A kinetic and dynamic analysis of Foxp3 induced in T cells by TGF-beta

TGF-beta induces Foxp3 expression in stimulated T cells. These Foxp3 cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3 regulatory T cells (natural regulatory T cells (nTreg)). We performed a single-cell analysis to better characterize the regulation of Foxp3 in iTreg in vitro and assess their dynamics after transfer in vivo. TGF-beta up-regulated Foxp3 in CD4(+)Foxp3 T cells only when added within a 2- to 3-day window of CD3/CD28 stimulation. Up to 90% conversion occurred, beginning after 1-2 days of treatment. Foxp3 expression strictly required TCR stimulation but not costimulation and was independent of cell cycling. Removal of TGF-beta led to a loss of Foxp3 expression after an approximately 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3 cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3 cells were detected by 28 days after transfer. However, some Foxp3 cells persisted even to this late time point, and these preferentially localized to the lymph nodes and bone marrow. CXCR4 was preferentially expressed on Foxp3 iTreg within the bone marrow, and CD62L was preferentially expressed on those in the lymph nodes. Like transferred nTreg and in contrast with revertant Foxp3 cells, Foxp3 iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in na?ve-stimulated T cells is transient in vitro, dependent on TGF-beta activity within a highly restricted window after activation and continuous TGF-beta presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.     

Structural plasticity and enzyme action: crystal structures of mycobacterium tuberculosis peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase cleaves the ester bond between tRNA and the attached peptide in peptidyl-tRNA in order to avoid the toxicity resulting from its accumulation and to free the tRNA available for further rounds in protein synthesis. The structure of the enzyme from Mycobacterium tuberculosis has been determined in three crystal forms. This structure and the structure of the enzyme from Escherichia coli in its crystal differ substantially on account of the binding of the C terminus of the E. coli enzyme to the peptide-binding site of a neighboring molecule in the crystal. A detailed examination of this difference led to an elucidation of the plasticity of the binding site of the enzyme. The peptide-binding site of the enzyme is a cleft between the body of the molecule and a polypeptide stretch involving a loop and a helix. This stretch is in the open conformation when the enzyme is in the free state as in the crystals of M. tuberculosis peptidyl-tRNA hydrolase. Furthermore, there is no physical continuity between the tRNA and the peptide-binding sites. The molecule in the E. coli crystal mimics the peptide-bound enzyme molecule. The peptide stretch referred to earlier now closes on the bound peptide. Concurrently, a channel connecting the tRNA and the peptide-binding site opens primarily through the concerted movement of two residues. Thus, the crystal structure of M. tuberculosis peptidyl-tRNA hydrolase when compared with the crystal structure of the E. coli enzyme, leads to a model of structural changes associated with enzyme action on the basis of the plasticity of the molecule.     

Incorporation of glycosylphosphatidylinositol-anchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles

The rapid worldwide spread of human immunodeficiency virus (HIV) mandates the development of successful vaccination strategies. Since live attenuated HIV is not accepted as a vaccine due to safety concerns, virus-like particles (VLPs) offer an attractive safe alternative because they lack the viral genome yet they are perceived by the immune system as a virus particle. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this we generated chimeric simian immunodeficiency virus (SIV) VLPs containing either glycosylphosphatidylinositol (GPI)-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity and ability to enhance immune responses in vivo. Immunization of mice with chimeric SIV VLPs containing GM-CSF induced SIV Env-specific antibodies as well as neutralizing activity at significantly higher levels than those induced by standard SIV VLPs, SIV VLPs containing CD40L, or standard VLPs mixed with soluble GM-CSF. In addition, mice immunized with chimeric SIV VLPs containing either GM-CSF or CD40L showed significantly increased CD4(+)- and CD8(+)-T-cell responses to SIV Env, compared to standard SIV VLPs. Taken together, these results demonstrate that the incorporation of immunostimulatory molecules enhances humoral and cellular immune responses. We propose that anchoring immunostimulatory molecules into SIV VLPs can be a promising approach to augmenting the efficacy of VLP antigens.     

A kinetic and dynamic analysis of Foxp3 induced in T cells by TGF-beta

TGF-beta induces Foxp3 expression in stimulated T cells. These Foxp3+ cells (induced regulatory T cells (iTreg)) share functional and therapeutic properties with thymic-derived Foxp3+ regulatory T cells (natural regulatory T cells (nTreg)). We performed a single-cell analysis to better characterize the regulation of Foxp3 in iTreg in vitro and assess their dynamics after transfer in vivo. TGF-beta up-regulated Foxp3 in CD4+Foxp3- T cells only when added within a 2- to 3-day window of CD3/CD28 stimulation. Up to 90% conversion occurred, beginning after 1-2 days of treatment. Foxp3 expression strictly required TCR stimulation but not costimulation and was independent of cell cycling. Removal of TGF-beta led to a loss of Foxp3 expression after an approximately 4-day lag. Most iTreg transferred into wild-type mice down-regulated Foxp3 within 2 days, and these Foxp3- cells were concentrated in the blood, spleen, lung, and liver. Few of the Foxp3- cells were detected by 28 days after transfer. However, some Foxp3+ cells persisted even to this late time point, and these preferentially localized to the lymph nodes and bone marrow. CXCR4 was preferentially expressed on Foxp3+ iTreg within the bone marrow, and CD62L was preferentially expressed on those in the lymph nodes. Like transferred nTreg and in contrast with revertant Foxp3- cells, Foxp3+ iTreg retained CD25 and glucocorticoid-induced TNFR family-related gene. Thus, Foxp3 expression in na?ve-stimulated T cells is transient in vitro, dependent on TGF-beta activity within a highly restricted window after activation and continuous TGF-beta presence. In vivo, a subset of transferred iTreg persist long term, potentially providing a lasting source for regulatory activity after therapeutic administration.     

Y-chromosomal insights into the genetic impact of the caste system in India

The caste system has persisted in Indian Hindu society for around 3,500 years. Like the Y chromosome, caste is defined at birth, and males cannot change their caste. In order to investigate the genetic consequences of this system, we have analysed male-lineage variation in a sample of 227 Indian men of known caste, 141 from the Jaunpur district of Uttar Pradesh and 86 from the rest of India. We typed 131 Y-chromosomal binary markers and 16 microsatellites. We find striking evidence for male substructure: in particular, Brahmins and Kshatriyas (but not other castes) from Jaunpur each show low diversity and the predominance of a single distinct cluster of haplotypes. These findings confirm the genetic isolation and drift within the Jaunpur upper castes, which are likely to result from founder effects and social factors. In the other castes, there may be either larger effective population sizes, or less strict isolation, or both. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Tatiana Zerjal and Arpita Pandya contributed equally to this work.     

Comparative sequence analysis of acid sensitive/resistance proteins in Escherichia coli and Shigella flexneri

The molecular basis for the survival of bacteria under extreme conditions in which growth is inhibited is a question of great current interest. A preliminary study was carried out to determine residue pattern conservation among the antiporters of enteric bacteria, responsible for extreme acid sensitivity especially in Escherichia coli and Shigella flexneri. Here we found the molecular evidence that proved the relationship between E. coli and S. flexneri. Multiple sequence alignment of the gadC coded acid sensitive antiporter showed many conserved residue patterns at regular intervals at the N-terminal region. It was observed that as the alignment approaches towards the C-terminal, the number of conserved residues decreases, indicating that the N-terminal region of this protein has much active role when compared to the carboxyl terminal. The motif, FHLVFFLLLGG, is well conserved within the entire gadC coded protein at the amino terminal. The motif is also partially conserved among other antiporters (which are not coded by gadC) but involved in acid sensitive/resistance mechanism. Phylogenetic cluster analysis proves the relationship of Escherichia coli and Shigella flexneri. The gadC coded proteins are converged as a clade and diverged from other antiporters belongs to the amino acid-polyamine-organocation (APC) superfamily.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Detached and attached Arabidopsis leaf assays reveal distinctive defense responses against hemibiotrophic Colletotrichum spp

The agriculturally important genus Colletotrichum is an emerging model pathogen for studying defense in Arabidopsis. During the process of screening for novel pathogenic Colletotrichum isolates on Arabidopsis, we found significant differences in defense responses between detached and attached leaf assays. A near-adapted isolate Colletotrichum linicola A1 could launch a typical infection only on detached, but not attached, Arabidopsis leaves. Remarkably, resistance gene-like locus RCH1-mediated resistance in intact plants also was compromised in detached leaves during the attacks with the virulent reference isolate C. higginsianum. The differences in symptom development between the detached leaf and intact plant assays were further confirmed on defense-defective mutants following inoculation with C. higginsianum, where the greatest inconsistency occurred on ethylene-insensitive mutants. In intact Arabidopsis plants, both the salicylic acid- and ethylene-dependent pathways were required for resistance to C. higginsianum and were associated with induced expression of pathogenesis-related genes PR1 and PDF1.2. In contrast, disease symptom development in detached leaves appeared to be uncoupled from these defense pathways and more closely associated with senescence: an observation substantiated by coordinated gene expression analysis and disease symptom development, and chemically and genetically mimicking senescence.