OpenStructure: an integrated software framework for computational structural biology.

Research projects in structural biology increasingly rely on combinations of heterogeneous sources of information, e.g. evolutionary information from multiple sequence alignments, experimental evidence in the form of density maps and proximity constraints from proteomics experiments. The OpenStructure software framework, which allows the seamless integration of information of different origin, has previously been introduced. The software consists of C++ libraries which are fully accessible from the Python programming language. Additionally, the framework provides a sophisticated graphics module that interactively displays molecular structures and density maps in three dimensions. In this work, the latest developments in the OpenStructure framework are outlined. The extensive capabilities of the framework will be illustrated using short code examples that show how information from molecular-structure coordinates can be combined with sequence data and/or density maps. The framework has been released under the LGPL version 3 license and is available for download from http://www.openstructure.org.

LFA-1 antagonism inhibits early infiltration of endogenous memory CD8 T cells into cardiac allografts and donor-reactive T cell priming.

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC-mismatched cardiac allografts and produce IFN-γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti-LFA-1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti-LFA-1 mAb given to C57BL/6 6 (H-2(b)) recipients of A/J (H-2(a)) heart grafts on days -1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN-γ-induced genes. Donor-specific T cells producing IFN-γ were at low/undetectable numbers in spleens of anti-LFA-1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti-LFA-1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti-LFA-1 mAb inhibits early donor-reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.

Role of TNFalpha in early chemokine production and leukocyte infiltration into heart allografts.

The acute phase cytokines IL-1beta, IL-6 and TNFalpha are produced early during inflammatory processes, including ischemia-reperfusion. The appearance and role of these cytokines in the early inflammation following reperfusion of grafts remain poorly defined. This study investigated the role of TNFalpha in the induction of early leukocyte infiltration into vascularized heart allografts. TNFalpha and IL-6 mRNA levels reached an initial peak 3 h posttransplant and a second peak at 9-12 h with equivalent levels in iso- and allografts. A single dose of anti-TNFalpha mAb given at reperfusion decreased neutrophil and macrophage chemoattractant levels and early neutrophil, macrophage and memory CD8 T-cell infiltration into allografts. Anti-TNFalpha mAb also extended graft survival from 8.6+/-0.6 days to 14.1+/-0.8 days. When assessed on day 7 posttransplant, the number of donor-reactive CD8 T cells producing IFN-gamma in the spleen was reduced almost 70% in recipients treated with anti-TNFalpha mAb. Whereas anti-CD154 mAb prolonged survival to day 21, administration of anti-TNFalpha and anti-CD154 mAb delayed rejection to day 32 and resulted in long-term (>80 days) survival of 40% of the heart allografts. These data implicate TNFalpha as an important mediator of early inflammatory events in allografts that undermine graft survival.

Effector functions of donor-reactive CD8 memory T cells are dependent on ICOS induced during division in cardiac grafts.

Alloreactive T-cell memory is present in every transplant recipient and endangers graft survival. Even in the absence of known sensitizing exposures, heterologous immunity and homeostatic T-cell proliferation generate 'endogenous' memory T cells with donor-reactivity. We have recently shown that endogenous donor-reactive CD8 memory T cells infiltrate murine cardiac allografts within hours of reperfusion and amplify early posttransplant inflammation by producing IFN-gamma. Here, we have tested the role of ICOS co-stimulation in eliciting effector function from these memory T cells. ICOS is not expressed on the cell surface of circulating CD8 memory T cells but is rapidly upregulated during cell division within the allograft parenchyma. Donor-reactive CD8 memory T-cell infiltration, proliferation and ICOS expression are regulated by donor class I MHC molecule expression. ICOS blockade significantly reduced IFN-gamma production and other proinflammatory functions of the activated CD8 memory T cells. Our data demonstrate that this induction of ICOS expression within peripheral tissues is an important feature of CD8 memory T-cell activation and identify ICOS as a specific target for neutralizing proinflammatory functions of endogenous CD8 memory T cells.

CCR5 is required for regulation of alloreactive T-cell responses to single class II MHC-mismatched murine cardiac grafts.

The effector CD4 T-cell response in wild-type C57BL/6 recipients of single class II MHC-disparate B6.H-2(bm12) cardiac allografts is restricted by CD4(+)CD25(+) regulatory T cells (Tregs) resulting in long-term allograft survival. To investigate the role chemokine receptors might play in Treg function, this study tested the requirement for CCR5 on Tregs to suppress the alloimmune response in C57BL/6 recipients of B6.H-2(bm12) cardiac allografts. In contrast to the long-term survival of B6.H-2(bm12) allografts in wild-type recipients (>100 days), the allografts were acutely rejected within 25 days in CCR5(-/-) recipients with intense infiltration of CD4 T cells. Numbers and duration of donor-reactive CD4 T cells producing IFN-gamma and IL-4 were markedly increased in spleens of B6.CCR5(-/-) versus wild-type recipients. Wild-type and B6.CCR5(-/-) mice had equivalent numbers of splenic FoxP3(+) Tregs before and following transplantation, and these Tregs were equivalently suppressive in vitro. However, diminished numbers of FoxP3(+) Tregs infiltrated B6.H-2(bm12) allografts in B6.CCR5(-/-) recipients. Adoptive transfer of wild-type, but not CCR5-deficient, CD4(+)CD25(+) Tregs to CCR5(-/-) recipients restored long-term survival of B6.H-2(bm12) cardiac grafts. Collectively, these results indicate that CCR5 expression is required for the regulatory functions of Tregs that restrict alloreactive CD4 T-cell responses to single class II MHC-mismatched cardiac allografts.

Donor-reactive CD8 memory T cells infiltrate cardiac allografts within 24-h posttransplant in naive recipients.

Normal immune responses stimulated by pathogenic and environmental antigens generate memory T cells that react with donor antigens and no currently used immunosuppressive drug completely inhibits memory T-cell function. While donor-reactive memory T cells clearly compromise graft outcomes, mechanisms utilized by memory T cells to promote rejection are largely unknown. In this study, we investigated how early endogenous memory cells infiltrate and express effector function in cardiac allografts. Endogenous CD8 memory T cells in nonsensitized recipients distinguish syngeneic versus allogeneic cardiac allografts within 24 h of reperfusion. CD8-dependent production of IFN-gamma and CXCL9/Mig was observed 24 to 72 h posttransplant in allografts but not isografts. CXCL9 was produced by donor cells in response to IFN-gamma made by recipient CD8 T cells reactive to donor class I major histocompatibility complex (MHC) molecules. Activated CD8 T cells were detected in allografts at least 3 days before donor-specific effector T cells producing IFN-gamma were detected in the recipient spleen. Early inflammation mediated by donor-reactive CD8 memory T cells greatly enhanced primed effector T-cell infiltration into allografts. These results suggest that strategies for optimal inhibition of alloimmunity should include neutralization of infiltrating CD8 memory T cells within a very narrow window after transplantation.

The 5A structure of heterologously expressed plant aquaporin SoPIP2;1.

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.

Oxidation of zinc-binding cysteine residues in transcription factor proteins.

Recent results on the oxidation of cysteine residues that bind zinc in transcription factors and their analogous peptides and in related proteins and model systems are reviewed. Two classes of oxidants, the transition metals and dioxygen, hydrogen peroxide, and related species, are considered, and the role of metal ions in suppressing or enhancing Cys oxidation is a major focus. Cysteines in the zinc-bound structures of transcription factors are less susceptible to oxidation than in the metal-free form, and this appears to correlate with reduced accessibility of the thiolates to oxidants. Substitution of other metal ions for Zn(II) increases the rate of Cys oxidation, apparently through increased oxidant accessibility. Reactions that result in reversible or irreversible oxidation of these zinc-binding cysteines under biological conditions are identified in the context of deleterious implications for gene expression.