RESUMEN
Although preventive screening, brief intervention and referral to treatment for adolescent substance use is recommended by the American Academy of Pediatrics, primary care providers inconsistently address substance use with their pediatric patients (AAP Committee on Practice and Ambulatory Medicine and AAP Bright Futures Periodicity Schedule Workgroup, 2017). Further research on provider perceptions about addressing adolescent substance use may help identify and address some barriers to screening. However, there are few validated measures of provider perceptions toward patient substance, and none are specific to pediatric patients. This study (conducted in Maryland, 2015-2017) examines the internal consistency and factor structure of an adapted measure to assess perceptions of adolescent substance use. Internal consistency was assessed using responses from a sample of 276 healthcare practitioners (87.7% women, 12.3% men). Their professions included the following: Certified Medical Assistants (10.9%), Registered Nurses (17.8%), Nurse Practitioners (8.3%), Physician Assistants (3.6%), Medical Doctors (13.8%), Clinical Therapists (10.9%) and Other (21.0%). A four-factor solution was identified and initial evidence suggests the adapted measure is appropriate for use with health care providers. A subsample of 181 participants who reported direct interaction with adolescent patients in a provider role was also used to assess convergent validity with self-reported screening practices and effectiveness. Provider-reported frequency of alcohol and drug use assessment for pediatric patients was significantly related to positive perceptions about adolescent substance use on all subscales. The adapted measure could prove useful for assessing provider readiness to receive adolescent substance use screening training and could be further adapted to include items unique to adolescent care, including parental involvement.
RESUMEN
BACKGROUND: Cerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia. METHODS: Male Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test. RESULTS: Qualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7. In parallel, aminopeptidase N and cytosolic alanyl-aminopeptidase activities remained unchanged. CONCLUSIONS: Distinct expression, localization and activity patterns of proline- and alanine-specific proteases indicate their involvement in ischemia-triggered inflammation and neurodegeneration. Consistently, IPC1755, a non-selective protease inhibitor, revealed a significant reduction of cortical lesions after transient cerebral ischemia and may suggest dipeptidyl peptidase IV, aminopeptidase N and proteases with similar substrate specificity as potentially therapy-relevant targets.
Asunto(s)
Isquemia Encefálica/enzimología , Antígenos CD13/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/tratamiento farmacológico , Antígenos CD13/genética , Infarto Cerebral/enzimología , Infarto Cerebral/etiología , Dipeptidil Peptidasa 4/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Lateralidad Funcional , Proteína Ácida Fibrilar de la Glía/metabolismo , Glicoesfingolípidos/uso terapéutico , Masculino , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Reactive oxygen species (ROS) are key players in ischemia-induced neurodegeneration. We investigated whether hippocampal neurons may lack sufficient redox-buffering capacity to protect against ROS attacks. Using organotypic hippocampal slice cultures (OHSCs) transiently exposed to oxygen and glucose deprivation (OGD) and gerbils suffering from a two-vessel occlusion (2VO) as complementary ex vivo and in vivo models, we have elucidated whether the intrinsic redox systems interfere with ischemia-induced neurodegeneration. Cell- type-specific immunohistological staining of hippocampal slice cultures revealed that pyramidal neurons, in contrast to astrocytes and microglia, express free thiols only weakly. In addition, free thiol levels were extensively decreased throughout the hippocampal formation immediately after OGD, but recovered within 24 hr after reperfusion. In parallel, progressive glia activation and proliferation were observed. Increased neuronal exposure to ROS was monitored by dihydroethidium oxidation in hippocampal pyramidal cell layers immediately after OGD. Coadministration of reduction equivalents (α-lipoic acid) and thiol-stimulating agents (enalapril, ambroxol) decreased ischemia-induced neuronal damage in OGD-treated OHSCs and in gerbils exposed to 2VO, whereas single drug applications remained ineffective. In summary, limited redox buffering capacities of pyramidal neurons may underlie their exceptional vulnerability to cerebral ischemia. Consistently, multidrug treatments supporting endogenous redox systems may offer a strategy to promote valid neuroprotection.
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Isquemia Encefálica/patología , Neuronas/patología , Oxidación-Reducción , Daño por Reperfusión/patología , Animales , Muerte Celular , Modelos Animales de Enfermedad , Etidio/análogos & derivados , Etidio/metabolismo , Fluoresceínas/metabolismo , Gerbillinae , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosa/deficiencia , Glicoproteínas/metabolismo , Hipocampo/citología , Hipoxia , Lectinas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Rodaminas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Ácido Tióctico/farmacología , VersicanosRESUMEN
Dipeptidyl peptidase IV (DP IV)/CD26 and aminopeptidase N (APN)/CD13 family enzymes control T cell functions. We have previously defined these peptidases as targets to treat autoimmune disease, but the underlying mechanism is unclear. Here, we determined the effect of enzymatic inhibitors on chemotaxis by CD4+ effector T (Teff) cells. Exposure of Teff cells to the inhibitor of DP IV activity, Lys[Z(NO2)]-pyrrolidide (LZNP) and the inhibitor of APN activity, actinonin has no effect on chemotaxis or unstimulated cell migration, even at high inhibitor concentrations. LZNP and actinonin also fail to suppress migration of unfractionated lymph node cells, excluding paracrine action through other leukocyte subsets. In contrast, inhibition of DP IV and APN activities selectively suppresses lymphocyte functions including proliferation and production of the T helper type (Th)1 cytokine IFN-γ, the Th17 cytokine IL-17, as well as TNF-α, and ameliorates autoimmunity in vivo. The present results combined with previous studies suggest that LZNP and actinonin do not prevent migration of pathogenic Teff cells into target tissues, but rather suppress disease through inhibitor induced release of TGF-ß by T cells at the site of inflammation.
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Antígenos CD13/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Dipeptidil Peptidasa 4/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/inmunología , Quimiocina CXCL12/metabolismo , Quimiotaxis/efectos de los fármacos , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/enzimología , Encefalomielitis Autoinmune Experimental/inmunología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Ácidos Hidroxámicos/farmacología , Activación de Linfocitos/efectos de los fármacos , Lisina/análogos & derivados , Lisina/farmacología , Ratones , Ratones Endogámicos C57BL , Pirroles/farmacología , Pirrolidinas/farmacología , Tiazoles/farmacología , Transgenes/genéticaRESUMEN
The cellular dipeptidyl peptidase IV (DPIV, E.C.3.4.14.5, CD26) is a type II membrane peptidase with various physio-logical functions. Our main knowledge on DPIV comes from studies of soluble DPIV which plays a role in regulation of glucose homeostasis by inactivation of the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic poly-peptide. It has been reported that membrane-bound DPIV plays a crucial role in the immune system and in other tissues and cells, but the knowledge on the action of cellular DPIV and its regulation is limited. In this study, we show particularly for immune cells that DPIV and not DP8 or DP9 is the most potent member of the DPIV family in regulating cellular immune functions. Moreover, we provide evidence that soluble and cellular DPIV differ in functions and hand-ling of substrates and inhibitors owing to the different accessibility of peptide substrates to the two access paths of DPIV. The different functions are based on the favored access path of the central pore of cellular DPIV and a special central pore binding site which assists substrate access to the active site of the enzyme. The newly discovered central pore binding site mediates an autosterical regulation of cellular DPIV and is its most crucial target site to regulate cellular functions such as growth and cytokine production. Neuropeptide Y (NPY) processing by cellular DPIV was found to be inhibited by ligands which interact with the central pore binding site. This finding suggests a crucial role of the immunosuppressive cytokine NPY in the function of DPIV in growth regulation.
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Dipeptidil Peptidasa 4/química , Dipeptidil Peptidasa 4/metabolismo , Dominio Catalítico , Línea Celular , Citocinas/metabolismo , ADN/biosíntesis , Inhibidores de la Dipeptidil-Peptidasa IV/química , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Humanos , Linfocitos/enzimología , Modelos Moleculares , Neuropéptido Y/metabolismoRESUMEN
Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, 'peptidase-targeted Immunoregulation' (PETIR™), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR™ in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR™ represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.
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Antígenos CD13/antagonistas & inhibidores , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Animales , Línea Celular , ADN/biosíntesis , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos , Linfocitos T/efectos de los fármacosRESUMEN
The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)2-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO2)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.
Asunto(s)
Dipeptidasas/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Espacio Intracelular/metabolismo , Animales , Línea Celular , ADN/biosíntesis , Dipeptidil Peptidasa 4/genética , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Colorantes Fluorescentes/metabolismo , Humanos , Indoles/metabolismo , Isoleucina/análogos & derivados , Isoleucina/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Ratones , Ratones Noqueados , Neuropéptido Y/metabolismo , Pirrolidinas/metabolismo , Especificidad por Sustrato , Linfocitos T/enzimologíaRESUMEN
BACKGROUND: Dipeptidyl peptidase IV (DP IV, CD26) and DP IV-like enzymes, such as dipeptidyl peptidase II (DP II), dipeptidyl peptidase 8 (DP8), and dipeptidyl peptidase 9 (DP9), have been recognized to regulate T lymphocyte activation. Lys[Z(NO2)]-thiazolidide (LZNT) and Lys[Z(NO2)]-pyrrolidide (LZNP), non-selective inhibitors of DP IV-like activity known to target DP IV as well as DP II, DP8, and DP9, suppress T lymphocyte proliferation in vitro. Moreover, these inhibitors are capable of attenuating the severity of autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and experimental arthritis, a model of human rheumatoid arthritis, in vivo, particularly in combination with inhibitors of aminopeptidase N (APN, CD13) enzymatic activity. METHODS: Here, we studied the influence of non-selective and selective inhibitors of DP IV-like enzymes on DNA synthesis in mitogen-stimulated splenocytes from wild-type C57BL/6 mice and DP IV/CD26-knockout (DP IV/CD26-KO) mice. RESULTS: LZNT and LZNP, the non-selective inhibitors of DP IV-like activity, suppressed the DNA synthesis in stimulated splenocytes from wild-type and DP IV/ CD26-KO mice to a comparable extent. Further, a selective inhibitor of DP8/DP9 activity was capable of suppressing DNA synthesis in mitogen-stimulated splenocytes of both wild-type and knockout mice to the same extent. In contrast, selective inhibitors of DP IV and DP II lacked this suppressive activity. CONCLUSIONS: Our data support the hypothesis that DP8 and/or DP9 represent additional pharmacological targets for the suppression of T cell proliferation and for anti-inflammatory therapy.
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Dipeptidil Peptidasa 4/deficiencia , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Proliferación Celular/efectos de los fármacos , ADN/biosíntesis , Dipeptidil Peptidasa 4/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Modelos Animales de Enfermedad , Lisina/análogos & derivados , Lisina/química , Lisina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirrolidinas/química , Pirrolidinas/farmacología , Bazo/citología , Bazo/inmunología , Linfocitos T/efectos de los fármacos , Tiazoles/química , Tiazoles/farmacologíaRESUMEN
BACKGROUND: In the past, different research groups could show that treatment of immune cells with inhibitors of post-proline splitting dipeptidyl aminopeptidases leads to functional changes in the immune system consistent with immunosuppression. This is due to the inhibition of proliferation of lymphocytes and the production of inflammatory cytokines of the TH1, TH2, and TH17, cells as well as the induction of immunosuppressive cytokines, such as transforming growth factor-beta1 (TGF-beta1) and interleukin (IL)-1RA. Until recently, most of the effects of these inhibitors on immune functions were attributed to the inhibition of dipeptidyl aminopeptidase IV (DPIV/CD26). With the identification of new peptidases of the DPIV family (DASH) with the same or similar substrate specificity [fibroblast activation protein (FAP), DP8/9], the question arose whether and to what extent the inhibition of intracellularly localized enzymes, DP8 and DP9, contribute to the observed immunosuppression. In addition, members of the aminopeptidase N (APN) family are also involved in the regulation of immune functions. Hence, the concept of a combined targeting of both families of peptidases for treatment of inflammatory diseases is a promising strategy. RESULTS/CONCLUSIONS: Summarizing data obtained from the usage of different non-selective and selective inhibitors of DPIV, DP8/9, FAP, and DPII, this review provides evidence that in addition to DPIV, DP8/9 also regulate the immune response via modulation of cell cycle progression and cytokine production. The strongest and most consistent effects in vitro were, however, observed with non-selective inhibitors for the suppression of DNA synthesis and cytokine production. Similar effects were provoked by APN inhibitors, which were also found to suppress DNA synthesis and the production of inflammatory cytokines in vitro. However, different mechanisms and signaling pathways appear to mediate the cellular effects resulting from the inhibition of either APN or DPIV family members. In particular, members of the APN family uniquely influence the function of CD4+CD25+ regulatory T-cells. Consequently, the concomitant inhibition of both APN and DPIV enzyme families by means of two separate inhibitors or by binary inhibitors with specificity for both enzyme families (PETIR, peptidase targeted immunoregulation) synergistically affects immune cells on the level of cell cycle regulation, suppression of TH1, TH2, and TH17 cytokines as well as the activation of regulatory T-cells. Besides leukocytes, dermal cells as sebocytes, keratinocytes, and fibroblasts are also targeted by these inhibitors. This strongly suggests a broad potential of the multiple anti-inflammatory effects of PETIR in treatment of chronic inflammatory diseases, such as autoimmune diseases, allergies, and transplant rejections, as well as of inflammatory skin diseases, such as acne, psoriasis, rosacea or atopic dermatitis. The first active dual inhibitor, IP10.C8, has been developed by IMTM for the treatment of inflammatory skin diseases and has just entered the first phase II study.
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Antígenos CD13/inmunología , Dipeptidil Peptidasa 4/inmunología , Animales , Inhibidores de la Dipeptidil-Peptidasa IV , Humanos , Inhibidores de Proteasas/farmacología , Enfermedades de la Piel/tratamiento farmacológico , Enfermedades de la Piel/inmunologíaRESUMEN
Inflammatory bowel diseases (IBD) are driven by imbalances in innate and acquired immune response. In IBD two dysregulated T cell subsets are in the focus of interest: activated effector T cells and regulatory T cells. These T cell subsets are characterized by a strong expression of the ectopeptidases dipeptidyl peptidase IV (DPIV /CD26) and aminopeptidase N (APN/CD13), which are thought to a role in the control of immune activation and in regulating cellular communication by hydrolyzing bioactive polypeptides. Since inhibitors of both enzymes were shown to be effective in limiting immune activation processes in vitro as well as in vivo, they emerged as new drug candidates for the treatment of diseases associated with an imbalanced T cell response, such as IBD. In this review we intent to throw light on the putative role of DPIV, APN and related enzymes in the regulation of immune and non-immune processes in inflammatory bowel diseases, on possible benefits from peptidase inhibitor therapy in these diseases as well on the gaps of knowledge in this field.
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Antígenos CD13/antagonistas & inhibidores , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/inmunología , Tracto Gastrointestinal/efectos de los fármacos , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Sustancia P/fisiología , Subgrupos de Linfocitos T/inmunología , Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
Telmisartan, in addition to blocking angiotensin (Ang) II type 1 receptor (AT(1)R), activates peroxisome proliferator activated receptor gamma (PPARgamma) signaling that interferes with nitric oxide (NO) system. Because aging of endothelial cells (ECs) is hallmarked by a reduction in NO synthesis, we hypothesized that telmisartan increases NO formation by regulated asymmetrical dimethylarginine (ADMA)-dimethylarginine dimethylaminohydrolase (DDAH)-system through blocking AT(1)R and activating PPARgamma signaling. To test this hypothesis, ECs were cultured with telmisartan, eprosartan, Ang II, and GW9662 (PPARgamma antagonist) until the twelfth passage. During the process of aging, PPARgamma protein expression decreased significantly, whereas the expression of AT(1)R increased. Telmisartan reversed these effects and dose-dependently decreased reactive oxygen species and 8-iso-prostaglandin (PG) F(2alpha) formation. This effect was associated with an upregulated activity and protein expression of DDAH, accompanied by a decrease in ADMA concentration, an increase in NO metabolites, and delayed senescence. Blockade of PPARgamma signaling by GW9662 or PPARgamma small-interference RNA prevented the effect of telmisartan on ADMA-DDAH-NO system. Coincubation with Ang II did not affect the effect of telmisartan-delayed senescence, whereas Ang II itself accelerated endothelial aging. Moreover, AT(1)R blocker eprosartan that did not influence PPARgamma protein expression had no effect on ADMA system and senescence. We have demonstrated that telmisartan mainly by activating PPARgamma signaling can alter the catabolism and release of ADMA as an important cardiovascular risk factor. We therefore propose that telmisartan translationally and posttranslationally upregulated DDAH expression via activation of PPARgamma signaling, causing ADMA to diminish and increase NO synthesis sufficient to delay senescence.
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Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Arginina/análogos & derivados , Bencimidazoles/farmacología , Benzoatos/farmacología , Senescencia Celular/efectos de los fármacos , Óxido Nítrico/metabolismo , PPAR gamma/fisiología , Receptor de Angiotensina Tipo 1/fisiología , Acrilatos/farmacología , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Angiotensina II/farmacología , Anilidas/farmacología , Arginina/metabolismo , Células Cultivadas , Senescencia Celular/fisiología , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Imidazoles/farmacología , Estrés Oxidativo/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , PPAR gamma/genética , Receptor de Angiotensina Tipo 1/genética , Transducción de Señal/fisiología , Telmisartán , Tiofenos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system. Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) play a regulatory role in T cell activation and represent potential targets for the treatment of inflammatory disorders. Synthetic inhibitors of DP IV and/or APN enzymatic activity induce production of the immunosuppressive cytokine TGF-beta1 and subsequently suppress DNA synthesis and Th1 cytokine production of activated human T cells. Compelling evidence has demonstrated that IL-17-producing CD4 cells (Th17) are a major contributor to the pathogenesis of autoimmune inflammation. Here, we report that inhibitors of DP IV-like activity as well as of APN activity inhibit IL-17 production in activated human and mouse T cells. Combining inhibitors of DP IV and APN increases the suppressive effect on T cell specific IL-17 production in vitro compared to a single peptidase inhibitor. In the following, we summarize the evidence for the role of both ectoenzymes in T cell activation in vitro and in vivo and provide a rationale for the use of combined or dual ectopeptidase inhibitors to treat autoimmune diseases like MS.
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Antígenos CD13/biosíntesis , Dipeptidil Peptidasa 4/biosíntesis , Encefalomielitis/metabolismo , Esclerosis Múltiple/inmunología , Linfocitos T/inmunología , Animales , Apoptosis , Antígenos CD13/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Inflamación , Interleucina-17/metabolismo , Activación de Linfocitos , Espectrometría de Masas/métodos , Esclerosis Múltiple/enzimología , Péptido Hidrolasas/química , Linfocitos T/metabolismoRESUMEN
Skin cells express dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN) and their related molecules of the DP IV-like family DP2, DP6, DP8, DP9 and fibroblast activation protein (FAP), as well as the cytoplasmic alanyl aminopeptidase (cAAP). The inhibitors of DP IV-like activity, Lys(Z(NO2))-thiazolidide (LZNT) and Lys(Z(NO2))-pyrrolidide (LZNP), and the APN inhibitors actinonin and bestatin affect proliferation, differentiation and cytokine production in sebocytes and keratinocytes, which are involved in the initiation of acne. Furthermore, they suppress proliferation of Propionibacterium acnes-stimulated T cells ex vivo and induce an anti-inflammatory cytokine profile. In the mouse tail model of psoriasis they have a pro-differentiative effect. In addition, these inhibitors suppress skin fibroblast proliferation, whereas only inhibition of DP IV-like activity decreases TGF-beta1 expression and abrogates the TGF-beta1 mediated stimulatory effects on TGF-beta1 and fibronectin production, collagen synthesis and matrix deposition in these cells. Targeting enzyme activity of DP IV and APN and their related molecules might be a novel approach for the treatment of acne, psoriasis or keloids.
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Acné Vulgar/tratamiento farmacológico , Antígenos CD13/farmacología , Dipeptidil Peptidasa 4/fisiología , Enfermedades de la Piel/metabolismo , Animales , Antígenos CD13/química , Cricetinae , Dipeptidil Peptidasa 4/química , Inhibidores Enzimáticos/farmacología , Fibrosis , Humanos , Inflamación , Mesocricetus , Ratones , Modelos Biológicos , Psoriasis/terapia , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/metabolismoRESUMEN
The ectopeptidases dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) are known to regulate T cell activation. Since selective inhibitors of DP IV and APN suppress DNA synthesis and cytokine production of stimulated T cells in a TGF-beta1-dependent manner, we tested whether combined application of DP IV and APN inhibitors enhances this immunomodulatory effect. The results show that simultaneous application of DP IV and APN inhibitors significantly suppressed DNA synthesis in mitogen- or anti-CD3-stimulated human T cells in vitro when compared to the use of a single DP IV or APN inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting both DP IV and APN led to a potent treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). This review summarizes the evidence for the role of both enzymes in T cell activation in vitro and in vivo and provides a rationale for using combined and dual peptidase inhibitors to treat autoimmune diseases like MS.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Antígenos CD13/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV , Animales , Antígenos CD13/inmunología , Dipeptidil Peptidasa 4/inmunología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/enzimología , Inflamación/inmunología , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéuticoRESUMEN
Inhibitors of alanyl-aminopeptidase e.g. phebestin increase the expression of transforming growth factor (TGF)-beta1 in mononuclear cells. We investigated whether phebestin also produced this effect in CD4+CD25+ T-cells and whether phebestin-treated CD4+CD25+ T-cells were capable of ameliorating acute colitis in mice. The suppressive activity of mouse CD4+CD25+ T-cells was assessed in vitro by co-culture with splenocytes. mRNA expression associated with the suppressive phenotype was determined in vitro and in vivo. The in vivo role of phebestin-exposed CD4+CD25+ T-cells was studied in sodium dextran sulfate-induced acute colitis in mice. The proliferation of activated effector T-cells or splenocytes in vitro was inversely correlated with the number of CD4+CD25+ T-cells. Phebestin pre-treatment substantially enhanced the suppressive activity of these cells and increased expression levels of TGF-beta1 and FoxP3. Furthermore, transfer of CD4+CD25+ T-cells exposed to phebestin for a short time ex vivo significantly reduced the mouse colitis disease activity index. We conclude that aminopeptidase inhibitors support the suppressive activity as well as TGF-beta1 and FoxP3 expression of natural regulatory T-cells.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Antígenos CD4/metabolismo , Colitis/enzimología , Factores de Transcripción Forkhead/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Linfocitos T Reguladores/enzimología , Factor de Crecimiento Transformador beta1/genética , Animales , Colitis/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The ectopeptidases Dipeptidylpeptidase IV and Alanyl-Aminopeptidase N, strongly expressed by both, activated and regulatory T cells were shown to co-operate in T cell regulation. Based on the findings that DPIV and APN inhibitors induce the TGF-beta1 and IL-10 production and a suppression of T helper cell proliferation in parallel, and that particularly APN inhibitors amplify the suppressing activity of regulatory T cells, both peptidases represent a promising target complex for treatment of diseases associated with an imbalanced T cell response, such as inflammatory bowel diseases (IBD). The aim of the present study was to analyze the therapeutic potential of DPIV and APN inhibitors in vivo in a mouse model of colitis. Balb/c mice received 3% (w/v) dextran sulphate sodium with the drinking water for 7 days. After onset of colitis symptoms, inhibitor treatment started at day 3. Disease activity index (DAI) was assessed daily, supplemented by histological and immunological analysis. While the DPIV inhibitor Lys-[Z(NO])(2)]-pyrrolidide or the APN-inhibitor Actinonin alone had marked but no significant therapeutic effects, the simultaneous administration of both inhibitors reduced colitis activity in comparison to placebo treated mice, significantly (DAI 4.8 vs. 7.7, p<0.005). A newly developed compound IP12.C6 with inhibitory capacity toward both enzymes significantly attenuated the clinical manifestation of colitis (DAI 3.2 vs. 7.6, p<0.0001). TGF-beta mRNA was found to be up-regulated in colon tissue of inhibitor-treated animals. In summary our results strongly suggest that combined DPIV and APN inhibition by synthetic inhibitors represents a novel and efficient approach for the pharmacological therapy of IBD by triggering endogenous immunosuppressive mechanisms.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Inhibidores de la Dipeptidil-Peptidasa IV , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Animales , Peso Corporal/efectos de los fármacos , Colitis/sangre , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Citocinas/sangre , Sulfato de Dextran/farmacología , Quimioterapia Combinada , Femenino , Factores de Transcripción Forkhead/genética , Expresión Génica/efectos de los fármacos , Ácidos Hidroxámicos/uso terapéutico , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/inducido químicamente , Lisina/análogos & derivados , Lisina/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Pirrolidinas/uso terapéutico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The ectoenzymes dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, CD13) have been implicated in the regulation of T cell activation and function. Both DP IV and APN serve as targets of efficient enzymatic inhibitors which induce autocrine production of TGF-beta1 and subsequent suppression of T cell proliferation and cytokine release. Here, we tested the hypothesis that the simultaneous inhibition of DP IV and APN enzymatic activity on leukocytes potentiates the anti-inflammatory effect of single DP IV or APN inhibitors. Our data show that the combined application of DP IV and APN inhibitors increased suppression of DNA synthesis in human peripheral blood mononuclear cells and isolated T cells in vitro when compared to the use of a single ectopeptidase inhibitor. Moreover, the combined action of DP IV and APN inhibitors markedly increased TGF-beta1 production associated with the observed immunosuppressive effects. In vivo, targeting DP IV and APN provided a potent therapeutic approach for the treatment of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Taken together, our study suggests that combined DP IV and APN inhibition on pathogenic T cells represents a novel and efficient therapy for autoimmune disease of the central nervous system by a mechanism that involves an active TGF-beta1-mediated anti-inflammatory effect at the site of pathology.
Asunto(s)
Antígenos CD13/antagonistas & inhibidores , Inhibidores de la Dipeptidil-Peptidasa IV , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inhibidores de Proteasas/uso terapéutico , Linfocitos T/efectos de los fármacos , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Ácidos Hidroxámicos/farmacología , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/efectos de los fármacos , Lisina/análogos & derivados , Lisina/farmacología , Ratones , Ratones Endogámicos , Proteína Proteolipídica de la Mielina/inmunología , Fragmentos de Péptidos/inmunología , Fitohemaglutininas/farmacología , Mitógenos de Phytolacca americana/farmacología , Inhibidores de Proteasas/farmacología , Pirrolidinas/farmacología , Linfocitos T/metabolismo , Tiazoles/farmacología , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , VacunaciónRESUMEN
We investigated here the effect of l-arginine on asymmetric dimethylarginine (ADMA) or homocysteine-accelerated endothelial aging. Endothelial cells were cultured in medium containing 70micromol/L arginine until fourteenth passage. ADMA, dl-homocysteine, and l-arginine were replaced every 48h starting at the fourth passage. ADMA or homocysteine inhibited significantly the population doublings (PD) and accelerated the process of aging. Co-incubation with l-arginine enhanced PD, inhibited senescence associated beta-galactosidase activity, and increased telomerase activity. This effect was associated with an increase in NO synthesis and NO synthase protein expression. Furthermore, l-arginine-induced NO formation was accompanied by a reduction in oxidative stress and an increase in protein expression and enzyme activity of heme oxygenase (HO)-1. The NO synthase inhibitor l-NAME completely abolished the effect of l-arginine on ADMA or homocysteine-accelerated aging. These findings demonstrate that l-arginine prevents the onset of endothelial aging in ADMA or homocysteine-treated cells by increasing NO formation and consequently the induction of HO-1. This might provide a new strategy to delay ADMA or homocysteine-accelerated aging.