Toxin Subunit Research Articles

Inhibitory and Injury-Protection Effects of O-Glycan on Gastric Epithelial Cells Infected with Helicobacter pylori.

ABSTRACTHelicobacter pylori (H. pylori) is an important pathogen that can cause gastric cancer. Multiple adhesion molecules mediated H. pylori adherence to cells is the initial step in the infection of host cells. H. pylori cholesterol-α-glucosyltransferase (CGT) recognizes and extracts cholesterol from cell membranes to destroy lipid raft structure, further promotes H. pylori adhesion to gastric epithelial cells. O-Glycan, a substance secreted by the deep gastric mucosa, can competitively inhibit CGT activity and may serve as an important factor to prevent H. pylori colonization in the deep gastric mucosa. However, the inhibitory and injury-protection effects of O-Glycan against H. pylori infection has not been well investigated. In this study, we found that O-Glycan significantly inhibited the relative urease content in the coinfection system. In the presence of O-glycan, the injury of GES-1 cells in H. pylori persistent infection model was attenuated and the cell viability was increased. We use fluorescein isothiocyanate-conjugated cholera toxin subunit B (FITC-CTX-B) to detect lipid rafts on gastric epithelial cells and observed that O-glycan can protect H. pylori from damaging lipid raft structures on cell membranes. In addition, transcriptome data showed that O-glycan treatment significantly reduced the activation of inflammatory cancer transformation pathway caused by H. pylori infection. Our results suggest that O-Glycan is able to inhibit H. pylori persistent infection of gastric epithelial cells, reduce the damage caused by H. pylori, and could serve as a potential medicine to treat patients infected with H. pylori.

COLOCALIZATION AND REMODELING OF HEART- AND LUNG-PROJECTING STELLATE GANGLION NEURONS IN BLEOMYCIN-INDUCED PULMONARY FIBROSIS

COLOCALIZATION AND REMODELING OF HEART- AND LUNG-PROJECTING STELLATE GANGLION NEURONS IN BLEOMYCIN-INDUCED PULMONARY FIBROSIS

A Multi-Specific DARPin Potently Neutralizes Shiga Toxin 2 via Simultaneous Modulation of Both Toxin Subunits.

Shiga toxin-producing E. coli (STEC) is a common cause of bloody diarrhea. The pathology of STEC infection derives from two exotoxins—Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2)—that are secreted by STEC in the gut, from where they are systemically absorbed, causing severe kidney damage leading to hemolytic uremic syndrome (HUS). Currently, there is no effective treatment for HUS, and only supportive care is recommended. We report the engineering of a panel of designed ankyrin repeat proteins (DARPin) with potent neutralization activity against Stx2a, the major subtype associated with HUS. The best dimeric DARPin, SD5, created via a combination of directed evolution and rational design, neutralizes Stx2a with a half maximal effective concentration (EC50) of 0.61 nM in vitro. The two monomeric DARPin constituents of SD5 exhibit complementary functions—SHT targets the enzymatic A subunit of Stx2a and inhibits the toxin’s catalytic activity, while DARPin #3 binds the B subunit, based on the cryo-EM study, and induces a novel conformational change in the B subunit that distorts its five-fold symmetry and presumably interferes with toxin attachment to target cells. SD5 was fused to an albumin-binding DARPin, and the resulting trimeric DARPin DA1-SD5 efficiently protects mice in a toxin challenge model, pointing to a high potential of this DARPin as a therapeutic for STEC infection. Finally, the unprecedented toxin conformational change induced by DARPin #3 represents a novel mode of action for neutralizing Stx2 toxicity and reveals new targets for future drug development.

CATR Tracing Approach to Identify Individual Intermediary Neurons Based on Their Input and Output: A Proof-of-Concept Study Connecting Cerebellum and Central Hubs Implicated in Developmental Disorders.

Over the past decades, it has become increasingly clear that many neurodevelopmental disorders can be characterized by aberrations in the neuro-anatomical connectome of intermediary hubs. Yet, despite the advent in unidirectional transsynaptic tracing technologies, we are still lacking an efficient approach to identify individual neurons based on both their precise input and output relations, hampering our ability to elucidate the precise connectome in both the healthy and diseased condition. Here, we bridge this gap by combining anterograde transsynaptic- and retrograde (cATR) tracing in Ai14 reporter mice, using adeno-associated virus serotype 1 expressing Cre and cholera toxin subunit B as the anterograde and retrograde tracer, respectively. We have applied this innovative approach to selectively identify individual neurons in the brainstem that do not only receive input from one or more of the cerebellar nuclei (CN), but also project to the primary motor cortex (M1), the amygdala or the ventral tegmental area (VTA). Cells directly connecting CN to M1 were found mainly in the thalamus, while a large diversity of midbrain and brainstem areas connected the CN to the amygdala or VTA. Our data highlight that cATR allows for specific, yet brain-wide, identification of individual neurons that mediate information from a cerebellar nucleus to the cerebral cortex, amygdala or VTA via a disynaptic pathway. Given that the identified neurons in healthy subjects can be readily quantified, our data also form a solid foundation to make numerical comparisons with mouse mutants suffering from aberrations in their connectome due to a neurodevelopmental disorder.

Designing of Chimeric Vaccine against Canine Distemper Virus Targeting Hemaglutanin Protein

The canine distemper virus is highly contagious and affects dogs' respiratory systems. The virus belongs to the paramyxoviridae family and order Mononegavirales. This class of viruses contains a negative-strand RNA. This virus also affects raccoons, foxes, and other animals. The current study aims to design a vaccine against the virus employing reverse vaccinology. The target candidate for the vaccine design is a surface protein called Hemagglutinin. Viral hemagglutinin protein sequences were retrieved from the Uniprot database, and conserved regions were identified. Possible B-cell epitope regions were identified using the ABCpred server. These epitopes were analyzed for allergenic and antigenic properties using the Allergen FP server and VaxiJen v2.0 server. The epitopes, which were antigenic and non-allergenic, were screened for T cell epitopes using NetMHC and NetMHC2 servers. The toxicity of the selected peptides was evaluated using the Toxinpred server. The epitopes were further screened for transmembrane helices and signal peptides employing TMHMM v. 2.0 and SignalP 4.1 servers, respectively. The epitopes were then checked for the parameters using the ProtParam tool. Finally, the solubility of the epitopes was determined using the SOLPro server. Using the selected epitopes, a chimeric vaccine construct was constructed with the peptides by linking the peptides with the GPGPG linker to the cholera toxin subunit B. The chimeric vaccine was modeled using the Robetta server, and codon optimization of the construct was performed using the JCAT tool.

Siderophore Immunization Restricted Colonization of Adherent-Invasive Escherichia coli and Ameliorated Experimental Colitis.

ABSTRACTInflammatory bowel diseases (IBD) are characterized by chronic inflammation of the gastrointestinal tract and profound alterations to the gut microbiome. Adherent-invasive Escherichia coli (AIEC) is a mucosa-associated pathobiont that colonizes the gut of patients with Crohn’s disease, a form of IBD. Because AIEC exacerbates gut inflammation, strategies to reduce the AIEC bloom during colitis are highly desirable. To thrive in the inflamed gut, Enterobacteriaceae acquire the essential metal nutrient iron by producing and releasing siderophores. Here, we implemented an immunization-based strategy to target the siderophores enterobactin and its glucosylated derivative salmochelin to reduce the AIEC bloom in the inflamed gut. Using chemical (dextran sulfate sodium) and genetic (Il10−/− mice) IBD mouse models, we showed that immunization with enterobactin conjugated to the mucosal adjuvant cholera toxin subunit B potently elicited mucosal and serum antibodies against these siderophores. Siderophore-immunized mice exhibited lower AIEC gut colonization, diminished AIEC association with the gut mucosa, and reduced colitis severity. Moreover, Peyer’s patches and the colonic lamina propria harbored enterobactin-specific B cells that could be identified by flow cytometry. The beneficial effect of siderophore immunization was primarily B cell-dependent because immunized muMT−/− mice, which lack mature B lymphocytes, were not protected during AIEC infection. Collectively, our study identified siderophores as a potential therapeutic target to reduce AIEC colonization and its association with the gut mucosa, which ultimately may reduce colitis exacerbation. Moreover, this work provides the foundation for developing monoclonal antibodies against siderophores, which could provide a narrow-spectrum strategy to target the AIEC bloom in Crohn’s disease patients.

Transplantation of NEP1-40 and NT-3 Gene-Co-Transduced Neural Stem Cells Improves Function and Neurogenesis after Spinal Cord Injury in a Rat Model.

Spinal cord injury (SCI) generally results in necrosis, scarring, cavitation, and a release of inhibitory molecules of the nervous system, which lead to disruption of neurotransmission and impede nerve fiber regeneration. This study was intended to evaluate the therapeutic efficacy rates of the transplantation of NEP1-40- and NT-3 gene-co-transduced neural stem cells (NSCs) in a rat model of SCI. Ninety Sprague-Dawley rats were subdivided randomly into six groups: sham-operated, SCI model, SCI + NSCs-NC, SCI + NEP1-40-NSCs, SCI + NT-3-NSCs, and SCI + NEP1-40/NT-3-NSCs. Motor function at different time points was evaluated using the Basso, Beattie, and Bresnahan locomotor activity scoring system (BBB). At 8 weeks post-transplantation, histological analysis, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, immunofluorescent assay, immunocytochemical staining, and cholera toxin subunit B (CTB) retrograde tracing were performed. BBB scores of the co-transduction group significantly surpassed those of other transplantation groups and of the SCI-model group after 2 weeks post-transplantation. The apoptotic rate of neurocytes was significantly lower in the co-transduction group than in other experimental groups. Expression of NF-200, MBP, and ChAT was significantly higher in the SCI + NEP1-40/NT-3-NSCs group than in other transplantation groups, whereas the expression of GFAP and GAD67 was the second lowest after the sham-operated group. CTB retrograde tracing showed that CTB-positive neural fibers on the caudal side of the hemisected site were more numerous in the SCI + NEP1-40/NT-3-NSCs group than in other experimental groups. Transplantation of NEP1-40- and NT-3-gene-co-transduced NSCs can modify the protein expression following acute SCI and promote neuron formation and axonal regeneration, thus having a neuroprotective effect. Furthermore, this effect surpasses that of transplantation of single-gene-transduced NSCs. Transplantation of NEP1-40- and NT-3-gene-co-transduced NSCs is effective at the neural recovery of the rat model of SCI and may be a novel strategy for clinical treatment of SCI.

Microglial infiltration mediates cognitive dysfunction in rat models of hypothalamic obesity via a hypothalamic-hippocampal circuit involving the lateral hypothalamic area.

This study aimed to explore the mechanism underlying cognitive dysfunction mediated by the lateral hypothalamic area (LHA) in a hypothalamic-hippocampal circuit in rats with lesion-induced hypothalamic obesity (HO). The HO model was established by electrically lesioning the hypothalamic nuclei. The open field (OP) test, Morris water maze (MWM), novel object recognition (NOR), and novel object location memory (NLM) tests were used to evaluate changes in cognition due to alterations in the hypothalamic-hippocampal circuit. Western blotting, immunohistochemical staining, and cholera toxin subunit B conjugated with Alexa Fluor 488 (CTB488) reverse tracer technology were used to determine synaptophysin (SYN), postsynaptic density protein 95 (PSD95), ionized calcium binding adaptor molecule 1 (Iba1), neuronal nuclear protein (NeuN), and Caspase3 expression levels and the hypothalamic-hippocampal circuit. In HO rats, severe obesity was associated with cognitive dysfunction after the lesion of the hypothalamus. Furthermore, neuronal apoptosis and activated microglia in the downstream of the lesion area (the LHA) induced microglial infiltration into the intact hippocampus via the LHA-hippocampal circuit, and the synapses engulfment in the hippocampus may be the underlying mechanism by which the remodeled microglial mediates memory impairments in HO rats. The HO rats exhibited microglial infiltration and synapse loss into the hippocampus from the lesioned LHA via the hypothalamic-hippocampal circuit. The underlying mechanisms of memory function may be related to the circuit.

Inter-Ligand STD NMR: An Efficient 1D NMR Approach to Probe Relative Orientation of Ligands in a Multi-Subsite Protein Binding Pocket.

In recent years, Saturation Transfer Difference NMR (STD NMR) has been proven to be a powerful and versatile ligand-based NMR technique to elucidate crucial aspects in the investigation of protein-ligand complexes. Novel STD NMR approaches relying on “multi-frequency” irradiation have enabled us to even elucidate specific ligand-amino acid interactions and explore the binding of fragments in previously unknown binding subsites. Exploring multi-subsite protein binding pockets is especially important in Fragment Based Drug Discovery (FBDD) to design leads of increased specificity and efficacy. We hereby propose a novel multi-frequency STD NMR approach based on direct irradiation of one of the ligands in a multi-ligand binding process, to probe the vicinity and explore the relative orientation of fragments in adjacent binding sub-sites, which we called Inter-Ligand STD NMR (IL-STD NMR). We proved its applicability on (i) a standard protein-ligand system commonly used for ligand-observed NMR benchmarking: Naproxen as bound to Bovine Serum Albumin, and (ii) the biologically relevant system of Cholera Toxin Subunit B and two inhibitors adjacently bound within the GM1 binding site. Relative to Inter-Ligand NOE (ILOE), the current state-of-the-art methodology to probe relative orientations of adjacent ligands, IL-STD NMR requires about one tenth of the experimental time and protein consumption, making it a competitive methodology with the potential to be applied in the pharmaceutical industries.

Small sensory spinal lesions that affect hand function in monkeys greatly alter primary afferent and motor neuron connections in the cord.

Small sensory spinal injuries induce plasticity across the neuraxis, but little is understood about their effect on segmental connections or motor neuron (MN) function. Here, we begin to address this at two levels. First, we compared afferent input distributions from the skin and muscles of the digits with corresponding MN pools to determine their spatial relationship, in both the normal state and 4-6 months after a unilateral dorsal root/dorsal column lesion (DRL/DCL), affecting digits 1-3. Second, we looked at specific changes to MN inputs and membrane properties that likely impact functional recovery. Monkeys received a targeted unilateral DRL/DCL, and 4-6 months later, cholera toxin subunit B (CT-B) was injected bilaterally into either the distal pads of digits 1-3, or related intrinsic hand muscles, to label inputs to the cord, and corresponding MNs. In controls (unlesioned side), cutaneous and proprioceptive afferents from digits 1-3 showed different distribution patterns but similar rostrocaudal spread within the dorsal horn from C1 to T2. In contrast, MNs were distributed across just two segments (C7-8). Following the lesion, sensory inputs were significantly diminished across all 10 segments, though this did not alter MN distributions. Afferent and monoamine inputs, as well as KCC2 cotransporters, were also significantly altered on the cell membrane of CT-B labeled MNs postlesion. In contrast, inhibitory neurotransmission and perineuronal net integrity were not altered at this prechronic timepoint. Our findings indicate that even a small sensory injury can significantly impact sensory and motor spinal neurons and provide new insight into the complex process of recovery.

Intranasal and Intramuscular Immunization with Outer Membrane Vesicles from Serogroup C Meningococci Induced Functional Antibodies and Immunologic Memory

ABSTRACT Background Immunization is the key to prevent invasive meningococcal disease (IMD), caused by Neisseria meningitidis. Outer membrane vesicles (OMVs) can be used as meningococcal antigens. Methods Isogenic mice A/Sn (H2a) were immunized with low antigenic doses of OMVs of an N. meningitidis C:2a:P1.5 strain, via intranasal/intramuscular route, adjuvanted by cholera toxin subunit B (CTB) or via intramuscular route only, adjuvanted by aluminium hydroxide (AH). Mice were followed until old age and humoral and cellular responses were assessed by ELISA, Immunoblotting, Dot-blot, Serum-bactericidal assay, Immunohistochemistry and ELISpot. Results OMV+CTB and OMV+AH groups presented statistically higher antibodies titers, which persisted until middle and old ages. IgG isotypes point to a Th2 type of response. Avidity indexes were considered high, regardless of adjuvant use, but only groups immunized with OMVs and adjuvants (OMV+CTB and OMV+AH) presented bactericidal activity. The antibodies recognized antigens of molecular weights attributed to porin and cross-reactivity proteins. Although the spleen of old mice did not present differences in immunohistochemistry marking of CD68+, CD4+, CD79+ and CD25+ cells, splenocytes of immune groups secreted IL-4 and IL-17 when stimulated with OMVs and meningococcal C polysaccharide. Conclusion We concluded that both adjuvants, CTB and AH, improved the immunogenicity of low doses of OMVs and contributed to a persistent immune response. Even though AH is well established in the vaccinology area, CTB seems to be a promising adjuvant candidate for meningococcal vaccines: it is suitable for mucosal delivery and supports a Th2 type of response. Therefore, OMVs are still a relevant vaccine platform.

Development and applications of sialoglycan-recognizing probes (SGRPs) with defined specificities: exploring the dynamic mammalian sialoglycome.

Glycans that are abundantly displayed on vertebrate cell surface and secreted molecules are often capped with terminal sialic acids (Sias). These diverse 9-carbon-backbone monosaccharides are involved in numerous intrinsic biological processes. They also interact with commensals and pathogens, while undergoing dynamic changes in time and space, often influenced by environmental conditions. However, most of this sialoglycan complexity and variation remains poorly characterized by conventional techniques, which often tend to destroy or overlook crucial aspects of Sia diversity and/or fail to elucidate native structures in biological systems, i.e. in the intact sialome. To date, in situ detection and analysis of sialoglycans has largely relied on the use of plant lectins, sialidases, or antibodies, whose preferences (with certain exceptions) are limited and/or uncertain. We took advantage of naturally evolved microbial molecules (bacterial adhesins, toxin subunits, and viral hemagglutinin-esterases) that recognize sialoglycans with defined specificity to delineate 9 classes of sialoglycan recognizing probes (SGRPs: SGRP1-SGRP9) that can be used to explore mammalian sialome changes in a simple and systematic manner, using techniques common in most laboratories. SGRP candidates with specificity defined by sialoglycan microarray studies were engineered as tagged probes, each with a corresponding nonbinding mutant probe as a simple and reliable negative control. The optimized panel of SGRPs can be used in methods commonly available in most bioscience labs, such as ELISA, western blot, flow cytometry, and histochemistry. To demonstrate the utility of this approach, we provide examples of sialoglycome differences in tissues from C57BL/6 wild-type mice and human-like Cmah-/- mice.

Top-down projections of the prefrontal cortex to the ventral tegmental area, laterodorsal tegmental nucleus, and median raphe nucleus.

Anatomical and functional evidence suggests that the PFC is fairly unique among all cortical regions, as it not only receives input from, but also robustly projects back to mesopontine monoaminergic and cholinergic cell groups. Thus, the PFC is in position to exert a powerful top-down control over several state-setting modulatory transmitter systems that are critically involved in the domains of arousal, motivation, reward/aversion, working memory, mood regulation, and stress processing. Regarding this scenario, the origin of cortical afferents to the ventral tegmental area (VTA), laterodorsal tegmental nucleus (LDTg), and median raphe nucleus (MnR) was here compared in rats, using the retrograde tracer cholera toxin subunit b (CTb). CTb injections into VTA, LDTg, or MnR produced retrograde labeling in the cortical mantle, which was mostly confined to frontal polar, medial, orbital, and lateral PFC subdivisions, along with anterior- and mid-cingulate areas. Remarkably, in all of the three groups, retrograde labeling was densest in layer V pyramidal neurons of the infralimbic, prelimbic, medial/ventral orbital and frontal polar cortex. Moreover, a lambda-shaped region around the apex of the rostral pole of the nucleus accumbens stood out as heavily labeled, mainly after injections into the lateral VTA and LDTg. In general, retrograde PFC labeling was strongest following injections into MnR and weakest following injections into VTA. Altogether, our findings reveal a fairly similar set of prefrontal afferents to VTA, LDTg, and MnR, further supporting an eminent functional role of the PFC as a controller of major state-setting mesopontine modulatory transmitter systems.

Elaboration of a Toxigenic Vibrio cholerae Typing Scheme Based on Bioinformatiсs Analysis Data

Introduction: The current stage of the seventh cholera pandemic is characterized by the emergence of novel Vibrio cholerae gene variants, gradually replacing their predecessors and occupying a dominant position in the etiology of the disease. Determining their epidemic potential by identifying the number of genetic markers is unsuitable for operational analysis. Thus, the development of a method for differentiating pathogens based on PCR detection of a limited number of markers seems relevant. Objective: To create a database of whole genome V. cholerae sequences containing different alleles of cheA3 (VCA1095) and rtxA genes based on bioinformatics analysis data and to elaborate a simple and informative toxigenic vibrio typing scheme. Materials and methods: The NCBI database-extracted results of whole genome sequencing of 3,309 strains of Vibrio cholerae isolated in 1962–2021 were used for the analysis. The software was developed in Java. Results: The bioinformatics analysis of the database of whole genome V. cholerae sequences, including 3,309 genomes of third wave strains, enabled us to divide them into three groups: “pre-Haitian”, “Haitian”, and “post-Haitian”. All of them contained alleles of the genes of toxin-co-regulated tcpACIRS101 pili and the MARTX rtxA4 cytotoxin with a null mutation that caused a premature stop codon. However, in the “pre-Haitian” strains, the gene of the cholera toxin subunit B of the classical ctxB1 type and the prototype gene of histidine kinase cheA3 (VCA1095) were always detected, which in PCR formed a 95 bp long amplicon and was designated as VCA1095-95. In the “Haitian” strains, a deletion of 8 bp occurred in this gene, and the PCR amplicon was shortened to 87 bp (VCA1095-87). Its mandatory combination with the ctxB7 allele was revealed. The “post-Haitian” strains contained an even shorter rtxA4a allele due to the deletion of 60 bp in the proximal part. Conclusion: Since the analysis of a large number of genomes revealed strict correlations between certain alleles in each group, we consider it possible to use only two markers for operational analysis, i.e. alleles of the cheA3 and rtxA genes. The typing scheme based on their PCR detection can be used to facilitate determination of the epidemic potential of newly isolated cultures.

Ingenious Action of Vibrio cholerae Neuraminidase Recruiting Additional GM1 Cholera Toxin Receptors for Primary Human Colon Epithelial Cells.

For five decades it has been known that the pentamer of B subunits (choleragenoid) of the cholera toxin (CT) of Vibrio cholerae binds with high preference to the ganglioside GM1 (II3Neu5Ac-Gg4Cer). However, the exact structures of CT-binding GM1 lipoforms of primary human colon epithelial cells (pHCoEpiCs) have not yet been described in detail. The same holds true for generating further GM1 receptor molecules from higher sialylated gangliosides with a GM1 core through the neuraminidase of V. cholerae. To avoid the artificial incorporation of exogenous gangliosides from animal serum harboring GM1 and higher sialylated ganglio-series gangliosides, pHCoEpiCs were cultured in serum-free medium. Thin-layer chromatography overlay binding assays using a choleragenoid combined with electrospray ionization mass spectrometry revealed GM1 lipoforms with sphingosine (d18:1) as the sole sphingoid base linked to C14:0, C16:0, C18:0 or C20:0 fatty acyl chains forming the ceramide (Cer) moieties of the main choleragenoid-binding GM1 species. Desialylation of GD1a (IV3Neu5Ac,II3Neu5Ac-Gg4Cer) and GT1b (IV3Neu5Ac,II3(Neu5Ac)2-Gg4Cer) of pHCoEpiCs by V. cholerae neuraminidase was observed. GD1a-derived GM1 species with stable sphingosine (d18:1) and saturated fatty acyl chains varying in chain length from C16:0 up to C22:0 could be identified, indicating the ingenious interplay between CT and the neuraminidase of V. cholerae recruiting additional GM1 receptors of pHCoEpiCs.

Effect of electroacupuncture on ocular surface sensory neuralgia and expression of P2X3 receptor and PKC in cornea and trigeminal ganglion of dry eye guinea pigs

To observe the effect of electroacupuncture (EA) on ocular surface sensory neuralgia and the expression of P2X3 receptor (P2X3R) and protein kinase C(PKC)in cornea and trigeminal ganglion (TG) in dry eye disease (DED) guinea pigs, so as to explore its mechanism underlying improvement of ocular surface sensory neuralgia in DED. Male British tricolor short haired guinea pigs were randomly divided into control, model, medication (pranoprofen), EA and sham acupuncture groups, with 8 guinea pigs in each group. The dry eye model was induced by subcutaneous injection of scopolamine hydrobromide solution (0.6 mg/0.2 mL,once daily) for 10 d. Guinea pigs in the medication group were treated by applying pranoprofen eye drops to eyes, 1 drop for one eye each time, three times a day. Guinea pigs of the EA group received EA stimulation (4 Hz/20 Hz,1 mA) of bilateral "Cuanzhu" (BL2) and "Taiyang" (HN5) and acupuncture at "Jingming" (BL1) "Sizhukong" (TE23), "Tongziliao" (GB1) for 15 min, once a day. Guinea pigs in the sham acupuncture group received blunt stimu-lation at the surface of the same acupoint with the tip of the acupuncture needle, once a day. All the treatments were conducted for 14 d. The corneal epithelium fluorescein staining score (0-3 points) was given according to the number of fluorescence-positive dots and flake-like coloration, the corneal mechanical perception thread (CMPT) detected using a corneal perception meter, and the palpebral fissure height measured. The number of sensory neurons in the cornea and TG was determined by using cholera toxin subunit B conjugated with Alexa Fluor 488 fluorescence labelling, and the expression levels of P2X3R and PKC in the cornea and TG detected by using immunohistochemistry and Western blot, separately. Compared with the control group, the corneal fluorescein staining score, immunoactivity and expression of P2X3R proteins in both cornea and TG, PKC proteins in TG were significantly increased (P<0.01), whereas the CMPT and the height of palpebral fissure and the number of TG neurons significantly decreased in the model group (P<0.05,P<0.01). In comparison with the model group, the fluorescein staining score in the medication and EA groups, the immunoactivity and expression of P2X3R in cornea and TG in the EA group, and that of TG PKC in the EA group and the sham acupuncture groups were significantly decreased (P<0.05, P<0.01), while the height of palpebral fissure and CMPT after EA and the number of labelling TG sensory neurons were remarkably increased in the EA group (P<0.01) rather than in the medication and sham acupuncture groups (P>0.05). EA can alleviate the damage of corneal epithelium and sensory neurons in dry eye model guinea pigs, which may be related to its functions in down-regu-lating the expression of P2X3R and PKC in the cornea and TG.

Recombinant BCG-LTAK63 Vaccine Candidate for Tuberculosis Induces an Inflammatory Profile in Human Macrophages.

Tuberculosis (TB) is one of the top 10 leading causes of death worldwide. The recombinant BCG strain expressing the genetically detoxified A subunit of the thermolabile toxin from Escherichia coli (LTAK63) adjuvant (rBCG-LTAK63) has previously been shown to confer superior protection and immunogenicity compared to BCG in a murine TB infection model. To further investigate the immunological mechanisms induced by rBCG-LTAK63, we evaluated the immune responses induced by rBCG-LTAK63, BCG, and Mycobacterium tuberculosis (Mtb) H37Rv strains in experimental infections of primary human M1 and M2 macrophages at the transcriptomic and cytokine secretion levels. The rBCG-LTAK63-infected M1 macrophages more profoundly upregulated interferon-inducible genes such as IFIT3, OAS3, and antimicrobial gene CXCL9 compared to BCG, and induced higher levels of inflammatory cytokines such as IL-12(p70), TNF-β, and IL-15. The rBCG-LTAK63-infected M2 macrophages more extensively upregulated transcripts of inflammation-related genes, TAP1, GBP1, SLAMF7, TNIP1, and IL6, and induced higher levels of cytokines related to inflammation and tissue repair, MCP-3 and EGF, as compared to BCG. Thus, our data revealed an important signature of immune responses induced in human macrophages by rBCG-LTAK63 associated with increased inflammation, activation, and tissue repair, which may be correlated with a protective immune response against TB.

How, where and when is SPINK3 bound and removed from mouse sperm?

Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein-lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.

Interleukin-6 signaling requires EHD1-mediated alteration of membrane rafts.

Interleukin-6 (IL-6) is involved in many inflammatory diseases. IL-6 binds to membrane-bound IL-6 receptor α (IL-6Rα) (classic signaling) or soluble IL-6Rα (trans-signaling); this complex then associates with the signal-transducing membrane protein gp130. IL-6Rα and gp130 float on membrane (i.e., lipid) rafts; however, how membrane rafts regulate IL-6 signaling remains unclear. Here, we demonstrate that both IL-6 classic signaling and trans-signaling depend on membrane cholesterol, an essential raft component. Super-resolution fluorescence imaging using perfringolysin O D4 fragments that selectively bind to high cholesterol concentrations revealed that IL-6 and hyper-IL-6, a fusion protein of IL-6 and soluble IL-6Rα, induce the alteration of membrane rafts. IL-6 and hyper-IL-6 induced D4-positive raft (D4 raft) formation without affecting cholera toxin subunit B (CTB)-positive rafts (CTB rafts). Receptor clustering of IL-6Rα and gp130 and STAT3 phosphorylation occurred in D4 rafts. These results indicate that D4 rafts serve as platforms for the assembly of functional IL-6 receptor complexes. We found that Eps15 homology domain-containing protein 1 (EHD1) mediates the formation of functional IL-6 receptor complexes through D4 rafts. Overall, we uncover a novel regulatory mechanism of the EHD1-mediated alteration of membrane raft in IL-6 signaling.

Oral MucoRice-CTB vaccine is safe and immunogenic in healthy US adults

MucoRice-CTB is a promising cold-chain-free oral cholera vaccine candidate. Here, we report a double-blind, randomized, placebo-controlled, phase I study conducted in the USA in which vaccination with the 6-g dose of MucoRice-CTB induced cross-reactive antigen-specific antibodies against the B subunit of cholera toxin (CTB) and enterotoxigenic Escherichia coli heat-labile enterotoxin without inducing serious adverse events. This dosage was acceptably safe and tolerable in healthy men and women. In addition, it induced a CTB-specific IgA response in the saliva of two of the nine treated subjects; in one subject, the immunological kinetics of the salivary IgA were similar to those of the serum CTB-specific IgA. Antibodies from three of the five responders to the vaccine prevented CTB from binding its GM1 ganglioside receptor. These results are consistent with those of the phase I study in Japan, suggesting that oral MucoRice-CTB induces neutralizing antibodies against diarrheal toxins regardless of ethnicity.