Pertussis Toxin Research Articles

A Chronic Increase in Blood-Brain Barrier Permeability Facilitates Intraneuronal Deposition of Exogenous Bloodborne Amyloid-Beta1-42 Peptide in the Brain and Leads to Alzheimer's Disease-Relevant Cognitive Changes in a Mouse Model.

Increased blood-brain barrier (BBB) permeability and amyloid-β (Aβ) peptides (especially Aβ1-42) (Aβ42) have been linked to Alzheimer's disease (AD) pathogenesis, but the nature of their involvement in AD-related neuropathological changes leading to cognitive changes remains poorly understood. To test the hypothesis that chronic extravasation of bloodborne Aβ42 peptide and brain-reactive autoantibodies and their entry into the brain parenchyma via a permeable BBB contribute to AD-related pathological changes and cognitive changes in a mouse model. The BBB was rendered chronically permeable through repeated injections of Pertussis toxin (PT), and soluble monomeric, fluorescein isothiocyanate (FITC)-labeled or unlabeled Aβ42 was injected into the tail-vein of 10-month-old male CD1 mice at designated intervals spanning ∼3 months. Acquisition of learned behaviors and long-term retention were assessed via a battery of cognitive and behavioral tests and linked to neuropathological changes. Mice injected with both PT and Aβ42 demonstrated a preferential deficit in the capacity for long-term retention and an increased susceptibility to interference in selective attention compared to mice exposed to PT or saline only. Immunohistochemical analyses revealed increased BBB permeability and entry of bloodborne Aβ42 and immunoglobulin G (IgG) into the brain parenchyma, selective neuronal binding of IgG and neuronal accumulation of Aβ42 in animals injected with both PT and Aβ42 compared to controls. Results highlight the potential synergistic role of BBB compromise and the influx of bloodborne Aβ42 into the brain in both the initiation and progression of neuropathologic and cognitive changes associated with AD.

Comparison of Bordetella pertussis Antibody Levels in Pregnant Women and Umbilical Cord Blood: A Multicenter Study.

In countries where pertussis vaccination is not administered during pregnancy, the determination of pertussis antibody levels in pregnant women is very important in terms of knowing the current seroepidemiology and potential strategies for immunizations. We included 396 pregnant women who were admitted to 4 different obstetrics and gynecology clinics. Anti-Bordetella pertussis toxin (PT) IgG and anti-Bordetella pertussis filamentous hemagglutinin IgG levels in maternal and cord blood pairs were determined by the ELISA method. Venous blood serum anti-PT level was below 5 IU/mL in 58.8%, 5-40 IU/mL in 34.8%, 40-100 IU/mL in 5.1% and >100 IU/mL in 1.3% of pregnant women. Cord blood serum anti-PT level was below 5 IU/mL in 47.7%, 5-40 IU/mL in 44.5%, 40-100 IU/mL in 6.8% and >100 IU/mL in 1% of pregnant women. In our study, the anti-PT level was found below 40 IU/mL in 93.6% of pregnant women and 92.2% of cord blood. Our study found the anti-filamentous hemagglutinin level below 40 IU/mL in 81% of pregnant women and 66.2% of cord blood. Although it is known that pertussis causes serious morbidity and mortality in young infants all over the world and that the most effective and reliable way to prevent it is vaccination of pregnant women, it is a remarkable contradiction that pertussis vaccination rates and therefore seropositivity rates in pregnant women are very low.

Role of autoreactive Tc17 cells in the pathogenesis of experimental autoimmune encephalomyelitis

AbstractBackgroundThe pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE—an animal model of MS) is primarily mediated by T cells. However, recent studies have only focused on interleukin (IL)‐17‐secreting CD4+ T‐helper cells, also known as Th17 cells. This study aimed to compare Th17 cells and IL‐17‐secreting CD8+ T‐cytotoxic cells (Tc17) in the context of MS/EAE.MethodsFemale C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein peptides 35–55 (MOG35–55), pertussis toxin, and complete Freund's adjuvant to establish the EAE animal model. T cells were isolated from the spleen (12–14 days postimmunization). CD4+ and CD8+ T cells were purified using isolation kit and then differentiated into Th17 and Tc17, respectively, using MOG35–55 and IL‐23. The secretion levels of interferon‐γ (IFN‐γ) and IL‐17 were measured via enzyme‐linked immunosorbent assay using cultured CD4+ and CD8+ T cell supernatants. The pathogenicity of Tc17 and Th17 cells was assessed through adoptive transfer (tEAE), with the clinical course assessed using an EAE score (0–5). Hematoxylin and eosin as well as Luxol fast blue staining were used to examine the spinal cord. Purified CD8+ CD3+ and CD4+ CD3+ cells differentiated into Tc17 and Th17 cells, respectively, were stimulated with MOG35–55 peptide for proliferation assays.ResultsThe results showed that Tc17 cells (15,951 ± 1985 vs. 55,709 ± 4196 cpm; p < 0.050) exhibited a weaker response to highest dose (20 μg/mL) MOG35–55 than Th17 cells. However, this response was not dependent on Th17 cells. After the 48 h stimulation, at the highest dose (20 μg/mL) of MOG35–55. Tc17 cells secreted lower levels of IFN‐γ (280.00 ± 15.00 vs. 556.67 ± 15.28 pg/mL, p < 0.050) and IL‐17 (102.67 ± 5.86 pg/mL vs. 288.33 ± 12.58 pg/mL; p < 0.050) than Th17 cells. Similar patterns were observed for IFN‐γ secretion at 96 and 144 h. Furthermore, Tc17 cell‐induced tEAE mice exhibited similar EAE scores to Th17 cell‐induced tEAE mice and also showed similar inflammation and demyelination.ConclusionThe degree of pathogenicity of Tc17 cells in EAE is lower than that of Th17 cells. Future investigation on different immune cells and EAE models is warranted to determine the mechanisms underlying MS.

β2-Adrenergic Regulation of the Neuromuscular Transmission and Its Lipid-Dependent Switch.

β2-Adrenoceptors (β2-ARs) are the most abundant subtype of adrenergic receptors in skeletal muscles. Their activation via a stabilization of postsynaptic architecture has beneficial effects in certain models of neuromuscular disorders. However, the ability of β2-ARs to regulate neuromuscular transmission at the presynaptic level is poorly understood. Using electrophysiological recordings and fluorescent FM dyes, we found that β2-AR activation with fenoterol enhanced an involvement of synaptic vesicles in exocytosis and neurotransmitter release during intense activity at the neuromuscular junctions of mouse diaphragm. This was accompanied by an improvement of contractile responses to phrenic nerve stimulation (but not direct stimulation of the muscle fibers) at moderate-to-high frequencies. β2-ARs mainly reside in lipid microdomains enriched with cholesterol and sphingomyelin. The latter is hydrolyzed by sphingomyelinases, whose upregulation occurs in many conditions characterized by muscle atrophy and sympathetic nerve hyperactivity. Sphingomyelinase treatment reversed the effects of β2-AR agonist on the neurotransmitter release and synaptic vesicle recruitment to the exocytosis during intense activity. Inhibition of Gi protein with pertussis toxin completely prevented the sphingomyelinase-mediated inversion in the β2-AR agonist action. Note that lipid raft disrupting enzyme cholesterol oxidase had the same effect on β2-AR agonist-mediated changes in neurotransmission as sphingomyelinase. Thus, β2-AR agonist fenoterol augmented recruitment and release of synaptic vesicles during intense activity in the diaphragm neuromuscular junctions. Sphingomyelin hydrolysis inversed the effects of β2-AR agonist on neurotransmission probably via switching to Gi protein-dependent signaling. This phenomenon may reflect a dependence of the β2-AR signaling on lipid raft integrity in the neuromuscular junctions.

Stable Levels of Antibodies Against Unrelated Toxoid Vaccines After COVID-19: COVID-19 Infection Does Not Affect Toxoid Vaccine Antibody Levels.

Lymphopenia is common in COVID-19. This has raised concerns that COVID-19 could affect the immune system akin to measles infection, which causes immune amnesia and a reduction in protective antibodies. We recruited COVID-19 patients (n = 59) in Helsinki, Finland, and collected plasma samples on 2 to 3 occasions during and after infection. We measured IgG antibodies to diphtheria toxin, tetanus toxoid, and pertussis toxin, along with total IgG, SARS-CoV-2 spike protein IgG, and neutralizing antibodies. We also surveyed the participants for up to 17 months for long-term impaired olfaction as a proxy for prolonged post-acute COVID-19 symptoms. No significant differences were found in the unrelated vaccine responses while the serological response against COVID-19 was appropriate. During the acute phase of the disease, the SARSCoV-2 IgG levels were lower in outpatients when compared to inpatients. SARS-CoV-2 serology kinetics matched expectations. In the acute phase, anti-tetanus and anti-diphtheria IgG levels were lower in patients with prolonged impaired olfaction during follow up than in those without. We could not detect significant decline in overall humoral immunity during or after COVID-19 infection. In severe COVID-19, there appears to be a temporary decline in total IgG levels.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model.

Since the 2000s, sporadic outbreaks of whooping cough have been reported in advanced countries, where the acellular pertussis vaccination rate is relatively high, and in developing countries. Small-scale whooping cough has also continued in many countries, due in part to the waning of immune protection after childhood vaccination, necessitating the development of an improved pertussis vaccine and vaccination program. Currently, two different production platforms are being actively pursued in Korea; one is based on the aP (acellular pertussis) vaccine purified from B. pertussis containing pertussis toxoid (PT), filamentous hemagglutin (FHA) and pertactin (PRN), and the other is based on the recombinant aP (raP), containing genetically detoxified pertussis toxin ADP-ribosyltransferase subunit 1 (PtxS1), FHA, and PRN domain, expressed and purified from recombinant E. coli. aP components were further combined with diphtheria and tetanus vaccine components as a prototype DTaP vaccine by GC Pharma (GC DTaP vaccine). We evaluated and compared the immunogenicity and the protective efficacy of aP and raP vaccines in an experimental murine challenge model: humoral immunity in serum, IgA secretion in nasal lavage, bacterial clearance after challenge, PTx (pertussis toxin) CHO cell neutralization titer, cytokine secretion in spleen single cell, and tissue resident memory CD4+ T cell (CD4+ TRM cell) in lung tissues. In humoral immunogenicity, GC DTaP vaccines showed high titers for PT and PRN and showed similar patterns in nasal lavage and IL-5 cytokine secretions. The GC DTaP vaccine and the control vaccine showed equivalent results in bacterial clearance after challenge, PTx CHO cell neutralization assay, and CD4+ TRM cell. In contrast, the recombinant raP vaccine exhibited strong antibody responses for FHA and PRN, albeit with low antibody level of PT and low titer in PTx CHO neutralization assay, as compared to control and GC DTaP vaccines. The raP vaccine provided a sterile lung bacterial clearance comparable to a commercial control vaccine after the experimental challenge in murine model. Moreover, raP exhibited a strong cytokine response and CD4+ TRM cell in lung tissue, comparable or superior to the experimental and commercial DTaP vaccinated groups. Contingent on improving the biophysical stability and humoral response to PT, the raP vaccine warrants further development as an effective alternative to aP vaccines for the control of a pertussis outbreak.

Phosphodiesterases Mediate the Augmentation of Myogenic Constriction by Inhibitory G Protein Signaling and is Negatively Modulated by the Dual Action of RGS2 and 5.

G protein regulation by regulators of G protein signaling (RGS) proteins play a key role in vascular tone maintenance. The loss of Gi/o and Gq/11 regulation by RGS2 and RGS5 in non-pregnant mice is implicated in augmented vascular tone and decreased uterine blood flow (UBF). RGS2 and 5 are closely related and co-expressed in uterine arteries (UA). However, whether and how RGS2 and 5 coordinate their regulatory activities to finetune G protein signaling and regulate vascular tone are unclear. Here, we determined how the integrated activity of RGS2 and 5 modulates vascular tone to promote UBF. Using ultrasonography and pressure myography, we examined uterine hemodynamics and myogenic tone (MT) of UA of wild type (WT), Rgs2-/-, Rgs5-/-, and Rgs2/5 dbKO mice. We found that MT was reduced in Rgs5-/- relative to WT or Rgs2-/- UA. Activating Gi/o with dopamine increased, whereas exogenous cAMP decreased MT in Rgs5-/- UA to levels in WT UA. Dual deletion of Rgs2 and 5 abolished the reduced MT due to the absence of Rgs5 and enhanced dopamine-induced Gi/o effects in Rgs2/5 dbKO UA. Conversely, and as in WT UA, Gi/o inhibition with pertussis toxin or exogenous cAMP decreased MT in Rgs2/5 dbKO to levels in Rgs5-/- UA. Inhibition of phosphodiesterases (PDE) concentration-dependently decreased and normalized MT in all genotypes, and blocked dopamine-induced MT augmentation in Rgs2-/-, Rgs5-/-, and Rgs2/5 dbKO UA. We conclude that Gi/o augments UA MT in the absence of RGS2 by a novel mechanism involving PDE-mediated inhibition of cAMP-dependent vasodilatation..

Role of Formyl Peptide Receptors and β-Arrestin-1 in suPAR Signal Transduction in Mouse Podocytes: Interactions with αVβ3-Integrin.

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and acute kidney injuries. suPAR can trigger transduction cascades in podocytes by outside-in activation of αVβ3-integrin, but there is evidence that the functional cell surface response element is actually a complex of different types of receptors, which may also include the receptor for advanced glycation end-products (RAGE) and formyl peptide receptors (FPRs). Here we observed that ROS accumulation and Src activation could be evoked by continuous 24 h exposure to either suPAR or the FPR agonist fMLF. Responses to suPAR and fMLF were completely blocked by either the FPR antagonist WRW4 or by the αV-integrin inhibitor cilengitide. Moreover, endogenous podocyte mouse Fpr1 co-immunoprecipitates with β3-integrin, suggesting that these receptors occur as a complex on the cell surface. suPAR- and fMLF-evoked activation of Src and ROS differed in time course. Thus, robust pertussis toxin (PTX)-sensitive responses were evoked by 60 min exposures to fMLF but not to suPAR. By contrast, responses to 24 h exposures to either suPAR or fMLF were PTX-resistant and were instead abolished by knockdown of β-arrestin-1 (BAR1). FPRs, integrins, and RAGE (along with various Toll-like receptors) can all function as pattern-recognition receptors that respond to "danger signals" associated with infections and tissue injury. The fact that podocytes express such a wide array of pattern-recognition receptors suggests that the glomerular filter is designed to change its function under certain conditions, possibly to facilitate clearance of toxic macromolecules.

The Chaperonin TRiC/CCT Inhibitor HSF1A Protects Cells from Intoxication with Pertussis Toxin.

Pertussis toxin (PT) is a bacterial AB5-toxin produced by Bordetella pertussis and a major molecular determinant of pertussis, also known as whooping cough, a highly contagious respiratory disease. In this study, we investigate the protective effects of the chaperonin TRiC/CCT inhibitor, HSF1A, against PT-induced cell intoxication. TRiC/CCT is a chaperonin complex that facilitates the correct folding of proteins, preventing misfolding and aggregation, and maintaining cellular protein homeostasis. Previous research has demonstrated the significance of TRiC/CCT in the functionality of the Clostridioides difficile TcdB AB-toxin. Our findings reveal that HSF1A effectively reduces the levels of ADP-ribosylated Gαi, the specific substrate of PT, in PT-treated cells, without interfering with enzyme activity in vitro or the cellular binding of PT. Additionally, our study uncovers a novel interaction between PTS1 and the chaperonin complex subunit CCT5, which correlates with reduced PTS1 signaling in cells upon HSF1A treatment. Importantly, HSF1A mitigates the adverse effects of PT on cAMP signaling in cellular systems. These results provide valuable insights into the mechanisms of PT uptake and suggest a promising starting point for the development of innovative therapeutic strategies to counteract pertussis toxin-mediated pathogenicity.

Butyrate, Valerate, and Niacin Ameliorate Anaphylaxis by Suppressing IgE-Dependent Mast Cell Activation: Roles of GPR109A, PGE2, and Epigenetic Regulation.

Short-chain fatty acids (SCFAs) are produced by the intestinal microbiota during the fermentation of dietary fibers as secondary metabolites. Several recent studies reported that SCFAs modulate the development and function of immune-related cells. However, the molecular mechanisms by which SCFAs regulate mast cells (MCs) remain unclear. In the current study, we analyzed the function and gene expression of mouse MCs in the presence of SCFAs invitro and invivo. We found that the oral administration of valerate or butyrate ameliorated passive systemic anaphylaxis and passive cutaneous anaphylaxis in mice. The majority of SCFAs, particularly propionate, butyrate, valerate, and isovalerate, suppressed the IgE-mediated degranulation of bone marrow-derived MCs, which were eliminated by the Gi protein inhibitor pertussis toxin and by the knockdown of Gpr109a. A treatment with the HDAC inhibitor trichostatin A also suppressed IgE-mediated MC activation and reduced the surface expression level of FcεRI on MCs. Acetylsalicylic acid and indomethacin attenuated the suppressive effects of SCFAs on degranulation. The degranulation degree was significantly reduced by PGE2 but not by PGD2. Furthermore, SCFAs enhanced PGE2 release from stimulated MCs. The SCFA-mediated amelioration of anaphylaxis was exacerbated by COX inhibitors and an EP3 antagonist, but not by an EP4 antagonist. The administration of niacin, a ligand of GPR109A, alleviated the symptoms of passive cutaneous anaphylaxis, which was inhibited by cyclooxygenase inhibitors and the EP3 antagonist. We conclude that SCFAs suppress IgE-mediated activation of MCs invivo and invitro involving GPR109A, PGE2, and epigenetic regulation.

Intranasal sensitization model of alopecia areata using pertussis toxin as adjuvant.

Nasopharyngeal Bordetella pertussis (BP) colonization is common, with about 5% of individuals having PCR evidence of subclinical BP infection on nasal swab, even in countries with high vaccination rates. BP secretes pertussis toxin (PTx). PTx is an adjuvant commonly used to induce autoimmunity in multiple animal models of human disease. Colocalization of PTx and myelin from myelinated nerves in the nasopharynx may lead to host sensitization to myelin with subsequent autoimmune pathology. C57BL/6J female adult mice were given varied doses and schedules of intranasal PTx, MOG35-55 antigen, or controls to test whether intranasal administration of PTx and myelin oligodendrocyte peptide (MOG35-55) could induce experimental autoimmune encephalomyelitis (EAE) in mice. While we observed systemic cell-mediated immunity against MOG35-55, we did not observe EAE. Unexpectedly, many mice developed alopecia. We systematically investigated this finding. Patchy alopecia developed in 36.4% of mice with the optimized protocol. Pathology consistent with alopecia areata was confirmed histologically by documenting concomitant reduced anagen phase and increased telogen phase hair follicles (HFs) in biopsies from patches of hair loss in mice with alopecia. We also found reduced CD200 staining and increased CD3+T cells surrounding the HFs of mice with alopecia compared to the mice without alopecia, indicating HF Immune Privilege (HFIP) collapse. Systemic immune responses were also found, with increased proportions of activated T cells and B cells, as well as MHCII+ dendritic cells in peripheral blood and/or splenocytes. Finally, in mice initially exposed to intranasal MOG35-55 and PTx in combination, but not to either agent alone, splenocytes were shown to proliferate after in vitro stimulation by MOG35-55. Consistent with prior investigations, PTx exhibited a dose-response effect on immune cell induction and phenotype, with the lowest PTx dose failing to induce autoimmunity, the highest PTx dose suppressing autoimmunity, and intermediate doses optimizing autoimmunity. We propose that this is the first report of an autoimmune disease in an animal model triggered by colocalization of intranasal PTx and autoantigen. This model parallels a natural exposure and potential intranasal sensitization-to-pathology paradigm and supports the plausibility that nasopharyngeal subclinical BP colonization is a cause of alopecia areata.

Intravitreal application of stem cells and their products does not ameliorate experimental autoimmune uveitis

Aims/Purpose: Stem cell therapy has demonstrated potential therapeutic benefits in various medical disciplines. This study evaluated therapeutic effects of mesenchymal stem cells (MSCs) or their by‐products on intraocular inflammation in experimental autoimmune uveitis (EAU) model.Methods: EAU was induced in C57BL/6J mouse strain through administration of IRBP, Freund's adjuvant and pertussis toxin. Conditioned medium was procured through cultivation of MSCs in serum‐free medium, while MSC supernatant was obtained via MSC centrifugation and membrane removal. Either conditioned medium or MSC supernatant was intravitreally administered unilaterally, typically on day 14 post‐EAU induction. Ocular fundus of mice was documented regularly. Experiments were generally concluded on day 28, with eyes processed for FACS and PCR analysis. Infiltration of retina by inflammatory cells (F4/80, CD11c, CD45, CD80, CD86, CD206) and gene expression of pro‐inflammatory factors (Gfap, Iba‐1, IL‐1, iNOS) were quantified.Results: Across all experiments, clinical grading, FACS, and PCR analysis indicated exacerbation of intraocular inflammation. Post‐intravitreal administration of MSC by‐products into eyes with EAU, increased infiltration of retina by inflammatory cells and upregulation in gene expression of pro‐inflammatory factors were observed. These findings were corroborated in vitro, where co‐culturing of retina with EAU in presence of MSCs or their supernatant did not significantly reduce gene expression of pro‐inflammatory factors. Upsurge in pro‐inflammatory factors was also noted in control experiments on healthy eyes post‐intravitreal injection of no substance, serum‐free medium, PBS, or conditioned medium.Conclusions: No improvement in EAU was observed following application of MSC products, both in vivo and in vitro. Instead, inflammation was more severe. Trauma induced by intravitreal application itself significantly influenced outcome.Financial support: GA UK 309621, SVV UK 260631.

Safety assessments of recombinant DTaP vaccines developed in South Korea.

Pertussis bacteria have many pathogenic and virulent antigens and severe adverse reactions have occurred when using inactivated whole-cell pertussis vaccines. Therefore, inactivated acellular pertussis (aP) vaccines and genetically detoxified recombinant pertussis (rP) vaccines are being developed. The aim of this study was to assess the safety profile of a novel rP vaccine under development in comparison to commercial diphtheria-tetanus-acellular pertussis (DTaP) vaccines. The two positive control DTaP vaccines (two- and tri-components aP vaccines) and two experimental recombinant DTaP (rDTaP) vaccine (two- and tri-components aP vaccines adsorbed to either aluminum hydroxide or purified oat beta-glucan) were used. Temperature histamine sensitization test (HIST), indirect Chinese hamster ovary (CHO) cell cluster assay, mouse-weight-gain (MWG) test, leukocytosis promoting (LP) test, and intramuscular inflammatory cytokine assay of the injection site performed for safety assessments. HIST results showed absence of residual pertussis toxin (PTx) in both control and experimental DTaP vaccine groups, whereas in groups immunized with tri-components vaccines, the experimental tri-components rDTaP absorbed to alum showed an ultra-small amount of 0.0066 IU/mL. CHO cell clustering was observed from 4 IU/mL in all groups. LP tests showed that neutrophils and lymphocytes were in the normal range in all groups immunized with the two components vaccine. However, in the tri-components control DTaP vaccine group, as well as two- and tri-components rDTaP with beta-glucan group, a higher monocyte count was observed 3 days after vaccination, although less than 2 times the normal range. In the MWG test, both groups showed changes less than 20% in body temperature and body weight before the after the final immunizations. Inflammatory cytokines within the muscle at the injection site on day 3 after intramuscular injection revealed no significant response in all groups. There were no findings associated with residual PTx, and no significant differences in both local and systemic adverse reactions in the novel rDTaP vaccine compared to existing available DTaP vaccines. The results suggest that the novel rDTaP vaccine is safe.

Brain‐derived neurotrophic factor alleviates clinical symptoms of experimental autoimmune uveoretinitis and supports retinal regeneration

Purpose: Brain‐derived neurotrophic factor (BDNF) is one of the most important neurotrophins that has a fundamental role in the regulation of neuronal growth, proliferation, and regeneration, as well as an important modulatory effect on the immune cells. In addition, BDNF potentiates the neurodifferentiation of Müller cells (MCs), thus supporting retinal regeneration. Therefore, we aimed to investigate the anti‐inflammatory effect of BDNF in experimental autoimmune uveoretinitis (EAU), a severe neuroinflammatory condition, and whether BDNF could facilitate the regenerative capacity of MCs in EAU.Methods: EAU in mice was induced by s.c. administration of retinal antigen IRBP1‐20 emulsified in a complete Freud's adjuvant, followed by i.v. administration of Pertussis toxin. BDNF was administered daily in the form of eye drops from day 8 to day 14 after immunization. On day 14, fundoscopy was performed to assess clinical signs of EAU. Mice were then euthanized, and their eyes and retinas were isolated for further Western blot, RT‐qPCR, flow cytometry, and immunohistochemistry analyses.Results: The treatment with BDNF significantly improved the clinical manifestations of EAU, including optic disc oedema, vasculitis, and inflammatory infiltrates in the retina. BDNF significantly decreased the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ myeloid cells into the retina and also suppressed the expression of several pro‐inflammatory cytokines and chemokines. In addition, BDNF administration significantly increased the expression of markers of neurogenesis in retinal MCs.Conclusions: Together, our results demonstrate that BDNF has important anti‐inflammatory and neurogenic properties and that it may serve as a promising target for developing novel therapeutic strategies for neurodegenerative and neuroinflammatory diseases of the retina.Support.This study was supported by the following grant projects: GAUK 378421, 260 648/SVV/2023, and COOPERATIO Neurosciences.

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to wild-type mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Genomic characterization of Bordetella pertussis in South Africa, 2015-2019.

Pertussis remains a public health concern in South Africa, with an increase in reported cases and outbreaks in recent years. Whole genome sequencing was performed on 32 Bordetella pertussis isolates sourced from three different surveillance programmes in South Africa between 2015 and 2019. Genome sequences were characterized using multilocus sequence typing, vaccine antigen genes (ptxP, ptxA, ptxB, prn and fimH) and overall genome structure. All isolates were sequence type 2 and harboured the pertussis toxin promoter allele ptxP3. The dominant genotype was ptxP3-ptxA1-ptxB2-prn2-fimH2 (31/32, 96.9 %), with no pertactin-deficient or other mutations in vaccine antigen genes identified. Amongst 21 isolates yielding closed genome assemblies, eight distinct genome structures were detected, with 61.9 % (13/21) of the isolates exhibiting three predominant structures. Increases in case numbers are probably not due to evolutionary changes in the genome but possibly due to other factors such as the cyclical nature of B. pertussis disease, waning immunity due to the use of acellular vaccines and/or population immunity gaps.

G protein‑mediated EGFR transactivation is a common mechanism through which the CXCL12 receptors, CXCR4 and CXCR7, control human cancer cell migration.

C‑X‑C motif chemokine 12 (CXCL12) promotes metastasis of several tumors by affecting cell migration and invasion via its receptors, C‑X‑C chemokine receptor type (CXCR)4 and CXCR7. Current therapeutic approaches focus on the selective inactivation of either CXCR4 or CXCR7 in patients with cancer. Alternative strategies may emerge from the analysis of downstream events that mediate the migratory effects of CXCL12 in cancer cells. While CXCR4 activates cell signaling through both G proteins and arrestins, CXCR7 is believed to preferentially signal through arrestins. The present study analyzed the CXCL12‑dependent chemotaxis of A549, C33A, DLD‑1, MDA‑MB‑231 and PC‑3 cells, in which either the activity of G proteins, EGFR or Src kinase was inhibited pharmacologically or the expression of arrestins was inhibited by RNA interference. The results demonstrated that CXCL12‑induced migration of A549, C33A, DLD‑1, MDA‑MB‑231 and PC‑3 cells was attenuated by the Gαi/o‑inhibitor pertussis toxin (PTX), but was unaffected by small interfering RNA‑mediated gene silencing of β‑arrestin1/2. In particular, the sensitivity of DLD‑1 migration to PTX was unexpected, as it is solely dependent on the non‑classical chemokine receptor, CXCR7. Furthermore, chemotactic responses to CXCL12 were additionally prevented by inhibiting EGFR activity via AG1478 and Src kinase activity via Src inhibitor‑1. In conclusion, the results of the present study suggest that G protein‑ and Src‑dependent transactivation of EGFR is a common mechanism through which CXCL12‑bound CXCR4 and/or CXCR7 control cancer cell migration and metastasis. These findings highlight EGFR as a potential therapeutic target that interferes with CXCL12‑induced cancer expansion.

Butyrate reduces adherent-invasive E. coli-evoked disruption of epithelial mitochondrial morphology and barrier function: involvement of free fatty acid receptor 3

ABSTRACT Gut bacteria provide benefits to the host and have been implicated in inflammatory bowel disease (IBD), where adherent-invasive E. coli (AIEC) pathobionts (e.g., strain LF82) are associated with Crohn’s disease. E. coli-LF82 causes fragmentation of the epithelial mitochondrial network, leading to increased epithelial permeability. We hypothesized that butyrate would limit the epithelial mitochondrial disruption caused by E. coli-LF82. Human colonic organoids and the T84 epithelial cell line infected with E. coli-LF82 (MOI = 100, 4 h) showed a significant increase in mitochondrial network fission that was reduced by butyrate (10 mM) co-treatment. Butyrate reduced the loss of mitochondrial membrane potential caused by E. coli-LF82 and increased expression of PGC-1 α mRNA, the master regulator of mitochondrial biogenesis. Metabolomics revealed that butyrate significantly altered E. coli-LF82 central carbon metabolism leading to diminished glucose uptake and increased succinate secretion. Correlating with preservation of mitochondrial network form/function, butyrate reduced E. coli-LF82 transcytosis across T84-cell monolayers. The use of the G-protein inhibitor, pertussis toxin, implicated GPCR signaling as critical to the effect of butyrate, and the free fatty acid receptor three (FFAR3, GPR41) agonist, AR420626, reproduced butyrate’s effect in terms of ameliorating the loss of barrier function and reducing the mitochondrial fragmentation observed in E. coli-LF82 infected T84-cells and organoids. These data indicate that butyrate helps maintain epithelial mitochondrial form/function when challenged by E. coli-LF82 and that this occurs, at least in part, via FFAR3. Thus, loss of butyrate-producing bacteria in IBD in the context of pathobionts would contribute to loss of epithelial mitochondrial and barrier functions that could evoke disease and/or exaggerate a low-grade inflammation.

PEPITEM Treatment Ameliorates EAE in Mice by Reducing CNS Inflammation, Leukocyte Infiltration, Demyelination, and Proinflammatory Cytokine Production.

To investigate the effect of the therapeutic treatment of the immunopeptide, peptide inhibitor of trans-endothelial migration (PEPITEM) on the severity of disease in a mouse model of experimental autoimmune encephalomyelitis (EAE) as a model for human multiple sclerosis (MS), a series of experiments were conducted. Using C57BL/6 female mice, we dosed the PEPITEM in the EAE model via IP after observing the first sign of inflammation. The disease was induced using MOG35-55 and complete Freund's adjuvants augmented with pertussis toxin. The EAE score was recorded daily until the end of the experiment (21 days). The histological and immunohistochemistry analysis was conducted on the spinal cord sections. A Western blot analysis was performed to measure the protein concentration of MBP, MAP-2, and N-Cadherin, and ELISA kits were used to measure IL-17 and FOXP3 in the serum and spinal cord lysate. The therapeutic treatment with PEPITEM reduced the CNS infiltration of T cells, and decreased levels of the protein concertations of MBP, MAP-2, and N-Cadherin were observed, in addition to reduced concertations of IL-17 and FOXP3. Using PEPITEM alleviated the severity of the symptoms in the EAE model. Our study revealed the potential of PEPITEM to control inflammation in MS patients and to reduce the harmful effects of synthetic drugs.

Effective and safe transfer of maternal antibodies persisting two months postpartum following maternal immunization with different doses of recombinant pertussis-containing vaccines

IntroductionRecombinant acellular pertussis (ap) vaccines containing genetically inactivated pertussis toxin (PTgen) and filamentous hemagglutinin (FHA) with or without tetanus (TT) and diphtheria (DT) vaccines (Td) were found safe and immunogenic in non-pregnant and pregnant women. We report here maternal antibody transfer and safety data in mothers and neonates. MethodsThis is the follow up of a phase 2 trial in 2019 among 400 pregnant women who randomly received one dose of recombinant pertussis-only vaccine containing 1 µg PTgen and 1 µg FHA (ap1gen), or Td combined with ap1gen (Tdap1gen), or with 2 µg PTgen and 5 µg FHA (Tdap2gen), or with 5 µg PTgen and 5 µg FHA (TdaP5gen, Boostagen®, BioNet, Thailand) or chemically-inactivated acellular pertussis comparator (Tdap8chem, Boostrix™, GSK, Belgium), either in the second or third trimester of gestation. IgG against PT, FHA, TT and DT were assessed by ELISA, PT-neutralizing antibodies (PTNA) by Chinese Hamster Ovary cell assay and safety outcomes at delivery in mothers and at birth. ResultsAnti-PT and anti-FHA geometric mean concentration (GMC) ratio between infants at birth and mothers at delivery was above 1 in all groups. PT GMC in infants at birth were ≥30 IU/mL in all groups with the highest titers in infants found in TdaP5gen group at birth (118.8 [95% CI 93.9–150.4]). At 2 months, PT GMC ratio to Tdap8chem (98.75% CI) was significantly higher for TdaP5gen (2.6 [1.7–4.0]) and comparable for other recombinant vaccines. No difference in PTNA titers at birth was observed between all groups nor between time of vaccination. Adverse events were comparable in all vaccine groups. ConclusionsBioNet licensed (TdaP5gen and Tdap2gen) and candidate vaccines (Tdap1gen and ap1gen) when given to pregnant women in the second or third trimester of gestation are safe and have induced passive pertussis immunity to infants.