Biotin Functionalized Self-Assembled Peptide Nanofiber as an Adjuvant for Immunomodulatory Response.

Biotinylated peptide amphiphile (Biotin-PA) nanofibers, are designed as a noncovalent binding location for antigens, which are adjuvants to enhance, accelerate, and prolong the immune response triggered by antigens. Presenting antigens on synthetic Biotin-PA nanofibers generated a higher immune response than the free antigens delivered with a cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN) (TLR9 agonist) adjuvant. Antigen attached Biotin-PA nanofibers trigger splenocytes to produce high levels of cytokines (IFN-γ, IL-12, TNF-α, and IL-6) and to exhibit a superior cross-presentation of the antigen. Both Biotin-PA nanofibers and CpG ODN induce a Th-1-biased IgG subclass response; however, delivering the antigen with Biotin-PA nanofibers induce significantly greater production of total IgG and subclasses of IgG compared to delivering the antigen with CpG ODN. Contrary to CpG ODN, Biotin-PA nanofibers also enhance antigen-specific splenocyte proliferation and increase the proportion of the antigen-specific CD8(+) T cells. Given their biodegradability and biocompatibility, Biotin-PA nanofibers have a significant potential in immunoengineering applications as a biomaterial for the delivery of a diverse set of antigens derived from intracellular pathogens, emerging viral diseases such as COVID-19, or cancer cells to induce humoral and cellular immune responses against the antigens.

Macrophages as delivery vehicles for anticancer agents.

The delivery of anticancer agents via passive approaches such as the enhanced permeability and retention effect is unlikely to achieve sufficient concentrations throughout the tumor volume for effective treatment. Cell-based delivery approaches using tumor tropic cells have the potential to overcome the limitations of passive approaches. Specifically, this review focuses on the use of monocytes/macrophages for the delivery of a variety of anticancer agents, including nanoparticles, chemotherapeutics and gene constructs. The efficacy of this delivery approach, both as monotherapy and in combination with light-based phototherapy modalities, has been demonstrated in numerous in vitro and animal studies, however, its clinical potential remains to be determined.

Understanding the Effect of Surface Chemistry of Mesoporous Silica Nanorods on Their Vaccine Adjuvant Potency.

Mesoporous silica nanoparticles are reported as adjuvants in nanovaccines in generating robust antigen-specific immunity. However, the effect of surface chemistry in initiating and modulating the immune response remains largely unexplored. In this study, mesoporous silica nanorods (MSNRs) are modified with NH2 and C18 groups to investigate the influence of surface functional groups (OH, NH2 , and C18 ) on their adjuvant efficacy. It is found that compared to OH and NH2 groups, the hydrophobic C18 modification significantly enhances antigen uptake by antigen presenting cells and endosomal-lysosomal escape in vitro, dendritic cells, and macrophages maturation ex vivo, and elicits secretion of interferon-γ level and antibody response in immunized mice. Moreover, bare MSNR and MSNRNH2 exhibit T-helper 2 biased immune response, while MSNRC18 shows a T-helper 1 biased immune response. These findings suggest that the surface chemistry of nanostructured adjuvants has profound impact on the immune response, which provides useful guidance for the design of effective nanomaterial based vaccines.

[In vitro anti-tumor effect of human dendritic cells vaccine induced by astragalus polysacharin: an experimental study].

OBJECTIVE: To explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS). METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay. RESULTS: The morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio. CONCLUSION: APS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

New conjugates of tuftsin and muramyl dipeptide as stimulators of human monocyte-derived dendritic cells.

Muramyl dipeptide (MDP) and tuftsin are known biologically active compound displaying a significant influence on various cell populations of innate immune response. MDP, as a fragment of bacterial cell wall, stimulates not only macrophages and monocytes, but also dendritic cells. In contrast, little is known about tuftsin influence on these cells. Therefore it seemed vital to access whether tuftsin or its derivatives conjugated with MDP could influence the activity of this subpopulation of antigen presenting cells (APC). Immature dendritic cells (iDCs) were derived from human monocytes through eight-day tissue culture supplemented with hrIL-4 and hrGM-CSF. On the day 9 DCs were stimulated with newly synthesized conjugates of tuftsin and muramyl dipeptide. The influence of the examined compounds on the activity and maturity of monocyte-derived DCs was estimated by flow cytometry analysis. The flow cytometry analysis revealed that tuftsin and some of its analogues do stimulate maturation and activity of DCs but to a lesser extend in comparison to MDP. The obtained results suggest further development of the experiments concerning the influence of MDP and tuftsin analogues on the activity of dendritic cells.

To forge a solid immune recognition.

Phagocytosis and innate immune responses to solid structures are topics of interest and debate. Alum, monosodium urate, calcium pyrophosphate dehydrate, silica and by extension all solid entities draw varying degrees of attention from phagocytes, such as antigen presenting cells. For some, innocuous soluble metabolites turn into fierce irritants upon crystallization, pointing to divergent signaling mechanisms of a given substance in its soluble and solid states. Over the years, many mechanisms have been proposed, including phagocytic receptors, toll like receptors, and NACHT-LRRs (NLRs), as well as several other protein structure mediated recognition of the solids. Is there a more general mechanism for sensing solids? In this perspective, I present an alternative view on the topic that membrane lipids can engage solid surfaces, and the binding intensity leads to cellular activation. I argue from the stands of evolution and biological necessity, as well as the progression of our understanding of cellular membranes and phagocytosis. The effort is to invite debate of the topic from a less familiar yet equally thrilling viewing angle.

Cinnamon extract suppresses experimental colitis through modulation of antigen-presenting cells.

AIM: To investigate the anti-inflammatory effects of cinnamon extract and elucidate its mechanisms for targeting the function of antigen presenting cells. METHODS: Cinnamon extract was used to treat murine macrophage cell line (Raw 264.7), mouse primary antigen-presenting cells (APCs, MHCII(+)) and CD11c(+) dendritic cells to analyze the effects of cinnamon extract on APC function. The mechanisms of action of cinnamon extract on APCs were investigated by analyzing cytokine production, and expression of MHC antigens and co-stimulatory molecules by quantitative real-time PCR and flow cytometry. In addition, the effect of cinnamon extract on antigen presentation capacity and APC-dependent T-cell differentiation were analyzed by [H(3)]-thymidine incorporation and cytokine analysis, respectively. To confirm the anti-inflammatory effects of cinnamon extract in vivo, cinnamon or PBS was orally administered to mice for 20 d followed by induction of experimental colitis with 2,4,6 trinitrobenzenesulfonic acid. The protective effects of cinnamon extract against experimental colitis were measured by checking clinical symptoms, histological analysis and cytokine expression profiles in inflamed tissue. RESULTS: Treatment with cinnamon extract inhibited maturation of MHCII(+) APCs or CD11c(+) dendritic cells (DCs) by suppressing expression of co-stimulatory molecules (B7.1, B7.2, ICOS-L), MHCII and cyclooxygenase (COX)-2. Cinnamon extract induced regulatory DCs (rDCs) that produce low levels of pro-inflammatory cytokines [interleukin (IL)-1ß, IL-6, IL-12, interferon (IFN)-γ and tumor necrosis factor (TNF)-α] while expressing high levels of immunoregulatory cytokines (IL-10 and transforming growth factor-ß). In addition, rDCs generated by cinnamon extract inhibited APC-dependent T-cell proliferation, and converted CD4(+) T cells into IL-10(high) CD4(+) T cells. Furthermore, oral administration of cinnamon extract inhibited development and progression of intestinal colitis by inhibiting expression of COX-2 and pro-inflammatory cytokines (IL-1ß, IFN-γ and TNF-α), while enhancing IL-10 levels. CONCLUSION: Our study suggests the potential of cinnamon extract as an anti-inflammatory agent by targeting the generation of regulatory APCs and IL-10(+) regulatory T cells.

Artificial dental pulp exposure injury up-regulates antigen-presenting cell-related molecules in rat central nervous system.

INTRODUCTION: Bacterial infection and resulting inflammation of the dental pulp might not only trigger neuroimmune interactions in this tissue but also sensitize the central nervous system (CNS) such as the thalamus via nociceptive neurons. Thus, immunopathologic changes in the rat thalamus that take place after pulp inflammation were investigated. METHODS: Pulp exposure was made in mandibular right first molars of 5-week-old Wistar rats. After 24 hours, the thalamus was retrieved and subjected to either immunohistochemistry for class II major histocompatibility complex (MHC) molecules and glial fibrillary acidic protein (GFAP) or mRNA expression analysis of antigen-presenting cell-related molecules and N-methyl-D-aspartate receptor 2D subunit (NR2D) by means of reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. RESULTS: At 24 hours after pulp exposure, the density of class II MHC molecule-expressing and GFAP-expressing cells was increased in the contralateral thalamus. Gene expression analysis revealed the up-regulation of class II MHC molecules, CD80, CD83, CD86, and NR2D in the contralateral thalamus, as compared with the ipsilateral thalamus. CONCLUSIONS: These results suggest the signal of pulp inflammation induces neuronal activation in the CNS.

A Toll-like receptor 2/6 agonist reduces allergic airway inflammation in chronic respiratory sensitisation to Timothy grass pollen antigens.

BACKGROUND: The hygiene hypothesis negatively correlates the microbial burden of the environment with the prevalence of T helper type 2 (Th2)-related disorders, e.g. allergy and asthma. This is explained by Th1 triggering through pathogen-associated molecular patterns via Toll-like receptors (TLRs). In this study, the biological effects of a TLR2/6 agonist as a potential treatment of allergic inflammation are explored. METHODS: In a model of chronic allergic airway inflammation induced by intranasal administration of Timothy grass pollen allergen extract, early TLR agonism and/or interferon (IFN)-gamma administration was compared to the therapeutic and immune-modulating effects of dexamethasone with regard to the cellular inflammation and cytokine profiles. RESULTS: Eosinophilic inflammation was clearly reduced by TLR2/6 agonism. This effect was also seen without simultaneous administration of IFN-gamma. However, lymphocyte counts were not affected among the different treatment groups. More precise determination of the lymphocyte-mediated immune reaction showed that TLR2/6 agonism induced neither CD4+foxp3+ regulatory T cells in draining lymph nodes nor a pronounced Th1 immune response. In contrast, dexamethasone reduced both sensitisation as well as allergic inflammation and, in addition, CD11c+ antigen-presenting cells in lymph nodes. Our data clearly point to the potential to rebalance Th2-skewed allergic immune responses by therapeutic TLR2/6 agonist administration. CONCLUSION: The use of the TLR2/6 agonist is a promising therapeutic approach in diseases with an imbalance in T cell responses, such as allergy and asthma.

LFA-1-mediated T cell costimulation through increased localization of TCR/class II complexes to the central supramolecular activation cluster and exclusion of CD45 from the immunological synapse.

T cell activation is associated with a dramatic reorganization of cell surface proteins and associated signaling components into discrete subdomains within the immunological synapse in T cell:APC conjugates. However, the signals that direct the localization of these proteins and the functional significance of this organization have not been established. In this study, we have used wild-type and LFA-1-deficient, DO11.10 TCR transgenic T cells to examine the role of LFA-1 in the formation of the immunological synapse. We found that coengagement of LFA-1 is not required for the formation of the central supramolecular activation cluster (cSMAC) region, but does increase the accumulation of TCR/class II complexes within the cSMAC. In addition, LFA-1 is required for the recruitment and localization of talin into the peripheral supramolecular activation cluster region and exclusion of CD45 from the synapse. The ability of LFA-1 to increase the amount of TCR engaged during synapse formation and segregate the phosphatase, CD45, from the synapse suggests that LFA-1 might enhance proximal TCR signaling. To test this, we combined flow cytometry-based cell adhesion and calcium-signaling assays and found that coengagement of LFA-1 significantly increased the magnitude of the intracellular calcium response following Ag presentation. These data support the idea that in addition to its important role on regulating T cell:APC adhesion, coengagement of LFA-1 can enhance T cell signaling, and suggest that this may be accomplished in part through the organization of proteins within the immunological synapse.

T cells stimulated by CD40L positive leukemic blasts-pulsed dendritic cells meet optimal functional requirements for adoptive T-cell therapy.

Adoptive T-cell immunotherapy may provide complementary therapy for childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In this study, we have analyzed the functional characteristics of anti-BCP-ALL effector T cells generated by co-culturing T lymphocytes and dendritic cells (DC) from allogeneic human stem cell transplantation (HSCT) donors. After 21-day co-culture with DC pulsed with CD40L+ apoptotic BCP-ALL blasts, T cells presented with both effector and central memory phenotype, and showed high and specific cytotoxic activity against leukemic cells (average lysis = 77%), mostly mediated by CD8+ T cells. Noticeably, growth of CD4 T cells was maintained (45% of total cells), which actively produced Th1 cytokines (IFN-gamma, TNF-alpha, IL-2), but not IL-4, IL-5 and IL-10. Anti-BCP-ALL T cells expressed CD49d and CXCR4 (implicated in the recruitment to bone marrow), and CD62L and CCR7 (involved in the migration to lymphoid organs). In accordance with this profile, T cells significantly migrated in response to the chemokines CXCL12 and CCL19. In conclusion, stimulation of T cells with CD40L+BCP-ALL cells-loaded DC not only elicited the generation of potent and specific anti-leukemic cytotoxic effectors, but also the differentiation of specific and functional Th-1 CD4 lymphocytes. These effectors are fully equipped to reach leukemia-infiltrated tissues and have characteristics to support and orchestrate the anti-tumor immune-response.

[Effects of large dose of Astragalus membranaceus on the dendritic cell induction of peripheral mononuclear cell and antigen presenting ability of dendritic cells in children with acute leukemia].

OBJECTIVE: To explore the effects of large dose of Astragalus membranaceus (Astragalus) on the dentritic cell (DC) induction in vitro and augumentation by peripheral mononuclear cell (MNC) and on antigen presenting ability of DC in children with acute leukemia. METHODS: Forty-four children with acute leukemia in complete remission stage were divided into two groups. Twenty patients in the Astragalus (90 g daily) group were treated with large dose of Astragalus (90 g daily) based on conventional chemotherapy for one month, while 24 patients in the control group received chemotherapy alone. MNC were extracted from peripheral blood by wall-sticking method and cultured with such cell factors as interleukin-4, gramulocyte macrophage colony stimulating factor, tumor necrosis factor-alpha for 7-8 days. Phenotype of DC was assayed by flow cytometry and antigen presenting ability of them was assayed by mixed lymphocyte reaction. RESULTS: There was no morphological difference in MNC induced DC between the two groups. The average number of DC in Astragalus group and control group was 4.4 x 10(6) / 2.5 x 10(6) MNC and 2.6 x 10(6) / 2.5 x 10(6) MNC, respectively, showing significant difference (P < 0.001). DC in Astragalus group could stimulate the proliferation of allogeneic lymphocytes strongly, showing significant difference when compared with that in the control group (P < 0.001). Conclusion Large dose of Astragalus could increase the DC induction of MNC and enhance the antigen presenting ability of DC in acute leukemia patients.

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen.

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.

T cell reactivity with allergoids: influence of the type of APC.

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.

Effects of capsaicin treatment on immunoglobulin secretion in the rat: further evidence for involvement of tachykinin-containing afferent nerves.

Neonatal capsaicin treatment has previously been shown to diminish the primary antibody response of adult rats to the subcutaneously administered T-dependent antigen, sheep red blood cells, as measured using a modification of the Cunningham plaque-forming cell assay technique. We have now studied the kinetics of this response in adult normal, neonatally capsaicin-pretreated and neonatally capsaicin-pretreated substance P-infused rats, and examined the effects of the tachykinin antagonist Spantide, on the plaque-forming cell response. Capsaicin pretreatment did not affect the antigen-specific plaque-forming cell response over the first 4 days following antigen injection. At days 5, 6 and 7 of the response, there was a statistically significant decrease in the number of plaque-forming cells secreting antigen-specific IgM, an effect not observed in capsaicin-pretreated rats which were given a subcutaneous infusion of substance P at the time of antigen injection. The tachykinin antagonist Spantide inhibited the plaque-forming cell response in normal rats after in vivo infusion at the time of antigen injection by more than 70%. This effect of Spantide was dose dependent, occurred with maximal effect at 10 microM, and appeared to be independent of any histamine-mediated action. The results of this study provide further evidence for a receptor-mediated immunomodulatory role of tachykinin-containing primary afferent nerves.