Biomedical subjects
Publications and source records attributed to Christopher A Hunter.
A supramolecular approach has been used to investigate the free energies of intermolecular aromatic stacking interactions. Chemical double mutant cycles have been used to measure the effect of a range of substituents on face-to-face stacking interactions with phenyl and pentafluorophenyl rings. Electrostatic effects dominate the trends in interaction energy.
Previous reports have focused on the ability of IL-27 to promote naive T cell responses but the present study reveals that surface expression of WSX-1, the ligand-specific component of the IL-27R, is low on these cells and that highest levels are found on effector and memory CD4(+) and CD8(+) T cells. Accordingly, during infection with Toxoplasma gondii, in vivo T cell activation is associated with enhanced expression of WSX-1, and, in vitro, TCR ligation can induce expression of WSX-1 regardless of the polarizing (Th1/Th2) environment present at the time of priming. However, while these data establish that mitogenic stimulation promotes expression of WSX-1 by T cells, activation of NK cells and NKT cells prompts a reduction in WSX-1 levels during acute toxoplasmosis. Together, with the finding that IL-2 can suppress expression of WSX-1 by activated CD4(+) T cells, these studies indicate that surface levels of the IL-27R can be regulated by positive and negative signals associated with lymphoid cell activation. Additionally, since high levels of WSX-1 are evident on resting NK cells, resting NKT cells, effector T cells, regulatory T cells, and memory T cells, the current work demonstrates that IL-27 can influence multiple effector cells of innate and adaptive immunity.
Three isomeric zinc bisporphyrins have been prepared by covalently linking together two aminoporphyrins with an isophthalic acid derivative. The porphyrins differ in the substitution pattern on the meso phenyl groups, that is, ortho, meta, or para. Titrations carried out by UV-visible and 1H NMR spectroscopy have been used to map out the stabilities and the stoichiometries of the complexes formed with 1,4-diazabicyclo[2.2.2]octane (DABCO) in chloroform. The ortho- and meta-substituted bisporphyrins form 1:1 intramolecular sandwich complexes. The para-substituted bisporphyrin cannot adopt the cofacial conformation required for this type of complex and forms a higher order 2:2 intermolecular assembly, which is stable over a wide range of DABCO concentrations.
During infection, CD4(+) Th cell responses polarize to become primarily Th1 or Th2. Th1 cells, which make IFN-gamma, are crucial for immunity to many bacterial and protozoal infections, whereas Th2 cells, which make IL-4, IL-5, and IL-13, are important for resistance to helminth infections. Polarized Th1 responses are induced by dendritic cells (DCs), which respond to pathogen-derived TLR ligands to produce IL-12 and related cytokines that are instrumental in Th1 cell outgrowth, and coordinately process and present Ag in the context of MHC class II to activate naive Th cells. In this study we show that in addition to providing positive signals for Th1 cell development, mouse DCs activated by TLR engagement can also provide a potent negative signal that prevents the development of Th2 cells. Production of this signal, which is not IL-12, IL-18, IL-23, IL-27, or IFN-gamma and is not provided via Th1 cells, is dependent upon a MyD88-dependent, TNF receptor-associated factor-6-independent signaling pathway in DCs. The signal is released from DCs in response to activation via TLR ligands and exerts an effect directly on Th cells rather than through a third-party cell. Our findings indicate that DCs can provide potent negative as well as positive instruction for Th response polarization, and that these instructional signals are distinct and independent.
Using calmodulin antagonism as a model, it is demonstrated that, under circumstances in which binding sites are motionally independent, it is possible to create bifunctional ligands that bind with significant affinity enhancement over their monofunctional counterparts. Suitable head groups were identified by using a semiquantitative screen of monofunctional tryptophan analogs. Two bifunctional ligands, which contained two copies of the highest-affinity head group tethered by rigid linkers, were synthesized. The bifunctional ligands bound to calmodulin with a stoichiometry of 1:1 and with an affinity enhancement over their monofunctional counterparts; the latter bound with a stoichiometry of 2:1 ligand:protein. A lower limit to the effective concentrations of the domains of calmodulin relative to each other (0.2-2 mM) was determined. A comparable effective concentration was achieved for bifunctional ligands based on higher-affinity naphthalene sulphonamide derivatives.
IL-10 plays a vital role in controlling the inflammatory response during acute Toxoplasma gondii infection, however the production of IL-10 during the chronic phase of toxoplasmosis has been associated with parasite persistence. To address this paradox, the production and effect of IL-10 in the brain during toxoplasmic encephalitis (TE) was investigated. Analysis of brain mononuclear cells (BMNC) from chronically infected mice revealed that infiltrating macrophages and CD4(+) T cells were the major sources of IL-10. Endogenous levels of IL-10 inhibited the production of IL-12, IFN-gamma, TNF-alpha, and IL-6 from both hematopoetic and non-hematopoetic cells in the brain, as well as anti-microbial activity of astrocytes. Furthermore, IL-10-/- mice that progressed to the chronic phase of infection had equivalent parasite burden to WT mice but developed a lethal inflammatory response within the brain characterized by increased numbers of CD4(+) T cells and macrophages, and elevated production of inflammatory cytokines. Finally, partial depletion of CD4(+) T cells decreased the severity of the inflammation in the brain and allowed IL-10-/- mice to survive infection. Together these results point to a vital role for IL-10 in the control of CD4(+) T cell mediated inflammation in the brain during TE.
Understanding the factors that influence T helper 1 (T(H)1)- and T(H)2-cell responses has been one of the main focuses of immunology for almost 20 years. Whereas the central role of interleukin-12 (IL-12) in the generation of T(H)1 cells has long been appreciated, subsequent studies indicated that IL-23 and IL-27, two cytokines that are closely related to IL-12, also regulate T(H)1-cell responses. However, as discussed in this article, it is now recognized that the ability of IL-23 to stimulate a unique T-cell subset to produce IL-17 has a dominant role in autoimmune inflammation. By contrast, IL-27 has a role in limiting the intensity and duration of adaptive immune responses.
OBJECTIVE: To determine whether human CTLA4-Ig ([hu]CTLA4-Ig) inhibits costimulation-dependent lymphocyte proliferation in vitro, compare the effects of (hu)CTLA4-Ig with cyclosporine and steroids on CD4+ and CD8+ T-cell lymphocyte proliferation, and determine whether memory T-cell function remains intact in the presence of (hu)CTLA4-Ig. ANIMALS: 29 cats. PROCEDURE: Peripheral blood mononuclear cells (PBMCs) were stimulated with concanavalin A (costimulation-dependent mitogen) or phorbol 12-myristate 13-acetate and ionomycin (costimulation independent mitogens) alone or in the presence of (hu)CTLA4-Ig, cyclosporine, or dexamethasone; effects of these treatments on lymphocyte proliferation were assessed by incorporation of thymidine labeled with tritium or flow cytometry. Antigen-specific proliferation was determined by stimulating PBMCs from 2 healthy cats seropositive for Toxoplasma gondii with soluble Toxoplasma antigen alone or in the presence of (hu)CTLA4-Ig or cyclosporine. RESULTS: (hu)CTLA4-Ig inhibited costimulation-dependent lymphocyte proliferation in vitro but had no effect on costimulation-independent lymphocyte proliferation. Compared with mitogen alone, (hu)CTLA4-Ig caused a significant decrease in responder frequency and proliferative capacity of CD4+ T cells; however, the effect on CD8+ T cells was not significant. Cyclosporine alone or with dexamethasone had a significantly greater suppressive effect on responder frequency and proliferative capacity of CD4+ and CD8+ T cells, compared with (hu)CTLA4-Ig. Compared with cyclosporine, (hu)CTLA4-Ig appeared to have a sparing effect on antigen-specific proliferation of memory CD4+ and CD8+ T cells. CONCLUSIONS AND CLINICAL RELEVANCE: (hu)CTLA4-Ig selectively inhibited costimulation-dependent proliferation of lymphocytes in vitro and had a sparing effect on antigen-specific proliferation of memory cells. The specificity of its mechanism of action suggests that (hu)CTLA4-Ig may prevent allograft rejection but leave memory responses to previously encountered antigens intact.
There is increasing awareness that helminth infections can ameliorate proinflammatory conditions. In part, this is due to their inherent ability to induce Th2 and, perhaps, regulatory T cell responses. However, recent evidence indicates that helminths also have direct anti-inflammatory effects on innate immune responses. In this study, we address this issue and show that soluble molecules from the eggs of the helminth parasite Schistosoma mansoni (SEA) suppress LPS-induced activation of immature murine dendritic cells, including MHC class II, costimulatory molecule expression, and IL-12 production. SEA-augmented LPS-induced production of IL-10 is in part responsible for the observed reduction in LPS-induced IL-12 production. However, analyses of IL-10(-/-) DC revealed distinct IL-10-independent suppressive effects of SEA. IL-10-independent mechanisms are evident in the suppression of TLR ligand-induced MAPK and NF-kappaB signaling pathways. Microarray analyses demonstrate that SEA alone uniquely alters the expression of a small subset of genes that are not up-regulated during conventional TLR-induced DC maturation. In contrast, the effects of SEA on TLR ligand-induced DC activation were striking: when mixed with LPS, SEA significantly affects the expression of >100 LPS-regulated genes. These findings indicate that SEA exerts potent anti-inflammatory effects by directly regulating the ability of DC to respond to TLR ligands.
Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1-/- mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.
A database of the structural properties of all 32,896 unique DNA octamer sequences has been calculated, including information on stability, the minimum energy conformation and flexibility. The contents of the database have been analysed using a variety of Euclidean distance similarity measures. A global comparison of sequence similarity with structural similarity shows that the structural properties of DNA are much less diverse than the sequences, and that DNA sequence space is larger and more diverse than DNA structure space. Thus, there are many very different sequences that have very similar structural properties, and this may be useful for identifying DNA motifs that have similar functional properties that are not apparent from the sequences. On the other hand, there are also small numbers of almost identical sequences that have very different structural properties, and these could give rise to false-positives in methods used to identify function based on sequence alignment. A simple validation test demonstrates that structural similarity can differentiate between promoter and non-promoter DNA. Combining structural and sequence similarity improves promoter recall beyond that possible using either similarity measure alone, demonstrating that there is indeed information available in the structure of double-helical DNA that is not readily apparent from the sequence.
Molecular recognition events in solution are affected by many different factors that have hampered the development of an understanding of intermolecular interactions at a quantitative level. Our tendency is to partition these effects into discrete phenomenological fields that are classified, named, and divorced: aromatic interactions, cation-pi interactions, CH-O hydrogen bonds, short strong hydrogen bonds, and hydrophobic interactions to name a few.1 To progress in the field, we need to develop an integrated quantitative appreciation of the relative magnitudes of all of the different effects that might influence the molecular recognition behavior of a given system. In an effort to navigate undergraduates through the vast and sometimes contradictory literature on the subject, I have developed an approach that treats theoretical ideas and experimental observations about intermolecular interactions in the gas phase, the solid state, and solution from a single simplistic viewpoint. The key features are outlined here, and although many of the ideas will be familiar, the aim is to provide a semiquantitative thermodynamic ranking of these effects in solution at room temperature.
IL-27 is a recently identified heterodimeric cytokine produced in response to microbial and host derived inflammatory cues. Initial studies indicated that IL-27 promotes the generation of Th1 responses required for resistance to intracellular infection and unveiled the molecular mechanisms mediating this effect. However, subsequent work uncovered a role for IL-27 in the suppression of Th1 and Th2 responses. Thus, by discussing its pleotropic functions in the context of infection-induced immunity and by drawing parallels to fellow IL-6/IL-12 family cytokines, this review will attempt to reconcile the pro- and anti-inflammatory effects of IL-27.
The ability of NK and T cells to produce IFN-gamma is critical for resistance to numerous intracellular pathogens but the kinetics of these responses differ. Consistent with this is a requirement for naive T cells to become activated and undergo proliferation-dependent epigenetic changes to the IFN-gamma locus that allow them to produce IFN-gamma. The data presented here reveal that unlike T cells, murine NK cells produce IFN-gamma under conditions of short-term cytokine stimulation, and these events are independent of proliferation and cell cycle progression. Furthermore, analysis of the IFN-gamma locus in NK cells reveals that this locus is constitutively demethylated. The finding that NK cells do not need to remodel the IFN-gamma locus to produce IFN-gamma, either because they do not exhibit epigenetic repression or they have undergone prior remodeling during development, provides a molecular basis for the innate and adaptive regulation of the production of this cytokine.
IL-23 and IL-12 are heterodimeric cytokines which share the p40 subunit, but which have unique second subunits, IL-23p19 and IL-12p35. Since p40 is required for the development of the Th1 type response necessary for resistance to Toxoplasma gondii, studies were performed to assess the role of IL-23 in resistance to this pathogen. Increased levels of IL-23 were detected in mice infected with T. gondii and in vitro stimulation of dendritic cells with this pathogen resulted in increased levels of mRNA for this cytokine. To address the role of IL-23 in resistance to T. gondii, mice lacking the p40 subunit (common to IL-12 and IL-23) and mice that lack IL-12 p35 (specific for IL-12) were infected and their responses were compared. These studies revealed that p40(-/-) mice rapidly succumbed to toxoplasmosis, while p35(-/-) mice displayed enhanced resistance though they eventually succumbed to this infection. In addition, the administration of IL-23 to p40(-/-) mice infected with T. gondii resulted in a decreased parasite burden and enhanced resistance. However, the enhanced resistance of p35(-/-) mice or p40(-/-) mice treated with IL-23 was not associated with increased production of IFN-gamma. When IL-23p19(-/-) mice were infected with T. gondii these mice developed normal T cell responses and controlled parasite replication to the same extent as wild-type mice. Together, these studies indicate that IL-12, not IL-23, plays a dominant role in resistance to toxoplasmosis but, in the absence of IL-12, IL-23 can provide a limited mechanism of resistance to this infection.
The recent identification of IL-27 (IL-27p28/EBV-induced gene 3) and IL-27R (WSX-1/gp130) has provided new insights for the biology of IL-6/IL-12 family cytokines. Initial studies indicated that IL-27 can directly regulate T cell functions and suggested an important role for it in promoting Th1 type responses. However, subsequent studies have revealed that IL-27R signaling influences a variety of immune cell types and can inhibit either Th1 or Th2 type responses. Though elucidation of the Jak/STAT signaling pathways activated by IL-27R ligation has unveiled some of the molecular mechanisms used by IL-27 to promote inflammation, little is known about the anti-inflammatory activities of this cytokine. Thus, the aim of this review is to discuss the pleotropic nature of the IL-27/IL-27R interaction and attempt to reconcile the pro- and anti-inflammatory properties of this immunomodulator.
Chemical double mutant cycles have been used to quantify the interactions of halogens with the faces of aromatic rings in chloroform. The halogens are forced over the face of an aromatic ring by an array of hydrogen-bonding interactions that lock the complexes in a single, well-defined conformation. These interactions can also be engineered into the crystal structures of simpler model compounds, but experiments in solution show that the halogen-aromatic interactions observed in the solid state are all unfavourable, regardless of whether the aromatic rings contain electron-withdrawing or electron-donating substituents. The halogen-aromatic interactions are repulsive by 1-3 kJ mol(-1). The interactions with fluorine are slightly less favourable than with chlorine and bromine.
There are conflicting reports on the requirements for the IL-27-WSX-1 pathway in the development of Th type 1 responses and resistance to intracellular pathogens; although early IFN-gamma production and resistance to Leishmania major are impaired in the absence of WSX-1 signaling, WSX-1(-/-) mice generate robust IFN-gamma responses and control infection with other intracellular protozoan pathogens. In this report, we resolve these conflicting observations and demonstrate that, in the absence of IL-4, WSX-1 is not required for early IFN-gamma production and control of L. major. Thus, the requirement for WSX-1 signaling in Th type 1 cell differentiation is restricted to conditions in which IL-4 is produced.