Humanization of T cell-mediated immunity in mice.

Despite the enormous promise of T cell therapies, the isolation and study of human T cell receptors (TCRs) of dedicated specificity remains a major challenge. To overcome this limitation, we generated mice with a genetically humanized system of T cell immunity. We used VelociGene technology to replace the murine TCRαß variable regions, along with regions encoding the extracellular domains of co-receptors CD4 and CD8, and major histocompatibility complex (MHC) class I and II, with corresponding human sequences. The resulting "VelociT" mice have normal myeloid and lymphoid immune cell populations, including thymic and peripheral αß T cell subsets comparable with wild-type mice. VelociT mice expressed a diverse TCR repertoire, mounted functional T cell responses to lymphocytic choriomeningitis virus infection, and could develop experimental autoimmune encephalomyelitis. Immunization of VelociT mice with human tumor-associated peptide antigens generated robust, antigen-specific responses and led to identification of a TCR against tumor antigen New York esophageal squamous cell carcinoma-1 with potent antitumor activity. These studies demonstrate that VelociT mice mount clinically relevant T cell responses to both MHC-I­ and MHC-II­restricted antigens, providing a powerful new model for analyzing T cell function in human disease. Moreover, VelociT mice are a new platform for de novo discovery of therapeutic human TCRs.

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs.

In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a lacZ reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.

Changing sedation practices in the intensive care unit--protocol implementation, multifaceted multidisciplinary approach and teamwork.

INTRODUCTION: Sedation protocols have demonstrated effectiveness in improving ICU sedation practices. However, the importance of multifaceted multidisciplinary approach on the success of such protocols has not been fully examined. METHODS: The study was conducted in a tertiary care medical-surgical ICU as a prospective, 4-pronged, observational study describing a quality improvement initiative that employs 2 types of controlled comparisons: a "before and after" comparison related to intense education of ICU clinicians and nurses about sedation and analgesia in the ICU, and a comparison of protocolized versus non-protocolized care. Patients were assigned alternatively to receive sedation by a goal-directed protocol using the Riker Sedation-Agitation Scale (SAS) or by standard practice. A multifaceted multidisciplinary educational program was initiated including the use of point of use reminders, directed educational efforts, and opinion leaders. This included several lectures and in-services and the routine availability of at least one member of this group to answer questions. We included all consecutive patients receiving mechanical ventilation, who were judged by their treating team to require intravenous sedation. MEASUREMENTS AND MAIN RESULTS: The following data was collected: demographics, Acute Physiology and Chronic Health Evaluation (APACHE) II score and Simplified Acute Physiology score (SAPS) II, daily doses of analgesics and sedatives, duration of mechanical ventilation, ICU length of stay (LOS) and ventilator associated pneumonia (VAP) incidence. To examine the effect of the multifaceted multidisciplinary approach, we compared the first 3 months to the second 3 months in the following 4 groups: G1 no protocol group in the first 3 months, G2 protocol group in first 3 months, G3 no protocol group in the second 3 months, G4 protocol group in the second 3 months. After ICU day 3, SAS in the groups G2, G3 and G4 became higher than in G1 reflecting "lighter" levels of sedation. There were significant reductions in the use of analgesics and sedatives in the protocol group after 3 months. This was associated with a reduction in VAP rate and trends towards shorter mechanical ventilation duration and hospital length of stay (LOS). CONCLUSIONS: The implementation of a multifaceted multidisciplinary approach including the use of point of use reminders, directed educational efforts, and opinion leaders along with sedation protocol led to significant changes in sedation practices and improvement in patients' outcomes. Such approach appears to be critical for the success of ICU sedation protocol.

Discovery and evaluation of highly active imidotitanium ethylene polymerisation catalysts using high throughput catalyst screening.

A family of ca. 50 imidotitanium precatalysts [Ti(NR)(Me(3)[9]aneN(3))Cl(2)](R = alkyl or aryl; Me(3)[9]aneN(3)= 1,4,7-trimethyltriazacyclononane) were prepared in good yields using semi-automated procedures; high-throughput screening techniques identified seven highly active ethylene polymerisation precatalysts with activities in the range ca. 3 400 to 10 000 kg(PE) mol(-1) h(-1) bar(-1).

Studies of a nickel-based single-molecule magnet.

A cyclic complex [Ni(12)(chp)(12)(O(2)CMe)(12)(thf)(6)(H(2)O)(6)] (1) has been synthesised and studied (chp=6-chloro-2-pyridonate). Complex 1 exhibits ferromagnetic exchange between the S=1 centres, giving an S=12 spin ground state. Detailed studies demonstrate that it is a single-molecule magnet with an energy barrier of approximately 10 K for reorientation of magnetisation. Resonant quantum tunnelling is also observed. The field between resonances allows accurate measurement of D, which is 0.067 K. Inelastic neutron scattering studies have allowed exchange parameters to be derived accurately, which was impossible from susceptibility data alone. Three exchange interactions are required: two ferromagnetic nearest neighbour interactions of approximately 11 and 2 cm(-1) and an anti-ferromagnetic next nearest neighbour interaction of -0.9 cm(-1).

Dinuclear and Hexanuclear Iron(III) Oxide Complexes with a Bis(bipyridine) Ligand: A New [Fe(6)(&mgr;(3)-O)(4)](10+) Core.

The use of the bis(bipyridine) ligand L (1,2-bis(2,2'-bipyridyl-6-yl)ethane) has yielded new dinuclear and hexanuclear complexes. The FeCl(3)/NaO(2)CPh/L (4:4:1) reaction system in MeCN gives red-brown [Fe(6)O(4)Cl(4)(O(2)CPh)(4)L(2)][FeCl(4)](2) (1). The same reaction system in a 3:3:1 ratio in MeOH gives orange [Fe(2)(OMe)(2)Cl(2)(O(2)CPh)L][FeCl(4)] (2). Complex 1.2MeCN: monoclinic, P2(1)/a, a = 15.317(2) Å, b = 18.303(3) Å, c = 16.168(3) Å, beta = 108.91(1) degrees, and Z = 2. Complex 2: triclinic, P&onemacr;, a = 14.099(6) Å, b = 18.510(7) Å, c = 7.108(3) Å, alpha = 96.77(2) degrees, beta = 99.45(2) degrees, gamma = 81.16(2) degrees, and Z = 2. The cation of 1 consists of a near-planar [Fe(6)(&mgr;(3)-O)(4)](10+) core that can be described as three edge-fused [Fe(2)O(2)] rhombs to which are attached two additional Fe atoms. The cation of 2 contains a [Fe(2)(&mgr;-OMe)(2)(&mgr;-O(2)CPh)](3+) core. In both cations, the L group acts as a bridging ligand across an Fe(2) unit, with the bpy rings essentially parallel. Variable-temperature solid-state magnetic-susceptibility studies of 1 and 2 in the 2.00-300 K range reveal that for both complexes the data are consistent with an S = 0 cation and S = (5)/(2) [FeCl(4)](-) anions. These conclusions were confirmed by magnetization vs field studies in the 2.00-4.00 K and 10.0-50.0 kG ranges. Fitting of the data for 2 to the appropriate theoretical equation for an equimolar composition of Fe(2) cations and [FeCl(4)](-) anions allowed the exchange interaction in the cation to be determined as J = -10.5 cm(-)(1) (H = -2JS(1)S(2)) with g held at 2.00. The obtained J value is consistent with that predicted by a previously published magnetostructural relationship.