The Impact of mRNA Technology in Regenerative Therapy: Lessons for Oral Tissue Regeneration.

Oral tissue regeneration following chronic diseases and injuries is limited by the natural endogenous wound-healing process. Current regenerative approaches implement exogenous systems, including stem cells, scaffolds, growth factors, and plasmid DNA/viral vectors, that induce variable clinical outcomes. An innovative approach that is safe, effective, and inexpensive is needed. The lipid nanoparticle-encapsulated nucleoside-modified messenger RNA (mRNA) platform has proven to be a successful vaccine modality against coronavirus disease 2019, demonstrating safety and high efficacy in humans. The same fundamental technology platform could be applied to facilitate the development of mRNA-based regenerative therapy. While the platform has not yet been studied in the field of oral tissue regeneration, mRNA therapeutics encoding growth factors have been evaluated and demonstrated promising findings in various models of soft and hard tissue regeneration such as myocardial infarction, diabetic wound healing, and calvarial and femoral bone defects. Because restoration of both soft and hard tissues is crucial to oral tissue physiology, this new therapeutic modality may help to overcome challenges associated with the reconstruction of the unique and complex architecture of oral tissues. This review discusses mRNA therapeutics with an emphasis on findings and lessons in different regenerative animal models, and it speculates how we can apply mRNA-based platforms for oral tissue regeneration.

Cigarette smoke extract modulates human beta-defensin-2 and interleukin-8 expression in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Human gingival epithelial cells (HGECs) are continually exposed to oral bacteria and to other harmful agents. Their responses to stimuli are critical in maintaining periodontal homeostasis. The aim of this study was to investigate the modulating effect of cigarette smoke extract (CSE) on the innate immune responses of HGECs. MATERIAL AND METHODS: Toll-like receptor (TLR) expression of HGECs was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The effect of CSE or nicotine on the expression of the antimicrobial peptide human beta-defensin-2 (hBD-2) and the pro-inflammatory cytokine interleukin (IL)-8 in stimulated HGEC cultures was evaluated by RT-PCR and enzyme-linked immunosorbent assay. RESULTS: The HGECs expressed mRNA of TLRs 1, 2, 3, 5, 6, 9, 10, and minimally of TLR4, but not of TLRs 7 or 8. Stimulation of HGECs with highly purified TLR2, 3 or 5 ligands led to expression of hBD-2 and of IL-8. Enhancement of hBD-2 and IL-8 was observed in HGECs after combined stimulation with Porphyromonas gingivalis lipopolysaccharide (TLR2 ligand) and tumour necrosis factor-alpha, compared with stimulation using either agent alone. After CSE exposure, hBD-2 expression was markedly reduced in stimulated HGEC cultures, whereas IL-8 expression was markedly increased. These effects were also observed, but were markedly attenuated, upon nicotine treatment. CONCLUSION: Human gingival epithelial cells play a critical role in orchestrating the innate immune responses of periodontal tissue via TLR signalling. Our results represent the first demonstration that CSE can modulate HGEC function by suppressing hBD-2 and enhancing IL-8 production, and this may be, in part, a possible mechanism which promotes periodontal disease.

Preclinical evaluation of the safety and immunogenicity of a vaccine consisting of Plasmodium falciparum liver-stage antigen 1 with adjuvant AS01B administered alone or concurrently with the RTS,S/AS01B vaccine in rhesus primates.

Several lines of evidence suggest that targeting pre-erythrocytic-stage parasites for malaria vaccine development can provide sterile immunity. The objectives of this study were (i) to evaluate preclinically the safety and immunogenicity of a new recombinant pre-erythrocytic-stage antigen, liver-stage antigen 1 (LSA1), in nonhuman primates; and (ii) to investigate the potential for immune interference between LSA1 and the leading malaria vaccine candidate, RTS,S, by comparing the immune responses after single-antigen vaccination to responses after simultaneous administration of both antigens at separate sites. Using a rhesus monkey model, we found that LSA1 formulated with the GlaxoSmithKline proprietary adjuvant system AS01B (LSA1/AS01B) was safe and immunogenic, inducing high titers of antigen-specific antibody and CD4+ T-cell responses, as monitored by the production of interleukin-2 and gamma interferon, using intracellular cytokine staining. RTS,S/AS01B vaccination was well tolerated and demonstrated robust antibody and moderate CD4+ T-cell responses to circumsporozoite protein (CSP) and HBsAg. Positive CD8+ T-cell responses to HBsAg were detected, whereas the responses to CSP and LSA1 were negligible. For both LSA1/AS01B and RTS,S/AS01B, no statistically significant differences were observed between individual and concurrent administration in the magnitude or duration of antibody and T-cell responses. Our results revealed that both pre-erythrocytic-stage antigens were safe and immunogenic, administered either separately or simultaneously to rhesus monkeys, and that no significant immune cross interference occurred with concurrent separate-site administration. The comparison of the profiles of immune responses induced by separate-site and single-site vaccinations with LSA1 and RTS,S warrants further investigation.

Effects of IL-17 on human gingival fibroblasts.

Interleukin (IL)-17 is present in inflammatory periodontal lesions, thus suggesting a role in mediating inflammation. We tested the hypothesis that IL-17, especially when combined with interferon (IFN)-gamma, may modulate the responses of human gingival fibroblasts (HGFs). IL-17 induced IL-8 and minimal intercellular adhesion molecule (ICAM)-1 expression. It had no effect on expression of HLA-DR, CD40, or the immune-suppressive enzyme indoleamine 2,3-dioxygenase (IDO). The effects of IL-17 on HGFs were compared with those of IFN-gamma. Unlike IL-17, IFN-gamma augmented the expression of HLA-DR, ICAM-1, and IDO, but not IL-8. Thus, IL-17 and IFN-gamma induce different HGF responses when administered separately. Interestingly, when IL-17 and IFN-gamma were combined, marked enhancement of ICAM-1, IL-8, and IDO expression by HGFs was observed. These findings suggest that IL-17, especially when combined with IFN-gamma, could play an important role in immune modulation through stimulation of HGFs in periodontal disease.

Increased expression of adhesive proteins on leukocytes by TNF alpha.

To further elucidate the role of tumor necrosis factor alpha (TNF alpha) in cell adhesion, we investigated the effect of TNF alpha on the expression of surface adhesive protein. After treatment with TNF alpha for 1 h, the increased expression of surface adhesive proteins (beta subunit) was observed on granulocytes but not on monocytes or lymphocytes. The expression of Mac-1 (3,4-fold increase) was consistently enhanced more than p 150,95 (1.4-fold increase) and LFA-1 expression was unchanged. Dose-response and time course studies indicated a parallel relationship between TNF alpha-increased expression of surface adhesive proteins and TNF alpha-induced granulocyte adhesion. The anti-inflammatory drug Dexamethasone suppressed both TNF alpha-induced granulocyte adhesion and TNF alpha-induced expression of surface adhesive proteins. The inhibition of granulocyte adhesion correlated with the reduction of surface adhesive protein expression. The data suggest that one contributing factor in the mechanism by which Dexamethasone inhibited TNF alpha-induced granulocyte adhesion may be diminished expression of surface adhesive proteins.

Binding of tumor necrosis factor alpha (TNF-alpha) to high-affinity receptors on polymorphonuclear cells.

The effects of tumor necrosis factor alpha (TNF-alpha) on human polymorphonuclear (PMN) cells were investigated. We found that 125I-TNF-alpha bound specifically to high-affinity receptors on PMN cells. At 4 degrees C, the binding occurred rapidly and reached steady state after 20 min. The Scatchard plot showed a single class of high-affinity receptors with approximately 2200 receptors/cell and a dissociation constant of 2 x 10(-10) M. There was a linear relationship between TNF-alpha binding and TNF-alpha-induced PMN cell adherence. The concentration of TNF-alpha required to achieve approximately 50% of maximum binding was also approximately the concentration required to reach 50% cell adherence. Auranofin was shown to inhibit TNF-alpha-induced PMN cell adherence at a dose of 5-10 micrograms/ml. This inhibitory effect was not due to the inhibition of TNF-alpha binding to PMN cells by the drug. These observations may have clinical implications.

Whole blood cultures to assess the immunostimulatory activities of CpG oligodeoxynucleotides.

Specially designed oligodeoxynucleotide (ODN) sequences known as 'CpG' ODNs elicit innate and acquired immune responses. In general, screening of new CpG ODNs has been conducted by conventional lymphoproliferative assays or expression of activation markers in peripheral blood mononuclear cell (PBMC) cultures. Here, we compared conventional in vitro human PBMC assays with whole blood assays for screening the immunostimulatory properties of CpG ODNs. Commercially available DNA preparations and mycobacterial-based adjuvants were used as comparators. Activation was assessed by flow cytometry and cytokine production. CpG ODNs, identified by four-letter codes, consisted of 2006 (strong human cell stimulant), 1826 (strong murine cell stimulant), 1840 (weak immunostimulant), and 2041, a non-CpG ODN. In both test systems, and in accordance with previous reports, 2006 was an effective up-regulator of CD40 on human dendritic cells (DC1, DC2), monocytes, and B cells, and of CD69 on NK cells. In contrast to murine cells exposed to CpG ODNs, IL-12 (p40) and IFN-gamma production in human immune cells was negligible, but greatly enhanced by adding GM-CSF. Like 2006, two comparator mycobacterial adjuvant formulations activated DC1, DC2, monocytes and natural killer (NK) cells, but only 2006 had a strong effect on B cells. The usefulness of the whole blood assay was further demonstrated by studies in small volumes of umbilical cord mononuclear cells, that like adult blood cells, showed up-regulation of CD40 expression on B cells, DC, and monocytes, and CD69 on NK cells. The whole blood assay, in conjunction with flow cytometry, is useful for assessing the immunological properties of CpG ODN sequences.

Isolation and characterization of rhesus blood dendritic cells using flow cytometry.

Recognition of dendritic cells (DCs) as initiators and modulators of immune responses and growing use of rhesus monkeys for the preclinical optimization of vaccine formulations prompted characterization of the phenotype and function of isolated rhesus peripheral blood DCs. We developed a flow cytometric method to directly identify and isolate DCs from rhesus peripheral blood whereby a T cell depleted population negative for CD3, CD14, CD16 and CD20 but positive for CD83 yielded a cell population with surface markers, morphology, and a cytokine profile similar to human myeloid DCs. Rhesus blood DCs were more effective than monocytes and B cells in mixed lymphocyte reactions and in the presentation of recombinant malaria blood stage antigen MSP-1((42)) to autologous T cells. The ability to isolate rhesus blood DC from peripheral blood should be a useful tool for immunological investigations.

Plasmodium falciparum pigment induces monocytes to release high levels of tumor necrosis factor-alpha and interleukin-1 beta.

We show that high levels of tumor necrosis factor-alpha (TNF-alpha) activity were consistently detected when monocytes were cocultured with Plasmodium falciparum schizont stage-parasitized erythrocytes that subsequently ruptured. Isolated pigment recovered from ruptured schizonts was found to specifically induce monocyte release of high levels of TNF-alpha and interleukin-1 beta (IL-1 beta). Particulate free-culture supernatant that contained various soluble parasite macromolecules induced relatively low levels of TNF-alpha and IL-1 beta. When isolated pigment was treated with protease, the monokine inducing-activity was abolished. Isolated pigment prepared from different natural isolates of P. falciparum stimulated variable levels of monokine production. We propose that in vivo, malaria pigment from parasites sequestered in the host microvasculature is a physiologically relevant moiety that interacts with monocytes and stimulates the release of TNF-alpha and IL-1 beta. These observations suggest that malaria pigment may be a virulence factor in the monokine-mediated induction of organ-specific and systemic pathophysiology in falciparum malaria.

Regulation of leukocyte adhesion molecules CD11b/CD18 and leukocyte adhesion molecule-1 on phagocytic cells activated by malaria pigment.

There is increasing evidence that inappropriate immune activation induced by parasite products occurs in malaria disease. To further elucidate the role of Plasmodium falciparum-derived products on host immune activation, we studied the expression of leukocyte adhesion molecules (CD11b/CD18 and LAM-1) on neutrophils and monocytes in response to malaria pigment using flow cytometry. Exposure of leukocytes to isolated malaria pigment derived from ruptured schizonts resulted in significant up-regulation of CD11b/CD18 expression and down-regulation of LAM-1 on both neutrophils and monocytes. In contrast, culture supernatants (pigment free) from ruptured schizonts did not alter the expression of CD11b/CD18 and LAM-1. The increase of CD11b/CD18 and the loss of LAM-1 expression occurred simultaneously with the earliest response detected at 10 min and a plateau reached by 60 min. The effect of malaria pigment on leukocyte adhesion molecules was inhibited by EDTA in a dose-dependent manner. Phagocytosis of malaria pigment was also suppressed by EDTA. This observation suggests that phagocytosis of malaria pigment may be a prerequisite for the effect of malaria pigment on the regulation of CD11b/CD18 and LAM-1 expression. Regulation of leukocyte adhesion molecules through up-regulation of CD11b/CD18 and down-regulation of LAM-1 by malaria pigment could promote leukocyte adherence to endothelium in vivo. This increased adherence of malaria pigment-activated leukocytes might induce cytokine (tumor necrosis factor alpha and interleukin-1beta)-mediated increases in capillary permeability resulting in local tissue edema, and a cytokine-mediated increase in adhesion molecule expression causing vascular clogging by adherent red blood cells, and in severe disease by adherent leukocytes.

Monocyte activation by Porphyromonas gingivalis LPS in aggressive periodontitis with the use of whole-blood cultures.

In this study, we re-visited the issue of hyper-responsiveness of monocytes to bacterial lipopolysaccharide (LPS) in aggressive periodontitis patients. We used whole-blood cultures to compare monocyte activation by Porphyromonas gingivalis LPS between Thai subjects with generalized aggressive periodontitis and those without periodontitis. Upon stimulation with P. gingivalis LPS, expression of co-stimulatory molecules on monocytes and expression of CD69 on NK and gamma delta T-cells were analyzed by flow cytometry, and the production of interleukin-1 beta and prostaglandin E(2) was monitored by ELISA. LPS stimulation resulted in a dose-dependent up-regulation of CD40, CD80, and CD86 on monocytes, and up-regulation of CD69 on NK cells and gamma delta T-cells in both the periodontitis and non-periodontitis groups. The levels of activation markers and the mediator production after LPS stimulation were quite similar for both groups. In conclusion, we did not observe hyper-responsiveness of monocytes to P. gingivalis LPS challenge in Thai patients with aggressive periodontitis.

The immune modulation of B-cell responses by Porphyromonas ginginvalis and interleukin-10.

BACKGROUND: Polyclonal B-cell activation induced by periodontopathic bacteria has been cited as being important for elevated numbers of B cells, but the role of bacteria in the pathogenesis of periodontal disease remains unknown. In this study, we used an in vitro model to investigate the activation of immune cells by the periodontopathic bacterium Porphyromonas gingivalis in healthy subjects. METHODS: Peripheral blood mononuclear cells (PBMC) or purified subsets of lymphocytes were stimulated with sonicated extracts of P. gingivalis for 24 hours. Cells were harvested and monitored for expression of CD69 by flow cytometry. Cytokine production (IL-10, IL-12, and IL-15) in P. gingivalis-stimulated PBMC cultures was measured by ELISA. To identify IL-10 producer cells, a cell depletion experiment was used and confirmed by the ability of the purified cell population to produce IL-10. To evaluate the effect of P. gingivalis and IL-10, the proliferative response of purified B cells was assessed by [3H] thymidine uptake. RESULTS: PBMC cultured with P. gingivalis led to a large number of activated B and natural killer (NK) cells as monitored by CD69 expression. When positively sorted cells were used, the bacterium itself could directly activate only B cells but not NK cells, alphabeta, and gammadelta T cells. Measurement of B-cell regulatory cytokine production in P. gingivalis-stimulated PBMC cultures revealed a large amount of IL-10 but no detectable IL-12 or IL-15; the major producing cells were monocytes, not B cells or alphabeta T cells. When IL-10 was added to B cells in the presence of bacteria, significantly increased B-cell proliferative responses were observed. CONCLUSIONS: These results suggest that P. gingivalis, both directly and indirectly via macrophage IL-10, may play an important role in polyclonal B-cell activation associated with periodontal disease.

Evaluation of the safety and immunogenicity of Plasmodium falciparum apical membrane antigen 1, merozoite surface protein 1 or RTS,S vaccines with adjuvant system AS02A administered alone or concurrently in rhesus monkeys.

In an effort to broaden the immune response induced by the RTS,S/AS02(A),vaccine, we have evaluated the immunogenicity of the RTS,S antigen when combined with MSP1(42) and with AMA1, antigens derived from the asexual blood stage. The objectives of this study were (i) to determine whether MSP1(42) and AMA1 vaccines formulated with the AS02(A) Adjuvant System were safe and immunogenic in the rhesus monkey model; (ii) to investigate whether MSP1(42) or AMA1 induced immune interference to each other, or to RTS,S, when added singly or in combinations at a single injection site; (iii) in the event of immune interference, to determine if this could be reduced when antigens were administered at separate sites. We found that MSP1(42) and AMA1 were safe and immunogenic, eliciting antibodies, and Th1 and Th2 responses using IFN-gamma and IL-5 as markers. When malaria antigens were delivered together in one formulation, MSP1(42) and RTS,S reduced AMA1-specific antibody responses as measured by ELISA however, only MSP1(42) lowered parasite growth inhibitory activity of anti-AMA1 antibodies as measured by in vitro growth inhibition assay. Unlike RTS,S, MSP1(42) significantly reduced AMA1 IFN-gamma and IL-5 responses. MSP1(42) suppression of AMA1 IFN-gamma responses was not seen in animals receiving RTS,S+AMA1+MSP1(42) suggesting that RTS,S restored IFN-gamma responses. Conversely, AMA1 had no effect on MSP1(42) antibody and IFN-gamma and IL-5 responses. Neither AMA1 alone or combined with MSP1(42) affected RTS,S antibody or IFN-gamma and IL-5 responses. Immune interference by MSP1(42) on AMA1 antibody responses was also evident when AMA1, MSP1(42) and RTS,S were administered concurrently at separate sites. These results suggest that immune interference may be complex and should be considered for the design of multi-antigen, multi-stage vaccines against malaria.

Potentiation of cytotoxic effect of lymphotoxin by anti-cancer drugs and elevated temperatures.

The cytotoxic lymphokine, lymphotoxin (LT), has been shown to possess antitumor effect in vitro and in vivo. We examined the effect of the combination of partially purified LT with anti-cancer drugs and elevated temperatures on mouse transformed fibroblast cell line, L-929, and two human carcinoma of the cervix cell lines, HeLa and ME180. The cells were treated for 7 hr with Adriamycin, cisplatin, or bleomycin. These cells were then incubated for 24 hr in the presence of LT. At the end of the incubation period, cytotoxicity was measured by the neutral red dye uptake assay. There was 10- to 47-fold potentiation of cytotoxicity of LT on L-929 cells. The potentiation of cytotoxicity on human carcinoma of cervix cell lines ranged from 3- to 23-fold. L-929 cells and ME180 cells were incubated for 7 hr at 40 or 42 degrees C followed by 24 hr of incubation in the presence of LT. The elevated temperature treatment also enhanced (5- to 9-fold) the cytotoxic effect of LT. DNA, RNA, and protein syntheses of the ME180 cells was measured following incubation at 42 degrees C. It was observed that all three parameters were suppressed by incubation at this temperature. It was, therefore, possible that the repair of LT damaged cells was hampered by the elevated temperature treatment. It is suggested that LT may have a potential as an anti-tumor agent in combination with selected therapeutic drugs and hyperthermia.

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha).

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.

Cellular origin of human lymphotoxin and its purification.

The ability of various subsets of human mononuclear cells to produce human lymphotoxin (LT) was examined. Peripheral blood mononuclear cells were separated into OKT4+, OKT8+, and Leu-11a+ subpopulations by flow cytometry. Both OKT4+ and OKT8+ cells produced LT upon phytohemagglutinin (PHA) stimulation, but the OKT4+ T cells were the major source of LT. In contrast, Leu-11a+ cells failed to produce LT. The LT production and the proliferative response to PHA, of OKT4+ and OKT8+ cells, were inhibited by prostaglandin E2 and histamine. The LT, derived from PHA-stimulated peripheral blood lymphocytes, was purified by a procedure involving Blue-agarose, Con A-Sepharose chromatography, preparative gradient polyacrylamide gel electrophoresis and preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The LT activity was recovered from SDS gel with major activity peak in the Mr 76,000 region and the minor activity peak in the Mr 24,000 region. The LT derived from 1788 lymphoblastoid cell line also showed heterogeneity on SDS gel. The activity was recovered from two peaks in the region of Mr 70,000 and 20,000.

Activation of gammadelta T cells in malaria: interaction of cytokines and a schizont-associated Plasmodium falciparum antigen.

A soluble Plasmodium falciparum antigen that specifically stimulates gammadelta T cells has been found associated predominantly with schizonts rather than ring forms, trophozoites, or gametocytes. This schizont-associated antigen (SAA) is resistant to protease digestion, is anionic at pH 8.5, is heat- and pH-resistant, and contains a phosphate group(s) that is crucial for biologic activity. Partially purified SAA induced proliferative responses and interferon-gamma production by gammadelta T cells. These stimulatory effects were greatly enhanced by monocyte-derived cytokines, interleukin (IL)-10, IL-12, and IL-1beta, but not by tumor necrosis factor-alpha. Taken together, these results suggest that concurrent stimulation of gammadelta T cells by SAA and by cytokines released from activated monocytes (IL-10, IL-12, IL-1beta) may represent the major mechanism underlying the selective activation of gammadelta T cells that is consistently observed in clinical cases of P. falciparum infection.

Human dendritic cells are activated by dengue virus infection: enhancement by gamma interferon and implications for disease pathogenesis.

The ability of dendritic cells (DCs) to shape the adaptive immune response to viral infection is mediated largely by their maturation and activation state as determined by the surface expression of HLA molecules, costimulatory molecules, and cytokine production. Dengue is an emerging arboviral disease where the severity of illness is influenced by the adaptive immune response to the virus. In this report, we have demonstrated that dengue virus infects and replicates in immature human myeloid DCs. Exposure to live dengue virus led to maturation and activation of both the infected and surrounding, uninfected DCs and stimulated production of tumor necrosis factor alpha (TNF-alpha) and alpha interferon (IFN-alpha). Activation of the dengue virus-infected DCs was blunted compared to the surrounding, uninfected DCs, and dengue virus infection induced low-level release of interleukin-12 p70 (IL-12 p70), a key cytokine in the development of cell-mediated immunity (CMI). Upon the addition of IFN-gamma, there was enhanced activation of dengue virus-infected DCs and enhanced dengue virus-induced IL-12 p70 release. The data suggest a model whereby DCs are the early, primary target of dengue virus in natural infection and the vigor of CMI is modulated by the relative presence or absence of IFN-gamma in the microenvironment surrounding the virus-infected DCs. These findings are relevant to understanding the pathogenesis of dengue hemorrhagic fever and the design of new vaccination and therapeutic strategies.