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1.
Exp Dermatol ; 33(5): e15099, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38794814

RESUMO

Suitable human models for the development and characterization of topical compounds for inflammatory skin diseases such as atopic dermatitis are not readily available to date. We describe here the development of a translational model involving healthy human skin mimicking major aspects of AD and its application for the characterization of topical Janus kinase inhibitors. Full thickness human abdominal skin obtained from plastic surgery stimulated in vitro with IL4 and IL13 shows molecular features of AD. This is evidenced by STAT6 phosphorylation assessed by immunohistochemistry and analysis of skin lysates. Broad transcriptome changes assessed by AmpliSeq followed by gene set variation analysis showed a consistent upregulation of gene signatures characterizing AD in this model. Topical application of experimental formulations of compounds targeting the JAK pathway to full thickness skin normalizes the molecular features of AD induced by IL4 and IL13 stimulation. The inhibitory effects of topical JAK inhibitors on molecular features of AD are supported by pharmacokinetic analysis. The model described here is suited for the characterization of topical compounds for AD and has the potential to be extended to other inflammatory skin diseases and pathophysiological pathways.


Assuntos
Dermatite Atópica , Inibidores de Janus Quinases , Pele , Humanos , Dermatite Atópica/tratamento farmacológico , Pele/metabolismo , Pele/efeitos dos fármacos , Inibidores de Janus Quinases/farmacologia , Fator de Transcrição STAT6/metabolismo , Interleucina-4/metabolismo , Interleucina-13/metabolismo , Fosforilação , Transcriptoma , Modelos Biológicos , Pirimidinas/farmacologia , Administração Tópica , Piperidinas
2.
Eur J Pharmacol ; 945: 175533, 2023 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-36690055

RESUMO

BACKGROUND: Histamine has been postulated to play a role in atopic dermatitis via histamine receptor 4, mediating pruritic and inflammatory effects. The H4R antagonist adriforant (PF-3893787 or ZPL389) indicated clinical efficacy in a Ph2a study in atopic dermatitis. Preclinical investigations of adriforant had been scarce as experiments in transfectants with H4R from several species suggested partial agonism, not seen in human cells. OBJECTIVE: During the Ph2b trial in AD, we performed experiments to understand the pharmacology of adriforant in primary murine cells and in vivo models. We assessed its effects on ERK phosphorylation and transcriptional changes in bone marrow-derived mast cells, histamine-dependent Ca2+ flux in neurons and histamine-induced itch response. In addition, its impact on MC903-induced skin inflammation was evaluated. RESULTS: We show that, contrary to transfectants, adriforant is a competitive antagonist of the murine histamine receptor 4, antagonizes histamine-induced ERK phosphorylation, normalizes histamine-induced transcriptional changes in mast cells and reduces histamine-dependent Ca2+ flux in neurons. Administration to mice reduces acute histamine-induced itch response. In addition, adriforant ameliorates inflammation in the mouse MC903 model. CONCLUSIONS: Our results suggest that functional inhibition of histamine receptor 4 by adriforant reduces itch and inflammation in vivo. The effects observed in mice, however, did not translate to clinical efficacy in patients as the Ph2b clinical trial with adriforant did not meet pre-specified efficacy endpoints. Given the complex pathogenesis of AD, antagonism of histamine receptor 4 alone appears insufficient to reduce disease severity in AD patients, despite the effects seen in mouse models.


Assuntos
Dermatite Atópica , Humanos , Camundongos , Animais , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/induzido quimicamente , Histamina/farmacologia , Prurido/induzido quimicamente , Prurido/tratamento farmacológico , Receptores Histamínicos , Inflamação/tratamento farmacológico , Pele
3.
Clin Transl Immunology ; 10(3): e1255, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33747510

RESUMO

OBJECTIVE: Ligelizumab is a humanised IgG1 anti-IgE antibody that binds IgE with higher affinity than omalizumab. Ligelizumab had greater efficacy than omalizumab on inhaled and skin allergen provocation responses in mild allergic asthma. This multi-centre, randomised, double-blind study was designed to test ligelizumab in severe asthma patients not adequately controlled with high-dose inhaled corticoids plus long-acting ß2-agonist. METHODS: Patients received 16 weeks ligelizumab (240 mg q2w), omalizumab or placebo subcutaneously, and ACQ-7 was measured as primary outcome at Week 16. In addition, the study generated dose-ranging data of ligelizumab and safety data. RESULTS: A total of 471 patients, age 47.4 ± 13.36 years, were included in the study. Treatment with ligelizumab did not significantly improve asthma control (ACQ-7) and exacerbation rates compared to omalizumab and placebo. Therefore, primary and secondary objectives of the study were not met. The compound was well tolerated, and the safety profile showed no new safety findings. Pharmacokinetic data demonstrated faster clearance and lower serum concentrations of ligelizumab than historical omalizumab data, and exploratory in vitro data showed differential IgE blocking properties relative to FcεRI and FcεRII/CD23 between the two compounds. CONCLUSION: Ligelizumab failed to demonstrate superiority over placebo or omalizumab. Although ligelizumab is more potent than omalizumab at inhibiting IgE binding to the high-affinity FcεRI, there is differential IgE blocking properties relative to FcεRI and FcεRII/CD23 between the two compounds. Therefore, the data suggest that different anti-IgE antibodies might be selectively efficacious for different IgE-mediated diseases.

5.
Bioorg Med Chem Lett ; 25(20): 4642-7, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26320624

RESUMO

Design and optimization of benzo- and pyrido-thiazoles/isothiazoles are reported leading to the discovery of the potent, orally bioavailable Syk inhibitor 5, which was found to be active in a rat PK/PD model. Compound 5 showed acceptable overall kinase selectivity. However, in addition to Syk it also inhibited Aurora kinase in enzymatic and cellular settings leading to findings in the micronucleus assay. As a consequence, compound 5 was not further pursued.


Assuntos
Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Relação Dose-Resposta a Droga , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Quinase Syk , Tiazóis/administração & dosagem , Tiazóis/química
6.
J Med Chem ; 58(4): 1950-63, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25633741
7.
Bioorg Med Chem Lett ; 24(19): 4617-4621, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25217444

RESUMO

Jak3, together with Jak1, is involved in signal transduction initiated by cytokines signaling through the common gamma chain which are important in immune homeostasis and immune pathologies. Based on genetic evidence Jak3 has been considered to be an attractive target for immunosuppression. The Jak inhibitor tofacitinib (CP-690,550) which is an approved drug for rheumatoid arthritis was originally introduced as a selective Jak3 inhibitor, however, it also inhibits Jak1 and Jak2. The search for new selective Jak3 inhibitors has yielded several compounds whose profiles will be reviewed here. Implications on Jak3 as a therapeutic target are also discussed.


Assuntos
Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Humanos , Janus Quinase 3/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
9.
Blood ; 121(20): 4101-9, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23558016

RESUMO

It is not known how naive B cells compute divergent chemoattractant signals of the T-cell area and B-cell follicles during in vivo migration. Here, we used two-photon microscopy of peripheral lymph nodes (PLNs) to analyze the prototype G-protein-coupled receptors (GPCRs) CXCR4, CXCR5, and CCR7 during B-cell migration, as well as the integrin LFA-1 for stromal guidance. CXCR4 and CCR7 did not influence parenchymal B-cell motility and distribution, despite their role during B-cell arrest in venules. In contrast, CXCR5 played a nonredundant role in B-cell motility in follicles and in the T-cell area. B-cell migration in the T-cell area followed a random guided walk model, arguing against directed migration in vivo. LFA-1, but not α4 integrins, contributed to B-cell motility in PLNs. However, stromal network guidance was LFA-1 independent, uncoupling integrin-dependent migration from stromal attachment. Finally, we observed that despite a 20-fold reduction of chemokine expression in virus-challenged PLNs, CXCR5 remained essential for B-cell screening of antigen-presenting cells. Our data provide an overview of the contribution of prototype GPCRs and integrins during naive B-cell migration and shed light on the local chemokine availability that these cells compute.


Assuntos
Linfócitos B/fisiologia , Comunicação Celular/fisiologia , Quimiocinas/fisiologia , Quimiotaxia de Leucócito/genética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Receptores CCR7/fisiologia , Receptores CXCR4/fisiologia , Receptores CXCR5/fisiologia , Células Estromais/fisiologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Comunicação Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Quimiocinas/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Feminino , Deleção de Genes , Antígeno-1 Associado à Função Linfocitária/fisiologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores CCR7/genética , Receptores CCR7/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Receptores CXCR5/genética , Receptores CXCR5/metabolismo , Células Estromais/metabolismo
10.
Chem Biol ; 18(3): 314-23, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21439476

RESUMO

Genetic deficiency of Jak3 leads to abrogation of signal transduction through the common gamma chain (γc) and thus to immunodeficiency suggesting that specific inhibition of Jak3 kinase may result in immunosuppression. Jak1 cooperates with Jak3 in signaling through γc-containing receptors. Unexpectedly, a Jak3-selective inhibitor was less efficient in abolishing STAT5 phosphorylation than pan-Jak inhibitors. We therefore explored the roles of Jak1 and Jak3 kinase functionality in signaling using a reconstituted system. The presence of kinase-inactive Jak1 but not kinase-inactive Jak3 resulted in complete abolishment of STAT5 phosphorylation. Specific inhibition of the "analog-sensitive" mutant AS-Jak1 but not AS-Jak3 by the ATP-competitive analog 1NM-PP1 abrogated IL-2 signaling, corroborating the data with the selective Jak3 inhibitor. Jak1 thus plays a dominant role over Jak3 and these data challenge the notion that selective ATP-competitive Jak3 kinase inhibitors will be effective.


Assuntos
Janus Quinase 1/metabolismo , Janus Quinase 3/metabolismo , Receptores de Citocinas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Humanos , Interleucina-2/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 1/genética , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/genética , Camundongos , Mutação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Receptores de Citocinas/química , Fator de Transcrição STAT5/metabolismo
11.
J Med Chem ; 54(1): 284-8, 2011 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-21155605

RESUMO

We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.


Assuntos
Indóis/síntese química , Janus Quinase 3/antagonistas & inibidores , Maleimidas/síntese química , Linhagem Celular , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Humanos , Imunossupressores/síntese química , Imunossupressores/química , Imunossupressores/farmacologia , Indóis/química , Indóis/farmacologia , Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/química , Maleimidas/química , Maleimidas/farmacologia , Modelos Moleculares , Estrutura Molecular , Fosforilação , Piperidinas , Pirimidinas/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT5/metabolismo , Relação Estrutura-Atividade
12.
Cell Transplant ; 19(11): 1369-81, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20977829

RESUMO

Diabetic patients suffer from impaired wound healing, characterized by only modest angiogenesis and cell proliferation. Stem cells may stimulate healing, but little is known about the kinetics of mobilization and function of bone marrow progenitor cells (BM-PCs) during diabetic wound repair. The objective of this study was to investigate the kinetics of BM-PC mobilization and their role during early diabetic wound repair in diabetic db/db mice. After wounding, circulating hematopoietic stem cells (Lin(-)c-Kit(+)Sca-1(+)) stably increased in the periphery and lymphoid tissue of db/db mice compared to unwounded controls. Peripheral endothelial progenitor cells (CD34(+)VEGFR(+)) were 2.5- and 3.5-fold increased on days 6 and 10 after wounding, respectively. Targeting the CXCR4-CXCL12 axis induced an increased release and engraftment of endogenous BM-PCs that was paralleled by an increased expression of CXCL12/SDF-1α in the wounds. Increased levels of peripheral and engrafted BM-PCs corresponded to stimulated angiogenesis and cell proliferation, while the addition of an agonist (GM-CSF) or an antagonist (ACK2) did not further modulate wound healing. Macroscopic histological correlations showed that increased levels of stem cells corresponded to higher levels of wound reepithelialization. After wounding, a natural release of endogenous BM-PCs was shown in diabetic mice, but only low levels of these cells homed in the healing tissue. Higher levels of CXCL12/SDF-1α and circulating stem cells were required to enhance their engraftment and biological effects. Despite controversial data about the functional impairment of diabetic BM-PCs, in this model our data showed a residual capacity of these cells to trigger angiogenesis and cell proliferation.


Assuntos
Células da Medula Óssea/citologia , Diabetes Mellitus Tipo 2/complicações , Células-Tronco/fisiologia , Cicatrização/fisiologia , Animais , Proliferação de Células , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Ferimentos e Lesões/complicações , Ferimentos e Lesões/terapia
13.
PLoS One ; 5(2): e9175, 2010 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-20161793

RESUMO

BACKGROUND: CXCR7 (RDC1), the recently discovered second receptor for CXCL12, is phylogenetically closely related to chemokine receptors, but fails to couple to G-proteins and to induce typical chemokine receptor mediated cellular responses. The function of CXCR7 is controversial. Some studies suggest a signaling activity in mammalian cells and zebrafish embryos, while others indicate a decoy activity in fish. Here we investigated the two propositions in human tissues. METHODOLOGY/PRINCIPAL FINDINGS: We provide evidence and mechanistic insight that CXCR7 acts as specific scavenger for CXCL12 and CXCL11 mediating effective ligand internalization and targeting of the chemokine cargo for degradation. Consistently, CXCR7 continuously cycles between the plasma membrane and intracellular compartments in the absence and presence of ligand, both in mammalian cells and in zebrafish. In accordance with the proposed activity as a scavenger receptor CXCR7-dependent chemokine degradation does not become saturated with increasing ligand concentrations. Active CXCL12 sequestration by CXCR7 is demonstrated in adult mouse heart valves and human umbilical vein endothelium. CONCLUSIONS/SIGNIFICANCE: The finding that CXCR7 specifically scavenges CXCL12 suggests a critical function of the receptor in modulating the activity of the ubiquitously expressed CXCR4 in development and tumor formation. Scavenger activity of CXCR7 might also be important for the fine tuning of the mobility of hematopoietic cells in the bone marrow and lymphoid organs.


Assuntos
Quimiocina CXCL11/metabolismo , Quimiocina CXCL12/metabolismo , Receptores CXCR/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Quimiocina CXCL11/genética , Quimiocina CXCL12/genética , Embrião não Mamífero/metabolismo , Endocitose/fisiologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Miocárdio/metabolismo , Receptores CXCR/genética , Receptores CXCR/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Peixe-Zebra
14.
Genesis ; 47(8): 545-58, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19517561

RESUMO

The chemokine receptors CCR2 and CCR5 represent potential novel therapeutic targets to treat important inflammatory and infectious diseases, including atherosclerosis and HIV infection. To study the functions of both receptors in vivo, we aimed to generate Ccr2/Ccr5 double-deficient mice. As these genes are separated by <20 kb, they were inactivated consecutively by two rounds of gene targeting in embryonic stem (ES) cells. Thereby neomycin and hygromycin selection cassettes flanked by four identical loxP recognition sequences for Cre recombinase were integrated into the ES cell genome together with EGFP and DsRed2 reporter genes. Both selection cassettes could be deleted in vitro by transiently transfecting ES cells with Cre expression vectors. However, after blastocyst microinjection these cells yielded only weak chimeras, and germline transmission was not achieved. Therefore, Ccr2/Ccr5 double-deficient mice were generated from ES cells still carrying both selection cassettes. Microinjection of zygotes with a recombinant fusion protein consisting of maltose-binding protein and Cre (MBP-Cre) allowed the selective deletion of both cassettes. All sequences in between and both reporter genes were left intact. Deletion of both selection cassettes resulted in enhanced DsRed2 reporter gene expression. Cre protein microinjection of zygotes represents a novel approach to perform complex recombination tasks.


Assuntos
Genes Reporter , Integrases/administração & dosagem , Receptores CCR2/genética , Receptores CCR5/genética , Zigoto , Animais , Sequência de Bases , Primers do DNA , Células-Tronco Embrionárias/metabolismo , Deleção de Genes , Vetores Genéticos , Células Germinativas , Hibridização in Situ Fluorescente , Integrases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microinjeções , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Med Chem ; 51(24): 7915-20, 2008 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19053768

RESUMO

The interaction of the chemokine receptor CXCR4 with its ligand CXCL12 is involved in many biological processes such as hematopoesis, migration of immune cells, as well as in cancer metastasis. CXCR4 also mediates the infection of T-cells with X4-tropic HIV functioning as a coreceptor for the viral envelope protein gp120. Here, we describe highly potent, selective CXCR4 inhibitors that block CXCR4/CXCL12 interactions in vitro and in vivo as well as the infection of target cells by X4-tropic HIV.


Assuntos
Receptores CXCR4/química , Tioureia/química , Administração Oral , Animais , Disponibilidade Biológica , Química Farmacêutica/métodos , Quimiocina CXCL12/química , Quimiocinas/metabolismo , Desenho de Fármacos , Proteína gp120 do Envelope de HIV/química , Humanos , Concentração Inibidora 50 , Modelos Químicos , Ratos , Receptores CXCR4/antagonistas & inibidores , Linfócitos T/metabolismo , Linfócitos T/virologia
16.
Int Immunopharmacol ; 6(13-14): 1902-10, 2006 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-17161343

RESUMO

The sphingosine-1-phosphate receptor agonist FTY720 induces lymphopenia by inhibiting lymphocyte egress from thymus and lymph nodes. The immediate effect of the drug on T cells in blood and lymphoid tissues is well documented, however effects on peripheral T cell sub-populations have not been studied. We therefore analyzed the changes in T cell subset compositions in liver, lung, kidney, spleen, lymph nodes and blood induced by FTY720-treatment using 9-parameter flow cytometry. In untreated mice, naive T cells were present in all peripheral organs. Naive T cells were depleted from peripheral organs within 3 days by FTY720, and with slower kinetics from lymphoid organs. Antigen-experienced T cell subsets were less affected by FTY720-treatment and substantial numbers were retained in the periphery. The proportion of CD8(+)CD44(+)CD43(+) Gr-1(+) effector memory cells increased after FTY720-treatment, while that of CD8(+)CD44(+)CD62L(+) central memory cells was unchanged. Our data demonstrate that naive T cells pass peripheral tissues as part of their default recirculation pathway. FTY720 treatment primarily affects the recirculation of naive and central memory cells, both of which re-circulate through lymph nodes on a regular basis, but does not influence effector memory cells. This suggests that treatment with FTY720 may not interfere with immune functions mediated locally by tissue-resident peripheral effector/memory T cells.


Assuntos
Depleção Linfocítica , Tecido Linfoide/efeitos dos fármacos , Propilenoglicóis/farmacologia , Esfingosina/análogos & derivados , Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD/análise , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Contagem de Células , Movimento Celular/efeitos dos fármacos , Feminino , Cloridrato de Fingolimode , Citometria de Fluxo , Imunossupressores/farmacologia , Rim/citologia , Rim/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Linfonodos/citologia , Linfonodos/efeitos dos fármacos , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos C57BL , Esfingosina/farmacologia , Baço/citologia , Baço/efeitos dos fármacos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia
17.
J Invest Dermatol ; 126(9): 2057-64, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16778803

RESUMO

CCR5 is one of the major inflammatory chemokine receptors with potential therapeutical applications in humans. However, the redundancy of chemokines and their receptors, and the species specificity of chemokine receptor antagonists pose challenges to understanding of the role they play in pharmacological situations. To address this question, we used a humanized severe combined immunodeficient mouse model grafted with human skin and autologous leukocytes, and evaluated the effect of a blocking antibody against human CCR5, on CCL5-induced cutaneous leukocyte recruitment in vivo. At baseline, CCL5 induced a significant recruitment of T cells mainly of the memory phenotype, of monocytes/macrophages, eosinophils, and IFN-gamma(+) but not IL-4(+) and IL-5(+) cells. In vivo, anti-CCR5 antibody was able to almost completely inhibit the recruitment of monocytes/macrophages and T-helper (Th)1-type cells to inhibit partially the attraction of memory T cells, but had no effect on eosinophil infiltration, although all these cell types express other CCL5 binding chemokine receptors than CCR5. These results indicate that the in vivo environment regulates target cell specificity of CCL5 leading to differential cell recruitment, suggesting that antagonizing CCR5 receptor may be of therapeutic value in diseases such as acquired immuno deficiency syndrome, where CCL5/CCR5, monocytes, and Th1-type cells play a predominant role.


Assuntos
Movimento Celular/imunologia , Quimiocinas CC/imunologia , Imunoterapia/métodos , Receptores CCR5/imunologia , Transplante de Pele/imunologia , Células Th1/imunologia , Animais , Anticorpos/farmacologia , Quimiocina CCL5 , Modelos Animais de Doenças , Eosinófilos/citologia , Eosinófilos/imunologia , Humanos , Memória Imunológica , Interferon gama/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos SCID , Monócitos/citologia , Monócitos/imunologia , Mutação , Receptores CCR5/genética , Células Th1/citologia , Células Th1/metabolismo , Células Th2/citologia , Células Th2/imunologia , Transplante Heterólogo/imunologia
18.
Cytometry A ; 69(6): 533-40, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16646049

RESUMO

BACKGROUND: The analysis of cells from multiple experimental groups by multiparameter flow cytometry leads to the generation of complex data sets, for which adequate analysis tools are not commonly available. We report here that software designed for transcriptomics applications can be used in multiparameter flow cytometry. METHODS: Lymphocytes isolated from nine different mouse organs were stained and subjected to 10-parameter flow cytometry. The resulting data set contained 594 different T cell subsets per organ per mouse and was organized into a so-called flow cytometry array (FCA). RESULTS: Computation of a hierarchical tree revealed that lymph nodes and spleen were populated by similar T cell subsets, while T cells from peripheral organs displayed a diverse subset composition. Furthermore, organ-specific T cell subsets were identified. CONCLUSIONS: This new FCA concept in flow cytomics proved to be a valuable tool for the fast and unbiased analysis of complex multiparameter flow cytometry data sets. It can be used for assessing disease progression and therapeutic intervention, and for the association of disease-related biomarkers on the protein level.


Assuntos
Citometria de Fluxo/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Subpopulações de Linfócitos T/classificação , Animais , Interpretação Estatística de Dados , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Especificidade de Órgãos
19.
Bioorg Med Chem Lett ; 16(1): 108-12, 2006 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16236504

RESUMO

The design, synthesis, and the biological evaluation of 2-benzamido-pyrimidines as novel IKK inhibitors are described. By optimization of the lead compound 3, compounds 16 and 24 are identified as good inhibitors of IKK2 with IC(50) values of 40 and 25 nM, respectively. Compound 16 also demonstrated significant in vivo activity in an acute model of cytokine release.


Assuntos
Química Farmacêutica/métodos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinase I-kappa B/antagonistas & inibidores , Pirimidinas/síntese química , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Concentração Inibidora 50 , Modelos Químicos , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade
20.
Eur J Immunol ; 34(9): 2568-78, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15307189

RESUMO

Experimental and human organ transplant studies suggest an important role for chemokine (C-C-motif) receptor-5 (CCR5) in the development of acute and chronic allograft rejection. Because early transplant damage can predispose allografts to chronic dysfunction, we sought to identify potential pathophysiologic mechanisms leading to allograft damage by using wild-type and Ccr5-deficient mice as recipients of fully MHC-mismatched heart and carotid-artery allografts. Gene expression in rejecting heart allografts was analyzed 2 and 6 days after transplantation using Affymetrix GeneChips. Microarray analysis led to identification of four metalloproteinase genes [matrix metalloproteinase (Mmp)3, Mmp12, Mmp13 and a disintegrin and metalloprotease domain (Adam)8] with significantly diminished intragraft mRNA expression in Ccr5-deficient mice at day 6. Accordingly, allografts from Ccr5-deficient mice showed less tissue remodeling and hence better preservation of the myocardial architecture compared with allografts from wild-type recipients. Moreover, survival of cardiac allografts was significantly increased in Ccr5-deficient mice. Carotid artery allografts from Ccr5-deficient recipients showed better tissue preservation, and significant reduction of neointima formation and CD3+ T cell infiltration. Ccr5 appears to play an important role in transplant-associated arteriosclerosis that may involve metalloproteinase-mediated vessel wall remodeling. We conclude that early tissue remodeling may be a critical feature in the predisposition of allografts to the development of chronic dysfunction.


Assuntos
Antígenos CD/genética , Arteriosclerose/prevenção & controle , Colagenases/genética , Transplante de Coração/efeitos adversos , Metaloproteinase 3 da Matriz/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Receptores CCR5/fisiologia , Proteínas ADAM , Animais , Artérias Carótidas/transplante , Ciclosporina/farmacologia , Metaloproteinase 12 da Matriz , Metaloproteinase 13 da Matriz , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante Homólogo
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