The nucleus of the solitary tract (NTS) regulates life‐sustaining functions ranging from appetite and digestion to heart rate and breathing. It is also the brain's primary sensory nucleus for ...visceral sensations relevant to symptoms in medical and psychiatric disorders. To better understand which neurons may exert top‐down control over the NTS, here we provide a brain‐wide map of all neurons that project axons directly to the caudal, viscerosensory NTS, focusing on a medial subregion with aldosterone‐sensitive HSD2 neurons. Injecting an axonal tracer (cholera toxin b) into the NTS produces a similar pattern of retrograde labeling in rats and mice. The paraventricular hypothalamic nucleus (PVH), lateral hypothalamic area, and central nucleus of the amygdala (CeA) contain the densest concentrations of NTS‐projecting neurons. PVH afferents are glutamatergic (express Slc17a6/Vglut2) and are distinct from neuroendocrine PVH neurons. CeA afferents are GABAergic (express Slc32a1/Vgat) and are distributed largely in the medial CeA subdivision. Other retrogradely labeled neurons are located in a variety of brain regions, including the cerebral cortex (insular and infralimbic areas), bed nucleus of the stria terminalis, periaqueductal gray, Barrington's nucleus, Kölliker‐Fuse nucleus, hindbrain reticular formation, and rostral NTS. Similar patterns of retrograde labeling result from tracer injections into different NTS subdivisions, with dual retrograde tracing revealing that many afferent neurons project axon collaterals to both the lateral and medial NTS subdivisions. This information provides a roadmap for studying descending axonal projections that may influence visceromotor systems and visceral “mind–body” symptoms.
Rats and mice have similar patterns of retrogradely labeled neurons that project axons to the NTS.
Afferent neurons in the hypothalamus are glutamatergic, while those in the amygdala are GABAergic.
In the paraventricular hypothalamic nucleus, NTS afferent neurons are distinct from neuroendocrine neurons.
Injecting retrograde tracers into the medial and lateral NTS subdivisions produce similar patterns of retrograde labeling.
Macromolecular drugs inefficiently cross membranes to reach their cytosolic targets. They require drug delivery vectors to facilitate their translocation across the plasma membrane or escape from ...endosomes. Optimization of these vectors has however been hindered by the difficulty to accurately measure cytosolic arrival. We have developed an exceptionally sensitive and robust assay for the relative or absolute quantification of this step. The assay is based on benzylguanine and biotin modifications on a drug delivery vector of interest, which allow, respectively, for selective covalent capture in the cytosol with a SNAP‐tag fusion protein and for quantification at picomolar sensitivity. The assay was validated by determining the absolute numbers of cytosolic molecules for two drug delivery vectors: the B‐subunit of Shiga toxin and the cell‐penetrating peptide TAT. We expect this assay to favor delivery vector optimization and the understanding of the enigmatic translocation process.
Macromolecular drug delivery vectors must translocate at the plasma membrane or escape from endosomes to reach the cytosol. This very inefficient process requires optimization, which is hindered by the difficulty to accurately measure cytosolic arrival. An exceptionally sensitive and robust assay to quantify this step was developed. The assay was validated by determining the absolute numbers of two macromolecular vectors reaching the cytosol.
Abstract
Objectives
FMF is the most frequent autoinflammatory disease and is associated in most patients with bi-allelic MEFV mutations. MEFV encodes Pyrin, an inflammasome sensor activated following ...RhoGTPase inhibition. The functional consequences of MEFV mutations on the ability of Pyrin variants to act as inflammasome sensors are largely unknown. The aim of this study was to assess whether MEFV mutations affect the ability of Pyrin to detect RhoGTPase inhibition and other inflammasome stimuli.
Methods
IL-1β and IL-18 released by monocytes from healthy donors (HDs) and FMF patients were measured upon specific engagement of the Pyrin, NLRP3 and NLRC4 inflammasomes. Cell death kinetics following Pyrin activation was monitored in real time.
Results
Monocytes from FMF patients secreted significantly more IL-1β and IL-18 and died significantly faster than HD monocytes in response to low concentrations of Clostridium difficile toxin B (TcdB), a Pyrin-activating stimulus. Monocytes from patients bearing two MEFV exon 10 pathogenic variants displayed an increased Pyrin inflammasome response compared with monocytes from patients with a single exon 10 pathogenic variant indicating a gene-dosage effect. Using a short priming step, the response of monocytes from FMF patients to NLRP3- and NLRC4-activating stimuli was normal indicating that MEFV mutations trigger a specific hypersensitivity of monocytes to low doses of a Pyrin-engaging stimulus.
Conclusion
Contrary to the NLRP3 mutations described in cryopyrin-associated periodic syndrome, FMF-associated MEFV mutations do not lead to a constitutive activation of Pyrin. Rather, FMF-associated mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome without affecting other canonical inflammasomes.
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Peptide-based vaccines have the potential to overcome the limitations of classical vaccines; however, their use is hampered by a lack of carriers and adjuvants suitable for human use. ...In this study, an efficient self-adjuvanting peptide vaccine delivery system was developed based on the ionic interactions between cationic trimethyl chitosan (TMC) and a peptide antigen coupled with synthetically defined anionic α-poly-(l-glutamic acid) (PGA). The antigen, possessing a conserved B-cell epitope derived from the group A streptococcus (GAS) pathogen and a universal T-helper epitope, was conjugated to PGA using cycloaddition reaction. The produced anionic conjugate formed nanoparticles (NP-1) through interaction with cationic TMC. These NP-1 induced higher systemic and mucosal antibody titers compared to antigen adjuvanted with standard mucosal adjuvant cholera toxin B subunit or antigen mixed with TMC. The produced serum antibodies were also opsonic against clinically isolated GAS strains. Further, a reduction in bacterial burden was observed in nasal secretions, pharyngeal surface and nasopharyngeal-associated lymphoid tissue of mice immunized with NP-1 in GAS challenge studies. Thus, conjugation of defined-length anionic polymer to peptide antigen as a means of formulating ionic interaction-based nanoparticles with cationic polymer is a promising strategy for peptide antigen delivery.
A self-adjuvanting delivery system is required for peptide vaccines to enhance antigen delivery to immune cells and generate systemic and mucosal immunity. Herein, we developed a novel self-adjuvanting nanoparticulate delivery system for peptide antigens by combining polymer-conjugation and complexation strategies. We conjugated peptide antigen with anionic α-poly-(l-glutamic acid) that in turn, formed nanoparticles with cationic trimethyl chitosan by ionic interactions, without using external crosslinker. On intranasal administration to mice, these nanoparticles induced systemic and mucosal immunity, at low dose. Additionally, nanoparticles provided protection to vaccinated mice against group A streptococcus infection. Thus, this concept should be particularly useful in developing nanoparticles for the delivery of peptide antigens.
induces antibiotic-associated diarrhea due to the release of toxin A (TcdA) and toxin B (TcdB), the latter being its main virulence factor. The epidemic strain NAP1/027 has an increased virulence ...attributed to different factors. We compared cellular intoxication by TcdB
with that by the reference strain VPI 10463 (TcdB
). In a mouse ligated intestinal loop model, TcdB
induced higher neutrophil recruitment, cytokine release, and epithelial damage than TcdB
. Both toxins modified the same panel of small GTPases and exhibited similar in vitro autoprocessing kinetics. On the basis of sequence variations in the frizzled-binding domain (FBD), we reasoned that TcdB
and TcdB
might have different receptor specificities. To test this possibility, we used a TcdB from a NAP1 variant strain (TcdB
) unable to glucosylate RhoA but with the same receptor-binding domains as TcdB
. Cells were preincubated with TcdB
to block cellular receptors, prior to intoxication with either TcdB
or TcdB
. Preincubation with TcdB
blocked RhoA glucosylation by TcdB
but not by TcdB
, indicating that the toxins use different host factors for cell entry. This crucial difference might explain the increased biological activity of TcdB
in the intestine, representing a contributing factor for the increased virulence of the NAP1/027 strain.
Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how ...they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.
•The retrograde tracer CTB labeled PL, IL, and vHPC efferents to the hamster amygdala.•Dominants have elevated c-Fos in the PL and IL during acute social defeat.•Dominants activate PL and IL neural ...projections to the amygdala during social defeat.
Stress is a contributing factor in the etiology of several mood and anxiety disorders, and social defeat models are used to investigate the biological basis of stress-related psychopathologies. Male Syrian hamsters are highly aggressive and territorial, but after social defeat they exhibit a conditioned defeat (CD) response which is characterized by increased submissive behavior and a failure to defend their home territory against a smaller, non-aggressive intruder. Hamsters with dominant social status show increased c-Fos expression in the infralimbic (IL) cortex following social defeat and display a reduced CD response at testing compared to subordinates and controls. In this study, we tested the prediction that dominants would show increased defeat-induced neural activity in IL, but not prelimbic (PL) or ventral hippocampus (vHPC), neurons that send efferent projections to the basolateral amygdala (BLA) compared to subordinates. We performed dual immunohistochemistry for c-Fos and cholera toxin B (CTB) and found that dominants display a significantly greater proportion of double-labeled c-Fos + CTB cells in both the IL and PL. Furthermore, dominants display more c-Fos-positive cells in both the IL and PL, but not vHPC, compared to subordinates. These findings suggest that dominant hamsters selectively activate IL and PL, but not vHPC, projections to the amygdala during social defeat, which may be responsible for their reduced CD response. This project extends our understanding of the neural circuits underlying resistance to social stress, which is an important step toward delineating a circuit-based approach for the prevention and treatment of stress-related psychopathologies.
Clostridium difficile is an opportunistic pathogen that establishes in the colon when the gut microbiota are disrupted by antibiotics or disease. C. difficile infection (CDI) is largely caused by two ...virulence factors, TcdA and TcdB. Here, we report a 3.87-Å-resolution crystal structure of TcdB holotoxin that captures a unique conformation of TcdB at endosomal pH. Complementary biophysical studies suggest that the C-terminal combined repetitive oligopeptides (CROPs) domain of TcdB is dynamic and can sample open and closed conformations that may facilitate modulation of TcdB activity in response to environmental and cellular cues during intoxication. Furthermore, we report three crystal structures of TcdB-antibody complexes that reveal how antibodies could specifically inhibit the activities of individual TcdB domains. Our studies provide novel insight into the structure and function of TcdB holotoxin and identify intrinsic vulnerabilities that could be exploited to develop new therapeutics and vaccines for the treatment of CDI.
The laminar and topographical organization of connections between the hippocampal formation and parahippocampal regions was investigated in the rabbit following in vivo injection of cholera toxin B ...subunit as a retro‐ and antero‐grade tracer and biotinylated dextran amine as an anterograde tracer. We confirmed several connectional features different from those of the rat, that is, the rabbit presubiculum received abundant afferents from CA1 and had many reciprocal connections with the entorhinal cortex. On the other hand, we identified many similarities with the rat: both the CA1 and subicular afferents that originated from the entorhinal cortex were abundant; moreover, the presubiculum received many inputs from the subiculum and sent massive projections to the entorhinal cortex. By plotting retrograde and anterograde labels in two‐dimensional unfolded maps of the entire hippocampal and parahippocampal regions, we found that each group of entorhinal cells that project to CA1, subiculum, and presubiculum, and also the termination of the presubiculo‐entorhinal projection, was distributed in band‐like zones in layers II–III, extending across the medial and lateral entorhinal cortex. Our results suggest that the rabbit has a basic connectivity that is common with that of the rat, and also has additional hippocampal–presubicular and entorhino–presubicular connections that may reflect functional evolution in learning and memory.
We identified several connectional features different from those of the rat, that is, the rabbit presubiculum received abundant afferents from CA1 and had many reciprocal connections with the entorhinal cortex. Our results suggest that the rabbit has basic connectivity in common with the rat (black arrows), and also has additional hippocampal‐parahippocampal connections (blue arrows).
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