IL-2 mRNA Research Articles

Biomimetic calcium carbonate nanoparticles delivered IL-12 mRNA for targeted glioblastoma sono-immunotherapy by ultrasound-induced necroptosis.

Glioblastoma (GBM) is the most aggressive brain tumor, which owns the characteristics of high recurrence, low survival rate and poor prognosis because of the existence of blood brain barrier (BBB) and complicated brain tumor microenvironment. Currently, immunotherapy has attracted much attention on account of favorable therapeutic effect. In this study, we designed a cRGD-modified cancer cell membrane (CM) coated calcium carbonate nanoparticle to deliver interleukin-12 messenger RNA (IL-12 mRNA@cRGD-CM-CaCO3 NPs). The cRGD-modified CM as the shell can endow the nanoparticles with BBB crossing and tumor homing/homotypic targeting effect in the brain tumor microenvironment. IL-12 mRNA-loaded calcium carbonate nanoparticles as the core allow synergistic immunotherapy of necroptosis-induced immune response and IL-12 mRNA transfection under ultrasound irradiation. The as-prepared biomimetic nanoparticles showed superior target and immunotherapeutic outcomes, suggesting that this biomimetic nanoplatform provides a feasible strategy for promoting BBB-penetrating and antitumor immunity.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Upregulation of CD86 and IL-12 by rhododendrol in THP-1 cells cocultured with melanocytes through ROS and ATP

BackgroundThe tyrosinase inhibitor rhododendrol (RD), used as a skin whitening agent, reportedly has the potential to induce leukoderma. ObjectiveAlthough an immune response toward melanocytes was demonstrated to be involved in leukoderma, the molecular mechanism is not fully understood. MethodsWe hypothesized that if RD is a pro-hapten and tyrosinase-oxidized RD metabolites are melanocyte-specific sensitizers, the sensitizing process could be reproduced by the human cell line activation test (h-CLAT) cocultured with melanocytes (h-CLATw/M) composed of human DC THP-1 cells and melanoma SK-MEL-37 cells. Cell surface expression, ROS generation and ATP release, mRNA expression, and the effects of several inhibitors were examined. ResultsWhen RD was added to the h-CLATw/M, the expression of cell-surface CD86 and IL-12 mRNA was greatly enhanced in THP-1 cells compared with those in the h-CLAT. The rapid death of melanoma cells was induced, with ROS generation and ATP release subsequently being greatly enhanced, resulting in the cooperative upregulation of CD86 and IL-12. Consistent with those observations, an ROS inhibitor, ATP receptor P2X7 antagonist, or PERK inhibitor antagonized the upregulation. CD86 upregulation was similarly observed with another leukoderma-inducible tyrosinase inhibitor, raspberry ketone, but not with the leukoderma noninducible skin-whitening agents ascorbic acid and tranexamic acid. ConclusionRD is a pro-hapten sensitizer dependent on tyrosinase that induces ROS generation and ATP release from melanocytes for CD86 and IL-12 upregulation in DCs, possibly leading to the generation of tyrosinase-specific cytotoxic T lymphocytes. The coculture system h-CLATw/M may be useful for predicting the sensitizing potential to induce leukoderma.

Hypoxia inducible factor-1α mediates the mechanism of the Hedgehog pathway in tendinopathy repair by Asperosaponin VI.

Our previous study found that asperosaponin VI (ASA VI) has a positive effect on the repair of tendinopathy. However, its molecular biological mechanism is unclear. To investigate the role of hypoxia inducible factor-1α (HIF-1α) in mediating the hedgehog (Hh) pathway in tendinopathy repair by ASA VI. A total of 36 2-month-old female SD rats were classified into the normal group (NG, n=10) and tendinopathy model group (n=26). The tendinopathy model group was further divided into the model group (MG), ASA VI group (AG), and triamcinolone acetonide+lidocaine group (TG). Compared with those in the MG group, IL-1 mRNA was significantly downregulated and IL-4 and IL-10 were increased in the AG group (P<0.01). The mRNA expression levels of MMP3, TIMP3, VEGF-A, KDR, and VWF mRNA decreased (P<0.01). Immunofluorescence staining revealed that CD31/endomucin levels were significantly attenuated. Scx, Mkx, EYA1, EYA2, COL1, COL3, and TNC mRNA levels showed significant differences (P<0.01). Immunofluorescence staining suggested the upregulation of Scx and the downregulation of Sox9. Shh, Ptch1, Smo, Gli1, Cyc-D1, Cyc-E1, and c-Myc mRNA levels were downregulated (P<0.01). The protein expression levels of Gli 1, Shh, and Ptch1 decreased significantly (P<0.01). The immunofluorescence staining levels of Shh, Ptch, and Gli 1 significantly decreased. ASA VI inhibits local vascular hyperproliferation and downregulates the HIF-1α/Hh pathway to promote the tendinous differentiation of tendon stem/progenitor cells and the repair of tendinopathy. The effect of ASA VI on HIF-1α levels may be an effective target in the treatment of tendinopathy.

Effects of Acute Cold Stress after Intermittent Cold Stimulation on Immune-Related Molecules, Intestinal Barrier Genes, and Heat Shock Proteins in Broiler Ileum.

Cold stress will have a negative impact on animal welfare and health. In order to explore the effect of intermittent cold stimulation training on the cold resistance of broilers. Immune-related and intestinal barrier genes were detected before and after acute cold stress (ACS), aiming to find an optimal cold stimulation training method. A total of 240 1-day-old Ross broilers (Gallus) were divided into three groups (G1, G2, and G3), each with 5 replicates (16 chickens each replicate). The broilers of G1 were raised at normal temperature, while the broilers of G2 and G3 were treated with cold stimulation at 3 °C lower than the G1 for 3 h and 6 h from 15 to 35 d, respectively, at one-day intervals. At 50 d, the ambient temperature for all groups was reduced to 10 °C for six hours. The results demonstrated that before ACS, IL6, IL17, TLR21, and HSP40 mRNA levels in G3 were apparently down-regulated (p < 0.05), while IL8 and Claudin-1 mRNA levels were significantly up-regulated compared with G1 (p < 0.05). After ACS, IL2, IL6, and IL8 expression levels in G3 were lower than those in G2 (p < 0.05). Compared to G2, Claudin-1, HSP90 mRNA levels, HSP40, and HSP70 protein levels were increased in G3 (p < 0.05). The mRNA levels of TLR5, Mucin2, and Claudin-1 in G2 and IL6, IL8, and TLR4 in G3 were down-regulated after ACS, while IL2, IL6, and IL17 mRNA levels in G2 and HSP40 protein levels in G3 were up-regulated after ACS (p < 0.05). Comprehensive investigation shows that cold stimulation at 3 °C lower than the normal feeding temperature for six hours at one day intervals can enhanced immune function and maintain the stability of intestinal barrier function to lessen the adverse effects on ACS in broilers.

Echinococcus granulosus Protoscoleces-Derived Exosome-like Vesicles and Egr-miR-277a-3p Promote Dendritic Cell Maturation and Differentiation.

Cystic echinococcosis, a major parasitic disease caused by Echinococcus granulosus, seriously threatens human health. The excretory–secretory (ES) products of E. granulosus can induce immune tolerance in dendritic cells (DCs) to downregulate the host’s immune response; however, the effect of exosomes in the ES products on the DCs has remained unclear. This study showed that E. granulosus protoscoleces-derived exosome-like vesicles (PSC-ELVs) could be internalized by bone marrow-derived dendritic cells (BMDCs), allowing for the delivery of the parasite microRNAs to the BMDCs. Moreover, PSC-ELVs induced BMDCs to produce the proinflammatory cytokinesinterleukin (IL)-6, IL-12, IL-β, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). PSC-ELVs also upregulated the BMDCs surface marker major histocompatibility complex class II (MHC II), as well as costimulatory molecules CD40, CD80, and CD86. PSC-ELV-derived egr-miR-277a-3p upregulated the IL-6, IL-12, and TNF-α mRNA levels in BMDCs. Moreover, egr-miR-277a-3p directly targeted Nfkb1 (encoding nuclear factor kappa B 1) to significantly suppress the mRNA and protein levels of NF-κB1 in BMDCs, while the expression of NF-κB p65 significantly increased, suggesting that egr-miR-277a-3p induces the production of proinflammatory cytokines by the modification of the NF-kB p65/p50 ratio in BMDCs. These results demonstrated that PSC-ELVs and egr-miR-277a-3p might enhance DCs maturation and differentiation in a cross-species manner, which in turn may modulate the host immune responses and offer a new approach to echinococcosis prevention and treatment.

Selenium-chitosan alleviates the toxic effects of Zearalenone on antioxidant and immune function in mice.

This study assessed the protective effects of selenium-chitosan (SC) against antioxidant and immune function-related damage induced by zearalenone (ZEN) in mice. In total, 150 female mice were allotted to five groups for a 30-day study. Control mice were fed a basal diet. Mice in the ZEN, ZEN-Se1, ZEN-Se2 and ZEN-Se3 groups were fed the basal diet supplemented with same dose of ZEN (2 mg/kg) and different doses of SC, 0.0, 0.2, 0.4 and 0.6 mg/kg, respectively (calculated by selenium). After 30 days, the total antioxidant capacity (T-AOC) level, glutathione peroxidase (GSH-Px) activity, total superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) content in plasma and liver, as well as Con A-induced splenocyte proliferation, plasma interleukins concentrations and liver interleukin mRNA expression levels were determined. The plasma and liver GSH-Px activities, liver T-AOC levels, Con A-induced splenocyte proliferation, interleukin (IL) contents and mRNA expression levels in the ZEN group were significantly lower than in the control group (P < 0.01 or P < 0.05), whereas plasma and liver MDA contents in the ZEN group were significantly higher than in the control group (P < 0.01 or P < 0.05). Additionally, plasma and liver GSH-Px activities, liver T-AOC levels, Con A-induced splenocyte proliferation, IL-1β, IL-17A, IL-2 and IL-6 contents and mRNA expression levels in ZEN+Se2 and ZEN+Se3 groups were significantly higher than in the ZEN group (P < 0.01 or P < 0.05), whereas plasma and liver MDA contents in the ZEN+Se2 and ZEN+Se3 groups were significantly lower than in the ZEN group (P < 0.01 or P < 0.05). The plasma and liver GSH-Px activities, Con A-induced splenocyte proliferation, IL-1β and IL-6 contents, IL-2 and IL-17A mRNA expression levels in the ZEN+Se1 group were also significantly higher than in the ZEN group (P < 0.01 or P < 0.05), whereas the plasma MDA content in the ZEN+Se1 group was also significantly lower than in the ZEN group (P < 0.01). Thus, SC may alleviate antioxidant function-related damage and immunosuppression induced by ZEN in mice.

Effect of live Eimeria vaccination or salinomycin on growth and immune status in broiler chickens receiving in-feed inclusion of gelatin and vitamin E

This experiment determined if 2% of gelatin, to improve the levels of proline and glycine in the diet, and 70 mg/kg of vitamin E supplementation would relieve the impaired performance of male Cobb broilers vaccinated for coccidiosis. Half of the chicks were vaccinated via water (live oocysts), while the other half received medication (salinomycin) in the feed until 35 d of age. The effects of coccidiosis vaccine on performance and mRNA levels of genes involved in mucin synthesis, cytokines, trefoil family factor-2 (TFF2), and metabolic processes (CD36) in the jejunum of broilers were measured. Vaccination negatively affected performance in the first 21 d; however, the inclusion of gelatin and vitamin E reduced this negative response. Additionally, supplementation with these nutrients led to an improvement in broilers receiving the coccidiostat (P < 0.05). From 21 to 35 d, birds treated with gelatin and coccidiosis vaccine experienced better body weight gain than birds without gelatin and vitamin E (P < 0.05). Vaccinated chickens had decreased body weight and decreased anti-inflammatory cytokine expression. Furthermore, they had increased inflammatory cytokine expression, mucin 2 expression, and TFF2 compared to salinomycin-fed broilers (P < 0.05). Transcripts for IL-1B, IFN-y, MUC2, TFF2 were decreased while mRNAs for IL-4 and IL-10 increased in salinomycin-fed broilers compared to vaccinated broilers (P < 0.05). In conclusion, broilers vaccinated against coccidiosis increase their pro-inflammatory immune status and mucin expression compared to broilers receiving salinomycin. These events may contribute to lower performance in vaccinated broiler chicks. Moreover, vitamin E and gelatin can minimize the vaccine's negative immune effects and promote better performance.

Oceanimonas sp. BPMS22-derived protein protease inhibitor induces anti-leishmanial immune responses through macrophage M2 to M1 repolarization

The present study aimed to validate the potential of a novel serine protein protease inhibitor (PPI), purified from marine Oceanimonas sp. BPMS22, induced M2 to M1 repolarization of the macrophages to treat visceral leishmaniasis (VL). Peptide mass fingerprint of the purified trypsin digested PPI peptide was obtained using matrix-assisted laser desorption ionization-time of flight combined with tandem mass spectrometry (MALDI-TOF MS/MS) and the sequence was used to construct a 3D protein model by homology modelling. The IC50 of PPI were 25.28 ± 1.675 μg/mL and 0.415 ± 0.015 μg/mL against promastigotes and intracellular amastigotes, respectively, indicating the host-directed therapy using PPI. The PPI enhanced the effector molecule i.e., nitric oxide (NO), and dampened the arginase activity in a dose-dependent manner. In vitro studies revealed that the BPMS22-derived PPI significantly (p < 0.05) decreased the mRNA expressions of M2 markers (FIZZ-1, YM-1, CD206, Arg-1) and increased the mRNA expressions of M1 markers (iNOS, IL-1β, IL-12) in rIL-4 + rIL-10 induced M2 macrophages. Interestingly, the BPMS22-derived PPI also significantly (p < 0.05) decreased the FIZZ-1, YM-1, CD206, and Arg-1; significantly (p < 0.05) increased iNOS, IL-12, and IFN-γ mRNA expression in L. donovani -infected murine macrophages, alongside the decreased parasite load in it. Hence, PPI has the potential to repolarize the cytokines (rIL-4 + rIL-10) pre-stimulated and L. donovani-infected M2 macrophages to M1 phenotype in vitro. A decrease in parasite burden after treatment with PPI indicated the acceleration of the parasite killing by enhancing the macrophage effector functions. Further, in vivo PPI treatment reduced hepatic and splenic Leishman donovan units (LDU) up to 93.34 % and 87.63 %, respectively. This was followed by a surge in pro-inflammatory cytokines and dampening anti-inflammatory cytokines (p < 0.01), which exhibited anti-VL immunity. These observations might open new perspectives on PPI in macrophage repolarization to treat VL.

Dietary β‐sitosterol supplementation enhanced intestinal immune function of large yellow croaker ( Larimichthys crocea ) infected with Aeromonas hydrophila

An 80-day feeding trial was performed to investigate the effect of dietary β-sitosterol supplementation on intestinal immune function in large yellow croaker. 540 fish (initial average weight: 189.74 ± 0.31 g) were fed six diets containing graded levels of β-sitosterol (0.0%, 0.5%, 1.0%, 1.5%, 2.0% and 2.5%) and then challenged with Aeromonas hydrophila for 10 days. Results demonstrated that dietary β-sitosterol (1) improved growth performance, attenuated enteritis morbidity and intestinal histopathological lesions; (2) increased lysozyme and acid phosphatase activities, complement 3 and complement 4 contents as well as IgM contents (not in the proximal intestine [PI]) and up-regulated LEAP-2C (rather than LEAP-2A) and hepcidin mRNA expressions in the intestine; (3) down-regulated pro-inflammatory cytokines IL-1β (not in PI), IL-2, IL-6, IL-8, IFN-γ and IFITM mRNA expressions partly associating with NF-κB signalling and up-regulated anti-inflammatory cytokines IL-4/13A (not IL-4/13B), IL-11 and TGF-β mRNA expressions partly associating with TOR signalling in the intestine. Finally, based on percentage weight gain, enteritis morbidity and lysozyme activities in three intestinal segments, the optimal dietary β-sitosterol supplementation levels were estimated to be 1.44%, 2.03%, 2.11%, 1.94% and 1.69% diets respectively. In summary, the optimal dietary β-sitosterol supplementation improved immune function in the intestine of large yellow croaker.

Increased sIL-2Rα leads to obstruction of IL-2 biological function and Treg cells differentiation in SLE patients via binding to IL-2.

BackgroundThe decrease of IL-2 level is believed to play an important role in the disease occurrence and development of SLE, but the relevant mechanisms have not been fully clarified. Many studies have found that the level of soluble interleukin 2 receptor α (sIL-2Rα) in SLE patients is significantly increased. Considering the fact that sIL-2Rα has the ability to bind IL-2, we want to know whether the increased sIL-2Rα has some impact on the level and function of IL-2 in SLE patients.MethodsNew onset SLE patients, treated SLE patients and healthy volunteers were recruited. The levels of serum IL-2, IL-2 mRNA in CD3+ T cells and serum sIL-2Rα were detected and compared in these subjects. Two mixed solid-phase sandwich ELISA system were designed to measure exclusively the heterodimers complex of sIL-2Rα/IL-2. The sera from SLE patients were pretreated with or without immune complex dissociation solution and detected for IL-2 levels. IL-2 standard or serum from HCs were used to co-incubate with recombinant sIL-2Rα or serum samples with high levels of sIL-2Rα and detected for IL-2 levels by ELISA. The inhibitory effect of sIL-2Rα on IL-2 biological activity was investigated by CTLL-2 cell proliferation assay. The frequencies and absolute counts of Treg cells were detected by flow cytometry before and after the addition of recombinant sIL-2Rα.ResultsThe levels of serum IL-2 in SLE patients were significantly decreased and negatively correlated with SLEDAI. However, there was no significant difference in IL-2 mRNA levels in CD3+ T cells between SLE patients and healthy controls. The levels of serum sIL-2Rα in SLE patients were significantly increased, positively correlated with the SLEDAI and negatively correlated with the levels of serum IL-2. sIL-2Rα was shown to bind to IL-2 to form immune complex, resulting in false reduction in the detection level of serum IL-2 and significant decrease in biological activity of IL-2. The increase of sIL-2Rα was demonstrated to be one of the important mechanisms for the obstruction of Treg cells differentiation in SLE patients.ConclusionIncreased serum sIL-2Rα can bind to IL-2, leading to obstruction of IL-2 activity and Treg cells differentiation.

Effect of Qing Chang oral liquid on the treatment of artificially infected chicken coccidiosis and the cellular immunity.

Qing Chang oral liquid (QOL) is a veterinary drug, which mainly composed of Artemisiae annuae herba, Dichroae radix, Agrimonia pilosa and Sanguisorbae radix. This study aims to explore the effect of Qing Chang Oral Liquid (QOL) on the treatment effect of artificially infected chicken coccidiosis and to the cellular immunity. Healthy Roman chickens were randomly divided into five groups: blank group, model group, QOL high-, medium- and low-dose groups. All the groups were orally administered with 1 × 104 sporulated oocysts (except the blank group). After 5 days of oral administration, the high-, medium- and low-dose groups of QOL were added to the drinking water at 2.4, 1.8 and 1.2ml/kg, respectively. The blank and model groups were fed normally, and this experiment lasted for 7 days. The clinical signs were observed, and the relative weight gain, survival rate, cecum lesion score and oocyst value were measured to evaluate the effect of QOL. Meanwhile, the peripheral blood T-lymphocyte subsets and cecal IL-2, IL-17, IFN-γ mRNA expression were detected by flow cytometry and fluorescent quantitative PCR. The chickens in the model group were in poor mental state, gathered together, had loose stools or bloody stools and had less food intake and less exercise. The chicken mental state improved, the food intake and drinking water increased, and the faeces are normal in the high-, medium- and low-dose groups, especially in the high-dose group, the anti-coccidial indexes were up to 173.08. No significant differences were observed (p > 0.05) in the peripheral blood CD3+ , CD3+ CD4+ between the experimental groups. Compared with the blank group, there were different degrees of increase in each dose drug group of the cecal IFN-γ, IL-2 and IL-17 mRNA expression, but the high-dose group was significantly reduced compared with the model group (p < 0.01), but there was no significant difference (p > 0.05). This study suggests that QOL has positive anti-coccidial effect but has no obvious effect on the cellular immunity.

Effect of Dietary Lactose Supplementation on Growth Performance and Intestinal Epithelium Functions in Weaned Pigs Challenged by Rotavirus.

Simple SummaryLactose is a kind of carbohydrate that exists in mammal milk. It has some physiological functions, such as providing energy, regulating gut microbiota, and affecting immunity. Rotavirus (RV) is the main pathogen that induces severe diarrhea in piglets, which impairs their growth and development. In this study, we investigated whether different levels (4% and 6%) of dietary lactose supplementation alleviates RV-induced diarrhea in weaned piglets. The results showed that lactose administration relieved the negative effect of RV on growth, which was derived from the improvement of nutrient utilization, gut barrier function, and immunity. Moreover, supplementing 6% lactose in the diets had a tendency to alleviate diarrhea in RV-infected piglets. Thus, we suggest that the diet of weaned piglets should be supplemented with more than 4% lactose (especially in the early period of weaning) if the cost of feed can be afforded.The purpose of this study was to investigate whether dietary lactose supplementation relieves rotavirus (RV)-induced diarrhea and gut dysfunction. Thirty-six crossbred weaned piglets were randomly allocated into three groups and fed diets containing 0, 4%, and 6% lactose for 20 days. On Day 15, half of the piglets in each group were orally infused with RV. RV infection impaired growth performance; induced severe diarrhea; decreased serum D-xylose concentration and morphology and sIgA level of jejunal mucosa; downregulated MUC1, MUC2, occludin, Bcl-2, IL-4, pBD3, pBD2, and pBD1 mRNA expression of jejunal mucosa and/or mesenteric lymph nodes; upregulated Bax, caspase-3, IL-2, IFN-γ, and IFN-β mRNA expression of jejunal mucosa and/or mesenteric lymph nodes; and damaged microbiota and metabolites of cecal digesta in weaned piglets (p < 0.05). Dietary lactose supplementation improved nutrient digestibility and growth performance and relieved the negative influence of RV challenge on intestinal barrier function, mRNA expression of cytokines, and host defense peptides of jejunal mucosa and/or mesenteric lymph nodes in weaned piglets (p < 0.05). Dietary administration of 6% lactose tended to relieve diarrhea (p = 0.07). These results suggest that lactose in feed increases growth performance and has a tendency to alleviate RV-induced diarrhea, derived from the improvement of nutrient utilization, gut barrier function, and immunity.

Latent, Early or Late Human Herpes Virus-6B Expression in Adult Mesial Temporal Lobe Epilepsy: Association of Virus Life Cycle with Inflammatory Cytokines in Brain Tissue and Cerebral Spinal Fluid

Background: Human herpes virus-6B (HHV-6B) was suggested as an important etiologic factor of mesial temporal lobe epilepsy, while the mechanism is still unknown. Here, we aimed to analyze antigens representing latent, early and late HHV-6B infection and the association with inflammatory cytokines in brain tissue and cerebral spinal fluid (CSF) from MTLE patients with HHV-6B-positivity. Methods: Nested polymerase chain reaction (nPCR), real-time PCR, immunohistochemistry (IHC) and suspension bead array for cytokines were performed. Results: Nested polymerase chain reaction (nPCR) in brain tissue revealed HHV-6B DNA in 19 of 49 MTLE patients (39%) and 1 of 19 controls (5%) (P < 0.001), but not in CSF. ICH showed HHV-6B early antigen (P41) positivity in 3 patients (6%), late antigen (gp116/54/64) positivity in 5 patients (10%), latent antigen (U94) positivity in 8 patients (16%), and multiple antigen (early and late or/and latent) positivity in 9 patients (18%). None of these HHV-6B related proteins were found positive in control brain tissue. PCR revealed significant up-regulation of IL-1a, IL-2 and IL-7 mRNA levels in the brain tissue from MTLE patients expressing early antigens compared to those expressing late, latent, multiple antigens, negative antigens and the controls. Suspension bead array of the CSF confirmed significant up-regulation of IL-1a and IL-7 protein expression from MTLE patients expressing early antigens compared to the other groups. Conclusions: Our finding suggests HHV-6B is a common etiologic agent of MTLE. Different virus life cycle may play an important modifying role in inflammatory biology that warrants further investigation. Though virus DNA is difficult detected in CSF, up-regulation of IL-1a and IL-7 in CSF indicates the two cytokines may be taken as indirect biomarker of HHV-6B infection.

Eosinophilic Esophagitis: Cytokines Expression and Fibrotic Markers in Comparison to Celiac Disease.

Introduction: Eosinophilic esophagitis (EoE) is now recognized as the main inflammatory condition that leads to fibrosis, unlike other chronic inflammatory gastrointestinal diseases, such as celiac disease. The aim of our study is to characterize the collagen deposition and cytokine expression involved in the fibrogenic response in patients affected by EoE in comparison to celiac disease. Materials and Methods: Consecutive patients with a clinical suspicion of untreated EoE or active celiac disease were enrolled. In the control group, patients with negative upper endoscopy were included. Total RNA was isolated from biopsy specimens using a commercial kit (SV Total RNA Isolation System, Promega Italia Srl). Quantitative real-time PCR (qRT-PCR) was performed in triplicate using a StepOne™ Real-Time PCR instrument (Thermo Fisher Scientific, Monza, Italy). mRNA encoding for inflammatory molecules: interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 13 (IL-13), and fibrotic markers: transforming growth factor beta 1 (TGF-β), mitogen-activated protein kinase kinase kinase 7 (MAP3K7), serpin family E member 1 (SERPINE1), were quantified using TaqMan Gene Expression Assays (Applied Biosystems). RESULTS. In EoE, the qPCR analysis showed an increase in all the inflammatory cytokines. Both IL-5 and Il-3 mRNA expression resulted in a statistically significant increase in oesophageal mucosa with respect to the celiac duodenum, while no differences were present in IL-4 expression. TGF-β expression was similar to the controls in the mid esophagus but reduced in the distal EoE esophagus (RQ: 0.46 ± 0.1). MAP3K7 expression was reduced in the mid esophagus (RQ: 0.59 ± 0.3) and increased in the distal esophagus (RQ: 1.75 ± 0.6). In turn, the expression of SERPINE1 was increased in both segments and was higher in the mid than in the distal esophagus (RQ: 5.25 ± 3.9, 1.92 ± 0.9, respectively). Collagen deposition was greater in the distal esophagus compared to the mid esophagus [18.1% ± 8 vs. 1.3% ± 1; p = 0.008]. Conclusions: The present study confirms the esophageal fibrotic involution involving the distal esophagus and shows that the inflammatory pathway in EoE is peculiar to this disease and different from other chronic inflammatory gastrointestinal disorders such as celiac disease.

Effect of Curcumin as Feed Supplement on Immune Response and Pathological Changes of Broilers Exposed to Aflatoxin B1.

In this study, we examined the protective effects of curcumin against the AFB1-induced immune response of and pathological changes in broilers. Histopathology examinations showed that at day 28, AFB1 (5 mg/kg) exposure leads to severe histological changes in the spleen, thymus and bursa of Fabricius with a decrease in the number and karyoplasmic area ratio of plasma cells. Curcumin alleviated the AFB1-induced immune organs’ damage as well as the changes in plasma cells in a dose-dependent manner. RT-PCR data showed that AFB1 significantly downregulated the IL-2 and IFN-γ mRNA expression levels in the thymus, spleen and bursa of Fabricius. However, curcumin supplementation improved the AFB1-induced immune organs’ damage via upregulated cytokines’ expression. Intriguingly, similar trends were noticed in abnormal morphological changes and the immune response at day 35 after the withdrawal of AFB1 and curcumin from the diet, suggesting the protective effects and immunomodulatory function against AFB1 in broilers. The current study provides a scientific experimental basis for the application of curcumin as a therapeutic drug or additive in animal husbandry productive practice.

2020 Research Grant. Mitochondrial targeted protection following incomplete spinal cord injury with noninvasive technology

BACKGROUND CONTEXT Most of the traumatic spinal cord injury (SCI) are treatable, but despite advances in rehabilitation strategies continued impairment in sensorimotor function persists and does not return to pre-injury levels. Following the initial trauma there is a secondary cascade of events characterized by damage to the vasculature of the spinal cord and, therefore, reducing local oxygen delivery to the mitochondria. The main source of ROS (reactive oxygen species; free radicals) production is the mitochondrial electron transport chain. During tissue injury, calcium is released, which activates mitochondrial proteins. The hyperactivate electron transport chain now generates a mitochondrial membrane potential that is above the optimal range. This, in turn, exponentially increases ROS production, which then initiates cellular death cascades causing further damage to the spinal cord, such as inhibiting neuronal differentiation. PURPOSE The purpose of this study was to use a noninvasive infrared red-light therapy (IRL) which reduces the mitochondrial hyperactivity show immediately after SCI. We hypothesized that IRL will reduce mitochondrial hyperactivity which will result in lower ROS production at the epicenter of SCI. METHODS A between-subjects design was used where male Sprague-Dawley rats (3 months old) were randomized into three groups (sham, SCI and SCI + IRL). A balloon catheter model of spinal cord compression was used to induce SCI. The IRL treatment was for 2 hours under anesthesia. RESULTS We found that IRL therapy significantly (p < 0.03) reduced ROS production at the epicenter of SCI. In addition, we found that IL-8 and IL-12 mRNA expression were significantly lower in the SCI + IRL relative to the SCI group, respectively. CONCLUSIONS The results of this pilot study indicated that IRL treatment reduced mitochondrial hyperactivity associated with the initial 24 hours following SCI. FDA DEVICE/DRUG STATUS This abstract does not discuss or include any applicable devices or drugs. Most of the traumatic spinal cord injury (SCI) are treatable, but despite advances in rehabilitation strategies continued impairment in sensorimotor function persists and does not return to pre-injury levels. Following the initial trauma there is a secondary cascade of events characterized by damage to the vasculature of the spinal cord and, therefore, reducing local oxygen delivery to the mitochondria. The main source of ROS (reactive oxygen species; free radicals) production is the mitochondrial electron transport chain. During tissue injury, calcium is released, which activates mitochondrial proteins. The hyperactivate electron transport chain now generates a mitochondrial membrane potential that is above the optimal range. This, in turn, exponentially increases ROS production, which then initiates cellular death cascades causing further damage to the spinal cord, such as inhibiting neuronal differentiation. The purpose of this study was to use a noninvasive infrared red-light therapy (IRL) which reduces the mitochondrial hyperactivity show immediately after SCI. We hypothesized that IRL will reduce mitochondrial hyperactivity which will result in lower ROS production at the epicenter of SCI. A between-subjects design was used where male Sprague-Dawley rats (3 months old) were randomized into three groups (sham, SCI and SCI + IRL). A balloon catheter model of spinal cord compression was used to induce SCI. The IRL treatment was for 2 hours under anesthesia. We found that IRL therapy significantly (p < 0.03) reduced ROS production at the epicenter of SCI. In addition, we found that IL-8 and IL-12 mRNA expression were significantly lower in the SCI + IRL relative to the SCI group, respectively. The results of this pilot study indicated that IRL treatment reduced mitochondrial hyperactivity associated with the initial 24 hours following SCI.

Abstract P2057: Long Non-coding Rna Bcyrn1 In Cardiosphere-derived Extracellular Vesicles Promotes Regulatory T Cell Proliferation, Migration And Activation: Implications For Cardioprotection

Background: Regulatory T (Treg) cells are increasingly implicated as modulators of myocardial ischemia-reperfusion (I/R) injury. We have previously found that extracellular vesicles isolated from human cardiosphere-derived cells (CDC-EVs) could promote Treg proliferation and augment production of the anti-inflammatory cytokine IL-10 in vivo, attenuating cardiac inflammation and functional decline. However, the mechanism by which CDC-EVs regulate Treg is largely unknown. In this study we assessed the hypothesis that long non-coding RNA (LncRNA) within CDC-EVs induces Treg proliferation, migration and IL-10 production, which mediates cardioprotection. Methods: BCYRN1 (Brain Cytoplasmic RNA 1) was identified as a LncRNA species in CDC-EVs using RNA sequencing. We assessed CDC-EVs and LncRNA BCYRN1 effects on Treg by measuring cell proliferation (Cell Counting Kit-8 assay), cell migration (trans-well migration assay), and the production of IL-10 (qPCR and ELISA assay). Additionally, RNA pulldown and luciferase assays were used to determine whether BCYRN1 functions as a miRNA sponge. In vivo , I/R injury was induced in 8-week-old C57BL/6 mouse by left anterior descending coronary artery ligation for 45 minutes followed by reperfusion. Fifteen minutes later, the mice received (i.v., via tail vein) CDC-EVs, CDC-EV BCYRN1 (overexpress BCYRN1 in CDC-EVs), or vehicle (PBS). Infarct size (by TTC staining) and infiltrating Treg (by flow cytometry) were assessed 72 hrs post I/R. Results: Exposure of human Treg to CDC-EVs resulted in Treg proliferation (3.00± 0.11 fold increase, N=5), migration (1.77 ± 0.10 fold increase, N=5), and production of IL-10 (qPCR: 2.60 ± 0.35 fold increase, N=6 and ELISA: 6.12 ± 0.78 fold increase, N=5). RNA sequencing of CDC-EV contents showed that BCYRN1 was one of the most highly enriched LncRNAs. RNA pulldown and luciferase assays showed that BCYRN1 contained binding sites for multiple miRNAs, including miR-138, miR-150 and miR-98 that repress the expression of ATG7, CCR6 and IL-10 mRNAs, respectively. These results reveal that BCYRN1 serves as a miRNA sponge, leading to Treg cell proliferation (via induction of ATG-7 dependent autophagy), CCR-6 dependent migration and production of IL-10. In the mouse I/R model (N=5, each group), CDC-EVs and CDC-EV BCYRN1 (but not vehicle) induced Treg infiltration, proliferation and IL10 production in the heart, with a concomitant decrease of infarct size. Conclusions: We have pinpointed BCYRN1 as a bioactive constituent of CDC-EVs, promoting Treg cell-mediated cardioprotection. BCYRN1 serves as a miRNA sponge to mediate its beneficial effects on Treg infiltration, proliferation, and IL-10 production in the heart. Thus, a single LncRNA mimics the effects of CDC-EV on Treg in vitro and in vivo . Our study provides a novel approach, targeting Treg by a defined LncRNA mined from CDC-EV, to treat myocardial infarction.

Recombinant Ehrlichia canis GP19 Protein as a Promising Vaccine Prototype Providing a Protective Immune Response in a Mouse Model

Simple summaryOne of the limitations of vaccine development against E. canis infection is the indefinite knowledge of the protective immunity in the host. In this study, recombinant protein GP19 was produced as a vaccine prototype, rGP19, for inducing protective immune responses in a mouse model against E. canis. Antibody responses against E. canis were evaluated and revealed that the immunized mice with rGP19 showed higher antibody levels than in adjuvant-immunized and naive mice, both pre- and post-challenging with E. canis. DNA from blood, liver, and spleen were extracted to determine ehrlichial loads. The rGP19-immunized mice showed significantly lower ehrlichial loads in blood, liver, and spleen DNA compared with adjuvant-immunized mice. This study also detected IFN-γ-producing CD4+ T cells in the rGP19-immunized mice and then were later infected with E. canis on day 14 of the post-infection period using flow cytometry. Additionally, Cytokine mRNA expression was investigated and revealed up-regulation of IFNG and IL1 mRNA expression in rGP19-immunized mice. The present study provides evidence of rGP19 that can eliminate E. canis by manipulating both humoral and cell-mediated immune responses in the laboratory animal model.The intracellular bacterium Ehrlichia canis is the causative pathogen of canine monocytic ehrlichiosis (CME) in dogs. Despite its veterinary and medical importance, there is currently no available vaccine against this pathogen. In this study, the recombinant GP19 (rGP19) was produced and used as a recombinant vaccine prototype in a mouse model against experimental E. canis infection. The efficacy of the rGP19 vaccine prototype in the part of stimulating B and T cell responses and conferring protection in mice later challenged with E. canis pathogen were evaluated. The rGP19-specific antibody response was evaluated by ELISA after E. canis challenge exposure (on days 0, 7, and 14 post-challenge), and demonstrated significantly higher mean antibody levels in rGP19-immunized mice compared with adjuvant-immunized and naive mice. Significantly lower ehrlichial loads in blood, liver, and spleen DNA samples were detected in the immunized mice with rGP19 by qPCR. The up-regulation of IFNG and IL1 mRNA expression were observed in mice immunized with rGP19. In addition, this study detected IFN-γ-producing memory CD4+ T cells in the rGP19-immunized mice and later infected with E. canis on day 14 post-infection period using flow cytometry. The present study provided a piece of evidence that rGP19 may eliminate E. canis by manipulating Th1 and B cell roles and demonstrated a promising strategy in vaccine development against E. canis infection in the definitive host for further study.

Eosinophils, but Not Type 2 Innate Lymphoid Cells, Are the Predominant Source of Interleukin 4 during the Innate Phase of Leishmania major Infection.

Interleukin (IL)-4 plays a central role in the initiation of a type 2 T helper cell (Th2) response, which leads to non-healing and progressive infections with the protozoan parasite Leishmania (L.) major. Here, we tested the hypothesis that type 2 innate lymphoid cells (ILC2), which promote the development of Th2 cells, form an important source of IL-4 early after intradermal or subcutaneous L. major infection. Lineage-marker negative CD90.2+CD127+PD1− ILC2 were readily detectable in the ear or foot skin, but hardly in the draining lymph nodes of both naïve and L. major-infected self-healing C57BL/6 and non-healing BALB/c mice and made up approximately 20% to 30% of all CD45+SiglecF− cells. Dermal ILC2 of C57BL/6 mice expressed the inducible T cell-costimulator (ICOS, CD278), whereas BALB/C ILC2 were positive for the stem cell antigen (Sca)-1. Within the first 5 days of infection, the absolute numbers of ILC2 did not significantly change in the dermis, which is in line with the unaltered expression of cytokines activating (IL-18, IL-25, IL-33, TSLP) or inhibiting ILC2 (IL-27, IFN-γ). At day 5 to 6 post infection, we observed an upregulation of IL-4, but not of IL-5, IL-10 or IL-13 mRNA. Using IL-4-reporter (4get) mice, we found that the production of IL-4 by C57BL/6 or BALB/c mice was largely restricted to CD45+SiglecF+ cells of high granularity, i.e., eosinophils. From these data, we conclude that eosinophils, but not ILC2, are a major innate source of IL-4 at the skin site of L. major infection.