Identification of the SPLUNC1 ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airway epithelial cultures.

The epithelial sodium channel (ENaC) is responsible for Na(+) and fluid absorption across colon, kidney, and airway epithelia. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its NH2-terminal region lacks structure. Here we found that an 18 amino acid peptide, S18, which corresponded to residues G22-A39 of the SPLUNC1 NH2 terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold), while SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the βENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid (ASL) height was abnormally low (4.2 ± 0.6 μm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 μm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.

Role of innate immunity in cardiac inflammation after myocardial infarction.

Over the past two decades, inflammation has emerged as a key pathophysiological process during myocardial infarction. It develops consecutively to the activation of innate immune defense mechanisms, in response to the release of endogenous molecules by necrotic cells and the extracellular matrix. These danger signals are sensed by cellular receptors normally involved in antimicrobial defenses, including toll-like receptors and a subset of NOD-like receptors, which promote intracellular signaling dependent on nuclear factor kappaB and on the formation of the inflammasome. These mechanisms stimulate the expression of multiple inflammatory mediators and growth factors, sequentially inducing the recruitment of inflammatory cells, the clearance of injured tissue, angiogenesis, and the proliferation of fibroblasts, eventually resulting in scar formation and infarct healing. Dysregulation of these responses may result in continued cardiomyocyte loss, fibrosis beyond the limits of the infarcted area, reactive hypertrophy and chamber dilatation, a process termed adverse cardiac remodeling, leading to functional compromise and heart failure. This review presents the current state of knowledge on the process of immune activation within the infarcted myocardium and its consequences.

Gene expression profiling of isolated tumour cells from anaplastic large cell lymphomas: insights into its cellular origin, pathogenesis and relation to Hodgkin lymphoma.

Anaplastic large cell lymphoma (ALCL) is a main type of T-cell lymphomas and comprises three distinct entities: systemic anaplastic lymphoma kinase (ALK) positive, systemic ALK(-) and cutaneous ALK(-) ALCL (cALCL). Little is known about their pathogenesis and their cellular origin, and morphological and immunophenotypical overlap exists between ALK(-) ALCL and classical Hodgkin lymphoma (cHL). We conducted gene expression profiling of microdissected lymphoma cells of five ALK(+) and four ALK(-) systemic ALCL, seven cALCL and sixteen cHL, and of eight subsets of normal T and NK cells. The analysis supports a derivation of ALCL from activated T cells, but the lymphoma cells acquired a gene expression pattern hampering an assignment to a CD4(+), CD8(+) or CD30(+) T-cell origin. Indeed, ALCL display a down-modulation of many T-cell characteristic molecules. All ALCL types show significant expression of NFkappaB target genes and upregulation of genes involved in oncogenesis (e.g. EZH2). Surprisingly, few genes are differentially expressed between systemic and cALCL despite their different clinical behaviour, and between ALK(-) ALCL and cHL despite their different cellular origin. ALK(+) ALCL are characterized by expression of genes regulated by pathways constitutively activated by ALK. This study provides multiple novel insights into the molecular biology and pathogenesis of ALCL.

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease.

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): cellular localization, lesion-affected expression, and impaired regenerative axonal growth.

Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice.

Interactions between feather-degrading bacteria and an avian host : zebra finches (Taeniopygia guttata)

Abstract: Birds harbor a variety of bacteria on their plumage, some of which can degrade feathers in vitro. Whether these keratinolytic bacteria are active on live birds and can effect feather degradation on birds is debatable. The effect of such bacteria on the body condition and behavior of birds, is unknown. Using a community of feather-degrading bacteria (EB), we investigate the interaction between the activity and load of such bacteria, on the morphology, body condition, and behavior of zebra finches (Taeniopygia guttata). In Chapter 2, we find that the elevated loads of such microbes lead to a reduction in the expression of morphological traits, such as male bill color (a sexually selected trait) and uropygial gland volume, without reducing body mass, or evoking a cellular immune response. We also suggest the presence of a carotenoid based defense response in hosts, to such elevated loads of microbes and document a sex-based difference in the source of carotenoids used for such a response. In Chapter 3, we investigated the effect of EB loads on male mate choice of zebra finches, wherein male choice of females with elevated and un-altered bacterial loads, varied with male size. We found that larger males preferred females with higher bacterial load and smaller males preferred females with lower bacterial load. Chapter 4 demonstrates that the presence of melanin in feathers reduces the growth and activity of the community of feather-degrading bacteria (EB) and that the EB community can effect feather degradation in humid conditions, without broth. Additional results also demonstrate that the EB community consists of bacteria that can attach themselves to feathers on live birds and those that can live freely on avian plumage. Finally, chapter 5 demonstrates that the self-maintenance, social and sexual behaviors of birds are implicated in the infection and horizontal transmission of bacteria. It also suggests a linked oral - faecal - genital mode of transmission of pathogens in birds. These results demonstrate that differential loads of normal flora of vertebrate hosts can effect changes in their morphology and behavior. They also shed light on the role of feather-degrading bacteria in the evolution of melanin polymorphism in birds and suggest that bacteria can be active on live birds. This thesis also highlights the importance of social and, sexual behaviors of birds, in epidemiology. Résumé: Les Oiseaux ont dans leur plumage diverses bactéries dont certaines dégradent les plumes in vitro, néanmoins. Il n'est pas clair, au vu de précédentes études, si ces bactéries kératinolytiques sont actives sur des oiseaux vivants, et si celles-ci dégradent effectivement le plumage de leur hôte, L'effet de ces bactéries sur la condition corporelle ainsi que le comportement des oiseaux n'est pas connu. A l'aide d'une communauté de bactéries dégradant les plumes (EB), non pathogènes, nous examinons les interactions entre l'activité et la charge bactérienne sur la morphologie, la condition corporelle et le comportement du diamant mandarins (Taeniopygia guttata). Dans le chapitre 2, nous montrons qu'une charge élevée de ces microbes mène à une réduction de l'expression de certains traits morphologiques, tels que la couleur du bec chez le mâle (un trait soumis à sélection sexuelle), ainsi que le volume de la glande uropygienne, sans qu'il y ait une réduction de la masse corporelle, ni déclenchement d'une réponse immune cellulaire. Nos données suggèrent la présence d'une défense chez l'hôte à des charges élevées de bactéries basée sur la présence de caroténoïdes. Nous montrons, de plus une différence liée au sexe dans la source des caroténoïdes utilisé pour cette réponse. Dans le chapitre 3 nous examinons l'influence de la charge bactérienne EB sur le choix des mâles chez le diamant mandarins. Des femelles avec une charge bactérienne normale et augmentée sont choisies par les mâles et ce choix varie avec la taille des mâles. Nous avons mis en évidence que les grands mâles préfèrent les femelles avec une charge bactérienne plus élevée. Les petits mâles préfèrent les femelles avec une charge bactérienne réduite. Le chapitre 4 démontre que la présence de mélanine dans les plumes réduit la croissance et l'activité de la communauté de bactéries dégradant le plumage (EB), et que cette communauté EB peut dégrader les plumes dans des conditions humides, sans milieu de culture liquide. De plus nous montrons que cette communauté consiste en des bactéries qui peuvent s'attacher sur les plumes d'oiseaux vivants ainsi que des bactéries libres. Pour finir nous montrons dans le chapitre 5 que la maintenance corporelle, l'interaction sociale et le comportement sexuel de ces oiseaux sont impliqués dans l'infection et la transmission horizontale de ces bactéries. Nos données suggèrent une transmission orale-fécale-génitale des pathogènes chez les oiseaux. Ces résultats montrent que des charges différentes de la flore bactérienne habituelle et non pathogène de vertébrés peuvent affecter leur morphologie et leur comportement. Ils éclaircissent également le rôle des bactéries dégradant les plumes dans l'évolution du polymorphisme mélanique chez les oiseaux et suggèrent que ces bactéries peuvent être actives sur des oiseaux vivants. Cette thèse souligne également l'importance du comportement social et sexuel des oiseaux dans l'épidémiologie.

CREB-2, a cellular CRE-dependent transcription repressor, functions in association with Tax as an activator of the human T-cell leukemia virus type 1 promoter.

The Tax protein of the human T-cell leukemia virus type 1 (HTLV-1) has been implicated in human T-cell immortalization. The primary function of Tax is to transcriptionally activate the HTLV-1 promoter, but Tax is also known to stimulate expression of cellular genes. It has been reported to associate with several transcription factors, as well as proteins not involved in transcription. To better characterize potential cellular targets of Tax present in infected cells, a Saccharomyces cerevisiae two-hybrid screening was performed with a cDNA library constructed from the HTLV-1-infected MT2 cell line. From this study, we found 158 positive clones representing seven different cDNAs. We focused our attention on the cDNA encoding the transcription factor CREB-2. CREB-2 is an unconventional member of the ATF/CREB family in that it lacks a protein kinase A (PKA) phosphorylation site and has been reported to negatively regulate transcription from the cyclic AMP response element of the human enkephalin promoter. In this study, we demonstrate that CREB-2 cooperates with Tax to enhance viral transcription and that its basic-leucine zipper C-terminal domain is required for both in vitro and in vivo interactions with Tax. Our results confirm that the activation of the HTLV-1 promoter through Tax and factors of the ATF/CREB family is PKA independent.

Positive and negative regulation of cellular immune responses in physiologic conditions and diseases.

The immune system has evolved to allow robust responses against pathogens while avoiding autoimmunity. This is notably enabled by stimulatory and inhibitory signals which contribute to the regulation of immune responses. In the presence of a pathogen, a specific and effective immune response must be induced and this leads to antigen-specific T-cell proliferation, cytokines production, and induction of T-cell differentiation toward an effector phenotype. After clearance or control of the pathogen, the effector immune response must be terminated in order to avoid tissue damage and chronic inflammation and this process involves coinhibitory molecules. When the immune system fails to eliminate or control the pathogen, continuous stimulation of T cells prevents the full contraction and leads to the functional exhaustion of effector T cells. Several evidences both in vitro and in vivo suggest that this anergic state can be reverted by blocking the interactions between coinhibitory molecules and their ligands. The potential to revert exhausted or inactivated T-cell responses following selective blocking of their function made these markers interesting targets for therapeutic interventions in patients with persistent viral infections or cancer.

Iowa Department of Public Defense, Agency Performance Plan

Annual Report, Agency Performance Plan

Cell disposition of raltegravir and newer antiretrovirals in HIV-infected patients: high inter-individual variability in raltegravir cellular penetration.

Objectives The site of pharmacological activity of raltegravir is intracellular. Our aim was to determine the extent of raltegravir cellular penetration and whether raltegravir total plasma concentration (C(tot)) predicts cellular concentration (C(cell)). Methods Open-label, prospective, pharmacokinetic study on HIV-infected patients on a stable raltegravir-containing regimen. Plasma and peripheral blood mononuclear cells were simultaneously collected during a 12 h dosing interval after drug intake. C(tot) and C(cell) of raltegravir, darunavir, etravirine, maraviroc and ritonavir were measured by liquid chromatography coupled to tandem mass spectrometry after protein precipitation. Longitudinal mixed effects analysis was applied to the C(cell)/C(tot) ratio. Results Ten HIV-infected patients were included. The geometric mean (GM) raltegravir total plasma maximum concentration (C(max)), minimum concentration (C(min)) and area under the time-concentration curve from 0-12 h (AUC(0-12)) were 1068 ng/mL, 51.1 ng/mL and 4171 ng·h/mL, respectively. GM raltegravir cellular C(max), C(min) and AUC(0-12) were 27.5 ng/mL, 2.9 ng/mL and 165 ng·h/mL, respectively. Raltegravir C(cell) corresponded to 5.3% of C(tot) measured simultaneously. Both concentrations fluctuate in parallel, with C(cell)/C(tot) ratios remaining fairly constant for each patient without a significant time-related trend over the dosing interval. The AUC(cell)/AUC(tot) GM ratios for raltegravir, darunavir and etravirine were 0.039, 0.14 and 1.55, respectively. Conclusions Raltegravir C(cell) correlated with C(tot) (r = 0.86). Raltegravir penetration into cells is low overall (∼5% of plasma levels), with distinct raltegravir cellular penetration varying by as much as 15-fold between patients. The importance of this finding in the context of development of resistance to integrase inhibitors needs to be further investigated.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

Role of Endogenous GLP-1 and GIP in Beta Cell Compensatory Responses to Insulin Resistance and Cellular Stress.

Role of GLP-1 and GIP in beta cell compensatory responses to beta cell attack and insulin resistance were examined in C57BL/6 mice lacking functional receptors for GLP-1 and GIP. Mice were treated with multiple low dose streptozotocin or hydrocortisone. Islet parameters were assessed by immunohistochemistry and hormone measurements were determined by specific enzyme linked immunoassays. Wild-type streptozotocin controls exhibited severe diabetes, irregularly shaped islets with lymphocytic infiltration, decreased Ki67/TUNEL ratio with decreased beta cell and increased alpha cell areas. GLP-1 and GIP were co-expressed with glucagon and numbers of alpha cells mainly expressing GLP-1 were increased. In contrast, hydrocortisone treatment and induction of insulin resistance increased islet numbers and area, with enhanced beta cell replication, elevated mass of beta and alpha cells, together with co-expression of GLP-1 and GIP with glucagon in islets. The metabolic responses to streptozotocin in GLP-1RKO and GIPRKO mice were broadly similar to C57BL/6 controls, although decreases in islet numbers and size were more severe. In contrast, both groups of mice lacking functional incretin receptors displayed substantially impaired islet adaptations to insulin resistance induced by hydrocortisone, including marked curtailment of expansion of islet area, beta cell mass and islet number. Our observations cannot be explained by simple changes in circulating incretin concentrations, suggesting that intra-islet GLP-1 and GIP make a significant contribution to islet adaptation, particularly expansion of beta cell mass and compensatory islet compensation to hydrocortisone and insulin resistance.

Change in defense mechanisms and coping over the course of short-term dynamic psychotherapy for adjustment disorder.

Short-term dynamic psychotherapy (STDP) has rarely been investigated with regard to its underlying mechanisms of change, even if psychoanalytic theory informs us about several potential putative mechanisms of change in patients. Change in overall defensive functioning is one. In this study, we explored the role of overall defensive functioning, by comparing it on the process level with the neighbouring concept of overall coping functioning. A total of N=32 patients, mainly presenting adjustment disorder, were included in the study. The patients underwent STDP up to 40 sessions; three sessions per psychotherapy were transcribed and analyzed by using two observer-rating scales: Defense Mechanism Rating Scales (Perry, 1990) and Coping Action Patterns (Perry, Drapeau, Dunkley, & Blake, 2005). Hierarchical linear modeling was applied to model the change over the course of therapy and relate it to outcome. Results suggest that STDP has an effect on the target variable of overall defensive functioning, which was absent for overall coping functioning. Links with outcome confirm the importance of the effect. These results are discussed from methodological and clinical viewpoints.

Report of Recommendations to the Iowa Department of Public Defense, June 30, 2002

State Agency Audit Report