Different Stages Of Activation Research Articles

The Varkud Satellite Ribozyme: A Thirty-Year Journey through Biochemistry, Crystallography, and Computation.

The discovery of catalytic RNAs or ribozymes introduced a new class of enzymes to biology. In addition to their increasingly important roles in modern life, ribozymes are key players in the RNA World hypothesis, which posits that life started or flourished with RNA supporting both genetic and enzymatic functions. Therefore, investigations into the mechanisms of ribozyme function provide an exciting opportunity to examine the foundational principles of biological catalysis. Ribozymes are also attractive model systems to investigate the relationship between structure and function in RNA. Endonucleolytic ribozymes represent the largest class of catalytic RNA, of which the Varkud satellite (VS) ribozyme is structurally the most complex. The last ribozyme to be discovered by accident, the VS ribozyme had eluded structural determination for over two decades. When we solved the first crystal structures of the VS ribozyme, an extensive body of biochemical and biophysical data had accumulated over the years with which we could evaluate the functional relevance of the structure. Conversely, the structures provided a new perspective from which to reexamine the functional data and test new hypotheses. The VS ribozyme is organized in a modular fashion where independently folding domains assemble into the active conformation of the ribozyme via three-way junctions. Structures of the VS ribozyme in complex with its substrate at different stages of activation enabled us to map the structural reorganization of the substrate that must precede catalysis. In addition to defining the global architecture of the RNA, the essential interactions between the substrate and catalytic domains, and the rearrangements in the substrate prior to catalysis, these structures provided detailed snapshots of the ribozyme active site, revealing potential catalytic interactions. High resolution structures of the active site bolstered the view that the catalytic mechanism involved nucleobase-mediated general acid-base catalysis and uncovered additional catalytic interactions between the cleavage site and catalytic residues. Informed by the crystal structures of the VS ribozyme, an integrated experimental and computational approach identified the key players and essential interactions that define the active site of the ribozyme. This confluence of biochemical, structural, and computational studies revealed the catalytic mechanism of the ribozyme at unprecedented detail. Additionally, comparative analyses of the active site structures of the VS ribozyme and other nucleic acid-based endoribonucleases revealed common architectural motifs and strikingly similar catalytic strategies. In this Account, we document the progress of VS ribozyme research starting from its discovery and extending to the elucidation of its detailed catalytic mechanism 30 years later.

The Interactions of Parasite Calreticulin With Initial Complement Components: Consequences in Immunity and Virulence.

Because of its capacity to increase a physiologic inflammatory response, to stimulate phagocytosis, to promote cell lysis and to enhance pathogen immunogenicity, the complement system is a crucial component of both the innate and adaptive immune responses. However, many infectious agents resist the activation of this system by expressing or secreting proteins with a role as complement regulatory, mainly inhibitory, proteins. Trypanosoma cruzi, the causal agent of Chagas disease, a reemerging microbial ailment, possesses several virulence factors with capacity to inhibit complement at different stages of activation. T. cruzi calreticulin (TcCalr) is a highly-conserved, endoplasmic reticulum-resident chaperone that the parasite translocates to the extracellular environment, where it exerts a variety of functions. Among these functions, TcCalr binds C1, MBL and ficolins, thus inhibiting the classical and lectin pathways of complement at their earliest stages of activation. Moreover, the TcCalr/C1 interaction also mediates infectivity by mimicking a strategy used by apoptotic cells for their removal. More recently, it has been determined that these Calr strategies are also used by a variety of other parasites. In addition, as reviewed elsewhere, TcCalr inhibits angiogenesis, promotes wound healing and reduces tumor growth. Complement C1 is also involved in some of these properties. Knowledge on the role of virulence factors, such as TcCalr, and their interactions with complement components in host–parasite interactions, may lead toward the description of new anti-parasite therapies and prophylaxis.

Activated monocytes and markers of inflammation in newly diagnosed multiple sclerosis.

In multiple sclerosis (MS), the inflammation and demyelination of the central nervous system (CNS) develop in distinct ways. This makes diagnosing patients difficult, imperative to initiating early and proper treatment. Several common features exist, among them a profound infiltration of monocytes into the CNS mediating demyelination and tissue destruction. In the periphery, monocytes are divided into three subsets depending on expression of CD14 and CD16, representing different stages of activation and differentiation. To investigate their involvement in MS, peripheral blood mononuclear cells (PBMCs) from 61 patients with incipient, untreated MS and 22 symptomatic control (SC) patients as well as 6 patients with radiologically isolated syndrome (RIS) were characterized ex vivo. In addition, paired serum and cerebrospinal fluid (CSF) samples were analyzed with a panel of biomarkers. In PBMC samples, we demonstrate decreased levels of nonclassical monocytes with a concomitant significant decrease of human endogenous retrovirus (HERV) H3 envelope epitopes on this monocyte subset compared with SC and RIS. The observed HERV expression is present on nonclassical monocytes irrespective of MS and thus presumably a result of the inflammatory activation. For the other surface markers analyzed, we found significantly decreased expression between classical and nonclassical monocytes. In matched samples of CSF a highly significant increase in levels of soluble markers of activation and inflammation is shown, and notably this is not the case for the serum samples. Of the soluble markers investigated, interleukin (IL)‐12/IL‐23p40 had the highest discriminatory power in differentiating patients with MS from SC and RIS, almost comparable to the immunoglobulin G index.

Proteomic Profiling of Stem Cell Tissues during Regeneration of Deer Antler: A Model of Mammalian Organ Regeneration.

As the only known mammalian organ that can fully and annually regenerate, deer antler has significant advantages over lower-order animal models when investigating the control of stem-cell-based organ regeneration. Antler regeneration is known to be initiated and maintained by neural-crest-derived stem cells in different states of activation. Antler stem cells can therefore be used as a model to study proteins and pathways involved in the maintenance of a stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90, and CD105 were examined within the antler tip. Label-free quantification was performed to investigate the protein profiles of antler stem cells under different stages of activation and included dormant pedicle periosteum (DPP), antler growth center (GC), post-active stem cells from mid-beam antler periosteum (MAP), and deer facial periosteum (FP) as a control (n = 3 per group). PEAKS and IPA software were used to analyze the proteomic data. Our research confirmed the central role of stem cell activation in the development of this mammalian organ by localizing the MSC markers within the antler growth center. Label-free quantification revealed that the greatest number of unique proteins (87) was found in the growth center. There were only 12 proteins found with expression levels that significantly differed between DPP and FP. Protein profiles of these two groups indicated that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. The number of significantly regulated proteins among DPP, MAP, and GC was 153. Among them, the majority were upregulated in the growth center. Activation of antler stem cells was associated with many biological processes and signaling pathways, such as Hippo and canonical Wnt signaling. This work identifies the key pathways, molecular/cellular functions, and upstream regulators involved in mammal organ regeneration. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD016824.

Galectin-1 modulation of neutrophil reactive oxygen species production depends on the cell activation state

Here we report the effects of exogenous and endogenous galectin-1 (Gal-1) in modulating the functional responses of human and murine neutrophils at different stages of activation, i.e. naive, primed, and activated. Exposure to Gal-1 did not induce ROS production in either naive or N-formyl-methionyl-leucyl-phenylalanine-primed (fMLP; 10−9 M) neutrophils. However, Gal-1 elicited a concentration-dependent ROS production in neutrophils activated with fMLP at concentrations ranging from 10-8 M to 10-6 M. Additional fMLP (10-7 M) stimulation of fMLP-activated neutrophils increased ROS production, whose intensity was inversely related to the fMLP concentration used in the first activation step (10-8 M to 10-6 M), and was not influenced by the presence of Gal-1. Naive neutrophils treated with Gal-1 and then exposed to fMLP (10-6 M) or phorbol-12-myristate-13-acetate (10-7 M) produced less ROS, as compared to naive neutrophils not treated with Gal-1. Interestingly, these in vitro Gal-1 effects were associated with Gal-1 carbohydrate-binding activity and the ability to decrease FPR-1 (formyl peptide receptor 1) expression in naive human neutrophils. Conversely, positive ROS modulation by Gal-1 in activated neutrophils was not associated with FPR-1 expression but it was related to its carbohydrate recognition. In vitro, fMLP stimulation of Gal-1-/- mouse neutrophils produced more ROS than fMLP stimulation of Gal-1+/+ neutrophils and this effect may be associated with increased FPR-1 expression. Exogenous Gal-1 induced ROS production in Gal-1-/- mouse neutrophils more effectively than in Gal-1+/+ mouse neutrophils. Compared to Gal-1+/+ mice, Gal-1-/- mice exhibited lower bacterial load in the peritoneal fluid and peripheral blood, thus indicating a greater bactericidal activity in vivo. These findings demonstrate that endogenous Gal-1 restricts ROS generation that correlates with bacterial killing capacity in inflammatory neutrophils. Thus, endogenous and exogenous Gal-1 may either positively or negatively modulate the effector functions of neutrophils according to the cell activation stage.

Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry.

As checkpoint inhibitor immunotherapies gain traction among cancer researchers and clinicians, the need grows for assays that can definitively phenotype patient immune cells. Herein, we present an 8-color flow cytometry panel for lineage and immune checkpoint markers and validate it using healthy human donor peripheral blood mononuclear cells (PBMCs). Flow cytometry data was generated on a BD LSR Fortessa and supported by Luminex multiplex soluble immunoassay. Our data showed significant variation between donors at both baseline and different stages of activation, as well as a trend in increasing expression of checkpoint markers on stimulated CD4+ and CD8+ T-cells with time. Soluble immune checkpoint quantification assays revealed that LAG-3, TIM-3, CTLA-4, and PD-1 soluble isoforms are upregulated after stimulation. This 8-color flow cytometry panel, supported here by soluble immunoassay, can be used to identify and evaluate immune checkpoints on T-lymphocytes in cryopreserved human PBMC samples. This panel is ideal for characterizing checkpoint expression in clinical samples for which cryopreservation is necessary.

Influence of In-Bed Catalysis by Ash-Coated Olivine on Tar Formation in Steam Gasification of Biomass

The use of catalytic bed materials has become a state-of-the-art solution to control the concentration of tar in fluidized bed biomass steam gasifiers. Ash-coated olivine is commonly applied as bed material, owing to its relatively high catalytic activity towards tar species. However, the mechanisms and conversion pathways influenced by the ash-coated olivine when applied as an in-bed catalyst are still not well understood. The present work aims at proving that the ash-layered olivine prevents the formation of biomass-derived tar at an early stage of their formation. Tests with olivine at different stages of activation and at different temperatures are carried out in the Chalmers 2-4MWth DFB gasifier. Detailed characterization of the tar and light hydrocarbon fractions are presented and discussed in relation to the sources of aromatic species. It is concluded that the ash-coated olivine prevents the formation of aromatic tar species by promoting the steam reforming of early tar precursors. Gas-phase interactions of the early tar precursors and bed material contribute to the tar reduction observed. The results indicate that olivine interferes the cyclization routes involving C2H2 and C3 hydrocarbons.

Mechanism of Selective Enzyme Inhibition through Uncompetitive Regulation of an Allosteric Agonist.

Classical uncompetitive inhibitors are potent pharmacological modulators of enzyme function. Since they selectively target enzyme-substrate complexes (E:S), their inhibitory potency is amplified by increasing substrate concentrations. Recently, an unconventional uncompetitive inhibitor, called CE3F4R, was discovered for the exchange protein activated by cAMP isoform 1 (EPAC1). Unlike conventional uncompetitive inhibitors, CE3F4R is uncompetitive with respect to an allosteric effector, cAMP, as opposed to the substrate (i.e., CE3F4R targets the E:cAMP rather than the E:S complex). However, the mechanism of CE3F4R as an uncompetitive inhibitor is currently unknown. Here, we elucidate the mechanism of CE3F4R's action using NMR spectroscopy. Due to limited solubility and line broadening, which pose major challenges for traditional structural determination approaches, we resorted to a combination of protein- and ligand-based NMR experiments to comparatively analyze EPAC mutations, inhibitor analogs, and cyclic nucleotide derivatives that trap EPAC at different stages of activation. We discovered that CE3F4R binds within the EPAC cAMP-binding domain (CBD) at a subdomain interface distinct from the cAMP binding site, acting as a wedge that stabilizes a cAMP-bound mixed-intermediate. The mixed-intermediate includes attributes of both the apo/inactive and cAMP-bound/active states. In particular, the intermediate targeted by CE3F4R traps a CBD's hinge helix in its inactive conformation, locking EPAC into a closed domain topology that restricts substrate access to the catalytic domain. The proposed mechanism of action also explains the isoform selectivity of CE3F4R in terms of a single EPAC1 versus EPAC2 amino acid difference that destabilizes the active conformation of the hinge helix.

LaNi5 hydride powder flowability as a function of activation and hydrogen content

Volume expansion of hydrogen absorbing materials, together with inter-particle friction can cause tension accumulation in hydride containers during absorption. When hydride particles absorb hydrogen there is an important volume increase, in the order of 25%. Particles have to accommodate to the container geometry but this movement is opposed by inter-particle friction. Under certain conditions tensions can build up, compromising the mechanical integrity of the container. This phenomenon needs to be addressed at the design stage to avoid mechanical failure of the container.Flow behavior of powder materials is a relevant technological field, usually addressed by means of qualitative or quantitative flowability measuring devices. The rotating drum technique, while mainly qualitative, is well established and can be modified into a completely sealed unit. To determine the flowability of a hydride under different activation stages and hydrogen content levels we developed a rotating drum device that can be pressurized with hydrogen or depressurized. We report particle size evolution and flowability measurements of a hydrogen absorbing material (LaNi5) at different stages of activation for both absorbed and desorbed states. The results of the present study show that the flowability of LaNi5 in more dependent on the degree of activation of the sample than on hydrogen absorption state.

Abstract # 1813 G-alpha-s-protein coupled receptor signaling suppresses integrin-mediated cell adhesion of antigen-specific human CD8+ T cells

An efficient immune response requires a selective and strong interaction of T cells with endothelium or target cells. These interactions are strengthened by the rapid induction of beta2-integrin-mediated adhesion upon chemokine receptor or T-cell receptor (TCR) stimulation, respectively. We and others have shown that an increase in G-alpha-s-protein coupled receptor (GalphasPCR) signaling, such as via beta2-adrenergic receptors counteracts chemokine-induced beta2-integrin activation and promotes de-adhesion and mobilization of effector CD8+ T cells from the marginal pool into the circulation. We asked whether GalphasPCR agonists also inhibit integrin activation induced by TCR stimulation of human antigen-specific T cells. Using soluble multimeric ICAM1-Fc-F(ab)2 fluorescent complexes, we developed a flow cytometry assay to identify integrin activation on virus-specific CD8+ T cells following short-term stimulation and staining with cytomegalovirus, Epstein–Barr virus, or influenza virus peptide-major histocompatibility complex class I HLA-multimers. From the GalphasPCR agonists used, epinephrine, norepinephrine, prostaglandins E2/D2 and adenosine strongly down-modulated the beta2-integrin activation on all virus-specific T cells and subsets of different stages of activation, while dopamine, serotonin and histamine had no effects. This active down-modulation of integrin activation through GalphasPCR signaling might be an essential mechanism by which the immune response is suppressed in a variety of pathologies characterized by increased levels of catecholamines (e.g., sleep deprivation), prostaglandins (e.g., tumor growth), or adenosine (e.g., hypoxia).

Astaxanthin prevents and reverses the activation of mouse primary hepatic stellate cells

Activation of hepatic stellate cells (HSCs) is a critical step that leads to the development of liver fibrosis. We showed that astaxanthin (ASTX), a xanthophyll carotenoid, displays antifibrogenic effects in LX-2 cells, a human HSC cell line. In this study, we further determined the effect of ASTX on HSC activation and inactivation using primary HSCs from C57BL/6J mice. Quiescent and activated HSCs were incubated with ASTX (25μM) at different stages of activation. ASTX prevented the activation of quiescent HSCs, as evidenced by the presence of intracellular lipid droplets and reduction of α-smooth muscle actin, an HSC activation marker. Also, ASTX reverted activated HSCs to a quiescent phenotype with the reappearance of lipid droplets with a concomitant increase in lecithin retinol acyltransferase mRNA. Cellular accumulation of reactive oxygen species was significantly reduced by ASTX, which was attributable to a decrease in NADPH oxidase 2 expression. The antifibrogenic effect of ASTX was independent of nuclear erythroid 2-related factor 2 as it was observed in HSCs from wild-type and Nrf2−/− mice. In conclusion, ASTX inhibits HSC activation and reverts activated HSCs to a quiescent state. Further investigation is warranted to determine if ASTX effectively prevents the development of liver fibrosis.

G.P.200: The expression profile of developmental markers in diagnostic muscle biopsies indicates the presence of a “neurogenic muscle regeneration”

In standard muscle biopsy investigation regenerating fibers are commonly seen in dystrophic muscle, but not in neurogenic myopathy. Using immunohistochemistry these disease groups do differ in the expression of embryonic and fetal myosin heavy chain, but the significance of this is unknown. To learn more about regenerating processes in these groups we investigated the expression of several markers of satellite cells in different stages of activation, and of embryonic and fetal myosin heavy chain in muscle biopsies from patients with dystrophy and neurogenic myopathy, as well as in controls. Frozen sections from 10 patients with limb-girdle muscle dystrophies, 10 patients with motor neuron disease, 10 young and 10 elderly age matched control patients were selected. Immunohistological staining using following antibodies was performed: PAX7, N-CAM, MyoD1, myogenin, neonatal myosin heavy chain, developmental myosin heavy chain and spectrin. The same area in adjacent sections was photographed, followed by counting of fibers and satellite cells. The quotient of myogenin and PAX7 was used as an index for satellite cell activation, and the expressions of the variants of myosin heavy chain were compared between the groups. All biopsies were from the anterior tibial muscle. We found a significantly higher index of activated satellite cells in neurogenic myopathy than in dystrophic myopathy or controls. The higher index of satellite cell activation was paralleled by a higher expression of MyoD1 (indicating satellite cell proliferation). Embryonic myosin was more frequent in dystrophic compared to neurogenic myopathy. The parameters were low and did not differ between the young and elderly control biopsies. The results indicate that there is satellite cell activation in neurogenic myopathy exceeding the activation in dystrophies, and that regenerating fibers in this disease group may by pass the stage of expressing embryonic myosin.

CD34 und Alpha-smooth-Muscle-Actin-Keratozyten-Expression nach photodynamischer Inaktivierung (PDI)

Photodynamic inactivation (PDI) may be a potential treatment alternative in therapy-resistant infectious keratitis. PDI may eliminate the microorganisms from the infected cornea by damage caused through free oxygen radicals, or even by supporting different stages of activation of keratocytes and inflammatory cell response. The purpose of this study was to determine the impact of PDI on activation of human keratocytes in culture. Primary human keratocytes were isolated by digestion in collagenase A (1 mg/mL) from human corneal buttons, and cultured in DMEM/Ham's culture medium supplemented with 10% foetal calf serum. Keratocytes underwent illumination (670 nm) for 13 minutes following exposure to 0, 50, 150 and 250 nMol/ml concentrations of the photosensitizer chlorin e6 (Ce6) in the culture medium. Twenty-four hours after treatment CD34 and α-smooth-muscle actin expression of the cells was analysed using flow-cytometry (FACS). Using Ce6 or illumination only, α-smooth-muscle actin expression of the cells did not change significantly. Twenty-four hours after PDI the percentage of CD34-positive keratocytes did not change significantly using 50-250 nM Ce6, however, the percentage of α-smooth-muscle actin-positive keratocytes decreased significantly at 250 nM Ce6 (p = 0.01). As a short-term effect, PDI seems to inhibit myofibroblastic transformation of keratocytes, but does not have an impact on activation of CD34-positive keratocytes.With this impact PDI possibly may reduce the antimicrobial defence of keratocytes.

CD9 is a novel marker for plasma cell precursors in human germinal centers

The germinal center (GC) is the dynamic microenvironment where Ag-activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high affinity antibodies. B cells within the GC have great heterogeneity, containing B cells at different stages of activation and differentiation. However, there are few surface markers that allow subsets of GC-B cells to be distinguished. In the present study, we show that GC-B cells in human tonsils contain two distinct populations regarding CD9 expression; CD9− and CD9+ cells. CD9+ GC-B cells are functionally more differentiated towards PC based upon the following evidence; (1) CD9+ cells express higher levels of PC transcription factor, Blimp-1 while lower levels of B cell transcription factors, Bcl-6 and Pax-5, compared to CD9− cells, (2) CD9+ cells differentiate into plasmablasts faster than CD9− cells in the presence of cytokines that generate PC, and (3) CD9 expression was induced in CD9− GC-B cells under PC generating condition and gradually increased in the course of PC differentiation. Taken together, our data suggest that CD9 is a novel marker for a human GC-B cell subset that is committed to PC lineage.

Impact of photodynamic inactivation (PDI) on bFGF, HGF, KGF, TGFß1 and VEGF secretion of keratocytes in vitro

AbstractPurpose Photodynamic inactivation (PDI) may eliminate the microorganisms from the infected cornea by damages caused through free oxygen radicals, or even by supporting different stages of activation of keratocytes and inflammatory cell response. The purpose of this study was to determine the impact of PDI on bFGF, HGF, KGF, TGFß1 and VEGF secretion of keratocytes in vitro.Methods Primary human keratocytes were isolated by digestion in collagenase A (1 mg/ml) from human corneal buttons, and cultured in DMEM/Ham’s culture medium supplemented with 10% FCS. Keratocytes underwent illumination (670 nm) for 13 minutes following exposure to 100 nM concentration of photosensitizer chlorin e6 (Ce6) in the culture medium. One day after treatment, bFGF, HGF, KGF, TGFß1 and VEGF release of the cells was determined using enzyme‐linked immunosorbent assay (ELISA).Results One day after PDI, the secretion of bFGF was 1.58, of HGF 1.61, of KGF 0, of TGFß1 2.83 and of VEGF 9.01 pg/µg protein. The secretion of bFGF decreased (p=0.007) significantly one day after PDI, compared to controls. In HGF, KGF, TGFß1 and VEGF secretion no significant changes could be detected. Using Ce6 or illumination only, bFGF, HGF, KGF, TGFß1 and VEGF secretion of keratocytes did not change significantly.Conclusion As a short‐term effect, PDI decreases bFGF release of keratocytes in vitro. The altered secretion of these factors may play a role in the activation of keratocytes following PDI.

Effect of a small molecule BCL‐2 inhibitor on immune function and use with a recombinant vaccine

Small molecule BCL-2 inhibitors are being examined as monotherapy in phase I/II clinical trials for several types of tumors. However, few data are available about the effect of BCL-2 inhibitors on immune function. The aims of our study were to investigate the effect of a small molecule BCL-2 inhibitor on immune function and determine the most effective way of combining this inhibitor with a recombinant vaccine to treat tumors. The in vitro effect of the pan-BCL-2 inhibitor GX15-070 was assessed in mouse CD8 T lymphocytes at 2 different stages of activation as well as regulatory T lymphocytes (Treg). The in vivo effect of GX15-070 after recombinant vaccinia/fowlpox CEA-TRICOM vaccination was analyzed in tumor-infiltrating lymphocytes, and in splenocytes of mice bearing subcutaneous tumors. The therapeutic efficacy of such sequential therapy was measured as a reduction of pulmonary tumor nodules. Activated mature CD8 T lymphocytes were more resistant to GX15-070 as compared to early-activated cells. Treg function was significantly decreased after treatment with the BCL-2 inhibitor. In vivo, GX15-070 was given after vaccination so as to not negatively impact the induction of vaccine-mediated immunity, resulting in increased intratumoral activated CD8:Treg ratio and significant reduction of pulmonary tumor nodules. Our study is the first to show the effect of a small molecule BCL-2 inhibitor on the immune system and following a vaccine. It is also the first to demonstrate the efficacy of this sequence in reducing tumors in mouse models, providing a rationale for the design of combinational clinical studies.

Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

Glycosidic bond cleavage in deoxynucleotides — A density functional study

Density functional theory was used to study the glycosidic bond cleavage in deoxynucleotides with the main goal to determine the effects of the nucleobase, hydrogen bonding with the nucleobase, and the (bulk) environment on the reaction energetics. Since direct glycosidic bond cleavage is a high-energy process, two nucleophile models were considered (HCOO–···H2O and HO–), which represent different stages of activation of a water nucleophile. The glycosidic bond cleavage barriers were found to decrease, while the reaction exothermicity increases, with an increase in the nucleobase acidity. The gas-phase barriers and reaction energies for bond cleavage in all deoxynucleotides were found to be significantly affected by hydrogen-bonding interactions with the nucleobase (by up to 30 kJ mol–1 depending on the nucleophile). Although the barriers increase and reaction energies become less exothermic in enzymatic and aqueous environments, the effects of the bulk environment are similar in the presence and absence of small molecules bound to the nucleobase. Therefore, the effects of hydrogen bonding with the bases are approximately the same in all environments. Our results suggest that hydrogen bonding with the nucleobase may play an important role in the glycosidic bond cleavage in both pyrimidine and purine nucleotides in a variety of environments.

Cytokine profiling using monocytes/macrophages cultured on common biomaterials with a range of surface chemistries

Cytokines, chemokines, and growth factors were assayed from the supernatants of monocytes and macrophages cultured on common biomaterials with a range of surface chemistries. TNF-alpha, MCP-1, MIP-1alpha, IL-8, IL-6, IL-1beta, VEGF, IL-1ra, and IL-10 were measured from monocyte/macrophage cultures at different stages of activation and differentiation seeded onto polyethylene, polyurethane, expanded polytetrafluoroethylene, polymethyl methacrylate, and a hydrogel copolymer of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol acrylate in tissue culture polystyrene (TCPS) plates. Empty TCPS wells and organo-tin polyvinyl chloride served as "blanks" and positive controls, respectively. Results showed an overall increase in cytokine, chemokine, and growth factor production as monocytes are activated or differentiated into macrophages and that proinflammatory and anti-wound healing cytokines and chemokines dominate this profile. However, cytokine production was only modestly affected by the surface chemistry of these four stable and noncytotoxic biomaterials.

Assessing Activation Stage and Employing a “Next Steps” Approach to Supporting Patient Self-Management

Current approaches to chronic illness care seek to engage the patient as part of the care team. Yet, finding effective ways to engage patients in their own care and support patient self-management has been challenging. Instead of pushing patients to immediately adopt all recommended behavioral changes, many programs encourage small steps while working toward a larger goal. A strategy that first assessed the level of mastery, and then encouraged "next step" behaviors, may be more effective as the recommended steps would be calibrated to the patient's level of competency. In this analysis, we build on the previous research to determine whether there are behaviors that are more or less likely to be adopted at different stages of activation.