The dorsal root ganglion houses primary sensory neurons whose afferent fibers transmit peripheral information to the central nervous system. Sensory transduction relies on ion channels, including TRP channels, and ASICs, which convert various stimuli into neural signals. Ion channels expressed in primary sensory neurons, including those innervating skeletal muscle, play crucial roles not only in nociception but also in the regulation of cardiovascular reflexes. Insulin not only regulates systemic glucose homeostasis but also modulates ion channel sensitivity in sensory neurons through PI3K-PKC signaling. Therefore, this review outlines current evidence on insulin-mediated modulation of sensory ion channels and explores its implications for the pathophysiology of diabetic neuropathy and the abnormal exercise pressor reflex function observed in early-stage type 2 diabetes.

Background: Stimulation with allergen and allergen-specific IgE activates mast cells, leading to allergic reactions. We studied how the recently defined alarmin, Histamine-Releasing Factor (HRF), promotes this immune response. Methods: HRF reactivity to IgE and other Ab isotypes was analyzed by in-vitro binding assays and 3D-structure modelling. Extracellular and intracellular formations of HRF multimers were studied in aqueous solution and siRNA-treated cells, respectively. Effects of HRF dimers and multimers on in-vitro mast cell activation were measured in IgE-sensitized mast cells by stimulation with high or low valency antigen. In-vivo effects of HRF in allergic reactions were studied in passive anaphylaxis and food allergy models using WT or non-multimerizable HRF mutant mice as well as humanized mice. Results: HRF binds to a substantial subset of both antigen-driven and non-antigen-driven antibodies. Extracellular HRF proteins become non-enzymatically disulfide-linked dimers and multimers, whereas intracellular HRF requires oxidoreductases for dimerization. Surprisingly, while HRF dimers can enhance antigen/IgE-induced cytokine production, stronger signals by HRF multimers are required for degranulation. HRF multimers render HRF-reactive IgE-sensitized mast cells more sensitive and responsive to suboptimal antigen doses. In-vivo anaphylactic responses induced by passive sensitization with HRF-reactive IgE and subsequent antigen challenge are drastically reduced by blockade of HRF-IgE interactions and by HRF-Cys172Ala mutation in mutant mice. Enhancement of anaphylactic responses by HRF is more prominent with low-valency antigen than with supernaturally high-valency antigen. Conclusion: The results collectively demonstrate that HRF multimers are important for IgE-mediated optimal allergic reactions.

We have previously reported that insulin potentiates the response to mechanical stimuli in small dorsal root ganglion (DRG) neurons. However, the mechanisms underlying the insulin-induced potentiated responsiveness to mechanical stimulation in sensory neurons remain unclear. Transient receptor potential vanilloid 4 (TRPV4) is expressed as a mechanosensitive channel in DRG neurons and is activated by mechanical stimuli. We therefore hypothesized that insulin augments the response to mechanical stimulation in small DRG neurons by enhancing sensitization of TRPV4 channels. Colocalization of TRPV4, insulin receptor (IR), and the C-fiber marker peripherin in small DRG neurons was evaluated by immunofluorescence, demonstrating that 53 ± 10% of TRPV4-positive small DRG neurons were colocalized with IR and peripherin. In in vitro whole cell patch clamp recordings from cultured DRG neurons, mechanically activated currents were significantly increased 5 min after the application of insulin (P = 0.0137) and such augmentation was suppressed by TRPV4 antagonist HC067047. We further examined the impact of insulin on the expression of the IR signaling pathway proteins in cultured DRG neurons using western blotting. Akt was significantly increased in cultured DRG neurons incubated with insulin (phospho-Akt: P = 0.0007, phospho/total Akt ratio: P = 0.0183). Furthermore, blocking IR signaling kinases, phosphoinositide 3-kinase (PI3K), and PKC suppressed the insulin-induced augmentation in TRPV4 agonist-induced currents (PI3K: P = 0.0074, PKC: P = 0.0028). Collectively, our results suggest that insulin-induced potentiation of mechanical response in small DRG neurons occurs through enhanced sensitization of TRPV4 channels.NEW & NOTEWORTHY We investigated whether insulin potentiates the sensitization of TRPV4 channels to mechanical stimulation in DRG neurons. Insulin-induced enhancement of mechanical response was suppressed by blocking TRPV4 channels. Furthermore, blockade of insulin receptor signaling pathways, PI3K and PKC, inhibited insulin-induced sensitization of TRPV4 channels. Our results provide evidence that insulin-induced potentiation of mechanical sensation in small DRG neurons is mediated through enhancing the sensitization of TRPV4 channels via the insulin receptor signaling pathway.

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder leading to not only cognitive decline but also impaired somatosensory perception. Stimulation of skeletal muscle afferent fibers during muscle contraction increases blood pressure, known as the exercise pressor reflex (EPR). However, to date, it has not been elucidated whether AD alters the signal transduction associated with the EPR or whether AD alters cardiovascular responses to activation of muscle afferents. We hypothesized that EPR function is indeed altered in AD. Purpose: The aim of this investigation was to determine the cardiovascular responses to stimulation of muscle afferents in a rat model of sporadic AD. METHOD: To generate an sporadic AD rat model, streptozotocin (STZ, 3 mg/kg) was intracerebroventricularly injected into the lateral ventricle of male Sprague–Dawley rats (ICV-STZ, body weight (BW): 385 ± 31 g, n = 17). In parallel, a separate group of rats were treated with ICV saline as a vehicle control (CON, BW: 407 ± 44 g, n = 14). Spatial learning and memory function were assessed using the Morris Water Maze (MWM) behavioral test. During the MWM navigation test (multiple trials over a 4-day period), each rat was placed in a circular pool, and the time to reach a translucent platform recorded. In separate trials, mean arterial pressure (MAP) and heart rate (HR) responses to (1) 30-s passive stretch of the hindlimb muscle, (2) intra-arterial administration of capsaicin (0.3 µg/100 µL), and (3) tibial nerve stimulation at 10 x motor threshold (MT) and at 50 x MT were evaluated. All experiments were performed ten weeks after the administration of the test solution. Results: In the MWM test, the time to reach the platform was progressively shorter over the 4 days of testing in both groups (P < 0.001). As expected, ICV-STZ rats had a significantly longer time to reach the platform as compared to CON rats (P = 0.0046). Peak changes in MAP, HR, and developed tension in response to passive stretch were not different between CON and ICV-STZ rats. Similarly, the peak cardiovascular responses to stimulation by intra-arterial capsaicin injection did not change among groups. However, the peak pressor and cardioaccelerator responses to tibial nerve stimulation at both 10 x MT and 50 x MT were significantly suppressed in ICV-STZ rats compared to CON rats (main effect of group: ΔMAP: P = 0.0003, ΔHR: P = 0.0035; main effect of MT: ΔMAP: P = 0.0111, ΔHR: P = 0.2485). Conclusion: The findings suggest that in experimentally induced sporadic AD, decreased spatial learning and memory function is likewise accompanied by blunted cardiovascular responses to electrical, but not mechanical or chemical, stimulation of muscle afferents. Supported by NIH HL-151632 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Introduction:Individuals allergic to peanuts (PN) may show IgE cross-reactivity to tree nuts, especially walnuts (WN), which often complicates diagnosis. Vicilin-buried peptides (VBPs), short segments within the N-terminal vicilin leader sequence (LS), contribute to cross-reactivity due to their ubiquitous, highly conserved and stable α-hairpin structures. The binding patterns of cross-reactive IgE to linear and conformational epitopes of PN and WN LSs and constituent VBPs may serve as a model for understanding clinically symptomatic cross-reactivity. Methods:Serum samples (n = 30) from primarily oral food challenge-positive individuals with PN allergy (PNA, 33%), WN allergy (WNA, 47%), and PN and WN allergies (PWA, 20%) were collected. These sera and a monoclonal IgE antibody (6D12) were examined for IgE binding with microarrays of overlapping peptides from native Ara h 1 LS [AH1LS, Ara h 1.0101 (26-84)] and recombinant Jug r 2 LS [JR2LS, Jug r 2.0101 (1-173)] and via direct and competitive inhibition ELISA with intact LSs and constituent VBPs from PN (AH1.1) and WN (JR2.1, JR2.2, JR2.3). A mixed model analysis assessed the contribution of IgE binding patterns to VBPs in relation to PNA, WNA, or PWA status. Results:All three intact WN VBPs bound IgE at similar frequencies, with individual sera showing varying preferences for specific VBPs. AH1.1 was less recognized by WNA individuals but more frequently recognized by PNA and PWA subjects. WN VBPs were recognized by PNA sera samples at rates comparable to AH1.1. Our data indicates that each VBP can bind to one IgE molecule with high affinity. In a competitive inhibition ELISA, combining VBP competitors did not enhance inhibition compared to the dominant VBP, suggesting that both high- and low-affinity VBPs compete for the same monoclonal IgE in serum. This observation was mimicked by 6D12, a monoclonal IgE against JR2.1. Discussion:Cross-reactivity among VBPs most likely arises from monoclonal IgE binding to α-hairpin structures and their overlapping linear amino acid sequences. The combination of linear and conformational IgE binding patterns enabled us to differentiate between the WNA, PNA, and PWA groups in this study and may assist us in using AH1LS and JR2LS to distinguish PN and WN allergies in the future.

BACKGROUND:Recent studies have highlighted the deleterious role of high phosphate intake in hypertension via sympathetic overactivation, yet the underlying mechanisms remain unclear. Dietary phosphate loading triggers physiologic release of FGF23 (fibroblast growth factor-23) from the bone to maintain phosphate homeostasis. Both FGF23 and FGF receptors (FGFRs) are present in the central nervous system, but their role in neural control of blood pressure during phosphate loading is unknown. We investigated central FGF23/FGFR signaling in high-phosphate diet-induced sympathetic dysregulation of blood pressure in rats. METHODS:FGF23 protein levels were measured by immunoprecipitation, immunoblotting, and immunohistochemistry. FGF23 translocation into the brain was determined by injecting infrared-labeled FGF23 intravenously into anesthetized Sprague-Dawley rats. Mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) responses to hindlimb muscle contraction were measured in decerebrate Sprague-Dawley rats treated with either a normal 0.6% phosphate diet (NP) or a high 1.2% phosphate diet (HP) for 12 weeks before and after intracerebroventricular (ICV) administration of FGFR signaling inhibitors. RESULTS:Excess phosphate intake significantly increased FGF23 protein levels in the brainstem (HP versus NP, P=0.009) and cerebrospinal fluid (HP versus NP, P<0.001). Peripheral injection of infrared-labeled FGF23 showed clear entry into the choroid plexus and medulla oblongata. ICV administration of PD173074, a pan-FGFR(1-4) inhibitor, significantly attenuated the heightened RSNA (Δ=84±53 versus 32±25% [P<0.0001]) and MAP (Δ=35±14 versus 9±7 mm Hg [P<0.0001]) responses to muscle contraction in HP animals, but did not affect the RSNA and MAP responses during stimulation in NP animals (ΔRSNA=40±29 versus 30±22% and ΔMAP=18±13 versus 13±9 mm Hg before versus after ICV injection). ICV injection of BLU9931, a relatively selective FGFR4 inhibitor, also decreased the responses in HP rats only (∆RSNA=112±70 versus 65±46% [P=0.006] and ∆MAP=41±14 versus 20±14 mm Hg [P<0.0001] before versus after ICV injection). However, ICV administration of AZD4547, a FGFR1-3 inhibitor, and C-terminal FGF23 peptide, a competitive inhibitor of FGF23/FGFR/α-Klotho complex formation, did not alter the responses in either NP or HP animals. CONCLUSIONS:Our data reveal a novel pathophysiologic paradigm of high-phosphate diet-induced sympathoexcitation and hypertension by FGF23 crossing into the brain, possibly acting via FGFR4.

BACKGROUND:Human IgE mAbs recognizing peanut allergens have recently become available, but we lack a detailed understanding of how these IgEs target allergens. OBJECTIVE:We sought to determine the molecular details of the antibody-allergen interaction for a panel of clinically important human IgE mAbs and to develop strategies to disrupt disease causing antibody-allergen interactions. METHODS:We identified candidates from a panel of epitope binned human IgE mAbs that recognize 2 important and homologous peanut allergens, Ara h 2 and Ara h 6. Crystal structures were determined revealing the interfaces (antigenic sites) of exemplars of 5 common IgE bins. RESULTS:Among the common antigenic sites on Ara h 2 and Ara h 6, 2 sites (A and B) are highly conserved between the allergens, explaining the cross-reactivity of antibodies that recognize these sites. Three sites (C, D, and F) involve residues that are not conserved between the allergens. Of the 5 common sites, 3 sites (B, C, and D) involve residues that are near each other only when the allergens are properly folded, such that these sites are conformational. Two additional sites (sites A and F) involve largely linear stretches of amino acids. Site F targeted antibody, 38B7, binds to a peptide sequence DPYSPOHS, in which hydroxylation of the last proline is critical for binding. This sequence is repeated 2 or 3 times depending on the Ara h 2 isoform, enabling 38B7 to induce anaphylaxis as a single mAb, without a second antibody. We have mutated key residues in each site and created a panel of hypoallergens, having reduced IgE mAb binding and lacking the ability to induce anaphylaxis in our murine model. CONCLUSION:We created a structural map of the IgE antibody response to the most important peanut allergen proteins to enable the design of new allergy immunotherapies and vaccines.

Cardiovascular responses to exercise are exaggerated in chronic kidney disease (CKD) patients. Enhanced sympathetic activation is thought to play a role with the exercise pressor reflex (EPR), a reflex originating in contracting muscle, modulating this response. Previous studies suggest an overactive EPR in CKD patients as indicated by muscle sympathetic overactivation during static handgrip exercise. However, the role of the EPR could not be fully elucidated due to experimental constraints inherent to humans. The purpose of this study was to specifically test EPR function in a CKD animal model. Male Sprague-Dawley rats were assigned to a diet containing 0.25% adenine to induce CKD or a control diet. Mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) responses to activation of the EPR, including its functional components, the mechanoreflex and metaboreflex, were assessed in decerebrate, unanesthetized animals after feeding 10 to 14 weeks. Plasma creatinine was significantly higher in CKD rats compare to controls (1.80±0.78 vs. 0.34±0.02 mg·dl-1, P = 0.017). MAP and RSNA responses to muscle contraction (i.e., EPR activation) were potentiated in CKD rats compared to controls (Δ=36±19 vs. 17±8 mmHg, P = 0.014 and Δ=159±62 vs. 64±54 %, P = 0.004, respectively). Similarly, the pressor and sympathetic responses to passive muscle stretch (i.e., mechanoreflex stimulation) were significantly higher in CKD than in control animals. Intraarterial capsaicin administration (i.e., metaboreflex activation) induced an augmented pressor response in CKD rats, compared with controls. Our findings suggest that the EPR, stimulated by the mechano- and metaboreflex, is exaggerated in CKD.

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron®. Genetron® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. It is characterized by synaptic loss and the increase of amyloid β (Aβ) in the brain often detrimentally affecting function. Brainstem is the key central integration site for sensory input from working skeletal muscle. Stimulation of skeletal muscle afferent fibers during muscle contraction increases blood pressure. However, whether AD alters or preserves the central processing of peripheral sensory afferent signals remains to be elucidated. Thus, we tested the hypothesis that the magnitude of the pressor response is functionally altered in intracerebroventricular-streptozotocin injected rats (ICV-STZ). Streptozotocin (3 mg/kg) was intracerebroventricularly injected into the lateral ventricle of male Sprague-Dawley rats. In parallel, a separate group of rats were treated with ICV saline as a vehicle control. Spatial learning and memory function were assessed using the Morris Water Maze behavioral test. Results demonstrate that ICV-STZ rats had a significantly longer time to reach a target platform compared to controls (P = 0.0046). ICV-STZ injection also significantly increased brainstem Aβ1-40 (P = 0.0082), but not Aβ1-42 (P = 0.0744). Further, the peak pressor and cardioaccelerator responses to tibial nerve stimulation were significantly attenuated in ICV-STZ rats compared to controls (ΔMAP: P = 0.0003, ΔHR: P = 0.0035). The findings suggest that the cardiovascular responses to electrical stimulation of sensory afferents are blunted in ICV-STZ rats.

Evidence suggests that brain insulin availability acutely modulates arterial baroreflex function. However, little is known about the impact of blocking brain insulin receptor (IR) signaling on arterial baroreflex. We hypothesized that blockade of IR signaling in the brain acutely impairs arterial baroreflex function. Our hypothesis was tested using baroreflex open-loop analysis to evaluate the two subsystems of the arterial baroreflex: the carotid sinus pressure (CSP)-sympathetic nerve activity (SNA) relationship (the neural arc) and the SNA-arterial pressure (AP) relationship (the peripheral arc). In anesthetized healthy male rats, the bilateral carotid sinus baroreceptor regions were surgically isolated from the systemic circulation, and then CSP was changed stepwise from 60 to 180 mmHg before and over 120 min after lateral intracerebroventricular (ICV) administration of either artificial cerebrospinal fluid (control solution) or IR antagonist GSK1838705. ICV injection of GSK1838705 significantly decreased renal SNA (RSNA), AP, and heart rate during stepwise CSP input over a period of 120 min after administration (p < .05). The maximum gain of the neural arc was significantly reduced 120 min after ICV injection of GSK1838705 (p = .002). Furthermore, GSK1838705 significantly attenuated the operating-point RSNA (p = .025) and AP (p < .001) as estimated by the baroreflex equilibrium diagram. Moreover, 120-min baroreflex stimulation via stepwise CSP input significantly increased c-Fos expression in IR-positive neurons in medullary cardiovascular centers (p < .001). Our findings suggest that IR signaling in the brain can modulate AP regulation via alteration of the neural arc of the arterial baroreflex.

Allergen-specific immunotherapy represents the only method of achieving a lasting reduction in the severity of allergic symptoms. However, the need to expose patients to the allergens to which they are sensitized carries risks. One solution is to use denatured allergens whereby the structure of allergenic proteins is disrupted, preventing their recognition by immunoglobulin E (IgE) antibodies and thus reducing the risk of adverse reactions. Denaturation is often carried out by using chemical cross-linking to generate allergoids. Gold nanoclusters (AuNCs) are emerging as versatile tools in biotechnology due in part to their ability to conjugate a wide range of biological molecules. Previous works have described the formation of AuNC using egg allergens such as Gal d 4 (lysozyme), Gal d 2 (ovalbumin), and whole egg whites. In all cases, AuNC bioconjugation disrupted the protein structure, allowing for their use in biosensing applications. In this work, we hypothesize that these AuNC-allergen bioconjugates could be used to generate "Allergolds", chemically altered versions of allergenic proteins analogous to traditional allergoid formulations. Using spectroscopic techniques, we confirm that the formation of AuNC bioconjugates of the chicken egg Gal d 4 and Gal d 2 disrupts protein structure when generated from both purified protein and whole egg whites. This structural perturbation was found to be resilient to a range of chemical conditions and successfully disrupted recognition by human IgE. These results establish Allergolds as a potential tool for generating systematically denatured allergens from both purified proteins and biological extracts.

BACKGROUND:A major obstacle to the effective diagnosis of cephalosporin allergies is that the haptens, or segments of their molecular structures, which are responsible for the initiation of an immunogenic response, are unknown. OBJECTIVE:This study aimed to identify immunogenic moieties of cefazolin to accurately predict IgE-mediated allergy and cross-reactivity with other cephalosporin antibiotics. METHODS:Hapten immunogenicity analysis is performed using liposomal nanoallergens integrated in a cellular degranulation assay to quantify secreted allergic mediators. RBL-SX38 cells were primed with purified human monoclonal IgE or patient plasma samples before nanoallergen challenge. The monoclonal IgE priming consisted of dust mite- or peanut-specific negative controls and were compared against a cefazolin-specific monoclonal IgE. The plasma samples, in contrast, came from 3 drug allergy-negative control patients, or 2 cefazolin-allergic patients who provided 3 samples. RESULTS:Multiple forms of cefazolin are immunogenic when multivalently presented by nanoallergens, and there may be alternate nanoparticle formulations that can effectively diagnose IgE-mediated cefazolin allergy in patients. Additionally, common R1 or R2 groups in cephalosporin molecular structures do not singularly identify shared cross-reactivity. CONCLUSION:This study highlights an innovative method to reproducibly distinguish cefazolin-allergic from nonallergic patients using finely tuned cefazolin-hapten-presenting nanoallergens in conjunction with an in vitro cellular degranulation assay.

Background: Studies of human IgE and its targeted epitopes on allergens have been very limited. We established a method to immortalize IgE-encoding B cells from patients with allergy. Objective: We sought to develop an unbiased and comprehensive panel of peanut-specific human IgE mAbs to characterize key immunodominant antigenic regions and epitopes on peanut allergens to map molecular interactions responsible for inducing anaphylaxis. Methods: Using human hybridoma technology to immortalize IgE-encoding B cells from peripheral blood of subjects with severe peanut allergy, we generated a panel of naturally occurring human IgE mAbs in an unbiased manner. Isolated IgE mAbs were characterized extensively in allergen binding assays, peptide array analysis, antigenic mapping, binding kinetic analysis, serum blocking, skin testing inhibition, and functional assessment using human FC epsilon RI transgenic mice. Results: We created a large panel of 54 peanut-specific IgE mAbs, of which 63% were specific for Ara h 2 and/or Ara h 6. Pairs of IgE mAbs with the same antigen specificity but different binding sites were able to mediate passive systemic anaphylaxis in FC epsilon RI transgenic mice. A single mAb targeting the repetitive motif on Ara h 2 was able to induce degranulation and anaphylaxis on its own. IgG1 switch variant immunoglobulins of the IgE mAb inhibited binding of 30% to 60% of patients' IgE to peanut extract (ImmunoCAP) and reduced peanut extract-induced skin wheal sizes by 1.6 to 7.4 mm in patients with peanut allergy. Conclusion: We created a molecular map of the IgE antibody response to the most important peanut allergen proteins to enable the design of new allergy immunotherapies and vaccines. (J Allergy Clin Immunol 2025;155:1595-606.)