Defense mechanisms and coping patterns were assessed using validated observer-rated methodology based on transcribed, semistructured follow-along independent dynamic interviews. The results indicated that, whereas some patients in the active treatments changed on the symptomatic levels, some others remained unchanged during the course of their 2-year-long treatment. However, with regard to potential mechanisms of change in these patients, changes in defense mechanisms and coping patterns were revealed to be important processes over time in successful therapies and, to a lesser extent, in less successful treatments. No change was found either on outcome or on the process measure for the control condition, that is, clinical management. These results are discussed along with previous data comparing change in defense mechanisms and coping during the course of treatments.

Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas.

Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.

Wireless E911 Emergency Communications Fund Iowa Homeland Security and Emergency Management Division of the Iowa Department of Public Defense independent auditor's reports, basic financial statements and supplementary information, June 30, 2009.

Audit report on the Wireless E911 Emergency Communication Fund of the Iowa Homeland Security and Emergency Management Division of the Iowa Department of Public Defense for the year ended June 30, 2009

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

Atrial natriuretic peptide administered as intravenous infusion or bolus injection to patients with liver cirrhosis and ascites.

The effect of a synthetic atrial natriuretic peptide (h-ANP, 25 amino acids, Wy-47.663) on blood pressure, renal electrolyte excretion, plasma catecholamines, and plasma renin activity was studied in nine patients with cirrhosis of the liver and ascites. The peptide was infused intravenously at 24-h intervals for 2 h in groups of four patients each in two different doses (0.015 and 0.075 micrograms/kg/min or 0.06 and 0.3 micrograms/kg/min). A control experiment with the vehicle was performed in all patients. In three patients h-ANP (1 and 2 micrograms/kg i.v.) was administered as an intravenous bolus injection. Consistent falls in blood pressure were observed during h-ANP infusion only with the two higher doses. The two lower infused doses induced a consistent natriuresis; this renal response was abolished when the two larger doses were used. When given as a bolus, h-ANP had a natriuretic effect comparable to that of the two lower doses of infused h-ANP. Plasma catecholamines and plasma renin activity increased during infusion of the two higher doses of h-ANP. It thus appears that in patients with cirrhosis and ascites, the natriuretic effect of infused h-ANP decreases rather than increases when the doses are raised. Bolus administration of h-ANP may be less prone to trigger counterbalancing responses and side-effects.

Subconjunctival Injection of XG-102, a JNK Inhibitor Peptide, in Patients with Intraocular Inflammation: A Safety and Tolerability Study.

Abstract Purpose: We aimed to investigate the safety, tolerability, and systemic diffusion of a single escalating dose of XG-102 (a 31-D-amino-acid peptide inhibiting JNK pathway activation), administered subconjunctivally in the treatment of post-surgery or post-trauma intraocular inflammation. Methods: This is a dose-escalating, tolerance Phase Ib study. Twenty patients with post-surgery or post-traumatic intraocular inflammation were assigned to 1 of the 4 dose escalating (45, 90, 450, or 900 μg XG-102) groups of 5 patients each. Patients were evaluated at 24, 48 h, 8, and 28 days following the administration of XG-102, including laboratory tests, standard eye examinations, vital signs, and occurrence of adverse events. A single plasma quantification of XG-102 was performed 30 min after administration, according to previous pharmacokinetics studies performed on volunteers. Results: A total of 17 non-serious adverse events, considered unrelated to the study treatment, were reported for 10 patients. The adverse event incidence was not related to the drug dose. All patients experienced a decrease in intraocular inflammation as of 24 h post-administration and this decrease was sustained up to 28 days thereafter. No patient required local injection or systemic administration of corticoids following the administration of XG-102. XG-102 was undetectable in the first 3 dose groups. In the fourth-dose group (900 μg) the XG-102 plasma levels were above the limit of detection for 3 patients and above the limit of quantification for 1 patient. Conclusions: In this first clinical trial using XG-102, administered as a single subconjunctival injection as adjunct therapy, in patients with recent post-surgery or post-trauma intraocular inflammation is safe and well tolerated. Further studies are required to evaluate its efficacy.

Report on the Iowa Department of Public Defense for the year ended June 30, 2009

Report on the Iowa Department of Public Defense for the year ended June 30, 2009

Molecular modeling of peptide-MHC class I complexes : applications to peptide based immunotherapy

Summary The specific CD8+ T cell immune response against tumors relies on the recognition by the T cell receptor (TCR) on cytotoxic T lymphocytes (CTL) of antigenic peptides bound to the class I major histocompatibility complex (MHC) molecule. Such tumor associated antigenic peptides are the focus of tumor immunotherapy with peptide vaccines. The strategy for obtaining an improved immune response often involves the design of modified tumor associated antigenic peptides. Such modifications aim at creating higher affinity and/or degradation resistant peptides and require precise structures of the peptide-MHC class I complex. In addition, the modified peptide must be cross-recognized by CTLs specific for the parental peptide, i.e. preserve the structure of the epitope. Detailed structural information on the modified peptide in complex with MHC is necessary for such predictions. In this thesis, the main focus is the development of theoretical in silico methods for prediction of both structure and cross-reactivity of peptide-MHC class I complexes. Applications of these methods in the context of immunotherapy are also presented. First, a theoretical method for structure prediction of peptide-MHC class I complexes is developed and validated. The approach is based on a molecular dynamics protocol to sample the conformational space of the peptide in its MHC environment. The sampled conformers are evaluated using conformational free energy calculations. The method, which is evaluated for its ability to reproduce 41 X-ray crystallographic structures of different peptide-MHC class I complexes, shows an overall prediction success of 83%. Importantly, in the clinically highly relevant subset of peptide-HLAA*0201 complexes, the prediction success is 100%. Based on these structure predictions, a theoretical approach for prediction of cross-reactivity is developed and validated. This method involves the generation of quantitative structure-activity relationships using three-dimensional molecular descriptors and a genetic neural network. The generated relationships are highly predictive as proved by high cross-validated correlation coefficients (0.78-0.79). Together, the here developed theoretical methods open the door for efficient rational design of improved peptides to be used in immunotherapy. Résumé La réponse immunitaire spécifique contre des tumeurs dépend de la reconnaissance par les récepteurs des cellules T CD8+ de peptides antigéniques présentés par les complexes majeurs d'histocompatibilité (CMH) de classe I. Ces peptides sont utilisés comme cible dans l'immunothérapie par vaccins peptidiques. Afin d'augmenter la réponse immunitaire, les peptides sont modifiés de façon à améliorer l'affinité et/ou la résistance à la dégradation. Ceci nécessite de connaître la structure tridimensionnelle des complexes peptide-CMH. De plus, les peptides modifiés doivent être reconnus par des cellules T spécifiques du peptide natif. La structure de l'épitope doit donc être préservée et des structures détaillées des complexes peptide-CMH sont nécessaires. Dans cette thèse, le thème central est le développement des méthodes computationnelles de prédiction des structures des complexes peptide-CMH classe I et de la reconnaissance croisée. Des applications de ces méthodes de prédiction à l'immunothérapie sont également présentées. Premièrement, une méthode théorique de prédiction des structures des complexes peptide-CMH classe I est développée et validée. Cette méthode est basée sur un échantillonnage de l'espace conformationnel du peptide dans le contexte du récepteur CMH classe I par dynamique moléculaire. Les conformations sont évaluées par leurs énergies libres conformationnelles. La méthode est validée par sa capacité à reproduire 41 structures des complexes peptide-CMH classe I obtenues par cristallographie aux rayons X. Le succès prédictif général est de 83%. Pour le sous-groupe HLA-A*0201 de complexes de grande importance pour l'immunothérapie, ce succès est de 100%. Deuxièmement, à partir de ces structures prédites in silico, une méthode théorique de prédiction de la reconnaissance croisée est développée et validée. Celle-ci consiste à générer des relations structure-activité quantitatives en utilisant des descripteurs moléculaires tridimensionnels et un réseau de neurones couplé à un algorithme génétique. Les relations générées montrent une capacité de prédiction remarquable avec des valeurs de coefficients de corrélation de validation croisée élevées (0.78-0.79). Les méthodes théoriques développées dans le cadre de cette thèse ouvrent la voie du design de vaccins peptidiques améliorés.

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis.

Psoriasis is a common T-cell-mediated skin disease with 2-3% prevalence worldwide. Psoriasis is considered to be an autoimmune disease, but the precise nature of the autoantigens triggering T-cell activation remains poorly understood. Here we find that two-thirds of patients with moderate-to-severe plaque psoriasis harbour CD4(+) and/or CD8(+) T cells specific for LL37, an antimicrobial peptide (AMP) overexpressed in psoriatic skin and reported to trigger activation of innate immune cells. LL37-specific T cells produce IFN-γ, and CD4(+) T cells also produce Th17 cytokines. LL37-specific T cells can infiltrate lesional skin and may be tracked in patients blood by tetramers staining. Presence of circulating LL37-specific T cells correlates significantly with disease activity, suggesting a contribution to disease pathogenesis. Thus, we uncover a role of LL37 as a T-cell autoantigen in psoriasis and provide evidence for a role of AMPs in both innate and adaptive immune cell activation.

Optimal activation of tumor-reactive T cells by selected antigenic peptide analogues.

Many mechanisms have been proposed to explain why immune responses against human tumor antigens are generally ineffective. For example, tumor cells have been shown to develop active immune evasion mechanisms. Another possibility is that tumor antigens are unable to optimally stimulate tumor-specific T cells. In this study we have used HLA-A2/Melan-A peptide tetramers to directly isolate antigen-specific CD8(+) T cells from tumor-infiltrated lymph nodes. This allowed us to quantify the activation requirements of a representative polyclonal yet monospecific tumor-reactive T cell population. The results obtained from quantitative assays of intracellular Ca(2+) mobilization, TCR down-regulation, cytokine production and induction of effector cell differentiation indicate that the naturally produced Melan-A peptides are weak agonists and are clearly suboptimal for T cell activation. In contrast, optimal T cell activation was obtained by stimulation with recently defined peptide analogues. These findings provide a molecular basis for the low immunogenicity of tumor cells and suggest that patient immunization with full agonist peptide analogues may be essential for stimulation and maintenance of anti-tumor T cell responses in vivo.

Wireless E911 Emergency Communications Fund Iowa Homeland Security and Emergency Management Division of the Iowa Department of Public Defense independent auditor's reports, basic financial statements and supplementary information, June 30, 2010.

Audit report on the Wireless E911 Emergency Communications Fund of the Iowa Homeland Security and Emergency Management Division of the Iowa Department of Public Defense for the year ended June 30, 2010

Lymphocyte specificity to protein antigens. III. Capacity of low responder mice to beef cytochrome c to respond to a peptide fragment of the molecule.

Lymph node cells derived from A.TH or A.TL mice primed with beef cytochrome c show striking patterns of reactivity when assayed in vitro for antigen-induced T cell proliferation. Whereas cells from A.TH mice respond specifically to beef cytochrome c or peptides composed of amino acids 1-65 and 81-104, cells from A.TL mice respond neither to beef cytochrome c nor to peptide 1-65, but proliferate following exposure to either peptide 81-104 or to a cytochrome c hybrid molecule in which the N-terminal peptide of beef (1-65) was substituted by a similar peptide obtained from rabbit cytochrome c. Thus, T cells from mice phenotypically unresponsive to beef cytochrome may, in fact, contain populations of lymphocytes capable of responding to a unique peptide, the response to which is totally inhibited when the same fragment is presented in the sequence of the intact protein.

Molecular mechanisms implicated in NF-κB activation and antiviral defense

SUMMARY Nuclear factor kappa B (NF-κB) transcription factors control many aspects of cell fate through induction of inflammatory, immune or survival molecules. We have identified two novel proteins, named receptor interacting protein (RIP)-4 and caspase recruitment domain (CARD) adaptor inducing interferon-β (Cardif), which activate NF-κB. Further, we have found that Cardif plays a prominent antiviral function. Antiviral innate immunity is mounted upon recognition by the host of virally associated structures like double-stranded (ds) RNA, which constitutes a viral replication product of many viruses within infected cells. dsRNA, depending on its subcellular localization, can be sensed by two separate arms of host defense. Firstly, Toll-like receptor (TLR)-3, a member of the type I transmembrane TLR family, recognizes endosomally-located dsRNA. Secondly, cytoplasmic dsRNA is detected by the recently identified RNA helicase retinoic acid inducible gene I (RIG-I). Triggering of TLR3- and RIG-I-dependent pathways results in the activation of the transcription factors NF-κB and Interferon regulatory factor (IRF)-3, which cooperatively transduce antiviral immune responses. We have demonstrated that RIP1, a kinase previously shown to be required for TNF signaling, transmits TLR3-dependent NF-κB activation. Further we have identified and characterized Cardif as an essential adaptor transmitting RIG-I-mediated antiviral responses, including activation of NF-κB and IRF3. In addition, we showed that Cardif is cleaved and inactivated by a serine protease of hepatitis C virus, and therefore may represent an attractive target for this virus to escape innate immune responses. RESUME Les facteurs de transcription "nuclear factor kappa B" (NF-κB) contrôlent divers aspects du devenir cellulaire à travers l'induction de molécules inflammatoires, immunitaires ou de survie. Nous avons identifié deux nouvelles protéines, nommées "receptor interacting protein" (RIP)-4 et "caspase recruitment domain (CARD) adaptor inducing interferon-β" (Cardif), qui activent NF-κB. En outre, nous avons trouvé que Cardif joue un rôle antiviral crucial. L'immunité innée antivirale s'établit au moment de la reconnaissance par l'hôte de structures virales, comme l'ARN double brin, qui constitue un produit de réplication de beaucoup de virus à l'intérieur de cellules infectées. L'ARN double brin, dépendant de sa localisation subcellulaire, peut être détecté par deux branches de défense distinctes. Premièrement, le récepteur transmembranaire "Toll-like" (TLR), TLR3, reconnaît l'ARN double brin lorsque localisé dans les endosomes. Deuxièmement, l'ARN double brin cytoplasmique est reconnu par l'ARN hélicase récemment décrite "retinoic acid inducible gene I" (RIG-I). Le déclenchement de voies dépendantes de TLR3 et RIG-I active les facteurs de transcription NF-κB et IRF3, qui coopèrent afin de transduire des réponses immunitaires antivirales. Nous avons démontré que RIP1, une kinase décrite précédemment dans le signalement du TNF, transmet l'activation de NF-κB dépendante de TLR3. De plus, nous avons identifié et caractérisé Cardif comme un adapteur essentiel transmettant les réponses antivirales médiées par RIG-I, qui incluent l'activation de NF-κB et IRF3. De surcroît, Cardif est clivé et inactivé par une sérine protéase du virus de l'hépatite C, et ainsi pourrait représenter une cible attractive pour ce virus afin d'échapper aux réponses immunitaires innées.