Mouse Spleen Cells Research Articles

NB-UVB irradiation attenuates inflammatory response in psoriasis.

Psoriasis is a chronic inflammatory disease characterized by immunological imbalance and vasodilation. Many triggering factors for psoriasis initiate inflammation via the activation of NF‐κB. Narrow‐band ultraviolet B (NB‐UVB) irradiation can be used as a general treatment for psoriasis, although the molecular mechanism has not yet been determined. The aim of this study was to elucidate the potential molecular mechanism of NB‐UVB irradiation therapy on psoriasis. We collected serum samples from patients with psoriasis and healthy control, and detected the expression of inflammatory factors by ELISA. In addition, we established mouse model of psoriasis. After different doses of NB‐UVB irradiation, the proportion of CD4+, CD8+, and CD11c+ cells in mouse spleen was detected by flow cytometry. Meanwhile, the expression of inflammatory factors in the damaged skin of mice was detected by RT‐PCR and Western blot analysis, and mouse serum levels of inflammatory factors were detected by ELISA. Our results showed that NB‐UVB irradiation regulated the expression of inflammatory factors in psoriasis patients. In mice, high‐dose NB‐UVB irradiation effectively eliminated IMQ‐induced psoriasis‐like dermatitis and inhibited the expression of pro‐inflammatory factors. In conclusion, our results indicate that NB‐UVB irradiation could regulate the expression of inflammatory factors and attenuate psoriasis plaques.

Antioxidant activity and immune-enhancing effect of aqueous leaf Elaeagnus multiflora Thunb

In this study, we aimed to investigate the antioxidant and immunopotentiating effects of leaves of cherry elaeagnus (Elaeagnus multiflora, Em). The leaf, stem, and root of Em have been used in Kampo medicine. There are data on the therapeutic effects of Em fruit, but no information on its leaves. We thus investigated the antioxidant and immunopotentiating effects of Em leaf extract. Balb/c mouse spleen cells were treated with concanavalin A and hot aqueous Em leaf extract (50, 100, and 200 μg/mL), and the effects on spleen cell proliferation and the secretion of interleukin (IL)-2, IL-4, and IL-10 were evaluated. A concentration-dependent increase in the secretion of IL-2 and IL-4 cytokines was observed, with concentrations of 1.86 pg/mL of IL-2 and 37.63 pg/mL of IL-4 when cells were treated with 200 μg/mL of the extract. Natural killer (NK) cell activity was determined based on a co-culture of spleen cells and Yac-1 cells. NK activity gradually increased in a concentration-dependent manne. Further, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity was 58.5% and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity was 78.2% with an extract concentration of 200 μg/mL. The high antioxidant activity of the hot water extract of Em leaves suggests their potential therapeutic value and warrants further studies.

Monoclonal Antibody Against N Terminal 439-451 Epitopes of Anti-Mullerian Hormone and Its Properties

To prepare the specific monoclonal antibody against the N-terminal specific epitope peptide of anti-mullerian hormone (AMH) and to identify its specificity. Using bioinformatics analysis software to predict the specific peptide fragment of AMH. Then synthesized four antigenic epitope peptide segments of mature N-terminal region of AMH as the screening target antigen. Synthesized AMH wholegene.Using the prokaryotic expression system to abtain recombinant AMH protein. Immunized BALB/c mice with the recombinant AMH, and prepared mouse spleen cells for fusing with SP/20 cells. Preparation of AMH monoclonal antibody by hybridoma technology. The monoclonal antibodies against AMH were screened by using four N-terminal epitope peptides (1: 439-451 RGRDPRGPGRAQ, 2: 273-285 PPRPSAELEESPP, 3: 42-54 DLDWPPGSPQEPL, 4: 494-506 WPQSDRNPRYGNH) as antigens, and indirect ELISA and Western blot were used to identify the antigen binding characteristics of the selected monoclonal antibodies. Two hybridoma cell lines with stable anti-AMH-1 and anti-AMH-2 antibody activities were screened. The two antibodies were named anti-AMH-1 and anti-AMH-2 respectively. The antibody titers were 1∶12 000 and 1∶1 600 after purification. Western blot confirmed that the two McAbs recognized different antigens. Anti-AMH-1 could not only recognize the N-terminal 439-451 epitope peptide of AMH, but also recognize the amino acid sequence of recombinant AMH, as well as the ovarian tissue. Anti-AMH-2 could recognize recombinant AMH and ovarian tissue. Two monoclonal antibodies against N-terminal specific epitopes of human AMH were successfully constructed.

Evaluation the Incidence of Genotoxic Effects of Artificial Food Favoring Additives in Bone Marrow Cells and Spleen Cells in Mice


 Genetic material is the most important component of cells because it contains the genetic information; hence any disruption to the structure chromosome of cells could lead to very bad results. Genotoxicity use to evaluate the safety of any chemical compounds on genetic materials. Artificial food flavoring additive are chemical substances to produce specific placebo effects added to foods but impart specific flavor to it.
 The present study evaluates the genotoxic effect of artificial food flavoring additive on structure of chromosomes at three different concentrations (50%, 100%and 150%) on both bone marrow cells and spleen cells in mice for fourteen successive days. It was found that artificial food flavoring additive at concentration (50% and 100%) show not significant increase in total chromosomal aberration in both bone marrow cells and spleen cells when compare to negative control (p>0.05) meanwhile at concentration 150% it causes a significant increase when compare to negative control (p<0.05) .The results have been showed that artificial food flavoring additive had a genotoxic effect at (50%, 100% and 150%).

Evaluation the Incidence of Genotoxic Effects of Artificial Food Favoring Additives in Bone Marrow Cells and Spleen Cells in Mice


 Genetic material is the most important component of cells because it contains the genetic information; hence any disruption to the structure chromosome of cells could lead to very bad results. Genotoxicity use to evaluate the safety of any chemical compounds on genetic materials. Artificial food flavoring additive are chemical substances to produce specific placebo effects added to foods but impart specific flavor to it.
 The present study evaluates the genotoxic effect of artificial food flavoring additive on structure of chromosomes at three different concentrations (50%, 100%and 150%) on both bone marrow cells and spleen cells in mice for fourteen successive days. It was found that artificial food flavoring additive at concentration (50% and 100%) show not significant increase in total chromosomal aberration in both bone marrow cells and spleen cells when compare to negative control (p>0.05) meanwhile at concentration 150% it causes a significant increase when compare to negative control (p<0.05) .The results have been showed that artificial food flavoring additive had a genotoxic effect at (50%, 100% and 150%).

Key role of macrophages in tolerance induction via T regulatory type 1 (Tr1) cells.

T regulatory type 1 (Tr1) cells are a class of regulatory T cells (Tregs ) participating in peripheral tolerance, hence the rationale behind their testing in clinical trials in different disease settings. One of their applications is tolerance induction to allogeneic islets for long-term diabetes-free survival. Currently the cellular and molecular mechanisms that promote Tr1-cell induction in vivo remain poorly understood. We employed a mouse model of transplant tolerance where treatment with granulocyte colony-stimulating factor (G-CSF)/rapamycin induces permanent engraftment of allogeneic pancreatic islets in C57BL/6 mice via Tr1 cells. The innate composition of graft and spleen cells in tolerant mice was analyzed by flow cytometry. Graft phagocytic cells were co-cultured with CD4+ T cells in vitro to test their ability to induce Tr1-cell induction. Graft phagocytic cells were depleted in vivo at different time-points during G-CSF/rapamycin treatment, to identify their role in Tr1-cell induction and consequently in graft survival. In the spleen, the site of Tr1-cell induction, no differences in the frequencies of macrophages or dendritic cells (DC) were observed. In the graft, the site of antigen uptake, a high proportion of macrophages and not DC was detected in tolerant but not in rejecting mice. Graft-infiltrating macrophages of G-CSF/rapamycin-treated mice had an M2 phenotype, characterized by higher CD206 expression and interleukin (IL)-10 production, whereas splenic macrophages only had an increased CD206 expression. Graft-infiltrating cells from G-CSF/rapamycin-treated mice-induced Tr1-cell expansion in vitro. Furthermore, Tr1-cell induction was perturbed upon in-vivo depletion of phagocytic cells, early and not late during treatment, leading to graft loss suggesting that macrophages play a key role in tolerance induction mediated by Tr1 cells. Taken together, in this mouse model of Tr1-cell induced tolerance to allogeneic islets, M2 macrophages infiltrating the graft upon G-CSF/rapamycin treatment are key for Tr1-cell induction. This work provides mechanistic insight into pharmacologically induced Tr1-cell expansion in vivo in this stringent model of allogeneic transplantation.

Development of an in vivo model to assess immune and angiogenic responses to metallic implants using Humanized TLR-4 mice

Abstract Metal on metal(MOM) hips often fail due to inappropriate inflammation and angiogenesis which may be due to localized concentrations of metal ions and/or MOM wear particles in the joint. Critically, human, but not mouse TLR-4 receptors, are responsive to nickel, cobalt and MOM particles. Therefore, we are using humanized, hTLR-4 mice in these studies. MOM hips are predominately cobalt, so our initial experiments were focused upon this ion. Spleen cells of hTLR -4 mice were exposed to 3 concentrations of CoCl2 (2.5μM, 25μM, 250μM) for 3 and 6 days. The cultures were analyzed for endothelial cell markers, (MADCAM and CD31)and T cell markers, (CD4 and CD8). A dose dependent induction of endothelial markers was seen on day 3 with a dose dependent reduction on day 6, with no changes in CD4 or CD8 expression. Next, spleen cells of TLR-4 mice were exposed to 3 different ratios of cell to MOM particles (1:5, 1:50, 1:500 per cell) for 3 and 6 days. Endothelial markers were induced, but it was not dose dependent, however the endothelial cell numbers increased between 3 and 6 days. Again, T cell markers were not altered. To assess the in-vivo responses to MOM particles, nude mice were implanted with angioreactors containing MOM particles (2.5×105 and 2.5 × 106). On day 12, the angioreactors were removed and the was a dose dependent increase in endothelial cells. Currently, we are extending the MOM angiogenesis studies in hTLR-4 mice. In sum, both CoCl2 and MOM particles induced endothelial cells and angiogenesis. The hTLR-4 mouse model will greatly aid in the evaluating the angiogenic and immunotoxicological profile of ions and MOM wear debris, particularly during menopause. Funded by FDA Office of Women’s Health.

Removal of Fc Glycans from [89Zr]Zr-DFO-Anti-CD8 Prevents Peripheral Depletion of CD8+ T Cells.

The N-linked biantennary glycans on the heavy chain of immunoglobulin G (IgG) antibodies (mAbs) are instrumental in the recognition of the Fc region by Fc-gamma receptors (FcγR). In the case of full-length mAb-based imaging tracers targeting immune cell populations, these Fc:FcγR interactions can potentially deplete effector cells responsible for tumor clearance. To bypass this problem, we hypothesize that the enzymatic removal of the Fc glycans will disrupt Fc:FcγR interactions and spare tracer-targeted immune cells from depletion during immunopositron emission tomography (immunoPET) imaging. Herein, we compared the in vitro and in vivo properties of 89Zr-radiolabeled CD8-specific murine mAb (anti-CD8wt, clone 2.43), a well-known depleting mAb, and its deglycosylated counterpart (anti-CD8degly). Deglycosylation was achieved via enzymatic treatment with the peptide: N-glycosidase F (PNGaseF). Both anti-CD8wt and anti-CD8degly mAbs were conjugated to p-SCN-Bn-desferrioxamine (DFO) and labeled with 89Zr. Bindings of both DFO-conjugated mAbs to FcγR and CD8+ splenocytes were compared. In vivo imaging and distribution studies were conducted to examine the specificity and pharmacokinetics of the radioimmunoconjugates in tumor-naive and CT26 colorectal tumor-bearing mice. Ex vivo analysis of CD8+ T cell population in spleens and tumors obtained postimaging were measured via flow cytometry and qRT-PCR. The removal of the Fc glycans from anti-CD8wt was confirmed via SDS-PAGE. A reduction in FcγR interaction was exhibited by DFO-anti-CD8degly, while its binding to CD8 remained unchanged. Tissue distribution showed similar pharmacokinetics of [89Zr]Zr-DFO-anti-CD8degly and the wt radioimmunoconjugate. In vivo blocking studies further demonstrated retained specificity of the deglycosylated radiotracer for CD8. From the imaging studies, no difference in accumulation in both spleens and tumors was observed between both radiotracers. Results from the flow cytometry analysis confirmed depletion of CD8+ T cells in spleens of mice administered with DFO-anti-CD8wt, whereas an increase in CD8+ T cells was shown with DFO-anti-CD8degly. No statistically significant difference in tumor infiltrating CD8+ T cells was observed in cohorts administered with the probes when compared to control unmodulated mice. CD8 mRNA levels from excised tumors showed increased transcripts of the antigen in mice administered with [89Zr]Zr-DFO-anti-CD8degly compared to mice imaged with [89Zr]Zr-DFO-anti-CD8wt. In conclusion, the removal of Fc glycans offers a straightforward approach to develop full length antibody-based imaging probes specifically for detecting CD8+ immune molecules with no consequential depletion of their target cell population in peripheral tissues.

Concentration-dependent effect of silymarin on concanavalin A-stimulated mouse spleen cells in vitro

AbstractAims: Silymarin (SIL), a mixture of phenolic compounds, has a pleiotropic mode of action on various cell types, including immune cells. In this study, we investigated the concentration-dependent effect of SIL on proliferation of concanavalin A (CoA)-stimulated mouse spleen T lymphocytes, their viability, and secretion of IFN-g and IL-4 cytokinesex vivoin relation to gene expressions of transcription factors nuclear factor kappa B and Foxp3. In addition, metabolic activity of T cells was determined as changes in the mitochondrial membrane potential and apoptosis.Material/Methods: Isolated splenocytes were stimulated with lectin CoA and treated with SIL atthe concentrations of 5, 10, 20, and 40 μg/ml for 70 h and unstimulated cells served as the control. Cultures of splenocytes were evaluated for proliferation index following BrdU incorporation and viability of cells after trypan blue staining. Gene expressions of transcription factors and cytokines were assessed using real-time PCR, whereas ELISA test was applied to measure cytokine secretion. Mitochondrial membrane potential and apoptosis were determined by flow cytometry.Results: We demonstrated that CoA-activated mouse spleen T lymphocytes show different susceptibilities to low (£10 μg/ml) and higher (20 and 40 μg/ml) SIL concentrations. Low concentrations resulted in increased proliferation, cytokine secretion, and mitochondrial membrane potential and reduced transition of cells to apoptosis. High concentration of SIL had the opposite effect without exerting significant cytotoxicity and upregulated genes for cytokines and transcription factors on mRNA level. It is possible that individual subpopulations of T cells induced by CoA were differentially affected by the various SIL concentrations and the dose of 40 μg/ml had the profound suppressive effect. This correlated with the highest expression of Foxp3 factor, indicating that this dose stimulated preferential differentiation to Tregs lymphocytes.Conclusions: Treatment with suitable doses of SIL can provide potential benefits in the modulation of host immune functions in various diseases.

Influence of ZnO Nanoparticles on Candida albicans of Human Male Pleural Fluid

The utilization of metal oxide nanoparticles, especially zinc oxide, is of a great importance in the medical field because of its physical and chemical properties as well as its antimicrobial potential effects. In our study, the ZnO nanoparticles were synthesized by the precipitation method where pH=14. ZnO nanoparticles were characterized by ultraviolet–visible spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM) and atomic force microscope (AFM). Antifungal activity of the ZnO was tested against candida albicans. The results showed that C. albicans (15 samples) became resistant to the fungal activity after testing its sensitivity to several types of fungal antibiotics. UV-vis spectroscopy, XRD, TEM and AFM showed that this precipitation synthetic method can produce a good quality of ZnO nanoparticles with sizes in the range of nanometer scale. In ZnO NPs (calcined), the inhibition zone diameter of C. albicans was 11 mm at a ZnO NPs concentration of 800 mg/ml, while no inhibition zones were formed at the other concentrations (0.25, 0.5, 1, 2, 10, 50 and 500 mg/ml). Using ZnO NPs (not calcined), the inhibition zone diameter of C. albicans was 24 mm at a concentration of 800 mg/ml, while no inhibition zones were observed at the other concentrations (0.25, 0.5, 1, 2, 10, 50 and 500 mg/ml). In addition, a toxicity test was performed on mice and proved that ZnO NPs are effective against C. albicans with a toxic effect on liver and spleen cells in rats. The aim of this research was to characterize the in vitro activity of ZnO nanoparticles synthesized by the precipitation method against C. albicans of human male pleural fluid using the well diffusion method as well as their toxic effects on both liver and spleen cells in mice.

Immune Regulation of TNFAIP3 in Psoriasis through Its Association with Th1 and Th17 Cell Differentiation and p38 Activation.

Background Psoriasis is an immune-mediated chronic inflammatory skin disorder in which the dysregulation of immune cells plays an important role in its development. Tumor necrosis factor- (TNF-) α antagonists affect the immune repertoire, while TNF-α-induced protein 3 (TNFAIP3) has a protective role against the deleterious effects of inflammation and participates in immune regulation. Objective We investigated the immune regulation of TNFAIP3 in the pathogenesis of psoriasis and determined whether it is involved in the antipsoriatic effect of TNF-α antagonists. Methods mRNA levels were evaluated in blood from patients with moderate-to-severe psoriasis. The effects of TNF-α antagonists were examined in a mouse imiquimod- (IMQ-) induced psoriasis-like dermatitis model. In the mouse model, TNFAIP3 mRNA expression was determined using RT-PCR. Serum levels of IL-17A, IL-23, IFN-γ, TNF-α, phosphorylated ERK1/2, p38, and JNK were measured using ELISA. The proportion of Th1 and Th17 cells in mouse spleens was analyzed using flow cytometry. Results mRNA expression levels of TNFAIP3 in the blood were significantly lower in patients with moderate and severe psoriasis (mean ± SD = 0.44 ± 0.25) compared with normal subjects (mean ± SD = 1.00 ± 0.82) (P < 0.01). In the mouse model, IMQ downregulated TNFAIP3 expression levels, which were increased after TNF-α antagonist treatment (P < 0.05). Serum levels of Th17 cytokines (IL-17A and IL-23) and Th1 cytokines (IFN-γ and TNF-α) were significantly higher in the IMQ and IMQ/rat IgG1 groups compared with the control group, and the application of TNF-α antagonists significantly decreased the levels of inflammatory cytokines (P < 0.01). Notably, phosphorylated p38 levels were increased in the IMQ and IMQ/rat IgG1 groups compared with the control group but were downregulated by treatment with TNF-α antagonists (P < 0.05). Th1 and Th17 cells were significantly increased in the IMQ group compared with the control group (P < 0.01). Conclusion TNFAIP3 downregulation associated with Th1 and Th17 cell differentiation and p38 activation might contribute in part to the mechanism of immune dysfunction in psoriasis. TNF-α antagonists might partly exert their effects on psoriasis via this pathway.

Evaluation of in vitro Cytotoxic Effects of Especifico Pessoa Phytotherapic Tincture on Ehrlich Tumor Cells and Mice Spleen Cells

Especifico Pessoa (EP) is traditionally used for the treatment of snakebite envenoming. The traditional use of EP and its properties have been reported. In this study, we evaluated the in vitro cytotoxic effects of EP on Ehrlich tumor and mice spleen cells. Cytotoxicity assay was carried out by using Trypan blue exclusion method. Spleen cell suspension was prepared (n=2) with RPMI medium and tumor cell suspension was prepared from ascitic fluid of Ehrlich tumor-bearing mice (n=1); both the suspensions contained 4 x 106 cells mL-1. Pure EP or EP diluted in RPMI (1:2; 1:4) was used. The results were expressed as percentage of cell viability and demonstrate that EP is toxic to Ehrlich cells at all concentrations (Control: 96.42 ± 3.40; Pure: 1.55 ± 2.91; 1:2: 4.85 ± 5.04; 1:4: 13.39 ± 5.08), but nontoxic to spleen cells in at the lowest dilution (Control: 72.86 ± 13.79; Pure: 13.52 ± 6,36; 1:2: 41.36 ± 13.51; 1:4: 56.59 ± 8.62). Therefore, the results demonstrate that EP has cytotoxic effects, depending on the dose and the cell line evaluated.

Effects of Cadmium and Acanthopanax senticosus Extract on the Activities of Immune Cells in Mice Spleen

Cadmium is an environmental hazard which suppress humoral and cellular immune response of immune cells. Acanthopanax senticosus (Rupr. et Maxim.) Harms (AS) is a widely used adaptogen containing triterpenoid saponins, flavonoids and polysaccharides. AS improves immune response enhancing activities of immune cells. The aim of this study was to investigate the ability of liquid extract of AS to moderate immunotoxic effects of cadmium. The effects of AS extract on accumulation of Cd as well as on levels of splenic immune cells after chronic treatment with CdCl2 were investigated. BALB/c mice were periodically injected intraperitoneal (i.p.) for 6 weeks with CdCl2 and AS extract solutions of two different concentrations in deionized water: 0.05 LD50 AS; 0.10 LD50 AS; 0.05 LD50 Cd2+; 0.05 LD50 Cd2+ and 0.05 LD50 AS; 0.05 LD50 Cd2+ and 0.10 LD50 AS. Control group mice were periodically injected i.p. with the saline solution. The levels of macrophages, T lymphocytes and B lymphocytes were determined by immunohistochemistry using, respectively, CD68, CD3 and CD20 antibodies. The results showed that periodical injection of CdCl2 and CdCl2 together with AS caused increase of Cd2+ concentration in the spleen as compared to control and AS groups. Injections of cadmium increased the number of macrophages. AS extract and/or Cd2+ did not change the number of T lymphocytes. AS extract significant increase the number of B lymphocytes in comparison to control group, furthermore the number of B lymphocytes was higher in Cd+AS 0.1 LD50 group than in mice injected with CdCl2 only.

Streptococcus thermophilus ST285 Alters Pro-Inflammatory to Anti-Inflammatory Cytokine Secretion against Multiple Sclerosis Peptide in Mice.

Probiotic bacteria have beneficial effects to the development and maintenance of a healthy microflora that subsequently has health benefits to humans. Some of the health benefits attributed to probiotics have been noted to be via their immune modulatory properties suppressing inflammatory conditions. Hence, probiotics have become prominent in recent years of investigation with regard to their health benefits. As such, in the current study, we determined the effects of Streptococcus thermophilus to agonist MBP83–99 peptide immunized mouse spleen cells. It was noted that Streptococcus thermophilus induced a significant increase in the expression of anti-inflammatory IL-4, IL-5, IL-10 cytokines, and decreased the secretion of pro-inflammatory IL-1β and IFN-γ. Regular consumption of Streptococcus thermophilus may therefore be beneficial in the management and treatment of autoimmune diseases such as multiple sclerosis.

Immunogenicity Assessment of Rift Valley Fever Virus Virus-Like Particles in BALB/c Mice.

Rift Valley fever (RVF) is an acute, febrile zoonotic disease that is caused by the RVF virus (RVFV) and is spread by arthropod vectors. Virus-like particle (VLP) vaccines, which have the advantages of strong immunogenicity and safety, play an important role in the prevention of this disease. VLPs for RVFV were successfully prepared by our research group using a baculovirus-insect cell expression system. To study the immunogenicity of these RVFV VLPs, a correct 3rd or 4th generation recombinant baculovirus, rBac-N-G, was identified and used to infect Sf9 cells, which were cultured in suspension at a large scale. Subsequently, cell debris was removed by centrifugation, and the VLPs were concentrated by ultracentrifugation and purified using a sucrose gradient, after which they were used to immunize BALB/c mice by intramuscular injection. The results showed that the RVFV VLPs prepared by our research group could effectively induce mice to produce RVFV neutralizing antibodies and that the prepared VLPs could stimulate mouse spleen cells to produce high levels of the cytokines IL-4 and IFN-γ. Moreover, the proportion of lymphocytes producing IL-4 and IFN-γ in the spleen of mice immunized with RVFV VLPs was significantly increased. Therefore, the RVFV VLPs prepared in this study had strong immunogenicity and could effectively activate humoral and cellular immunity in mice. This study lays a solid foundation for the development of RVFV VLP vaccine candidates and promotes the healthy development of animal husbandry and human public health.

In vitro cytotoxicity of Duguetia sp extracts on Ehrlich tumor cells

The genus Duguetia , of the Annonaceae family, includes around 100 species that have been used in folk medicine to treat several diseases. Its phytochemical compounds have been researched for their antifungal, antioxidant, antigenotoxic, anti-inflammatory and cytotoxic properties. Therefore, the objective of the present study was to conduct a preliminary study on the biological effects of Duguetia sp extracts by evaluating their in vitro cytotoxic effect on different cell types. To prepare the methanolic extract (ME), the leaves were macerated with 80% methanol. Part of the ME was dissolved in 10% phosphoric acid, and the acidic aqueous solution was partitioned with dichloromethane. The organic phase was evaporated to obtain an acetogenin-rich extract (ACE). The extracts were diluted in RPMI culture medium, supplemented with 20% fetal bovine serum, and added to Erhlich tumor cells or mice spleen cells at different concentrations: the ACE extract at 1.35 mg mL -1 , 0.67 mg mL -1 or 0,34 mg mL -1 , and the ME extract at 1.40 mg mL -1 , 0.70 mg mL -1 or 0,35 mg mL -1 . After 24 h, cytotoxicity analysis was performed using the Trypan Blue exclusion method. The results demonstrated that the extracts are cytotoxic at all concentrations to Ehrlich tumor cells and mice spleen cells. According to the results, we concluded that the extracts of Duguetia sp had a cytotoxic effect on the studied cells in vitro. This was the first study to report the biological effect of this plant genus in this type of cells; however, further study is needed to determine the current species used and the compounds present in the extracts.

Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response.

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4+ T cells, rather than CD8+ T cells or CD19+ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFβ1, leading to an increase in a highly suppressive CD25+ FoxP3+ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4+ , CD8+ , and IFN-γ+ expressing cell infiltration. These findings point to a unique CD4+ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

The effects of short-term calorie restriction on mutations in the spleen cells of infant-irradiated mice.

The risk of cancer due to exposure to ionizing radiation is higher in infants than in adults. In a previous study, the effect of adult-onset calorie restriction (CR) on carcinogenesis in mice after early-life exposure to X-rays was examined (Shang, Y, Kakinuma, S, Yamauchi, K, et al. Cancer prevention by adult-onset calorie restriction after infant exposure to ionizing radiation in B6C3F1 male mice. Int J Cancer. 2014; 135: 1038-47). The results showed that the tumor frequency was reduced in the CR group. However, the mechanism of tumor suppression by CR is not yet clear. In this study, we examined the effects of CR on radiation-induced mutations using gpt delta mice, which are useful to analyze mutations in various tissues throughout the whole body. Infant male mice (1-week old) were exposed to 3.8 Gy X-rays and fed a control (95 kcal/week/mouse) or CR (65 kcal/week/mouse) diet from adult stage (7-weeks old). Mice were sacrificed at the age of 7 weeks, 8 weeks and 100 days, and organs (spleen, liver, lung, thymus) were harvested. Mutations at the gpt gene in the DNA from the spleen were analyzed by using a gpt assay protocol that detects primarily point mutations in the gpt gene. The results showed that mutation frequencies were decreased in CR groups compared with non-CR groups. Sequence analysis of the gpt gene in mutants revealed a reduction in the G:C to T:A transversion in CR groups. Since it is known that 8-oxoguanine could result in this base substitution and that CR has an effect of reducing oxidative stress, these results indicate that the suppression of oxidative stress by CR is the cause of the reduction of this transversion.

Lactobacillus rhamnosus GG alleviates β-conglycinin-induced allergy by regulating the T cell receptor signaling pathway.

Currently, the need for safe and effective methods for relieving allergies is an important concern. In this study, we evaluated the role of Lactobacillus rhamnosus GG (LGG) in alleviating β-conglycinin (β-CG)-induced allergies and elucidated the related molecular mechanisms. Typical allergy symptoms and inflammatory factors in the serum showed that LGG intervention effectively alleviated β-CG induced allergy in mice, which was better than natural recovery (NR). Intestinal villi were restored and lower levels of CD4+ T cells infiltrated after LGG intervention. We evaluated whether LGG intervention weakened the proliferation ability of the spleen cells of allergic mice, balancing between T/B cells and Th1/Th2 and Th17/Treg cytokines. Transcriptome analysis revealed that 4106 differentially expressed mRNAs were identified by comparing the LGG group and β-CG group, and 546 differentially expressed mRNAs were identified by comparing the LGG group and NR group. KEGG pathway analysis identified that the T cell receptor (TCR) signaling pathway was significantly enriched upon LGG intervention, and the upregulated Ifnar2 and the downregulated Tgfbr2, Il13r2 and Il4ra were further validated by qPCR analysis. Therefore, the above results fully revealed the important role of LGG in alleviating β-CG-induced allergies.

A Novel Surface Modification and Immobilization Method of Anti-CD25 Antibody on Nonwoven Fabric Filter Removing Regulatory T Cells Selectively.

Anti-CD25 antibodies were immobilized on polypropylene (PP) nonwoven fabrics to specifically remove mouse regulatory T cells (Tregs) from mouse spleen cells. PP fibers were coated with peptide nanosheets, which were prepared by self-assembling of a mixture of X-poly(sarcosine)-b-(l-Leu-Aib)6 (X: glycolic acid or a phenylboronic acid) and Y-poly(sarcosine)-b-(d-Leu-Aib)6 (Y: glycolic acid or diazirine derivative). Anti-CD25 antibodies were immobilized by covalent linking between the sugar moiety of the antibody and the phenylboronic acid group on the peptide nanosheet. The removal rate of mouse Tregs from the mouse spleen cells was more than 95% only by passing the filters, while the nonspecific removal rates of other cells were less than 15%. The coating of peptide nanosheets on PP fibers was very effective to provide a suitable environment for the immobilized antibody to interact with the counterpart cells while the coating suppressed nonspecific adsorption of other cells.