Splenocyte Proliferation Research Articles

A Self-Amplifying Human Papillomavirus 16 Vaccine Candidate Delivered by Tobacco Mosaic Virus-Like Particles.

Virus-like particles (VLPs) are naturally occurring delivery platforms with potential for mRNA vaccines that can be used as an alternative to lipid nanoparticles. Here we describe a self-amplifying mRNA vaccine based on tobacco mosaic virus (TMV) expressing a mutated E7 protein from human papillomavirus 16 (HPV16). E7 is an early gene that plays a central role in viral replication and the oncogenic transformation of host cells, but nononcogenic mutant E7 proteins can suppress this activity. Immunization studies involving the delivery of self-amplifying mutant E7 mRNA packaged with TMV coat proteins confirmed the elicitation of E7-specific IgG antibodies. Additional in vitro splenocyte proliferation and cytokine profiling assays indicated the activation of humoral and cellular immune responses. We conclude that TMV particles are suitable for the delivery of mRNA vaccines and can preserve their integrity and functionality in vitro and in vivo.

Nitric oxide-producing monocyte-myeloid suppressor cells expand and accumulate in the spleen and mesenteric lymph nodes of Yersinia enterocolitica-infected mice.

Yersinia enterocolitica (Ye) is a Gram-negative bacterium that causes gastrointestinal infections. The myeloid-derived suppressor cells (MDSCs) constitute a cellular population with the capacity of inducing the specific suppression of T cells. Although there is evidence supporting the role of MDSCs in controlling the immune responses in several bacterial infections, its role during Ye infection has not yet been reported. Therefore, the purpose of the present work was to analyze MDSCs after oral Ye infection. C57BL/6 wild-type mice were infected with Ye WAP-314 serotype O:8. The proliferation of splenocytes and mesenteric lymph nodes (MLN) cells was measured as well as the levels of cytokines and nitric oxide (NO) in culture supernatants. The frequency and subsets of MDSCs were analyzed in the intestinal mucosa and spleen by flow cytometry. Furthermore, monocytic-MDSCs (Mo-MDSCs) and polymorphonuclear-MDSCs (PMN-MDSCs) were purified from the spleen of infected mice and their suppressor activity was evaluated in co-cultures with purified T cells. we observed a marked expansion of CD11b+Gr-1+ cells, a phenotype consistent with MDSCs, in the spleen and intestinal mucosa of Ye-infected mice. Interestingly, a robust proliferation of splenocytes and MLN cells was observed only when the MDSCs were depleted or the NO production was blocked. In addition, we determined that only Mo-MDSCs had the ability to suppress T-cell proliferation. Our results highlight a mechanism by which Ye may induce suppression of the immune responses. We suggest that NO-producing Mo-MDSCs expand and accumulate in MLN and spleen of Ye-infected mice. These cells can then suppress the T-cell function without interfering with the anti-bacterial effector response. Instead, these immature myeloid cells may perform an important function in regulating the inflammatory response and protecting affected tissues.

Mannose targeting of poly(lactic-co-glycolic acid): a promising approach for improving sublingual allergen-specific immunotherapy

Objective One of the most effective treatments for allergic respiratory diseases is allergen-specific sublingual immunotherapy (SLIT). While, mannose targeting has been applied in various immunostimulatory approaches, but it has not been investigated in sublingual allergen-specific immunosuppressive treatment. This study assesses mannose targeting for the ovalbumin (Ova) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles(NPs). Methods The emulsion-solvent evaporation method was employed for the synthesis of PLGA NPs containing Ova, and subsequently they attached to D-mannose. Ova-sensitized mice underwent treatment in different ways: subcutaneous administration of 10 µg Ova, sublingual administration of 5 and 10 µg Ova loaded in PLGA NPs, 5 and 10 µg Ova loaded in mannose-targeted PLGA NPs, 10 µg Ova, and 10 µg Ova loaded in dendritic cell-specific aptamer-attached PLGA NPs. Serum Ova-specific IgE and IgG2a levels, as well as IFN-γ, IL-4, IL-10, and IL-17a levels in the supernatant of Ova-stimulated splenocytes were measured. Splenocyte proliferation was assessed using an MTT assay, and also lung histological examinations, and nasal lavage fluid cell counting were performed. Results Ova-specific IgE, IL-4, IL-17a levels, eosinophil cell count, and splenocyte proliferation were remarkably reduced in the mice treated with mannose or aptamer targeted NPs compared to other groups. Also, IL-10 and IFN-γ levels were remarkably increased in the targeted NPs groups. Conclusion Our findings indicated that mannose targeting of PLGA NPs could decrease allergen dose and improve immunomodulatory effects of SLIT. However, this approach suggests an effective formulation for SLIT in the mice model, further studies with common allergens are needed for application in humans.

Development of fast-dissolving sublingual nanofibers containing allergen and curcumin for immune response modulation in a mouse model of allergic rhinitis.

Curcumin (CUR) has been considered a potential therapeutic agent for allergic reactions due to its antioxidant and anti-inflammatory activities. Nanofibers have attracted increasing attention in drug delivery. The aim of this study was to investigate the combined therapeutic effects of curcumin and allergen in nanofiber-based treatments in order to increase the effectiveness of sublingual immunotherapy (SLIT) efficacy in a mouse model of allergic rhinitis. Nanofibers containing CUR (1.25% and 2.5%) and ovalbumin 2% (OVA) as an allergen were prepared via electrospinning and characterized. BALB/c mice were sensitized with OVA to the induced allergic rhinitis model. SLIT with free and/or nanofibers was carried out. IL-4, INF-γ, and IgE serum levels were measured using ELISA. Splenocyte proliferation was evaluated by the MTT assay. Lung and nasal histological examinations and nasal lavage fluid (NALF) cell counting were carried out. Nanofibers containing 1.25% CUR and 2% OVA were chosen as the optimal formulations. SLIT treatment with the CUR and OVA nanofiber co-administration led to a significantly decreased serum IgE. Nanofiber containing 2.5 µg of CUR/mouse combined with OVA nanofiber showed a significant decrease in IL-4 and an increase in IFN-γ compared to other groups. NALF assessment showed a significant decrease in specific cell and eosinophil counts in the treated nanofiber groups. The histopathological results of NAL in the optimal formulations were near normal, with diminished cellular infiltration and inflammation. Our findings suggest that co-sublingual administration of allergen and CUR nanofibers can be considered as potential immunomodulatory agents.

Immunomodulatory properties of splenocytes treated with chlorpromazine in experimental aggression

Aggressive behavior is considered to be as one of the central symptoms of many neuropsychiatric disorders. It is a serious medical and biological problem associated both with high frequency and severity of manifestations and lack of selective correction means. The purpose of this study was to evaluate the functional phenotype of immune cells treated with chlorpromazine in aggressive animals in vitro, as well as the effect of transplantation of these cells on syngeneic aggressive recipient’s immune cells functional activity. Aggressive behavior was formed in active mice as a result of repeated experience of victories in agonistic interactions with animals with a submissive partner. Further, aggressive animals were isolated into individual cells and used as donors and recipients of immune cells. Immune cells were obtained under sterile conditions from suspension of spleen cells precultured with chlorpromazine. The level of spontaneous and mitogen-induced cell proliferation was assessed using a standard method of radioactive label incorporation. The quantitative content of cytokines in samples of treated with chlorpromazine cell cultures supernatant was determined by enzyme immunoassay using appropriate test systems. Further aggressive syngeneic recipients were intravenously injected with splenocytes precultured with chlorpromazine. The control group of animals was injected with splenocytes precultured under similar conditions, without the chlorpromazine addition. The recipients were assessed for the spleen cells proliferative activity and the intensity of humoral and cellular immune response using standard methods, by the number of antibody-forming spleen cells and severity of a delayed-type hypersensitivity reaction in response to T-dependent antigen introduction. It was found that treatment with chlorpromazine in vitro suppressed mitogen-stimulated proliferation of splenocytes in aggressive mice, without changing of spontaneous proliferation. It was also accompanied by some cytokines production decrease: IL-6, IL-2 and IFNã. When studying the humoral and cellular immune response intensity in aggressive recipients after transplantation of donor’s syngeneic splenocytes treated with chlorpromazine, a decrease in the intensity of humoral immune response was recorded. Transplantation of donor’s splenocytes treated with chlorpromazine was also accompanied by a decrease in spontaneous and mitogen-stimulated proliferation of splenocytes from aggressive recipients. Thus, the obtained data indicate the inhibitory effect of chlorpromazine on immune cell’s functional activity in aggressive mice, as well as the positive immunomodulatory effect of chlorpromazine-treated immune cells transplantation at aggressive behavior in experimental animals.

In-utero transfer of decidualized endometrial stromal cells increases the frequency of regulatory T cells and normalizes the abortion rate in the CBA/J × DBA/2 abortion model.

Failure to adequate decidualization leads to adverse pregnancy outcomes including pregnancy loss. Although there are plenty of reports underscoring immune dysfunction as the main cause of abortion in CBA/J females mated with DBA/2 males (CBA/J × DBA/2), little is known about the potential role of impaired endometrial decidualization. Endometrial stromal cells (ESCs) from CBA/J mice were in-vitro decidualized, and the proteome profile of the secretome was investigated by membrane-based array. CBA/J mice were perfused In-utero with either decidualized ESCs (C×D/D), undecidualized ESCs (C×D/ND), or PBS (C×D/P) 12 days before mating with DBA/2 males. Control mice were not manipulated and were mated with male DBA/2 (C×D) or Balb/c (C×B) mice. On day 13.5 of pregnancy, reproductive parameters were measured. In-vivo tracking of EdU-labeled ESCs was performed using fluorescence microscopy. The frequency of regulatory T cells (Tregs) in paraaortic/renal and inguinal lymph nodes was measured by flow cytometry. The proliferation of pregnant CBA/J splenocytes in response to stimulation with DBA/2 splenocytes was assessed by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometry. In C×D/D mice, the resorption rate was reduced to match that seen in the C×B group. Intrauterine perfused ESCs appeared in uterine stroma after 2 days, which remained there for at least 12 days. There was no difference in the number of implantation sites and embryo weight across all groups. The frequency of Tregs in the inguinal lymph nodes was similar across all groups, but it increased in the paraaortic/renal lymph nodes of C×D/D mice to the level found in C×B mice. No significant changes were observed in the proliferation of splenocytes from pregnant C×D/D compared to those of the C×D group in response to stimulation with DBA/2 splenocytes. Decidualization of ESCs was associated with a profound alteration in ESC secretome exemplified by alteration in proteins involved in extracellular matrix (ECM) remodeling, response to inflammation, senescence, and immune cell trafficking. Our results showed that the deficiency of Tregs is not the primary driver of abortion in the CBA/J × DBA/2 model and provided evidence that impaired endometrial decidualization probably triggers endometrial immune dysfunction and abortion in this model.

Investigation of Folate-Functionalized Magnetic-Gold Nanoparticles Based Targeted Drug Delivery for Liver: In Vitro, In Vivo and Docking Studies.

Magnetic nanoparticles used for targeted drug administration present a promising approach in cancer treatment owing to its notable advantages, such as targeted and enhanced encapsulation ability and improved bio protection compared with conventional drug delivery methods. Au shell-iron core nanoparticles (Fe3O4@Au) were manufactured by a chemical process, coated with dextran to encapsulate curcumin, and functionalized for precision drug delivery using folic acid to combat liver cancer. Dynamic light scattering, scanning electron microscopy, transmission electron microscopy, vibrational spectroscopy, and magnetometry were applied to assess the synthesis of the Fe3O4@Au-DEX-CU-FA compound. The mean size, zeta potential, and polydispersity of Fe3O4@Au-DEX-CU-FA were 63.3 ± 2.33 nm, -68.3 ± 1.78 mV, and 0.041 ± 0.008, respectively. Molecular docking models were created to examine the relationship between Fe3O4@Au-CU and BCL-XL, BAK, and to identify potential binding sites. The loading efficiency and release profile tests examined the medication delivery system's ability. MTT assay was subsequently utilized to determine the optimal dosage and therapeutic efficacy of Fe3O4@Au-DEX-CU-FA on cancer SNU-449 and healthy THLE-2 cell lines. Flow cytometry demonstrated that Fe3O4@Au-DEX-CU-FA effectively induced cancer cell death. Fe3O4@Au-DEX-FA showed a regulated release profile of free curcumin at 37 °C and pH values of 7.4 and 5.4. Real-time PCR revealed increased BAK expression and decreased BCL-XL expression. Nude tumor-bearing mice were used for in vivo experiments. Fe3O4@Au-DEX-CU-FA treatment dramatically reduced the swelling size compared with free CU and control treatments. It also resulted in a longer lifespan, expanded splenocyte proliferation, increased IFN-γ levels, and decreased IL-4 levels. The regular cells showed no cytotoxic effect compared with the cancer type, confirming that Fe3O4@Au-DEX-CU-FA maintained its potent anticancer actions. The data suggests that Fe3O4@Au-DEX-CU-FA possesses a promising potential as a therapeutic agent for combating tumors.

The Effects of Mesenchymal Stem Cells Loaded with Oncolytic Coxsackievirus A21 on Mouse Models of Colorectal Cancer.

Cancer is a major cause of death worldwide. Colorectal cancer is the second most common type. Additional treatments like chemotherapy and radiation therapy may be recommended. Developing new techniques is vital due to drug resistance and a lack of targeted therapies. In this study, the effects of mesenchymal stem cells (MSCs) loaded with oncolytic Coxsackievirus A21 (CVA21) on a mouse model of CRC were investigated. The therapeutic potency of MSCs loaded with oncolytic CVA21 were evaluated in an experimental mouse model of colorectal cancer which received an injection CT26 cells per mouse subcutaneously. Splenocyte proliferation index, lactate dehydrogenase (LDH) assay, nitric oxide (NO) production assessment, and cytokine assay (IFN-γ, IL-4, IL-10, and TGF-β) in the splenocyte supernatant were all used to evaluate the impact of MSCs loaded with CVA21. The results of this study showed that the treatment of a mouse model of colorectal cancer with MSCs loaded with oncolytic CVA21 could significantly suppress the tumor growth, which was accompanied by stimulation of splenocytes proliferation index, an increase of NO and LDH. Also, MSCs loaded with oncolytic CVA21 increased the secretion of IFN-γ and decreased the secretion of IL-4, IL-10, and TGF-β. The results of the current study suggest that MSCs loaded with oncolytic CVA21 therapy for the CRC mouse model may have some potential advantages. On the other hand, the results of the study showed that, in addition to activating the acquired immune system, the use of MSCs loaded with oncolytic CVA21 also stimulates the innate immune system by increasing level of nitric oxide.

Adipose tissue–derived mesenchymal stem cells promote the vascularization of pancreatic islets transplanted into decellularized pancreatic skeletons

We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization.Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue–derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein–expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system.In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence–positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule.Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue.

Immunization with the glutathione S-transferase Sj26GST with Chi-CpG NP against Schistosoma japonicum in mice

Schistosomiasis caused by Schistosoma japonicum (S. japonicum) is a major public health problem in the Philippines, China and Indonesia. In this study, the immunopotentiator CpG-ODN was encapsulated within chitosan nanoparticles (Chi NPs) to create a combination adjuvant (Chi-CpG NP). This approach was employed to enhance the immunogenicity of 26 kDa glutathione S-transferase (Sj26GST) from S. japonicum through intranasal immunization. The results demonstrated higher levels of specific anti-Sj26GST antibodies and Sj26GST-specific splenocyte proliferation compared to mice that were immunized with Sj26GST + Chi-CpG NP. Cytokine analysis of splenocytes revealed that the Sj26GST + Chi-CpG NP induced a slight Th1-biased immune response, with increased production of IFN-γ by CD4+ T-cells in the spleen. Subsequently, mice were intradermally inoculated with 1 × 107 organisms in the Coeliac cavity. The bacterial organ burden detected in the liver of immunized mice suggested that Sj26GST + Chi-CpG NP enhances protective immunity to inhibit S. japonicum colonization. Therefore, Sj26GST + Chi-CpG NP vaccination enhances Sj26GST-specific immunogenicity and provides protection against S. japonicum.

Sustained release of stem cell secretome from nano-villi chitosan microspheres for effective treatment of atopic dermatitis

Canine atopic dermatitis (AD) arises from hypersensitive immune reactions. AD symptoms entail severe pruritus and skin inflammation, with frequent relapses. Consequently, AD patients require continuous management, imposing financial burdens and mental fatigue on pet owners. In this study, we aimed to investigate the therapeutic relevance of secretome from canine adipose tissue-derived mesenchymal stem cells (MSCs), especially after encapsulation in nano-villi chitosan microspheres (CS-MS) to expect improved efficacy. Conditioned media (CM) from MSCs significantly inhibited the proliferation of splenocytes, induced the generation of regulatory T cells, and decreased mast cell degranulation. We found that beneficial soluble factors known to reduce AD symptoms, including transforming growth factor-beta 1, were detectable after sequential concentration and lyophilization of CM. The CS-MS, developed by a phase inversion regeneration method, showed high loading and sustained release of the secretome. Local injection of secretome-loaded CS-MS (ST/SC-MS) effectively reduced clinical severity compared to groups treated with secretome. Histological analysis revealed that ST/SC-MS potently suppressed epidermal hyperplasia, immunocyte infiltration and mast cell activation in the lesion. Taken together, this study presents a novel therapeutic approach exhibiting more potent and prolonged immunoregulatory efficacy of MSC secretome for canine AD treatment.

Sanfeng Tongqiao Dripping Pills alleviate House Dust Mite-induced allergic rhinitis in mice by inhibiting Th2 differentiation and repairing the nasal epithelial barrier

BackgroundSanfeng Tongqiao Dripping Pills (SFTQ) has clinically demonstrated a promising therapeutic effect on allergic rhinitis (AR). However, the active ingredients and underlying mechanisms of SFTQ remain unclear. PurposeExploring the effects, mechanisms, and active ingredients of SFTQ in the treatment of AR is valuable. Study designThe mechanisms of SFTQ and its active ingredients in treating AR were investigated through in vivo and in vitro studies. MethodsA HDM-induced AR model was established in BALB/c mice. The effects of SFTQ in treating AR were evaluated by AR-like symptoms, EOS count, and pathological changes in the nasal tissue in vivo. The effects of SFTQ active components on epithelial cells (ECs) were evaluated in Poly(I:C) and TNF-α co-stimulated human nasal ECs (RPMI-2650). Additionally, the effects of SFTQ active components on splenocytes proliferation and Th cell differentiation were assessed. A co-culture system of ECs and T lymphocytes was established to investigate the impact of Th2 cells on the structure and function of ECs. The effects of SFTQ ingredients on ECs, T lymphocytes, and the HDM-induced AR model were further confirmed through in vivo and in vivo studies, respectively. ResultsSFTQ significantly alleviated AR-like symptoms and pathological changes in the nasal tissue of AR mice. The treatment elevated the expression of Occludin and E-cadherin in the nasal epithelium and reduced the percentage of Th2 cells in cervical lymph nodes (CLN). Among the active compounds of SFTQ, L-Menthone and Pulegone notably downregulated IL-33 levels in activated ECs, while Hesperetin significantly decreased TSLP and IL-33 levels. In the co-culture system of ECs and Th2 cells, exposure to Baicalin, Wogonin, and Pulegone increased the TEER value of ECs, while notably inhibiting the production of TSLP and IL-33. Furthermore, in HDM-induced AR mice, treatments with Baicalin, Luteolin, and Hesperetin effectively inhibited AR-like symptoms. Additionally, Luteolin and Hesperetin significantly reduced the inflammatory cells infiltration and the population of Th2 cells in AR mice. ConclusionSFTQ and its active ingredients effectively alleviated HDM-induced AR in mice by inhibiting Th2 cell differentiation and repairing the nasal epithelial barrier. Our study can provide a scientific basis for SFTQ to be used in clinical treatment of AR.

Immune Boosting Effect of Limosilactobacillus Reuteri in Immunocompetent C57BL/6J Mice.

Probiotics are well-known to be directly or indirectly involved in the host immune system. In this study, we analyzed the immune-boosting effects of lactic acid bacteria, including Limosilactobacillus and Lactococcus, in immunocompetent C57BL/6J mice. Three different lactic acid bacteria strains were orally administered to C57BL/6J mice for 8 weeks. Then, liver, spleen, and whole blood were harvested after sacrificing the animals. There were no significant changes in whole-body weight, weight of organs, or complete blood cell count by oral administration of lactic acid bacteria. The frequencies of CD3+, CD4+, and CD8+ T cells were significantly increased in the Limosilactobacillus reuteri MG5462 group compared to control. The frequency of NK1.1+ cells was significantly increased in the Lactococcus lactis MG5474 group compared to control. On the other hand, splenocyte proliferations and natural killer cytotoxicity did not differ between groups. In addition, the MG5462 group had a significant increase in the production of TNF-α compared to the control, which is consistent with the upregulation of T cells in the MG5462 group. Therefore, Limosilactobacillus reuteri could be a functional food additive to boost immunity by positively affecting T cell populations.

MIL-53(Al)–oil/water emulsion composite as an adjuvant promotes immune responses to an inactivated pseudorabies virus vaccine in mice and pigs

Although vaccination with inactivated vaccines is a popular preventive method against pseudorabies virus (PRV) infection, inactivated vaccines have poor protection efficiency because of their weak immunogenicity. The development of an effective adjuvant is urgently needed to improve the efficacy of inactivated PRV vaccines. In this study, a promising nanocomposite adjuvant named as MIL@A–SW01–C was developed by combining polyacrylic acid-coated metal–organic framework MIL-53(Al) (MIL@A) and squalene (oil)-in-water emulsion (SW01) and then mixing it with a carbomer solution. One part of the MIL@A was loaded onto the oil/water interface of SW01 emulsion via hydrophobic interaction and coordination, while another part was dispersed in the continuous water phase using carbomer. MIL@A–SW01–C showed good biocompatibility, high PRV (antigen)-loading capability, and sustained antigen release. Furthermore, the MIL@A–SW01–C adjuvanted PRV vaccine induced high specific serum antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response compared with commercial adjuvants, such as alum and biphasic 201. In the mouse challenge experiment, two- and one-shot vaccinations resulted in survival rates of 73.3 % and 86.7 %, respectively. After one-shot vaccination, the host animal pigs were also challenged with wild PRV. A protection rate of 100 % was achieved, which was much higher than that observed with commercial adjuvants. This study not only establishes the superiority of MIL@A–SW01–C composite nanoadjuvant for inactivated PRV vaccine in mice and pigs but also presents an effective method for developing promising nanoadjuvants. Statement of significanceWe have developed a nanocomposite of MIL-53(Al) and oil-in-water emulsion (MIL@A–SW01–C) as a promising adjuvant for the inactivated PRV vaccines. MIL@A–SW01–C has good biocompatibility, high PRV (antigen) loading capability, and prolonged antigen release. The developed nanoadjuvant induced much higher specific IgG antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response than commercial adjuvants alum and biphasic 201. In mouse challenge experiments, survival rates of 73.3 % and 86.7 % were achieved from two-shot and one-shot vaccinations, respectively. At the same time, a protection rate of 100 % was achieved with the host animal pigs challenged with wild PRV.

Study of humoral and cellular immunity following the immunization of C57Bl/6 mice with a prototype of the inactivated Chikungunya vaccine

Introduction. Chikungunya virus infection is a problem for the health care system of affected regions due to the lack of specific prevention, as well as effective antiviral drugs. The critical role of cellular immunity in viral control and clearance for the Chikungunya fever has been demonstrated in many studies. Therefore, effective stimulation of not only humoral but also cellular immunity is of undeniable importance when assessing the effectiveness of a potential vaccine for the prevention of this infection. The aim of the present study was to investigate the formation of protective immunity after administration of a drug containing inactivated Chikungunya virus (CHIKV) to C57Bl/6 mice. Materials and methods. Inactivated CHIKV (concentrations of 10 μg and 40 μg) was administered intramuscularly to C57Bl/6 mice twice with an interval of 14 days. Indicators of humoral immunity were assessed by ELISA and neutralization test (NT), cellular immunity — by the production of IFN-γ and splenocyte proliferation in vitro. The concentration of cytokines IL-1, IL-2, IL-6, IL-10, IL-12p70 and TNF was determined by ELISA. When assessing the protective immunity in animals, CHIKV was injected into the dorsal surface of the foot of the right hind paw at a dose of 2.89 ± 0.10 lg TCD50 in a volume of 20 μl. Results. The most pronounced immune response was noted to the administration of 40 μg of inactivated CHIKV, which was manifested in the balanced production of the studied cytokines, the formation of specific humoral (according to the results of ELISA and NT) and cellular — specific proliferation of splenocytes in vitro and production of IFN-γ. When assessing efficacy, the development of foot edema in immunized animals was significantly lower than in animals in the control group. Discussion. CHIKV, inactivated by beta-propiolactone, had pronounced immunogenic properties. The balance of production of pro- and anti-inflammatory cytokines, as well as the Th1/Th2 immune response, characterized the formation of adaptive immunity in mice without a pronounced inflammatory response. The formation of a specific humoral and cellular immune response has been demonstrated. A study of protection in a non-lethal animal model confirmed the efficacy of the inactivated vaccine. Conclusion. Double administration of the inactivated CHIKV vaccine at a dose of 40 μg to C57Bl/6 mice demonstrated pronounced immunogenicity, which allows us to evaluate it as a promising prophylactic vaccine.

A xanthan gum and carbomer-codispersed divalent manganese ion-loaded tannic acid nanoparticle adjuvanted inactivated pseudorabies virus vaccine induces balanced humoral and cellular immune responses

Adjuvants including aluminum adjuvant (Alum) and oil-water emulsion have been widely used in inactivated pseudorabies virus (PRV) vaccines to improve their performance, however, they are not sufficient to protect from PRV infection because of the weak immune response and poor Th1-type immune response. Divalent manganese ion (Mn2+) has been reported to increase the cellular immune response significantly. In this work, a xanthan gum and carbomer-dispersed Mn2+-loaded tannic acid-polyethylene glycol (TPMnXC) nanoparticle colloid is developed and used as an adjuvant to improve the performance of the inactivated PRV vaccine. The good in vitro and in vivo biocompatibility of the developed TPMnXC colloid has been confirmed by the cell viability assay, erythrocyte hemolysis, blood routine analysis, and histological analysis of mouse organs and injection site. The TPMnXC-adjuvanted inactivated PRV vaccine (TPMnXC@PRV) significantly promotes higher and more balanced immune responses indicating with an increased specific total IgG antibody and IgG2a/IgG1 ratio, efficient splenocytes proliferation, and elevated Th1- and Th2-type cytokine secretion than those of control groups. Wild PRV challenge experiment is performed using mice as a model animal, achieving a protection rate of up to 86.67 %, which is much higher than those observed from the commercial Alum. This work not only demonstrates the high potentiality of TPMnXC in practical applications but also provides a new way to develop the Mn2+-loaded nanoadjuvant for veterinary vaccines.

Modulating effects of heat-killed and live Limosilactobacillus reuteri PSC102 on the immune response and gut microbiota of cyclophosphamide-treated rats.

In the present study, we investigated the potential immunomodulatory effects of heat-killed (hLR) and live Limosilactobacillus reuteri PSC102 (LR; formerly Lactobacillus reuteri PSC102) in RAW264.7 macrophage cells and Sprague-Dawley rats. RAW264.7 murine macrophage cells were stimulated with hLR and LR for 24 h. Cyclophosphamide (CTX)-induced immunosuppressed Sprague-Dawley rats were orally administered with three doses of hLR (L-Low, M-Medium, and H-High) and LR for 3 weeks. The phagocytic capacity, production of nitric oxide (NO), and expression of cytokines in RAW264.7 cells were measured, and the different parameters of immunity in rats were determined. hLR and LR treatments promoted phagocytic activity and induced the production of NO and the expression of iNOS, TNF-α, IL-1β, IL-6, and Cox-2 in macrophage cells. In the in vivo experiment, hLR and LR treatments significantly increased the immune organ indices, alleviated the spleen injury, and ameliorated the number of white blood cells, granulocytes, lymphocytes, and mid-range absolute counts in immunosuppressive rats. hLR and LR increased neutrophil migration and phagocytosis, splenocyte proliferation, and T lymphocyte subsets (CD4+, CD8+, CD45RA+, and CD28+). The levels of immune factors (IL-2, IL-4, IL-6, IL-10, IL-12A, TNF-α, and IFN-γ) in the hLR and LR groups were upregulated compared with those in the CTX-treatment group. hLR and LR treatments could also modulate the gut microbiota composition, thereby increasing the relative abundance of Bacteroidetes and Firmicutes but decreasing the level of Proteobacteria. hLR and LR protected against CTX-induced adverse reactions by modulating the immune response and gut microbiota composition. Therefore, they could be used as potential immunomodulatory agents.

In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 μM but had an impact at 2 μM, and induced zebrafish embryotoxicity at 20 μM, but not at 2 or 0.2 μM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue.

Porcine Brain Enzyme Hydrolysate Enhances Immune Function and Antioxidant Defense via Modulation of Gut Microbiota in a Cyclophosphamide-Induced Immunodeficiency Model.

This study examined how consuming porcine brain enzyme hydrolysate (PBEH) affects the immune function and composition of the gut microbiota in an immunodeficient animal model. Male Wistar rats aged 6 weeks were fed casein (control), 100 mg/kg body weight (BW), red ginseng extract (positive-control), and 6, 13, and 26 mg PBEH per kg BW (PBEH-L, PBEH-M, and PBEH-H, respectively) daily for 4 weeks. At 30 min after consuming assigned compounds, they were orally administered cyclophosphamide (CTX; 5 mg/kg BW), an immunosuppressive agent, to suppress the immune system by inhibiting the proliferation of lymphocytes. The normal-control rats were fed casein and water instead of CTX. Natural killer cell activity and splenocyte proliferation induced by 1 μg/mL lipopolysaccharide were lower in the control group than the normal-control group, and they significantly increased with PBEH consumption, particularly at high doses. The PBEH consumption increased dose-dependently in the Th1/Th2 ratio compared to the control. The lipid peroxide contents were lower in the PBEH group than in the control group. Moreover, PBEH m and PBEH-H consumption mitigated white pulp cell damage, reduced red pulp congestion, and increased spleen mast cells in the histological analysis. Intestinal microbiota composition demonstrated differences between the groups at the genus levels, with Akkermansia being more abundant in the control group than the normal-control group and the PBEH-H group showing a decrease. However, Bifidobacterium decreased in the control group but increased in the PBEH-H group. The β-diversity revealed distinct microbial communities of PBEH and positive-control groups compared to the control group (p < 0.05). The metagenome predictions revealed that PBEH-H influenced amino acid metabolism, antioxidant defense, insulin sensitivity, and longevity pathways. In conclusion, PBEH-H intake boosted immune responses and reduced lipid peroxides by modulating gut microbiota composition. These findings suggest that PBEH-H has the potential as a dietary supplement for improving immune function and gut health in individuals with immunodeficiency.

Antioxidant Properties of Brassica Rapa L. Seeds and Immunostimulant Effect on Immunity Broilers Cells (Gallus Gallus Domesticus)

Background: In the past few decades, researchers have focused on finding the benefits of natural substances derived from plants. Objective: This study aimed to evaluate the use of Brassica rapa seeds in poultry feed as an antioxidant and immunostimulant of host defenses. Methods: We prepared three extracts using ethanol, Ethyl Acetate, and water. Spectrophotometric methods determined the total phenolic and flavonoid content in the three extracts. Antioxidant activity was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and β-Carotene bleaching methods. An assessment of their immunostimulant activities in vitro was performed on chicken immunity cell proliferation (splenocytes, thymocytes, and Bursa cells) and IgY production. method: We prepared three different extracts using ethanol, Ethyl Acetate, and water. The total phenolic and flavonoid content in the three extracts was determined by spectrophotometric methods.Antioxidant activity has been assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP), 2,2&amp;#039;-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals and β-Carotene bleaching methods. Assessment of their immunostimulant activities in vitro was tested on chicken immunity cell proliferation (splenocytes, thymocytes, and Bursa cells) and IgY production Results: The total phenolic contents ranged from 462.5 to 794.8 mg /g of extract, the order of TPC was as follows: EtOAc &gt; EtOH &gt;Water. Flavonoid contents were, 63.7 mg in EtOH extract, 81.2 mg in EtOAc extract, and 108.7 mg/g Extract in aqueous extract. By DPPH, the IC50s of EtOH, EtOAc, and water, were 1.8μg/ml, 2.4μg/ml, and 1.5μg/ml, respectively. Using the Ferric- Reducing Antioxidant Power (FRAP), we remarked that the EtOH and EtOAc extracts have important antioxidant powers. The ABTS assay indicated that EtOH and water had the highest activities with IC50s of 0.19 and 0.07, respectively. Finally, by the β-carotene bleaching test, we observed that the IC50 of the EtOH, EtOAc, and water were 62.1 μg/mL; 72.7 μg/mL, and 45.8 μg/mL, respectively. The results obtained indicate that Brassica aqueous extract stimulates humoral immunity by stimulating splenocyte (B and T-lymphocytes) and Bursa cell (B-lymphocyte) proliferation by more than 200% of response vs control. In addition, the aqueous extract highly stimulated the function of bursa cells by 208% of the reaction. In the same conditions, we recorded a stimulation of cellular immunity mediated by thymocytes by an increase in cell proliferation (352.7% of response) implicated in virus protection. These extracts also possessed an antimicrobial effect against diverse microorganisms such as coliforms and Staphylococcus. The FT-IR spectrum indicated that the hydroxyl group (phenol), hydroxybenzoic acid family, carbohydrate molecules such as glucopyranose, a carbonyl ester group, hydrocarbon chains (alkyls groups)) were present. result: The total phenolic contents ranged from 462.5 to 794.8 mg /g of Extract, the order of TPC was as follows: EtOAc &amp;gt; EtOH &amp;gt;Water. Flavonoid contents were, 63.7 mg in EtOH extract, 81.2 mg in EtOAc extract, and 108.7 mg/g Extract in aqueous extract.By DPPH, the IC50s of EtOH, EtOAc, and water, were respectively 1.8µg/ml, 2.4µg/ml, and 1.5µg/ml. Using the ferric-reducing antioxidant power (FRAP) we remarked that the EtOH and EtOAc extracts have important antioxidant powers. The ABTS assay indicated that EtOH and water had the highest activities with IC50s of 0.19 and 0.07 respectively. Finally, by the β-carotene bleaching test, we observed that the IC50 of the EtOH, EtOAc, and water were 62.1 µg/mL; 72.7 µg/mL, and 45.8 µg/mL respectively. The results obtained indicate that Brassica aqueous extract stimulates humoral immunity by stimulating splenocyte (B and T-lymphocytes) and Bursa cell (B-lymphocyte) proliferation by more than 200% of response vs control. In addition, the aqueous extract stimulated highly the function of bursa cells by 208% of the response. In the same conditions, we recorded a stimulation of cellular immunity mediated by thymocytes by an increase in cell proliferation (352.7% of response) implicated in virus protection. These extracts possessed also an antimicrobial effect against diverse microorganisms such as coliforms and Staphylococcus. The FT-IR spectrum indicated that the hydroxyl group (phenol), hydroxybenzoic acid family, carbohydrate molecules such as glucopyranose, a carbonyl ester group, hydrocarbon chains (alkyls groups)) were present. Conclusion: This result could have interesting applications in the poultry feed industry to enhance the performance of animal development.