Strategies for Liver Transplantation Tolerance.

Liver transplant (LT) recipients require life-long immunosuppression (IS) therapy to preserve allograft function. The risks of chronic IS include an increased frequency of malignancy, infection, renal impairment, and other systemic toxicities. Despite advances in IS, long-term LT outcomes have not been improved over the past three decades.

De novo therapy with everolimus and reduced-exposure cyclosporine following pediatric kidney transplantation: a prospective, multicenter, 12-month study.

Prospective data regarding the de novo use of everolimus following kidney transplantation in children are sparse. In a prospective, 12-month, single-arm, open-label study, pediatric kidney transplant patients received everolimus (target trough concentration ≥3 ng/mL) with reduced-exposure CsA and corticosteroids, with or without basiliximab induction. Sixteen of the 18 patients completed the study on-treatment.

Everolimus with reduced tacrolimus improves renal function in de novo liver transplant recipients: a randomized controlled trial.

In a prospective, multicenter, open-label study, de novo liver transplant patients were randomized at day 30±5 to (i) everolimus initiation with tacrolimus elimination (TAC Elimination) (ii) everolimus initiation with reduced-exposure tacrolimus (EVR+Reduced TAC) or (iii) standard-exposure tacrolimus (TAC Control). Randomization to TAC Elimination was terminated prematurely due to a higher rate of treated biopsy-proven acute rejection (tBPAR). EVR+Reduced TAC was noninferior to TAC Control for the primary efficacy endpoint (tBPAR, graft loss or death at 12 months posttransplantation): 6.

Efficacy and safety of SBR759, a novel calcium-free, iron(III)-based phosphate binder, in Asian patients undergoing hemodialysis: A 12-week, randomized, open-label, dose-titration study versus sevelamer hydrochloride.

Aim: SBR759 is a calcium-free, polymeric, iron(III)-based oral phosphate binder, in development for the treatment of hyperphosphatemia. The efficacy and safety of SBR759 was compared with sevelamer hydrochloride in chronic kidney dialysis patients on hemodialysis.

Methods: Japanese and Taiwanese hyperphosphatemic patients who were on hemodialysis (n = 203) received starting doses of 3.

Everolimus with reduced cyclosporine versus MMF with standard cyclosporine in de novo heart transplant recipients.

Background: Pharmacokinetic modeling supports trough monitoring of everolimus, but prospective data comparing this approach versus mycophenolate mofetil (MMF) in de novo cardiac transplant recipients are currently unavailable.

Methods: In a 12-month multicenter open-label study, cardiac transplant patients received everolimus (trough level 3-8 ng/mL) with reduced cyclosporine A (CsA) or MMF (3 g/day) with standard CsA, both with corticosteroids+/-induction therapy.

Results: In total, 176 patients were randomized (everolimus 92, MMF 84).

Multicenter trial of everolimus in pediatric renal transplant recipients: results at three year.

There are few prospective clinical trials of mTOR inhibitors (or proliferation signal inhibitors) combined with CNI inhibitors in de novo pediatric renal transplantation. Results reported here are from a multicenter, open-label study in de novo pediatric renal transplant patients ( View Article and Find Full Text PDF

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells.

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites.

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin.

Optimization of the anti-(human CD3) immunotoxin DT389-scFv(UCHT1) N-terminal sequence to yield a homogeneous protein.

The production and regulatory approval processes for biopharmaceuticals require detailed characterization of potential products. Therapeutic proteins should preferably be homogeneous, although limited, reproducible, heterogeneity may be tolerated. A diphtheria toxin-based anti-(human CD3) immunotoxin, DT389-scFv(UCHT1), was expressed in Escherichia coli and purified following refolding [DT389 corresponds to amino acids 1-389 of diphtheria toxin, scFv is single-chain variable-region antibody fragment and UCHT1is an anti-(human CD3) monoclonal antibody].

Influence of relative binding affinity on efficacy in a panel of anti-CD3 scFv immunotoxins.

The in vitro cell killing potency of an immunotoxin reflects the aggregate of several independent biochemical properties. These include antigen binding affinity; internalization rate, intracellular processing and intrinsic toxin domain potency. This study examines the influence of antigen binding affinity on potency in various immunotoxin fusion proteins where target antigen binding is mediated by single chain antibody variable region fragments (scFv).

A high-capacity alkaline phosphatase reporter system for the rapid analysis of specificity and relative affinity of peptides from phage-display libraries.

We describe a novel reporter enzyme cassette system which enables the analysis of large numbers of linear and cyclic peptides in terms of their binding to a specific target molecule. In this system, peptides selected for target binding from random peptide phage-display libraries are expressed as cloned fusion proteins with bacterial alkaline phosphatase. The binding specificity and relative affinity of each peptide-enzyme fusion protein is then evaluated in a target-specific ELISA.

A human immunoglobulin (Ig)A calpha3 domain motif directs polymeric Ig receptor-mediated secretion.

Polymeric immunoglobulins provide immunological protection at mucosal surfaces to which they are specifically transported by the polymeric immunoglobulin receptor (pIgR). Using a panel of human IgA1/IgG1 constant region "domain swap" mutants, the binding site for the pIgR on dimeric IgA (dIgA) was localized to the Calpha3 domain. Selection of random peptides for pIgR binding and comparison with the IgA sequence suggested amino acids 402-410 (QEPSQGTTT), in a predicted exposed loop of the Calpha3 domain, as a potential binding site.

Localization of the binding site for the monocyte immunoglobulin (Ig) A-Fc receptor (CD89) to the domain boundary between Calpha2 and Calpha3 in human IgA1.

Immunoglobulin (Ig) A serves as the first line of humoral defense at all mucosal surfaces and is present in large quantities of blood. In playing its role in humoral immunity, IgA interacts with a variety of effector molecules present both in serum and on the surfaces of immune and inflammatory cells. To study these interactions, we previously established expression of human IgA1 in insect cells using recombinant baculoviruses and showed that the expressed antibody is a structurally and functionally intact polypeptide useful for examining the molecular properties of IgA.

Cloning and characterisation of TPO autoantibodies using combinatorial phage display libraries.

Thyroid lymphocyte RNA from a Hashimoto patient with high serum levels of autoantibodies to thyroid peroxidase (TPO) was used to construct a phage display antibody library in the phagemid vector pComb3. The library (100,000cfu) encoded IgG1 heavy chains together with kappa light chains. Selection of the phages displaying TPO antibody on TPO-coated ELISA plates yielded a phage population enriched for surface expression of TPO antibody Fabs.

Cloning of a human autoimmune response: preparation and sequencing of a human anti-thyroglobulin autoantibody using a combinatorial approach.

Thyroid lymphocyte RNA from a Hashimoto patient exhibiting high titre serum IgG autoantibodies against thyroglobulin (Tg) has been used to construct a Fab library in phage lambda. Screening of this library with radioiodinated Tg has permitted the cloning of an anti-Tg antibody (MH52) with an affinity of 4.5 x 10(9) molar-1 as determined by inhibition ELISA.

A protein kinase C pseudosubstrate peptide inhibits phosphorylation of the CD3 antigen in streptolysin-O-permeabilized human T lymphocytes.

Activation of human T lymphocytes leads to the phosphorylation of the CD3-antigen gamma polypeptide. We have investigated a possible role for protein kinase C (PKC) in mediating this phosphorylation event by using T cells permeabilized with streptolysin-O in the presence of 120 mM-K+ buffers containing Ca2+-EGTA. The gamma-chain was phosphorylated by [gamma-32P]ATP in permeabilized T lymphoblasts in the presence of phorbol 12,13-dibutyrate (Pdbu) or phytohaemagglutinin (PHA).

The association of type 1, type 2A and type 2B phosphatases with the human T lymphocyte plasma membrane.

Several putative plasma-membrane-associated components of the T-lymphocyte signal-transduction pathway are phosphorylated during the initial events of cellular activation. Little is known about the control of dephosphorylation of these components. We have shown by immunoblotting that the type 1 phosphatase, the type 2A phosphatase and type 2B phosphatase (calcineurin) are associated with the plasma membrane of normal human T lymphoblasts and the human T leukaemic cell line Jurkat 6.

Evidence that a GTP binding protein regulates phosphorylation of the CD3 antigen in human T lymphocytes.

The role of guanine nucleotide binding regulatory proteins (G proteins) in the regulation of phosphorylation of the gamma subunit of the CD3 antigen has been examined. CD3 gamma chain phosphorylation in isolated T cell microsomes was stimulated by the G protein activator guanosine 5'-0 thiotriphosphate (GTP gamma S), but cyclic adenosine monophosphate and guanosine 5'-diphosphate were ineffective at inducing gamma chain phosphorylation. The effect of GTP gamma S was rapid and transient; a half maximal effect was observed with 50 microM of the nucleotide.

The human T3 gamma chain is phosphorylated at serine 126 in response to T lymphocyte activation.

The gamma subunit of the human T lymphocyte T3 antigen is rapidly phosphorylated on serine residues in vivo during the initiation of T cell activation by a polyclonal mitogen (Phaseolus vulgaris phytohemagglutinin), an activator of protein kinase C (phorbol 12,13-dibutyrate), and an elevator of intracellular calcium (ionomycin). The sites of phosphorylation were identified by comparing tryptic peptide analyses of T3 gamma chains labeled in vivo with various synthetic peptides, corresponding to portions of the cytoplasmic domain of the gamma chain that had been labeled in vitro using purified protein kinase C. Two sites, serines 123 and 126, were phosphorylated in response to ionomycin, whereas a single site, serine 126, was phosphorylated when T lymphocytes were stimulated by P.

A consensus amino-acid sequence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding proteins.

A group of calcium-binding proteins which bind to biomembranes has recently been identified in widely different cells and tissues (refs 1-7, reviewed in ref. 8). Three of these proteins (p70, p36 and p32.

Homology between the subunits of S100 and a 10kDa polypeptide associated with p36 of pig lymphocytes.

The polypeptide of about 10,000-Mr which is associated with the p36 polypeptide of pig mesenteric lymph node lymphocytes has a N-terminal 51 amino acid sequence that is 56 and 42% homologous with the N-terminii of the alpha and beta subunits respectively of S100.