Montree Jaturanpinyo

Antigen-Specific Suppression of Inflammatory Arthritis Using Liposomes

Existing therapies for rheumatoid arthritis and other autoimmune diseases are not Ag specific, which increases the likelihood of systemic toxicity. We show that egg phosphatidylcholine liposomes loaded with Ag (OVA or methylated BSA) and a lipophilic NF-κB inhibitor (curcumin, quercetin, or Bay11-7082) suppress preexisting immune responses in an Ag-specific manner. We injected loaded liposomes into mice primed with Ag or into mice suffering from Ag-induced inflammatory arthritis. The liposomes targeted APCs in situ, suppressing the cells’ responsiveness to NF-κB and inducing Ag-specific FoxP3+ regulatory T cells. This regulatory mechanism suppressed effector T cell responses and the clinical signs of full-blown Ag-induced arthritis. Thus, liposomes encapsulate Ags and NF-κB inhibitors stably and efficiently and could be readily adapted to deliver Ags and inhibitors for Ag-specific suppression of other autoimmune and allergic diseases.

Pectin nanoparticle enhances cytotoxicity of methotrexate against hepG2 cells

Objective: This work has aimed to develop methotrexate-conjugated pectin nanoparticle for delivering a cytotoxic drug to hepatic cancer cell. Methods: Methotrexate was conjugated to pectin by carbodiimide chemistry. Nanoparticles of pectin conjugated with methotrexate (MTX) were then fabricated by using ionotropic gelation. The size, shape and surface charge were characterized by dynamic light scattering and microscopic method. Cytotoxicity of MTX-pectin nanoparticle was monitored by MTT assay. Results: Methotrexate-pectin nanoparticle was successfully formulated. Drug release study indicated that MTX-NP exhibited sustained drug release at pH 7.4. Sustained release of methotrexate may enable methotrexate-pectin nanoparticle as a controlled drug delivery system. Cytotoxicity study confirmed the activity of the drug incorporated in nanoparticles. In addition, the cytotoxicity of methotrexate was increased when conjugated to pectin nanoparticles, compared to free methotrexate. Conclusions: This study verified that pectin can deliver methotrexate to hepatic cancer cell and provide sustained drug delivery. The cytotoxicity of methotrexate was enhanced when methotrexate was conjugated to pectin indicating the improved drug delivery to cancer cell.

Development of delayed-release proliposomes tablets for oral protein drug delivery

Context: One among many attempts to improve oral protein drug delivery was utilizing the colloidal drug carriers particularly liposomes. Objective: The purpose was to develop proliposomes of bovine serum albumin (BSA) in the form of granules and delayed-release tablets by using simple tablet manufacturing process. Materials and methods: BSA proliposomes granules were prepared by spraying 7:3 (w/w) – lecithin:cholesterol solution mixture onto BSA-mannitol granules rotating in a glass coating pan. BSA proliposomes granules were directly compressed into tablets and subsequently coated with Eudragit® L100 film. The physical properties and stability in gastrointestinal fluids of delayed-release BSA proliposomes tablets as well as reconstituted liposomes were assessed. Results: The BSA proliposomes tablets disintegrated readily and the obtained reconstituted BSA liposomes exhibited multilamellar vesicles, the size and entrapment efficiency of which were around 2–3 µm and 10–14%, respectively. The delayed-release BSA proliposomes tablets were found to be relatively stable in United States Pharmacopoeia (USP) simulated gastric and intestinal fluids. Increase in amount of BSA in granules resulted in the increase in entrapment efficiency and loading capacity. Discussion: The Fourier transform infrared spectroscopy (FTIR) results indicated increase in α-helix structure of BSA entrapped in liposomes. 31P phosphorous nuclear magnetic resonance spectroscopy (31P-NMR) spectrum indicated interaction between BSA molecules and phosphoric acid polar groups of bilayers membrane. Conclusion: The delayed-release BSA proliposomes tablets developed could completely be reconstituted into liposomes with sufficient resistance to the hostile environment in gastrointestinal tract.

Phospholipid vesicle–bound lysozyme to enhance permeability in human intestinal cells

Background: Oral peptide and protein drug delivery still remain the area of challenges for pharmaceutical scientists due to their low stability and permeability in gastrointestinal (GI) tract. In this study phospholipid vesicle–bound lysozyme were prepared and assessed for their physicochemical properties, secondary structure, and permeation across Caco-2 cells. Results: Lysozyme was found to be substantially bound onto negatively charged vesicles via electrostatic interaction as evidenced by zeta potential measurements regardless of cholesterol content. In contrast, the size of phospholipid vesicle–bound lysozyme became larger with the increasing cholesterol content. The secondary structure of vesicle-bound lysozyme examined by FTIR was unchanged compared to that in buffer solution. The apparent permeability of vesicle-bound lysozyme across Caco-2 cells monolayer was significantly enhanced with a size dependent manner compared to that of solution. Conclusion: The permeation across Caco-2 cell monolayers of phospholipid vesicle–bound lysozyme was demonstrated to be significantly enhanced with a size-dependent manner.

Targeting Antigen-loaded Liposomes to Myeloid Antigen Presenting Cells

Induction of immune cell death is one of the many mechanisms used by tumors to evade immune recognition. Here we assessed for the presence of spontaneous apoptosis in blood dendritic cells (DC; LinHLA-DR+ cells) from patients with breast cancer. We document the presence of a significantly (p < 0.05) higher proportion of apoptotic (Annexin-V+ and TUNEL+) blood DC in patients with early stage breast cancer (Stage I-II; n ¼ 13) compared to healthy volunteers (n ¼ 15). We examined the role of tumor products on this phenomenon and show that supernatants derived from breast cancer lines induce apoptosis of blood DC in PBMC cultures. Aiming to identify factors that protect these cells from apoptosis, we then compared a range of clinically available maturation stimuli including, inflammatory cytokines (TNF- a, IL-1 b, IL-6 and PGE 2; CC); synthetic double stranded RNA (poly I:C) and soluble CD40 ligand. While inflammatory cytokines and poly I:C induced robust phenotypic maturation, they failed to protect blood DC from apoptosis. In contrast, CD40 stimulation induced strong up-regulation of the antigen presenting machinery, secretion of IL-12 and protected blood DC through sustained expression of Bcl-2. Exogenous IL-12 also protected blood DC from apoptosis through sustained expression of Bcl-2, suggesting that CD40L-protection could be mediated, at least in part, through IL-12 secretion. Cumulatively our results demonstrate spontaneous apoptosis of blood DC in patients with breast cancer and confirm that ex vivo conditioning of blood DC can protect them from tumor-induced apoptosis.Diverse infectious and inflammatory environmental triggers, through unknown mechanisms, initiate autoimmune disease in genetically predisposed individuals. Here we show that IL-1b, a key cytokine mediator of the inflammatory response, suppresses CD25+CD4+ regulatory T cell function. Surprisingly, suppression by IL-1b occurs only where antigen is presented simultaneously to CD25+CD4+ T cells and to CD25CD4+ antigen-specific effector T cells. Further, NOD mice show an intrinsic over-production of IL-1 that contributes to reduced CD25+CD4+ regulatory T cell function. Thus, inflammation or constitutive over-expression of IL-1b in a genetically predisposed host can initiate a positive feedback loop licensing autoantigen-specific effector cells to inhibit the regulatory T cells maintaining tolerance to self.Dendritic cells (DC) are the potent antigen presenting cells which modulate T cell responses to self or non-self antigens. DC play a significant role in the pathogenesis of autoimmune diseases, inflammation and infection, but also in the maintenance of tolerance. NF-kappaB, particularly RelB is a crucial pathway for myeloid DC differentiation and functional maturation. While the current paradigm is that mature, nuclear RelB+ DC prime T cells for immunity/autoimmunity and immature DC for tolerance, RelB-deficient mice paradoxically develop generalised systemic autoimmune inflammatory disease with myelopoiesis and splenomegaly. Previous studies suggested abnormal DC differentiation in healthy relatives of type 1 diabetes (t1dm) patients. Therefore, we compared NF- kB activation in monocyte-derived DC from t1dm and non-t1dm controls in response to LPS. While resting DC appeared normal, DC from 6 out of 7 t1dm patients but no t2dm or rheumatoid arthritis patients failed to translocate NF- kB subunits to the nucleus in response to LPS, along with a failure to up-regulate expression of cell surface CD40 and MHC class I. NF- kB subunit mRNA increased normally in t1dm DC after LPS. Both the classical or non-canonical NF- kB pathways were affected as both TNF-a and CD40 stimulation led to a similarly abnormal NF- kB response. In contrast, expression of phosphorylated p38 MAPK and pro-inflammatory cytokine production was intact. These abnormalities in NF- kB activation appear to be generally and specifically applicable at a post-translational level in t1dm, and have the capacity to profoundly influence immunoregulation in affected individuals.The delivery of exogenous antigen to antigen presenting cells (APC) for processing and presentation is the first step in the generation of immune responses. We have utilized mannan or mannose as a vehicle to target protein and peptide antigens to mannose binding receptors on antigen presenting cells. In these studies antigen (protein or peptides) conjugated to oxidized mannan (OxMan) generated cellular immune responses in mice whilst antigen conjugated to reduced mannan (RedMan) gave humoral immune responses. These differential immune responses are due to the ability of OxMan to deliver exogenous conjugated antigen to the cytoplasm of APC (macrophages and DC). We have now used OxMan and RedMan to deliver DNA and RNA to APC. Mannan conjugates of poly-lysine (PLL) and polyethyleneimine (PEI) successfully complexed with DNA and RNA as evidenced by retardation on agarose gel electrophoresis. OxMan-PEI or PLL complexed with DNA or RNA transfected mannose receptor expressing J774 cells as well as bone marrow-derived dendritic cells. Mice immunized with OxMan-PLL-DNA conjugate were protected from a challenge of OVA expressing tumour cells. The combination of mannose receptor targeting and immunomodulating properties of OxMan results in an excellent adjuvant/delivery system.Cancer immunotherapy trials conducted over the last few years have concentrated on the analysis of immunological markers of response to antigenically well defined vaccines. Improvements in the quantity or quality of T cell responses, in a patient population, are proposed to allow a rational basis for improved clinical outcomes. However, using current methodologies, only weak immune correlates of clinical response have been found. Further, little attention has been given to other patient or tumour characteristics predisposing to favourable clinical responses following immunotherapy. Establishing such correlates is fundamental to understanding how vaccines work. We have followed this line of investigation with a matured, autologous, dendritic cell/irradiated melanoma cell vaccine for Stage IV melanoma patientsMost of the skin grafts from (K14hGH.FVB C57BL/6) F1 mice, which express foreign antigen (human growth hormone, hGH) in skin keratinocytes driven by keratin 14 promoter, were spontaneously rejected by syngeneic wild type F1 recipients and hGH-specific immune responses such as antibody and hGHspecific T cells were generated in these recipients. Interestingly, a 2nd F1 hGH-expressing skin graft was rejected by graft primed recipients, but was not rejected from such recipients if CD4+ or CD8+ T cells were depleted prior to the placement of the 2nd graft. Surprisingly, this 2nd graft retained healthy even after CD4+ or CD8+ T cells were allowed to recover so that the animal could reject a freshly placed 3rd F1 hGH-expressing graft. Furthermore, inflammatory response induced by topical treatment with imiquimod could lead to the rejection of some well-healed 2nd grafts. This result indicates that both CD4+ and CD8+ T cells are required for the rejection and the ability of effector T cells to reject a graft is determined by local factors in the graft which are presumably determined by inflammation induced by surgery or imiquimod treatment. Taken together, our results suggest that in addition to CD4+ and CD8+ T cells, local environmental factors induced by inflammation are also crucial for effector T cell functions leading to graft destruction. The understanding of these local factors will lead to more effective immunotherapy for established, epithelial cancer in the future.Inhibition of NFkB by the compound Bay 11–7082 (Bay) induces tolerogenic properties in dendritic cells (DC). While activation of NFkB can be induced by reactive oxygen species (ROS) and thiol/disulfide redox states, the consequences of NFkB blockade on ROS/redox state is not known. To generate immature DC, monocytes were cultured in GM-CSF and IL-4 (with or without Bay) for 48 h. Genes potentially involved in redox regulation were determined using microarray technology and validated using FACS, real-time PCR or western blotting. ROS were measured using two fluorescent dyes DHR-123 and DHE (to detect H2O2 or O2 respectively). We found increased expression of genes associated with reductants such as thioredoxin reductase (TrxR1) and glutathione (GSH), although those associated with the breakdown of H2O2 such as glutathione peroxidase, peroxiredoxins and catalase were decreased. Interestingly, Bay-treated DC produced less ROS in comparison to control DC under basal conditions and following stimulation with various pro-oxidants. In conclusion, Bay-treated DC display not only tolerogenic properties but also an intracellular reducing environment and an impaired ability to produce ROS. We are currently investigating whether exogenous ROS can interfere with the tolerogenic properties of Bay-treated DC.