Rice bran oil ameliorates inflammatory responses by enhancing mitochondrial respiration in murine macrophages.

Previous studies have revealed the anti-inflammatory properties of rice bran oil (RBO), but the detailed mechanisms are poorly understood. Recent studies on the molecular/cellular anti-inflammatory mechanisms of dietary components have demonstrated that mitochondrial respiration plays a key role in macrophage functioning. Since dietary lipids are major substrates for mitochondrial respiration through ß-oxidation, the current study examined whether RBO regulates inflammatory responses by modulating mitochondrial energy metabolism. Palm oil (PO), enriched with palmitic acid which are known to be effectively taken up by cells and used for oxidative phosphorylation, served as a positive control. In the in vitro model of LPS-stimulated RAW 264.7 murine cells, the levels of pro-inflammatory cytokines (IL-6 and TNF-α) in the culture supernatant were significantly reduced by RBO treatment. In contrast, secretion of the anti-inflammatory cytokine IL-10 was upregulated by RBO. Transcription of genes encoding inflammatory mediator molecules (COX-2 and iNOS) and expression of activation markers (CD80, CD86, and MHC-II) in LPS-stimulated RAW 264.7 cells were suppressed by RBO. Mitochondrial respiration (as assessed by an extracellular flux analyzer) increased upon RBO treatment, as the basal respiration, maximal respiration, ATP production, and spare respiratory capacity were upregulated. In an in vivo study, C57BL/6 mice were fed a negative control diet containing corn oil (CO), PO, or RBO for 4 weeks, and bone marrow-derived macrophages (BMDM) were isolated from their tibias and femurs. In pro-inflammatory M1-polarized BMDM (M1-BMDM), the RBO-induced suppression of IL-6 and TNF-α was recapitulated in vivo. Mitochondrial respiration in M1-BMDM also increased following the RBO intervention and the PO control treatment as compared to CO fed negative control. Overall, the current study for the first time demonstrates that RBO regulates inflammatory responses in murine macrophages by upregulating mitochondrial respiration. Further clinical studies are required to validate the animal study.

Gel Phase 1,2-Distearoyl-sn-glycero-3-phosphocholine-Based Liposomes Are Superior to Fluid Phase Liposomes at Augmenting Both Antigen Presentation on Major Histocompatibility Complex Class II and Costimulatory Molecule Display by Dendritic Cells in Vitro.

Lipid-based nanoparticles have in recent years attracted increasing attention as pharmaceutical carriers. In particular, reports of them having inherent adjuvant properties combined with their ability to protect antigen from degradation make them suitable as vaccine vectors. However, the physicochemical profile of an ideal nanoparticle for vaccine delivery is still poorly defined. Here, we used an in vitro dendritic cell assay to assess the immunogenicity of a variety of liposome formulations as vaccine carriers and adjuvants. Using flow cytometry, we investigated liposome-assisted antigen presentation as well as the expression of relevant costimulatory molecules on the cell surface. Cytokine secretion was further evaluated with an enzyme-linked immunosorbent assay (ELISA). We show that liposomes can successfully enhance antigen presentation and maturation of dendritic cells, as compared to vaccine fusion protein (CTA1-3Eα-DD) administered alone. In particular, the lipid phase state of the membrane was found to greatly influence the vaccine antigen processing by dendritic cells. As compared to their fluid phase counterparts, gel phase liposomes were more efficient at improving antigen presentation. They were also superior at upregulating the costimulatory molecules CD80 and CD86 as well as increasing the release of the cytokines IL-6 and IL-1ß. Taken together, we demonstrate that gel phase liposomes, while nonimmunogenic on their own, significantly enhance the antigen-presenting ability of dendritic cells and appear to be a promising way forward to improve vaccine immunogenicity.

Marina crystal minerals (MCM) activate human dendritic cells to induce CD4+ and CD8+ T cell responses in vitro.

Marina crystal minerals (MCM) are a mixture that contains crystallized minerals along with trace elements extracted from seawater. It is a nutritional supplement that is capable of enhancing natural killer (NK) cell activity and increasing T and B cell proliferation in humans post ingestion. However, its effect on dendritic cells (DCs), the cells that bridge innate and adaptive immunity, is not yet known. In this study, we examine the stimulatory effects of MCM on DCs' maturation and function in vitro. Human monocyte-derived DCs were treated with MCM at two different concentrations (10 and 20 µg/mL) for 24 h. Results showed that MCM treatment activated DCs in a dose-dependent fashion. It caused the upregulation of costimulatory molecules CD80, CD86, and HLA-DR, and prompted the production of DC cytokines, including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and IL-1ß, and chemokines (monocyte chemotactic protein-1 (MCP-1)) and interferon-gamma-inducible protein-10 (IP-10). In addition, activated DCs primed CD4+ T cells to secrete significant amounts of interferon gamma (IFN-γ), and they also stimulated CD8+ T cells to express higher amounts of CD107a. These results indicate that MCM is a potentially powerful adjuvant, from natural materials, that activates human DCs in vitro and therefore may suggest its possible use in immune-based therapies against cancer and viral infections.

PeptoSomes for Vaccination: Combining Antigen and Adjuvant in Polypept(o)ide-Based Polymersomes.

In this work, the first vaccine is reported based on a PeptoSome, which contains a model antigen (SIINFEKL) and adjuvant (CpG). PeptoSomes are polypept(o)ide-based polymersomes built of a block-copolymer with polysarcosine (PSar) as the hydrophilic block (X n = 111) and poly(benzyl-glutamic acid) (PGlu(OBn)) as the hydrophobic one (X n = 46). The polypept(o)ide is obtained with low dispersity index of 1.32 by controlled ring-opening polymerization. Vesicle formation by dual centrifugation technique allows for loading of vesicles up to 40 mol%. PeptoSomes are characterized by multiangle dynamic light scattering, static light scattering, and cryogenic transmission electron microscopy (cryoTEM). The PeptoSomes have a hydrodynamic radius of 39.2 nm with a low dispersity (µ 2 = 0.1). The ρ-ratio R g /R h of 0.95 already indicates that vesicles are formed, which can be confirmed by cryoTEM. Loaded PeptoSomes deliver the antigen (SIINFEKL) and an adjuvant (CpG) simultaneously into dendritic cells (DCs). Upon cellular uptake, dendritic cells are stimulated and activated, which leads to expression of cluster of differentiation CD80, CD86, and MHCII, but induces excretion of proinflammatory cytokines (e.g., TNFα). Furthermore, DC-mediated antigen-specific T-cell proliferation is achieved, thus underlining the enormous potential of PeptoSomes as a versatile platform for vaccination.

Piperlongumine Suppresses Dendritic Cell Maturation by Reducing Production of Reactive Oxygen Species and Has Therapeutic Potential for Rheumatoid Arthritis.

Piperlongumine (PLM) is a natural product from the plant Piper longum that inhibits platelet aggregation, atherosclerosis plaque formation, and tumor cell growth. It has potential value in immunomodulation and the management of autoimmune diseases. In this study, we investigated the role of PLM in regulating the differentiation and maturation of dendritic cells (DCs), a critical regulator of immune tolerance, and evaluated its clinical effects in a rheumatoid arthritis mouse model. We found that PLM treatment reduced LPS-induced murine bone marrow-derived DC maturation, characterized by reduced expression of CD80/86, secretion of MCP-1, IL-12p70, IL-6, TNFα, IFN-γ, and IL-23, and reduced alloproliferation of T cells; however, PLM does not affect cell differentiation. Furthermore, PLM reduced intracellular reactive oxygen species (ROS) production by DCs and inhibited the activation of p38, JNK, NF-κB, and PI3K/Akt signaling pathways. Conversely, PLM increased the expression of GSTP1 and carbonyl reductase 1, two enzymes that counteract ROS effects. ROS inhibition by exogenous N-acetyl-l-cysteine suppressed DC maturation. PLM treatment improved the severity of arthritis and reduced in vivo splenic DC maturation, collagen-specific CD4(+) T cell responses, and ROS production in mice with collagen-induced arthritis. Taken together, these results suggest that PLM inhibits DC maturation by reducing intracellular ROS production and has potential as a therapeutic agent for rheumatoid arthritis.

[Experimental study on anti-tumor effect of xihuang pill and its immune clearance function].

OBJECTIVE: To discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms. METHOD: Walker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA. RESULT: The tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05). CONCLUSION: Xihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.

IGRP and insulin vaccination induce CD8+ T cell-mediated autoimmune diabetes in the RIP-CD80GP mouse.

Autoimmune diabetes is characterized by autoantigen-specific T cell-mediated destruction of pancreatic islet beta cells, and CD8(+) T cells are key players during this process. We assessed whether the bitransgenic RIP-CD80 x RIP-LCMV-GP (RIP-CD80GP) mice may be a versatile antigen-specific model of inducible CD8(+) T cell-mediated autoimmune diabetes. Antigen-encoding DNA, peptide-loaded dendritic cells and antigen plus incomplete Freund's adjuvant were used for vaccination. Of 14 pancreatic proteins tested by DNA vaccination, murine pre-proinsulin 2 (100% of mice; median time after vaccination, 60 days) and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) (77%, 58 days) could induce diabetes. Vaccination with DNA encoding for zinc transporter 8, Ia-2, Ia-2ß, glutamic acid decarboxylase 67 (Gad67), chromogranin A, insulinoma amyloid polypeptide and homeobox protein Nkx-2.2 induced diabetes development in 25-33% of mice. Vaccination with DNA encoding for Gad65, secretogranin 5, pancreas/duodenum homeobox protein 1 (Pdx1), carboxyl ester lipase, glucagon and control hepatitis B surface antigen (HBsAg) induced diabetes in <20% of mice. Diabetes induction efficiency could be increased by DNA vaccination with a vector encoding a ubiquitin-antigen fusion construct. Diabetic mice had florid T cell islet infiltration. CD8(+) T cell targets of IGRP were identified with a peptide library-based enzyme-linked immunospot assay, and diabetes could also be induced by vaccination with major histocompatibility complex (MHC) class I-restricted IGRP peptides loaded on mature dendritic cells. Vaccination with antigen plus incomplete Freund's adjuvant, which can prevent diabetes in other models, led to rapid diabetes development in the RIP-CD80GP mouse. We conclude that RIP-CD80GP mice are a versatile model of antigen specific autoimmune diabetes and may complement existing mouse models of autoimmune diabetes for evaluating CD8(+) T cell-targeted prevention strategies.

Evaluating combinations of costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy.

Combinatory strategies are becoming of increasing interest in cancer immunotherapy. Costimulation by individual members of the immunoglobulin-like (Ig)- and TNF superfamily have already shown promising antitumor potential, thus prompting the exploration of their synergistic abilities in combinatorial approaches. Here, we pursued a targeted strategy with antibody-fusion proteins composed of a tumor-directed antibody and the extracellular domain of the costimulatory ligand B7.1, 4-1BBL, OX40L, GITRL or LIGHT, respectively. Costimulatory activity was assessed in an experimental setting where initial T cell activation was induced by a bispecific antibody (tumor-related antigen × CD3). Advantage of combined targeted costimulation was shown for either B7.1 or 4-1BBL with OX40L, GITRL, LIGHT and 4-1BBL in terms of T cell proliferation and IFN-γ release. Since encouraging results were obtained by the combination of B7.1 and 4-1BBL, we adapted the model system for a time-shift setting. Here, enhanced proliferation and granzyme B expression as well as reduced PD-1 expression on the T cell population demonstrated the benefit of costimulation-assisted restimulation. Finally, the antitumor potential of this combinatorial setting was confirmed in vivo in a lung metastasis mouse model. Thus, combinatorial approaches with costimulatory antibody-ligand fusion proteins seem a promising strategy to be further investigated for cancer immunotherapy.

Prevention of autoimmune diabetes and induction of ß-cell proliferation in NOD mice by hyperbaric oxygen therapy.

We evaluated the effects of hyperbaric oxygen therapy (HOT) on autoimmune diabetes development in nonobese diabetic (NOD) mice. Animals received no treatment or daily 60-min HOT 100% oxygen (HOT-100%) at 2.0 atmospheres absolute and were monitored for diabetes onset, insulitis, infiltrating cells, immune cell function, and ß-cell apoptosis and proliferation. Cyclophosphamide-induced diabetes onset was reduced from 85.3% in controls to 48% after HOT-100% (P < 0.005) and paralleled by lower insulitis. Spontaneous diabetes incidence reduced from 85% in controls to 65% in HOT-100% (P = 0.01). Prediabetic mice receiving HOT-100% showed lower insulitis scores, reduced T-cell proliferation upon stimulation in vitro (P < 0.03), increased CD62L expression in T cells (P < 0.04), reduced costimulation markers (CD40, DC80, and CD86), and reduced major histocompatibility complex class II expression in dendritic cells (DCs) (P < 0.025), compared with controls. After autoimmunity was established, HOT was less effective. HOT-100% yielded reduced apoptosis (transferase-mediated dUTP nick-end labeling-positive insulin-positive cells; P < 0.01) and increased proliferation (bromodeoxyuridine incorporation; P < 0.001) of insulin-positive cells compared with controls. HOT reduces autoimmune diabetes incidence in NOD mice via increased resting T cells and reduced activation of DCs with preservation of ß-cell mass resulting from decreased apoptosis and increased proliferation. The safety profile and noninvasiveness makes HOT an appealing adjuvant therapy for diabetes prevention and intervention trials.

Immunomodulatory studies of a bioactive fraction from the fruit of Prunus cerasus in BALB/c mice.

In order to evaluate the role of ethyl acetate fraction (PNRS-EtOAC) obtained from the Prunus cerasus fruit in the modulation of immune responses, detailed studies were carried out using a panel of in vivo assays. Oral administration of PNRS-EtOAC (25-100 mg/kg) stimulated the IgM and IgG titre expressed in the form of hemagglutination antibody (HA) titre. Further, it elicited a dose related increase in the delayed type hypersensitivity reaction (DTH) after 24 and 48 h in BALB/c mice. Besides augmenting the humoral and cell mediated immune response, the concentration of cytokines (IFN-γ, IL-4, and TNF-α) in serum with respect to T cell interactions, i.e. the proliferation of lymphocytes were significantly increased at 50 mg/kg compared with the control. The results in these studies demonstrated the immunostimulatory effect of PNRS-EtOAC in a dose-dependent manner with respect to the macrophage activation possibly expressing the phagocytosis and nitrite production by the enhancement of TNF-α production as a mode of action.

Differential effects of chlorogenic acid on various immunological parameters relevant to rheumatoid arthritis.

Despite chlorogenic acid (CGA) being widely present in nature, particularly in the human diet, there is very little information regarding its pharmacological activities. The present investigation was carried out to investigate the antiarthritic activities of this compound in adjuvant induced-arthritis in male Wistar rats, and to explore the underlying mechanisms of actions in view of immunological responses. We observed that CGA effectively controlled the total (CD3) and differentiated (CD4 and CD8) T cells count at the dose of 40 mg/kg. We also assessed the effect on co-stimulatory molecules (CD28, CD80/86) and found that CGA efficiently suppressed CD80/86 but failed to bring any changes in the CD28 count, whereas ibuprofen (standard drug) resulted in highly significant inhibition of both. We next examined the effect on CD4⁺ T cells specific Th1/Th2 cytokines by flow cytometry and observed that CGA suppressed the Th1 cytokines in a highly significant manner but elevated Th2 cytokines with dose dependence. Results of the present investigation suggest that CGA is a potent antiarthritic agent.

Abatacept mechanism of action: concordance with its clinical profile.

The double and simultaneous molecular interaction between antigen-presentig cells (APC) and T lymphocytes is essential for the optimal activation of the immunological response and requires the participation of two membrane receptor groups. Abatacept is a fusion protein that selectively modulates one of these two ways, by binding to CD80 and CD86 receptors on APC. In this way, the drug inhibits T cell activation, selectively blocking the specific interaction of CD80/CD86 receptors to CD28 and, therefore, inhibiting T cell proliferation and B cell immunological response. This pharmacological action results in the normalization of inflammatory mediators in rheumatoid arthritis patients and in a safe and efficacious clinical response. Abatacept in combination with methotrexate prevents the progression of joint damage and improves physical function in rheumatoid arthritis patients.

A novel monoclonal antibody against human CD80 and its immune protection in a mouse lupus-like disease.

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), is an attractive means to induce antigen-specific peripheral tolerance in autoimmune disease and organ transplantation. In this study, we generated and characterized a monoclonal antibody (Clone 4E5) against human CD80. 4E5 could recognize both human and mouse CD80 and suppress mixed lymphocyte reaction in vitro. To investigate their potency for clinical use, we further administrated 4E5 to a mouse lupus-like disease model (C57BL/J6) induced by Pristane. 4E5 could inhibit the immune response and attenuate the severity of lupus-like disease. The data showed 4E5 function and suggested that blockade of CD80/CD28 co-stimulatory signal pathway with 4E5 is a promising strategy to decelerate the progression of lupus-like disease and other autoimmune diseases.

Bromelain treatment leads to maturation of monocyte-derived dendritic cells but cannot replace PGE2 in a cocktail of IL-1ß, IL-6, TNF-α and PGE2.

Immunotherapy using dendritic cells (DC) has shown promising results. However, the use of an appropriate DC population is critical for the outcome of this treatment, and the search for an optimal DC subset is still ongoing. The DC used in immunotherapy today are usually matured with a cytokine cocktail consisting of TNF-α, IL-1ß, IL-6 and PGE(2). These cells have deficits in their cytokine production, particularly IL-12p70, mainly because of the presence of PGE(2). Bromelain is a pineapple stem extract containing a mixture of proteases that has been used clinically in adjuvant cancer treatment. In this study, we analysed the effect of bromelain on human monocyte-derived DC. We added bromelain to the cytokine cocktail and modified cytokine cocktails with either no PGE(2) or reduced amounts of PGE(2), respectively. Combining bromelain with the cytokine cocktails containing PGE(2) resulted in an increased surface expression of CD83, CD80 and CD86. The chemokine receptor CCR7 was also considerably upregulated in these DC populations compared with DC treated with the cytokine cocktail alone. Removal or reduction of PGE(2) from the cytokine cocktail did not increase the IL-12p70 secretion from stimulated DC, and addition of bromelain to the different cytokine cocktails resulted in only a minor increase in IL-12p70 production. Moreover, combining bromelain with the cytokine cocktails did not improve the T cell stimulatory capacity of the generated DC populations. In conclusion, bromelain treatment of monocyte-derived DC does not improve the functional quality compared with the standard cytokine cocktail.

[Effect of polypeptide extract from scorpion venom (PESV) on immune escape of Lewis lung carcinomas].

OBJECTIVE: To study the effects of polypeptide extract from scorpion venom (PESV) on immune escape of Lewis lung carcinomas (LLC) and its mechanism. METHOD: Forty C57BL/6J mice were inoculated with LLC cells suspension (1 x 10(7) cells/ mL) in right armpit subcutaneously. The tumor-bearing mice were randomly divided into two groups: the control group and the PESV group. PESV was intragastrically subjected to the mice of the experimental group for 18 days. The tumor volume and tumor inhibitory rate were determined. The expression levels of VEGF,TGF-beta1 and IL-10 in tumor microenvironment were determined by immunohisto-chemistry-staining and ELISA. Surface co-stimulatory molecules CD80 and CD86 of tumor infiltrating dendritic cells (DC) were determined by immunohistochemistry-staining and flow cytometry. RESULT: The growth inhibitory rate of PESV was 56. 60%. The expression levels of VEGF,TGF-beta1 and IL-10 were decreased in tumor and serum, while the expression of co-stimulatory molecules CD80 and CD86 on DC were increased in tumor. Compared with the control group, the differences were all significant (P < 6.05). CONCLUSION: PESV was effective in recovering immuno-surveillance and intervening immune escape of lung cancer through multi-pathway. And its effects might be attained by decreasing the level of VEGF, TGF-beta1 and IL-10 in tumor microenvironment and increasing the expression of co-stimulatory molecules CD80 and CD86 on DC.

B7-H1 expression on non-B and non-T cells promotes distinct effects on T- and B-cell responses in autoimmune arthritis.

The immune system has developed several regulatory mechanisms to maintain homeostasis of adaptive immune responses. T-cell programmed death (PD)-1 recognition of B7-H1 (PD-L1) expressed on APC and non-lymphoid tissue regulates T-cell activation. We show that B7-H1(-/-) mice exhibit exacerbated proteoglycan (PG)-induced arthritis and increased Th-1 CD4(+) T-cell responses. Unexpectedly, the PG-specific antibody response in B7-H1(-/-) mice was diminished. A reduction in the number of peanut agglutinin(+) GC coincided with a decrease in CD19(+) GL-7(+) CD95(+) GC B cells that was a result of increased caspase-induced apoptosis. The percent of CD38(+) CD138(+) emerging plasma cells was decreased. B7-H1(-/-) mice exhibited an increased frequency of CD4(+) PD-1(hi) CXCR5(hi) ICOS(hi) CD62L(lo) T follicular helper cells that displayed a hyperactive phenotype with increased expression of mRNA transcripts for Bcl6, IL-21, and the apoptosis-inducer molecule FasL. In cell transfer of B7-H1(-/-) cells into SCID mice, non-B and non-T cells were sufficient to normalize the antibody response, T-cell hyperactivity, and the development of PG-induced arthritis. These findings indicate that B7-H1 on non-B and non-T cells signals through PD-1 on T effector cells to prevent excessive activation and reduce autoimmune arthritis. Furthermore, these findings demonstrate a novel role for B7-H1 expression in promoting B-cell survival by regulating the activation of T follicular helper cell.

[Effects of phenylethanoid glycosides from seeds of Plantago asiatica on maturation of dendritic cells].

OBJECTIVE: To elucidate the immunomodulatory mechanism of phenylethanoid glycosides from the seeds of Plantago asiatica by testing its effects on the maturing of murine bone marrow derived dendritic cells (DCs). METHOD: Monocytes generated from bone marrow of Balb/cj mouse were cultured for 6 days in complete RPMI 1640 medium containing 10% FBS, rmGM-CSF and rmIL-4.50 mg x L(-1) acteoside or isoacteoside was added to cells on day 6 of culture for 24 h. The surface molecules expression level of DCs and their phagocytose ability were analysis by flow cytometry. RESULT: Both acteoside and isoacteoside could increase the expression of CD11c, CD86, MHC II and CD80 on DCs surface. The ability of unstimulated DCs to uptake FITC-dextran was higher than that of phenylethanoid glycosides or LPS treated DCs. CONCLUSION: Both acteoside and isoacteoside could induce maturation of murine dendritic cells.

Lactoferrin modulation of BCG-infected dendritic cell functions.

Lactoferrin, an 80-kDa iron-binding protein with immune modulating properties, is a unique adjuvant component able to enhance efficacy of the existing Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine to protect against murine model of tuberculosis. Although identified as having effects on macrophage presentation events, lactoferrin's capability to modulate dendritic cells (DCs) function when loaded with BCG antigens has not been previously recognized. In this study, the potential of lactoferrin to modulate surface expression of MHC II, CD80, CD86 and CD40 from bone marrow-derived dendritic cells (BMDCs) was examined. Generally, lactoferrin decreased pro-inflammatory cytokines [tumor necrosis factor (TNF)-alpha, IL-6 and IL-12p40] and chemokines [macrophage inflammatory protein (MIP)-1alpha and MIP-2] and increased regulatory cytokine, transforming growth factor-beta1 and a T-cell chemotatic factor, monocyte chemotactic protein-1, from uninfected or BCG-infected BMDCs. Culturing BCG-infected BMDCs with lactoferrin also enhanced their ability to respond to IFN-gamma activation through up-regulation of maturation markers: MHC I, MHC II and the ratio of CD86:CD80 surface expression. Furthermore, lactoferrin-exposed BCG-infected DCs increased stimulation of BCG-specific CD3(+)CD4(+) splenocytes, as defined by increasing IFN-gamma production. Finally, BCG-/lactoferrin-vaccinated mice possessed an increased pool of BCG antigen-specific IFN-gamma producing CD3(+)CD4(+)CD62L(-) splenocytes. These studies suggest a mechanism in which lactoferrin may exert adjuvant activity by enhancing DC function to promote generation of antigen-specific T cells.

Induction of Treg by monocyte-derived DC modulated by vitamin D3 or dexamethasone: differential role for PD-L1.

Specific therapy with modulated DC may restore immunological tolerance, thereby obviating the need for chronic immunosuppression in transplantation or autoimmunity. In this study we compared the tolerizing capacity of dexamethasone (Dex)- and 1 alpha,25-dihydroxyvitamin D3 (VD3)-modulated DC. Treatment of monocytes with either VD3 or Dex resulted in DC with stable, semi-mature phenotypes compared with standard DC, with intermediate levels of co-stimulatory and MHC class II molecules, which remained unaltered after subsequent pro-inflammatory stimulation. IL-12p70 secretion was lost by VD3- and Dex-DC, whereas IL-10 secretion was unaffected. VD3-DC distinctly produced large amounts of TNF-alpha. Both VD3- and Dex-DC possessed the capacity to convert CD4 T cells into IL-10-secreting Treg potently suppressing the proliferation of responder T cells. However, only Treg induced by VD3-DC exhibited antigen specificity. VD3-, but not Dex-, DC expressed significant high levels of PD-L1 (programmed death-1 ligand), upon activation. Blockade of PD-L1 during priming redirected T cells to produce IFN-gamma instead of IL-10 and abolished acquisition of regulatory capacity. Our findings demonstrate that both VD3- and Dex-DC possess durable but differential tolerogenic features, acting via different mechanisms. Both are potentially useful to specifically down-regulate unwanted immune responses and induce immune tolerance. These modulated DC appear suitable as adjuvant in antigen-specific clinical vaccination intervention strategies.

Molecular insight into the immune up-regulatory properties of the leaf extract of Ashwagandha and identification of Th1 immunostimulatory chemical entity.

Withania somnifera, commonly called Ashwagandha in the Indian traditional system of medicine has been reported for several pharmacological activities. This study demonstrates, for the first time, the potential role of the chemically standardized leaf extract of W. somnifera (WSL) and it's identified component in activating immune system. WSL enhanced Th1 cytokine IFN-gamma expression in Con A primed splenocytes in vitro. When given orally for 2 weeks to BALB/c mice immunized with emulsion of OVA in Freund's adjuvant (OVA-FCA), it caused dose-dependent proliferation of T cells and improved their ability to secrete IL-2 and IFN-gamma, but moderately down-regulated Th2 cytokine IL-4. Flow cytometric analysis of lymphocyte surface markers of T cells CD3(+), CD4(+) and CD8(+), and B cells CD19(+) indicated prominent enhancement in proliferation and differentiation of lymphocytes. Further, the effect of WSL in immunized mice elicited up-regulation of beta-integrins LFA (CD11a) and Mac-1 (CD11b) in splenocytes. Co-stimulatory molecules CD80 and CD86 that are important secondary signals for the activation of immune system elicited remarkable enhanced expression when observed in spleen-derived macrophages isolated from WSL treated mice. Chemical standardization of WSL suggested that the withanolide 2,3 dihydro-3-sulphonile withanone is a major constituent of WSL responsible for skewing to Th1 immune polarization by stimulating the expression of IFN-gamma and B cell switch over to secrete IgG2a while simultaneously enhancing the expression of co-stimulatory molecules and integrins. These studies demonstrate the possible usefulness of WSL and its major constituent WSL-2 as Th1 immune adjuvants for chronic infectious ailments where patients suffer from weakened Th1 immunity.