Inflammation-Targeting Mesenchymal Stem Cells Combined with Photothermal Treatment Attenuate Severe Joint Inflammation.

Current clinical therapeutic efficacy for the treatment of osteo- and rheumatoid-arthritis is obviously limited. Although mesenchymal stem cells (MSCs) are considered as a source of promising regenerative therapy, un-modified or genetically engineered MSCs injected in vivo restrict their clinical utility because of the low drug efficacy and unpredicted side effect, respectively. Herein, a strategy to enhance the migration efficacy of MSCs to inflamed joints via an inflammation-mediated education process is demonstrated. To reinforce the limited anti-inflammatory activity of MSCs, gold nanostar loaded with triamcinolone is conjugated to MSC. Furthermore, near-infrared laser-assisted photothermal therapy (PTT) induced by gold nanostar significantly elevates the anti-inflammatory efficacy of the developed drugs, even in advanced stage arthritis model. An immunological regulation mechanism study of PTT is first suggested in this study; the expression of the interleukin 22 receptor, implicated in the pathogenesis of arthritis, is downregulated in T lymphocytes by PTT, and Th17 differentiation from naïve CD4 T cell is inhibited. Collectively, inflammation-targeting MSCs conjugated with triamcinolone-loaded gold nanostar (Edu-MSCs-AuS-TA) promote the repolarization of macrophages and decrease neutrophil recruitment in joints. In addition, Edu-MSCs-AuS-TA significantly alleviate arthritis-associated pain, improve general locomotor activity, and more importantly, induce cartilage regeneration even for severe stages of arthritis model.

Biological testing of chitosan-collagen-based porous scaffolds loaded with PLGA/Triamcinolone microspheres for ameliorating endoscopic dissection-related stenosis in oesophagus.

OBJECTIVES: Endoscopic submucosal dissection (ESD), a preferential approach for early oesophageal neoplasms, inevitably results in oesophageal strictures in patients. Clinical use of glucocorticoids through submucosal injection is beneficial for inhibiting oesophageal stricture following injury; however, it also has limitations, such as dose loss and perforation. Hence, alternatives to glucocorticoid therapy should be developed. METHODS: A novel porous composite scaffold, ChCo-TAMS, composed of chitosan, collagen-I and triamcinolone acetonide (TA) loaded into poly (lactic-co-glycolic) acid (PLGA) microspheres (TAMS), was successfully constructed and subjected to biological testing to ameliorate oesophageal ESD-related stenosis. RESULTS: The synthesized biomaterials displayed unique properties in inhibiting the activation of macrophages, chemokine-mediated cell recruitment and fibrogenesis of fibroblasts. Further application of the scaffolds in the rat dermal defect and porcine oesophageal ESD model showed that these novel scaffolds played a robust role in inhibiting wound contracture and oesophageal ESD strictures. CONCLUSIONS: The developed composite scaffolds provide a promising clinical medical device for the prevention of post-operative oesophageal stricture.

Graft polymerization of guar gum with acryl amide irradiated by microwaves for colonic drug delivery.

This article is aimed to discuss the modification of guar gum through microwave irradiation by varying the time of irradiation. The characterization of the modified products was carried out using FTIR spectroscopic analysis. The FT-IR spectrum of the pure guar gum (GG) sample showed a broad peak at 3298 cm(-1) while the modified GG sample displayed a peak at 1541 cm(-1) which was absent in the crude sample. The X-ray diffraction (XRD) analysis confirmed the increase in crystallinity due to grafting of the sample with polyacrylamide (GG-g-PAM). Scanning electron microscope (SEM) images revealed that granular form of guar gum was changed into fibrillar structure after grafting. Thermo-gravimetric analysis of the modified samples was also carried out and discussed. The role of guar gum as a matrix for controlled release of drug triamcinolone was evaluated. The GG-acrylamide grafted samples presented a correlation between drug release and time of microwave exposure. The results revealed that such modified product has potential applications in colonic drug delivery system.

Triamcinolone and intraocular sustained-release delivery systems in diabetic retinopathy.

Diabetic retinopathy (DR) still represents one of the leading causes of vision loss worldwide. Since this condition affects the posterior segment of the eye, topical application of ophthalmic medicines is of limited benefit, considering that they seldom reach therapeutic levels in the affected tissues. Systemic medications can be insufficient due to the eye's immunoprivileged condition and existence of both inner and outer blood-retinal barriers, which place limitations on the potential role of this route of administration for retinal diseases. In this setting, intraocular therapies have emerged as novel and vital tools in the ophthalmologist's armamentarium against DR, allowing for maximization of drug efficacy and limited risk of systemic side effects. Intravitreal injections of triamcinolone acetonide have been widely used for treating DR particularly in the 21(st) century. Other agents targeting molecules, such as anti-vascular endothelial growth factor, have also demonstrated a potential therapeutic role for treatment. Recent advances in ocular drug delivery methods have led to the development of intraocular implants, which help to provide prolonged treatment with controlled drug release. Moreover, they may add some potential advantages over traditional intraocular injections by delivering certain rates of drug directly to the site of action, amplifying the drug's half-life, contributing in the minimization of peak plasma levels of the drug, and avoiding the side effects associated with repeated intravitreal injections.

Glucocorticoid receptor heterozygosity combined with lack of receptor auto-induction causes glucocorticoid resistance in Jurkat acute lymphoblastic leukemia cells.

Glucocorticoids (GC) induce apoptosis in malignant lymphoblasts, but the mechanism of this process as well as that of the clinically important GC resistance is unknown. We investigated GC resistance in Jurkat T-ALL cells in which ectopic GC receptor (GR) restores GC sensitivity, suggesting deficient GR expression. Jurkat cells expressed one wild-type and one mutated (R477H) GR allele. GR(R477H) ligand-binding-dependent nuclear import, as revealed by live-cell microscopy of YFP-tagged GR, was unaffected. Transactivation and transrepression were markedly impaired; however, GR(R477H) did not act in a dominant-negative manner, that is, did not prevent cell death, when introduced into a GC-sensitive cell line by retroviral gene transfer. Contrary to another GR heterozygous, but GC-sensitive, T-ALL model (CCRF-CEM), Jurkats expressed lower basal GR levels and did not auto-induce their GR, as revealed by 'real-time' RT-PCR and immunoblotting. Absent GR auto-induction could not be restored by transgenic GR and, hence, was not caused by reduced basal GR levels. Thus, inactivation of one GR gene results in haploinsufficiency if associated with lack of GR auto-induction.

Detection and quantitation of fatty acid acyl conjugates of triamcinolone acetonide via gas chromatography-electron-capture negative-ion mass spectrometry.

The inherent electron-capture properties of triamcinolone acetonide (TAA) fatty acid conjugates were exploited for development of a GC-MS technique for quantitation of C21 long-chain fatty esters of TAA synthesized in BEAS-2B cells, an immortalized airway epithelium cell line. TAA esters extracted from BEAS-2B cells were purified and detected via selected ion monitoring of the molecular anions generated from the TAA esters under electron-capture negative-ion mass spectrometric conditions. Standard curves were linear over a range of 0.0 to >4.5 ng/mg protein with r(2) values = 1. Levels of TAA conjugates extracted from BEAS-2B treated with 10(-5)M TAA for 24h ranged from 0.024 to 0.301 ng/mg protein. Further evidence for confirmation of the identity of TAA fatty esters formed in BEAS-2B cells was obtained via selected reaction monitoring. The transition monitored was formation of the carboxy anion generated from each of the respective molecular anions of the TAA esters during collision-induced decomposition. These findings indicate that the GC-MS analysis is suitable for studies of the kinetics of the TAA fatty acid conjugates formation in vitro and may be directly applicable to determination of the kinetics of TAA fatty acid conjugation in vivo.

Inhibition of glucocorticoid receptor binding by nitric oxide.

Septic shock is a dangerous condition with high mortality rates. In sepsis, the inducible form of nitric oxide (NO) synthase is induced, releasing high amounts of NO. Glucocorticoids have potent anti-inflammatory properties and are very effective in inhibiting the induction of this enzyme if administered before the shock onset. It is known that glucocorticoid receptor (GR) has critical cysteine residues for steroid binding in its hormone-binding and DNA-binding domains. It has also been reported that NO reacts with ---SH groups, forming S-nitrosothiols. Therefore, we examined the potential effect of NO on the ligand-binding ability of GR. NO donors (S-nitroso-acetyl-DL-penicillamine, S-nitroso-DL-penicillamine, or S-nitroso-glutathione) decreased, in a time- and dose-dependent manner, the binding of [3H]triamcinolone to immunoprecipitated GR from mouse L929 fibroblasts. The nonnitrosylated parent molecules, N-acetyl-DL-penicillamine, and reduced gluthatione were without effect. Scatchard plots revealed that the number of ligand binding sites and Kd were reduced (50%) by NO donors. Western blot analysis ruled out the possibility that dissociation of GR/heat shock protein 90 heterocomplex or decrease in GR protein would account for the inhibitory effect of NO. Decreased ligand binding to GR was found when NO donors were incubated with intact fibroblasts. Incubation with NO donors also decreased the steroid-induced reduction in [3H]uridine incorporation into RNA. All of these NO effects were inhibited by the thiol-protecting agent dithiothreitol. Therefore, S-nitrosylation of critical ---SH groups in GR by NO with consequent decreases in binding and affinity may be the mechanisms which explain the failure of glucocorticoids to exert their anti-inflammatory effects in septic shock.

An in-vitro study of the effect of hydrocolloid patch occlusion on the penetration of triamcinolone acetonide through skin in man.

The aim of this study was to evaluate the effect of occlusion using hydrocolloid-containing patches on in-vitro triamcinolone acetonide (TACA) penetration of the epidermis while monitoring the uptake of water by the patches as a result of transepidermal water loss. The hydrocolloid patches were a laminate of a pressure-sensitive hydrophobic adhesive (containing a dispersion of 39% of either pectin or carmellose sodium) and a polyethylene film. The diffusion of a representative corticosteroid (TACA) through isolated epidermal sheet was shown to depend on the site from which the skin was removed. The two patch-types exhibited markedly different hydration rates when applied to the membranes. For example, after 96 h the carmellose sodium patch showed ten times the weight increase of the pectin patch. Epidermal diffusion rates were, however, similar, both showing a 3-4-fold enhancement over unoccluded conditions. The increase in TACA diffusion with the patches can be explained by the increase in skin hydration that occurs during occlusion. Despite the large differences in transepidermal water transfer through the epidermal membranes with the two types of hydrocolloid patch, however, this level of stratum corneum hydration was apparently similar. As the rate of diffusion was also independent of hydrocolloid patch component, it seems possible that the hydrophobic component of the patch matrix may also influence the level of skin hydration and consequent drug diffusion.

Temporary loss of glucocorticoid receptor-mediated regulation of gene expression in heat-shocked cells.

The effect of heat shock on the transcriptional activity of glucocorticoid receptor was assessed using HeLa cells stably transfected with the chloramphenicol acetyltransferase (CAT) gene the transcription of which is controlled by two glucocorticoid-responsive elements placed directly upstream of a core promoter. Heat shock inactivated the high-affinity glucocorticoid binding capacity of the cells and nullified the rate of accumulation of CAT mRNA in the presence of hormone. Hormonal responsiveness was restored on return to normal temperature concomitantly with recovery of high-affinity glucocorticoid binding capacity. Heat inactivation of the receptor was coincident with loss of its solubility and apparently unrelated to receptor degradation.

[Medicinal activation and inhibition of function of glucocorticoid receptors as the basis for regulation of the glucocorticoid effect]. / Lekarstvennoe aktivirovanie i ingibirovanie funktsii gliukokorticoidnykh retseptorov kak osnova reguliatsii gliukokortikoidnogo éffekta.

On the basis of Scatchard and Lineweaver-Burk analysis, it was demonstrated that a series of drugs either activated or inhibited the function of types II and III glucocorticoid receptors. Analgine (0.04-10.0 mM) and sodium salicylate (12.5-50.0 mM) suppress the type II glucocorticoid receptor function of rat liver cytosol. Maradol (5.0 mM) increases the type II glucocorticoid receptors density but decreases the measurable constant for the [3H]acetonide triamsinolone interaction with type II glucocorticoid receptors. Analgine (1.25-10.0 mM) and sodium salicylate (0.62-10.0 mM) increase the type III glucocorticoid receptor function of rat liver cytosol. Maradol (0.25-1.0 mM) suppresses the type III glucocorticoid receptor function. The mechanism of regulation of the glucocorticoid effect by nonsteroid drugs influencing upon the function of types II and III glucocorticoid receptors is discussed.

Advantages of the scanning ion microscopy for mapping halogen corticoids in normal and transformed cells in culture.

The intra-cellular distribution of eight halogen glucocorticoids was investigated by ion microscopy in two cellular varieties of cultured non-cancer cells (fibroblast 3T3) and cancer cells (human breast tumor cells MCF-7). Two types of ion microscopy helped to determine this distribution, a direct imaging ion microscope (SMI 300) with low spatial resolution, and a scanning ion microscope (IMS4F), featuring high resolution, serving to obtain maps representing the intra-cellular distribution of the fluorine elements and drugs present in these monolayer cultured cells. The fluorine images representative of the drugs containing fluorine showed that these drugs are essentially concentrated in the cell nuclei. In these nuclei, the distribution of these drugs is different from that of heterochromatin and of the nucleolus.

Glucocorticoid receptor DNA-binding specificity is increased by the organization of DNA in nucleosomes.

A DNA fragment containing glucocorticoid receptor binding sites in the mouse mammary tumor virus promoter was reconstituted in vitro with histones to form nucleosome cores, which become positioned on the DNA fragment in a sequence-specific manner. Glucocorticoid receptor binding to specific DNA sequences was analyzed by quantitative DNase I footprinting. The receptor interacted with surprisingly high affinity with one of the binding sites in the reconstituted promoter, although it was reduced by a factor of approximately 2 compared with the same site in protein-free DNA. By contrast, the affinity for random genomic nucleosomal sites was drastically reduced compared with histone-free DNA. Thus, reconstituting the promoter in vitro resulted in a 60- to 70-fold increase in binding specificity. Such an increase in selective binding may help to explain the ability of glucocorticoid receptor to effectively locate its target sites in chromatin.

Characterization of nuclear corticosteroid receptors in rat adipocytes. Regional variations and modulatory effects of hormones.

The corticosteroid receptor was investigated in isolated rat adipocytes with a new technique which characterizes the corticosteroid receptors that can be activated and tightly bound to the nucleus. The binding reaction with [3H]triamcinolone was performed with intact isolated adipocytes and the radioactivity associated with nucleus was subsequently determined after cell lysis. Scatchard analysis revealed a homogeneous class of nuclear corticosteroid receptors in rat epididymal adipocytes with an apparent Kd of 4.93 +/- 1.5 nM and a Bmax of 21.8 +/- 6.6 fmol/10(6) cells corresponding to about 13,000 receptors per nucleus. The corticosteroid binding exhibited regional variations in isolated adipocytes. The highest receptor number was found in epididymal adipocytes (Bmax 25.8 +/- 3.9 fmol/10(6) cells) whereas there were significantly lower nuclear binding sites in perirenal adipocytes (16.5 +/- 5.5 fmol/10(6) cells) (P less than 0.05) and subcutaneous adipocytes (4.8 +/- 1.5 fmol/10(6) cells) (P less than 0.01). The apparent affinity in the three fat depots were similar with Kd values about 4 nM. The nuclear corticosteroid receptor in adipocytes was steroid specific, as neither unlabelled estradiol nor testosterone were able to displace the [3H]triamcinolone binding at concentrations up to 100 microM. However, unlabelled progesterone and promegestrone (R5020) were able to compete with triamcinolone-binding (by 50-80%). In order to investigate whether the nuclear corticosteroid binding in adipocytes were under influence of other hormones we examined the effects of lipolytic and antilipolytic compounds on the binding. Preincubation with isoproterenol and dibutryl-cAMP for 1 h was able to decrease the corticosteroid binding by 30-50%. However, the antilipolytic hormone insulin had no effect in preincubations performed for up to 2 h. In conclusion, high affinity nuclear corticosteroid receptors were found in rat adipocytes. These receptors exhibited regional variations and were modulated by lipolytic hormones.

Identification of an epitope shared by the DNA-binding domain of glucocorticoid receptor and the B chain of insulin.

A monoclonal antibody (8G11-C6) generated by an auto-anti-idiotypic route and directed to a site near the ligand-binding site of the glucocorticoid receptor also binds to native insulin and the B chain of insulin but not to the A chain of insulin. The glucocorticoid receptor and the B chain of insulin, therefore, share a cross-reacting epitope. Examination of the primary sequences of the two proteins revealed a limited number of regions of identity or close homology. Several peptides representative of those regions were synthesized. A heptapeptide sequence of the B chain of insulin with homology to a sequence in the first "zinc finger" of the DNA-binding domain of the glucocorticoid receptor was identified as the cross-reactive epitope. This heptapeptide sequence is restricted to and highly conserved among insulins of various species. Homologous sequences are found in the DNA-binding domains of most steroid receptors and related DNA-binding proteins. Consistent with this is the finding that 8G11-C6 inhibits the binding of glucocorticoid receptor to DNA-cellulose.

Specificity of the dexamethasone-induced steroid receptor in Tetrahymena.

The dexamethasone-induced steroid receptor of Tetrahymena pyriformis specifically binds triamcinolone, which is itself a fluorinated glucocorticoid. It also binds dihydro-epi-androsterone (DHEA) but no or very little testosterone, digoxin or ouabain. It follows that the specificity of the induced steroid receptor of Tetrahymena may only partially be comparable with that of the mammalian steroid receptor.

Stability and transformation of the glucocorticoid receptor under acidic conditions.

The [3H]triamcinolone acetonide ([3H]TA)-binding ability of the rat liver glucocorticoid receptor (GR) was investigated under acidic conditions, ranging from pH 2 to 7.3. Both in the presence and absence of 10 mM molybdate, the [3H]TA-binding ability decreased below pH 6.5 and was almost completely lost below pH 5, pH 5.9 +/- 0.1 giving 50% [3H]TA-binding. The binding ability was recovered when the pH of the cytosol was reversed to 7.3 or the precipitate obtained on acidification was dissolved in a buffer of pH 7.3. Moreover, in the absence of molybdate, the [3H]TA-GR complexes formed at pH 7.3 remained unchanged until pH 5. Then they decreased, pH 3.9 +/- 0.1 giving 50% binding, and completely disappeared at pH 3. [3H]TA-binding activity recovered from the precipitate also decreased in a similar pH region (a 50% decrease in binding being observed at pH 4.2 +/- 0.04). These results suggest that rat liver GR is rather resistant under acidic conditions and that it exists in a peculiar state below pH 5.9 to approximately 4 as to its ligand binding property: unoccupied GR has no [3H]TA-binding ability but [3H]TA-GR complexes once formed at neutral pH do not dissociate. [3H]TA-GR complexes recovered from the precipitate at pH 5 had a Stokes radius of 7.5 nm, little DNA-cellulose-binding ability and sedimented at 8.6S on glycerol gradient centrifugation, indicating that the receptor existed in a nontransformed state. In addition, both occupied and unoccupied GR were transformed at about pH 4, their being 50% transformation. This transformation was accompanied by irreversible denaturation of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of growth of Candida spp. in the presence of various glucocorticoids on the adherence to human buccal epithelial cells.

In vitro culturing of three different yeast species with a number of glucocorticoids altered their adherence ability in two ways: Incubation with dexamethasone and triamcinolone acetonide promoted the adherence in general (the increase in adherence ranged between 17% and 44%), whilst growth in the presence of cortisone acetate or hydrocortisone blocked the adherence (inhibition ranged from 16% to 32%). No statistical difference in the adherence capabilities of different growth phases of C. albicans noted, and the effects of glucocorticoids persisted irrespective of the phase of growth used. An attempt to explain the differences in adherence of the Candida spp. investigated, in the presence of various steroids, on the basis of variation in their structural configurations and/or steroid-receptor interaction is given.