Cholera Toxin B Research Articles

Spinally projecting noradrenergic neurons of the locus coeruleus display resistance to AAV2retro-mediated transduction.

BackgroundThe locus coeruleus (LC) is the principal source of noradrenaline (NA) in the central nervous system. Projection neurons in the ventral portion of the LC project to the spinal cord and are considered the main source of spinal NA. To understand the precise physiology of this pathway, it is important to have tools that allow specific genetic access to these descending projections. AAV2retro serotype vectors are a potential tool to transduce these neurons via their axon terminals in the spinal cord, and thereby limit the expression of genetic material to the spinal projections from the LC. Here, we assess the suitability of AAV2retro to target these neurons and investigate strategies to increase their labelling efficiency.ResultsWe show that the neurons in the LC that project to the spinal dorsal horn are largely resistant to transduction with AAV2retro serotype vectors. Compared to Cholera toxin B (CTb) tracing, AAV2retro.eGFP labelled far fewer neurons within the LC and surrounding regions, particularly within neurons that express tyrosine hydroxylase (TH), the rate-limiting enzyme for NA synthesis. We also show that the sensitivity for transduction of this projection can be increased using AAV2retro.eGFP.cre in ROSA26tdTom reporter mice (23% increase), with a higher proportion of the newly revealed neurons expressing TH compared to those directly labelled with AAV2retro containing an eGFP expression sequence.ConclusionThese tracing studies identify limitations in AAV2retro-mediated retrograde transduction of a subset of projection neurons, specifically those that express NA and project to the spinal cord. This is likely to have implications for the study of NA-containing projections as well as other types of projection neuron in the central nervous system.

Governor Vessel Electro-Acupuncture Promotes the Intrinsic Growth Ability of Spinal Neurons through Activating Calcitonin Gene-Related Peptide/α-Calcium/Calmodulin-Dependent Protein Kinase/Neurotrophin-3 Pathway after Spinal Cord Injury.

Spinal cord injury (SCI) invariably results in neuronal death and failure of axonal regeneration. This is attributed mainly to the hostile microenvironment and the poor intrinsic regrowth capacity of the injured spinal neurons. We have reported previously that electro-acupuncture on Governor Vessel acupoints (GV-EA) can promote neuronal survival and axonal regeneration of injured spinal cord. However, the underlying mechanism for this has remained uncertain. The present study aimed to explore the neural afferent pathway of GV-EA stimulation and the possible mechanism by which GV-EA can activate the intrinsic growth ability of injured spinal neurons. By cholera toxin B (CTB) retrograde labeling, immunostaining, and enzyme-linked immunosorbent assay (ELISA), we showed here that GV-EA could stimulate the spinal nerve branches of the dorsal root ganglion cells. This would then increase the release of calcitonin gene-related peptide (CGRP) from the afferent terminals in the spinal cord. It is of note that the effect was abrogated after dorsal rhizotomy. Additionally, both in vivo and in vitro results showed that CGRP would act on the post-synaptic spinal cord neurons and triggered the synthesis and secretion of neurotrophin-3 (NT-3) by activating the calcitonin gene-related peptide (CGRP)/ receptor activity-modifying protein (RAMP)1/calcium/calmodulin-dependent protein kinase (αCaMKII) pathway. Remarkably, the observed effect was prevented by the dorsal rhizotomy and the blockers of the CGRP/RAMP1/αCaMKII pathway. More importantly, increase in NT-3 promoted the survival, axonal regrowth, and synaptic maintenance of spinal cord neurons in the injured spinal cord. Therefore, it is concluded that increase in NT-3 production is one of the mechanisms by which GV-EA can activate the intrinsic growth ability of spinal neurons after SCI. The experimental results have reinforced the theoretical basis of GV-EA for its clinical efficacy in patients with SCI.

Frontal cortex stroke-induced impairment in spatial working memory on the trial-unique nonmatching-to-location task in mice

Stroke-induced cognitive impairments are of significant concern, however mechanisms that underpin these impairments remain poorly understood and researched. To further characterise cognitive impairments in our frontal cortex stroke model, and to align our assessments with what is used clinically, we tested young C57BL/6J mice trained in operant touchscreen chambers to complete the trial-unique nonmatched-to-location (TUNL) task. Based on baseline performance, animals were given either stroke (n = 12) or sham (n = 12) surgery using a photothrombosis model, bilaterally targeting the frontal cortex. Upon recovery, post-stroke spatial working memory was assessed by varying the degree of separation and delay within TUNL trials. Seven weeks after surgery, animals received a prelimbic injection of the retrograde tracer cholera toxin B (CTB) to access thalamo-PFC connectivity. Tissue was then processed histologically and immunohistochemically to assess infarct volume, astrogliosis and thalamocortical connectivity. Assessment of TUNL probes revealed sensitivity to a frontal cortex stroke (separation: p = 0.0003, delay: p < 0.0001), with stroke animals taking significantly longer (p = 0.0170) during reacquisition of the TUNL task, relative to shams. CTB-positive cell counts revealed a stroke-induced loss of thalamo-PFC connectivity. In addition, quantification of reactive astrogliosis revealed a positive correlation between the degree of astrogliosis expanding into white matter tracts and the development of cognitive impairments. This study reveals a stroke-induced impairment in mice completing the TUNL task. Our findings also demonstrate a significant loss of thalamo-PFC connections and a correlation between white matter reactive astrogliosis and cognitive impairment. Future experiments will investigate therapeutic interventions in the hope of promoting functional improvement in cognition.

Alternative Approaches to Oral Tolerance Induction to Factor FVIII

A serious complication in the treatment X-linked bleeding disorder hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), which compromise traditional replacement therapy. We previously developed an Oral immunotherapy (OTI) based on repeated uptake of a mixture of lettuce plant cells transgenic for heavy chain (HC) or C2 domain of human FVIII fused to cholera toxin B (CTB) subunit [Blood 124:1659; Plant Biotechnol J. 16:1148]. Fusion proteins were transgenically expressed in the chloroplasts. Repeated oral uptake of a mixture of freeze-dried powder of lettuce cells accomplished antigen delivery to the immune system of the small intestine by targeting of the GM1 receptor that is highly expressed on the surface of the gut epithelium, resulting in induction of regulatory T cells (Treg) that suppress inhibitor formation upon subsequent intravenous (iv) FVIII replacement therapy. An alternative to oral antigen delivery is the oral delivery of immune modulatory antibodies. Here, we compared the plant cell-based method with oral delivery of anti-CD3, which has been successful in murine models of autoimmune disease and is currently evaluated in clinical trials. Unlike in iv administration, oral anti-CD3 does not systemically deplete T cells. Hemophilia A BALB/c mice (F8 e16 gene deletion) received oral gavage of a mixture of CTB-FVIII-HC/-C2 (1.5 µg/antigen) expressing lettuce leaf cells 2x/week for 9 weeks. Starting at 4 weeks into the experiment, 1 IU/mouse of BDD-FVIII (Xyntha) was given iv, once per week for 5 weeks. Alternatively, following a published protocol that was successful in other models, 5 µg of hamster anti-murine CD3 was given by oral gavage daily for 5 straight days, followed by 5 weekly iv injections of BDD-FVIII. Control animals (no OTI) developed inhibitors with an average titer of 18 ± 3 BU/ml (n=16). Of these, 88% were high-titer (i.e &amp;gt;5 BU/ml, up to 43 BU/ml). Inhibitor formation was significantly reduced in plant cell-treated mice (10 ± 2.5 BU/ml, n=17), with 47% showing no or low-titer inhibitors (&amp;lt;5 BU/ml). Interestingly, inhibitor formation in anti-CD3 treated mice showed a unique pattern, with two distinct outcomes. One subset of animals developed inhibitors of 0-15 BU/ml (on average 2.5-fold lower than control group), while the second subset showed increased titers of 41-89 BU/ml. There were no animals with intermediate titers. In general, FVIII-specific IgG titers followed the same patterns as inhibitor responses. When the 2 approaches (plant cells and anti-CD3) were combined, 50% of mice developed &amp;lt;5 BU/ml (as compared to 36% for anti-CD3 only). However, this did not represent an improvement over giving lettuce cells only. Finally, we tested anti-CD3 Fab fragment in place of FL anti-CD3. While a daily dose of 5 µg anti-CD3 Fab/mouse was unsuccessful in suppressing inhibitor formation, a dose of 0.5 µg marginally decrease titers. In summary, oral anti-CD3 delivery was not as effective in reducing inhibitor formation as plant cells expressing CTB-FVIII antigen fusions. While FL anti-CD3 showed promise in a subset of animals, it also posed a risk of an increased response in others, which complicates development of this approach. Distinct from all other treatment groups, lettuce-fed mice showed elevated frequencies in Foxp3+ Treg, a reduction in Teff cells, and the highest increase in LAP+ Treg. Further studies on induced CD4+LAP+FoxP3- Treg cells from lettuce-fed mice showed high expression of ICOS, CD69 and Ki-67, but not CTLA-4. Among the different subsets of Treg, LAP+ T cell contained the highest proportion of IL-10 expressing cells, and oral antigen delivery induced IL-10+LAP+ Treg, which are likely key to the suppression of the immune response against FVIII. Disclosures Daniell: Takeda Pharmaceuticals: Patents &amp; Royalties. Herzog:Takeda Pharmaceuticals: Patents &amp; Royalties.

Age-Related Alterations in Prelimbic Cortical Neuron Arc Expression Vary by Behavioral State and Cortical Layer.

Prefrontal cortical and medial temporal lobe connectivity is critical for higher cognitive functions that decline in older adults. Likewise, these cortical areas are among the first to show anatomical, functional, and biochemical alterations in advanced age. The prelimbic subregion of the prefrontal cortex and the perirhinal cortex of the medial temporal lobe are densely reciprocally connected and well-characterized as undergoing age-related neurobiological changes that correlate with behavioral impairment. Despite this fact, it remains to be determined how changes within these brain regions manifest as alterations in their functional connectivity. In our previous work, we observed an increased probability of age-related dysfunction for perirhinal cortical neurons that projected to the prefrontal cortex in old rats compared to neurons that were not identified as projection neurons. The current study was designed to investigate the extent to which aged prelimbic cortical neurons also had altered patterns of Arc expression during behavior, and if this was more evident in those cells that had long-range projections to the perirhinal cortex. The expression patterns of the immediate-early gene Arc were quantified in behaviorally characterized rats that also received the retrograde tracer cholera toxin B (CTB) in the perirhinal cortex to identify projection neurons to this region. As in our previous work, the current study found that CTB+ cells were more active than those that did not have the tracer. Moreover, there were age-related reductions in prelimbic cortical neuron Arc expression that correlated with a reduced ability of aged rats to multitask. Unlike the perirhinal cortex, however, the age-related reduction in Arc expression was equally likely in CTB+ and CTB− negative cells. Thus, the selective vulnerability of neurons with long-range projections to dysfunction in old age may be a unique feature of the perirhinal cortex. Together, these observations identify a mechanism involving prelimbic-perirhinal cortical circuit disruption in cognitive aging.

Activity of a vmPFC-DRN Pathway Corresponds With Resistance to Acute Social Defeat Stress.

The ventromedial prefrontal cortex (vmPFC) plays a critical role in stress resilience through top-down inhibition of key stress-sensitive limbic and hindbrain structures, including the dorsal raphe nucleus (DRN). In a model of experience-dependent stress resistance, socially dominant Syrian hamsters display fewer signs of anxiety following acute social defeat when compared to subordinate or control counterparts. Further, dominants activate vmPFC neurons to a greater degree during stress than do subordinates and become stress-vulnerable following pharmacological inhibition of the vmPFC. Dominants also display fewer stress-activated DRN neurons than subordinates do, suggesting that dominance experience gates activation of vmPFC neurons that inhibit the DRN during social defeat stress. To test whether social dominance alters stress-induced activity of a vmPFC-DRN pathway, we injected a retrograde tracer, cholera toxin B (CTB), into the DRN of dominant, subordinate, and control hamsters and used a dual-label immunohistochemical approach to identify vmPFC neurons co-labeled with CTB and the defeat-induced expression of an immediate early gene, cFos. Results indicate that dominant hamsters display more cFos+ and dual-labeled cells in layers V/VI of infralimbic and prelimbic subregions of the vmPFC compared to other animals. Furthermore, vmPFC-DRN activation corresponded directly with proactive behavioral strategies during defeat, which is indicative of stress resilience. Together, results suggest that recruiting the vmPFC-DRN pathway during acute stress corresponds with resistance to the effects of social defeat in dominant hamsters. Overall, these findings indicate that a monosynaptic vmPFC-DRN pathway can be engaged in an experience-dependent manner, which has implications for behavioral interventions aimed at alleviating stress-related psychopathologies.

Pim-1 Protects Retinal Ganglion Cells by Enhancing Their Regenerative Ability Following Optic Nerve Crush.

Provirus integration site Moloney murine leukemia virus (Pim-1) is a proto-oncogene reported to be associated with cell proliferation, differentiation and survival. This study was to explore the neuroprotective role of Pim-1 in a rat model subjected to optic nerve crush (ONC), and discuss its related molecules in improving the intrinsic regeneration ability of retinal ganglion cells (RGCs). Immunofluorescence staining showed that AAV2-Pim-1 infected 71% RGCs and some amacrine cells in the retina. Real-time PCR and Western blotting showed that retina infection with AAV2-Pim-1 up-regulated the Pim-1 mRNA and protein expressions compared with AAV2-GFP group. Hematoxylin-Eosin (HE) staining, γ-synuclein immunohistochemistry, Cholera toxin B (CTB) tracing and TUNEL showed that RGCs transduction with AAV2-Pim-1 prior to ONC promoted the survival of damaged RGCs and decreased cell apoptosis. RITC anterograde labeling showed that Pim-1 overexpression increased axon regeneration and promoted the recovery of visual function by pupillary light reflex and flash visual evoked potential. Western blotting showed that Pim-1 overexpression up-regulated the expression of Stat3, p-Stat3, Akt1, p-Akt1, Akt2 and p-Akt2, as well as βIII-tubulin, GAP-43 and 4E-BP1, and downregulated the expression of SOCS1 and SOCS3, Cleaved caspase 3, Bad and Bax. These results demonstrate that Pim-1 exerted a neuroprotective effect by promoting nerve regeneration and functional recovery of RGCs. In addition, it enhanced the intrinsic regeneration capacity of RGCs after ONC by activating Stat3, Akt1 and Akt2 pathways, and inhibiting the mitochondrial apoptosis pathways. These findings suggest that Pim-1 may prove to be a potential therapeutic target for the clinical treatment of optic nerve injury.

BDNF in Phrenic Motor Neurons: Effects of Cervical Spinal Injury and Intermittent Hypoxia

Cervical spinal cord injury (cSCI) compromises breathing. Since respiratory impairment increases mortality, there is a critical need for new treatments to enhance breathing function after cSCI. One emerging strategy to enhance breathing function after cSCI is to elicit spinal respiratory plasticity via exposure to acute intermittent hypoxia (AIH). Brain derived neurotrophic factor (BDNF) is both necessary and sufficient for AIH‐induced phrenic motor plasticity. Repetitive AIH exposure enhances BDNF expression within phrenic motor neurons in spinally intact rats, but its effects after cSCI are not well known. In contrast to the beneficial effects of AIH, chronic intermittent hypoxia (CIH), similar to that experienced during sleep apnea elicits neuropathology (including inflammation) that may undermine plasticity. The balance between the therapeutic vs pathogenic effects of intermittent hypoxia is a matter of “dose.” Since the impact of various intermittent hypoxia protocols on phrenic motor neuron BDNF expression in phrenic motor neurons has not been investigated, we tested the hypothesis that BDNF expression in phrenic motor neurons is enhanced by repetitive AIH, but not CIH, in spinally intact and injured rats. BDNF expression was assessed in the phrenic motor neurons of male rats with and without C2 hemisection (C2Hx; 12 wks post‐injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min 10.5% O2 episodes per day; 5‐min normoxic intervals); 3) mild CIH (5‐min 10.5% O2 episodes, 5‐min intervals; 8 hrs/day); and 4) moderate CIH (2‐min 10.5% O2 episodes, 2‐min intervals; 8 hrs/day). Rats received intrapleural Cholera toxin B (CtB) injections 14 days before C2Hx to label phrenic motor neurons. After the study, spinal cords were harvested and BDNF expression assessed via immunofluorescence using a custom MATLAB algorithm to determine BDNF optical density within identified phrenic motor neurons and in a region of interest (50 mm radius) around the center of gravity of CtB labeled neurons. Spinally intact and injured rats have differential responses to acute and chronic intermittent hypoxia. Whereas spinally intact rats exposed to daily AIH increase BDNF expression within and around phrenic motor neurons; there were no effects of mild or moderate CIH. In contrast, BDNF within and around phrenic motor neurons in spinal cord injured rats was unaffected by daily AIH, and was highest after moderate CIH. Thus, whereas only daily AIH elicits neurochemical plasticity in intact rats, similar effects were observed only with moderate CIH after C2Hx. These data support the idea that the “injured spinal cord is a new spinal cord,” and highlight the potential of intermittent hypoxia preconditioning to enhance the capacity for BDNF‐dependent phrenic motor plasticity and (possibly) functional recovery after cSCI.Support or Funding InformationAPS HEARST (JRO), SPARC (OT2OD023854) NIH T32 HD043730 (LLA) and HL134621 (MNK), NIH K12 HD055929 (EGR) and the UF McKnight Brain Institute

Epigenetic Mechanisms Underlying Organic Solute Transporter β Repression in Colorectal Cancer.

Colorectal cancer (CRC) is known to be the third most common cancer disease and the fourth-leading cause of cancer-related deaths worldwide. Bile acid, especially deoxycholic acid and lithocholic acid, were revealed to play an important role during carcinogenesis of CRC. In this study, we found organic solute transporter β (OSTβ), an important subunit of a bile acid export transporter OSTα-OSTβ, was noticeably downregulated in CRC. The decline of OSTβ expression in CRC was determined by Western blot and real-time polymerase chain reaction (RT-PCR), whereas chromatin immunoprecipitation (ChIP) was used to evaluate the histone acetylation state at the OSTβ promoter region in vivo and in vitro. CRC cell lines HT29 and HCT15 were treated with trichostation A (TSA) for the subsequent determination, including RT-PCR, small interfering RNA (siRNA) knockdown, ChIP, and dual-luciferase reporter gene assay, to find out which histone acetyltransferases and deacetylases exactly participated in regulation. We demonstrated that after TSA treatment, OSTβ expression increased noticeably because of upregulated H3K27Ac state at OSTβ promoter region. We found that stimulating the expression of p300 with CTB (Cholera Toxin B subunit, an activator of p300) and inhibiting p300 expression with C646 (an inhibitor of p300) or siRNA designed for p300 could control OSTβ expression through modulating H3K27Ac state at OSTβ promoter region. Therefore, downregulated expression of p300 in CRC may cause low expression of OSTβ in CRC via epigenetic regulation. Generally, we revealed a novel epigenetic mechanism underlying OSTβ repression in CRC, hoping this mechanism would help us to understand and inhibit carcinogenesis of CRC. SIGNIFICANCE STATEMENT: Organic solute transporter β (OSTβ) expression is lower in colon cancer tissues compared with adjacent normal tissues. We revealed the epigenetic mechanisms of it and proved that p300 controls OSTβ expression through modulating H3K27Ac state at OSTβ promoter region and hence causes low expression of OSTβ in colorectal cancer.

Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration.

In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.

Immunization of rabbits with recombinant Clostridium perfringens alpha toxins CPA-C and CTB-CPA-C in a bicistronic design expression system confers strong protection against challenge

The Clostridium perfringens alpha toxin (CPA), encoded by the plc gene, is the causative pathogen of gas gangrene, which is a lethal infection. In this study, we used an E. coli system for the efficient production of recombinant proteins and developed a bicistronic design (BCD) expression construct consisting of two copies of the C-terminal (247–370) domain of the alpha toxin (CPA-C) in the first cistron, followed by Cholera Toxin B (CTB) linked with another two copies of CPA-C in the second cistron that is controlled by a single promoter. Rabbits were immunized twice with purified proteins (rCPA-C rCTB-CPA-C) produced in the BCD expression system, with an inactivated recombinant E. coli vaccine (RE), C. perfringens formaldehyde-inactivated alpha toxoid (FA-CPA) and C. perfringensl-lysine/formaldehyde alpha toxoid (LF-CPA) vaccines. Following the second vaccination, 0.1 mL of pooled sera of the RE-vaccinated rabbits could neutralize 12× mouse LD100 (100% lethal dose) of CPA, while that of the rCPA-C rCTB-CPA-C-vaccinated rabbits could neutralize 6× mouse LD100 of CPA. Antibody titers against CPA were also assessed by ELISA, reaching titers as high as 1:2048000 in the RE group; this was significantly higher compared to the C. perfringens alpha toxoid vaccinated groups (FA-CPA and LF-CPA). Rabbits from all vaccinated groups were completely protected from a 2× rabbit LD100 of CPA challenge. These results demonstrate that the recombinant proteins are able to induce a strong immune responses, indicating that they may be potentially utilized as targets for novel vaccines specifically against the C. perfringens alpha toxin.

Super-Resolution Microscopy Reveals Local Accumulation of Plasma Membrane Gangliosides at Neisseria meningitidis Invasion Sites.

Neisseria meningitidis (meningococcus) is a Gram-negative bacterium responsible for epidemic meningitis and sepsis worldwide. A critical step in the development of meningitis is the interaction of bacteria with cells forming the blood-cerebrospinal fluid barrier, which requires tight adhesion of the pathogen to highly specialized brain endothelial cells. Two endothelial receptors, CD147 and the β2-adrenergic receptor, have been found to be sequentially recruited by meningococci involving the interaction with type IV pilus. Despite the identification of cellular key players in bacterial adhesion the detailed mechanism of invasion is still poorly understood. Here, we investigated cellular dynamics and mobility of the type IV pilus receptor CD147 upon treatment with pili enriched fractions and specific antibodies directed against two extracellular Ig-like domains in living human brain microvascular endothelial cells. Modulation of CD147 mobility after ligand binding revealed by single-molecule tracking experiments demonstrates receptor activation and indicates plasma membrane rearrangements. Exploiting the binding of Shiga (STxB) and Cholera toxin B (CTxB) subunits to the two native plasma membrane sphingolipids globotriaosylceramide (Gb3) and raft-associated monosialotetrahexosylganglioside GM1, respectively, we investigated their involvement in bacterial invasion by super-resolution microscopy. Structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM) unraveled accumulation and coating of meningococci with GM1 upon cellular uptake. Blocking of CTxB binding sites did not impair bacterial adhesion but dramatically reduced bacterial invasion efficiency. In addition, cell cycle arrest in G1 phase induced by serum starvation led to an overall increase of GM1 molecules in the plasma membrane and consequently also in bacterial invasion efficiency. Our results will help to understand downstream signaling events after initial type IV pilus-host cell interactions and thus have general impact on the development of new therapeutics targeting key molecules involved in infection.

Oral administration of recombinant Bacillus subtilis spores expressing Helicobacter pylori neutrophil-activating protein suppresses peanut allergy via up-regulation of Tregs.

Helicobacter pylori neutrophil-activating protein (NAP) is an immune modulator with anti-Th2 inflammation activity that can be used to prevent IgE-mediated allergic reactions. Cholera toxin B (CTB) is a mucosal adjuvant that can induce antigen tolerance. Bacillus subtilis spores are an ideal vehicle for the oral delivery of heterologous antigens. We investigated the therapeutic effect of recombinant NAP Bsubtilis spores on peanut allergies in a mouse model. Female C3H/HeJ mice were sensitized and challenged with peanut extract by oral administration. Before challenge, recombinant NAP and CTB-NAP (CNAP) spores were orally administered to sensitized mice for 4weeks. Faecal peanut-specific IgA and serum-specific IgE, IgG1, and IgG2a levels were measured, and the intestinal microbiota was analysed. Mice were intraperitoneally injected with anti-CD25 antibodies for regulatory T cell (Treg) depletion to evaluate the efficacy of Tregs in preventing peanut allergy. After challenge, anaphylactic reactions, plasma histamine, Tregs, and splenocyte interleukin (IL)-10, IL-4, IL-5 and interferon-γ (IFN-γ) levels were evaluated. After 4weeks of recombinant spore treatment, faecal IgA levels and serum IgG2a levels were increased, while serum IgG1 and IgE levels were reduced. Intestinal microbiota analysis revealed that CNAP spores increased the taxonomic abundance of Firmicutes at the phylum level and Clostridia at the class level. After challenge, the administration of NAP or CNAP spores to mice was found to ameliorate anaphylactic reactions and decrease plasma histamine levels. Administration of NAP or CNAP spores also enhanced IL-10 and IFN-γ secretion, and suppressed IL-4 and IL-5 secretion. The protective effect of CNAP spores was more pronounced than that of NAP spores; this therapeutic effect was lost after Treg depletion. Recombinant NAP spores successfully suppressed Th2 inflammation via the up-regulation of Tregs; this may serve as a novel therapeutic approach for treating food allergies.

Enhanced paracellular delivery of vaccine by hydrogel microparticles-mediated reversible tight junction opening for effective oral immunization

The current conventional injectable vaccines face several drawbacks such as inconvenience and ineffectiveness in mucosal immunization. Therefore, the current development of effective oral vaccines is vital to enable the generation of dual systemic and mucosal immunity. In the present study, we examine the potential of pH-responsive bacterial nanocellulose/polyacrylic acid (BNC/PAA) hydrogel microparticles (MPs) as an oral vaccine carrier. In-vitro entrapment efficiency and release study of Ovalbumin (Ova) demonstrated that as high as 72% of Ova were entrapped in the hydrogel, and the release of loaded Ova was pH-dependent. The released Ova remained structurally conserved as evident by Western blot and circular dichroism. Hydrogel MPs reduced the TEER measurement of HT29MTX/Caco2/Raji B triple co-culture monolayer by reversibly opening the tight junctions (TJs) as shown in the TEM images. The ligated ileal loop assay revealed that hydrogel MPs could facilitate the penetration of FITC-Ova into the Peyer's patches in small intestine. Ova and cholera toxin B (CTB) were utilized in in-vivo oral immunization as model antigen and mucosal adjuvant. The in-vivo immunization revealed mice orally administered with Ova and CTB-loaded hydrogel MPs generated significantly higher level of serum anti-Ova IgG and mucosal anti-Ova IgA in the intestinal washes, compared to intramuscular administrated Ova. These results conclude that BNC/PAA hydrogel MPs is a potential oral vaccine carrier for effective oral immunization.

Glutamatergic input varies with phrenic motor neuron size.

Like all skeletal muscles, the diaphragm muscle accomplishes a range of motor behaviors by recruiting different motor unit types in an orderly fashion. Recruitment of phrenic motor neurons (PhMNs) is generally assumed to be based primarily on the intrinsic properties of PhMNs with an equal distribution of descending excitatory inputs to all PhMNs. However, differences in presynaptic excitatory input across PhMNs of varying sizes could also contribute to the orderly recruitment pattern. In the spinal cord of Sprague-Dawley rats, we retrogradely labeled PhMNs using cholera toxin B (CTB) and validated a robust confocal imaging-based technique that utilizes semiautomated processing to identify presynaptic glutamatergic (Glu) terminals within a defined distance around the somal membrane of PhMNs of varying size. Our results revealed an ~10% higher density of Glu terminals at PhMNs in the lower tertile of somal surface area. These smaller PhMNs are likely recruited first to accomplish lower force ventilatory behaviors of the diaphragm as compared with larger PhMNs in the upper tertile that are recruited to accomplish higher force expulsive behaviors. These results suggest that differences in excitatory synaptic input to PhMNs may also contribute to the orderly recruitment of diaphragm motor units.NEW & NOTEWORTHY The distribution of excitatory glutamatergic synaptic input to phrenic motor neurons differs across motor neurons of varying size. These findings support the size principle of motor unit recruitment that underlies graded force generation in a muscle, which is based on intrinsic electrophysiological properties of motor neurons resulting from differences in somal surface area. A higher density of glutamatergic inputs at smaller, more excitable motor neurons substantiates the earlier and more frequent recruitment of these units.

Assessment of disease specific immune responses in enteric diseases using dried blood spot (DBS).

BackgroundBlood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV).Methodology/Principle findingsWe processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively.ConclusionOur results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings.

Characterization of glutamatergic VTA neural population responses to aversive and rewarding conditioning in freely-moving mice

The Ventral Tegmental Area (VTA) is a midbrain structure known to integrate aversive and rewarding stimuli, but little is known about the role of VTA glutamatergic (VGluT2) neurons in these functions. Direct activation of VGluT2 soma evokes rewarding behaviors, while activation of their downstream projections evokes aversive behaviors. To facilitate our understanding of these conflicting properties, we recorded calcium signals from VTAVGluT2+ neurons using fiber photometry in VGluT2-cre mice to investigate how this population was recruited by aversive and rewarding stimulation, both during unconditioned and conditioned protocols. Our results revealed that, as a population, VTAVGluT2+ neurons responded similarly to unconditioned-aversive and unconditioned-rewarding stimulation. During aversive and rewarding conditioning, the CS-evoked responses gradually increased across trials whilst the US-evoked response remained stable. Retrieval 24 h after conditioning, during which mice received only CS presentation, resulted in VTAVGluT2+ neurons strongly responding to CS presentation and to the expected-US but only for aversive conditioning. To help understand these differences based on VTAVGluT2+ neuronal networks, the inputs and outputs of VTAVGluT2+ neurons were investigated using Cholera Toxin B (CTB) and rabies virus. Based on our results, we propose that the divergent VTAVGluT2+ neuronal responses to aversion and reward conditioning may be partly due to the existence of VTAVGluT2+ subpopulations that are characterized by their connectivity.

A hydrogel engineered to deliver minocycline locally to the injured cervical spinal cord protects respiratory neural circuitry and preserves diaphragm function

We tested a biomaterial-based approach to preserve the critical phrenic motor circuitry that controls diaphragm function by locally delivering minocycline hydrochloride (MH) following cervical spinal cord injury (SCI). MH is a clinically-available antibiotic and anti-inflammatory drug that targets a broad range of secondary injury mechanisms via its anti-inflammatory, anti-oxidant and anti-apoptotic properties. However, MH is only neuroprotective at high concentrations that cannot be achieved by systemic administration, which limits its clinical efficacy. We have developed a hydrogel-based MH delivery system that can be injected into the intrathecal space for local delivery of high concentrations of MH, without damaging spinal cord tissue. Implantation of MH hydrogel after unilateral level-C4/5 contusion SCI robustly preserved diaphragm function, as assessed by in vivo recordings of compound muscle action potential (CMAP) and electromyography (EMG) amplitudes. MH hydrogel also decreased lesion size and degeneration of cervical motor neuron somata, demonstrating its central neuroprotective effects within the injured cervical spinal cord. Furthermore, MH hydrogel significantly preserved diaphragm innervation by the axons of phrenic motor neurons (PhMNs), as assessed by both detailed neuromuscular junction (NMJ) morphological analysis and retrograde PhMN labeling from the diaphragm using cholera toxin B (CTB). In conclusion, our findings demonstrate that local MH hydrogel delivery to the injured cervical spinal cord is effective in preserving respiratory function after SCI by protecting the important neural circuitry that controls diaphragm activation.

Comparison of different immunization routes on the immune responses induced by Mycobacterium tuberculosis ESAT-6/CFP-10 recombinant protein

According to some difficulties against tuberculosis (TB) vaccination, development of new TB vaccines has been noted in recent years. Selection of proper route for vaccination is one of the most important factors for induction of good immune responses. Hence, in this study, the effects of different administration routes, including intranasal (I.N), subcutaneous (S.C) and intramuscular (I.M) on immune responses against Mycobacterium tuberculosis ESAT-6/CFP-10 recombinant protein has been considered.Recombinant ESAT-6/CFP-10 protein with or without adjuvant (MF59 or cholera toxin B (CTB)) was administered by three routes of I.M, I.N and S.C to mice for three times. Then, the levels of specific antibodies, lymphocyte proliferation and IFN-γ/IL-5 cytokine profile have been carried out to evaluate the humoral and cellular responses.The results showed that the titers of specific antibodies were quickly elevated in S.C and I.M groups after first immunization. Otherwise, the raise of antibody has delay in the I.N immunized animals. The levels of IFN-γ and lymphocyte proliferation have been increased in all of vaccinated groups. However, the I.N immunized mice have lower levels of IL-5 production.Based on our finding, the ESAT-6/CFP-10 recombinant protein is a potent stimulator of immune responses in all of three immunization strategies. However intranasal administration of this antigen has tended to reinforcement of cellular immune responses.

Effect of Sedentary Conditions on the Rostrocaudal Expression of the NMDA NR1 Receptor Subunit in Bulbospinal C1 and non‐C1 Neurons of the Rat Rostral Ventrolateral Medulla (RVLM)

Bulbospinal RVLM neurons directly innervate sympathetic preganglionic cells and therefore play important roles in the regulation of sympathetic nerve activity and blood pressure. RVLM neurons are divided phenotypically into catecholaminergic (C1) and non‐catecholaminergic (non‐C1).Previous studies from our laboratory reported that the RVLM of sedentary compared to physically active rats demonstrates greater structural and functional neuroplasticity in a subregion‐specific manner. For example, RVLM neurons from sedentary versus physically active rats exhibit greater dendritic branching and elicit greater sympathoexcitation upon direct stimulation with glutamate in rostral versus caudal RVLM. Bulbospinal RVLM neurons also express NMDA receptors and could contribute to inactivity‐induced neuroplasticity and enhanced sympathetic activation.The purpose of the present study was to test the hypothesis that sedentary conditions increase NMDA NR1 subunit expression in the RVLM in a subregion‐dependent manner. Male Sprague‐Dawley rats were sedentary (no running wheels) or physically active (free access to running wheels) for 10–12 wks (n=6 ea). Spinal cord injections of cholera toxin B (CTB)‐labeled bulbospinal RVLM neurons. We examined coronal sections for triple‐label immunofluorescence by laser confocal microscopy, and quantified CTB‐positive C1 and non‐C1 neurons expressing the NR1 receptor subunit.As expected from previous work, significantly more bulbospinal C1 neurons were positive for NR1 in the caudal RVLM compared to rostral RVLM in both experimental groups (p&lt;0.001). Also as expected, more non‐C1 neurons immunoreactive for NR1 were in the rostral compared to caudal RVLM (p&lt;0.001). Interestingly, sedentary rats demonstrated a strong trend for a higher percentage of C1 cells and significantly lower percentage of non‐C1 cells positive for NR1 in rostral RVLM when compared to active animals (p=0.067 and p=0.0048, respectively). In contrast, there were no overt differences between sedentary versus active rats in the percentage of C1 and non‐C1 cells positive for NR1 in caudal RVLM.Preliminary western blotting data showed lower expression of PSD95 (an excitatory post‐synaptic density protein) in sedentary versus active animals (0.89±0.15 vs. 1.64±0.24, PSD95/GAPDH, respectively), suggesting differential synaptic/extrasynaptic localization of glutamate receptors, including NR1, in sedentary and active groups.The greater number of NR1‐positive, bulbospinal C1 neurons in rostral RVLM of sedentary rats is consistent with our hypothesis and previous data showing enhanced SNA following activation of glutamate receptors in sedentary vs. active rats. Sedentary conditions may also enhance excitation of RVLM neurons by altering the composition of NMDAR subunits, their synaptic/extrasynaptic localization, trafficking, or expression of synapse‐related proteins and/or transporters.Support or Funding InformationHL096787‐08; AHA25810010This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.