JBC Focus on PI3-Kinase with Echelon

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M204924200 on August 9, 2002

J. Biol. Chem., Vol. 277, Issue 42, 39343-39349, October 18, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/42/39343    most recent
M204924200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ma, L.
Right arrow Articles by Giannoukakis, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ma, L.
Right arrow Articles by Giannoukakis, N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Immunosuppressive Effects of Glucosamine*

Linlin MaDagger , William A. Rudert§, Jo HarnahaDagger , Marietta Wright§, Jennifer Machen§, Robert Lakomy§, Shiguang QianDagger , Lina LuDagger , Paul D. Robbins, Massimo Trucco§||, and Nick Giannoukakis||**DaggerDagger

From the Dagger  Department of Surgery, T. E. Starzl Transplantation Institute, the Departments of  Molecular Genetics and Biochemistry and ** Pathology, and the || Diabetes Institute, University of Pittsburgh School of Medicine, and the § Department of Pediatrics, Division of Immunogenetics, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania 15213

Received for publication, May 19, 2002, and in revised form, August 6, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitro as well as allogeneic mixed leukocyte reactivity in a dose-dependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glucosamine is a naturally occurring sugar that is synthesized by virtually all cells. Upon uptake, glucose is immediately phosphorylated and enzymatically converted into a series of substrates that will be either converted into glycogen, lipids, and proteins or used to generate ATP and CO2. 2-3% of glucose 6-phosphate, the immediate intracellular glucose derivative following uptake, is diverted into a pathway known as the hexosamine biosynthesis pathway (1, 2). The rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase is responsible for the commitment of glucose derivatives to the pathway, ultimately resulting in the formation of glycoprotein precursors (3). Glucosamine is not secreted outside of cells, but exogenously added glucosamine is taken up by glucose transporters (GLUT-2 and GLUT-4) and then phosphorylated (4, 5).

In 1953, Quastel and Cantero (6) demonstrated that glucosamine possesses tumor-inhibiting activity. Since then, a number of reports have confirmed the tumoricidal activity of glucosamine (7-16). Glucosamine has been shown to inhibit nucleic acid and protein biosynthesis and irreversible damage to organelles in tumor cells, but not in normal cells (8-15, 17-20). In addition, glucosamine also inhibits platelet aggregation and ATP release induced by Staphylococcus aureus, ADP, epinephrine, and collagen (21). The mechanisms by which glucosamine acts are not completely clear; however, it has been shown to alter the ultrastructure of plasma and intracellular membranes (9, 22), to inhibit membrane transport of nucleosides (14, 15), and reportedly to shift its distribution from glycoproteins to glycolipids (22).

O'Neill and Parish (23) demonstrated that monosaccharides, especially amino sugars, are able to inhibit cytotoxic T-lymphocyte function in culture, preventing target lysis in a cytotoxic T-lymphocyte clone-specific manner. Yagita et al. further demonstrated that free hexosamines are able to inhibit natural killer cell cytotoxicity in culture (24, 25) and that hexosamine release by tumors can, in part, explain escape of tumors from immune cell lysis (24). Since then, other than the observation that amiprilose, a synthetic monosaccharide, is able to attenuate T-cell activation in a dose-specific manner (26), no other work has been published on the utility of sugar derivatives, especially amino sugar derivatives (glucosamine, mannosamine, lactosamine, and fructosamine), as immunoregulatory agents. However, a recent report by Gouze et al. (27) demonstrated glucosamine-dependent inhibition of NF-kappa B activity in rat chondrocytes and IL-1beta 1 bioactivity by up-regulation of the type II IL-1 decoy receptor.

Glucosamine has received considerable attention in a number of studies over the past 5 years as an agent that may be beneficial for arthritis (28-31). Sold mainly over-the-counter in various formulations (glucosamine sulfate and glucosamine sulfate with chondroitin sulfate), manufacturers suggest that the beneficial effects of their compounds are due to the reconstruction of joint cartilage, one of the constituents of which is glucosamine in the form of glycoproteins of structural proteoglycans. A recent study in humans demonstrated beneficial effects of glucosamine in arthritis, although no firm conclusions could be made (28). The actual mechanism by which glucosamine may benefit the patient remains unknown, although a very recent investigation suggests that it may interfere with pro-inflammatory cytokine action on human chondrocytes (32).

However, glucosamine induces insulin resistance in the absence of high glucose or glutamine (2) as well as insulin resistance in isolated rat muscle (33). Glucosamine has also been shown to modulate the effects of insulin and glucose on pyruvate kinase (34), glycogen synthase (33, 34), and transforming growth factor-alpha (35) gene expression. A short-term exposure of cultured rat adipocytes to glucosamine decreases GLUT-4 activity, and longer, 16-h incubations result in decreased GLUT-4 cell surface levels (36). Furthermore, glucosamine infusion can induce insulin resistance in normoglycemic (but not hyperglycemic) rats, and this is accompanied by impaired GLUT-4 translocation to the cell surface of skeletal muscle in response to insulin (37). Insulin sensitivity in rat cardiac muscle and liver has also been shown to be affected by glucosamine infusion (38). More importantly, acute glucosamine infusion into humans has been demonstrated to mimic some of the metabolic aspects of insulin resistance in human type II diabetes mellitus (39).

To unravel the mechanisms involved in the possible beneficial effects of glucosamine, despite its reported effects on insulin sensitivity, we have begun to examine the effects of glucosamine on immune cell activation and inflammatory processes. In initial studies, we have discovered that glucosamine addition to immune cells in vitro prevented both their activation and their ability to initiate the mixed leukocyte reaction. More importantly, a single daily intravenous injection of glucosamine was able to prolong cardiac allograft survival in mice. Based on these data, we suggest that glucosamine alone or in its different formulations could be considered as a novel immunosuppressive agent with potential clinical utility.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Reporter T-cell Lines-- The Jurkat T-lymphoblast cell line used in this study is a human T-lymphocyte precursor cell line that responds identically to nonspecific, non-antigenic stimulation signals and is available from American Type Culture Collection (ATCC TIB-152) (40, 41). It has been extensively used to identify and unravel T-cell activation and signaling pathways (42-53). To test the effects on T-cell activation of a number of agents, we designed appropriate Jurkat-based reporter cell lines. We engineered the Jurkat cell line to stably express the beta -galactosidase gene under the control of four tandemly arranged NFAT cis-acting enhancer elements derived from the NFAT-PathDetect reporter plasmid (Stratagene, La Jolla, CA). NFAT describes the family of intracellular transcription factors that are among the very first to be activated in response to T-cell antigen receptor ligation or nonspecific signals (54-58). NFAT quickly translocates to the nucleus and activates the IL-2 gene, one of a number of genes whose products are important for maintaining and propagating the T-cell activation signal. The engineered Jurkat T-cells, which we term JLZB (beta -galactosidase reporter), express beta -galactosidase when exposed to factors that engage their T-cell receptors in a specific manner or to chemical nonspecific factors such as phorbol 12-myristate 13-acetate (PMA) and ionomycin. Cultures were exposed to hygromycin (Invitrogen) to select stably transfected clones. Individual clones were isolated and tested for NFAT inducibility. In a similar fashion, we also created a stable Jurkat cell line constitutively expressing the green fluorescent protein (GFP) under the control of the SRalpha promoter (consisting of the SV40 early promoter and the R-U5 segment of the human T-cell leukemia virus type 1 long terminal repeat). We designated this cell line as JSR-GFP. Stably transfected JSR-GFP clones were selected with 0.25 mg/ml Zeocin (Invitrogen). To determine promoter activation, we cultured 3 × 106 cells of each clone selected overnight in 1 ml of ionomycin (1 µg/ml; Calbiochem) and PMA (25 ng/ml; Sigma) in RPMI 1640 medium, 10% fetal bovine serum, and antibiotics (Invitrogen); and the next day, we quantitated the amount of beta -galactosidase using a commercially available kit (Tropix Inc., Bedford, MA) or by FACS analysis of JSR-GFP clones. We selected the clones exhibiting the highest inducibility for further experimentation.

Effects of Glucosamine on Reporter Cell Activation-- To examine the effects of glucosamine on T-cell activation, we added pure glucosamine (Sigma) dissolved in PBS (Sigma) to cultures of JLZB or JSR-GFP cells in RPMI 1640 medium, 10% fetal bovine serum, and antibiotics. Glucosamine was added to final concentrations ranging from 1 µM to 10 mM for 18-24 h in the presence or absence of PMA and ionomycin at activating concentrations. As a control, we incubated parallel cultures in equal concentrations of galactosamine and mannosamine (both purchased from Sigma). Cells were collected and centrifuged at 600 × g, and the pellets were washed extensively with PBS. The cells were subsequently lysed in reporter gene lysis buffer (Promega) or directly analyzed by FACS (JSR-GFP cells) in a FACSVantage SE flow cytometer (BD Biosciences). Reporter cell activation was determined by assessing the level of beta -galactosidase activity in the lysates of JLZB cells using the kit from Tropix Inc. Concurrently, secreted IL-2 levels were determined in the supernatant of the cells using a commercially available enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Cell viability in all instances and following treatment was measured using a commercially available reagent based on the 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS) assay (CellTiter96 Aqueous, Promega).

To examine the reversibility of the effects of glucosamine on reporter gene activity, JLZB cells were first stimulated with PMA and ionomycin in the presence or absence of 10 mM glucosamine. 24 h later, the cells were washed; the media were replaced; and the cells were once again stimulated with PMA and ionomycin. beta -Galactosidase activity was determined as described above.

Measurement of ATP Levels in Glucosamine-treated Jurkat T-cells-- To determine the effects of glucosamine administration on ATP levels in cultured Jurkat T-cells, we measured ATP indirectly using a commercially available reagent (ENLITEN, Promega). The assay is based on the ATP-dependent conversion of luciferin to oxyluciferin, AMP, pyrophosphate, carbon dioxide, and light, measured in a luminometer (560 nm). The intensity of emitted light is directly proportional to the amount of ATP present in the sample. Jurkat T-cells were stimulated with PMA and ionomycin as described above in the presence or absence of 10 mM glucosamine for 18 h. The cells were extensively washed, and equal numbers of cells from each treatment group as well as untreated cells were then lysed. The lysate was cleared by centrifugation, and an aliquot of the supernatant was used to assess ATP levels using the ENLITEN reagent.

Comparison of the Effects of Glucosamine with Those of Cyclosporin A and Tacrolimus on Reporter Cell Activation-- The effects of glucosamine were compared with those of immunosuppressive agents in clinical use (cyclosporin A and tacrolimus (FK-506)) and examined using the JLZB reporter cell line. Following the addition of glucosamine, galactosamine, or mannosamine (at different final concentrations ranging from 10 nM to 10 mM), cyclosporin A (Sandimmune, 100 ng/ml; Novartis, Basel, Switzerland), or tacrolimus (10 ng/ml; Prograf, Fujisawa, Deerfield, IL) to 3 × 106 cells, PMA and ionomycin were added, and the cells were incubated for 18-24 h. The next day, the culture supernatants were collected, and the cells were lysed. beta -Galactosidase activity was determined as described above.

Effects of Glucosamine on Dendritic Cell Activation-- 5-8-week-old mice were obtained from Jackson Laboratories (Bar Harbor, ME). C57/BL10 mouse bone marrow-derived dendritic cells were obtained from bone marrow progenitors cultured with granulocyte/macrophage colony-stimulating factor and IL-4 (R&D Systems) essentially as described (59, 60). By this method, the purity of dendritic cells is usually >85% as assessed by FACS analysis for dendritic cell surface markers (CD86, CD80, CD40, DEC-205, and class I and II major histocompatibility complexes; purchased from BD Biosciences) (59, 60). 1 × 105 dendritic cells were treated with glucosamine, galactosamine, or mannosamine (all at a final concentration of 10 mM) or PBS for 18-24 h and then stimulated with 25 µg/ml lipopolysaccharide (LPS; Sigma) for another 18-24 h. At the end of the stimulation, the culture supernatants were assessed for nitrite production, a marker of dendritic cell activation, using the Griess reagent method (Promega).

Effects of Glucosamine on Allogeneic MLR in Culture-- Irradiated C57/BL10 splenocytes and C3H/HeJ mouse T-lymphocytes (Jackson Laboratories) were co-cultured in the presence or absence of glucosamine, galactosamine, or mannosamine (2.5, 5, and 10 mM final concentrations) or medium alone for 5 days. At the end of the co-culture, tritiated thymidine was added for 18 h, and the radioactivity incorporated into the cells was determined by liquid scintillation counting.

Effects of Glucosamine on Cardiac Allograft Survival-- To examine the effects of glucosamine on cardiac allograft survival, heterotopic transplantation of C3H/HeJ hearts into C57/BL10 mice was performed as described (59). There is complete mismatch at the major histocompatibility complex between these two species. Each recipient was conditioned with one intravenous injection of 8, 20, or 40 µmol of glucosamine 1 day prior to transplantation and one daily intravenous injection of 40 µmol of glucosamine beginning on the 2nd day following transplantation until the end of the experiment (defined as the time of transplant rejection). To compare the efficacy of glucosamine with that of immunosuppressive agents in clinical use, we also treated a subset of mice with FK-506 (0.5 mg/kg/day) or cyclosporin A (10 mg/kg/day) once a day for 7 days following transplantation alone or in combination with different amounts of glucosamine. As a control, we also gave a single daily intravenous injection of 40 µmol of mannosamine or galactosamine to parallel sets of mice. Rejection of the transplant was determined to be the time at which palpable beating of the heart ceased.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Glucosamine on T-cell and Dendritic Cell Activation-- The effects of glucosamine on immune cell activation in vitro were first examined. Three parameters were evaluated: T-cell activation, dendritic cell activation, and allogeneic MLR. To assess T-cell activation, we added glucosamine to Jurkat T-lymphoblasts stably transfected with the beta -galactosidase gene under the control of NFAT cis-acting enhancer elements in tandem (JLZB cell line). Fig. 1A shows that the addition of 10 mM glucosamine to JLZB reporter cells for 18 h in the presence of chemical activators of T-cell function (PMA and ionomycin) resulted in a nearly complete prevention of expression of beta -galactosidase as assessed by beta -galactosidase activity. Additionally, Fig. 1B shows a significant suppression of IL-2 production by the same cells in the culture supernatants. To compare the efficacy of glucosamine with that of commonly used immunosuppressive agents, we assessed T-cell activation in the JLZB reporter cell line in the presence of cyclosporin A or tacrolimus (FK-506). Fig. 1A shows that glucosamine at concentrations as low as 500 µM was as effective as clinical doses of cyclosporin (100 ng/ml) and FK-506 (10 ng/ml) in suppressing the activation of the JLZB reporter cell line. The results also demonstrate that glucosamine inhibited NFAT-dependent transcription stimulated by PMA and ionomycin at similar levels compared with cyclosporin.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 1.   A, glucosamine prevents NFAT-driven beta -galactosidase expression in JLZB cells. beta -Galactosidase expression was significantly suppressed in JLZB cells treated with glucosamine and exposed to activating concentrations of PMA and ionomycin for 18-24 h. The bars represent beta -galactosidase activity (shown as relative light units (RLU)) in JLZB cells cultured in 10 mM glucosamine (Gn), cyclosporin A (CsA), or FK-506 (FK) for 18-24 h and then stimulated for an additional 18-24 h with activating concentrations of PMA and ionomycin (P+I). The last three bars show the beta -galactosidase activity of JLZB cells treated with PMA and ionomycin in the presence of different concentrations of glucosamine. The results shown are the means ± S.E. of three independent experiments performed in triplicate. Ctrl, control untreated cells. B, glucosamine suppression of IL-2 production. JLZB cells were treated with glucosamine and activating concentrations of PMA and ionomycin. 18-24 h later, IL-2 levels were measured by enzyme-linked immunosorbent assay. The bars represent detectable IL-2 in triplicate determinations, and the error bars represent the S.E. IL-2 levels were below the limit of assay detection where no bars are present. C, comparison of glucosamine with other amine sugars in suppressing IL-2 production in T-cells. Galactosamine (Gl) and mannosamine (Mn) were added to wild-type Jurkat cells at a final concentration of 10 mM. After an overnight culture, PMA/ionomycin was added, and IL-2 levels in the supernatants were assessed the day after by enzyme-linked immunosorbent assay. The bars represent the amount of secreted IL-2 in pg/ml, and the error bars represent the S.E. The results shown are representative of three separate experiments performed in triplicate. D, effect of glucosamine on metabolic viability of T-cells in culture. Cell viability was assessed in cultures treated with glucosamine and in parallel cultures further exposed to PMA/ionomycin. A490 (OD490) is directly proportional to the metabolically viable cell number. The results are representative of two separate experiments performed in triplicate, and the error bars indicate the S.E. WT Jurkat, wild-type Jurkat cells. E, reversibility of the effect of glucosamine on T-cells in culture. NFAT reporter activity is shown (in relative light units) in JLZB cell lysates. Cells were previously treated with glucosamine (10 mM), extensively washed with PBS, cultured overnight in normal growth medium, and then stimulated with PMA/ionomycin. The error bars represent the S.E. The data shown are from two separate experiments performed in triplicate. F, glucosamine does not suppress gene expression from a constitutive gene promoter. Glucosamine was added to JSR-GFP cells, followed by PMA/ionomycin stimulation. Fluorescence levels were quantitated by FACS. The bars represent the mean fluorescence intensity (MFI) of gated cells that excluded dead cells (propidium iodide-positive). The FACS profiles are shown on the right. Each bar represents the pooled average of triplicate cultures. G, glucosamine does not significantly alter ATP levels in Jurkat T-cells in culture. Glucosamine-treated Jurkat T-cells were stimulated with PMA and ionomycin; and 18 h later, the cleared lysates were assayed for ATP levels by an indirect luminometric assay. The bars represent the mean of the relative light intensity (in relative light units) measured in a microplate luminometer (triplicate determinations). The error bars indicate the S.E.

To control for potential nonspecific effects of glucosamine, we examined the effects of other aminated sugars (galactosamine and mannosamine) on IL-2 production by Jurkat T-cells in response to PMA and ionomycin. Fig. 1C demonstrates that galactosamine was unable to prevent IL-2 production in response to a PMA/ionomycin challenge. Interestingly, mannosamine was able to significantly prevent IL-2 production, but not at levels achievable by glucosamine (100 ± 20 ng/ml IL-2 in Jurkat cell supernatants versus 50 ± 36 ng/ml in mannosamine- and glucosamine-treated cells following PMA/ionomycin stimulation). To rule out the possibility that glucosamine exerted a nonspecific toxic effect on Jurkat T-cells, we examined cell viability based on MTS assessment using a commercially available reagent (CellTiter96 Aqueous, Promega). This assay determines metabolically viable cells, which convert the MTS substrate into an aqueous soluble formazan by dehydrogenases found in metabolically active cells. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture. Fig. 1D shows that no significant differences were observed in the viability of untreated and glucosamine-treated wild-type and JLZB cells. The observed decrease in viability of PMA/ionomycin-treated cells could be attributable to activation-induced cell death, very likely mediated through Fas/Fas ligand interactions, because one mechanism by which the response of activated T-cells is terminated is by fratricidal Fas/Fas ligand-induced apoptosis.

To rule out the possibility that glucosamine induces irreversible changes in the response of JLZB cells to PMA/ionomycin, we first cultured JLZB cells overnight in the presence of 10 mM glucosamine. The cells were then washed twice with PBS, and fresh medium was added. PMA and ionomycin were added 24 h thereafter. Fig. 1E shows that these JLZB cells responded appropriately to PMA/ionomycin, demonstrating that the effects of glucosamine are reversible. Finally, to show that glucosamine-induced suppression of NFAT promoter element-driven lacZ reporter expression is specific for NFAT-dependent transactivation and not a nonspecific effect on general promoter activity, we tested the effects of glucosamine on JSR-GFP cells, stably transfected Jurkat cells with the constitutive SRalpha promoter driving expression of a GFP cassette. Fig. 1F presents data from FACS analysis of untreated and glucosamine-treated cells in the form of mean fluorescence intensity and shows GFP expression in JSR-GFP cells treated with glucosamine following PMA/ionomycin stimulation. The fluorescence profiles of glucosamine-free cell cultures and glucosamine-treated cultures following PMA/ionomycin stimulation were identical, as illustrated in Fig. 1F. It is worth noting that, in prior experiments whose data are illustrated above (Fig. 1A), beta -galactosidase gene expression under the control of NFAT elements was suppressed by glucosamine, in contrast with what was observed in the same cell background in which a reporter gene was driven by a "constitutive" promoter (SRalpha ). To determine whether glucosamine affected the levels of ATP, we measured luciferin conversion into light in Jurkat T-cells cultured in 10 mM glucosamine overnight with or without PMA/ionomycin activation. Fig. 1G demonstrates that ATP levels in the supernatants of cells treated with glucosamine (10 mM final concentration) with or without PMA/ionomycin stimulation were no different from those in cells that were not exposed to the sugar. Finally, another indication of normal metabolic activity of glucosamine-treated cells was that constitutive expression of GFP in JSR-GFP cells and inducible lacZ expression in JLZB cells were easily inhibited by cycloheximide, but not by 10 mM glucosamine (data not shown).

Dendritic cells are the most potent immunostimulatory cells yet characterized. They are at the center of a complex immune cell network and define, if not dictate, the nature of the immune response and its strength. Dendritic cells can either traffic through tissues or exist within tissues as resident cells. Upon local disruption of tissue integrity by foreign pathogen invasion or by endogenous changes, dendritic cells acquire molecules by a variety of uptake pathways and migrate to the local lymphoid organs, where they engage naive T-cells via class I and II major histocompatibility complex/T-cell receptor interactions and activate the proliferation of the engaged T-cells (61, 62). In culture, dendritic cell activation can be ascertained by the levels of LPS-induced nitrite production, reflecting the activation of the inducible nitric-oxide synthase gene through the NF-kappa B pathway (63, 64). Fig. 2 shows the nitrite output of dendritic cells treated with LPS in the presence or absence of 10 mM glucosamine in an 18-h incubation. Nitrite levels were significantly reduced in dendritic cells treated with LPS in the presence of glucosamine.


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 2.   Glucosamine suppression of dendritic cell activation. The addition of LPS for 18-24 h to bone marrow-derived dendritic cell (Dc) cultures treated with glucosamine (Gn) did not result in nitrite production at the levels detected in cultures from LPS-treated dendritic cells not treated with glucosamine.

Another indicator of dendritic cell function is the MLR, where T-cell responder proliferation is measured as an index of the number of stimulator antigen-presenting cells. We examined the effect of glucosamine addition in an allogeneic MLR. Fig. 3 demonstrates a dose-dependent glucosamine suppression of allogeneic MLR. The addition of 10 mM glucosamine at the outset of the co-culture completely prevented the allogeneic MLR, whereas lower doses achieved partial, yet significant suppression.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3.   Glucosamine suppression of allogeneic MLR. Co-culture of irradiated bone marrow-derived dendritic cells with purified allogeneic T-cells in the presence of glucosamine resulted in a dose-dependent suppression of T-cell proliferation. The stimulator cell number remained constant, whereas the responder cell number varied as shown on the x axis. The results shown are representative of three independent experiments. C3H, C3H/HeJ; B10, C57/BL10.

Effects of Glucosamine on Cardiac Allograft Survival-- Finally, we examined whether glucosamine treatment can facilitate allograft survival. Table I illustrates the median survival time of allogeneic cardiac allografts in mice treated intravenously with 40 mmol of glucosamine 1 day prior to transplantation and one daily intravenous injection of 40 mmol of glucosamine beginning on the 2nd day following transplantation until the end of the experiment. Table I compares the efficacy of daily glucosamine injections and a 7-day treatment with conventionally used immunosuppressive agents (FK-506 and cyclosporin A). Allograft survival was significantly increased in the glucosamine-treated mice compared with untreated controls (median survival time = 18.7 versus 10.0 days; p < 0.05). Glucosamine also appeared to be efficacious compared with standard pharmacological agents in current clinical use.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Median survival time of cardiac allografts in mice treated with glucosamine
The survival of allografts in mice treated with glucosamine, cyclosporin A, or FK-506, is compared. Data are shown as median survival times (MST). The differences are statistically significant as assessed by log-rank analysis (p < 0.05) and by two-way analysis of variance. p < 0.0001 (2-way analysis of variance), Glucosamine treatment compared with no treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have shown that glucosamine is an efficacious agent that can suppress the activation of T-cells and dendritic cells, two crucial cells involved in immune responses. The suppressive effect was specific and reversible and could not be attributed to cell death. Furthermore, the effect was most potently achieved by glucosamine in comparison with other amine sugars. Galactosamine had no effect; and mannosamine, although able to achieve a significant effect, was not able to achieve suppression levels comparable to those of glucosamine. Other sugars tested (glucose, mannose, fructose, fructosamine, and lactosamine) were unable to prevent PMA/ionomycin-induced activation of Jurkat or JLZB cells as assessed by beta -galactosidase activity in JLZB lysates or IL-2 production (data not shown). Finally, the lack of an effect of glucosamine on a constitutive promoter (SRalpha ) compared with NFAT reporter elements demonstrates that glucosamine acts by interfering with specific, although as yet unidentified, molecular signaling pathways. Although a number of studies in tumor cell culture and adipocytes have demonstrated that glucosamine can affect the ATP and uridine pools (14, 15, 17-20, 22, 65), our data demonstrate that, in Jurkat T-cells, ATP levels were not affected at the maximal concentration of glucosamine used (10 mM). We were unable, however, to reliably measure uridine levels or uridine glucosamine metabolites even by high pressure liquid chromatography analysis (data not shown). Although we do not believe that depletion of uridine pools accounts for the glucosamine effects we observed, a more formal examination in the context of mechanistic studies of the effects of glucosamine is warranted. Concurrently, we do not believe that the responses we observed in the MLR were due to impotent counter-receptor interactions (i.e. major histocompatibility complex/T-cell receptor, B7/CD28) consequent to glucosamine-modulated underglycosylation of immunostimulatory molecules at the surface of the dendritic and T-cells in co-culture, as shown in a previous study (66).

We suggest that glucosamine contributes to or is itself a direct substrate of glycosyltransferases involved in post-translational modifications of signal transduction pathways, which in T-cells involve the NFAT family of transcription factors. Post-translational modification of proteins, especially transcription factors, by O-linked glycosylation is plausible as a means of regulating cell growth and function. A number of studies have demonstrated that O-linked glycosylation is an important post-translational modification of proteins and may affect protein function to the same degree as protein phosphorylation (reviewed in Refs. 67-69). Proteins as diverse as nucleoporins, RNA polymerase II, SP1, estrogen receptor, eukaryotic initiation factor-2, cytoskeletal proteins, p53, and nitric-oxide synthase are O-glycosylated, and their function is altered depending on state of glycosylation (reviewed in Refs. 67-70). Our data demonstrate that NFAT-dependent reporter gene expression is affected by glucosamine and raise the possibility that NFAT transcription factors and/or other proteins that culminate in NFAT translocation into the nucleus may be targets of O-glycosylation, which negatively regulates their activity.

Another potential mechanism of glucosamine actions could affect the oxidation state of cells. Kaneto et al. (71) demonstrated a glucosamine-dependent enhancement of hydrogen peroxide levels in pancreatic beta -cells and concluded that activation of the hexosamine pathway leads to deterioration of beta -cell function through the induction of oxidative stress rather than O-linked glycosylation. They also observed a significant decrease in the DNA-binding activity of PDX-1, an important beta -cell transcription factor. However, our data indicate that it is highly unlikely that oxidative stress accounts for the suppression of the T-cell and dendritic cell activation we observed. Nonetheless, mechanisms suggested by the data of Kaneto et al. are currently under investigation.

Our results also indicate that glucosamine is an effective agent in prolonging allogeneic cardiac allograft survival in mice. One could potentially extrapolate from these data to partly explain the reported beneficial effects of glucosamine in arthritis. By suppressing immune activity, synovial tissue could regenerate unimpeded. The absence of soluble mediators of inflammation and pain generated by immune cells at the site of cartilage erosion could provide the patient with an increased sense of well being that may or may not result in any remarkable long-term therapeutic effect. In over-the-counter formulations, glucosamine dosage in capsule form does not usually surpass 1.5 g/day. Therefore, glucosamine ingested in capsule form may not result in critical tissue levels required to achieve optimal therapeutic effect. This may be due to very quick clearance following oral ingestion, short half-life, and/or suboptimal dose. Carefully controlled studies evaluating clearance, half-life, and maximal safe dosage to achieve measurable anti-inflammatory and immunosuppressive effects will very likely yield answers to the potential to translate our findings to humans.

Glucosamine has been demonstrated, however, to induce insulin resistance in a variety of models, including humans (36-39, 72-75); and therefore, any anti-inflammatory or immunosuppressive benefit will have to be carefully considered with this in mind. It is noteworthy, however, that continuous infusion of glucosamine is necessary to achieve a state of insulin resistance in humans (39). It is possible that transient glucosamine exposure could achieve immunosuppression without inducing or promoting insulin resistance. This possibility requires careful examination.

One other point that our data raise is the possibility that users of glucosamine may be at risk for subtle compromises of immune responses. Whether this is a real concern remains to be determined, but the widespread availability of this compound warrants carefully controlled studies to assess any potential risk that long-term use may confer on immune activity in humans.

Finally, if our data can be clinically translated, it may be possible to use glucosamine formulations alone or in conjunction with conventional immunosuppressive agents at doses much lower than in current clinical use to achieve long-term transplant survival with a significantly decreased risk of the toxicity that is often associated with conventional pharmacological agents. Mechanistically, the glucosamine mode of action in immune cells remains to be determined and is under active investigation in our laboratory.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pathology, Diabetes Inst., University of Pittsburgh School of Medicine, Rangos Research Center 5102, 3460 Fifth Ave., Pittsburgh, PA 15213. Tel.: 412-692-8127; Fax: 412-692-8131; E-mail: Ngiann1@pitt.edu.

Published, JBC Papers in Press, August 9, 2002, DOI 10.1074/jbc.M204924200

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; NFAT, nuclear factor of activated T-cells; PMA, phorbol 12-myristate 13-acetate; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; PBS, phosphate-buffered saline; LPS, lipopolysaccharide; MLR, mixed leukocyte reaction/reactivity; MTS, 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Rossetti, L. (2000) Endocrinology 141, 1922-1925[Free Full Text]
2. Marshall, S., Bacote, V., and Traxinger, R. R. (1991) J. Biol. Chem. 266, 4706-4712[Abstract/Free Full Text]
3. Hart, G. W., Greis, K. D., Dong, L. Y., Blomberg, M. A., Chou, T. Y., Jiang, M. S., Roquemore, E. P., Snow, D. M., Kreppel, L. K., Cole, R. N., Comer, F. I., Arnold, C. S., and Hayes, B. K. (1995) Adv. Exp. Med. Biol. 376, 115-123[Medline] [Order article via Infotrieve]
4. Uldry, M., Ibberson, M., Hosokawa, M., and Thorens, B. (2002) FEBS Lett. 524, 199-203[CrossRef][Medline] [Order article via Infotrieve]
5. Rencurel, F., Waeber, G., Antoine, B., Rocchiccioli, F., Maulard, P., Girard, J., and Leturque, A. (1996) Biochem. J. 314, 903-909
6. Quastel, J. H., and Cantero, A. (1953) Nature 171, 252-254[CrossRef][Medline] [Order article via Infotrieve]
7. Bekesi, J. G., and Winzler, R. J. (1970) Cancer Res. 30, 2905-2912[Abstract/Free Full Text]
8. Molnar, Z., and Bekesi, J. G. (1972) Cancer Res. 32, 756-765[Abstract/Free Full Text]
9. Molnar, Z., and Bekesi, J. G. (1972) Cancer Res. 32, 380-389[Abstract/Free Full Text]
10. Bosmann, H. B. (1971) Biochim. Biophys. Acta 240, 74-93[Medline] [Order article via Infotrieve]
11. Bosmann, H. B. (1972) Biochem. Pharmacol. 21, 1977-1988[CrossRef][Medline] [Order article via Infotrieve]
12. Fare, G., Sammons, D. C., Seabourne, F. A., and Woodhouse, D. L. (1967) Nature 213, 308-309[CrossRef][Medline] [Order article via Infotrieve]
13. Fjelde, A., Sorkin, A., and Rhodes, J. M. (1956) Exp. Cell Res. 10, 88-98[CrossRef][Medline] [Order article via Infotrieve]
14. Friedman, S. J., Trotter, C. D., Kimball, T., and Skehan, P. J. (1977) Cancer Res. 37, 1141-1146[Abstract/Free Full Text]
15. Friedman, S. J., Kimball, T., Trotter, C. D., and Skehan, P. J. (1977) Cancer Res. 37, 1068-1074[Abstract/Free Full Text]
16. Rubin, A., Springer, G. F., and Hogue, M. J. (1954) Cancer Res. 14, 456-458[Free Full Text]
17. Plagemann, P. G. (1971) J. Cell. Physiol. 77, 241-248[CrossRef][Medline] [Order article via Infotrieve]
18. Plagemann, P. G. (1971) J. Cell. Physiol. 77, 213-240[CrossRef][Medline] [Order article via Infotrieve]
19. Plagemann, P. G., and Erbe, J. (1973) Cancer Res. 33, 482-492[Abstract/Free Full Text]
20. Scholtissek, C. (1972) Biochim. Biophys. Acta 277, 459-465[Medline] [Order article via Infotrieve]
21. Bertram, J., Ragatz, B. H., Baldwin, W., and Iatrides, P. G. (1981) Thromb. Res. 23, 301-307[CrossRef][Medline] [Order article via Infotrieve]
22. Friedman, S. J., and Skehan, P. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1172-1176[Abstract/Free Full Text]
23. O'Neill, H. C., and Parish, C. R. (1988) Immunology 64, 181-184[Medline] [Order article via Infotrieve]
24. Yagita, M., Seppo, A., Renkonen, O., and Saksela, E. (1993) Cancer Res. 53, 5600-5604[Abstract/Free Full Text]
25. Yagita, M., Nakajima, M., and Saksela, E. (1989) Cell. Immunol. 122, 83-95[CrossRef][Medline] [Order article via Infotrieve]
26. Chang, D. M. (1995) Immunopharmacol. Immunotoxicol. 17, 437-450[Medline] [Order article via Infotrieve]
27. Gouze, J. N., Bianchi, A., Becuwe, P., Dauca, M., Netter, P., Magdalou, J., Terlain, B., and Bordji, K. (2002) FEBS Lett. 510, 166-170[CrossRef][Medline] [Order article via Infotrieve]
28. Reginster, J. Y., Deroisy, R., Rovati, L. C., Lee, R. L., Lejeune, E., Bruyere, O., Giacovelli, G., Henrotin, Y., Dacre, J. E., and Gossett, C. (2001) Lancet 357, 251-256[CrossRef][Medline] [Order article via Infotrieve]
29. McAlindon, T. E., LaValley, M. P., Gulin, J. P., and Felson, D. T. (2000) J. Am. Med. Assoc. 283, 1469-1475[Abstract/Free Full Text]
30. Rovati, L. C., Annefeld, M., Giacovelli, G., Schmid, K., and Setnikar, I. (1999) Lancet 354, 1640[Medline] [Order article via Infotrieve]; Discussion 1641-1642
31. Lippiello, L., Woodward, J., Karpman, R., and Hammad, T. A. (2000) Clin. Orthop. Relat. Res. 381, 229-240
32. Shikhman, A. R., Kuhn, K., Alaaeddine, N., and Lotz, M. (2001) J. Immunol. 166, 5155-5160[Abstract/Free Full Text]
33. Robinson, K. A., Sens, D. A., and Buse, M. G. (1993) Diabetes 42, 1333-1346[Abstract]
34. Traxinger, R. R., and Marshall, S. (1992) J. Biol. Chem. 267, 9718-9723[Abstract/Free Full Text]
35. Daniels, M. C., Kansal, P., Smith, T. M., Paterson, A. J., Kudlow, J. E., and McClain, D. A. (1993) Mol. Endocrinol. 7, 1041-1048[Abstract]
36. Chen, H., Ing, B. L., Robinson, K. A., Feagin, A. C., Buse, M. G., and Quon, M. J. (1997) Mol. Cell. Endocrinol. 135, 67-77[CrossRef][Medline] [Order article via Infotrieve]
37. Rossetti, L., Hawkins, M., Chen, W., Gindi, J., and Barzilai, N. (1995) J. Clin. Invest. 96, 132-140[Medline] [Order article via Infotrieve]
38. Virkamaki, A., Daniels, M. C., Hamalainen, S., Utriainen, T., McClain, D., and Yki-Jarvinen, H. (1997) Endocrinology 138, 2501-2507[Abstract/Free Full Text]
39. Monauni, T., Zenti, M. G., Cretti, A., Daniels, M. C., Targher, G., Caruso, B., Caputo, M., McClain, D., Del Prato, S., Giaccari, A., Muggeo, M., Bonora, E., and Bonadonna, R. C. (2000) Diabetes 49, 926-935[Abstract]
40. Cassel, D. L., Hoxie, J. A., and Cooper, R. A. (1983) Cancer Res. 43, 4582-4586[Abstract/Free Full Text]
41. Nordmann, R., Andersen, E., Trussardi, R., and Mazer, N. A. (1989) Biochemistry 28, 1791-1797[CrossRef][Medline] [Order article via Infotrieve]
42. Aussel, C., Pelassy, C., Mary, D., Choquet, D., and Rossi, B. (1990) Immunopharmacology 20, 97-103[CrossRef][Medline] [Order article via Infotrieve]
43. Ng, J., Gustavsson, J., Jondal, M., and Andersson, T. (1990) Biochim. Biophys. Acta 1053, 97-105[Medline] [Order article via Infotrieve]
44. Jin, Y. J., Kaplan, D. R., White, M., Spagnoli, G. C., Roberts, T. M., and Reinherz, E. L. (1990) J. Immunol. 144, 647-652[Abstract]
45. Papadogiannakis, N., Nordstrom, T. E., Andersson, L. C., and Wolff, C. H. (1989) Eur. J. Immunol. 19, 1953-1956[Medline] [Order article via Infotrieve]
46. Nagata, K., Ohtani, K., Nakamura, M., and Sugamura, K. (1989) J. Virol. 63, 3220-3226[Abstract/Free Full Text]
47. Koretzky, G. A., Kohmetscher, M. A., Kadleck, T., and Weiss, A. (1992) J. Immunol. 149, 1138-1142[Abstract]
48. Williams, T. M., Moolten, D. M., Makni, H., Kim, H. W., Kant, J. A., and Kamoun, M. (1992) J. Immunol. 148, 2609-2616[Abstract]
49. Soula, M., Fagard, R., and Fischer, S. (1992) Int. Immunol. 4, 295-299[Abstract/Free Full Text]
50. Levine, B. L., May, W. S., Tyler, P. G., and Hess, A. D. (1991) J. Immunol. 147, 3474-3481[Abstract]
51. Ley, S. C., Davies, A. A., Druker, B., and Crumpton, M. J. (1991) Eur. J. Immunol. 21, 2203-2209[Medline] [Order article via Infotrieve]
52. Feuillard, J., Gouy, H., Bismuth, G., Lee, L. M., Debre, P., and Korner, M. (1991) Cytokine 3, 257-265[CrossRef][Medline] [Order article via Infotrieve]
53. Cavallo, R., Jemma, C., and Merlino, C. (1991) G. Batteriol. Virol. Immunol. 84, 11-14
54. Li, C., Lai, C. F., Sigman, D. S., and Gaynor, R. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7739-7743[Abstract/Free Full Text]
55. Verweij, C. L., Guidos, C., and Crabtree, G. R. (1990) J. Biol. Chem. 265, 15788-15795[Abstract/Free Full Text]
56. Yaseen, N. R., Park, J., Kerppola, T., Curran, T., and Sharma, S. (1994) Mol. Cell. Biol. 14, 6886-6895[Abstract/Free Full Text]
57. Boise, L. H., Petryniak, B., Mao, X., June, C. H., Wang, C. Y., Lindsten, T., Bravo, R., Kovary, K., Leiden, J. M., and Thompson, C. B. (1993) Mol. Cell. Biol. 13, 1911-1919[Abstract/Free Full Text]
58. Rooney, J. W., Sun, Y. L., Glimcher, L. H., and Hoey, T. (1995) Mol. Cell. Biol. 15, 6299-6310[Abstract]
59. Fu, F., Li, Y., Qian, S., Lu, L., Chambers, F., Starzl, T. E., Fung, J. J., and Thomson, A. W. (1996) Transplantation 62, 659-665[CrossRef][Medline] [Order article via Infotrieve]
60. Lu, L., McCaslin, D., Starzl, T. E., and Thomson, A. W. (1995) Transplantation 60, 1539-1545[Medline] [Order article via Infotrieve]
61. Lanzavecchia, A., and Sallusto, F. (2001) Cell 106, 263-266[CrossRef][Medline] [Order article via Infotrieve]
62. Rescigno, M., and Borrow, P. (2001) Cell 106, 267-270[CrossRef][Medline] [Order article via Infotrieve]
63. Lu, L., Bonham, C. A., Chambers, F. G., Watkins, S. C., Hoffman, R. A., Simmons, R. L., and Thomson, A. W. (1996) J. Immunol. 157, 3577-3586[Abstract]
64. Hirschowitz, E. A., Weaver, J. D., Hidalgo, G. E., and Doherty, D. E. (2000) Gene Ther. 7, 1112-1120[CrossRef][Medline] [Order article via Infotrieve]
65. Hresko, R. C., Heimberg, H., Chi, M. M., and Mueckler, M. (1998) J. Biol. Chem. 273, 20658-20668[Abstract/Free Full Text]
66. Irahara, M., Kamada, M., Mori, T., and Sudo, T. (1987) Immunobiology 174, 190-199[Medline] [Order article via Infotrieve]
67. Comer, F. I., and Hart, G. W. (2001) Biochemistry 40, 7845-7852[CrossRef][Medline] [Order article via Infotrieve]
68. Comer, F. I., and Hart, G. W. (2000) J. Biol. Chem. 275, 29179-29182[Free Full Text]
69. Comer, F. I., and Hart, G. W. (1999) Biochim. Biophys. Acta 1473, 161-171[Medline] [Order article via Infotrieve]
70. Du, X. L., Edelstein, D., Rossetti, L., Fantus, I. G., Goldberg, H., Ziyadeh, F., Wu, J., and Brownlee, M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12222-12226[Abstract/Free Full Text]
71. Kaneto, H., Xu, G., Song, K. H., Suzuma, K., Bonner-Weir, S., Sharma, A., and Weir, G. C. (2001) J. Biol. Chem. 276, 31099-31104[Abstract/Free Full Text]
72. Hawkins, M., Barzilai, N., Liu, R., Hu, M., Chen, W., and Rossetti, L. (1997) J. Clin. Invest. 99, 2173-2182[Medline] [Order article via Infotrieve]
73. Patti, M. E., Virkamaki, A., Landaker, E. J., Kahn, C. R., and Yki-Jarvinen, H. (1999) Diabetes 48, 1562-1571[Abstract]
74. Ciaraldi, T. P., Carter, L., Nikoulina, S., Mudaliar, S., McClain, D. A., and Henry, R. R. (1999) Endocrinology 140, 3971-3980[Abstract/Free Full Text]
75. McClain, D. A., Alexander, T., Cooksey, R. C., and Considine, R. V. (2000) Endocrinology 141, 1999-2002[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. Xing, W. Feng, L. G. Not, A. P. Miller, Y. Zhang, Y.-F. Chen, E. Majid-Hassan, J. C. Chatham, and S. Oparil
Increased protein O-GlcNAc modification inhibits inflammatory and neointimal responses to acute endoluminal arterial injury
Am J Physiol Heart Circ Physiol, July 1, 2008; 295(1): H335 - H342.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Grigorian, S.-U. Lee, W. Tian, I.-J. Chen, G. Gao, R. Mendelsohn, J. W. Dennis, and M. Demetriou
Control of T Cell-mediated Autoimmunity by Metabolite Flux to N-Glycan Biosynthesis
J. Biol. Chem., July 6, 2007; 282(27): 20027 - 20035.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
J.-T. Chen, J.-B. Liang, C.-L. Chou, M.-W. Chien, R.-C. Shyu, P.-I Chou, and D.-W. Lu
Glucosamine Sulfate Inhibits TNF-{alpha} and IFN-{gamma}-Induced Production of ICAM-1 in Human Retinal Pigment Epithelial Cells In Vitro
Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 664 - 672.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
B A Biggee, C M Blinn, T E McAlindon, M Nuite, and J E Silbert
Low levels of human serum glucosamine after ingestion of glucosamine sulphate relative to capability for peripheral effectiveness
Ann Rheum Dis, February 1, 2006; 65(2): 222 - 226.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G.-X. Zhang, S. Yu, B. Gran, and A. Rostami
Glucosamine Abrogates the Acute Phase of Experimental Autoimmune Encephalomyelitis by Induction of Th2 Response
J. Immunol., December 1, 2005; 175(11): 7202 - 7208.
[Abstract] [Full Text] [PDF]