Sec22b-dependent antigen cross-presentation is a significant contributor of T cell priming during infection with the parasite Trypanosoma cruzi.

Antigen cross-presentation is a vital mechanism of dendritic cells and other antigen presenting cells to orchestrate the priming of cytotoxic responses towards killing of infected or cancer cells. In this process, exogenous antigens are internalized by dendritic cells, processed, loaded onto MHC class I molecules and presented to CD8+ T cells to activate them. Sec22b is an ER-Golgi Intermediate Compartment resident SNARE protein that, in partnership with sintaxin4, coordinates the recruitment of the transporter associated with antigen processing protein and the peptide loading complex to phagosomes, where antigenic peptides that have been proteolyzed in the cytosol are loaded in MHC class I molecules and transported to the cell membrane. The silencing of Sec22b in dendritic cells primary cultures and conditionally in dendritic cells of C57BL/6 mice, critically impairs antigen cross-presentation, but neither affects other antigen presentation routes nor cytokine production and secretion. Mice with Sec22b conditionally silenced in dendritic cells (Sec22b-/-) show deficient priming of CD8+ T lymphocytes, fail to control tumor growth, and are resistant to anti-checkpoint immunotherapy. In this work, we show that Sec22b-/- mice elicit a deficient specific CD8+ T cell response when challenged with sublethal doses of Trypanosoma cruzi trypomastigotes that is associated with increased blood parasitemia and diminished survival.

Intranasal trans-sialidase-based vaccine against Trypanosoma cruzi triggers a mixed cytokine profile in the nasopharynx-associated lymphoid tissue and confers local and systemic immunogenicity.

Trypanosoma cruzi, the agent of Chagas disease, can infect through conjunctive or oral mucosas. Therefore, the induction of mucosal immunity by vaccination is relevant not only to trigger local protection but also to stimulate both humoral and cell-mediated responses in systemic sites to control parasite dissemination. In a previous study, we demonstrated that a nasal vaccine based on a Trans-sialidase (TS) fragment plus the mucosal STING agonist c-di-AMP, was highly immunogenic and elicited prophylactic capacity. However, the immune profile induced by TS-based nasal vaccines at the nasopharyngeal-associated lymphoid tissue (NALT), the target site of nasal immunization, remains unknown. Hence, we analyzed the NALT cytokine expression generated by a TS-based vaccine plus c-di-AMP (TSdA+c-di-AMP) and their association with mucosal and systemic immunogenicity. The vaccine was administered intranasally, in 3 doses separated by 15 days each other. Control groups received TSdA, c-di-AMP, or the vehicle in a similar schedule. We demonstrated that female BALB/c mice immunized intranasally with TSdA+c-di-AMP boosted NALT expression of IFN-γ and IL-6, as well as IFN-ß and TGF-ß. TSdA+c-di-AMP increased TSdA-specific IgA secretion in the nasal passages and also in the distal intestinal mucosa. Moreover, T and B-lymphocytes from NALT-draining cervical lymph nodes and spleen showed an intense proliferation after ex-vivo stimulation with TSdA. Intranasal administration of TSdA+c-di-AMP provokes an enhancement of TSdA-specific IgG2a and IgG1 plasma antibodies, accompanied by an increase IgG2a/IgG1 ratio, indicative of a Th1-biased profile. In addition, immune plasma derived from TSdA+c-di-AMP vaccinated mice exhibit in-vivo and ex-vivo protective capacity. Lastly, TSdA+c-di-AMP nasal vaccine also promotes intense footpad swelling after local TSdA challenge. Our data support that TSdA+c-di-AMP nasal vaccine triggers a NALT mixed pattern of cytokines that were clearly associated with an evident mucosal and systemic immunogenicity. These data are useful for further understanding the immune responses elicited by the NALT following intranasal immunization and the rational design of TS-based vaccination strategies for prophylaxis against T. cruzi.

Trypanosoma cruzi, Chagas disease and cancer: putting together the pieces of a complex puzzle.

Considering the extensive and widespread impact on individuals, cancer can presently be categorized as a pandemic. In many instances, the development of tumors has been linked to endemic microbe infections. Among parasitic infections, Trypanosoma cruzi stands out as one of the most extensively discussed protozoans in the literature that explores the association between diseases of parasite origin and cancer. However, the effective association remains an unsolved paradox. Both the parasite, along with protozoan-derived molecules, and the associated antiparasitic immune response can induce alterations in various host cell pathways, leading to modifications in cell cycle, metabolism, glycosylation, DNA mutations, or changes in neuronal signaling. Furthermore, the presence of the parasite can trigger cell death or a senescent phenotype and modulate the immune system, the metastatic cascade, and the formation of new blood vessels. The interaction among the parasite (and its molecules), the host, and cancer undoubtedly encompasses various mechanisms that operate differentially depending on the context. Remarkably, contrary to expectations, the evidence tilts the balance toward inhibiting tumor growth or resisting tumor development. This effect is primarily observed in malignant cells, rather than normal cells, indicating a selective or specific component. Nevertheless, nonspecific bystander mechanisms, such as T. cruzi's adjuvancy or the presence of proinflammatory cytokines, may also play a significant role in this phenomenon. This work aims to elucidate this complex scenario by synthesizing the main findings presented in the literature and by proposing new questions and answers, thereby adding pieces to this challenging puzzle.

Immunization With Lipopolysaccharide-Activated Dendritic Cells Generates a Specific CD8+ T Cell Response That Confers Partial Protection Against Infection With Trypanosoma cruzi.

Lipopolysaccharide (LPS) induces the activation of dendritic cells (DCs) throughout the engagement of toll-like receptor 4. LPS-activated DCs show increased capacity to process and present pathogen-derived antigens to activate naïve T cells. DCs-based vaccines have been successfully used to treat some cancer types, and lately transferred to the field of infectious diseases, in particular against HIV. However, there is no vaccine or DC therapy for any parasitic disease that is currently available. The immune response against Trypanosoma cruzi substantially relies on T cells, and both CD4+ and CD8+ T lymphocytes are required to control parasite growth. Here, we develop a vaccination strategy based on DCs derived from bone marrow, activated with LPS and loaded with TsKb20, an immunodominant epitope of the trans-sialidase family of proteins. We extensively characterized the CD8+ T cell response generated after immunization and compared three different readouts: a tetramer staining, ELISpot and Activation-Induced Marker (AIM) assays. To our knowledge, this work shows for the first time a proper set of T cell markers to evaluate specific CD8+ T cell responses in mice. We also show that our immunization scheme confers protection against T. cruzi, augmenting survival and reducing parasite burden in female but not male mice. We conclude that the immunization with LPS-activated DCs has the potential to prime significant CD8+ T cell responses in C57BL/6 mice independently of the sex, but this response will only be effective in female, possibly due to mice sexual dimorphisms in the response generated against T. cruzi.

Nasal immunization with a L. lactis-derived trans-sialidase antigen plus c-di-AMP protects against acute oral T. cruzi infection.

The new generation of vaccines for Chagas disease, are focused to induce both humoral and cellular response to effectively control Trypanosoma cruzi parasites. The administration of vaccine formulations intranasally has the advantage over parenteral routes that can induce a specific response at mucosal and systemic levels. This study aimed to evaluate and compare the immunogenicity and prophylactic effectiveness of two Trans-sialidase (TS)-based mucosal vaccines against T. cruzi administered intranasally. Vaccines consisted of a recombinant fragment of TS expressed in Lactococcus lactis formulated in two different adjuvants. The first, was an immunostimulant particle (ISPA, an ISCOMATRIX-like adjuvant), while the second was the dinucleotide c-di-AMP, which have shown immunostimulant properties at the mucosal level. BALB/c mice were immunized intranasally (3 doses, one every two weeks) with each formulation (TS + ISPA or TS + c-di-AMP) and with TS alone or vehicle (saline solution) as controls. Fifteen days after the last immunization, both TS + ISPA or TS + c-di-AMP induced an evident systemic humoral and cellular response, as judged by the increased plasma anti-TS IgG2a titers and IgG2a/IgG1 ratio and enhanced cellular response against TS. Plasma derived antibodies from TS + c-di-AMP also inhibit in vitro the invasion capacity of T. cruzi. Furthermore, specific secretory IgA was more enhanced in TS + c-di-AMP group. Protective efficacy was proved in vaccinated animals by an oral T. cruzi-challenge. Parasitemia control was only achieved by animals vaccinated with TS + c-di-AMP, despite all vaccinates groups showed enhanced CD8+IFN-γ+ T cell numbers. In addition, it was reflected during the acute phase in a significant reduction of tissue parasite load, clinical manifestations and diminished tissue damage. The better prophylactic capacity elicited by TS + c-di-AMP was related to the induction of neutralizing plasma antibodies and augmented levels of mucosal IgA since TS + ISPA and TS + c-di-AMP groups displayed similar immunogenicity and CD8+IFN-γ+ T-cell response. Therefore, TS + c-di-AMP formulation appears as a promising strategy for prophylaxis against T. cruzi.

New strategies in ion channel screening for drug discovery: are there ways to improve its productivity?

INTRODUCTION: From a drug discovery point of view, ion channels are very interesting and challenging targets. Over the past decade, great efforts have been made in developing platforms for patch clamp-based high-quality screening of ion channels in discovering new drug candidates as well for evaluating their safety profiles. Indeed, the automated patch clamp (APC) has recently reached the data throughput requirements of high-throughput screening (HTS) allowing for new screening strategies with ion channel active compounds. AREAS COVERED: This editorial article comments on the past and present developments of APC-based drug screening. Furthermore, it also looks at the implications of APC technology meeting HTS-standards as well as its use in compound safety evaluation. EXPERT OPINION: In the imminent future, we will see a paradigm shift in ion channel drug screening toward using APC-based platforms for primary drug library screens. This way, the redundancy of the drug discovery process and the risk of false-negatives could be drastically reduced. Furthermore, cardiac safety can be addressed early, avoiding late-phase withdrawals with promising drug candidates. It is our firm belief that APC-based ion channel HTS will facilitate the discovery of candidates, which otherwise would have not been found, and shorten the drug development cycle, saving time and cost.

HTS techniques for patch clamp-based ion channel screening - advances and economy.

INTRODUCTION: Ten years ago, the first publication appeared showing patch clamp recordings performed on a planar glass chip instead of using a conventional patch clamp pipette. "Going planar" proved to revolutionize ion channel drug screening as we know it, by allowing high quality measurements of ion channels and their effectors at a higher throughput and at the same time de-skilling the highly laborious technique. Over the years, platforms evolved in response to user requirements regarding experimental features, data handling plus storage, and suitable target diversity. AREAS COVERED: This article gives a snapshot image of patch clamp-based ion channel screening with focus on platforms developed to meet requirements of high-throughput screening environments. The commercially available platforms are described, along with their benefits and drawbacks in ion channel drug screening. EXPERT OPINION: Automated patch clamp (APC) platforms allow faster investigation of a larger number of ion channel active compounds or cell clones than previously possible. Since patch clamp is the only method allowing direct, real-time measurements of ion channel activity, APC holds the promise of picking up high quality leads, where they otherwise would have been overseen using indirect methods. In addition, drug candidate safety profiling can be performed earlier in the drug discovery process, avoiding late-phase compound withdrawal due to safety liability issues, which is highly costly and inefficient.

Insulin-secreting INS-1E cells express functional TRPV1 channels.

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for ß-cells, and in primary ß-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca (2+) concentration ([Ca (2+)]i) in the INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca (2+)]i in a concentration-dependent manner, and the [Ca (2+)]i increase was dependent on extracellular Ca (2+). AM404 also increased [Ca (2+)]i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca (2+)]i increases. Capsaicin did not increase [Ca (2+)]i in the primary ß-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary ß-cells, express functional TRPV1 channels.

Port-a-patch and patchliner: high fidelity electrophysiology for secondary screening and safety pharmacology.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the world's smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanion's higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Planar patch clamp: advances in electrophysiology.

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes.

Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.

Automated ion channel screening: patch clamping made easy.

Efficient high resolution techniques are required for screening efforts and research targeting ion channels. The conventional patch clamp technique, a high resolution but low efficiency technique, has been established for 25 years. Recent advances have opened up new possibilities for automated patch clamping. This new technology meets the need of drug developers for higher throughput and facilitates new experimental approaches in ion channel research. Specifically, Nanion's electrophysiology workstations, the Port-a-Patch and the Patchliner, have been successfully introduced as high-quality automated patch clamp platforms for industry as well as academic users. Both platforms give high quality patch clamp recordings, capable of true giga-seals and stable recordings, accessible to the user without the need for years of practical training. They also offer sophisticated experimental possibilities, such as accurate and fast ligand application, temperature control and internal solution exchange. This article describes the chip-based patch clamp technology and its usefulness in ion channel drug screening and academic research.

Controlling desensitized states in ligand-receptor interaction studies with cyclic scanning patch-clamp protocols.

Ligand-gated ion channels are important control elements in regulation of cellular activities, and increasing evidence demonstrates their role as therapeutic targets. The receptors display complex desensitization kinetics, occurring on vastly different time scales. This is not only important in biology and pharmacology but might also be of technological significance since populations of receptors under microfluidic control can function analogously to DRAM memory circuits. Using a novel microfluidic method, and computer modeling of the receptor state distributions, we here demonstrate that GABAA receptor populations can be controlled to display high or low EC50 values, depending on input function (i.e., the exact pattern of agonist application). The sensitivity of the receptors can be tuned up to 40-fold (beta-alanine) by the particular agonist exposure pattern. By combining patch-clamp experiments with computer modeling of receptor state distributions, we can control the assembly of receptors in desensitized states. The technique described can be used as an analytical tool to study the effect of desensitization on the activity of ion channel effectors. We describe the differential blocking effect of the competitive antagonist bicuculline on the high- and low-EC50 GABAA receptor preparations and conclude that the inhibition is dramatically dependent on how the different desensitized states are populated. Furthermore, we show that both GABA and beta-alanine, two agonists with different affinity but similar efficacy, induce the same type of desensitization behavior and memory effects in GABAA receptors.

Contribution of AMPA and NMDA receptors to early and late phases of LTP in hippocampal slices.

Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor mediated responses were investigated in rat hippocampal slices under 4h of long-term potentiation (LTP) expression. A modified medium containing the NMDA receptor antagonist AP5 and low concentration of Mg(2+) was used to monitor isolated AMPA responses. NMDA components were determined from composite excitatory postsynaptic potentials (EPSPs) under brief (15-20 min) wash-out of AP5. LTP was induced in a medium with low concentration of AP5, resulting in an about two-fold larger increase of the AMPA component than of the NMDA component at both 1h and 4h after induction. Similar results were obtained if LTP was induced in "normal Mg(2+)" and the NMDA components were assessed at the end of experiment, from either composite or isolated NMDA EPSPs, with or without blockade of GABAergic inhibition. It is generally believed that LTP undergoes biochemical and/or structural conversions during the first few hours. Our study, however, shows constant expression of LTP, at least in terms of AMPA versus NMDA components, during this time. The data support the notion that LTP initiates as a predominant amplification of AMPA receptors and remains so for at least 4h.

A biohybrid dynamic random access memory.

We report that GABA(A) receptors in a patch-clamped biological cell form a short-term memory circuit when integrated with a scanning-probe microfluidic device. Laminar patterns of receptor activators (agonists) provided by the microfluidic device define and periodically update the data input which is read and stored by the receptors as state distributions (based on intrinsic multistate kinetics). The memory is discharged over time and lasts for seconds to minutes depending on the input function. The function of the memory can be represented by an equivalent electronic circuit with striking similarity in function to a dynamic random access memory (DRAM) used in electronic computers. Multiplexed biohybrid memories may form the basis of large-scale integrated biocomputational/sensor devices with the curious ability to use chemical signals including odorants, neurotransmitters, chemical and biological warfare agents, and many more as input signals.

Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation.

A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased at a high voltage. Specifically, alkaline phosphatase and proteases were detected in single NG108-15 cells using fluorescein diphosphate and casein BODIPY FL, respectively. The intracellular 1,4,5-inositol triphosphate (IP3) and ryanodine receptors were detected after EFC introduction of the selective receptor agonists IP3 and cyclic adenosine diphosphate ribose (cADPr), respectively. Receptor activation in both cases resulted in increased cytosolic concentrations of free calcium ions that were measured using the calcium-ion-selective probe, fluo-3. The effect of cADPr could be blocked by coadministration of the ryanodine receptor antagonist ruthenium red. Furthermore, electroporation of a plurality of cells grown in microwell structures (100 x 100 x 45 microm) molded in PDMS is demonstrated. The methods and systems described using an EFC for electroporation and delivery of protein markers, ligands, and substrates might be useful in high-throughput screening of intracellular targets, with applications in proteomics and phenotype profiling, as well as in drug discovery.