Spontaneous Ocular Autoimmunity in Mice Expressing a Transgenic T Cell Receptor Specific to Retina: A Tool to Dissect Mechanisms of Uveitis.

The "classical" EAU model induced by immunization of mice with the retinal protein IRBP or its peptides has been very useful to study basic mechanisms of ocular inflammation, but is inadequate for some types of studies due to the need for active immunization in the context of strong bacterial adjuvants. We generated transgenic (Tg) mice on the B10.RIII background that express a T cell receptor (TCR) specific for IRBP161-180. Three strains of TCR Tg mice were established. Spontaneous uveitis developed in two of the three strains by 2-3 months of age. Susceptibility correlated with a higher copy number of the transgenic TCR and a higher proportion of TCR Tg T cells in the peripheral repertoire. Even in mice with uveitis, peripheral IRBP-specific CD4(+) T cells displayed mostly a naïve phenotype. In contrast, T cells infiltrating uveitic eyes mostly showed an effector/memory phenotype, and included Th1, Th17 as well as T regulatory cells. These mice thus provide a new and distinct model of uveitis from the "classical" EAU, and may represent some types of uveitis more faithfully. Importantly, this new transgenic model of uveitis can serve as a template for therapeutic manipulations, and as a source of naïve retina-specific T cells for a variety of basic and pre-clinical studies. Several examples of such studies will be discussed.

Enhanced T-cell activation and differentiation in lymphocytes from transgenic mice expressing ubiquitination-resistant 2KR LAT molecules.

Linker for activation of T cells (LAT) is critical for the propagation of T-cell signals upon T-cell receptor (TCR) activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KR LAT) resulted in decreased LAT ubiquitination and elevated T-cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T-cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KR LAT. On TCR stimulation of T cells from these mice, proximal signaling and cytokine production was elevated in 2KR versus wild-type (WT) LAT mice. Enhanced cytolytic activity as well as T-helper responses were observed on LAT expression, which were further elevated by 2KR LAT expression. Despite greater T-effector function, WT or 2KR LAT expression did not have any effect on clearance of certain pathogens or tumors. Our data support the model that lack of tumor clearance is due to increased differentiation and acquisition of effector phenotype that is associated with suboptimal immunity in an immunotherapy model. Thus, our data further reinforce the role of LAT ubiquitination in TCR signaling and uncovers a novel role for LAT in driving T-cell differentiation.

A new approach to an old problem: synthetic biology tools for human disease and metabolism.

The field of synthetic biology seeks to develop engineering principles for biological systems. Along these lines, synthetic biology can address human metabolic disease through the development of genetic approaches to the study and modification of metabolism. The re-engineering of natural metabolic states provides fundamental understanding of the integrated components underlying dysfunctional metabolism. Alternatively, the development of biological devices that can both sense and affect metabolic states could render unique control over disease states. In this chapter, we discuss the advancement of synthetic biological approaches to monitoring and engineering metabolism, as well as prospects for synthetic biology's future role in the prevention and treatment of metabolic disease.

Nuclear origins of cell-to-cell variability.

During the past decade, it has become increasingly evident that there is variation in the transcriptome of genetically identical cells, even when grown in homogenous environments. This cell-to-cell variability has been shown to have a central role in processes ranging from stem cell differentiation to chemotherapy resistance. Given that many genes display extensive heterogeneity in their messenger RNA (mRNA) abundance on a per cell basis, understanding the nuclear sources of this variability is important for our fundamental grasp of nuclear function and stands to have clinical manifestations. In this chapter, we assess the contribution of different transcription regimes, nuclear architecture dynamics, RNA polymerase elongation, and gene copy number to transcriptome heterogeneity. We also discuss techniques that can be used to quantify single-cell mRNA abundance and conclude by commenting on future research directions.

Molecular systems biology in drug development.

Molecular systems biology seeks to explain the behavior of complex cellular systems through a multicomponent analysis. We illustrate this approach and its relevance to drug development by reviewing two examples of the interplay between cellular processes and drugs: the internalization and recycling of oncogenic receptors, their ligands and therapeutic antibodies; and strategies for discovering drugs that affect intracellular protein kinase pathways.

Regulation of sterol metabolism in Candida albicans by the UPC2 gene.

Candida albicans is an important pathogenic fungus of humans, causing a range of infections. These infections are usually treated with antifungal drugs that target sterol metabolism. Resistance to these antifungals can result from overexpression of sterol biosynthetic genes. Therefore it is of interest to understand transcriptional regulation of sterol biosynthesis in C. albicans. Recently two reports [Silver, Oliver and White (2004) Eukaryot. Cell 3, 1391-1397; MacPherson, Akache, Weber, De Deken, Raymond and Turcotte (2005) Antimicrob. Agents Chemother. 49, 1745-1752] have identified and characterized a single C. albicans transcription factor gene UPC2 that regulates sterol metabolism. The details of both characterizations are compared and contrasted. These reports extend our understanding of sterol regulation in this important human pathogen.

The mantle flow field beneath western North America.

Although motions at the surface of tectonic plates are well determined, the accompanying horizontal mantle flow is not. We have combined observations of surface deformation and upper mantle seismic anisotropy to estimate this flow field for western North America. We find that the mantle velocity is 5.5 +/- 1.5 centimeters per year due east in a hot spot reference frame, nearly opposite to the direction of North American plate motion (west-southwest). The flow is only weakly coupled to the motion of the surface plate, producing a small drag force. This flow field is probably due to heterogeneity in mantle density associated with the former Farallon oceanic plate beneath North America.

Pediatric sedation for procedures titrated to a desired degree of immobility results in unpredictable depth of sedation.

OBJECTIVE: To test the hypothesis that the need to attain immobility during pediatric sedation for procedures determines the depth of sedation, which cannot always be predicted. DESIGN: A retrospective review of sedation documents of 301 consecutive sedations of pediatric patients undergoing various procedures SETTING: Division of Critical Care sedation service within a children's hospital. MEASUREMENTS AND MAIN RESULTS: The medical records and sedation forms of our most recent 301 consecutive sedations were retrospectively reviewed. Based on the data gathered, the patients were categorized according to their achieved level of immobility, their level of consciousness according to the definitions of the American Academy of Pediatrics, the procedures for which sedation was administered, and the sedatives used. A total of 125 males and 89 females received 301 sedations. Their ages ranged from 22 days to 29 years (mean 7 y + 6 y). We recognized four categories of immobility for procedures. In category 1, some motion was allowed during painless and noninvasive procedures to the extent that it did not risk the patient nor hinder the successful performance of the procedures. In category 2, the patients were kept motionless during painless and noninvasive procedures. In category 3, the patients were kept motionless during painful and invasive procedures with the addition of local anesthetic. In category 4, the patients remained motionless throughout their painful or invasive procedure without the use of local anesthetics. There were 32, 10, 156 and 103 sedations in each category, respectively. Conscious sedation (CS) was observed in six sedations (19%) in category 1 of immobility; it was observed in none (0%) in category 2, in 4 sedations (2.6%) in category 3, and in 1 sedation (1%) in category 4. Deep sedation (DS) was noted in 26 category 1 sedations (81%), in 10 category 2 sedations (100%), in 136 category 3 sedations (87%), and in 63 category 4 sedations (61%). General anesthesia (GA) was only observed in categories 3 and 4 in 16 sedations (10%) and 39 sedations (38%), respectively. Intravenous (IV) ketamine, as a single agent or in combination with other agents, was the most frequently used sedative (88%) followed by IV benzodiazepines (64%), propofol (39%), opiates (15%), and barbiturates (5%). A total of 59 (19%) adverse events were encountered during the 301 sedations. In categories 1 and 2, no adverse event (0%) was encountered. In category 3, 19 adverse events took place (32%), and 40 adverse events (68%) (P< 0.05) occurred in category 4. CONCLUSIONS: Pediatric sedation results in 4 categories of immobility. Complete immobility during painful and invasive procedures is associated with a higher incidence of adverse events. The depth of sedation (ie, CS, DS, or GA) required to achieve each category of immobility is unpredictable and varies from patient to patient. Thus, granting a limited sedation authority (conscious sedation only) to physicians may be of limited practical value.

The conserved npl4 protein complex mediates proteasome-dependent membrane-bound transcription factor activation.

Proteolytic activation of membrane-bound transcription factors has emerged as an important mechanism for the regulation of gene expression. Two membrane-bound transcription factors regulated in this manner are the Saccharomyces cerevisiae proteins Mga2p and Spt23p, which direct transcription of the Delta9-fatty acid desaturase gene OLE1. We now show that a membrane-associated complex containing the highly conserved Npl4p, Ufd1p, and Cdc48p proteins mediates the proteasome-regulated cleavage of Mga2p and Spt23p. Mutations in NPL4, UFD1, and CDC48 cause a block in Mga2p and Spt23p processing, with concomitant loss of OLE1 expression. Taken together, our data indicate that the Npl4 complex may serve to target the proteasome to the ubiquitinated endoplasmic reticulum membrane-bound proteins Mga2p and Spt23p. Given the recent finding that NPL4 is allelic to the ERAD gene HRD4, we further propose that this NPL4 function extends to all endoplasmic reticulum-membrane-associated targets of the proteasome.

Localization of yeast telomeres to the nuclear periphery is separable from transcriptional repression and telomere stability functions.

The left telomere of Saccharomyces chromosome VII was often localized near the nuclear periphery, even in cells lacking the silencing proteins Sir3 or Hdf1. This association was lost in late mitotic cells and when transcription was induced through the telomeric tract. Although in silencing competent cells there was no correlation between the fraction of cells in which a telomeric gene was repressed and the fraction of cells in which it was localized to the periphery, no condition was found where the telomere was both silenced and away from the periphery. We conclude that localization of a telomere to the nuclear periphery is not sufficient for transcriptional repression nor does it affect the stability function of yeast telomeres.

Messenger RNAs are recruited for nuclear export during transcription.

Following transcription and processing, eukaryotic mRNAs are exported from the nucleus to the cytoplasm for translation. Here we present evidence that mRNAs are targeted for nuclear export cotranscriptionally. Combined mutations in the Saccharomyces cerevisiae hnRNP Npl3 and TATA-binding protein (TBP) block mRNA export, implying that cotranscriptional recruitment of Npl3 is required for efficient export of mRNA. Furthermore, Npl3 can be found in a complex with RNA Pol II, indicating that Npl3 associates with the transcription machinery. Finally, Npl3 is recruited to genes in a transcription dependent manner as determined by chromatin immunoprecipitation. Another mRNA export factor, Yra1, also associates with chromatin cotranscriptionally but appears to be recruited at a later step. Taken together, our results suggest that export factors are recruited to the sites of transcription to promote efficient mRNA export.

Pertussis toxin inhibits induction of tissue-specific autoimmune disease by disrupting G protein-coupled signals.

Pertussis toxin (PTX) has been used for many years as an adjuvant that promotes development of tissue-specific experimental autoimmune diseases such as experimental autoimmune encephalomyelitis, experimental autoimmune uveitis (EAU), and others. Enhancement of vascular permeability and of Th1 responses have been implicated in this effect. Here we report a surprising observation that, in a primed system, PTX can completely block the development of EAU. Disease was induced in B10.RIII mice by adoptive transfer of uveitogenic T cells, or by immunization with a uveitogenic peptide. A single injection of PTX concurrently with infusion of the uveitogenic T cells, or two injections 7 and 10 days after active immunization, completely blocked development of EAU. EAU also was prevented by a 1-h incubation in vitro of the uveitogenic T cells with PTX before infusing them into recipients. Uveitogenic T cells treated with PTX in vitro and lymphoid cells from mice treated with PTX in vivo failed to migrate to chemokines in a standard chemotaxis assay. Neither the isolated B-oligomer subunit of PTX that lacks ADP ribosyltransferase activity nor the related cholera toxin that ADP-ribosylates G(s) (but not G(i)) proteins blocked EAU induction or migration to chemokines. We conclude that PTX present at the time of cell migration to the target organ prevents EAU, and propose that it does so at least in part by disrupting signaling through G(i) protein-coupled receptors. Thus, the net effect of PTX on autoimmune disease would represent an integration of enhancing and inhibitory effects.

Anti-tumor activities of chondroitinase AC and chondroitinase B: inhibition of angiogenesis, proliferation and invasion.

In the current study, two specific glycosaminoglycan lyases, chondroitinase AC and chondroitinase B, were utilized to examine the roles of chondroitin sulfates and dermatan sulfate in tumor metastasis and angiogenesis. Melanoma cells (SK-MEL) or endothelial cells were treated with either medium or chondroitinase enzyme. Chondroitinase AC inhibited melanoma invasion and proliferation as well as endothelial proliferation and angiogenesis. Apoptosis of melanoma and endothelial cells, as measured by the activity of caspase-3, was also increased by chondroitinase AC, but not by chondroitinase B. Chondroitinase B inhibited endothelial and melanoma proliferation and invasion, but to a lesser extent than chondroitinase AC. Neither chondroitinase had a detectable effect on gelatinase secretion by melanoma cells. These results indicate that both chondroitin and dermatan sulfates regulate many cellular activities related to metastasis.

A survey of spinal and epidural techniques in adult cardiac surgery.

OBJECTIVE: To determine if a significant number of anesthesiologists are performing spinal and epidural techniques in adults undergoing cardiac surgery and if any neurologic injuries have been associated with these techniques. DESIGN: Anonymous survey of clinical practice. SETTING: Data collected via mail from members of the Society of Cardiovascular Anesthesiologists. PARTICIPANTS: Attending anesthesiologists. INTERVENTIONS: Anesthesiologists completed an anonymous survey of their use of spinal and epidural techniques in adult patients undergoing cardiac surgery. MEASUREMENTS AND MAIN RESULTS: Of 3974 anesthesiologists, 974 (24%) responded to the questionnaire; 892 are at institutions that perform cardiac surgery. Of the 892 responders, 68 (7.6%) reported they use spinal techniques, whereas 62 (7%) reported they use epidural techniques. Nine (1%) anesthesiologists reported they use both techniques. There were no reports of neurologic complications related to the use of spinal or epidural techniques performed by the anesthesiologists responding to the survey. CONCLUSIONS: A significant number of anesthesiologists are performing spinal and epidural techniques in adult patients undergoing cardiac surgery. Prospective, controlled trials should be performed to evaluate the benefits and risks of spinal and epidural techniques in this population.

Evaluating the validity of responsiveness to inhaled nitric oxide in pediatric patients with ARDS: an analytic tool.

OBJECTIVES: To determine whether improved oxygenation indicates a valid response to inhaled nitric oxide (iNO) therapy in patients with pediatric ARDS, and to establish an analytic tool to differentiate the iNO effects from those of other interactive factors in pediatric patients with ARDS. DESIGN: Consecutive case series evaluated by post hoc analysis tool. PATIENTS AND METHODS: Nineteen patients treated with iNO for ARDS or pulmonary hypertension were enrolled in our study. We evaluated the PaO(2)/fraction of inspired oxygen ratio (PF ratio), oxygenation index (OI), patient position (prone vs supine), PaCO(2), pH, and vasoactive drug support, and classified patients' responsiveness to iNO into three categories: (1) possible response, an increase in PF ratio, with no alteration of the aforementioned variables in a direction known to improve oxygenation; (2) nonspecific response, an increase in PF ratio with no increase in OI, and alteration of one or more of the other four criteria in a direction known to improve oxygenation; and (3) undetermined response, an increase in both the PF ratio and OI, indicating a deliberate augmentation in ventilator support. RESULTS: A total of 119 data points were evaluated. Fifty data points (42%) exhibited no response to iNO. Thirty-two data points (27%) were classified as having possible responses, 35 data points (29%) as nonspecific, and 2 data points (2%) as undetermined responses to the iNO treatment. CONCLUSIONS: In ARDS, improved oxygenation amid iNO treatment is multifactorial. In only 27% of our evaluated data points could the increase in PF ratio be attributed to iNO. We suggest that when clinically utilizing iNO, the interactive factors described by us should be taken into account for data analysis.

Preventing recurrent depression using cognitive therapy with and without a continuation phase: a randomized clinical trial.

BACKGROUND: Cognitive therapy (CT) may reduce depressive relapse and recurrence when patients learn and use the associated skills. Reported relapse and recurrence rates after CT discontinuation vary widely. The factors that determine when CT is preventive remain unidentified. We developed continuation-phase CT (C-CT) to teach responders skills to prevent relapse. This is the first randomized trial comparing CT with and without a continuation phase in responders to CT who were vulnerable, given their history of recurrent unipolar depression. METHODS: Patients aged 18 to 65 years (n = 156) with recurrent DSM-IV major depressive disorder (MDD) entered 20 sessions of acute-phase CT (A-CT). Unmedicated responders (ie, no MDD and 17-item Hamilton Rating Scale for Depression score < or =9; n = 84) were randomized to either 8 months (10 sessions) of C-CT or control (evaluation without CT). Follow-up lasted an additional 16 months. A clinician blind to assignment evaluated relapse and recurrence (ie, DSM-IV MDD). RESULTS: Over an 8-month period, C-CT significantly reduced relapse estimates more than control (10% vs 31%). Over 24 months, including the CT-free follow-up, age of onset and quality of remission during the late phase of A-CT each interacted with condition assignment to influence durability of effects. In patients with early-onset MDD, C-CT significantly reduced relapse and recurrence estimates (16% vs 67% in control). When patients had unstable remission during late A-CT, C-CT significantly reduced relapse and recurrence estimates to 37% (vs 62% in control). CONCLUSIONS: Findings suggest that 8 months of C-CT significantly reduces relapse and recurrence in the highest-risk patients with recurrent MDD. Risk factors influenced the necessity for C-CT.

Synthetic heparin pentasaccharide depolymerization by heparinase I: molecular and biological implications.

A synthetic pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 IU/mg) and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin pentasaccharides are resistant to the digestive effects of heparinase I. To investigate the effect of heparinase I on the AXa activity of pentasaccharide SR90107/ORG31540, graded concentrations (1.25-100 microg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was employed to determine the potential breakdown products of the pentasaccharide. The experiment with the pentasaccharide included incubation (37 degrees C) at 1 mg/ml and exposure to graded concentrations of heparinase I (0.125-1 U/ml). After 30 min of incubation, the enzymatic activity was stopped by heat treatment and the mixture was analyzed using high performance size exclusion chromatography (HPSEC). Heparinase I concentration-dependent cleavage of the pentasaccharide was evident. The breakdown products exhibited a mass of 1,034 d and 743 d, respectively, suggesting the generation of a trisaccharide and a disaccharide moiety. The extinction of a disaccharide moiety in the UV region was high, indicating the presence of a double bond in this molecule. These data clearly suggest that pentasaccharide SR90107/ORG31540 is digestible by heparinase I into its two components. Furthermore, these data support the hypothesis that heparinase I can be used as a neutralizing agent for pentasaccharide overdose. Additionally, a highly methylated analog of the previously mentioned synthetic pentasaccharide. SanOrg34006, which has also been subjected to similar experiments, has shown complete resistance to the depolymerizing function of heparinase I; therefore, its use may be appropriate in chronic situations as a long-acting form of the pentasaccharide.

The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants.

The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

Use of the A. victoria green fluorescent protein to study protein dynamics in vivo.

Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.