Indirect Recognition Research Articles

Enhancing the use of evidence-based decision-making processes to establish immunization policies and programmes at national level: what should be the roe of Pasteur Institutes?

In both developed and developing countries, the need for evidence-based decision-making in immunization programs has become crucial in light of multiple health priorities, limited human resources and logistical capacities, as well as the high cost of vaccines relative to limited public funds that are available. An important step that countries can take to encourage well-informed decision-making is to establish a group of national experts to advise the policy makers at the Ministry of Health and program managers for making decisions, including choices of new vaccines and needed adjustments to existing programs and schedules. So far, many countries have already constituted such National Immunization Technical Advisory Groups (NITAGs) while other countries are currently working towards their establishment with the support of technical partners such as WHO and AMP (SIVAC Initiative). Based on WHO recommendations, the composition of the advisory group should include two categories of members: core and non-core members. All core members should be independent and credible experts who serve in their own capacity and who do not represent the interests of a particular group. Core members only, should participate in advising and deciding on the final set of recommendations. Non-core members hold key positions with important government entities they represent (e.g. Outbreaks Surveillance Department, National Drugs Regulatory Authorities, National Public Health Laboratory etc.) and represent various important professional societies, and key technical partners (e.g. WHO and UNICEF). It is recommended that the committee be multidisciplinary and represent a broad range of expertise from the following disciplines: clinical medicine (pediatrics medicine, adult medicine, geriatrics), epidemiologists, infectious diseases specialists, microbiologists, public health, immunology, vaccinology. Pasteur Institutes have a crucial role to play in the NITAGs’ activities. They can either be sought to participate as non-core member or to send experts as core members. As non-core member, Pasteur Institutes provide data regarding burden of disease related to their laboratory surveillance activities. They will also provide specific information regarding immunology and safety issues related to vaccines. They can also provide information regarding emerging diseases and therefore influence the national research agenda by having the NITAG issuing specific recommendations. As core members, the experts from Pasteur Institutes should refrain from promoting the policies and views and products of the organization but this leads to an indirect recognition of the expertise of the institution. As shown in other countries, it also may conduct to NITAG formal request regarding surveys and studies related to vaccines to the institution. Among other countries, the SIVAC Initiative is currently supporting Cote d’Ivoire and Viet Nam in establishing and strengthening NITAGs. In both countries, Pasteur Institutes already participate actively to the process and should be able to share their experience with other institutes of the Pasteur network.

High Frequency of Alloantigen-Specific Regulatory T-Cells In Human Blood Allow Rapid Ex Vivo Expansion for Adoptive Therapy.

AbstractAbstract 3747Evidence from animal studies has demonstrated that allogeneic regulatory T (Treg) cells can be used as effective therapeutic tools in the prevention of allograft rejection and graft-vs-host disease (GVHD). However, translating Treg-based therapies from animal models to human clinical trials requires methods for the isolation and expansion of functional Treg. Loss of function has been a concern for Treg expanded broadly with anti-CD3 and CD28 antibody-coated beads. Based on preclinical data in rodents, allospecific Tregs are anticipated to be more potent and exert more selective immunoregulation than polyclonal Treg in the prevention or treatment of GVHD. Currently, no effective approach has been established for selective expansion of human allospecific Treg ex vivo. In this study, we show the selective direct and indirect recognition of alloantigen by specific human CD4+CD25+CD127− Tregs. CD4+CD25+CD127− Treg were freshly isolated from normal human blood, labeled with CFSE and stimulated by MHC (HLA)-mismatched APC's, in the presence of IL-2 and IL-15 to amplify the proliferative responses, and sirolimus to suppress other T cells. The precursor frequency of the antigen specific Tregs was calculated by Limiting Dilution Analysis and found to be 1/49 -1/138 among all Tregs. The expanded antigen specific Tregs with low CFSE content were in general greater than 90% by days 12–15 in culture. CFSE-low Tregs were sorted by flow cytometry and exhibited complete suppression against CD25-negative CD4 T cell responses up to ratios of 1:100, in contrast CFSE-high Tregs were not suppressive. Ex vivo expanded antigen specific Tregs maintained high Foxp3, retained lymphoid homing receptor CD62L and chemokine receptor CCR7 expression, suggesting that they are functional and able to migrate to lymphoid tissue in vivo. Among the APC's tested, DC was found to be better stimulus with potent suppressive potential when compared to T-cell stimulated PBMC's. Specific indirect allorecognition was elicited when Tregs were stimulated with autologous APC's pulsed with allogeneic cell lysate in the presence of sirolimus, IL-15 and IL-2. Precursor frequency of antigen specific Tregs with indirect allorecognition was 100 fold lower than the precursor frequency of Tregs with direct allospecificity. Highly suppressive antigen specific Tregs show a duplication time of approximately 24 hours in the presence alloantigen, cytokines and sirolimus, and can be expanded in vitro by 400 fold in a 12 day period. This magnitude of expansion predicts the feasibility of conducting translational clinical trials. Our data may provide a platform for the selective expansion of Tregs against minor histocompatibility antigens to prevent GVHD while sparing graft-vs.-leukemia effects. Disclosures:No relevant conflicts of interest to declare.

Use of structural DNA properties for the prediction of transcription-factor binding sites in Escherichia coli

Recognition of genomic binding sites by transcription factors can occur through base-specific recognition, or by recognition of variations within the structure of the DNA macromolecule. In this article, we investigate what information can be retrieved from local DNA structural properties that is relevant to transcription factor binding and that cannot be captured by the nucleotide sequence alone. More specifically, we explore the benefit of employing the structural characteristics of DNA to create binding-site models that encompass indirect recognition for the Escherichia coli model organism. We developed a novel methodology [Conditional Random fields of Smoothed Structural Data (CRoSSeD)], based on structural scales and conditional random fields to model and predict regulator binding sites. The value of relying on local structural-DNA properties is demonstrated by improved classifier performance on a large number of biological datasets, and by the detection of novel binding sites which could be validated by independent data sources, and which could not be identified using sequence data alone. We further show that the CRoSSeD-binding-site models can be related to the actual molecular mechanisms of the transcription factor DNA binding, and thus cannot only be used for prediction of novel sites, but might also give valuable insights into unknown binding mechanisms of transcription factors.

Activation of a Rac GTPase by the NLR Family Disease Resistance Protein Pit Plays a Critical Role in Rice Innate Immunity

The nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

The shape of the DNA minor groove directs binding by the DNA-bending protein Fis

The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of approximately 65 degrees , and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

Cross‐species divergence of the major recognition pathways of ubiquitylated substrates for ubiquitin/26S proteasome‐mediated proteolysis

The recognition of ubiquitylated substrates is an essential element of ubiquitin/26S proteasome-mediated proteolysis (UPP), which is mediated directly by the proteasome subunit RPN10 and/or RPN13, or indirectly by ubiquitin receptors containing ubiquitin-like and ubiquitin-associated domains. By pull-down and mutagenesis assays, we detected cross-species divergence of the major recognition pathways. RPN10 plays a major role in direct recognition in Arabidopsis and yeast based on the strong affinity for the long and K48-linked ubiquitin chains. In contrast, both the RPN10 and RPN13 homologs play major roles in humans. For indirect recognition, the RAD23 and DSK2 homologs (except for the human DSK2 homolog) are major receptors. The human RAD23 homolog is targeted to the 26S proteasome by the RPN10 and RPN13 homologs. In comparison, Arabidopsis uses UIM1 and UIM3 of RPN10 to bind DSK2 and RAD23, respectively. Yeast uses UIM in RPN10 and LRR in RPN1. Overall, multiple proteasome subunits are responsible for the direct and/or indirect recognition of ubiquitylated substrates in yeast and humans. In contrast, a single proteasome subunit, RPN10, is critical for both the direct and indirect recognition pathways in Arabidopsis. In agreement with these results, the accumulation of ubiquitylated substrates and severe pleiotropic phenotypes of vegetative and reproductive growth are associated with the loss of RPN10 function in an Arabidopsis T-DNA insertion mutant. This implies that the targeting and proteolysis of the critical regulators involved are affected. These results support a cross-species mechanistic and functional divergence of the major recognition pathways for ubiquitylated substrates of UPP.

Endoplasmic Reticulum Stress-Mediated Apoptosis Involved in Indirect Recognition Pathway Blockade Induces Long-Term Heart Allograft Survival

Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

DNA Shape Recognition by the Nucleoid Protein Fis and Its Role in Chromosome Compaction and DNA Recombination

The Fis nucleoid-associated DNA bending protein dynamically binds DNA in a largely sequence-neutral manner. Fis also forms stable complexes to DNA segments that share little sequence conservation. In order to understand how Fis is forming these specific DNA complexes and how it distorts DNA structure upon binding, we have determined X-ray crystal structures of 12 Fis-DNA complexes of variable affinities and stabilities. These structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the DNA minor groove and sequence-dependent induced fits over adjacent major groove interfaces to generate overall curvatures of 60-75°. In silico DNA modeling of Fis-bound and unbound DNA molecules suggests a model in which Fis initially selects binding sites based on their intrinsic minor groove shape and then bends the DNA to generate the bound structure. The structures show how binding of Fis leads to DNA compaction, and the location of residues involved in capturing DNA loops implies protein-protein interactions are required for loop stabilization. We have shown that DNA supercoiling and torsional energy associated with the Fis-bound DNA enhancer segment controls the rotational direction and number of Hin subunit translocations that mediate DNA exchange during Hin-catalyzed site-specific DNA inversion. The Fis-DNA structures, together with supporting experimental evidence, enable us to propose a new model for the structure of the Fis-bound Hin invertasome structure.

間接再認手続きによる言語的符号化困難な音列の潜在記憶の検出

Two experiments, using an indirect recognition procedure (Terasawa & Ohta, 1993) as an implicit memory task, were conducted to examine implicit memory for random tone sequences. The indirect recognition procedure involved two sessions. The second session was a general recognition experiment consisting of learning and a recognition test phase. The effects of the learning during the first session were examined based on the recognition performance in the second session. The interval between the sessions was 10 weeks for experiment 1 and 8 weeks for experiment 2. In each session, participants were required to rate their liking for each of the sequences presented. In the second session, participants were required to respond to an old-new recognition test about the items just presented. The targets and distractors in the test consisted of stimuli presented or not presented in the first session. Analyses of the hits and false alarms showed an effect of the number of presentations in the first session. This result indicates an effect of long lasting implicit memory for tone sequences.

Bacterial adaptation through distributed sensing of metabolic fluxes.

The recognition of carbon sources and the regulatory adjustments to recognized changes are of particular importance for bacterial survival in fluctuating environments. Despite a thorough knowledge base of Escherichia coli's central metabolism and its regulation, fundamental aspects of the employed sensing and regulatory adjustment mechanisms remain unclear. In this paper, using a differential equation model that couples enzymatic and transcriptional regulation of E. coli's central metabolism, we show that the interplay of known interactions explains in molecular-level detail the system-wide adjustments of metabolic operation between glycolytic and gluconeogenic carbon sources. We show that these adaptations are enabled by an indirect recognition of carbon sources through a mechanism we termed distributed sensing of intracellular metabolic fluxes. This mechanism uses two general motifs to establish flux-signaling metabolites, whose bindings to transcription factors form flux sensors. As these sensors are embedded in global feedback loop architectures, closed-loop self-regulation can emerge within metabolism itself and therefore, metabolic operation may adapt itself autonomously (not requiring upstream sensing and signaling) to fluctuating carbon sources.

Fungal effector proteins: past, present and future.

The pioneering research of Harold Flor on flax and the flax rust fungus culminated in his gene-for-gene hypothesis. It took nearly 50 years before the first fungal avirulence (Avr) gene in support of his hypothesis was cloned. Initially, fungal Avr genes were identified by reverse genetics and map-based cloning from model organisms, but, currently, the availability of many sequenced fungal genomes allows their cloning from additional fungi by a combination of comparative and functional genomics. It is believed that most Avr genes encode effectors that facilitate virulence by suppressing pathogen-associated molecular pattern-triggered immunity and induce effector-triggered immunity in plants containing cognate resistance proteins. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either on the plasma membrane or inside the plant cell. Indirect recognition of an effector (also known as the guard model) implies that the virulence target of an effector in the host (the guardee) is guarded by the resistance protein (the guard) that senses manipulation of the guardee, leading to activation of effector-triggered immunity. In this article, we review the literature on fungal effectors and some pathogen-associated molecular patterns, including those of some fungi for which no gene-for-gene relationship has been established.

Discrimination of offspring by indirect recognition in an egg-feeding dendrobatid frog, Oophaga pumilio

Offspring discrimination, the differential treatment of offspring and unrelated young, functions in numerous animal taxa to ensure that vital and costly parental care behaviours are appropriately directed. Discrimination can be facilitated either by direct (phenotypic) recognition of offspring or by indirect (nonphenotypic) recognition of offspring location. Offspring discrimination and recognition mechanisms have not been identified in an amphibian. In the strawberry poison frog, Oophaga pumilio, a dendrobatid frog with obligatory maternal provisioning behaviour, I tested whether mothers discriminate between offspring and unrelated young, whether they use direct or indirect recognition cues, and whether prior parental investment plays a contextual role in the differential treatment of young. Mother frogs utilized tadpole-rearing cups attached to tree trunks in wet tropical forest. After manipulating the identity, location and/or age of tadpoles in cups, I determined whether maternal provisioning behaviour was maintained by measuring tadpole growth and development. Mothers provisioned young regardless of tadpole identity, but were sensitive to location and did not provision tadpoles that were moved 2 cm to an adjacent cup. When given a choice between related and unrelated tadpoles in originally chosen or adjacent cups, mothers discriminated by location, but not by relatedness. Maternal provisioning behaviour persisted when a tadpole provisioned for 10 days was replaced with either an age-matched or newly hatched unrelated tadpole, so direct offspring recognition does not appear to be dependent on prior parental investment. Together, these results provide strong evidence that mother O. pumilio use indirect recognition cues to discriminate between offspring and unrelated offspring.

Comparative analysis of dendritic cells and anti-CD3/CD28 expanded regulatory T cells for application in transplantation

The containment of direct and indirect recognition of donor alloantigens by enhancement of the number and activity of regulatory T cells (Tregs) has been one of the promising approaches to achieve transplant tolerance. Two major methods, dendritic cell (DC) and anti-CD3/CD28 antibodies (Abs) have been introduced for ex vivo expansion of Tregs prior to their adoptive transfer. Here we compared the clinical advantage of using these methods of Tregs expansion by evaluating the nature and ability of expanded Tregs to abolish recipient alloreactive T-cell responses in vitro and in vivo. The ovalbumin (OVA)-specific Tregs isolated from DO11.10 mice were exposed to either Abs or syngeneic DC loaded with OVA peptide in the presence of exogenous IL-2 for a week. Using BABL/c as recipient and C57BL/6 as donor, the suppressive activity of these cells was examined. We found that DCs were much more efficient than Abs in expanding (9-fold versus 4-fold) and maintaining the viability (90% versus 35%) of purified Tregs. Interestingly, the Abs-expanded Tregs superbly contained the alloreactive T-cell proliferation and both Tregs were more suppressive when the Tregs cognate antigen and alloantigens were separately expressed on recipient and donor DCs (HVGD) rather than on recipient DC alone (GVHD). Importantly, however, DC-expanded Tregs maintained stable expression of Foxp3, survived longer and effectively contained the differentiation of alloreactive T cells into IFN-γ-producing effector cells both in vitro and in vivo. Our data suggests that DC-expanded Tregs provides a clinically advantageous means of preventing unwanted immune reactions to allografts.

A Family of Bacterial Cysteine Protease Type III Effectors Utilizes Acylation-dependent and -independent Strategies to Localize to Plasma Membranes

Bacterial phytopathogens employ a type III secretion system to deliver effector proteins into the plant cell to suppress defense pathways; however, the molecular mechanisms and subcellular localization strategies that drive effector function largely remain a mystery. Here, we demonstrate that the plant plasma membrane is the primary site for subcellular localization of the Pseudomonas syringae effector AvrPphB and five additional cysteine protease family members. AvrPphB and two AvrPphB-like effectors, ORF4 and NopT, autoproteolytically process following delivery into the plant cell to expose embedded sites for fatty acylation. Host-dependent lipidation of these three effectors directs plasma membrane localization and is required for the avirulence activity of AvrPphB. Surprisingly, the AvrPphB-like effectors RipT, HopC1, and HopN1 utilize an acylation-independent mechanism to localize to the cellular plasma membrane. Although some AvrPphB-like effectors employ acylation-independent localization strategies, others hijack the eukaryotic lipidation machinery to ensure plasma membrane localization, illustrating the diverse tactics employed by type III effectors to target specific subcellular compartments.

Historia Elastica

From scattered references in government documents and contemporary academic articles, this paper constructs a rough history of the wide and largely paralegal trade in Ficus elastica, or India-rubber, across the north-eastern frontier of British India. In order to avail of the resources beyond the revenue line, nearly every rubber firm operative in the area struck numerous informal arrangements with the chiefs of the local communities through a network of Marwari shops, which became public only after legal complications arose during community skirmishes or transfer of mahals.While evidences survive to suggest that these firms often enjoyed tacit support from the government, the firms’ indirect recognition of the territorial rights of these communities was also a constant source of irritation to the British administration, which deeply mourned the loss of revenue duties on the unregistered products. Confused discussions and half-hearted legal actions followed, entailing a peculiar legal regime and culture of extraction. The empirical details of this history may be helpful in rethinking the general nature of joint-stock capitalism in a ‘savage’ frontier.

Trajectories in teacher education: Recognising prior learning in practice

This article analyses the trajectories into teacher education of a group of child minders who are studying to become pre-school teachers. The specific focus is what impact their prior experiences and learning from pre-school have on their trajectories, and how these experiences and learning are recognised in the first year of teacher education. A situated learning perspective is applied, with a focus on participation and trajectories in the two communities of practice in which teacher education is situated – the university and the pre-school. The data consist of transcribed interviews with ten student teachers, and additional data are gathered from stakeholders in the program. The results show that prior experiences and learning are given implicit and indirect recognition. In the practice of the pre-school, the trajectory means a shift in identity, from child minder to student teacher and pre-school teacher. In the practice of the university, prior experiences and learning help students in their trajectories from peripheral to fuller participation.

Conferring indirect allospecificity on CD4+CD25+ Tregs by TCR gene transfer favors transplantation tolerance in mice

T cell responses to MHC-mismatched transplants can be mediated via direct recognition of allogeneic MHC molecules on the cells of the transplant or via recognition of allogeneic peptides presented on the surface of recipient APCs in recipient MHC molecules - a process known as indirect recognition. As CD4(+)CD25(+) Tregs play an important role in regulating alloresponses, we investigated whether mouse Tregs specific for allogeneic MHC molecules could be generated in vitro and could promote transplantation tolerance in immunocompetent recipient mice. Tregs able to directly recognize allogeneic MHC class II molecules (dTregs) were obtained by stimulating CD4(+)CD25(+) cells from C57BL/6 mice (H-2(b)) with allogeneic DCs from BALB/c mice (H-2(d)). To generate Tregs that indirectly recognized allogeneic MHC class II molecules, dTregs were retrovirally transduced with TCR genes conferring specificity for H-2K(d) presented by H-2A(b) MHC class II molecules. The dual direct and indirect allospecificity of the TCR-transduced Tregs was confirmed in vitro. In mice, TCR-transduced Tregs, but not dTregs, induced long-term survival of partially MHC-mismatched heart grafts when combined with short-term adjunctive immunosuppression. Further, although dTregs were only slightly less effective than TCR-transduced Tregs at inducing long-term survival of fully MHC-mismatched heart grafts, histologic analysis of long-surviving hearts demonstrated marked superiority of the TCR-transduced Tregs. Thus, Tregs specific for allogeneic MHC class II molecules are effective in promoting transplantation tolerance in mice, which suggests that such cells have clinical potential.

Induction of Indoleamine 2,3-Dioxygenase in Livers Following Hepatectomy Prolongs Survival of Allogeneic Hepatocytes After Transplantation

Objectives Indoleamine 2,3-dioxygenase (IDO), which catalyzes the breakdown of tryptophan into kyneurenine, has immunologic significance for the induction of maternal tolerance and liver allograft tolerance by inhibiting T-cell activation. In the present study, we compared survival of syngeneic or allogeneic hepatocytes in livers with or without hepatectomy. Subsequently, we investigated gene expression and localization of IDO in the recipient liver. Methods DA and Fisher 344 rats were used in the following experimental groups: group 1, DA hepatocytes transplanted into hepatectomized Fisher 344 rats; group 2, Fisher 344 hepatocytes transplanted into hepatectomized Fisher 344 rats; group 3, DA hepatocytes transplanted into nonhepatectomized Fisher 344 rats; and group 4, Fisher 344 hepatocytes transplanted into nonhepatectomized Fisher 344 rats. After transplantation, the surviving cells were evaluated on day 5. The IDO signal of the recipient liver was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Results In the hepatectomized groups subjected to allogeneic or syngeneic hepatocyte transplantation, the number of surviving hepatocytes was greater than in the nonhepatectomized group after transplantation. The IDO signals (RT-PCR) in the hepatectomized groups were stronger than those in the nonhepatectomized groups. Immunohistochemistry demonstrated that the IDO signal is located in liver antigen-presenting cells, such as Kupffer cells or dendritic cells, and not expressed in hepatocytes. Conclusions Our results demonstrated that IDO is induced in antigen-presenting cells of hepatectomized livers by which subsequently transplanted cells may be protected from rejection by inhibiting indirect or direct recognition of donor antigen and further T-cell activation.

Structural analysis of the genetic switch that regulates the expression of restriction-modification genes

Controller (C) proteins regulate the timing of the expression of restriction and modification (R–M) genes through a combination of positive and negative feedback circuits. A single dimer bound to the operator switches on transcription of the C-gene and the endonuclease gene; at higher concentrations, a second dimer bound adjacently switches off these genes. Here we report the first structure of a C protein–DNA operator complex, consisting of two C protein dimers bound to the native 35 bp operator sequence of the R–M system Esp1396I. The structure reveals a role for both direct and indirect DNA sequence recognition. The structure of the DNA in the complex is highly distorted, with severe compression of the minor groove resulting in a 50° bend within each operator site, together with a large expansion of the major groove in the centre of the DNA sequence. Cooperative binding between dimers governs the concentration-dependent activation–repression switch and arises, in part, from the interaction of Glu25 and Arg35 side chains at the dimer–dimer interface. Competition between Arg35 and an equivalent residue of the σ70 subunit of RNA polymerase for the Glu25 site underpins the switch from activation to repression of the endonuclease gene.

Immunological role of indoleamine 2,3‐dioxygenase in rat liver allograft rejection and tolerance

Indoleamine 2,3-dioxygenase (IDO) is expressed in the placenta and plays an essential role in maternal tolerance. Recent data showed that giving IDO inhibitor blocked liver allograft tolerance. However, the immunological role of IDO in rat liver allograft models has not been characterized. In the present study, the time-course of IDO expression and the localization of IDO were analyzed to address the role of IDO in the induction of tolerance. Rat orthotopic liver transplantations (OLT) were performed and IDO gene expression of OLT livers was analyzed. Immunohistochemistry was used to evaluate the localization of IDO-expressed cells in the liver. The IDO gene was detected in the allogeneic liver graft at the acute phase but the signal could not be detected when these OLT rats were treated with cyclosporinee A. The time-course of IDO gene expression in liver grafts of the spontaneous tolerant OLT model revealed that the IDO mRNA was expressed in both the rejection phase and the induction phase of tolerance, but the signal was gradually lowered during the maintenance phase of tolerance. Immunohistochemistry confirmed that the IDO protein was detected in antigen-presenting cells but not in hepatocytes. Our results demonstrated that IDO is induced in antigen-presenting cells of rat liver allografts under drug-free status, suggesting that indirect or direct recognition of donor antigen and further T-cell activation may be inhibited. IDO may act as a local immunosuppressive molecule to protect transplanted cells, tissues and organs from immune attack.