Spleen Cell Culture Supernatants Research Articles

Green tea polyphenols alleviate β-conglycinin-induced anaphylaxis by modulating gut microbiota in rats

Heretofore, there are no effective therapeutic interventions for food allergies induced by β-conglycinin, an major allergen in soybean. The objective of this study was to explore the alleviating effect and underlying mechanism of green tea polyphenols (GTPs) on β-conglycinin-induced anaphylaxis. Twenty-one-day-old weaned BN rats were sensitized by gavage of β-conglycin and then given drinking water containing 1% GTPs. The results showed that β-conglcyinin sensitized rats had increased serum IgE levels and IL-4 level in spleen cell culture supernatant compared with the control group. However, total IgE, β-conglycin-specific IgE and IL-4 levels were significantly decreased by GTPs, suggesting that it weakened Th2-type immune response and restored cytokine homeostasis. The impact of GTPs on gut microbiota mainly focused on the changes in specific microbes, characterized by a significant increase of Bacteroides and other beneficial bacteria, which would contribute to the recovery of gastrointestinal allergies. This study is the first to demonstrate that GTPs, as good immunomodulator, played an important role in alleviating soybean protein allergy, which were associated with the alterations in gut microbiota. These results contribute to the development and application of green tea and provided an effective nutritional therapy strategy for the prevention and treatment of allergic diseases caused by soybean allergens.

Troxerutin as an anti-inflammatory and anti-oxidative drug could ameliorate Type 1 diabetes complications in C57BL/6 mice

Type 1 diabetes (T1D) is a chronic autoimmune disease that even with insulin therapy, inflammatory complications will develop in the long term. 40 inbred C57BL/6 mice were randomly divided into four groups (n=10): Control group consisted of healthy mice receiving citrate buffer, Diabetic group included a group of diabetic mice, Diabetic+TX group was a group of diabetic mice treated with troxerutin (TX), and TX group was a group of healthy mice treated with TX. Two weeks after the final dose of streptozotocin (STZ), The cytokine levels were measured using ELISA in the culture supernatants of spleen cells after 72 hours. Radioimmunoassay was used to measure insulin and c-peptide levels. The fasting blood sugar (FBS) was measured by an automatic glucometer device. lymphocyte proliferation index was evaluated using MTT assay, myeloperoxidase (MPO) level was measured in serum and pathologic studies of the kidney and liver were performed. The levels of IL-1, IL-17, TNF-α and IFN-γ as well as MPO, FBS levels and proliferation index was significantly decreased in the treated diabetic group compared to the diabetic mice (p<0.05). plasma C-peptide and insulin significantly increased in treated diabetic group than in the diabetic mice (p<0.05). Histologically, in diabetic animals treated with Tx, inflammatory and degenerative processes in both kidney and liver tissues were alleviated significantly (p<0.05). According to the results, it was supported the anti-diabetic and anti-inflammatory effects of TX, however, more studies are needed to investigate the effects of TX and the dose-response relationship in this disease.

Immunogenicity of Chitosan-Based Non-invasive Vaccine Strategy Against Mycobacterium tuberculosis

Background: In spite of Bacillus Calmette-Guerin (BCG) vaccination, still tuberculosis caused by Mycobacterium tuberculosis remains a problematic impediment that requires perspective management. The aim of this study was to evaluate the immunogenicity of chitosan nanoparticles containing recombinant mycobacterial proteins and adjuvant in the Balb/C mice through a non-invasive nasal inhalation delivery route and measure the level of cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), and IL-17. Methods: Thirty mice in five different groups were vaccinated through inhalation with compounds set in different combinations. Two weeks after the last nasal delivery, IFN-γ, IL-4, and IL-17 were measured in spleen cell culture supernatants. Results: The IFN-γ and IL-17 concentrations were found to increase in the groups that received chitosan nanoparticles containing protein and adjuvant alone or as a BCG booster. Our study showed that the chitosan nanoparticle containing protein and adjuvant induced a Th1 response. However, the groups that first received BCG and then chitosan nanoparticles containing protein and adjuvant had the greatest Th1 response in terms of IFN-γ and IL-17 production in all the groups. Conclusion: Our findings showed that the vaccine designed to be administered through the nasal mucosa well stimulates cellular immunity and enhances the BCG vaccine’s effectiveness.

A Preliminary Study in Immune Response of BALB/c and C57BL/6 Mice with a Locally Allergic Rhinitis Model.

BALB/c and C57BL/6 mouse strains are commonly used in allergy research. The current study investigated the immunological differences between these two mouse strains with a locally allergic rhinitis model. Eighteen BALB/c and eighteen C57BL/6 mice received different doses of ovalbumin (OVA) intranasally for eight weeks (each mouse strain has three subgroups, 25 mg/mL group, 0.25 mg/mL group, and the PBS group). The allergic symptoms, OVA-specific serum antibody (IgE, IgG1, IgG2a), cytokines (IL-4, IFN-γ, IL-10) in the splenic culture supernatant, infiltrating eosinophils and goblet cells in local nasal mucosa were measured. RNA-seq technology was applied to detect differential gene expression in the local nasal mucosa. With the same dose of OVA stimulation, the exacerbation of allergic symptoms was more pronounced in C57BL/6 than in BALB/c. BALB/c serum IgE, IgG1, and IgG2a gradually increased, and C57BL/6 produced fewer serum antibodies IgE and IgG1, while IgG2a never increased. BALB/c spleen cell culture supernatant IL-4 and IL-10 increased with increasing dose, and IFN-γ increased significantly in the intermediate dose group, while IL-4, IL-10, and IFN-γ did not increase in C57BL/6. The infiltration of eosinophils and goblet cells in both mice was proportional to the dose, while C57BL/6 was elevated more than BALB/c. RNA-seq suggested that the innate immune response, immune system process function, Jun kinase (JNK) pathway, and MAPKK pathway were upregulated in C57BL/6 compared to BALB/c. The core genes responsible for the differential immune response in both mice with allergic rhinitis were Kng2, Kng1, Gnb3, Lpar3, Lpar1, Pik3r1, Pf4, Apob, Rps9, and Fbxo2. There are significant differences in the immunologic responses between BALB/c mice and C57BL/6 mice. BALB/c mice developed mild local allergic inflammatory reactions and strong systemic immune responses. In contrast, C57BL/6 mice had stronger local allergic inflammatory responses and relatively mild systemic immune responses. Different mice strains can be selected according to the research purpose.

PPD in HBsAg vaccine formulation suppressed IFN-γ and IL-4 cytokine responses and induced long-lived humoral immune responses: Results from 220-day monitoring of specific IgG responses.

Here, immune responsesand long-lived IgG responses of HBsAg-Alum, HBsAg-MF59, as well as HBsAg-MF59 were compared when formulated with PPD. BALB/cmice were vaccinatedsubcutaneously three times withatwo-week-interval. Then, specific IgG, long-lived IgG responses up to 220 days,andIgG1/IgG2aisotypes,andIFN-γ and IL-4 on spleen cell culture supernatant were assessed using ELISA. IFN-γ cytokine response between MF59- and Alum-adjuvanted vaccines did not show a significant difference. HBsAg-Alum revealed an increase in IL-4 cytokineversus HBsAg-MF59 at borderline(P=0.0553).In addition, HBsAg-MF59+PPD 10 µg showed a significant decrease in IL-4 and IFN-γ cytokinesversusHBsAg-MF59. Furthermore, HBsAg-MF59+PPD10 µg showed a significant increase intheIL-2/IL-4 ratioversusHBsAg-MF59 (P=0.0339).Specific IgG antibody showed a significant increase in HBsAg-MF59, as compared with HBsAg-Alum. Furthermore, HBsAg-MF59 plus PPD showed a significant increase in IgG responsesversusHBsAg-MF59 and HBsAg-Alum groups. Long-lived IgGresponses showeda significant increase in HBsAgMF59 versus HBsAg-Alum group and PPD in the HBsAg-MF59 vaccine formulation, resulting in a significant increase in IgG responses versus HBsAg-MF59 group. In addition,HBsAg-MF59 plus PPDsuppressed IgG1 response versus HBsAg-Alum. However, HBsAg-MF59 showed a significant increase in IgG2α versustheHBsAg-Alum group (P=0.0190). Immunization with HBsAg-MF59+PPD (10 µg) showed a significant increase versustheHBsAg-MF59 group (P=0.0040).IgG2a/IgG1 ratioinHBsAg-MF59+PPD1µgandHBsAg-MF59+PPD10 µggroups showed a significant increaseversusHBsAg-MF59 groups(P<0.0345). PPD leads toamore potent long-lived IgG responses intheHBsAg vaccine, highlighting its potentialasa component of a complex adjuvant.

Protection against genotype VII Newcastle disease virus challenge by a minicircle DNA vaccine coexpressing F protein and chicken IL-18 adjuvant

Genotype VII Newcastle disease virus (NDV) is still one of the most important virus threats severely affecting poultry production worldwide. Although inactivated vaccines are commercially available, there is still an urgent need to develop novel vaccine candidates for convenient and affordable vaccine application. Oral immunization using live attenuated bacteria such as Salmonella has recently attracted increasing interest, and in a previous study, we used a regulated delayed lysis Salmonella vector to deliver a DNA vaccine encoding the F protein and chicken IL-18 adjuvant together, named pYL23. To further improve its efficiency, we employed a novel in vivo minicircle DNA (mcDNA) platform to construct pYL58, which could maintain the complete plasmid during in vitro culture conditions and then transform into mcDNA in vivo whenever the plasmid was delivered by Salmonella into host cells. Compared with immunization with the parental strain harboring plasmid pYL23, immunization with Salmonella with pYL58 induced increased levels of serum IgY and mucosal sIgA in chickens, especially the intestinal and tracheal sIgA levels. Production of cytokines, including IL-4, IFN-γ, IL-18 and IFN-α, was also determined in serum and spleen cell culture supernatants after the 3rd immunization, and the results showed that the production of IFN-γ in the pYL58 group was significantly increased compared with that in the negative control group. Interestingly, compared with pYL23, significantly increased production of IFN-α in the cell supernatants from the pYL58 group was also observed. In addition, the CCK-8 assay results showed that the minicircle pYL58 significantly increased spleen cell proliferation. After virulent VII NDV challenge, pYL58 immunization could provide 70% protection compared with 50% protection in the pYL23 group, together with decreased virus titers in chicken lung samples at Day 5 and virus shedding at Days 3 and 5 post-challenge. This study demonstrated that the application of mcDNA technology dramatically increased the DNA vaccine efficiency, providing additional support for the use of our mcDNA platform in the veterinary field.

Chitosan nanoparticles containing fusion protein (Hspx-PPE44-EsxV) and resiquimod adjuvant (HPERC) as a novel booster vaccine for Mycobacterium tuberculosis.

This study attempted to explore the immunogenicity of chitosan nanoparticles containing fusion protein (Hspx-PPE44-EsxV; HPE) and resiquimod adjuvant (HPERC) in BALB/c mice. HPE was initially expressed in E. coli BL21 cells. HPE and resiquimod adjuvant were then encapsulated in chitosan nanoparticles (HPERC). One group of mice were subcutaneously vaccinated on days 0, 14, and 28 with HPERC, and the other group was primed with bacilli Calmette-Guérin (BCG) on day 0 and then boosted with HPERC on days 14 and 28. Two weeks after the last injection, IFN-γ, IL-4, and IL-17 in spleen cell culture supernatants, and IgG2a and IgG1 titers in sera were measured. HPERC size was 130.84 ± 12.08nm (n = 5). Zeta potential of HPERC was 29 ± 4 mv. The highest IFN-γ concentration was detected in BCG-primed mice that were boosted with HPERC. In addition, IL-17 production was significantly increased in all groups compared with that of control, except in those that received nanoparticle (NP), adjuvant (ADJ), NP/ADJ, and fusion protein (Hspx-PPE44-EsxV) (HPE). Comparison of IFN-γ and IL-4 concentration determined that Th1 was activated in BCG-primed and HPERC-boosted group in comparison to the other groups. No significant difference in concentration of IL-4 was observed between groups receiving HPERC and BCG-primed and HPERC-boosted group in comparison to group BCG. Concentrations of IgG2a and IgG1 also increased compared to the control group and the rate of IgG2a was higher compared to IgG1. Chitosan containing HPERC vaccine could induce a high level of specific cytokines in mice. The group of mice which first received BCG and then HPERC as booster vaccine could produce significant amounts of IFN-γ, IL-17, and IgG2a.

The Effects of Maternal Smoking on Pregnancy and Offspring: Possible Role for EGF?

Cigarette smoke exposure during pregnancy and lactation is associated with adverse pregnancy outcomes. Here, we investigated the effects of maternal smoke exposure on pregnancy and offspring immunity and explored whether, epidermal growth factor (EGF), an important growth-promoting factor in human colostrum and milk, might be a possible missing link in maternal smoke exposure and changes in infants’ immune responses. Pregnant BALB/c mice were exposed to either cigarette smoke or air during gestation and lactation, and effects on pulmonary inflammation in dams and immune responses in offspring were examined. Maternal smoke exposure increased airway hyperresponsiveness and accumulation of inflammatory cells in the lungs of pregnant dams compared to non-pregnant dams. The E-cadherin protein expression was reduced in mammary glands of cigarette smoke-exposed pregnant dams. EGF levels were higher in mammary glands and serum of smoke-exposed pregnant dams compared to air-exposed pregnant dams. Offspring from cigarette smoke-exposed dams exhibited elevated levels of IL-17A, MCP-1, IL-22, and IL-13 in anti-CD3 stimulated spleen cell culture supernatants. EGF levels were also increased in serum of offspring from smoke-exposed dams. A positive correlation was observed between serum EGF levels and neutrophil numbers in bronchoalveolar lavage fluid of the dams. Interestingly, IL-17A, MCP-1, IL-22, IL13, and IFN-γ levels in anti-CD3 stimulated spleen cell culture supernatants of male pups also showed a positive correlation with EGF serum levels. In summary, our results reveal that maternal smoke exposure predisposes dams to exacerbated airway inflammation and offspring to exacerbated immune responses and both phenomena are associated with elevated EGF concentrations.

Recombinant micro-exon gene 3 (MEG-3) antigens from Schistosoma mansoni failed to induce protection against infection but show potential for serological diagnosis

Sequence databases on Schistosoma mansoni have revealed micro-exon gene (MEGs) families. Many of these genes are highly expressed in parasite life cycle stages associated with the mammalian host infection and appear to be involved in immune evasion by schistosomes. So, we believe that MEG-coding proteins would make potential candidates for vaccine development or diagnosis for schistosomiasis. Here, we study MEG-3.2 and MEG-3.4, members of the MEG-3 family. Recombinant (r) proteins were produced and formulated with Freund's adjuvant for vaccination of mice. Immunization with recombinant MEG-3.2 or MEG-3.4 formulation generated high levels of IgG1 antibodies. Additionally, vaccination also induced a mixed Th1/Th2/Th17-type of response, since IFN-γ, IL-5 and IL-17 cytokines were detected in the supernatant of spleen cell cultures; however it failed to induce reduction in parasitic worm burden. Finally, the recombinant proteins were evaluated in a serological assay using human samples. Schistosome-infected individuals showed higher levels of both IgG and IgM against rMEG-3.2 compared to non-infected individuals, while only IgM anti-rMEG-3.4 antibodies were elevated in infected patients. Therefore, between both studied molecules, MEG-3.2 protein is the antigen that shows potential to compose a serological diagnosis test for schistosomiasis.

Juzentaihoto Suppresses Muscle Atrophy in Streptozotocin-Induced Diabetic Mice.

In diabetic patients, skeletal muscle atrophy occurs due to increased oxidative stress and inflammation. Skeletal muscle atrophy reduces the QOL of patients and worsens life prognosis. Therefore, development of preventive therapy for muscle atrophy in hyperglycemic state is eagerly awaited. Juzentaihoto is a medicinal herb that has a function to supplement physical strength, and it is expected to prevent muscle atrophy. To determine the preventive effect of juzentaihoto on muscle atrophy in hyperglycemic state, streptozotocin (STZ) was administered to induce diabetes in mice and the preventive effect of juzentaihoto was evaluated. Mice that received juzentaihoto extract (JTT) showed that the decrease in muscle fiber cross-sectional area in the gastrocnemius muscle was reversed. Additionally, the expression level of tumor necrosis factor α (TNF-α), an inflammatory cytokine, in serum decreased, and that of ubiquitin ligase (atrogin-1, muscle RING-finger protein-1) mRNA in skeletal muscle decreased. An anti-inflammatory cytokine interleukin-10 showed increased levels in the serum and increased levels in spleen cell culture supernatant collected from mice that received JTT. JTT had no effect on the blood glucose level. These results suggest that prophylactic administration of JTT to STZ-induced diabetic mice affects immune cells such as in spleen, causing an anti-inflammatory effect and inhibiting excessive activation of the ubiquitin-proteasome system, to reverse muscle atrophy.

Antigenicity and immunogenicity analysis of Xinjiang hemorrhagic fever virus Gc antigen fragment

Objective To express and purify two domains GcⅠ and GcⅡ of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein, and to study its immunogenicity and the effects on immune response in mice. Methods The prokaryotic expression plasmids of pET28a-GcⅠ and pET32a-GcⅡ were constructed and transformed into E. coli BL21, respectively. The expression and purification conditions of rGcⅠ and rGcⅡ proteins were optimized. The antigenicity of the fusion protein was detected by Western Blot and enzyme-linked immunosorbent assay (ELISA). BALB/c mice were immunized by protein immunization and DNA priming-protein boosting. The mice were randomly divided into 5 groups, including pVAX1-GcⅠ+rGcⅠ group, pVAX1-GcⅡ+rGcⅡ group, rGcⅠ group, rGcⅡ group and saline group (control group) with 7 mice in each group. The serum antibody titer of mice was detected by indirect ELISA, and the immune effect was evaluated by spleen T lymphocyte proliferation assay and cytokine content determination. Results The fusion proteins rGcⅠ and rGcⅡ were purified and obtained, which could react with positive serum of sheep and had good antigenicity. After three immunizations, the IgG levels in the serum of each experimental group were significantly higher than those in the control group (all P<0.001). The serum antibody titers of the experimental groups were reached above 1∶12 800. Among them, the concentration of Th2 type cytokine interleukin-4 (IL-4) in the spleen cell culture supernatant of rGcⅡ [(79.97±7.47) ng/L] and pVAX1-GcⅡ+rGcⅡ group [(61.43±9.27) ng/L] was significantly higher than (24.29±3.81) ng/L of the control group, respectively (all P<0.01). The highest mass concentration [(42.46±2.60) ng/L] of Th1 type cytokine interferon-γ (IFN-γ) was observed in the pVAX1-GcⅡ+rGcⅡ group, which was significantly higher than (20.33±1.67) ng/L of the control group, and the difference was statistically significant (P<0.001). That showed a significant antigen-specific splenic T lymphocyte proliferation (P<0.001). Conclusions The purified recombinant proteins rGcⅠand rGcⅡhave good immunogenicity, which can make the immune system T lymphocytes tend to Th2 response, and pVAX1-GcⅡ combined with recombinant protein GcⅡ can induce better antigen-specific immune effect. And pVAX1-GcⅡ combined with recombinant protein GcⅡ is expected to be used as vaccine candidates for the prevention and control of XHFV. Key words: Xinjiang hemorrhagic fever virus; Glycoprotein; Prokaryotic expression; Purification; Immunogenicity

Antiallergic Effect of Hizikia fusiformis in an Ovalbumin-Induced Allergic Rhinitis Mouse Model.

ObjectivesThe extract of Hizikia fusiformis is known to exhibit anticancer, antiatopic and antioxidant activities. We aimed to investigate the extract of H. fusiformis on allergic rhinitis inflammation in a mouse model.MethodsThe 4-week-old BALB/c mice were randomly assigned into four groups: group A, control group (n=9); group B, allergic rhinitis group (n=10); group C (n=10) received 300 mg/kg of H. fusiformis during nasal challenging period; group D (n=10) received 600 mg/kg of H. fusiformis during general sensitization period and 300 mg/kg of H. fusiformis during nasal challenging period. Allergic inflammation was made with ovalbumin (OVA) and alum then challenged intranasally with OVA. H. fusiformis was intraperitoneally administered 3 hours before the OVA administration. Allergic symptom score and the levels of immunoglobulin G1 (IgG1), IgG2a, OVA-specific IgE antibodies, levels of cytokines in the nasal mucosa and in spleen cell culture supernatant, such as tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-5, IL-13, and IL-10 were assessed. The percentage of regulatory T cell was analyzed by flow cytometry. Eosinophilic infiltration and goblet cell hyperplasia were also evaluated.ResultsH. fusiformis administered groups C and D showed significant inhibitory effects on nasal symptoms, IL-13 mRNA expression and eosinophil infiltration/goblet cell hyperplasia in the nasal tissue; OVA-specific IgE production in serum (P<0.05). In group D, H. fusiformis treatment downregulated IL-4, IL-5, IL-13, TNF-α, and IL-10 cytokine expression in splenocyte culture as well as significantly decreased IgG2a, IgG1 levels in serum compared with group B (P<0.05). However, the expressions of IL-5, interferon-γ and forkhead box P3 mRNA did not change in groups C and D.Conclusion H. fusiformis could induce antiallergic inflammation by suppressing the T-helper type 2 cytokine production (IL-13) locally and systemically, OVA-specific IgE formation, goblet cell hyperplasia, and eosinophilic infiltration in a mouse model of allergic rhinitis. Thus, H. fusiformis could be considered as a potential therapeutic agent in treating allergic rhinitis.

Enhancement of the effect of BCG vaccine against tuberculosis using DDA/TDB liposomes containing a fusion protein of HspX, PPE44, and EsxV

Tuberculosis has been a major health problem worldwide for years; therefore, it is important to develop and produce an effective vaccine against this disease. In this study, the immunogenicity of Mycobacterium tuberculosis fusion protein (FP) encapsulated in liposomes containing DDA/TDB was evaluated. The FP was expressed in E. coli BL21 and encapsulated in liposomal formulations. Three weeks after the last subcutaneous immunization, IFN-γ, IL-4, IL-17, and IL-12 in spleen cell culture supernatants, and IgG2a, IgG1, and IgG2b titres in sera were measured. The greatest IFN-γ and IL-12 interleukin concentrations were observed in the DDA/TDB/CHOL liposomes containing the FP. Initial injection with BCG improved the efficacy of the DDA/TDB/CHOL/FP vaccine. The IgG2a/IgG1 ratio was also high in the DDA/TDB/CHOL/FP group; furthermore, the IgG2a/IgG1 ratio was increased in the BCG-primed, DDA/TDB/CHOL/FP-boosted group, indicating induction of a cellular immune response. Our study showed that the FP-containing DDA/TDB/CHOL liposomes induced a Th1 response. However, the groups that first received BCG and then DDA/TDB/CHOL/FP had the greatest Th1 response in terms of IFN-γ and IL-12 production of all the groups. This suggests that these formulations enhance the BCG vaccine’s effectiveness.

Potentiation of the humoral immune response elicited by a commercial vaccine against bovine respiratory disease by Enterococcus faecalis CECT7121.

Vaccination against pathogens involved in bovine respiratory disease (BRD) is a useful tool to reduce the risk of this disease however, it has been observed that the commercially available vaccines only partially prevent the infections caused by Pasteurella multocida and Mannheimia haemolytica. Therefore, it is recommended to search for new adjuvant strategies to minimise the economic impact of this respiratory syndrome. A possibility to improve the conventional vaccine response is to modulate the immune system with probiotics, since there is accumulating evidence that certain immunomodulatory strains administered around the time of vaccination can potentiate the immune response. Considering veterinary vaccines are frequently tested in murine models, we have developed an immunisation schedule in BALB/c mice that allows us to study the immune response elicited by BRD vaccine. In order to evaluate a potential strategy to enhance vaccine efficacy, the adjuvant effect of Enterococcus faecalis CECT7121 on the murine specific humoral immune response elicited by a commercial vaccine against BRD was studied. Results indicate that the intragastric administration of E. faecalis CECT7121 was able to induce an increase in the specific antibody titres against the bacterial components of the BRD vaccines (P. multocida and M. haemolytica). The quality of the humoral immune response, in terms of antibody avidity, was also improved. Regarding the cellular immune response, although the BRD vaccination induced a low specific secretion of cytokines in the spleen cell culture supernatants, E. faecalis CECT7121-treated mice showed higher interferon-γ production than immunised control mice. Our results allowed us to conclude that the administration of E. faecalis CECT7121 could be employed as an adjuvant strategy to potentiate humoral immune responses.

T cells upon activation promote endothelin 1 production in monocytes via IFN-\u03b3 and TNF-\u03b1

Endothelin 1 (ET-1), mainly produced from vascular endothelial cells, induces vasoconstriction in physiological conditions. The endothelin receptor antagonist is among the most effective agents for pulmonary hypertension. However, little is known about the production source of ET-1 in inflammation and immunity. Here, we studied whether T cell-mediated ET-1 production system exists and operates independent of the production system in vascular endothelial cells. ET-1 production was readily detectable in the culture supernatant of human PBMCs and murine spleen cells stimulated with anti-CD3 antibody. Immunocytostaining showed that ET-1-producing cells emerged only in PBMCs stimulated with anti-CD3 antibody. Using the Transwell system, both murine and human monocytes sorted with magnetic beads in the inner chamber produced ET-1 when T cells were activated with antigen or anti-CD3 antibody in the outer chamber. This ET-1 production was inhibited by anti-IFN-γ and/or TNF-α antibody. Furthermore, monocytes purified from ETflox/flox;Tie2-Cre( + ) mice, which conditionally lack ET-1 in hematopoietic stem cells and vascular endothelial cells, did not produce ET-1 even when stimulated by antigen-specific T cell activation. This study demonstrates the existence of an immune-mediated ET-1 production induced by T cells upon activation through IFN-γ and TNF-α.

Enhanced efficacy of sublingual immunotherapy by liposome-mediated delivery of allergen.

Immunotherapy by sublingual administration of allergens provides high patient compliance and has emerged as an alternative to subcutaneous immunotherapy for the treatment of IgE-associated allergic diseases. However, sublingual immunotherapy (SLIT) can cause adverse events. Development of allergen delivery systems enabling more efficient delivery and hence lower allergen load might reduce the adverse events. In the present study, we have investigated neutral and cationic liposomes as delivery systems of ovalbumin (OVA), as a model allergen, in an OVA-induced allergic airway inflammation model. We investigated the liposome carriers’ ability to improve tolerance induction of antigens compared to the corresponding dose of free OVA. Mice were treated sublingually over 2 weeks with free or liposome encapsulated OVA followed by intraperitoneal injections and intranasal challenge. Mice sublingually treated with OVA-liposomes showed a significant reduction of airway eosinophilia and splenocyte proliferation in comparison to free OVA. A similar nonsignificant pattern was seen for OVA-specific IgE antibodies. In addition, reduced levels of interferon-γ and interleukin-5 were observed in spleen cell culture supernatants from OVA-liposome-treated mice compared to the sham-treated group. In conclusion, in vivo efficacy data showed that prophylactic SLIT with OVA-liposomes is significantly more effective in preventing allergic inflammation than the corresponding dose of free OVA.

THE EFFECT OF ALPHA-MANGOSTIN IN BALANCING THE RATIO OF CYTOKINES PRO-AND ANTI-INFLAMMATION-GAMMA (IFN-γ/IL-10) AND SEVERITY OF THE DISEASE IN MICE INFECTED WITH MYCOBACTERIUM TUBERCULOSIS MULTIDRUG-RESISTANT

ABSTRACTObjective: The objective of this study was observed to measure the effect of alpha-mangostin in balancing the ratio of interferon-gamma (IFN-γ) andinterleukin-10 (IL-10), and the severity of the disease in mice which infected with Mycobacterium tuberculosis multidrug-resistant (TB-MDR).Method: Infected BALB/c mice were consisted of five groups: Treated with anti-TB drugs+α-mangostin, treated with anti-TB drugs, given α-mangostinduring treatment, and control group. Cytokine levels of culture supernatant of spleen cells were measured by enzyme-linked immunosorbent assay.The number of bacterial colonies was derived from a primary cell culture of bronchoalveolar lavage. Statistical analysis was performed with Anova,Kruskal-Wallis test and correlation Pearson, and Spearman-rank test.Result: Median IFN-γ production was higher in mice, which given with α-mangostin during treatment is 1838.2 pg/ml and control is 1585.5 pg/mlcompared treated with anti-TB drugs+α-mangostin (1312 pg/ml) and anti-TB drugs (1429.3 pg/ml) (p&gt;0.05). The highest result production of medianIL-10 in the 3th group is (465.91 pg/ml) and the lowest in the control group is 195.29 pg/ml, p&gt;0.05. Median IFN-γ/IL-10 ratio of the 3th group verylow (3.94), it means the 3th group is experienced with severity of TB. Alpha-mangostin was decreased in severity of disease based on the number ofTB-MDR bacterial colonies (p≤0.05).Conclusion: α-mangostin have an effect on the balancing IFN-γ/IL-10 ratio and reduce a severity of TB-MDR with using immunomodulator.Keywords: Tuberculosis multidrug-resistant, Alpha-mangostin, Interferon-γ/interleukin-10 ratio, Number of tuberculosis multidrug-resistantbacterial colonies.

Inhibition of IL-4 but not IFN-γ production by splenocytes of mice immunized with ovalbumin after oral administration of 5-hydroxymethylfurfural

ABSTRACT5-Hydroxymethylfurfural (5-HMF) is one of the most important products of maillard reaction. This study sought to elucidate the immunomodulatory effect of 5-HMF by evaluating IFN-γ and IL-4 production in BALB/c mice sensitized with ovalbumin (OVA). Four groups of BALB/c mice (n = 5 for each group) including control, vehicle and two 5-HMF (188 and 750 mg/kg bw) treatment groups were studied. Interleukin-4(IL-4) and interferon gamma (IFN-γ) levels were measured in culture supernatants of spleen cells using enzyme-linked immunosorbent assay. It was found that the IL-4 level was significantly suppressed in 5-HMF treatment groups compared with the vehicle group (P < .05). Moreover, the IFN-γ/IL-4 ratio was significantly increased in a dose-independent manner compared with the vehicle group (P < .05). Oral administration of 5-HMF inhibited IL-4 but not IFN-γ production by splenocytes of mice immunized with OVA. These changes may reflect that 5-HMF could be a novel therapeutic approach for prevention of (Th2) cytokine-dominant disease.

Identification of α-tubulin, Der f 33, as a novel allergen from Dermatophagoides farinae

BackgroundHouse dust mites are an important source of indoor allergens. More than 30 allergens of Dermatophagoides farinae (D. farinae) have been identified. Yet there may be many other allergens in mites remain to be characterized. Methodsα-Tubulin (also named Der f 33) was cloned, expressed and purified. Reaction to specific-IgE, skin prick test and a mouse asthma model were employed to determine the allergenicity of Der f 33. ResultsThe recombinant Der f 33 reacted to the serum of patients with mite allergy. The positive rate of skin prick test (SPT) was 23.5%. In an asthma mouse model, Der f 33 induced the airway allergy-like responses. Moreover, serum specific IgE and IgG1, interleukin-4 (IL-4) from bronchoalveolar lavage fluid (BALF) and spleen cell culture supernatant were markedly increased. In addition, Der f 33 upregulated the CD80 and TNF-α levels in dendritic cells (DCs). ConclusionsDer f 33 is a novel allergen of D. farinae. It modulates the functions of DCs and induces airway allergy.

Emergence of long-lived autoreactive plasma cells in the spleen of primary warm auto-immune hemolytic anemia patients treated with rituximab

Primary warm autoimmune hemolytic anemia (wAIHA) is a rare autoimmune disease in which red blood cells are eliminated by IgG autoantibodies. We analyzed the antibody-secreting cells in the spleen and the peripheral blood of wAIHA patients in various contexts of treatment. Plasmablasts were observed in peripheral blood of newly diagnosed wAIHA patients and, accordingly, active germinal center reactions were present in the spleen of patients receiving short-term corticosteroid therapy. Long-term corticosteroid regimens markedly reduced this response while splenic plasma cells were able to persist, a fraction of them secreting anti-red blood cell IgG in vitro. In wAIHA patients treated by rituximab and who underwent splenectomy because of treatment failure, plasma cells were still present in the spleen, some of them being autoreactive. By using a set of diagnostic genes that allowed us to assess the plasma cell maturation stage, we observed that these cells displayed a long-lived program, differing from the one of plasma cells from healthy donors or from wAIHA patients with various immunosuppressant treatments, and more similar to the one of normal long-lived bone-marrow plasma cells. Interestingly, an increased level of B-cell activating factor (BAFF) was observed in the supernatant of spleen cell cultures from such rituximab-treated wAIHA patients. These results suggest, in line with our previous report on primary immune thrombocytopenia, that the B-cell depletion induced by rituximab promoted a suitable environment for the maturation and survival of auto-immune long-lived plasma cells in the spleen.