Counterselection against Dμ Is Mediated through Immunoglobulin (Ig)α-Igβ

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (Dμ). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Igα-Igβ, whereas the mechanism for counterselection against Dμ has not been determined. We have examined the role of the Igα-Igβ signal transducers in counterselection against Dμ using mice that lack Igβ. We found that Dμ expression is not selected against in developing B cells in Igβ mutant mice. Thus, the molecular mechanism for counterselection against Dμ in pre-B cells resembles positive selection in that it requires interaction between mDμ and Igα-Igβ.     

Allelic Exclusion in pTα-deficient Mice: No Evidence for Cell Surface Expression of Two T Cell Receptor (TCR)-β Chains, but Less Efficient Inhibition of Endogeneous Vβ→ (D)Jβ Rearrangements in the Presence of a Functional TCR-β Transgene

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-β chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-β chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-β rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-β rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-α (pTα) chain, a core component of the pre-TCR. Staining of CD3⁺ thymocytes and lymph node cells with antibodies specific for Vβ6 or Vβ8 and a pool of antibodies specific for most other Vβ elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTα-deficient mice expressing a functionally rearranged TCR-β transgene. Interestingly, although the transgenic TCR-β chain significantly influenced thymocyte development even in the absence of pTα, it was not able to inhibit fully endogeneous TCR-β rearrangements either in total thymocytes or in sorted CD25⁺ pre-T cells of pTα^−/− mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.     

The CD3-γδε and CD3-ζ/η Modules Are Each Essential for Allelic Exclusion at the T Cell Receptor β Locus but Are Both Dispensable for the Initiation of V to (D)J Recombination at the T Cell Receptor–β, –γ, and –δ Loci

The pre–T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-β chain. Recent experiments in pre-Tα chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-β locus. Using CD3-ε– and CD3-ζ/η–deficient mice harboring a productively rearranged TCR-β transgene, we showed that the CD3-γδε and CD3-ζ/η modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-β locus. Furthermore, using mutant mice lacking both the CD3-ε and CD3-ζ/η genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-β, TCR-γ, and TCR-δ loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-β locus first accessible to, and later insulated from, the action of the V(D)J recombinase.     

Suppressive Role of B Cells in Chronic Colitis of T Cell Receptor α Mutant Mice

The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-α^−/− mice was explored by creating double mutant mice (TCR-α^−/− × immunoglobulin (Ig)μ^−/−), which lack B cells. TCR-α^−/− × Igμ^−/− mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-α^−/− mice. Colitis was induced in recombination-activating gene-1 (RAG-1^−/−) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-α^−/− × Igμ^−/− mice. When purified B cells from TCR-α^−/− mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1^−/− mice. Administration of the purified Ig from TCR-α^−/− mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-α^−/− × Igμ^−/− mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-α^−/− × Igμ^−/− mice as compared to Igμ^−/− mice and TCR-α^−/− mice. Administration of the purified Ig from TCR-α^−/− mice into TCR-α^−/− × Igμ^−/− mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.     

Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca²⁺ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.     

Mutations in the Human λ5/14.1 Gene Result in B Cell Deficiency and Agammaglobulinemia

B cell precursors transiently express a pre–B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of λ5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igα and Igβ. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine λ5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for λ5/14.1. The maternal allele carried a premature stop codon in the first exon of λ5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the λ5/14.1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant λ5/14.1. These findings indicate that expression of the functional λ5/14.1 is critical for B cell development in the human.     

The Ataxia Telangiectasia mutated kinase controls Igκ allelic exclusion by inhibiting secondary Vκ-to-Jκ rearrangements

Allelic exclusion is enforced through the ability of antigen receptor chains expressed from one allele to signal feedback inhibition of V-to-(D)J recombination on the other allele. To achieve allelic exclusion by such means, only one allele can initiate V-to-(D)J recombination within the time required to signal feedback inhibition. DNA double-strand breaks (DSBs) induced by the RAG endonuclease during V(D)J recombination activate the Ataxia Telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) kinases. We demonstrate that ATM enforces Igκ allelic exclusion, and that RAG DSBs induced during Igκ recombination in primary pre–B cells signal through ATM, but not DNA-PK, to suppress initiation of additional Igκ rearrangements. ATM promotes high-density histone H2AX phosphorylation to create binding sites for MDC1, which functions with H2AX to amplify a subset of ATM-dependent signals. However, neither H2AX nor MDC1 is required for ATM to enforce Igκ allelic exclusion and suppress Igκ rearrangements. Upon activation in response to RAG Igκ cleavage, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by RAG DSBs during Igκ recombination transduce transient H2AX/MDC1-independent signals that suppress initiation of further Igκ rearrangements to control Igκ allelic exclusion.Assembly of Ig and TCR genes from variable (V), diversity (D), and joining (J) segments is the pervasive means by which antigen receptor (AgR) diversity is generated (Brady et al., 2010). V(D)J recombination is initiated by the RAG1/RAG2 (RAG) endonuclease that induces DNA double-strand breaks (DSBs) adjacent to participating segments (Schatz and Ji, 2011) and completed by DSB repair factors that process V(D)J coding ends (CEs) into coding joins (CJs; Helmink and Sleckman, 2012). AgR assembly occurs during and is required for lymphocyte differentiation. IgH genes are assembled through D_H-to-J_H recombination, followed by V_H-to-DJ_H rearrangements on one allele at a time in pro–B cells (Rajewsky, 1996). IgH chains expressed from in-frame V_HDJ_H joins can bind λ5/Vpre–B chains to form pre-BCRs that signal inhibition of V_H rearrangements, proliferation, and differentiation into pre–B cells (Rajewsky, 1996). The two-thirds of cells that assemble out-of-frame V_HDJ_H joins can attempt to assemble in-frame V_HDJ_H joins on the second allele (Rajewsky, 1996). Igκ genes are assembled from Vκ and Jκ segments on one allele at a time in G1 phase pre–B cells (Rajewsky, 1996). Igκ chains expressed from VκJκ joins can bind IgH chains to form κ⁺ BCRs that are subject to selection (Rajewsky, 1996; Nemazee, 2006). Non-autoreactive BCRs signal inhibition of Igκ recombination and differentiation into B cells (Nemazee, 2006). Autoreactive BCRs induce additional Igκ rearrangements that replace VκJκ complexes, a process known as Igκ editing (Nemazee, 2006). Pre–B cells that assemble out-of-frame VκJκ joins can attempt to assemble in-frame VκJκ joins on the other allele (Rajewsky, 1996).Most lymphocytes express surface AgR chains from a single allele. Allelic exclusion is enforced through the ability of Ig and TCR chains expressed from one allele to signal feedback inhibition of V-to-(D)J rearrangements on the second allele (Brady et al., 2010; Vettermann and Schlissel, 2010). To achieve allelic exclusion, only one allele can initiate V-to-(D)J recombination in the time required for feedback inhibition. V-to-(D)J recombination requires CTCF-mediated looping between RAG accessible V segments and RAG-bound D/J segments (Guo et al., 2011; Schatz and Swanson, 2011). In pre–B cells, Igκ loci replicate asynchronously and the early replicating allele is preferentially rendered accessible and selected for Igκ recombination (Mostoslavsky et al., 2001). The time between replication of Igκ loci might be sufficient to enable Igκ chains from the first allele to prevent Igκ rearrangements on the second allele. Yet experiments that show Igκ allelic exclusion is enforced by asynchronous replication between Igκ alleles have not been reported.The feedback model for allelic exclusion hypothesized that V(D)J recombination could activate transient intracellular signals that inhibit recombination on the second allele (Alt et al., 1980). RAG DSBs activate DNA-dependent protein kinase (DNA-PK), which forms an endonuclease with Artemis that processes CEs (Ma et al., 2002). RAG DSBs also activate Ataxia Telangiectasia mutated (ATM), which phosphorylates proteins to coordinate the cellular DSB response (Bredemeyer et al., 2006, 2008). In pre–B cells, RAG DSBs signal through ATM to initiate a genetic program that controls differentiation (Bredemeyer et al., 2008). ATM promotes high-density histone H2AX phosphorylation along RAG-cleaved loci (Savic et al., 2009). H2AX phosphorylation creates binding sites for MDC1, which retains activated ATM kinases around DSBs (Lou et al., 2006). The pools of activated ATM bound and not bound to H2AX/MDC1 exhibit different signaling capabilities (Celeste et al., 2002; Lou et al., 2006). In G1 phase cells, ATM promotes CJ formation independent of H2AX and MDC1 (Bredemeyer et al., 2006; Yin et al., 2009; Helmink et al., 2011). H2AX phosphorylation is detectable over only one Igκ locus in most pre–B cells including those with paired Igκ alleles (Hewitt et al., 2009). The fraction of cells with H2AX phosphorylation over both Igκ loci is fivefold higher in Atm^−/− mice relative to wild-type mice (Hewitt et al., 2009), suggesting that Igκ recombination initiates on a single paired allele and ATM bound to this allele acts on the other allele to prevent bi-allelic rearrangements (Hewitt et al., 2009). Detection of bi-allelic Igκ chromosome breaks in Atm-deficient pre–B cell lines provided support for this model (Hewitt et al., 2009).Here, we show in mice that inactivation of ATM causes a higher frequency of B cells expressing surface Igκ chains from both alleles. We show in primary pre–B cells that DSBs induced during Igκ recombination signal through ATM, but not DNA-PK, to suppress further Igκ rearrangements. Neither H2AX nor MDC1 is required for the ability of ATM to enforce Igκ allelic exclusion or inhibit Igκ recombination. Upon activation in response to RAG DSBs, ATM signals down-regulation of Gadd45α with concomitant repression of the Gadd45α targets Rag1 and Rag2. Our data indicate that ATM kinases activated by Igκ cleavage transduce transient H2AX/MDC1-independent signals that suppress further Igκ rearrangements and thereby enforce Igκ allelic exclusion.     

T Cell Development in Mice Lacking All T Cell Receptor ζ Family Members (ζ, η, and FcεRIγ)

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4⁺ and CD8⁺ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η^−/− and ζ/η^−/−–Fcγ^−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.     

Regulation of B Lymphocyte Development by the Truncated Immunoglobulin Heavy Chain Protein Dμ

The development of B lymphocytes from progenitor cells is dependent on the expression of a pre–B cell–specific receptor made up by a μ heavy chain associated with the surrogate light chains, immunoglobulin (Ig)α, and Igβ. A variant pre–B cell receptor can be formed in which the μ heavy chain is exchanged for a truncated μ chain denoted Dμ. To investigate the role of this receptor in the development of B cells, we have generated transgenic mice that express the Dμ protein in cells of the B lineage. Analysis of these mice reveal that Dμ expression leads to a partial block in B cell development at the early pre–B cell stage, probably by inhibiting V_H to D_HJ_H rearrangement. Furthermore, we provide evidence that Dμ induces V_L to J_L rearrangements.     

The Common Cytokine Receptor γ Chain Controls Survival of γ/δ T Cells

We have investigated the role of common γ chain (γ_c)-signaling pathways for the development of T cell receptor for antigen (TCR)-γ/δ T cells. TCR-γ/δ–bearing cells were absent from the adult thymus, spleen, and skin of γ_c-deficient (γ_c⁻) mice, whereas small numbers of thymocytes expressing low levels of TCR-γ/δ were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-γ/δ development through induction of V-J (variable-joining) rearrangements at the TCR-γ locus. In contrast, we detected clearly TCR-γ rearrangements in fetal thymi from γ_c⁻ mice (which fail to signal in response to IL-7) and reduced TCR-γ rearrangements in adult γ_c thymi. No gross defects in TCR-δ or TCR-β rearrangements were observed in γ_c⁻ mice of any age. Introduction of productively rearranged TCR Vγ1 or TCR Vγ1/Vδ6 transgenes onto mice bearing the γ_c mutation did not restore TCR-γ/δ development to normal levels suggesting that γ_c-dependent pathways provide additional signals to developing γ/δ T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from γ_c⁻ mice were dramatically reduced compared to γ_c⁺ transgenic littermates. We favor the concept that γ_c-dependent receptors are required for the maintenance of TCR-γ/δ cells and contribute to the completion of TCR-γ rearrangements primarily by promoting survival of cells committed to the TCR-γ/δ lineage.     

Apoptosis of Fashigh CD4+ Synovial T Cells by Borrelia-reactive Fas-ligandhigh γδ T Cells in Lyme Arthritis

The function of the minor subset of T lymphocytes bearing the γδ T cell antigen receptor is uncertain. Although some γδ T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of γδ cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore, synovial γδ T cell clones express elevated and sustained levels of the ligand for Fas (APO-1, CD95) compared to αβ T cells, and induce apoptosis of Fas^high CD4⁺ synovial lymphocytes. The findings suggest that γδ T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process.     

Catalyst-free chemoselective α-sulfenylation/β-thiolation for α,β-unsaturated carbonyl compounds

A novel, efficient, catalyst-free and product-controllable strategy has been developed for the chemoselective α-sulfenylation/β-thiolation of α,β-unsaturated carbonyl compounds. An aromatic sulfur group could be chemoselectively introduced at α- or β-position of carbonyls with different sulfur reagents under slightly changed reaction conditions. A series of desired products were obtained in moderate to excellent yields. Mechanistic studies revealed that B₂pin₂ played the key role in activating the transformation towards the β-thiolation of α,β-unsaturated carbonyl compounds. This transition-metal-catalyst-free method provides a convenient and efficient tool for the highly chemoselective preparation of α-thiolation or β-sulfenylation products of α,β-unsaturated carbonyl compounds.

This catalyst-free method provides a useful and efficient tool for the highly chemoselective preparation of α-thiolation or β-sulfenylation products of α,β-unsaturated carbonyl compounds.     

Positive Selection of γδ CTL by TL Antigen Expressed in the Thymus

To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL transgenic mouse strains by introducing Tla^a-3 of A strain origin with its own promoter onto a C3H background with no expression of TL in the thymus. These transgenic mouse strains, both of which express high levels of Tla^a-3-TL antigen in their thymus, were analyzed for their T cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response against TL was induced in Tg.Tla^a-3-1, Tg.Tla^a-3-2, and control C3H mice by skin grafts from H-2K^b/T3^b transgenic mice, Tg.Con.3-1, expressing T3^b-TL ubiquitously. Spleen cells from mice that had rejected the T3^b-TL positive skin grafts were restimulated in vitro with Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20% and 15% of Thy-1⁺ T cells derived from Tg.Tla^a-3-1 and Tg.Tla^a-3-2, respectively, expressed TCRγδ, whereas almost all those from C3H expressed TCRαβ. The MLC from Tg.Tla^a-3-2 and C3H demonstrated high CTL activity against TL, while those from Tg.Tla^a-3-1 had little or none. The generation of γδ CTL recognizing TL in Tg.Tla^a-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 γδ CTL clones were established from Tg.Tla^a-3-2, whereas none were obtained from C3H. Of the 14 γδ CTL clones, 8 were CD8⁺ and 6 were CD4⁻CD8⁻ double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCRγδ and CD3, and also by antibodies to CD8α and CD8β in CD8⁺ clones, showing that the activity was mediated by TCRγδ and coreceptors. The thymic origin of these γδ CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8αβ heterodimers in CD8⁺ clones on their surfaces and by the usage of TCR Vγ4 chains in 12 of the 14 clones. Taken together, these results suggest that Tla^a-3-TL antigen expressed in the thymus engages in positive selection of a sizable population of γδ T cells.     

Structural and electronic properties of α-, β-, γ-, and 6,6,18-graphdiyne sheets and nanotubes

α-, β-, γ- and 6,6,18-graphdiyne (GDYs) sheets, as well as the corresponding nanotubes (GDYNTs) are investigated systematically by using the self-consistent-field crystal orbital method. The calculations show that the GDYs and GDYNTs with different structures have different electronic properties. The α-GDY sheet is a conductor, while 2D β-, γ- and 6,6,18-GDYs are semiconductors. The carrier mobilities of β- and γ-GDY sheets in different directions are almost the same, indicating the isotropic transport characteristics. In addition, the electron mobility is in the order of 10⁶ cm² V⁻¹ s⁻¹ and it is two orders of magnitude larger than the hole mobility of 2D γ-GDY. However, α- and 6,6,18-GDY sheets have anisotropic mobilities, which are different along different directions. For the 1D tubes, the order of stability is γ-GDYNTs > 6,6,18-GDYNTs > β-GDYNTs > α-GDYNTs and is independent of the tube chirality and size. β- and γ-GDYNTs as well as zigzag α- and 6,6,18-GDYNTs are semiconductors with direct bandgaps, while armchair α-GDYNTs are metals, and armchair 6,6,18-GDYNTs change from semiconductors to metals with increasing tube size. The armchair β- and γ-GDYNTs are more favourable to transport holes, while the corresponding zigzag tubes prefer to transport electrons.

Theoretical investigation of α-, β-, γ- and 6,6,18-graphdiyne sheets as well as their corresponding nanotubes.     

NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB.     

Thermal stability and oxidation characteristics of α-pinene, β-pinene and α-pinene/β-pinene mixture

Turpentine is a renewable resource, has good combustion performance, and is considered to be a fuel or promising additive to diesel fuel. This is very important for the investigation of thermal stability and energy oxidation characteristics, because evaluation of energy or fuel quality assurance and use safety are necessary. The main components of turpentine are α-pinene and β-pinene, which have unsaturated double bonds and high chemical activity. By investigating their thermal stability and oxidation reaction characteristics, we know the chemical thermal properties and thermal explosion hazard of turpentine. In this present study, the thermal stability and oxidation characteristics of α-pinene, β-pinene and α-pinene/β-pinene mixture were investigated using a high sensitivity accelerating rate calorimeter (ARC) and C80 calorimeter. The important parameters of oxidation reaction and thermal stability were obtained from the temperature, pressure and exothermic behavior in chemical reaction. The results show that α-pinene and β-pinene are thermally stable without chemical reaction under a nitrogen atmosphere even when the temperature reaches 473 K. The initial exothermic temperature of the two pinenes and their mixture is 333–338 K, and the heat release (−ΔH) of their oxidation is 2745–2973 J g⁻¹. The oxidation activation energy (E_a) of α-pinene, β-pinene and α-pinene/β-pinene mixture is 116.25 kJ mol⁻¹, 121.85 kJ mol⁻¹, and 115.95 kJ mol⁻¹, respectively. There are three steps in the oxidation of pinenes: the first is the induction period of the oxidation reaction; the second is the main oxidation stage, and the pressure is reduced; the third is thermal decomposition to produce gas.

Turpentine is a renewable resource, has good combustion performance, and is considered to be a fuel or promising additive to diesel fuel.     

Assembly of Productive T Cell Receptor δ Variable Region Genes Exhibits Allelic Inclusion

The generation of a productive “in-frame” T cell receptor β (TCR β), immunoglobulin (Ig) heavy (H) or Ig light (L) chain variable region gene can result in the cessation of rearrangement of the alternate allele, a process referred to as allelic exclusion. This process ensures that most αβ T cells express a single TCR β chain and most B cells express single IgH and IgL chains. Assembly of TCR α and TCR γ chain variable region genes exhibit allelic inclusion and αβ and γδ T cells can express two TCR α or TCR γ chains, respectively. However, it was not known whether assembly of TCR δ variable regions genes is regulated in the context of allelic exclusion. To address this issue, we have analyzed TCR δ rearrangements in a panel of mouse splenic γδ T cell hybridomas. We find that, similar to TCR α and γ variable region genes, assembly of TCR δ variable region genes exhibits properties of allelic inclusion. These findings are discussed in the context of γδ T cell development and regulation of rearrangement of TCR δ genes.     

Virus-induced Transient Bone Marrow Aplasia: Major Role of Interferon-α/β during Acute Infection with the Noncytopathic Lymphocytic Choriomeningitis Virus

The hematologic consequences of infection with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were studied in wild-type mice with inherent variations in their interferon (IFN)-α/β responder ability and in mutant mice lacking α/β (IFN-α/β R^0/0) or γ IFN (IFN-γ R^0/0) receptors. During the first week of infection, wild type mice demonstrated a transient pancytopenia. Within a given genetic background, the extent of the blood cell abnormalities did not correlate with the virulence of the LCMV isolate but variations were detected between different mouse strains; they were found to depend on their IFN-α/β responder phenotype. Whereas IFN-γ R^0/0 mice were comparable to wild-type mice, IFN-α/β R^0/0 mice exhibited unchanged peripheral blood values during acute LCMV infection. In parallel, the bone marrow (BM) cellularity, the pluripotential and committed progenitor compartments were up to 30-fold reduced in wild type and IFN-γ R^0/0, but remained unchanged in IFN-α/β R^0/0 mice. Viral titers in BM 3 d after LCMV infection were similar in these mice, but antigen localization was different. Viral antigen was predominantly confined to stromal BM in normal mice and IFN-γ R^0/0 knockouts, whereas, in IFN-α/β R^0/0 mice, LCMV was detected in >90% of megakaryocytes and 10–15% of myeloid precursors, but not in erythroblasts. Although IFN-α/β efficiently prevented viral replication in potentially susceptible hematopoietic cells, even in overwhelming LCMV infection, unlimited virus multiplication in platelet and myeloid precursors in IFN-α/β R^0/0 mice did not interfere with the number of circulating blood cells. Natural killer (NK) cell expansion and activity in the BM was comparable on day 3 after infection in mutant and control mice. Adaptive immune responses did not play a major role because comparable kinetics of LCMV-induced pancytopenia and transient depletion of the pluripotential and committed progenitor compartments were observed in CD8^0/0 and CD4^0/0 mice, in mice depleted of NK cells, in lpr mice, and in perforin-deficient (P^0/0) mice lacking lytic NK cells. Thus, the reversible depression of hematopoiesis during early LCMV infection was not mediated by LCMV-WE–specific cytotoxic T lymphocyte, cytolysis, or secreted IFN-γ from virally induced NK cells but was a direct effect of IFN-α/β.     

Coordinated β-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for β-thalassemia

A primary challenge in lentiviral gene therapy of β-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVβ-shα2, that allows coordinated expression of the therapeutic β^A-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and β^A-T87Q-globin gene expression identical to the parent vector. Compared with the first β^A-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype.