Highly competent, non-exhausted CD8+ T cells continue to tightly control pathogen load throughout chronic Trypanosoma cruzi infection.

Trypanosoma cruzi infection is characterized by chronic parasitism of non-lymphoid tissues and is rarely eliminated despite potent adaptive immune responses. This failure to cure has frequently been attributed to a loss or impairment of anti-T. cruzi T cell responses over time, analogous to the T cell dysfunction described for other persistent infections. In this study, we have evaluated the role of CD8+ T cells during chronic T. cruzi infection (>100 dpi), with a focus on sites of pathogen persistence. Consistent with repetitive antigen exposure during chronic infection, parasite-specific CD8+ T cells from multiple organs expressed high levels of KLRG1, but exhibit a preferential accumulation of CD69+ cells in skeletal muscle, indicating recent antigen encounter in a niche for T. cruzi persistence. A significant proportion of CD8+ T cells in the muscle also produced IFNγ, TNFα and granzyme B in situ, an indication of their detection of and functional response to T. cruzi in vivo. CD8+ T cell function was crucial for the control of parasite burden during chronic infection as exacerbation of parasite load was observed upon depletion of this population. Attempts to improve T cell function by blocking PD-1 or IL-10, potential negative regulators of T cells, failed to increase IFNγ and TNFα production or to enhance T. cruzi clearance. These results highlight the capacity of the CD8+ T cell population to retain essential in vivo function despite chronic antigen stimulation and support a model in which CD8+ T cell dysfunction plays a negligible role in the ability of Trypanosoma cruzi to persist in mice.

Characterization of Lamprey IL-17 Family Members and Their Receptors.

IL-17 is an ancient cytokine implicated in a variety of immune defense reactions. We identified five members of the sea lamprey IL-17 family (IL-17D.1, IL-17D.2, IL-17E, IL-17B, and IL-17C) and six IL-17R genes (IL-17RA.1, IL-17RA.2, IL-17RA.3, IL-17RF, IL-17RE/RC, and IL-17RD), determined their relationship with mammalian orthologs, and examined their expression patterns and potential interactions to explore their roles in innate and adaptive immunity. The most highly expressed IL-17 family member is IL-17D.1 (mammalian IL-17D like), which was found to be preferentially expressed by epithelial cells of skin, intestine, and gills and by the two types of lamprey T-like cells. IL-17D.1 binding to rIL-17RA.1 and to the surface of IL-17RA.1-expressing B-like cells and monocytes of lamprey larvae was demonstrated, and treatment of lamprey blood cells with rIL-17D.1 protein enhanced transcription of genes expressed by the B-like cells. These findings suggest a potential role for IL-17 in coordinating the interactions between T-like cells and other cells of the adaptive and innate immune systems in jawless vertebrates.

Molecular basis of lipo-chitooligosaccharide recognition by the lysin motif receptor-like kinase LYR3 in legumes.

LYR3 [LysM (lysin motif) receptor-like kinase 3] of Medicago truncatula is a high-affinity binding protein for symbiotic LCO (lipo-chitooligosaccharide) signals, produced by rhizobia bacteria and arbuscular mycorrhizal fungi. The present study shows that LYR3 from several other legumes, but not from two Lupinus species which are incapable of forming the mycorrhizal symbiosis, bind LCOs with high affinity and discriminate them from COs (chitooligosaccharides). The biodiversity of these proteins and the lack of binding to the Lupinus proteins were used to identify features required for high-affinity LCO binding. Swapping experiments between each of the three LysMs of the extracellular domain of the M. truncatula and Lupinus angustifolius LYR3 proteins revealed the crucial role of the third LysM in LCO binding. Site-directed mutagenesis identified a tyrosine residue, highly conserved in all LYR3 LCO-binding proteins, which is essential for high-affinity binding. Molecular modelling suggests that it may be part of a hydrophobic tunnel able to accommodate the LCO acyl chain. The lack of conservation of these features in the binding site of plant LysM proteins binding COs provides a mechanistic explanation of how LCO recognition might differ from CO perception by structurally related LysM receptors.

Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominant trans-Sialidase-Specific CD8+ T Cells.

Trypanosoma cruzi infection drives the expansion of remarkably focused CD8(+) T cell responses targeting epitopes encoded by variant trans-sialidase (TS) genes. Infection of C57BL/6 mice with T. cruzi results in up to 40% of all CD8(+) T cells committed to recognition of the dominant TSKB20 and subdominant TSKB18 TS epitopes. However, despite this enormous response, these mice fail to clear T. cruzi infection and subsequently develop chronic disease. One possible reason for the failure to cure T. cruzi infection is that immunodomination by these TS-specific T cells may interfere with alternative CD8(+) T cell responses more capable of complete parasite elimination. To address this possibility, we created transgenic mice that are centrally tolerant to these immunodominant epitopes. Mice expressing TSKB20, TSKB18, or both epitopes controlled T. cruzi infection and developed effector CD8(+) T cells that maintained an activated phenotype. Memory CD8(+) T cells from drug-cured TSKB-transgenic mice rapidly responded to secondary T. cruzi infection. In the absence of the response to TSKB20 and TSKB18, immunodominance did not shift to other known subdominant epitopes despite the capacity of these mice to expand epitope-specific T cells specific for the model antigen ovalbumin expressed by engineered parasites. Thus, CD8(+) T cell responses tightly and robustly focused on a few epitopes within variant TS antigens appear to neither contribute to, nor detract from, the ability to control T. cruzi infection. These data also indicate that the relative position of an epitope within a CD8(+) immunodominance hierarchy does not predict its importance in pathogen control.

Epidermal and cortical roles of NFP and DMI3 in coordinating early steps of nodulation in Medicago truncatula.

Legumes have evolved the capacity to form a root nodule symbiosis with soil bacteria called rhizobia. The establishment of this symbiosis involves specific developmental events occurring both in the root epidermis (notably bacterial entry) and at a distance in the underlying root cortical cells (notably cell divisions leading to nodule organogenesis). The processes of bacterial entry and nodule organogenesis are tightly linked and both depend on rhizobial production of lipo-chitooligosaccharide molecules called Nod factors. However, how these events are coordinated remains poorly understood. Here, we have addressed the roles of two key symbiotic genes of Medicago truncatula, the lysin motif (LysM) domain-receptor like kinase gene NFP and the calcium- and calmodulin-dependent protein kinase gene DMI3, in the control of both nodule organogenesis and bacterial entry. By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that epidermal DMI3, but not NFP, is sufficient for infection thread formation in root hairs. Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation, whereas DMI3 is required in both cell layers for these processes. Our results therefore suggest that a signal, produced in the epidermis under the control of NFP and DMI3, is responsible for activating DMI3 in the cortex to trigger nodule organogenesis. We integrate these data to propose a new model for epidermal/cortical crosstalk during early steps of nodulation.

Contested Boundaries: psychiatry, disease, and diagnosis.

Since the 19th century, we have come to think of disease in terms of specific entities--entities defined and legitimated in terms of characteristic somatic mechanisms. Since the last third of that century, we have expanded would-be disease categories to include an ever-broader variety of emotional pain, idiosyncrasy, and culturally unsettling behaviors. Psychiatry has been the residuary legatee of these developments, developments that have always been contested at the ever-shifting boundary between disease and deviance, feeling and symptom, the random and the determined, the stigmatized and the value-free. Even in our era of reductionist hopes, psychopharmaceutical practice, and corporate strategies, the legitimacy of many putative disease categories will remain contested. The use of the specific disease entity model will always be a reductionist means to achieve necessarily holistic ends, both in terms of cultural norms and the needs of suffering individuals. Bureaucratic rigidities and stakeholder conflicts structure and intensify such boundary conflicts, as do the interests and activism of an interested lay public.

Delayed Activation of T Cells at the Site of Infection Facilitates the Establishment of Trypanosoma cruzi in Both Naive and Immune Hosts.

Although parasite entry through breaks in the skin or mucosa is one of the main routes of natural transmission of Trypanosoma cruzi, little is known about the host cell types initially invaded nor the ability of those host cells to initiate immune responses at the site of infection. To gain insights into these early events, we studied the fate of fluorescently tagged T. cruzi delivered subcutaneously in mouse footpads or ears. We demonstrate that the majority of parasites introduced into the skin initially proliferate there until 8 to 10 days postinfection, when the parasite load decreases. This decline in parasite numbers is dependent on the presence of an intact T cell compartment and on the ability of hosts to produce gamma interferon (IFN-γ). Many of the parasite-containing cells at the initial infection site display a macrophage/monocyte phenotype but with low expression of activation markers, suggesting that these cells provide an early niche for T. cruzi proliferation, rather than being active in parasite control. It is only after the first round of T. cruzi replication and release from host cells that signs of immune activation and control of parasites become apparent. The delay in the activation and failure to rapidly control parasite replication are observed even when T. cruzi-primed T cells are present, such as in chronically infected mice. This failure of a primed immune system to recognize and react prior to extensive parasite expansion at the infection site likely poses a significant challenge for the development of vaccines aiming to prevent T. cruzi infection. IMPORTANCE Trypanosoma cruzi, the parasite causing Chagas disease, usually infects through the mucosa or breaks in the skin, but little is known about the parasite's fate at the site of entry or the early events involving immune control there. Here, we track the local proliferation and subsequent dissemination of fluorescently tagged T. cruzi and the initial immune response at the point of entry. We show that T. cruzi preferentially infects innate immune cells in the skin and that the stimulation of an adaptive T cell response does not occur until after the release of parasites from this first round of infected host cells. This first immunologically "silent" proliferation occurs even in the presence of a strong immune T cell memory generated by previous infection. This capacity of T. cruzi to establish infections while avoiding initial immune recognition has important implications for the potential to develop vaccines to prevent T. cruzi infection.

CD8+ T cells specific for immunodominant trans-sialidase epitopes contribute to control of Trypanosoma cruzi infection but are not required for resistance.

CD8(+) T cells are essential for controlling Trypanosoma cruzi infection. During Brazil strain infection, C57BL/6 mice expand parasite-specific CD8(+) T cells recognizing the dominant TSKB20 (ANYKFTLV) and subdominant TSKB74 (VNYDFTLV) trans-sialidase gene (TS)-encoded epitopes with up to 40% of all CD8(+) T cells specific for these epitopes. Although this is one of the largest immunodominant T cell responses described for any infection, most mice fail to clear T. cruzi and subsequently develop chronic disease. To determine if immunodominant TS-specific CD8(+) T cells are necessary for resistance to infection, we epitope-tolerized mice by high-dose i.v. injections of TSKB20 or TSKB74 peptides. Tolerance induction led to deletion of TS-specific CD8(+) T cells but did not prevent the expansion of other effector CD8(+) T cell populations. Mice tolerized against either TSKB20 or TSKB74, or both epitopes simultaneously, exhibited transient increases in parasite loads, although ultimately they controlled the acute infection. Furthermore, BALB/c mice tolerized against the TSKD14 peptide effectively controlled acute T. cruzi infection. These data are consistent with the hypothesis that development of high-frequency CD8(+) T cell populations focused on TS-derived epitopes contributes to optimal control of acute infection but is not required for the development of immune resistance.

Identification of Glycan-Specific Variable Lymphocyte Receptors Using Yeast Surface Display and Glycan Microarrays.

The jawless vertebrates (lamprey and hagfish) evolved a novel adaptive immune system with many similarities to that found in the jawed vertebrates, including the production of antigen-specific circulating antibodies in response to immunization. However, the jawless vertebrates use leucine-rich repeat (LRR)-based antigen receptors termed variable lymphocyte receptors (VLRs) for immune recognition, instead of immunoglobulin (Ig)-based receptors. VLR genes are assembled in developing lymphocytes through a gene conversion-like process, in which hundreds of LRR gene segments are randomly selected as template donors to generate a large repertoire of distinct antigen receptors, similar to that found within the mammalian adaptive immune system. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus) for generating libraries of anti-carbohydrate (anti-glycan) variable lymphocyte receptor B, or VLRBs. The anti-carbohydrate VLRBs are isolated using a yeast surface display (YSD) expression platform and enriched by binding to glycan microarrays through the anti-glycan VLRB. This enables both the initial identification and enrichment of individual yeast clones against hundreds of glycans simultaneously. Through this enrichment strategy a broad array of glycan-specific VLRs can be isolated from the YSD library. Subsequently, the bound yeast cells are directly removed from the microarray, the VLR antibody clone is sequenced, and the end product is expressed as a VLR-IgG-Fc fusion protein that can be used for ELISA, Western blotting, flow cytometry, and immunomicroscopy. Thus, by combining yeast surface display with glycan microarray technology, we have developed a rapid, efficient, and novel method for generating chimeric VLR-IgG-Fc proteins that recognize a broad array of unique glycan structures with exquisite specificity.

Development of smart anti-glycan reagents using immunized lampreys.

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. We identified a wide variety of glycan-specific VLRBs detectable in lamprey plasma after immunization with whole fixed cells, tissue homogenates, and human milk. The cDNAs from lamprey lymphocytes were cloned into yeast surface display (YSD) libraries for enrichment by multiple methods. We generated VLRB-Ig chimeras, termed smart anti-glycan reagents (SAGRs), whose specificities were defined by microarray analysis and immunohistochemistry. 15 VLRB antibodies were discovered that discriminated between linkages, functional groups and unique presentations of the terminal glycan motif. The development of SAGRs will enhance future studies on glycan expression by providing sequenced, defined antibodies for a variety of research applications.

CD8+ T-Cell responses to Trypanosoma cruzi are highly focused on strain-variant trans-sialidase epitopes.

CD8+ T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8+ T cells in T. cruzi-infected mice and humans. This analysis demonstrates that in both hosts the CD8+ T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi-infected mice, with more than 30% of the CD8+ T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level.

Development of a GAL4-VP16/UAS trans-activation system for tissue specific expression in Medicago truncatula.

Promoters with tissue-specific activity are very useful to address cell-autonomous and non cell autonomous functions of candidate genes. Although this strategy is widely used in Arabidopsis thaliana, its use to study tissue-specific regulation of root symbiotic interactions in legumes has only started recently. Moreover, using tissue specific promoter activity to drive a GAL4-VP16 chimeric transcription factor that can bind short upstream activation sequences (UAS) is an efficient way to target and enhance the expression of any gene of interest. Here, we developed a collection of promoters with different root cell layers specific activities in Medicago truncatula and tested their abilities to drive the expression of a chimeric GAL4-VP16 transcription factor in a trans-activation UAS: ß-Glucuronidase (GUS) reporter gene system. By developing a binary vector devoted to modular Golden Gate cloning together with a collection of adapted tissue specific promoters and coding sequences we could test the activity of four of these promoters in trans-activation GAL4/UAS systems and compare them to "classical" promoter GUS fusions. Roots showing high levels of tissue specific expression of the GUS activity could be obtained with this trans-activation system. We therefore provide the legume community with new tools for efficient modular Golden Gate cloning, tissue specific expression and a trans-activation system. This study provides the ground work for future development of stable transgenic lines in Medicago truncatula.

A rice calcium- and calmodulin-dependent protein kinase restores nodulation to a legume mutant.

The Medicago truncatula DMI3 gene encodes a calcium- and calmodulin-dependent protein kinase (CCaMK) that is necessary for the establishment of both rhizobial and mycorrhizal symbioses. The two symbiotic signaling pathways diverge downstream of DMI3; therefore, it has been proposed that legumes have evolved a particular form of CCaMK, acting like a switch able both to discriminate between rhizobial and mycorrhizal calcium signatures and to trigger the appropriate downstream signaling pathway. To test this hypothesis, we examined whether a CCaMK gene from a nonlegume species was able to restore the rhizobial symbiotic properties of a M. truncatula dmi3 mutant. Our results show that a CCaMK gene from rice can restore nodule formation, indicating that CCaMKs from nonlegumes can interpret the calcium signature elicited by rhizobial Nod factors and activate the appropriate downstream target. The nodules did not contain bacteria, which suggests that DMI3 is also involved in the control of the infection process.

On the history of medicine in the United States, theory, health insurance, and psychiatry: an interview with Charles Rosenberg.

An interview with Charles Rosenberg conducted by Rafael Mantovani in November 2013 that addressed four topics. It first focused on the way in which Rosenberg perceived trends and directions in historical research on medicine in the United States during the second half of the twentieth century. The second focus was on his experience with other important historians who wrote about public health. Thirdly, he discussed his impressions about the current debate on health policy in his country. Finally, the last part explores some themes related to psychiatry and behavior control that have appeared in a number of his articles.

LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.