Renal Transplant Recipients Treated with Calcineurin-Inhibitors Lack Circulating Immature Transitional CD19+CD24hiCD38hi Regulatory B-Lymphocytes

BackgroundCD19⁺CD24^hiCD38^hi transitional immature B-lymphocytes have been demonstrated to play an important role in regulating the alloimmune response in transplant recipients. Here, we analyzed the effect of calcineurin inhibition on these peripherally circulating regulatory B-cells (Breg) in renal transplant recipients receiving cyclosporine A (CsA) or tacrolimus.MethodsPBMCs from healthy subjects (HS) (n = 16) and renal transplant recipients (n = 46) were isolated. Flow cytometry was performed for CD19, CD24, CD38 and IL-10 either after isolation or after 72 hours of co-culture in presence of PMA/Ionomycin and TLR9-ligand in presence or absence of increasing concentrations of tacrolimus or CsA.ResultsThe amount of CD19⁺ B-cells among lymphocytes was ∼9.1% in HS, ∼3.6% in CsA (n = 11, p<0.05) and ∼6.4% in TAC (n = 35, p<0.05) treated patients. Among B-cells, a distinct subset of Breg was found to be 4.7% in HS, 1.4% in tacrolimus treated patients and almost blunted in patients receiving CsA. Similarily, ∼4% of B-cells in HS and even fewer in CsA or tacrolimus treated patients produced IL-10 (0.5% and 1.5%, p<0.05) and this was confirmed both in non-transplanted CsA-treated healthy subjects and in in vitro co-culture experiments. Among 29 patients with <1% of Breg, 9 cases (31%) displayed an allograft rejection in contrast to only one case of rejection (6%) among 17 patients with >1%.ConclusionCalcineurin inhibitors reduce number and IL-10 production of Bregs in the peripheral circulation of both renal transplant recipients and non-transplanted healthy subjects. CNI induced Breg reduction is not restricted to a solid organ transplant setting and is not mediated by co-medication with steroids or MPA. A low proportion of Breg cells is associated with an elevated frequency of allograft rejection events.     

Muskoxen Modify Plant Abundance,Phenology, and Nitrogen Dynamics in a High Arctic Fen

Ecosystems - Herbivores are key drivers of vegetation dynamics in most ecosystems. However, the effect of high arctic herbivores on vegetation dynamics throughout a growing season is not well...     

Change in substrate specificity of cytotoxic necrotizing factor unmasks proteasome-independent down-regulation of constitutively active RhoA

Cytotoxic necrotizing factors CNF1 and CNF2 are produced by pathogenic Escherichia coli strains. They constitutively activate small GTPases of the Rho family by deamidation of a glutamine, which is crucial for GTP hydrolysis. Recently, a novel CNF (CNF(Y)) encompassing 65% identity to CNF1 has been identified in Yersinia pseudotuberculosis. In contrast to the E. coli toxins, which activate several isoforms of Rho family GTPases, CNF(Y) is a strong and selective activator of RhoA in vivo. By constructing chimeras between CNF1 and CNF(Y), we show that this substrate specificity is based on differences in the catalytic domains, whereas the receptor binding and translocation domains have no influence. We further define a loop element (L8) on the surface of the catalytic domains as important for substrate recognition. A single amino acid exchange in L8 is sufficient to shift substrate specificity of CNF1. Moreover, it is shown that RhoA activation by CNF1 is transient, which may be the consequence of the broader substrate specificity of the E. coli toxin, leading to cross-talk between the activated GTPases.     

Physicochemical and biological analysis of synthetic bacterial lipopeptides: validity of the concept of endotoxic conformation

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle.     

Computer-aided lead optimization: improved small-molecule inhibitor of the zinc endopeptidase of botulinum neurotoxin serotype A

Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA) endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K(i) (app) of 7+/-2.4 microM) using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K(i) (app) of 3.8+/-0.8 microM) with a relatively small increase in molecular weight (16 Da). The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.