Proliferation Of Spleen Cells Research Articles

Effects of Deletion of Macrophage ABCA7 on Lipid Metabolism and the Development of Atherosclerosis in the Presence and Absence of ABCA1

ABCA7, a close relative of ABCA1 which facilitates cholesterol efflux to lipid-poor apoproteins, has been implicated in macrophage lipid efflux and clearance of apoptotic cells in in vitro studies. In the current study, we investigated the in vivo effects of macrophage ABCA7 deficiency on lipid metabolism and atherosclerosis. Chimeras with dysfunctional ABCA7 in macrophages and other blood cells were generated by transplantation of bone marrow from ABCA7 knockout (KO) mice into irradiated low-density lipoprotein receptor (LDLr) KO mice. Unexpectedly, macrophage ABCA7 deficiency did not significantly affect atherosclerosis susceptibility of LDLr KO mice after 10 weeks Western-type diet feeding. However, ABCA7 deficiency was associated with 2-fold (p<0.05) higher macrophage ABCA1 mRNA expression levels. Combined disruption of ABCA1 and ABCA7 in bone-marrow-derived cells increased atherosclerotic lesion development (1.5-fold (p>0.05) as compared to wild type transplanted mice. However, single deletion of ABCA1 had a similar effect (1.8-fold, p<0.05). Macrophage foam cell accumulation in the peritoneal cavity was reduced in ABCA1/ABCA7 dKO transplanted animals as compared to single ABCA1 KO transplanted mice, which was associated with increased ABCG1 expression. Interestingly, spleens of ABCA1/ABCA7 double KO transplanted mice were significantly larger as compared to the other 3 groups and showed massive macrophage lipid accumulation, a reduction in CD3+ T-cells, and increased expression of key regulators of erythropoiesis. In conclusion, deletion of ABCA7 in bone marrow-derived cells does not affect atherogenesis in the arterial wall neither in the absence or presence of ABCA1. Interestingly, combined deletion of bone marrow ABCA1 and ABCA7 causes severe splenomegaly associated with cellular lipid accumulation, a reduction in splenic CD3+ T cells, and induced markers of erythropoeisis. Our data indicate that ABCA7 may play a role in T cell proliferation and erythropoeisis in spleen.

The Effect of Rosemary (Rosmarinus officinalis. L) Plant Extracts on the Immune Response and Lipid Profile in Mice

The hypolipidemic activity of leaves of rosemary (Rosmarinus officinalis), a herbal plant was studied. Three groups of BALB/c mice were treated as following: the first group was fed with normal diet (sugar &amp; water), the second group was fed with high cholesterol (HC) (2% by weight) and coconut oil (25% by weight), for 36 days, and the third group was fed with HC diet and given 100 mg/kg rosemary extract (10% w/v) during the last 15 days of treatment period. There was a significant decrease (P &lt; 0.001) in plasma total cholesterol (TC)(- 68.57%), low density lipoprotein (LDL) (- 56.34%), and triglycerides (TG) (- 182.61%). A significant increase in high density lipoprotein (HDL) (38.53%) was obtained in rosemary-fed mice compared to HC mice. In another experiment, the immunomodulatory activity of aqueous extract of R. officinalis was evaluated in BALB/c mice. Mice were treated with three doses of extract (10, 50, 100 mg/kg body weight) for 8 weeks. Humoral immunity against membrane proteins of sheep erythrocytes measured by ELISA showed that IgM (Immunoglobulin M) response significantly increased by 26.95%, 36.5%, 70.78% respectively, in mice fed with 10, 50, and 100 mg/kg of rosemary as compared to the control group (P &lt; 0.05). IgG (Immunoglobulin G) response increased significantly at all antibody titers, in mice fed with rosemary at doses of 10, 50, and 100 mg/kg. Meanwhile, there was no significant different in IgM and IgG responses between 50 and 100 mg/kg (P &gt; 0.05). On the other hand, concanavalin A-stimulated proliferation of spleen cells from mice fed with 100 mg rosemary extract was significantly higher by 57% than that of cells from the corresponding control animals.

Effect of dietary antimicrobials on immune status in broiler chickens.

This study evaluated the effects of dietary anticoccidial drugs plus antibiotic growth promoters (AGPs) on parameters of immunity in commercial broiler chickens. Day-old chicks were raised on used litter from a farm with endemic gangrenous dermatitis to simulate natural pathogen exposure and provided with diets containing decoquinate (DECX) or monensin (COBN) as anticoccidials plus bacitracin methylene disalicylate and roxarsone as AGPs. As a negative control, the chickens were fed with a non-supplemented diet. Immune parameters examined were concanavalin A (ConA)-stimulated spleen cell proliferation, intestine intraepithelial lymphocyte (IEL) and spleen cell subpopulations, and cytokine/chemokine mRNA levels in IELs and spleen cells. ConA-induced proliferation was decreased at 14 d post-hatch in DECX-treated chickens, and increased at 25 and 43 d in COBN-treated animals, compared with untreated controls. In DECX-treated birds, increased percentages of MHC2+ and CD4+ IELS were detected at 14 d, but decreased percentages of these cells were seen at 43 d, compared with untreated controls, while increased TCR2+ IELs were evident at the latter time. Dietary COBN was associated with decreased fractions of MHC2+ and CD4+ IELs and reduced percentages of MHC2+, BU1+, and TCR1+ spleen cells compared with controls. The levels of transcripts for interleukin-4 (IL-4), IL-6, IL-17F, IL-13, CXCLi2, interferon-γ (IFN-γ), and transforming growth factorβ4 were elevated in IELs, and those for IL-13, IL-17D, CXCLi2, and IFN-γ were increased in spleen cells, of DECX- and/or COBN-treated chickens compared with untreated controls. By contrast, IL-2 and IL-12 mRNAs in IELs, and IL-4, IL-12, and IL-17F transcripts in spleen cells, were decreased in DECX- and/or COBN-treated chickens compared with controls. These results suggest that DECX or COBN, in combination with bacitracin and roxarsone, modulate the development of the chicken post-hatch immune system.

Iron deficiency reduces serum and in vitro secretion of interleukin-4 in mice independent of altered spleen cell proliferation

Iron deficiency, a worldwide public health problem in children and adult women, impairs innate and cell-mediated immunity including interferon-γ secretion. Its effects on interleukin (IL)-4 have not been well investigated. Interleukin-4, a cytokine primarily secreted by TH2 lymphocytes, regulates B-cell proliferation and the switching of immunoglobulin (Ig)M to IgE subtypes; the latter is involved in the defense against helminth infection. Considering the fact that interferon-γ is a potent inhibitor of IL-4, we hypothesize that iron deficiency would increase the secretion of IL-4 and IgE. We measured IL-4 in serum and supernatant of concanavalin A and anti-CD3 antibody-treated spleen cells from iron-deficient, control, pair-fed DBA and C57BL/6 mice (20-24/group) and iron-replete mice for 3, 7, and 14 days (8-13/group). Feeding the low-iron diet (5 ppm vs 50 ppm for the control diet) for 2 months significantly reduced the mean levels of hemoglobin, hematocrit, liver iron stores, thymus weight, and induced splenomegaly in both strains of mice (P < .001). Iron deficiency, and not pair-feeding, reduced plasma IL-4 levels (P < .05), although it did not significantly affect IgE levels. Iron deficiency, especially when associated with thymus atrophy, reduced in vitro IL-4 secretion by activated spleen cells, cell proliferation, and percentage of CD4⁺IL-4⁺ cells (P < .05). Impaired cell proliferation did not fully explain reduced in vitro IL-4 secretion because iron-deficient mice with a normal thymus weight had a normal (3)H-thymidine uptake but decreased supernatant IL-4. It was likely due to low percentage of CD4⁺IL-4⁺. Iron repletion improved IL-4 measurements. Data suggest that iron deficiency has generalized negative effects on T-cell function. Unaltered plasma IgE may be due to other cytokines (ie, IL-13) that also modulate its secretion.

Effects of anticoccidial and antibiotic growth promoter programs on broiler performance and immune status

This study investigated the effects of various coccidiosis control programs in combination with antibiotic growth promoters (AGPs) on growth performance and host immune responses in broiler chickens. The coccidiosis programs that were investigated included in ovo coccidiosis vaccination (CVAC) with Inovocox or in-feed medication with diclazuril as Clinacox (CLIN) or salinomycin (SAL). The AGPs were virginiamycin or bacitracin methylene disalicylate plus roxarsone. As a negative control, chickens were non-vaccinated and fed with non-supplemented diets (NONE). All animals were exposed to used litter from a commercial broiler farm with confirmed contamination by Eimeria parasites to simulate in-field exposure to avian coccidiosis. Broiler body weights in the CVAC group were greater at 14 and 32days of age, but not at day 42, compared with the NONE, CLIN, and SAL groups. At day 14, the SAL group showed decreased body weight and reduced ConA-stimulated spleen cell proliferation compared with the CLIN and SAL groups. In contrast, at days 34 and 43, splenocyte proliferation was greater in the CVAC and CLIN groups compared with the NONE and SAL groups. Lymphocyte subpopulations and cytokine mRNA expression levels in the intestine and spleen were also altered by the denoted treatments. Collectively, these results suggest that in ovo coccidiosis vaccination or coccidiostat drug medication programs in combination with AGPs influences chicken growth and immune status in an Eimeria-contaminated environment.

Immunostimulatory effect by aqueous extract of Hizikia fusiforme in RAW 264.7 macrophage and whole spleen cells

Hizikia fusiforme is a commonly used food that possesses potent anti-bacterial, anti-fungal, and anti-inflammatory activities. The immunostimulatory activities of aqueous extract of Hizikia fusiforme (HFAE) in RAW 264.7 macrophages and whole spleen cells were investigated. HFAE activated RAW 264.7 macrophages to produce cytokines such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in a dose-dependent manner. In addition, HFAE induced the mRNA expression of TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophages. Moreover, HFAE stimulated proliferation of whole spleen cells and reference mitogen. Taken together, the results demonstrate that HFAE potently activates the immune function by regulating NO, TNF-α, IL-1β, and IL-6 in RAW 264.7 macrophage and promoting spleen cell proliferation.

Impact of Fresh or Used Litter on the Posthatch Immune System of Commercial Broilers

This study was carried out to investigate the effects of exposure of growing broiler chickens of commercial origin to used poultry litter on intestinal and systemic immune responses. The litter types evaluated were fresh wood shavings or used litter obtained from commercial poultry farms with or without a history of gangrenous dermatitis (GD). Immune parameters measured were serum nitric oxide (NO) levels, serum antibody titers against Eimeria or Clostridium perfringens, mitogen-induced spleen cell proliferation, and intestinal intraepithelial lymphocyte or splenic lymphocyte subpopulations. At 43 days posthatch, birds raised on used litter from a GD farm had higher serum NO levels and greater Eimeria or C. perfringens antibody levels compared with chickens raised on fresh litter or used, non-GD litter. Birds raised on non-GD and GD used litter had greater spleen cell mitogenic responses compared with chickens raised on fresh litter. Finally, spleen and intestinal lymphocyte subpopulations were increased or decreased depending on the litter type and the surface marker analyzed. Although it is likely that the presence of Eimeria oocysts and endemic viruses varies qualitatively and quantitatively between flocks and, by extension, varies between different used litter types, we believe that these data provide evidence that exposure of growing chicks to used poultry litter stimulates humoral and cell-mediated immune responses, presumably due to contact with contaminating enteric pathogens.

Correction of Murine SCID-X1 by Lentiviral Gene Therapy Using a Codon-optimized IL2RG Gene and Minimal Pretransplant Conditioning

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using γ-retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor γ gene (IL2RG) cDNA (coγc), regulated by its 1.1 kb promoter region (γcPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of Il2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and γcPr vectors. We conclude that SIN lentiviral gene therapy using coγc driven by the γcPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required.

Evaluation of DNA encoding acidic ribosomal protein P2 of Cryptosporidium parvum as a potential vaccine candidate for cryptosporidiosis

The Cryptosporidium parvum acidic ribosomal protein P2 (CpP2) is an important immunodominant marker in C. parvum infection. In this study, the CpP2 antigen was evaluated as a vaccine candidate using a DNA vaccine model in adult C57BL/6 IL-12 knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding CpP2 (CpP2-DNA) cloned into the pUMVC4b vector induced a significant anti-CpP2 IgG antibody response that was predominantly of the IgG1 isotype. Compared to control KO mice immunized with plasmid alone, CpP2-immunized mice demonstrated specific in vitro spleen cell proliferation as well as enhanced IFN-γ production to recombinant CpP2. Further, parasite loads in CpP2 DNA-immunized mice were compared to control mice challenged with C. parvum oocysts. Although a trend in reduction of infection was observed in the CpP2 DNA-immunized mice, differences between groups were not statistically significant. These results suggest that a DNA vaccine encoding the C. parvum P2 antigen is able to provide an effective means of eliciting humoral and cellular responses and has the potential to generate protective immunity against C. parvum infection but may require using alternative vectors or adjuvant to generate a more potent and balanced response.

Inhibition of colon tumor growth by IL-15 immunogene therapy

Interleukin-15 is a pleiotropic cytokine that has potential for cancer immunegene therapy. We previously reported the construction and characterization of IL-15 overexpression vectors pHi2-IL15-CMV-tat (L1) and pHi2-spIL15-CMV-tat (L3), as well as carcinoembryonic antigen promoter-amplified IL-15 expression plasmid vectors pHi2-IL15-CEA-tat (L2) and pHi2-spIL15-CEA-tat (L4). In the current study, we evaluated the expression and therapeutic efficacy of these vectors using a mouse colon carcinoma model. Plasmid vectors were transfected into SW480 and MCF-7 cells, and IL-15 overexpression was confirmed. IL-15 expressed by transfected tumor cells stimulated spleen cell proliferation in vitro. Intraperitoneal injection of plasmid pHi2-spIL15-CMV-tat (L3) into mice resulted in transgene expression by peritoneal mesothelial cells, inhibited CT-26 tumor growth and prolonged the survival of tumor-bearing mice. In addition, the in vivo transfection of plasmid pHi2-spIL15-CMV-tat (L3) via electroporation slowed the tumor formation of subcutaneously inoculated CT-26 cells. These data suggest that IL-15 overexpression achieves therapeutic effects in a mouse cancer model and that these gene transfer approaches should be further evaluated for use in the treatment of human cancers.

Mitogenic response and probiotic characteristics of lactic acid bacteria isolated from indigenously pickled vegetables and fermented beverages

Lactic acid bacteria from indigenous pickled vegetables and fermented beverages (fermented rice and Madhuca longifolia flowers) were isolated and investigated for their functional characteristics in vitro as potential new probiotic strains. Four isolates (all Lactobacillus spp.) selected on the basis of high tolerance to bile (0.2%) were identified by standard and molecular methods (16S rDNA) as L. helveticus, L. casei, L. delbrueckii and L. bulgaricus from pickled vegetables and fermented beverages respectively. These selected strains had antibiotic resistance, tolerance to artificial gastric juice and phenol (0.4%), enzymatic profile, and antagonistic activity against enteric pathogens (Enterobacter sakazakii, Salmonella typhimurium, Shigella flexneri 2a, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila). All strains survived well in artificial gastric juice at low pH (3.0) values for 4h, possessed bile salt hydrolase activity and were susceptible to most antibiotics including vancomycin. Additionally, the isolates exhibited high tolerance to phenol, high cell surface hydrophobicity (>60%) and induced proliferation of murine splenocytes. All the four strains of present study suppressed the Con A-stimulated proliferation of the mouse spleen cells, although L. casei had the strongest suppressive effect. The results of this study suggest a potential application of the strains (following human clinical trials), for developing probiotic foods.

English

The aim of this present study was to investigate the immunomodulatory and antioxidant activities of Siraitia grosvenorii polysaccharide (SGP) using a mouse model. The roles of regulation of SGP were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, fluorescence method and ELISA method. The chemical structures were analyzed by Fourier transform infrared spectroscopy (FTIR) and gas chromatography (GC). The results show that SGP promoted the proliferation of spleen cells and regulated the level of reactive oxygen species (ROS) in vitro . Furthermore, it significantly raised spleen and thymus indices and superoxidase dismutase activity and regulated the cytokine levels of spleen and thymus in mice in vivo . These findings suggest that SGP possesses immunomodulatory and antioxidant effects.

The tumor rejection effect of protein components from medicinal fungus

Medicinal fungi are renowned for their antitumor activity and have been used in oriental countries for thousands of years. Modern medicine has documented that polysaccharides are the main components responsible for antitumor activity in medicinal fungi. We reported that a water-soluble component Yt from Agrocybe aegerita exhibited tumor rejection activity. The component was determined to contain proteins (60%, two lectins, serine proteinase and several unknown proteins), low molecular weight compounds (40%) and trace polysaccharides. Yt showed high antitumor activity in several tumor cell lines. In tumor-bearing BALB/c mice, Yt treatment caused tumor (hepatocellular carcinoma H22 and sarcoma S180) rejection at a low concentration (2.5 mg/kg body weight) without significant toxicity. In a nude mouse model, Yt treatment did not induce significant changes in tumor size but prolonged the lifespan. In vivo data suggested that Yt induced tumor rejection by stimulating the immune response. Spleen cell proliferation was promoted ex vivo by Yt treatment. When mice were treated with Yt, splenocyte cytotoxic activity was enhanced and the mRNA level of cytokines was altered. Furthermore, the antitumor activity of Yt sharply declined when the proteins were degraded by proteinase K, which showed that proteins were the main antitumor component in Yt. Using the same isolation procedure, the components from three other medicinal fungi Cordyceps militaris, Lentinula edodes and Ganoderma lucidum demonstrated similar tumor rejection effects as Yt. All our findings indicated that besides polysaccharides, water-soluble protein components possessing high antitumor activity exist in medicinal fungi and require further study.

Development and characterization of mouse monoclonal antibodies reactive with chicken interleukin-2 receptor αlpha chain (CD25)

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 ( chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25 + spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25 + cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.

Characterization of antigen-binding and MHC class II-bearing T cells with suppressive activity in response to tolerogenic stimulus

Antigen specific non-responsiveness is generally developed through clonal deletion, anergy, and suppression. The term “suppression” is being considered as a functional immune deficit that can be adoptively transferred by regulatory/suppressor T cells. Following tolerance induction protocols the aim of the present study was to characterize the T cells involved in antigen-specific suppression. After defining the immunogenic and tolerogenic protocols in vitro and in vivo, it was shown that CD90+ cells from tolerogenic mice were able to reduce specific antibody production when adaptively transferred to immunized mice. These cells were shown to highly express CD25 and Foxp3, co-localizing with CD4 and MHC class II antigens (MHCII), while a small percentage of these cells (8–14%) was binding free antigen. Further isolation of CD90+MHCII+ and CD90+HSA+ from mice having received the tolerogenic treatment and adaptive transfer to immunized mice showed that CD90+MHCII+ cells were able to suppress antigen-specific response only when transferred along with the second antigenic challenge, while CD90+HSA+ cells were suppressive only when given along with the first antigenic challenge. The suppressive effect of these two sub-populations could also be obtained in in vitro spleen cell proliferation assays in response to the HSA antigenic stimulus. Both in vitro and in vivo tolerogenic treatments were shown to correlate with soluble MHCII production in culture supernatants or serum respectively. Increase of MHCII in the serum could only be detected upon adaptive transfer of CD90+HSA+ cells, whereas transfer of CD90+MHCII+ cells resulted in increased levels of IL-10 and IFN-γ in the serum. These results defined at least two different levels of suppression, one during the onset which was antigen-specific and MHC restricted, and another non-specific at the end of an immune response.

Modulation of immune and inflammatory responses on experimental arthritis following intraarticular gene transfer of tumor necrosis factor receptor-immunoglobulin Fc

In spite of popularity of TNF-α antagonist in the treatment of rheumatoid arthritis (RA), their modes of action are not fully understood. In the present study, we further explore the effects of gene transfer route of a TNF-α antagonist on arthritis. Recombinant adeno-associated virus 2 (rAAV2) encoding rat TNF receptor-immunoglobulin Fc (ratTNFR:Fc) fusion gene was injected intraarticularly in rats with collagen-induced arthritis (CIA). As revealed by examination of the clinical, radiographical, and histological aspects, local gene transfer of rAAV2/ratTNFR:Fc ameliorated the arthritis symptoms and inhibited the development of CIA. Compared with the vector control group, expressions of TNF-α, IL-1, and IFN-γ were down-regulated, and IL-10 release was up-regulated in the rAAV2/ratTNFR:Fc-treated group. Furthermore, administration of rAAV2/ratTNFR:Fc ameliorated the enlargement of spleen and significantly reduced spleen cell proliferation. Low level of nitric oxide (NO) in spleen was observed in CIA rats following the delivery of rAAV2/ratTNFR:Fc when compared to the vector control group. This study provides the evidence that intraarticular delivery of rAAV2/ratTNFR:Fc suppress the progression of arthritis by restoring the balance between pro-inflammatory and anti-inflammatory cytokines and inhibiting spleen cell proliferation. Our findings also implicate that the down-regulation of NO release on arthritis is involved in the anti-inflammatory mechanisms of TNF-α antagonist.

Immunomodulatory Effects of Eisenia bicyclis on Innate Immune Cells in Acute Exercise-Stress Rat Model

In this study we investigated effects of supplementation with ethyl acetate extracts of the brown alga Eisenia bicyclis on innate immune cells to evaluate the possibilities as an immunomoulator in exercise stress. Twenty male SD rats were divided into four groups and the treatments were as follows: A, no Eisenia bicyclis extract (EBE) (200 mg/kg) intake and maintained at rest ; B, no EBE intake and undergoing exercise ; C, EBE intake and undergoing exercise ; D, EBE intake and maintained at rest. After 5 weeks of oral supplementation, rats were undergoing intensive swimming exercises for 2 h and sacrificed to assess the effects on peritoneal macrophages, spleen cells and natural killer (NK) cells. We showed increasing effects on nitric oxide-inducible nitric oxide synthase (NO-iNOS) production by macrophages and no effects of NK tumoricidal activity and suppressive effects on spleen cell proliferation in exercise group. However, EBE supplementation suppressed NO-iNOS production by macrophages and increased NK tumoricidal activity and spleen cell proliferative response to mitogen in exercise group. Overall, these results that EBE supplementation has differential effects on innate immune response and could be useful as sports nutrition.

Plant Hsp90 Proteins Interact with B-Cells and Stimulate Their Proliferation

BackgroundThe molecular chaperone heat shock protein 90 (Hsp90) plays an important role in folding stabilization and activation of client proteins. Besides, Hsp90 of mammals and mammalian pathogens displays immunostimulatory properties. Here, we investigated the role of plant-derived Hsp90s as B-cell mitogens by measuring their proliferative responses in vitro.MethodologyPlant cytosolic Hsp90 isoforms from Arabidopsis thaliana (AtHsp81.2) and Nicotiana benthamiana (NbHsp90.3) were expressed in E. coli. Over-expression of recombinant plant Hsp90s (rpHsp90s) was confirmed by SDS-PAGE and western blot using and anti-AtHsp81.2 polyclonal anti-body. Both recombinant proteins were purified by Ni-NTA affinity chromatography and their identity confirmed by MALDI-TOF-TOF. Recombinant AtHsp81.2 and NbHsp90.3 proteins induced prominent proliferative responses in spleen cells form BALB/c mice. Polymyxin-B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate the rpHsp90-induced proliferation. In addition, in vitro incubation of spleen cells with rpHsp90 led to the expansion of CD19-bearing populations, suggesting a direct effect of these proteins on B lymphocytes. This effect was confirmed by immunofluorescence analysis, where a direct binding of rpHsp90 to B- but not to T-cells was observed in cells from BALB/c and C3H/HeN mice. Finally, we examined the involvement of Toll Like Receptor 4 (TLR4) molecules in the rpHsp90s induction of B-cell proliferation. Spleen cells from C3H/HeJ mice, which carry a point mutation in the cytoplasmic region of TLR4, responded poorly to prAtHsp90. However, the interaction between rpHsp90 and B-cells from C3H/HeJ mice was not altered, suggesting that the mutation on TLR4 would be affecting the signal cascade but not the rpHsp90-TLR4 receptor interaction.ConclusionsOur results show for the first time that spleen cell proliferation can be stimulated by a non-pathogen-derived Hsp90. Furthermore, our data provide a new example of a non-pathogen-derived ligand for TLRs.

Effect of FK506 on the immunosuppression of mesenchymal stem cells induced by interferon-γ and tumor necrosis factor-α

Objective To investigate the mechanism of immunosuppressant FK506 on the immunesuppression of mesenchymal stem cells ( MSCs) induced by interferon ( IFN) -γ and tumor necrosis factor (TNF)-α. Methods MSCs pretreated with inflammatory cytokines were co-cultured with mice spleen cells. After incubation with FK506 for 72 h, proliferation of spleen cells was measured by methyl thiazol tetrazolium (MTT) assay and the apoptosis was assessed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to detect the major molecule that mediated the immunosuppression of MSCs and the effect of FK506 on the molecule. Results MSCs pretreated with inflammatory cytokines could inhibit the proliferation of spleen cells and increase the apoptosis of spleen cells, and the apoptosis rate was (53. 5 ±2. 5) % , which was higher than that of MSCs pretreated with IFN-γ or TNF-α and control group ( P ＜0. 05), and IFN-γ and TNF-α upregulated inducible nitric oxide synthase (iNOS) expression in MSCs significantly, and this immunosuppression could also be antagonized by a specific inhibitor 1400W. However,FK506 could inhibit iNOS expression in MSCs pretreated with IFN-γ and TNF-α, the inhibition of spleen cells proliferation by MSCs was reduced significantly, and the apoptosis rate of spleen cells was reduced to (44. 3 ±3. 2)% (P＜0. 05). Conclusion FK506 inhibits the immunosuppression of MSCs through downregulation of iNOS expression in MSCs pretreated with IFN-γ and TNF-α. Key words: Mesenchymal stem cells; Inflammation; Immunosuppression; FK506

Structural determinants of the immunomodulatory properties of the C-terminal region of bovine β-casein

Bovine β-casein and its C-terminal sequence (191–209) were previously shown to possess immunomodulatory properties in studies on partially purified peptides from hydrolysates, where minor amounts of contaminants may affect the cellular response. The inhibitory effect of β-casein and of eight C-terminus synthetic β-casein peptides on mitogen-induced spleen cell proliferation was compared. Use of synthetic peptides allowed the unambiguous and accurate identification that a seven amino acid sequence at the C-terminus, including a PFP motif, was sufficient for immunomodulation. Substitution of the last proline (P206) in the PFP motif with D-Pro had a negative impact on the immunosuppressory activity of all these short peptides, whereas substitution of P206 with structural analogues of proline had almost no impact. A relationship was found between the immunomodulatory properties and the structural features of these peptides, as assessed by various spectroscopic approaches, indicating a role of structure in eliciting the immunomodulatory activity of these peptides.