Distinct cytokine-driven responses of activated blood gammadelta T cells: insights into unconventional T cell pleiotropy

Human Vgamma9/Vdelta2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3(+) cells. This "transitional response," occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in alphabeta T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vgamma9/Vdelta2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic alphabeta T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate gammadelta T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of gammadelta T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vgamma9/Vdelta2 TCR agonists.     

Synthesis and evaluation of alpha,beta-unsaturated alpha-aryl-substituted fosmidomycin analogues as DXR inhibitors

Fosmidomycin, which acts through inhibition of 1-deoxy-D-xylulose phosphate reductoisomerase (DXR) in the non-mevalonate pathway, represents a valuable recent addition to the armamentarium against uncomplicated malaria. In this paper, we describe the synthesis and biological evaluation of E- and Z-alpha,beta-unsaturated alpha-aryl-substituted analogues of FR900098, a fosmidomycin congener, utilizing a Stille or a Suzuki coupling to introduce the aryl group. In contrast with our expectations based on the promising activity earlier observed for several alpha-substituted fosmidomycin analogues, all synthesized analogues exhibited much lower binding affinity for DXR than fosmidomycin.     

Functional domains of the 50S subunit mature late in the assembly process

Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.     

Analysis of the association between CD40 and CD40 ligand polymorphisms and systemic sclerosis

Introduction

The aim of the present study was to investigate the possible role of CD40 and CD40 ligand (CD40LG) genes in the susceptibility and phenotype expression of systemic sclerosis (SSc).

Methods

In total, 2,670 SSc patients and 3,245 healthy individuals from four European populations (Spain, Germany, The Netherlands, and Italy) were included in the study. Five single-nucleotide polymorphisms (SNPs) of CD40 (rs1883832, rs4810485, rs1535045) and CD40LG (rs3092952, rs3092920) were genotyped by using a predesigned TaqMan allele-discrimination assay technology. Meta-analysis was assessed to determine whether an association exists between the genetic variants and SSc or its main clinical subtypes.

Results

No evidence of association between CD40 and CD40LG genes variants and susceptibility to SSc was observed. Similarly, no significant statistical differences were observed when SSc patients were stratified by the clinical subtypes, the serologic features, and pulmonary fibrosis.

Conclusions

Our results do not suggest an important role of CD40 and CD40LG gene polymorphisms in the susceptibility to or clinical expression of SSc.     

Pilot-scale conversion of lime-treated wheat straw into bioethanol: quality assessment of bioethanol and valorization of side streams by anaerobic digestion and combustion

Introduction  

The limited availability of fossil fuel sources, worldwide rising energy demands and anticipated climate changes attributed to an increase of greenhouse gasses are important driving forces for finding alternative energy sources. One approach to meeting the increasing energy demands and reduction of greenhouse gas emissions is by large-scale substitution of petrochemically derived transport fuels by the use of carbon dioxide-neutral biofuels, such as ethanol derived from lignocellulosic material.     

Crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, a crucial enzyme in the non-mevalonate pathway of isoprenoid biosynthesis.

We have solved the 2.5-A crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, an enzyme involved in the mevalonate-independent 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. The structure reveals that the enzyme is present as a homodimer. Each monomer displays a V-like shape and is composed of an amino-terminal dinucleotide binding domain, a connective domain, and a carboxyl-terminal four-helix bundle domain. The connective domain is responsible for dimerization and harbors most of the active site. The strictly conserved acidic residues Asp(150), Glu(152), Glu(231), and Glu(234) are clustered at the putative active site and are probably involved in the binding of divalent cations mandatory for enzyme activity. The connective and four-helix bundle domains show significant mobility upon superposition of the dinucleotide binding domains of the three conformational states present in the asymmetric unit of the crystal. A still more pronounced flexibility is observed for a loop spanning residues 186 to 216, which adopts two completely different conformations within the three protein conformers. A possible involvement of this loop in an induced fit during substrate binding is discussed.     

Prolonged (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells in macaques

Although phosphoantigen-specific Vgamma2Vdelta2 T cells appear to play a role in antimicrobial and anticancer immunity, mucosal immune responses and effector functions of these gammadelta T cells during infection or phospholigand treatment remain poorly characterized. In this study, we demonstrate that the microbial phosphoantigen (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) plus IL-2 treatment of macaques induced a prolonged major expansion of circulating Vgamma2Vdelta2 T cells that expressed CD8 and produced cytotoxic perforin during their peak expansion. Interestingly, HMBPP-activated Vgamma2Vdelta2 T cells underwent an extraordinary pulmonary accumulation, which lasted for 3-4 mo, although circulating Vgamma2Vdelta2 T cells had returned to baseline levels weeks prior. The Vgamma2Vdelta2 T cells that accumulated in the lung following HMBPP/IL-2 cotreatment displayed an effector memory phenotype, as follows: CCR5+CCR7-CD45RA-CD27+ and were able to re-recognize phosphoantigen and produce copious amounts of IFN-gamma up to 15 wk after treatment. Furthermore, the capacity of massively expanded Vgamma2Vdelta2 T cells to produce cytokines in vivo coincided with an increase in numbers of CD4+ and CD8+ alphabeta T cells after HMBPP/IL-2 cotreatment as well as substantial perforin expression by CD3+Vgamma2- T cells. Thus, the prolonged HMBPP-driven antimicrobial and cytotoxic responses of pulmonary and systemic Vgamma2Vdelta2 T cells may confer immunotherapeutics against infectious diseases and cancers.     

Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68.

Murine gammaherpesvirus 68 (gamma HV-68; also referred to as MHV-68) is a gammaherpesvirus which infects murid rodents. Previous studies showed that CD8 T cells are important for controlling gamma HV-68 replication during the first 2 weeks of infection and suggested a role for B cells in latent or persistent gamma HV-68 infection. To further define the importance of B cells and CD8 T cells during acute and chronic gamma HV-68 infection, we examined splenic infection in mice with null mutations in the transmembrane domain of the mu-heavy-chain constant region (MuMT; B-cell and antibody deficient) or in the beta2-microglobulin gene (beta2 -/-; CD8 deficient). Immunocompetent mice infected intraperitoneally with gamma HV-68 demonstrated peak splenic titers 9 to 10 days postinfection, cleared infectious virus 15 to 20 days postinfection, and harbored low levels of latent virus at 6 weeks postinfection. Beta2-/- mice showed peak splenic gamma HV-68 titers similar to those of normal mice but were unable to clear infectious virus completely from the spleen, demonstrating persistent infectious virus 6 weeks postinfection. These data indicate that CD8 T cells are important for clearing infectious gamma HV-68 from the spleen. Infected MuMT mice did not demonstrate detectable infectious gamma HV-68 in the spleen at any time after infection, indicating that mature B lymphocytes are necessary for acute splenic infection by gamma HV-68. Despite the lack of measurable acute infection, MuMT spleen cells harbored latent virus 6 weeks postinfection at a level about 100-fold higher than that in normal mice. These data demonstrate establishment of latency by a herpesvirus in an organ in the absence of acute viral replication in that organ. In addition, they demonstrate that gamma HV-68 can establish latency in a cell type other than mature B lymphocytes.     

Isoprenoid Pigments in Representatives of the Family <Emphasis Type="Italic">Microbacteriaceae</Emphasis>

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C₄₀-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and β-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 335–341.Original Russian Text Copyright © 2005 by Trutko, Dorofeeva, Evtushenko, Ostrovskii, Hintz, Wiesner, Jomaa, Baskunov, Akimenko.