RESUMEN
Dermcidin (DCD), an antimicrobial peptide with a broad spectrum of activity against bacteria such as Propionibacterum acnes, is expressed constitutively in sweat in the absence of stimulation due to injury or inflammation. The aim of this study was to determine the relationship between DCD expression and acne vulgaris associated with P. acnes. The antimicrobial activity of recombinant full-length DCD (50 µg/ml) was 97% against Escherichia coli and 100% against Staphylococcus aureus. Antimicrobial activity against P. acnes ranged from 68% at 50 µg/ml DCD to 83% at 270 µg/ml DCD. DCD concentration in sweat from patients with acne vulgaris (median 9.8 µg/ml, range 6.9-95.3 µg/ml) was significantly lower than in healthy subjects (median 136.7 µg/ml, range 45.4-201.6 µg/ml) (p = 0.001). DCD demonstrated concentration-dependent, but partial, microbicidal activity against P. acnes. These results suggest that reduced DCD concentration in sweat in patients with inflammatory acne may permit proliferation of P. acnes in pilosebaceous units, resulting in progression of inflammatory acne.
Asunto(s)
Acné Vulgar/metabolismo , Péptidos/análisis , Propionibacterium acnes/patogenicidad , Sudor/química , Acné Vulgar/diagnóstico , Acné Vulgar/microbiología , Adolescente , Adulto , Antibacterianos/farmacología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos/farmacología , Propionibacterium acnes/crecimiento & desarrollo , Propionibacterium acnes/efectos de la radiación , Proteínas Recombinantes/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Adulto JovenRESUMEN
Purpose: To evaluate the feasibility of using DARC (detection of apoptosing retinal cells) technology as a biomarker for preclinical assessment of glaucomatous damage in a non-human primate (NHP) model of ocular hypertension (OHT). Methods: Elevated intraocular pressure (IOP) was induced by applying a laser to the trabecular meshwork in each eye of NHPs. Changes in DARC counts in the retina, identified as fluorescent-tagged annexin V (ANX776)-positive cells, were evaluated together with optic nerve damage, assessed using spectral domain-optical coherence tomography. The pharmacokinetic properties of ANX776 in both healthy and OHT model monkeys were also examined. Results: Sustained elevation of IOP and subsequent thinning of the retinal nerve fiber layer thickness (RNFLT) around the optic nerve head were confirmed in the OHT model. Increases in DARC counts were also detected after IOP elevation. We identified a statistically significant relationship between cumulative DARC counts and reductions in RNFLT both globally and in each peripapillary sector. Intravenous administration of ANX776 increased blood annexin V in a dose-dependent manner, which was subsequently eliminated. Conclusions: This study revealed that DARC technology can effectively assess glaucomatous damage in an NHP OHT model. We obtained the fundamental data that could serve as a reference for developing preclinical models to evaluate the pharmacodynamics of neuroprotective agents using DARC technology in NHP OHT models. Translational Relevance: Our basic data in a monkey OHT model could be useful for future preclinical studies using DARC technology to estimate the pharmacodynamic response of neuroprotective agents.
Asunto(s)
Glaucoma , Fármacos Neuroprotectores , Hipertensión Ocular , Animales , Anexina A5 , Primates , ApoptosisRESUMEN
Hes genes are required to maintain diverse progenitor cell populations during embryonic development. Loss of Hes1 results in a spectrum of malformations of pharyngeal endoderm-derived organs, including the ultimobranchial body (progenitor of C cells), parathyroid, thymus and thyroid glands, together with highly penetrant C-cell aplasia (81%) and parathyroid aplasia (28%). The hypoplastic parathyroid and thymus are mostly located around the pharyngeal cavity, even at embryonic day (E) 15.5 to E18.5, indicating the failure of migration of the organs. To clarify the relationship between these phenotypes and neural crest cells, we examine fate mapping of neural crest cells colonized in pharyngeal arches in Hes1 null mutants by using the Wnt1-Cre/R26R reporter system. In null mutants, the number of neural crest cells labeled by X-gal staining is markedly decreased in the pharyngeal mesenchyme at E12.5 when the primordia of the thymus, parathyroid and ultimobranchial body migrate toward their destinations. Furthermore, phospho-Histone-H3-positive proliferating cells are reduced in number in the pharyngeal mesenchyme at this stage. Our data indicate that the development of pharyngeal organs and survival of neural-crest-derived mesenchyme in pharyngeal arches are critically dependent on Hes1. We propose that the defective survival of neural-crest-derived mesenchymal cells in pharyngeal arches directly or indirectly leads to deficiencies of pharyngeal organs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Cresta Neural/embriología , Faringe/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/deficiencia , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Región Branquial/citología , Región Branquial/metabolismo , Desarrollo Embrionario/genética , Proteínas de Homeodominio/genética , Células Madre Mesenquimatosas , Mesodermo/citología , Ratones , Ratones Noqueados , Organogénesis/genética , Organogénesis/fisiología , Glándulas Paratiroides/citología , Glándulas Paratiroides/embriología , Faringe/citología , Faringe/inervación , Timo/citología , Timo/embriología , Factor de Transcripción HES-1 , Cuerpo Ultimobranquial/citología , Cuerpo Ultimobranquial/embriologíaRESUMEN
The lack of the Hes1 gene leads to the failure of cranial neurulation due to the premature onset of neural differentiation. Hes1 homozygous null mutant mice displayed a neural tube closure defect, and exencephaly was induced at the mid/hindbrain boundary. In the mutant mesencephalon, the roof plate was not formed and therefore the ventricular zone showing cell proliferation was displaced to the brain surface. Furthermore, the telencephalon and ventral diencephalon were defective. Despite the severe defects of neurogenesis in null mutants, the mesencephalic dopaminergic (mesDA) neurons were specified at the midline of the ventral mesencephalon in close proximity to two important signal centers - floor plate and mid/hindbrain boundary (i.e., the isthmic organizer). Using mesDA neuronal markers, tyrosine hydroxylase (TH) and Pitx3, the development of mesDA neurons was studied in Hes1 null mice and compared with that in the wild type. At early stages, between embryonic day (E) 11.5 and E12.5, mesDA neurons were more numerous in null mutants than in the wild type. From E13.5 onward, however, the cell number and fiber density of mesDA neurons were decreased in the mutants. Their distribution pattern was also different from that of the wild type. In particular, mesDA neurons grew dorsally and invaded the rostral hindbrain. 5-HT neurons were also ectopically located in the mutant midbrain. Thus, the loss of Hes1 resulted in disturbances in the inductive and repulsive activities of the isthmic organizer. It is proposed that Hes1 plays a role in regulating the location and density of mesDA neurons.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Homeodominio/metabolismo , Mesencéfalo/embriología , Defectos del Tubo Neural/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Dopamina/metabolismo , Proteínas de Homeodominio/genética , Inmunohistoquímica , Mesencéfalo/citología , Ratones , Ratones Mutantes , Defectos del Tubo Neural/genética , Neuronas/fisiología , Neurulación/fisiología , Factor de Transcripción HES-1 , Factores de Transcripción/metabolismoRESUMEN
In patients with lepromatous leprosy, Mycobacterium leprae is often observed inside the human microvascular endothelial cells (HMVEC) surrounding Schwann cells (SC) at the site of lesions in the peripheral nerves. Based on this observation, it is considered that the nasal mucous may be the invasion pathway for M. leprae and HMVEC serve as an important reservoir for the bacteria before they invade SC. In light of previous research which revealed that Mce1A protein mediates bacterial invasion into nasal epithelial cells and HMVEC, we conducted a study to determine whether the invasion of M. leprae into HMVEC can be suppressed by blocking the Mce1A protein. In this study, we analyzed bacterial invasive activity by adding recombinant Escherichia coli, which express the active region (InvX:72 a.a.) of Mce1A protein on their external membrane, into cultured HMVEC, using the adhesin involved in the diffuse adherence mechanism. The number of bacteria that invaded into the cells was then measured by a colony counting method. The active region of Mce1A was divided into four sections, and hyperimmune antisera was prepared for each section for analyzing the inhibitory effect against invasion. The invasive activity was suppressed by antibodies against InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. This suggests that the InvX regions 1-24 a.a., 25-46 a.a. and 58-72 a.a. of Mce1A protein play an important role in the invasion of M. leprae into HMVEC and that it may be possible to suppress entry of M. leprae in HMVEC with antibodies against these regions.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Células Endoteliales/microbiología , Lepra/inmunología , Mycobacterium leprae/inmunología , Animales , Anticuerpos Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Línea Celular , Recuento de Colonia Microbiana , Humanos , Sueros Inmunes/inmunología , Sueros Inmunes/aislamiento & purificación , Lepra/microbiología , Lepra/prevención & control , Mycobacterium leprae/patogenicidad , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Leprosy is a chronic infection where the skin and peripheral nervous system is invaded by Mycobacterium leprae. The infection mechanism remains unknown in part because culture methods have not been established yet for M. leprae. Mce1A protein (442 aa) is coded by mce1A (1326 bp) of M. leprae. The Mce1A homolog in Mycobacterium tuberculosis is known to be associated with M. tuberculosis epithelial cell entry, and survival and multiplication within macrophages. Studies using recombinant proteins have indicated that Mce1A of M. leprae is also associated with epithelial cell entry. This study is aimed at identifying particular sequences within Mce1A associated with M. leprae epithelial cell entry. Recombinant proteins having N-terminus and C-terminus truncations of the Mce1A region of M. leprae were created in Escherichia coli. Entry activity of latex beads, coated with these truncated proteins (r-lep37 kDa and r-lep27 kDa), into HeLa cells was observed by electron microscopy. The entry activity was preserved even when 315 bp (105 aa) and 922 bp (308 aa) was truncated from the N-terminus and C-terminus, respectively. This 316-921 bp region was divided into three sub-regions: 316-531 bp (InvX), 532-753 bp (InvY), and 754-921 bp (InvZ). Each sub-region was cloned into an AIDA vector and expressed on the surface of E. coli. Entry of these E. coli into monolayer-cultured HeLa and RPMI2650 cells was observed by electron microscopy. Only E. coli harboring the InvX sub-region exhibited cell entry. InvX was further divided into 4 domains, InvXa-InvXd, containing sequences 1-24 aa, 25-46 aa, 47-57 aa, and 58-72 aa, respectively. Recombinant E. coli, expressing each of InvXa-InvXd on the surface, were treated with antibodies against these domains, then added to monolayer cultured RPMI cells. The effectiveness of these antibodies in preventing cell entry was studied by colony counting. Entry activity was suppressed by antibodies against InvXa, InvXb, and InvXd. This suggests that these three InvX domains of Mce1A are important for M. leprae invasion into nasal epithelial cells.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lepra/microbiología , Mycobacterium leprae/patogenicidad , Tabique Nasal/microbiología , Proteínas Bacterianas/genética , Línea Celular , Recuento de Colonia Microbiana , ADN Bacteriano/genética , Escherichia coli/genética , Vectores Genéticos/genética , Células HeLa , Humanos , Microesferas , Mycobacterium leprae/genética , Mycobacterium leprae/crecimiento & desarrollo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The long search of an animal model for leprosy were carried out as many researchers since the Mycobacterium leprae discovery by Dr. Hansen in 1874. The remarkable results were left after the development of the foot-pad method by Dr. Shepard in 1960. The introduction of the T-R mouse and athymic (nude) mouse for leprosy research, alsospontaneity examples of Hansen's disease was reported to armadillo, chimpanzee and mangabay monkey, and it was confirmed that Hansen's disease was the zoonosis. Although, We have established a congenic hypertensive nude rat, SHR/NCrj-rnu (SHR.F344-Foxn(rnu)), carrying nude (rnu) and hypertension genes. SHR/NCrj-rnu rats obtained showed high susceptibility to M. leprae and showed a characteristic disease with a progressive pattern of leproma formation. Also this hypertensive nude rat strain produce high level of IL-10. Therefore, congenic hypertensive nude rat may be useful for an animal model to leprosy.
Asunto(s)
Modelos Animales de Enfermedad , Lepra/historia , Ratas Endogámicas SHR , Ratas Desnudas , Animales , Historia del Siglo XX , Interleucina-10/biosíntesis , Lepra/microbiología , Lepra/patología , Ratones , Mycobacterium leprae/crecimiento & desarrollo , Ratas , Ratas Endogámicas SHR/metabolismo , Ratas Endogámicas SHR/microbiología , Ratas Desnudas/metabolismo , Ratas Desnudas/microbiologíaRESUMEN
Previous studies have shown that HDAC inhibitors selectively inhibit IL-2 gene expression, but the mechanism of this inhibition remains to be elucidated. It was recently reported that HDAC4, a component of the nuclear hormone receptor corepressor (N-CoR) complex, associates with the IL-2 promoter via the transcription factor myocyte enhancer factor 2 (MEF2). We therefore focused on the role of HDAC4/N-CoR complex in the transcriptional regulation of IL-2. Four approaches were used to characterize this role and to investigate the relation between the regulatory function of HDAC4/N-CoR complex and HDAC4-enzymatic activity: (i) HDAC4 silencing by RNA interference, (ii) overexpression of N-CoR repression domain 3 (RD3), (iii) overexpression of HDAC4 point mutants, and (iv) treatment with HDAC inhibitors. Here, we report that HDAC4 plays an essential role in IL-2 promoter activation, and that the formation of the HDAC4/N-CoR complex, which is closely related to HDAC4-enzymatic activity, might be involved in HDAC inhibitor-mediated inhibition of IL-2 gene expression. These observations indicate that the selective inhibition of HDAC4 or the interaction of HDAC4 with N-CoR is likely a potential target for the development of novel immunosuppressants.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Interleucina-2/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Represoras/antagonistas & inhibidores , Secuencia de Bases , Línea Celular , Cartilla de ADN , Silenciador del Gen , Histona Desacetilasas/genética , Historia del Siglo XV , Humanos , Mutación , Co-Represor 1 de Receptor Nuclear , Péptidos Cíclicos/farmacología , Regiones Promotoras Genéticas , ARN Interferente PequeñoRESUMEN
BACKGROUND: The transmission of Mycobacterium leprae, the causative pathogen of leprosy, has been postulated to occur mainly through upper respiratory route rather than skin-to-skin contact via minor injuries. The M. leprae genome contains mce1A gene, which encodes a putative mammalian cell entry protein. However, to date, there have been no functional analyses of the M. leprae mce1A gene product. OBJECTIVE: The aim of this study was to elucidate a possible relationship between this transmission mechanism and the mce1A gene product. METHODS: We analyzed the cell uptake activity in vitro of polystyrene latex beads coated with a purified recombinant (r-) protein expressed by a 849-bp locus within the mce1A gene. RESULTS: The r-protein promoted uptake of the beads into human nasal epithelial cells derived from nasal polyps, human bronchial epithelial cell line, normal human dermal fibroblasts, normal human microvascular endothelial cells and normal human keratinocytes cultured at 0.01 mM extracellular calcium concentration [Ca]; no uptake occurred with keratinocytes cultured at 1.2mM [Ca]. CONCLUSION: These results suggest that the mce1A gene product can mediate M. leprae entry into respiratory epithelial cells as their natural target cells, which may be the main mode of transmission. Endothelial cells, on the other hand, may serve as the reservoir of the bacilli for long-term infection. The M. leprae Mce1A protein has potential important implications for mode of transmission and pathogenesis of leprosy.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lepra/transmisión , Mycobacterium leprae/patogenicidad , Mucosa Nasal/metabolismo , Mucosa Respiratoria/metabolismo , Piel/metabolismo , Proteínas Bacterianas/genética , Calcio/metabolismo , Células Endoteliales/metabolismo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Queratinocitos/metabolismo , Lepra/metabolismo , Mycobacterium leprae/metabolismo , Mucosa Nasal/citología , Proteínas Recombinantes/metabolismo , Mucosa Respiratoria/citología , Piel/citologíaRESUMEN
Histone deacetylase inhibitors (HDAC inhibitors) are an emerging class of anticancer agents. To elucidate the mechanism of HDAC inhibitor-induced thrombocytopenia, we focused on the effects of HDAC inhibitors on megakaryocyte differentiation and performed Affymetrix GeneChip analysis of human megakaryocytic HEL cells treated with or without HDAC inhibitors. Here, we report that GATA-1 and 10 haematopoietic factors (SCL, NF-E2, EKLF, Pleckstrin, Thrombin-R, LMO2, PU.1, Fli-1, AML1, and TCF11) are transcriptionally repressed by HDAC inhibitors in a similar pattern (R>0.98), and putative GATA-1-binding sites are found in almost all promoters of these genes. In addition, luciferase reporter assays reveal that mutations of GATA-1-binding sites in the GATA-1 promoter abolish its sensitivity to HDAC inhibitor-mediated down-regulation in HEL cells. Further, this report also asserts that HDAC inhibitor increases megakaryocyte counts and inhibits GATA-1 gene expression in rat spleen. Together, these results suggest that HDAC inhibitors inhibit GATA-1 gene expression by decreasing the transactivation function of GATA-1 itself, and that this may in turn lead to a delay in megakaryocyte maturation and finally cause thrombocytopenia. Our findings may help our understanding of the molecular mechanism of HDAC inhibitor-mediated GATA-1 transcriptional repression and to reduce the risk of HDAC inhibitor-induced thrombocytopenia.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Factor de Transcripción GATA1/metabolismo , Hematopoyesis/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Megacariocitos/efectos de los fármacos , Trombocitopenia/metabolismo , Acetilación , Animales , Antineoplásicos/efectos adversos , Recuento de Células Sanguíneas , Western Blotting , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Inhibidores Enzimáticos/efectos adversos , Factor de Transcripción GATA1/genética , Perfilación de la Expresión Génica/métodos , Genes Reporteros , Hematopoyesis/genética , Factores de Crecimiento de Célula Hematopoyética/genética , Factores de Crecimiento de Célula Hematopoyética/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Luciferasas/genética , Masculino , Megacariocitos/enzimología , Megacariocitos/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos Cíclicos/farmacología , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Endogámicas Lew , Bazo/efectos de los fármacos , Bazo/metabolismo , Trombocitopenia/sangre , Trombocitopenia/inducido químicamente , Trombocitopenia/genética , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , TransfecciónRESUMEN
Interleukin (IL)-2 is an essential cytokine in T cell proliferation and homeostasis. The importance of IL-2 down-regulation in preventing acute rejection in organ transplantation and the development of autoimmune diseases has been demonstrated by the therapeutic usefulness of the widely used immunosuppressants cyclosporine A and FK506. Recently, a histone deacetylase (HDAC) inhibitor, FR235222, has been shown to inhibit IL-2 gene expression and to possess immunosuppressive activity in vivo. To elucidate the inhibitory mechanism of FR235222 in IL-2 gene expression, we performed Affymetrix GeneChip analysis of activated Jurkat cells treated with or without FR235222. Here, we show that many NF-kappaB-regulated genes are transcriptionally down-regulated by FR235222 in activated Jurkat cells. Further, luciferase reporter assays revealed that FR235222 selectively inhibits NF-kappaB activity without impairing NF-AT or AP-1 at the concentrations at which it potently inhibits IL-2 promoter activation. These results indicate that FR235222 inhibits IL-2 gene expression via a different mechanism to CsA and FK506, and that FR235222 has the ability to inhibit NF-kappaB activity, which may be partly related to the potent inhibition of IL-2 gene expression by FR235222. Our findings may help our understanding of the molecular mechanism of the inhibition of IL-2 gene expression by HDAC inhibitors and provide insight into the development of more effective and safer new immunosuppressants.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Inhibidores de Histona Desacetilasas , Interleucina-2/genética , Péptidos Cíclicos/farmacología , Transcripción Genética/efectos de los fármacos , ADN/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Inmunosupresores/farmacología , Células Jurkat , FN-kappa B/metabolismo , Unión Proteica , Subunidades de Proteína , Factores de Tiempo , Activación Transcripcional/efectos de los fármacosRESUMEN
Acridine orange (AO), a weakly basic fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured malignant melanoma cells during illumination with blue light. Immediately after the start of the illumination, the successive disruption of vesicles was observed as a flash of fluorescence, and shortly after that, blebs were formed on the plasma membrane. These cells died within 5 min. Vesicle disruption was completely inhibited when cells were treated with the vacuolar H(+)-ATPase inhibitor bafilomycin A1 followed by loading with AO, but not when bafilomycin A1 was treated after AO loading. Thus, the filling of AO in the vesicle, which is driven by vacuolar H(+)-ATPase, is initially required for vesicle disruption. In contrast, bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the blebs are formed independently of the vesicle disruption. Acute cell death was inhibited by treatment with bafilomycin A1 before but not after AO loading. Thus, AO- and blue light-induced acute cell death is associated with vesicle disruption rather than bleb formation. Both the vesicle disruption and the formation of plasma membrane blebs were inhibited by removal of oxygen from the cell environment and by singlet oxygen scavengers, sodium azide, ascorbic acid, and L-histidine, but not inhibited by the hydroxyl radical scavenger dimethyl thiourea. Acute cell death was also prevented by singlet oxygen scavengers but not by dimethyl thiourea. Thus, these phenomena are likely caused at least in part by the generation of singlet oxygen. The photosensitive features of plasma and vesicle membranes observed in the present study may be based on the use of the photodynamic effect, such as cancer therapy.
Asunto(s)
Membrana Celular/efectos de la radiación , Vesículas Citoplasmáticas/efectos de la radiación , Luz , Melanoma/radioterapia , Fototerapia/métodos , Naranja de Acridina , Muerte Celular/efectos de la radiación , Permeabilidad de la Membrana Celular/efectos de la radiación , Células Cultivadas , Humanos , Melanoma/patología , Microscopía Fluorescente , Oxígeno/fisiología , ATPasas de Translocación de Protón Vacuolares/fisiologíaRESUMEN
Trichophytosis, a common dermatophytosis, affects nearly 20-25% of the world's population. However, little is known about mechanisms for preventing colonization of Trichophyton on the skin. Dermcidin, an antimicrobial peptide that provides innate immunity to the skin and is constitutively secreted even in the absence of inflammatory stimulation, was studied to elucidate its antimycotic activity against Trichophyton. Recombinant dermcidin was determined to have antimycotic activity against Trichophyton rubrum, as evaluated by colony-forming unit (CFU) assays. The killing rate of dermcidin was 40.5% and 93.4% at 50 µg/mL (the average dermcidin concentration in healthy subjects) and 270 µg/mL, respectively. An effect of dermcidin treatment was found to be a reduction of the metabolic activity of Trichophyton as determined by nicotinamide adenine dinucleotide assay. Further, dermcidin concentrations in sweat of tinea pedis patients were found to be lower than those of healthy subjects. These findings suggest a mycostatic role for dermcidin, at normal sweat concentrations. Accordingly, we suspect that dermcidin, at normal sweat concentrations, inhibits growth of Trichophyton, where Trichophyton is subsequently eliminated in conjunction with epidermis turnover. Dermcidin, therefore, appears to play a role in the skin protection mechanism that prevents colonization of tinea pedis.
Asunto(s)
Péptidos/metabolismo , Tiña del Pie/inmunología , Trichophyton/efectos de los fármacos , Adulto , Estudios de Casos y Controles , Humanos , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Péptidos/aislamiento & purificación , Péptidos/farmacología , Proteínas Recombinantes/farmacología , Sudor/químicaRESUMEN
Histone deacetylase (HDAC) inhibitors repress interleukin-2 (IL-2) gene expression in T cells and possess immunosuppressive activity in vivo. In addition to its immunosuppressive activity, HDAC inhibitors block GATA binding protein-1 (GATA-1) gene expression in megakaryocytes and elicit thrombocytopenia. In this report we state that for a given immunosuppressive dose of HDAC inhibitor, the ratio of GATA-1 reporter gene activity relative to IL-2 reporter gene assay (G/I ratio of measured IC(50)) can be predictive of a HDAC inhibitor's thrombocytopenic effect. This study utilized nine HDAC inhibitors at a minimal effective dose in a rat heterotopic cardiac transplantation model and the resultant G/I ratios and platelet depletion rates were highly correlated (r=0.933). These results indicate that calculation of G/I ratio can be a novel method for selecting immunosuppressive HDAC inhibitor having minimal thrombocytopenic effect which will benefit the search for new immunosuppressants of greater safety and efficacy.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Inmunosupresores/farmacología , Trombocitopenia/inducido químicamente , Animales , Supervivencia Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/efectos adversos , Factor de Transcripción GATA1/genética , Genes Reporteros/efectos de los fármacos , Trasplante de Corazón/inmunología , Humanos , Inmunosupresores/efectos adversos , Interleucina-2/genética , Células Jurkat , Masculino , Plásmidos/genética , Ratas , Ratas Endogámicas Lew , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Relación Estructura-Actividad , Sales de Tetrazolio , Tiazoles , Trombocitopenia/sangre , Trasplante Heterotópico/inmunologíaRESUMEN
Mycobacterial mammalian cell entry protein 1A (Mce1A) is involved in the uptake of bacteria in non-phagocytic cells and also possibly in granuloma formation. However, it has not been clarified whether the interaction between mycobacterial Mce1A and epithelial cell induces chemokine and cytokine production which is required for granuloma formation. To this end, we infected A549 alveolar epithelial cells in vitro with E. coli expressing Mce1A on the cell surface and examined the resultant chemokine/cytokine production. Mce1A promoted bacterial adherence and internalization of E. coli into A549 cells, and these recombinant bacteria induced high levels of MCP-1 and IL-8 production, compared to E. coli harboring the plasmid vector alone. Chemokine production was enhanced by the internalization of recombinant E. coli expressing Mce1A because cytochalasin D treatment partially inhibited MCP-1 and IL-8 production. However, Mce1A-coated latex beads did not induce the chemokine production. These results suggest that although Mce1A does not induce production of chemokines, it may promote chemokine induction by augmenting the interaction between bacteria and epithelial cells.
Asunto(s)
Adhesión Bacteriana/inmunología , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Alveolos Pulmonares/inmunología , Tuberculosis/inmunología , Adhesión Bacteriana/efectos de los fármacos , Línea Celular , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Recuento de Colonia Microbiana , Citocalasina D/farmacología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/inmunología , Humanos , Interleucina-8/biosíntesis , Interleucina-8/genética , Interleucina-8/inmunología , Alveolos Pulmonares/citología , Alveolos Pulmonares/fisiología , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tuberculosis/genética , Regulación hacia ArribaRESUMEN
In order to know how caspases contribute to the intracellular fate of Mycobacterium tuberculosis and host cell death in the infected macrophages, we examined the effect of benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethane (z-VAD-fmk), a broad-spectrum caspase inhibitor, on the growth of M. tuberculosis H37Rv in RAW 264 cells. In the cells treated with z-VAD-fmk, activation of caspase-8, caspase-3/7, and caspase-9 was clearly suppressed, and DNA fragmentation of the infected cells was also reduced. Under this experimental condition, it was found that the treatment markedly inhibited bacterial growth inside macrophages. The infected cells appeared to undergo cell death of the necrosis type in the presence of z-VAD-fmk. We further found that z-VAD-fmk treatment resulted in the generation of intracellular reactive oxygen species (ROS) in the infected cells. By addition of a scavenger of ROS, the host cell necrosis was inhibited and the intracellular growth of H37Rv was significantly restored. Among inhibitors specific for each caspase, only the caspase-9-specific inhibitor enhanced the generation of ROS and induced necrosis of the infected cells. Furthermore, we found that severe necrosis was induced by infection with H37Rv but not H37Ra in the presence of z-VAD-fmk. Caspase-9 activation was also detected in H37Rv-infected cells, but H37Ra never induced such caspase-9 activation. These results indicated that caspase-9, which was activated by infection with virulent M. tuberculosis, contributed to the inhibition of necrosis of the infected host cells, presumably through suppression of intracellular ROS generation.
Asunto(s)
Caspasa 9/fisiología , Macrófagos/metabolismo , Macrófagos/microbiología , Mycobacterium tuberculosis/fisiología , Animales , Inhibidores de Caspasas , Técnicas de Cultivo de Célula , Activación Enzimática , Macrófagos/enzimología , Ratones , Necrosis/metabolismoAsunto(s)
Dermatosis Facial/epidemiología , Lupus Eritematoso Cutáneo/epidemiología , Adolescente , Adulto , Anciano , Niño , Dermatosis Facial/tratamiento farmacológico , Femenino , Humanos , Japón/epidemiología , Lupus Eritematoso Cutáneo/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tetraciclinas/uso terapéutico , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
FK614 is a structurally novel class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, with the mechanism of its insulin-sensitizing action most likely due to activation of PPARgamma. In this study, properties of FK614 for PPARgamma binding, ability to induce conformational change, and coactivator recruitment were investigated. FK614, rosiglitazone, and pioglitazone competed specific binding of [3H]rosiglitazone to PPARgamma with Ki values of 11 nM, 47 nM, and 1.3 microM, respectively. Limited trypsin digestion of PPARgamma with FK614 or rosiglitazone produced distinct patterns of digested polypeptides, suggesting that FK614 directly binds to PPARgamma but induces specific alterations in receptor conformation. FK614 induced interaction of PPARgamma with nuclear receptor coactivator CBP but of lower magnitude than rosiglitazone and pioglitazone. The estimated Kd values of FK614-, rosiglitazone-, and pioglitazone-PPARgamma complex to CBP peptide were 1.8, 0.64, and 0.72 microM, respectively, indicating FK614-PPARgamma complex exhibits a lower affinity for CBP peptide compared to other agonist-PPARgamma complexes. When tested the effect of FK614 on CBP recruitment induced by 9(S)-hydroxyoctadecadienoic acid, an endogenous ligand, FK614 negatively modulated PPARgamma activation. The unique properties of FK614 may underlie the molecular basis of ligand-dependent transcriptional modulation mediated by PPARgamma.
Asunto(s)
Bencimidazoles/metabolismo , Hipoglucemiantes/metabolismo , PPAR gamma/metabolismo , Bencimidazoles/farmacología , Glutatión/metabolismo , Hipoglucemiantes/farmacología , Cinética , Ligandos , Conformación Molecular , Péptido Hidrolasas/química , Pioglitazona , Plásmidos/genética , Rosiglitazona , Espectrometría de Fluorescencia , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacologíaRESUMEN
Adipogenesis is an important process for the improvement of insulin resistance by peroxisome proliferator-activated receptor (PPAR) gamma agonists, such as rosiglitazone and pioglitazone. FK614 [3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3-Hbenzimidazole-5-carboxamide] is a structurally novel class of PPARgamma agonist that improves insulin sensitivity in animal models of type 2 diabetes. Herein, we characterize FK614, a selective PPARgamma modulator (SPPARM) with differential properties affecting the regulation of fat cell function. FK614 behaves as a partial agonist in inducing the interaction of PPARgamma with both transcriptional coactivators, cAMP response element-binding protein-binding protein and steroid receptor coactivator-1, but as a full agonist with both PPAR-binding protein and PPAR-interacting protein, which are required for PPARgamma-mediated adipogenesis. In the differentiating 3T3-L1 adipocytes, the levels of adipose fatty acid-binding protein (aP2) mRNA expression and triglyceride accumulation induced by FK614 were as efficacious as those of rosiglitazone and pioglitazone. In contrast, the effect of FK614 on aP2 gene expression in mature adipocytes was less than that of the other PPARgamma agonists. Furthermore, the long-term treatment of mature adipocytes with rosiglitazone and pioglitazone reduced the expression of phosphodiesterase 3B, the down-regulation of which has an important role in the development of insulin resistance; however, FK614 had no such effect in mature adipocytes. Thus, FK614 behaves as an SPPARM with differential effects on the activation of PPARgamma at each stage of adipocyte differentiation. The stage-dependent selectivity of FK614 may contribute to its enhanced insulin sensitization in differentiating adipocytes and to reduced insulin resistance at the stage of adipocyte hypertrophy.