RESUMO
BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease of the skin and mucous membranes. This disease typically affects the elderly and presents with itch and localized or, most frequently, generalized bullous lesions. A subset of patients only develops excoriations, prurigo-like lesions, and eczematous and/or urticarial erythematous lesions. The disease, which is significantly associated with neurological disorders, has high morbidity and severely impacts the quality of life. OBJECTIVES AND METHODOLOGY: The Autoimmune blistering diseases Task Force of the European Academy of Dermatology and Venereology sought to update the guidelines for the management of BP based on new clinical information, and new evidence on diagnostic tools and interventions. The recommendations are either evidence-based or rely on expert opinion. The degree of consent among all task force members was included. RESULTS: Treatment depends on the severity of BP and patients' comorbidities. High-potency topical corticosteroids are recommended as the mainstay of treatment whenever possible. Oral prednisone at a dose of 0.5 mg/kg/day is a recommended alternative. In case of contraindications or resistance to corticosteroids, immunosuppressive therapies, such as methotrexate, azathioprine, mycophenolate mofetil or mycophenolate acid, may be recommended. The use of doxycycline and dapsone is controversial. They may be recommended, in particular, in patients with contraindications to oral corticosteroids. B-cell-depleting therapy and intravenous immunoglobulins may be considered in treatment-resistant cases. Omalizumab and dupilumab have recently shown promising results. The final version of the guideline was consented to by several patient organizations. CONCLUSIONS: The guidelines for the management of BP were updated. They summarize evidence- and expert-based recommendations useful in clinical practice.
Assuntos
Dermatologia , Penfigoide Bolhoso , Venereologia , Corticosteroides/uso terapêutico , Idoso , Vesícula/tratamento farmacológico , Humanos , Penfigoide Bolhoso/diagnóstico , Penfigoide Bolhoso/tratamento farmacológico , Qualidade de VidaRESUMO
BACKGROUND: Pemphigus encompasses a group of life-threatening autoimmune bullous diseases characterized by blisters and erosions of the mucous membranes and skin. Before the era of immunosuppressive treatment, pemphigus was almost always fatal. Due to its rarity, only few randomized controlled therapeutic trials are available. Recently, rituximab has been approved as first-line treatment for moderate and severe pemphigus vulgaris in Europe and the United States. OBJECTIVES: The Autoimmune blistering diseases Task Force of the European Academy of Dermatology and Venereology (EADV) has initiated a throughout update of the guideline for the management of patients with pemphigus. RESULTS: The guidelines for the management of pemphigus were updated, and the degree of consent among all task force members was included. The final version of the guideline was consented by the European Dermatology Forum (EDF) and several patient organizations.
Assuntos
Dermatologia , Guias como Assunto , Pênfigo , Venereologia , Academias e Institutos , Europa (Continente) , Humanos , Pênfigo/diagnóstico , Pênfigo/tratamento farmacológicoRESUMO
Autoinflammatory syndromes are characterized by an exaggerated activation of the innate immune system and frequently present with skin symptoms. In contrast to autoimmune disorders no specific autoantibodies or autoreactive immune cells are detected. Thus, the diagnosis is usually difficult and can only be made by a careful interpretation of anamnestic, clinical and laboratory parameters. In some hereditary autoimmune syndromes specific genetic mutations are described and can be helpful for the diagnosis. For treatment of these disorders both classic immunomodulatory drugs and specific cytokine inhibitors are used, mainly directed against interleukin1. Long-term therapy is generally required.
Assuntos
Doenças Autoimunes/imunologia , Doenças Hereditárias Autoinflamatórias , Hidradenite Supurativa/imunologia , Pioderma Gangrenoso/diagnóstico , Dermatopatias/imunologia , Doenças Autoimunes/diagnóstico , Citocinas , Hidradenite Supurativa/diagnóstico , Humanos , Interleucina-1 , Dermatopatias/etiologia , Dermatopatias/patologia , SíndromeRESUMO
BACKGROUND: Raynaud's phenomenon and the frequently ensuing digital ulcerations represent an early and very distressing symptom in patients with systemic sclerosis (scleroderma, SSc) causing significant limitations in the ability to work and quality of life. The use of vasoactive drugs (especially intravenous prostacyclin derivatives) is recommended to reduce the risk of hypoxic tissue damage up to the loss of fingers. METHODS: In order to obtain information about the current state of treatment of patients with prostacyclin derivatives in routine clinical life in Germany, a survey was conducted among the centers affiliated to the German Network for Systemic Scleroderma (DNSS). In addition, a separate patient survey was conducted by the schleroderma self-help group (Sklerodermie Selbsthilfe e.â¯V.), which only covered the symptoms Raynaud's syndrome, digital ulcers and the use of intravenous prostacyclin derivatives. RESULTS: Of the 433 patients surveyed 56% stated that they had already been treated with prostacyclin derivatives (iloprost/alprostadil) because of their illness and symptoms. A total of 61% received the treatment for severe Raynaud's phenomenon and 39% for digital ulcerations. Most respondents not only experienced an improvement in Raynaud's phenomenon and digital ulcers but also a significant improvement of limitations in everyday life. They also needed significantly less outside help and absenteeism from work was much lower. CONCLUSION: Patients consistently reported a positive effect of treatment with prostacyclin derivatives on Raynaud's phenomenon, acral ulcerations, pain and daily restrictions and felt well and safely cared for during inpatient treatment. These positive effects in the patients' perceptions provide crucial information supporting and confirming the current European and international treatment recommendations.
Assuntos
Epoprostenol , Doença de Raynaud , Escleroderma Sistêmico , Epoprostenol/análogos & derivados , Epoprostenol/uso terapêutico , Dedos/irrigação sanguínea , Alemanha , Humanos , Pacientes Internados , Qualidade de Vida , Doença de Raynaud/diagnóstico , Doença de Raynaud/tratamento farmacológico , Doença de Raynaud/epidemiologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/tratamento farmacológico , Pele/irrigação sanguíneaRESUMO
Raynaud's phenomenon (RP) is a frequent and painful vasospasm of small arteries localized in acral body regions (most frequently the fingers). The more frequent so-called primary RP is caused merely by a functional dysregulation of the tonus of vascular walls. In contrast, the rarer secondary RP is additionally associated with structural abnormalities of blood vessels. Knowledge of RP is important for rheumatologists because secondary RP can be associated with the presence or development of severe underlying diseases, especially with systemic sclerosis. Thus, the rheumatologist has to be aware of this condition. In this article the diagnostic procedures and the most important treatment approaches are summarized.
Assuntos
Doença de Raynaud , Escleroderma Sistêmico , Humanos , Doença de Raynaud/complicações , Doença de Raynaud/terapia , Reumatologistas , Escleroderma Sistêmico/complicaçõesRESUMO
Neurophilic urticarial dermatosis (NUD) is a new, important differential diagnosis of urticarial rashes. It is a clinical and histological response pattern that strongly correlates with systemic disease. Both autoinflammatory and autoimmune conditions can be present in patients with NUD. In this article the clinical and histological criteria of NUD, diagnostic considerations and the diseases most frequently associated with NUD are discussed.
Assuntos
Anti-Inflamatórios/uso terapêutico , Citocinas/sangue , Síndrome de Sweet/diagnóstico por imagem , Síndrome de Sweet/cirurgia , Urticária/diagnóstico , Urticária/terapia , Biomarcadores/sangue , Diagnóstico Diferencial , Humanos , Síndrome de Sweet/imunologia , Urticária/imunologiaRESUMO
The skin is one of the organs most frequently involved in vasculitides. Cutaneous vasculitis may present (1) part of a systemic vasculitis (e.g., IgA vasculitis), (2) a skin-restricted or skin-dominant variant of the corresponding systemic vasculitis without clinically apparent visceral involvement (e.g., cutaneous IgA vasculitis), or (3) a vasculitis occurring exclusively in the skin (e.g., erythema elevatun diutinum). The clinical symptoms of vasculitides are markedly determined by the size of the predominantly affected blood vessels. Systemic polyarteritis nodosa is regarded as a medium vessel vasculitis and is associated with multiple skin symptoms: (1) vasculitis of digital arteries with ensuing digital infarction, (2) livedo racemosa and subcutaneous nodules, and (3) in some patients even purpura and hemorrhagic macules due to additional small vessel vasculitis. In contrast, in its skin-restricted entity (i.e., cutaneous polyarteritis nodosa), the predominant symptoms are subcutaneous nodules surrounded by livedo racemosa, often on the lower legs. Among small-vessel vasculitides palpable purpura with predilection for the legs is a nearly pathognomonic feature of immune complex vasculitis. Variations in clinical symptoms indicate additional pathophysiological mechanisms or different vascultides: (1) ANCA-associated vasculitides often also entail nodules or sometimes livedo, (2) cryoglobulinemic vasculitis additionally may present with necrosis at cold exposed areas and involvement of vessels of various size, (3) small vessel vasculitis associated with systemic lupus erythematosus or rheumatoid arthritis shows predilection for additional sites (e.g., nailfolds) and also involvement of vessels beyond postcapillary venules, (4) recurrent macular vasculitis in hypergammaglobulinemia also occurs on dependent parts, but shows numerous small hemorrhagic macules instead of palpable purpura, (5) erythema elevatum diutinum begins with brightly red to violaceous plaques at extensor sites, followed by fibrotic nodules. Consequently, cutaneous symptoms provide pivotal clues for further diagnosis and ensuing management of vasculitides.
Assuntos
Dermoscopia/métodos , Dermatopatias Vasculares/diagnóstico , Dermatopatias Vasculares/imunologia , Vasculite/diagnóstico , Vasculite/imunologia , Diagnóstico Diferencial , HumanosRESUMO
BACKGROUND: α-Melanocyte-stimulating hormone (α-MSH) is a melanocortin peptide that increases skin pigmentation during ultraviolet light-mediated tanning. As α-MSH has been shown to possess anti-inflammatory effects, we assessed the clinical potential of a superpotent α-MSH analogue, afamelanotide (Nle(4)-D-Phe(7)-α-MSH), in patients with acne vulgaris, the most common inflammatory skin disorder. METHODS: Afamelanotide (16 mg) was given in a phase II open-label pilot study subcutaneously as a sustained-release resorbable implant formulation to 3 patients with mild-to-moderate facial acne vulgaris. Evaluation included lesion count, adverse effects and patient-reported outcome. Monitoring of laboratory parameters included differential blood counts, electrolytes, urine analysis, and liver and kidney function tests. Skin melanin density was measured by reflectance spectrophotometry. RESULTS: The total number as well as the number of inflammatory acne lesions declined in all patients 56 days after the first injection of afamelanotide. Life quality as measured by Dermatology Life Quality Index likewise improved in all 3 patients 56 days after the first injection of afamelanotide. There were no adverse effects except mild and short-term fatigue in one patient. All patients experienced increased pigmentation especially on the face. Clinically relevant changes in laboratory parameters were not detected. CONCLUSIONS: Afamelanotide appears to have anti-inflammatory effects in patients with mild-to-moderate acne vulgaris. Future trials are needed to confirm the anti-inflammatory action of this melanocortin analogue in patients with acne vulgaris.
Assuntos
Acne Vulgar/tratamento farmacológico , alfa-MSH/análogos & derivados , Acne Vulgar/fisiopatologia , Implantes de Medicamento , Humanos , Projetos Piloto , Qualidade de Vida , alfa-MSH/administração & dosagem , alfa-MSH/uso terapêuticoRESUMO
The principles of systemic glucocorticoid (GC) therapy in dermatology are reviewed. As a basis for an efficient GC therapy with few side effects the pharmacology, endogenous regulation as well as the mechanisms and side effects of GCs as well as their management are introduced. Modern therapeutic approaches such as circadian application and low-dose therapy are discussed as well as principles of tapering dosages and the most important indications for systemic GCs in dermatology.
Assuntos
Glucocorticoides/uso terapêutico , Dermatopatias/tratamento farmacológico , Adolescente , Insuficiência Adrenal/induzido quimicamente , Insuficiência Adrenal/prevenção & controle , Adulto , Idoso , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Cronofarmacoterapia , Feminino , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Glucocorticoides/farmacocinética , Humanos , Lactente , Recém-Nascido , Injeções Intramusculares , Gravidez , Pulsoterapia , Dermatopatias/sangueRESUMO
BACKGROUND: In 2005, a scoring system (CLASI, Cutaneous Lupus Erythematosus Disease Area and Severity Index) was developed for patients with cutaneous lupus erythematosus (CLE) to assess disease 'activity' and 'damage'. However, the CLASI does not give an accurate assessment of the severity in all disease subtypes. OBJECTIVES: The main objective of this study was to analyse critically the included parameters of the CLASI and to revise the activity and damage score taking into account various clinical features of the different subtypes of CLE. The revised CLASI (RCLASI) was also validated for use in clinical trials. Patients and methods A RCLASI was designed with regard to the anatomical region (i.e. face, chest, arms) and morphological aspects (i.e. erythema, scaling/hyperkeratosis, oedema/infiltration, scarring/atrophy) of skin lesions and evaluated by nine dermatologists who scored 12 patients with different subtypes of CLE to estimate inter- and intrarater reliability. RESULTS: Reliability studies demonstrated an intraclass correlation coefficient (ICC) for an inter-rater reliability of 0.89 for the activity score [95% confidence interval (CI) 0.79-0.96] and of 0.79 for the damage score (95% CI 0.62-0.92). The ICC for intrarater reliability for the activity score was 0.92 (95% CI 0.89-0.95) and the ICC for the damage score was 0.95 (95% CI 0.92-0.98). CONCLUSIONS: In the present study, a RCLASI was developed by experts, and reliability studies supported the validity and applicability of the revised scoring instrument for CLE. Thus, the RCLASI is a valuable instrument in multicentre studies and for the clinical evaluation of activity and damage in different disease subtypes.
Assuntos
Lúpus Eritematoso Cutâneo/classificação , Índice de Gravidade de Doença , Adulto , Idoso , Feminino , Humanos , Lúpus Eritematoso Cutâneo/patologia , Lúpus Eritematoso Cutâneo/fisiopatologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Phagocyte-derived S100 proteins are endogenous activators of innate immune responses. S100A12 binds to the receptor for advanced glycation end-products, while complexes of S100A8/S100A9 (myeloid-related proteins, MRP8/14; calprotectin) are ligands of toll-like receptor 4. These S100 proteins can be detected in stool. In the present study we analyse the release of S100A12 and MRP8/14 from intestinal tissue. Specimens from patients with Crohn's disease (CD; n = 30), ulcerative colitis (UC; n = 30), irritable bowel syndrome (IBS; n = 30) or without inflammation (n = 30) were obtained during endoscopy. After 24 h culture, S100A12 and MRP8/14 were analysed in supernatants. Endoscopic, histological, laboratory and clinical disease activity measures were documented. We found an increased spontaneous release of S100A12 from tissue in inflammatory bowel disease (IBD). The release of S100A12 into the supernatants was 28-fold enhanced in inflamed tissue when compared to non-inflamed tissue (mean 46.9 vs. 1.7 ng/ml, p < 0.0001). In active CD, release of S100A12 and MRP8/14 was strongly dependent on localization, with little release from sites of active ileal inflammation compared to colonic inflammation. This difference was more pronounced for S100A12 than for MRP8/14. S100A12 and MRP8/14 provoked up-regulation of adhesion molecules and chemokines on human intestinal microvascular endothelial cells (HIMECs) isolated from normal colonic tissue. The direct release of phagocyte-derived S100 proteins from inflamed tissues may reflect secretion from infiltrating neutrophils (S100A12) and also monocytes or epithelial cells (MRP8/14). Via activation of pattern recognition receptors, these proteins promote inflammation in intestinal tissue. The enhanced mucosal release can explain the correlation of fecal markers with disease activity in IBD.
Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Fagócitos/metabolismo , Proteínas S100/análise , Adulto , Biomarcadores/análise , Estudos de Casos e Controles , Células Cultivadas , Colite Ulcerativa/imunologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Colo , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Fezes/química , Feminino , Humanos , Íleo , Inflamação , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Molécula 1 de Adesão Intercelular/análise , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Fagócitos/patologia , Proteínas S100/metabolismo , Estatísticas não ParamétricasRESUMO
c-fos, which encodes a transcription factor of the AP-1 family, is a prototypical immediate-early gene induced by a number of proinflammatory cytokines including interleukin-1 (IL-1), the latter being an important regulator of skin homeostasis. Using the human keratinocyte cell line HaCaT as an in vitro model, we dissected the molecular pathways leading to IL-1-induced c-fos gene induction. Phosphorylation of the transcription factor cAMP response element binding protein (CREB) at Ser133 was found to be essential for IL-1-induced c-fos gene induction and was closely paralleled by protein kinase A (PKA) activation. In contrast to other cell types, the cyclooxygenase/prostaglandin pathway, known to activate the cAMP/PKA cascade, plays little, if any, role in c-fos expression downstream of the IL-1 receptor in keratinocytes. Simultaneous activation of several of the mitogen-activated protein kinase (MAPK) cascades occurred in response to IL-1, but each differentially contributed to c-fos induction by IL-1, with the p38/MAPK being the most crucial of all, the extracellular signal-regulated kinase pathway contributing in an additive manner and the Jun N-terminal kinase pathway playing little, if any, role. We also demonstrate that p38-dependent activation of mitogen- and stress-activated kinase 1 (MSK1), a CREB kinase, is a key step for c-fos gene activation by IL-1. Finally, we identify MSK1 as playing a positive role in the control of cell proliferation of both HaCaT keratinocytes and the A431 human epidermoid carcinoma line.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Interleucina-1/fisiologia , Queratinócitos/enzimologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/fisiologia , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Chlorocebus aethiops , Humanos , Queratinócitos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
In many acromegalic patients the paradoxical release of GH in response to TRH has been well documented, but the mechanisms underlying this phenomenon are not understood. It has been suggested that aberrant GH secretion may result from TRH endogenously synthesized by the adenoma. In 32 adenomas from acromegalic patients, TRH-like immunoreactivity (TRH-LI) was measured using 2 well characterized antisera. TRH-LI was not detectable in 10 samples, and in 19 samples, TRH-LI was measured only by the less specific antibody. With the TRH-specific antibody, TRH-LI was identified only in 3 samples, 2 of which contained exceedingly high concentrations (40 and 96 pg/mg tissue). In the latter 2 samples, prepro-TRH messenger ribonucleic acid was identified by Northern blot analysis, but not in the control tissue sample of a patient without pituitary disease and also not in the other adenomas analyzed by this technique. Transcripts of the TRH receptor were almost undetectable in all adenomas analyzed. For the TRH-degrading ectoenzyme, a potential regulator of TRH signals at adenohypophyseal target sites, transcripts were significantly expressed only in the TRH-producing adenomas. We conclude that the TRH-signaling elements examined are, in general, not directly involved in the mechanisms causing paradoxical GH secretion in acromegalic patients.
Assuntos
Acromegalia/fisiopatologia , Adenoma/química , Neoplasias Hipofisárias/química , Precursores de Proteínas/genética , Hormônio Liberador de Tireotropina/genética , Hormônio Liberador de Tireotropina/fisiologia , Acromegalia/etiologia , Adenoma/genética , Adenoma/patologia , Adenoma/cirurgia , Adulto , Idoso , Aminopeptidases/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/cirurgia , Ácido Pirrolidonocarboxílico/análogos & derivados , Receptores do Hormônio Liberador da Tireotropina/análise , Transdução de Sinais , Hormônio Liberador de Tireotropina/análiseRESUMO
Cell-cell interactions are important regulatory elements in anterior pituitary (AP) physiology. As model systems to study pituitary cell-cell interactions, AP cells kept either as monolayers or as organotypic reaggregate cultures were analyzed by differential display PCR. We identified six cDNA fragments (osteopontin (Opn), connective tissue growth factor (CTGF), alpha(v)-integrin, cathepsin H, lysozyme and O-acetyl GD(3) ganglioside synthase) that showed elevated expression in monolayers compared with reaggregate cultures and the AP. The adenohypophyseal mRNA expression of Opn and CTGF, two secreted signaling substances, was studied in more detail. In situ hybridization histochemistry revealed that Opn mRNA expression is restricted to a subpopulation of gonadotropes whereas CTGF hybridization signals could not be ascribed to any known cell type. Opn transcript levels were downregulated in the APs of lactating rats and decreased when rats received s.c. injections of 17beta-estradiol for 5 days. The mRNA expression was higher in male than in female rats and increased after gonadectomy. CTGF transcript levels were higher in male compared with female rats and were increased in pregnant rats and in rats treated for 5 days with triiodothyronine or dexamethasone. These results indicate that Opn and CTGF may be of physiological importance as local communication factors in the AP.
Assuntos
Substâncias de Crescimento/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Adeno-Hipófise/metabolismo , Sialoglicoproteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Northern Blotting/métodos , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Dexametasona/farmacologia , Feminino , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces/genética , Hibridização In Situ/métodos , Lactação , Masculino , Modelos Animais , Orquiectomia , Osteopontina , Ovariectomia , Reação em Cadeia da Polimerase/métodos , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Sialoglicoproteínas/genética , Tri-Iodotironina/farmacologiaRESUMO
Thyrotrophin-releasing hormone (TRH), a hypothalamic neuropeptide hormone and a putative neuromodulator/ neurotransmitter in the central nervous system is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a TRH-specific metallopeptidase localized on the surface of neuronal brain cells in culture and on lactotrophic cells of the pituitary. After succeeding in cloning the cDNA of TRH-DE we now report on the cellular distribution pattern of this enzyme in rat brain, spinal cord and pituitary gland using in situ hybridization histochemistry. In the pituitary, TRH-DE mRNA was found both in the anterior and the neural lobe but not in the intermediate lobe. After treatment with triiodothyronine (T3) a dramatic increase in the mRNA levels of the TRH-DE and a decrease in the intensity of the TRH receptor could be observed in the anterior lobe of the pituitary. In brain, TRH-DE transcripts were predominantly found in neo- and allocortical regions with strongest signals in the olfactory bulb, the piriform cortex, the cerebral cortex, the granular layer of the cerebellar cortex and the pyramidal cells of the Ammon's horn. In the diencephalon, the highest TRH-DE mRNA levels were observed in the medial habenulae followed by several hypothalamic subregions. In the mesencephalon and brainstem, moderate signals were present in the superior colliculi, substantia nigra, dorsal raphe and in the periolivar region. In the spinal cord, TRH-DE mRNA positive neurons were present in all layers. The very distinct distribution of TRH-DE in the brain and the hormonal regulation of the adenohypophyseal enzyme support the concept that this peptidase serves very specialized functions.
Assuntos
Aminopeptidases/genética , Sistema Nervoso Central/metabolismo , Hipófise/metabolismo , Animais , Northern Blotting , Expressão Gênica , Hibridização In Situ , Masculino , Ácido Pirrolidonocarboxílico/análogos & derivados , Ratos , Ratos Sprague-DawleyRESUMO
We describe the synthesis of a fluorescent dipeptide derivative, beta-Ala-Lys-Nepsilon-AMCA, which could be used as an excellent reporter molecule for studying the oligopeptide transport system in brain cell cultures. Fluorescence microscopic and immunocytochemical studies revealed that the reporter peptide specifically accumulated in astrocytes (type I and II) and O-2A progenitor cells but not in neurons or differentiated oligodendrocytes. In astroglia-rich cell culture the dipeptide derivative is taken up in unmetabolized form by an energy dependent, saturable process with apparent kinetic constants of KM = 28 microM and Vmax = 6 nmol x h(-1) x mg protein(-1) at pH 7.2. Competition studies revealed that the accumulation of beta-Ala-Lys-Nepsilon-AMCA is strongly inhibited by dipeptides and pseudopeptides such as bestatin, arphamenine A and B. The biochemical data indicated that the properties of this high-affinity oligopeptide carrier closely resemble those of the renal peptide transport system PepT2 and Northern blot analysis demonstrated that PepT2 mRNAis expressed in glial but not in neuronal cell cultures. In situ hybridization histochemistry also revealed a non-neuronal localization of PepT2 transcripts and a diffuse, widespread distribution of PepT2 signals throughout the entire rat brain. The selective accumulation of the fluorescent reporter molecule by brain cells under viable conditions may provide a useful tool for studying peptide uptake systems and other aspects of astroglial physiology.