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Porcine epidemic diarrhea virus (PEDV) is a harmful coronavirus infecting pigs, which is resulting in substantial financial losses in the global pig industry. The lack of effective vaccines or treatments underscores the pressing need for new antiviral strategies. Antimicrobial peptides (AMPs), specifically cathelicidins such as LL-37, have demonstrated promising activity against a range of viruses. This study aims to elucidate the antiviral mechanisms of cathelicidins by examining their inhibitory capabilities against PEDV in vitro. Four pig-derived antimicrobial peptides (PMAP-36, PMAP-23, PR-39, and PG-1), together with chicken-derived CATH-B1 and human-derived LL-37 were analyzed for their anti-PEDV activity. Flow cytometry and fluorescent microscopy confirmed that LL-37 and CATH-B1 had strong inhibitory effects at non-toxic concentrations of 5 and 10 µM, significantly reducing GFP-PEDV infection of Vero cells both in co- and pre-incubation setups. In contrast, none of the porcine peptides exhibited any inhibitory effects, even at higher doses. Fluorogenic LL-37 was shown to enter VERO cells, indicative of a possible immunomodulatory antiviral mode of action. However, transmission electron microscopy clearly indicated that both LL-37 and CATH-B1 affected virus morphology and caused aggregation of viral particles, showing that peptide-virus interaction caused reduced virus infectivity. In conclusion, this analysis highlights the potential of LL-37 and CATH-B1 as inhibitors against PEDV, suggesting promising directions for innovative therapeutic antiviral strategies., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Natural killer (NK) cells are cytotoxic lymphocytes that are present in the circulation but also in many organs including spleen and gut, where they play an important role in the defense against infections. Interaction of NK cells with target cells leads to degranulation, which results in the release of perforin and granzymes in the direct vicinity of the target cell. Chicken NK cells have many characteristics similar to their mammalian counterparts and based on similarities with studies on human NK cells, surface expression of CD107 was always presumed to correlate with granule release. However, proof of this degranulation or in fact the actual presence of perforin (PFN) and granzyme A (GrA) in chicken NK cells and their release upon activation is lacking. Therefore, the purpose of the present study was to determine the presence of perforin and granzyme A in primary chicken NK cells and to measure their release upon degranulation, as an additional tool to study the function of chicken NK cells. Using human specific antibodies against PFN and GrA in fluorescent and confocal microscopy resulted in staining in chicken NK cells. The presence of PFN and GrA was also confirmed by Western blot analyses and its gene expression by PCR. Stimulation of NK cells with the pectin SPE6 followed by flow cytometry resulted in reduced levels of intracellular PFN and GrA, suggesting release of PFN and GrA. Expression of PFN and GrA reversely correlated with increased surface expression of the lysosomal marker CD107. Finally it was shown that the supernatant of activated NK cells, containing the NK cell granule content including PFN and GrA, was able to kill Escherichia coli. This study correlates PFN and GrA release to activation of chicken NK cells and establishes an additional tool to study activity of cytotoxic lymphocytes in chickens., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Vaccination is commonly used to protect dogs against leptospirosis, however, memory immune responses induced by canine Leptospira vaccines have not been studied. In the present study, antibody and T cell mediated responses were assessed in dogs before and 2 weeks after annual revaccination with a commercial tetravalent Leptospira vaccine containing serogroups Canicola and Australis. Vaccination significantly increased average log 2 IgG titers from 6.50 to 8.41 in year 1, from 5.99 to 7.32 in year 2, from 5.32 to 8.32 in year 3 and from 5.32 to 7.82 in year 4. The CXCL-10 levels, induced by in vitro stimulation of PBMC with Canicola and Australis, respectively, significantly increased from 1039.05 pg/ml and 1037.38 pg/ml before vaccination to 2547.73 pg/ml and 2730.38 pg/ml after vaccination. IFN-γ levels increased from 85.60 pg/ml and 178.13 pg/ml before vaccination to 538.62 pg/ml and 210.97 pg/ml after vaccination. The percentage of proliferating CD4 + T cells in response to respective Leptospira strains significantly increased from 1.43 % and 1.25 % before vaccination to 24.11 % and 14.64 % after vaccination. Similar responses were also found in the CD8 + T cell subset. Vaccination also significantly enhanced the percentages of central memory CD4 + T cells from 12 % to 26.97 % and 27.65 %, central memory CD8 + T cells from 3 % to 9.47 % and 7.55 %, and effector CD8 + T cells from 3 % to 7.6 % and 6.42 %, as defined by the expression of CD45RA and CD62L, following stimulation with Canicola and Australis, respectively. Lastly, enhanced expression of the activation marker CD25 on T cells after vaccination was found. Together, our results show that next to IgG responses, also T cell responses are induced in dogs upon annual revaccination with a tetravalent Leptospira vaccine, potentially contributing to protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Safety and potency assessment for batch release testing of established vaccines still relies partly on animal tests. An important avenue to move to batch release without animal testing is the consistency approach. This approach is based on thorough characterization of the vaccine to identify critical quality attributes that inform the use of a comprehensive set of non-animal tests to release the vaccine, together with the principle that the quality of subsequent batches follows from their consistent production. Many vaccine antigens are by themselves not able to induce a protective immune response. The antigens are therefore administered together with adjuvant, most often by adsorption to aluminium salts. Adjuvant function is an important component of vaccine potency, and an important quality attribute of the final product. Aluminium adjuvants are capable of inducing NLRP3 inflammasome activation. The aim of this study was to develop and evaluate an in vitro assay for NLRP3 inflammasome activation by aluminium-adjuvanted vaccines. We evaluated the effects of Diphtheria-Tetanus-acellular Pertussis combination vaccines from two manufacturers and their respective adjuvants, aluminium phosphate (AP) and aluminium hydroxide (AH), in an in vitro assay for NLRP3 inflammasome activation. All vaccines and adjuvants tested showed a dose-dependent increase in IL-1β production and a concomitant decrease in cell viability, suggesting NLRP3 inflammasome activation. The results were analysed by benchmark dose modelling, showing a similar 50% effective dose (ED50) for the two vaccine batches and corresponding adjuvant of manufacturer A (AP), and a similar ED50 for the two vaccine batches and corresponding adjuvant of manufacturer B (AH). This suggests that NLRP3 inflammasome activation is determined by the adjuvant only. Repeated freeze-thaw cycles reduced the adjuvant biological activity of AH, but not AP. Inflammasome activation may be used to measure adjuvant biological activity as an important quality attribute for control or characterization of the adjuvant., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Most traditional vaccines are administered via the intramuscular route. Other routes of administration however, can induce equal or improved protective memory responses and might provide practical advantages such as needle-free immunization, dose sparing and induction of tissue-specific (mucosal) immunity. Here we explored the differences in immunological outcome after immunization with model antigens via two promising immunization routes (intradermal and intranasal) with or without the experimental adjuvant and TLR7/8-agonist R848. Because the adaptive immune response is largely determined by the local innate cells at the site of immunization, the effect of R848-adjuvation on local cellular recruitment, antigenic uptake by antigen-presenting cells and the initiation of the adaptive response were analyzed for the two routes of administration. We show a general immune-stimulating effect of R848 irrespective of the route of administration. This includes influx of neutrophils, macrophages and dendritic cells to the respective draining lymph nodes and an increase in antigen-positive antigen-presenting cells which leads for both intradermal and intranasal immunization to a mainly T H 1 response. Furthermore, both intranasal and intradermal R848-adjuvated immunization induces a local shift in DC subsets; frequencies of CD11b + DC increase whereas CD103 + DC decrease in relative abundance in the draining lymph node. In spite of these similarities, the outcome of immune responses differs for the respective immunization routes in both magnitude and cytokine profile. Via the intradermal route, the induced T-cell response is higher compared to that after intranasal immunization, which corresponds with the local higher uptake of antigen by antigen-presenting cells after intradermal immunization. Furthermore, R848-adjuvation enhances ex vivo IL-10 and IL-17 production after intranasal, but not intradermal, T-cell activation. Quite the opposite, intradermal immunization leads to a decrease in IL-10 production by the vaccine induced T-cells. This knowledge may lead to a more rational development of novel adjuvanted vaccines administered via non-traditional routes., (Copyright © 2018. Published by Elsevier Ltd.)

Vaccines often contain adjuvants to strengthen the response to the vaccine antigen. However, their modes of action at the site of injection (SOI) are poorly understood. Therefore, we assessed the local effects of adjuvant on the innate immune system in mice. We investigated the safe, widely used adjuvants MF59 and aluminum hydroxide (alum), as well as trehalose-6,6'-dibehenate (TDB), Complete Freund's Adjuvant (CFA) and the Toll-Like-Receptor-ligands lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam 3 CSK 4 ). We assessed muscle immune cell infiltration after adjuvant injection and observed 16h post immunization (hpi) an increased influx with CFA, MF59 and TDB, but not with alum, LPS or Pam 3 CSK 4 . An elevated influx with the latter three became visible only 72hpi. Contribution of granulocytes, macrophages and dendritic cells to the influx differed per adjuvant and in time. Adjuvants generally induced a local pro-inflammatory micro-milieu that was transient except for CFA and TDB. The gene expression of CXCL-1, CCL-2 and CCL-5, involved in recruitment of immune cells, varied per adjuvant and corresponded grossly with the observed influx of granulocytes and monocytes/macrophages. Muscles injected with CFA or MF59 (when co-injected with peptide) resulted in APC ex vivo capable to induce proliferation of peptide-specific T-cells. By adding in vitro an excess of peptide to the APC/T cell co-cultures, we observed an adjuvant-enhanced co-stimulation or antigen presentation by APC after CFA- but not MF59-injection. After TDB-injection this effect was observed only at 72hpi, but not 24hpi. Thus the cellular influx profile and the local cytokine and chemokine micro-milieu in the muscle were strongly influenced by the type of adjuvant. Additionally, the capacity of muscle APC to load and present antigen was affected by the adjuvant. These findings may assist the development of novel adjuvanted vaccines in a more rational manner., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Nasal vaccination is considered to be a promising alternative for parenteral vaccination against influenza virus as it is non-invasive and offers the opportunity to elicit strong antigen-specific responses both systemic and locally at the port of entry of the pathogen. Previous studies showed that non-living bacterium-like particles (BLPs) from the food-grade bacterium Lactococcus lactis are effective stimulators of local and systemic immune responses when administered intranasally. Moreover, in vitro, BLPs specifically interact with human Toll-like receptor 2 (TLR2), suggestive of a role for TLR2 dependent immune activation by BLPs., Methods: In the present study, we examined the role of TLR2 in vivo in immune activation after nasal administration of BLP mixed with split influenza vaccine (BLP-SV) of influenza A virus (IAV) using TLR2 knockout mice., Results: The systemic Th1 cell and subsequent B-cell responses induced after intranasal BLP-SV vaccination depended on the interaction of BLPs with TLR2. Notably, the BLP-SV-induced class switch to IgG2c depended on the interaction of BLP with TLR2. Local induced IAV-specific Th1 cell responses and the mucosal B-cell responses also depended on interaction of BLP with TLR2. Strongly reduced SIgA levels were observed in TLR2 knockout mice both in the nasal and vaginal lavages. In addition, detailed analysis of the T-cell response revealed that nasal BLP-SV vaccination promoted Th1/Th17 immune responses that coincided with increased IAV-specific IgG2c antibody production., Discussion: Altogether these results indicate that nasal BLP-SV vaccination induces IAV-specific T-cell and B-cell responses, both systemically and at the site of virus entry in a TLR2-dependent manner., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Rheumatoid arthritis (RA) is a chronic T-cell mediated autoimmune disease that affects primarily the joints. The induction of immune tolerance through antigen-specific therapies for the blockade of pathogenic CD4+ T cells constitutes a current focus of research. In this focus it is attempted to simultaneously activate multiple regulatory mechanisms, such as: apoptosis and regulatory T cells (Tregs). APL-1 is an altered peptide ligand derived from a novel CD4+ T-cell epitope of human heat-shock protein of 60kDa, an autoantigen involved in the pathogenesis of RA. Previously, we have reported that APL-1 induces CD4+ CD25(high)Foxp3+ Tregs in several systems. Here, we investigated the ability of APL-1 in inducing apoptosis in PBMCs from RA patients, who were classified as active or inactive according to their DAS28 score. APL-1 decreased the viability of PBMCs from active but not from inactive patients. DNA fragmentation assays and typical morphological features clearly demonstrated that APL-1 induced apoptosis in these cells. Activated CD4+ CD25+ T cells but not resting CD4+ CD25- T cells were identified as targets of APL-1. Furthermore, CD4+ T-cell responses to APL-1 were found to be dependent on antigen presentation via the HLA-DR molecule. Thus, APL-1 is a regulatory CD4+ T cell epitope which might modulate inflammatory immune responses in PBMCs from RA patients by inducing CD4+ CD25(high)Foxp3+ Tregs and apoptosis in activated CD4+ T cells. These results support further investigation of this candidate drug for the treatment of RA., (© 2013 Elsevier B.V. All rights reserved.)

Nasal vaccination is a promising, needle-free alternative to classical vaccination. Nanoparticulate delivery systems have been reported to overcome the poor immunogenicity of nasally administered soluble antigens, but the characteristics of the ideal particle are unknown. This study correlates differences in physicochemical characteristics of nanoparticles to their adjuvant effect, using ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) nanoparticles (PLGA NP), N-trimethyl chitosan (TMC) based NP (TMC NP) and TMC-coated PLGA NP (PLGA/TMC NP). PLGA NP and PLGA/TMC NP were prepared by emulsification/solvent extraction and TMC NP by ionic complexation. The NP were characterized physicochemically. Their toxicity and interaction with and stimulation of monocyte derived dendritic cells (DC) were tested in vitro. Furthermore, the residence time and the immunogenicity (serum IgG titers and secretory IgA levels in nasal washes) of the nasally applied OVA formulations were assessed in Balb/c mice. All NP were similar in size, whereas only PLGA NP carried a negative zeta potential. The NP were non-toxic to isolated nasal epithelium. Only TMC NP increased the nasal residence time of OVA compared to OVA administered in PBS and induced DC maturation. After i.m. administration all NP systems induced higher IgG titers than OVA alone, PLGA NP and TMC NP being superior to PLGA/TMC NP. Nasal immunization with the slow antigen releasing particles, PLGA NP and PLGA/TMC NP, did not induce detectable antibody titers. In contrast, nasal immunization with the positively charged, fast antigen releasing TMC NP led to high serum antibody titers and sIgA levels. In conclusion, particle charge and antigen release pattern of OVA-loaded NP has to be adapted to the intended route of administration. For nasal vaccination, TMC NP, releasing their content within several hours, being mucoadhesive and stimulating the maturation of DC, were superior to PLGA NP and PLGA/TMC NP which lacked some or all of these characteristics., ((c) 2010 Elsevier Ltd. All rights reserved.)

Chronically food-deprived male rats, when paired with a female rat in heat, were rewarded by receiving a food pellet following each intromission. Following castration there was a rapid decline in all aspects of male sexual behavior: after 3 weeks all sex behavior had stopped. There were no differences between the conditioned males and their yoked controls. Substitution with two doses of testosterone (through silastic implants) restored sexual behavior, but equally so in the conditioned and the control animals. Removal of the testosterone implant again caused a very rapid decline in sexual behavior, no differences between experimental and yoked control males. These results suggest that food, as a non-sexual stimulus, does not cause hungry male rats to continue to copulate for prolonged periods following castration. Furthermore, the combination of chronic food deprivation plus castration do summate with each other in the very rapid decline and cessation of male sexual performance.