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1.
Artigo em Inglês | MEDLINE | ID: mdl-36399889

RESUMO

Bimatoprost is a synthetic prostamide F2α analog that down-regulates adipogenesis in vitro. This effect has been attributed to participation in a negative feedback loop that regulates anandamide-induced adipogenesis. A follow-on investigation has now been conducted into the broader metabolic effects of bimatoprost using rats under both normal state and obesity-inducing conditions. Chronic bimatoprost administration attenuated weight gain in a dose dependent-manner in rats fed either standard [max effect -7%] or obesity-promoting diets [max effect -23%] over a 9-10 week period. Consistent with these findings, bimatoprost promoted satiety as measured by decreased food intake [max effect, -7%], gastric emptying [max effect, -33-50%] and decreased circulating concentrations of the gut hormones, ghrelin and GLP-1 [max effect, -33-50%]. Additionally, subcutaneous, and visceral fat mass were distinctly affected by treatment [-30% diet independent]. Taken together, these results suggest that bimatoprost regulates energy homeostasis through promoting satiety and a decrease in food intake. These newly reported activities of bimatoprost reveal an additional method of metabolic disease intervention for potential therapeutic exploitation.


Assuntos
Obesidade , Aumento de Peso , Ratos , Animais , Bimatoprost , Obesidade/tratamento farmacológico , Dieta , Adipogenia
2.
ACS Pharmacol Transl Sci ; 3(6): 1199-1210, 2020 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-33344897

RESUMO

It has previously been reported that a prototypical compound (AGN 211377), which blocks pro-inflammatory prostanoid receptors (DP1, DP2, EP1, EP4, FP, TP) and leaves open IP and EP2 receptors so that their anti-inflammatory properties could be exerted, produced superior inhibitory effects on cytokine release from human macrophages compared to cyclooxygenase (COX) inhibitors. This favorable activity profile translated into animal studies, with AGN 211377 exceeding the level of inhibition afforded by COX inhibition. AGN 211377 was not, however, a practical drug candidate, having poor bioavailability and cost of goods concerns. Compound 1 (designated AGN 225660) represents a second-generation compound with an entirely different "druggable" core structure. Such a dramatic change in chemical scaffold created uncertainty with respect to matching the effects of AGN 211377. AGN 225660 inhibited RANTES, IL-8, and MCP-1 secretion by at least 50%, from TNFα activated human macrophages. Although AGN 225660 reduced TNFα-evoked MCP-1 release from human monocyte-derived macrophages, it increased LPS-induced MCP-1 secretion (up to 2-fold) from human monocyte-derived dendritic cells. However, AGN 225660 inhibited the release of IL12p 70 and IL-23 from human monocyte-derived dendritic cells stimulated by LPS by more than 70%. This effect of AGN 225660 was reproduced in part by the prototype compound AGN 211377 and a combination of selective DP1, EP1, EP4, FP, and TP antagonists. These findings suggest important effects on T cell skewing and disease modification by this class of therapeutic agents. AGN 225660 exhibited good ocular bioavailability and was active in reducing ocular inflammation associated with phacoemulsification surgery, LPS, and arachidonic acid induced uveitis.

3.
Expert Opin Ther Targets ; 23(9): 755-771, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31456448

RESUMO

Introduction: In the past 30 years, only two drugs have received FDA approval for the treatment of androgenetic alopecia reflecting a lack of success in unraveling novel targets for pharmacological intervention. However, as our knowledge of hair biology improves, new signaling pathways and organogenesis processes are being uncovered which have the potential to yield more effective therapeutic modalities. Areas covered: This review focuses on potential targets for drug development to treat hair loss. The physiological processes underlying the promise of regenerative medicine to recreate new functional hair follicles in bald scalp are also examined. Expert opinion: The discovery of promising new targets may soon enable treatment options that modulate the hair cycle to preserve or extend the growth phase of the hair follicle. These new targets could also be leveraged to stimulate progenitor cells and morphogenic pathways to reactivate miniaturized follicles in bald scalp or to harness the potential of wound healing and embryogenic development as an emerging paradigm to generate new hair follicles in barren skin.


Assuntos
Alopecia/tratamento farmacológico , Desenvolvimento de Medicamentos , Folículo Piloso/efeitos dos fármacos , Alopecia/patologia , Animais , Aprovação de Drogas , Cabelo/crescimento & desenvolvimento , Humanos , Terapia de Alvo Molecular , Medicina Regenerativa , Transdução de Sinais
4.
J Ocul Pharmacol Ther ; 35(2): 98-105, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30547709

RESUMO

PURPOSE: The purpose of these studies was (1) to investigate the ability of human M1 phenotype macrophages to secrete vascular endothelial growth factor (VEGF) and the influence of prostacyclin receptor (IP) stimulation (2) to evaluate the contribution of the proangiogenic prostanoid prostacyclin to experimental choroidal neovascularization Methods: Human macrophages derived from primary blood mononuclear cells were functionally biased toward the M1 phenotype by using tumor necrosis factor α (TNFα). Experimental choroidal neovascularization was produced by laser photocoagulation. Antagonist drugs RO-3244794 (IP antagonist) and GW 627368 (EP4 antagonist) were administered according to an optimal dosing regimen that was predetermined by bioavailability studies. RESULTS: IP receptor stimulation had diametrically opposed effects on VEGF release compared with reported data on cytokine/chemokine secretion from human macrophages. For example, the IP agonist cicaprost stimulated VEGF secretion although it inhibits monocyte chemoattractant protein-1 (MCP-1) secretion: both would favor a proangiogenic effect. The IP receptor antagonist RO-3244794 produced an ∼20% statistically significant reduction in the neovascularized lesion area in the choroidal neovascularization model, which was a similar level to that produced by the EP4 antagonist GW 627368. Combining the 2 drugs produced a statistically significant reduction in neovascularization but only of slightly greater magnitude than that obtained with each antagonist administered alone. CONCLUSIONS: IP receptor stimulation potently and highly efficaciously promoted VEGF release from human M1 macrophages, indicating a possible contribution of the M1 macrophage subtype to VEGF-induced choroidal neovascularization. Studies in living animals suggest that prostacyclin and its target IP receptor contribute to choroidal neovascularization, although to a more modest extent than might have been expected.


Assuntos
Anti-Hipertensivos/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Epoprostenol/farmacologia , Macrófagos/efeitos dos fármacos , Soluções Oftálmicas/farmacologia , Animais , Células Cultivadas , Quimiocina CCL2/análise , Quimiocina CCL2/deficiência , Quimiocina CCL2/metabolismo , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Fatores de Crescimento do Endotélio Vascular/análise , Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
J Ocul Pharmacol Ther ; 33(3): 186-192, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28072560

RESUMO

PURPOSE: To investigate the therapeutic potential of a prostacyclin (IP) receptor agonist for ocular inflammation and the effect on immune cells. METHODS: The anti-inflammatory activities of cicaprost were determined in primary human monocyte-derived macrophages and human monocyte-derived dendritic cells (MoDC), as well as a lipopolysaccharides (LPS)-induced rat uveitis model. Multiple cytokine release was measured by utilizing Luminex Technology. Prostacyclin (IP) Receptor expression was detected by reverse transcription-polymerase chain receptor. Leukocyte infiltration and protein exudation in the rat uveitis model were measured using a hemocytometer and protein concentration by a NanoDrop instrument. RESULTS: Cicapost, an IP receptor agonist, potently inhibits proinflammatory chemokines/cytokine production not only from LPS- or TNFα (tumor necrosis factor-alpha)-induced primary human monocyte-derived macrophages, but also from LPS-stimulated MoDC. While constitutively expressed in macrophages, the IP receptor was inducible by LPS stimulation in MoDCs. In a LPS-induced rat uveitis model, cicaprost efficaciously prevents ocular inflammatory cell and protein leakage, as well as inflammatory cytokine release. CONCLUSION: The IP receptor agonist cicaprost is a potent anti-inflammatory agent, implicating that the tightly controlled PGI2/IP signaling pathway is important in regulating inflammation. This response could be harnessed in ocular inflammatory disease where steroids are currently the standard of care.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Células Dendríticas/efeitos dos fármacos , Modelos Animais de Doenças , Epoprostenol/análogos & derivados , Macrófagos/efeitos dos fármacos , Uveíte/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Citocinas/antagonistas & inibidores , Citocinas/biossíntese , Células Dendríticas/metabolismo , Epoprostenol/administração & dosagem , Epoprostenol/farmacologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos/metabolismo , Masculino , Ratos Endogâmicos Lew , Receptores de Epoprostenol/agonistas , Receptores de Epoprostenol/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Uveíte/induzido quimicamente
6.
FASEB J ; 31(1): 368-375, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27770021

RESUMO

The purpose of these studies was to test the hypothesis that a selected polypharmacological approach for treating the prostanoid-mediated component of inflammatory diseases would produce a therapeutic effect superior to global inhibition of prostaglandin (PG) biosynthesis by aspirin-like drugs. The compound studied was AGN 211377, which had been previously shown to produce a superior effect on cytokine release from human macrophages compared with cyclooxygenase (COX) inhibitors. AGN 211377 antagonizes prostanoid prostaglandin D2 (DP)1, DP2, prostaglandin E2 (EP)1, EP4, prostaglandin F2α, and thromboxane A2 receptors but not anti-inflammatory EP2, prostaglandin I2, or EP3 receptors. Established rodent models of ocular inflammatory diseases were used to determine therapeutic effects in living animals. The drugs were administered systemically after predetermination of their blood levels to ensure bioavailability at an appropriate dose level. Whereas compounds selective for a single prostanoid receptor typically exhibited modest but statistically significant inhibition, AGN 211377 profoundly inhibited S-antigen-induced uveitis and laser-induced retinal neovascularization. Consistent with previous polypharmacological studies on chemokine/cytokine release from human macrophages, the prostanoid EP1 receptor played a permissive role in suppressing neovascularization and inflammation in vivo Comparing AGN 211377 with a close structural congener lacking EP1 antagonism (AGN 197727), AGN 197727 was much less active than AGN 211377, but pronounced anti-inflammatory and angiostatic effects were achieved by adding the EP1 antagonist compound (SC-51322) to AGN 197727 in the systemic dosing regimen. Further, AGN 211377 produced superior anti-inflammatory activity compared with the nonsteroidal anti-inflammatory agent ketorolac. These results indicate the value of using a polypharmacological approach in the design of novel therapeutic agents in preference to compounds targeting a single receptor or enzyme. A compound such as AGN 211377 may represent more effective therapy than COX inhibitors in treating uveitis and ocular diseases where neovascularization is a significant part of the pathology.-Woodward, D. F., Wang, J. W., Ni, M., Bauer, A., Martos, J. L., Carling, R. W., Poloso, N. J. In vivo studies validating multitargeting of prostanoid receptors for achieving superior anti-inflammatory effects.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cinamatos/farmacologia , Neovascularização Patológica/prevenção & controle , Receptores de Prostaglandina/antagonistas & inibidores , Animais , Doenças Autoimunes , Sinalização do Cálcio , Lasers/efeitos adversos , Ratos , Ratos Endogâmicos Lew , Retina/patologia , Retina/efeitos da radiação , Vasos Retinianos/patologia , Vasos Retinianos/efeitos da radiação , Uveíte/tratamento farmacológico , Uveíte/etiologia
7.
PLoS One ; 11(11): e0166104, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27820856

RESUMO

Functional analysis of a clinical microbiome facilitates the elucidation of mechanisms by which microbiome perturbation can cause a phenotypic change in the patient. The direct approach for the analysis of the functional capacity of the microbiome is via shotgun metagenomics. An inexpensive method to estimate the functional capacity of a microbial community is through collecting 16S rRNA gene profiles then indirectly inferring the abundance of functional genes. This inference approach has been implemented in the PICRUSt and Tax4Fun software tools. However, those tools have important limitations since they rely on outdated functional databases and uncertain phylogenetic trees and require very specific data pre-processing protocols. Here we introduce Piphillin, a straightforward algorithm independent of any proposed phylogenetic tree, leveraging contemporary functional databases and not obliged to any singular data pre-processing protocol. When all three inference tools were evaluated against actual shotgun metagenomics, Piphillin was superior in predicting gene composition in human clinical samples compared to both PICRUSt and Tax4Fun (p<0.01 and p<0.001, respectively) and Piphillin's ability to predict disease associations with specific gene orthologs exhibited a 15% increase in balanced accuracy compared to PICRUSt. From laboratory animal samples, no performance advantage was observed for any one of the tools over the others and for environmental samples all produced unsatisfactory predictions. Our results demonstrate that functional inference using the direct method implemented in Piphillin is preferable for clinical biospecimens. Piphillin is publicly available for academic use at http://secondgenome.com/Piphillin.


Assuntos
Metagenoma/genética , Metagenômica/métodos , Microbiota/genética , Algoritmos , Bases de Dados Factuais , Humanos , Filogenia , RNA Ribossômico 16S , Software
8.
J Pharmacol Exp Ther ; 358(2): 173-80, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27217589

RESUMO

Prostaglandin E2 (PGE2)-2-glyceryl ester is a cyclo-oxygenase 2 product of the endocannabinoid 2-arachidonyl glycerol. It is claimed as pharmacologically novel, but this is complicated by rapid and irreversible isomerization to the 1(3) ester. For ocular studies, enzymatic hydrolysis of the ester moiety creates an additional complication. PG-glyceryl esters were stabilized to isomerization and hydrolysis by replacing the noncarbonyl O with NH, to form the serinolamide and propanediolamide as stable analogs of PG-2-glyceryl and PG-2-1(3) glyceryl esters, respectively. Intraocular pressure was measured in conscious dogs and conscious laser-induced ocular hypertensive monkeys. Pharmacological studies involved stable transfectants for each of the human recombinant prostanoid receptors and the isolated feline iris for prostamide activity. PGE2-serinolamide and PGE2- propanediolamide were essentially inactive at all receptors except the EP3 receptor (EC50, ∼500 nM). This obliged elucidation of EP3 receptor involvement in the intraocular pressure response to these PGE2-glycyerl ester analogs. Since the EP3 receptor agonists sulprostone and GR 63799 did not lower monkey intraocular pressure, a role for EP3 receptors in mediating the effects of PGE2-serinolamide and PGE2-propanediolamide is not indicated. PGE2-glyceryl ester (0.01% and 0.1%) substantially lowered intraocular pressure in monkeys. PGE2-propanediolamide was more efficacious than PGE2-serinolamide in lowering intraocular pressure in monkey eyes, but both appeared equieffective in dog eyes. PGE2-serinolamide dose-dependently (0.01- 0.1%) lowered intraocular pressure in both species, but PGF2 α-serinolamide was inactive. In conclusion, stable PGE2-glyceryl ester analogs lowered intraocular pressure. These findings are consistent with the presence of a PGE2-glyceryl ester-specific recognition site in the eye.


Assuntos
Dinoprostona/análogos & derivados , Pressão Intraocular/efeitos dos fármacos , Animais , Gatos , Dinoprostona/farmacologia , Cães , Feminino , Iris/efeitos dos fármacos , Iris/fisiologia , Macaca fascicularis , Masculino , Contração Muscular/efeitos dos fármacos
9.
J Biol Chem ; 288(32): 23307-21, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23801328

RESUMO

Lipid mediators variedly affect adipocyte differentiation. Anandamide stimulates adipogenesis via CB1 receptors and peroxisome proliferator-activated receptor γ. Anandamide may be converted by PTGS2 (COX2) and prostaglandin F synthases, such as prostamide/prostaglandin F synthase, to prostaglandin F2α ethanolamide (PGF2αEA), of which bimatoprost is a potent synthetic analog. PGF2αEA/bimatoprost act via prostaglandin F2αFP receptor/FP alt4 splicing variant heterodimers. We investigated whether prostamide signaling occurs in preadipocytes and controls adipogenesis. Exposure of mouse 3T3-L1 or human preadipocytes to PGF2αEA/bimatoprost during early differentiation inhibits adipogenesis. PGF2αEA is produced from anandamide in preadipocytes and much less so in differentiating adipocytes, which express much less PTGS2, FP, and its alt4 splicing variant. Selective antagonism of PGF2αEA receptors counteracts prostamide effects on adipogenesis, as does inhibition of ERK1/2 phosphorylation. Selective inhibition of PGF2αEA versus prostaglandin F2α biosynthesis accelerates adipogenesis. PGF2αEA levels are reduced in the white adipose tissue of high fat diet-fed mice where there is a high requirement for new adipocytes. Prostamides also inhibit zebrafish larval adipogenesis in vivo. We propose that prostamide signaling in preadipocytes is a novel anandamide-derived antiadipogenic mechanism.


Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Ácidos Araquidônicos/metabolismo , Dinoprostona/análogos & derivados , Endocanabinoides/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Alcamidas Poli-Insaturadas/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Ácidos Araquidônicos/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Dinoprostona/genética , Endocanabinoides/genética , Feminino , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
10.
Mol Immunol ; 54(3-4): 284-95, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23337716

RESUMO

Dendritic cells (DCs) are central players in coordinating immune responses, both innate and adaptive. While the role of lipid mediators in the immune response has been the subject of many investigations, the precise role of prostaglandins has often been plagued by contradictory studies. In this study, we examined the role of PGE(2) on human DC function. Although studies have suggested that PGE(2) specifically plays a role in DC motility and cytokine release profile, the precise receptor usage and signaling pathways involved remain unclear. In this report we found that irrespective of the human donor, monocyte-derived dendritic cells (MoDCs) express three of the four PGE(2) receptor subtypes (EP(2-4)), although only EP(2) and EP(4) were active with respect to cytokine production. Using selective EP receptor antagonists and agonists, we demonstrate that PGE(2) coordinates control of IL-23 release (a promoter of Th17, an autoimmune associated T cell subset) in a dose-dependent manner by differential use of EP(2) and EP(4) receptors in LPS-activated MoDCs. This is in contrast to IL-12, which is dose dependently inhibited by PGE(2) through both receptor subtypes. Low concentrations (∼1-10nM) of PGE(2) promoted IL-23 production via EP(4) receptors, while at higher (>50 nM), but still physiologically relevant concentrations, IL-23 is suppressed by an EP(2) dependent mechanism. These results can be explained by differential regulation of the common subunit, IL-12p40, and IL-23p19, by EP(2) and EP(4). By these means, PGE(2) can act as a regulatory switch of immune responses depending on its concentration in the microenvironment. In addition, we believe these results may also explain why seemingly conflicting biological functions assigned to PGE(2) have been reported in the literature, as the concentration of ligand (PGE(2)) fundamentally alters the nature of the response. This finding also highlights the potential of designing therapeutics which differentially target these receptors.


Assuntos
Células Dendríticas/imunologia , Dinoprostona/imunologia , Monócitos/imunologia , Receptores de Prostaglandina E Subtipo EP2/imunologia , Receptores de Prostaglandina E Subtipo EP4/imunologia , AMP Cíclico/imunologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Subunidade p40 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/metabolismo , Subunidade p19 da Interleucina-23/imunologia , Subunidade p19 da Interleucina-23/metabolismo , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Prostaglandina E Subtipo EP2/biossíntese , Receptores de Prostaglandina E Subtipo EP2/genética , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/biossíntese , Receptores de Prostaglandina E Subtipo EP4/genética , Receptores de Prostaglandina E Subtipo EP4/metabolismo
11.
J Immunol ; 177(8): 5451-8, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17015731

RESUMO

MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.


Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Microdomínios da Membrana , Peptídeos/imunologia , Ligação Proteica , Transporte Proteico
12.
J Biochem Biophys Methods ; 64(2): 99-109, 2005 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-16000225

RESUMO

A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Glicosilfosfatidilinositóis/química , Animais , Antígenos CD/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-1/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Hibridomas/metabolismo , Immunoblotting , Substâncias Macromoleculares/química , Octoxinol/farmacologia , Receptores de IgG/biossíntese , Solubilidade , Temperatura , Transfecção
13.
J Immunol ; 173(7): 4539-46, 2004 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-15383586

RESUMO

There have been many studies demonstrating that a portion of MHC class II molecules reside in detergent-insoluble membrane domains (commonly referred to as lipid rafts). We have proposed that the function of raft association is to concentrate specific MHC class II-peptide complexes in plasma membrane microdomains that can facilitate efficient T cell activation. We now show that MHC class II becomes lipid raft associated before binding antigenic peptides. Using pulse-chase radiolabeling techniques, we find that newly synthesized MHC class II and MHC class II-invariant chain complexes initially reside in a detergent-soluble membrane fraction and acquire detergent insolubility as they traffic to lysosomal Ag processing compartments. Monensin, an inhibitor of protein transport through the Golgi apparatus, blocks association of newly synthesized MHC class II with lipid rafts. Treatment of cells with leupeptin, which inhibits invariant chain degradation, leads to the accumulation of MHC class II in lipid rafts within the lysosome-like Ag-processing compartments. Raft fractionation of lysosomal membranes confirmed the presence of MHC class II in detergent-insoluble microdomains in Ag-processing compartments. These findings indicate that newly synthesized MHC class II complexes are directed to detergent-insoluble lipid raft microdomains before peptide loading, a process that may facilitate the loading of similar peptides on MHC class II complexes in these microdomains.


Assuntos
Antígenos HLA-D/metabolismo , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Apresentação de Antígeno/imunologia , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linhagem Celular Transformada , Cetomacrogol , Detergentes , Antígenos HLA-D/química , Células HeLa , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Líquido Intracelular/química , Lisossomos/imunologia , Lisossomos/metabolismo , Substâncias Macromoleculares , Microdomínios da Membrana/química , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transporte Proteico/imunologia , Solubilidade , Tensoativos
14.
Curr Opin Immunol ; 16(1): 103-7, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14734117

RESUMO

Activation of CD4(+) T cells requires the interaction of multiple T-cell receptors with MHC class II-peptide complexes on the surface of antigen-presenting cells (APCs). Recent studies have shown that MHC class II complexes are clustered in APC plasma membrane microdomains, thereby providing a mechanism for localized concentration of MHC class II-peptide complexes. The integrity of one type of APC membrane microdomain, the lipid raft, is important for antigen presentation to T cells. We propose a model in which the coordinated processes of MHC class II peptide loading and intracellular trafficking enhance T-cell activation by loading specific MHC class II-peptide complexes in discrete lipid raft microdomains.


Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe II/fisiologia , Microdomínios da Membrana/imunologia , Peptídeos/imunologia , Animais , Antígenos CD4/fisiologia , Proteínas de Membrana/metabolismo , Modelos Imunológicos , Peptídeos/metabolismo , Linfócitos T/imunologia
15.
J Immunol ; 170(3): 1329-38, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12538693

RESUMO

Activation of CD4(+) Th cells requires their cognate interaction with APCs bearing specific relevant MHC class II-peptide complexes. This cognate interaction culminates in the formation of an immunological synapse that contains the various proteins and lipids required for efficient T cell activation. We now show that APC lipid raft membrane microdomains contain specific class II-peptide complexes and serve as platforms that deliver these raft-associated class II molecules to the immunological synapse. APC rafts are required for T cell:APC conjugate formation and T cell activation at low densities of relevant class II-peptide complexes, a requirement that can be overcome at high class II-peptide density. Analysis of confocal microscopy images revealed that over time APC lipid rafts, raft-associated relevant class II-peptide complexes, and even immunologically irrelevant class II molecules accumulate at the immunological synapse. As the immunological synapse matures, relevant class II-peptide complexes are sorted to a central region of the interface, while irrelevant class II molecules are excluded from this site. We propose that T cell activation is facilitated by recruitment of MHC class II-peptide complexes to the immunological synapse by virtue of their constitutive association with lipid raft microdomains.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Ativação Linfocitária/imunologia , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo , Muramidase/metabolismo , Fragmentos de Peptídeos/metabolismo , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular/imunologia , Relação Dose-Resposta Imunológica , Antígenos de Histocompatibilidade Classe II/análise , Cooperação Linfocítica/imunologia , Camundongos , Camundongos Transgênicos , Muramidase/farmacologia , Fragmentos de Peptídeos/farmacologia , Linfócitos T/metabolismo , Células Tumorais Cultivadas
16.
Mol Immunol ; 38(11): 803-16, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11922938

RESUMO

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the generation and activation of dendritic cells (DCs), the most potent of antigen presenting cells (APCs). Tumors secreting GM-CSF have been shown to induce strong anti-tumor immune responses. In this report, we have constructed a glycosylphosphatidyl-inositol (GPI) anchored form of GM-CSF (GPI-GM-CSF). This protein subsequently was found expressed on the cell membrane and sensitive to phosphatidyl-inositol-specific phospholipase C (PIPLC), confirming that it is GPI-anchored. However, GM-CSF was also found in the culture supernatant of cells expressing GPI-GM-CSF. Inhibition studies using brefeldin A and para-formaldehyde fixation revealed that GM-CSF found in the supernatant was not secreted, but due to shedding or proteolytic cleavage. Accumulation of GM-CSF in the media from isolated membranes was time and temperature-dependent. The released portion represented 10-15% of all membrane-bound GM-CSF after 72h under culture conditions. GPI-GM-CSF retained functional activity to induce bone marrow cell proliferation and administration of GPI-GM-CSF expressing membranes induced the generation of DCs in vivo. These results demonstrate that GPI-anchored GM-CSF retains all functional activity of native GM-CSF while gaining the ability to attach to cell membranes. The ability of GPI-GM-CSF to be expressed on membranes and be partially released, can possibly lead to formation of a cytokine gradient, while retaining ability to target associated membrane antigens to DCs. This novel form of GM-CSF may have wide range of clinical applicability.


Assuntos
Membrana Celular/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Células da Medula Óssea/fisiologia , Células CHO , Divisão Celular , Cricetinae , Células Dendríticas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Transfecção
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