RESUMO
Temporary elevation of tumor temperature, also known as hyperthermia, is a safe and well-tolerated treatment modality. The efficacy of hyperthermia can be improved by efficient thermosensitizers, and various candidate drugs, including inhibitors of the heat stress response, have been explored in vitro and in animal models, but clinically relevant thermosensitizers are lacking. Here, we employ unbiased in silico approaches to uncover new mechanisms and compounds that could be leveraged to increase the thermosensitivity of cancer cells. We then focus on elesclomol, a well-performing compound, which amplifies cell killing by hyperthermia by 5- to 20-fold in cell lines and outperforms clinically applied chemotherapy when combined with hyperthermia in vitro. Surprisingly, our findings suggest that the thermosensitizing effects of elesclomol are independent of its previously reported modes of action but depend on copper shuttling. Importantly, we show that, like elesclomol, multiple other copper shuttlers can thermosensitize, suggesting that disturbing copper homeostasis could be a general strategy for improving the efficacy of hyperthermia.
Assuntos
Cobre , Hidrazinas , Neoplasias , Animais , Temperatura , Febre , Hipertermia , Neoplasias/tratamento farmacológicoRESUMO
BACKGROUND: Activated eosinophils cause major pathology in stable and exacerbating asthma; however, they can also display protective properties like an extracellular antiviral activity. Initial murine studies led us to further explore a potential intracellular antiviral activity by eosinophils. METHODS: To follow eosinophil-virus interaction, respiratory syncytial virus (RSV) and influenza virus were labeled with a fluorescent lipophilic dye (DiD). Interactions with eosinophils were visualized by confocal microscopy, electron microscopy, and flow cytometry. Eosinophil activation was assessed by both flow cytometry and ELISA. In a separate study, eosinophils were depleted in asthma patients using anti-IL-5 (mepolizumab), followed by a challenge with rhinovirus-16 (RV16). RESULTS: DiD-RSV and DiD-influenza rapidly adhered to human eosinophils and were internalized and inactivated (95% in ≤ 2 hours) as reflected by a reduced replication in epithelial cells. The capacity of eosinophils to capture virus was reduced up to 75% with increasing severity of asthma. Eosinophils were activated by virus in vitro and in vivo. In vivo this correlated with virus-induced loss of asthma control. CONCLUSIONS: This previously unrecognized and in asthma attenuated antiviral property provides a new perspective to eosinophils in asthma. This is indicative of an imbalance between protective and cytotoxic properties by eosinophils that may underlie asthma exacerbations.
Assuntos
Asma/etiologia , Eosinófilos/metabolismo , Viroses/complicações , Viroses/virologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Asma/diagnóstico , Asma/metabolismo , Modelos Animais de Doenças , Eosinófilos/patologia , Eosinófilos/ultraestrutura , Humanos , Vírus da Influenza A/fisiologia , Lectinas Tipo C/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Testes de Função RespiratóriaRESUMO
Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle.
Assuntos
Barreira Hematoencefálica/microbiologia , Infecções do Sistema Nervoso Central/patologia , Interações Hospedeiro-Patógeno , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium marinum/crescimento & desenvolvimento , Animais , Encéfalo/microbiologia , Modelos Animais de Doenças , Macrófagos/microbiologia , Peixe-ZebraRESUMO
Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.
Assuntos
Cápsulas Bacterianas/metabolismo , Infecções por Mycobacterium não Tuberculosas/metabolismo , Mycobacterium marinum/patogenicidade , Sistemas de Secreção Tipo VII/metabolismo , Virulência/fisiologia , Animais , Linhagem Celular , Cromatografia em Camada Fina , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Immunoblotting , Microscopia Eletrônica , Mycobacterium marinum/metabolismo , Fatores de Virulência/metabolismo , Peixe-ZebraRESUMO
Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP+ osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm2; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.
Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Gelsolina/deficiência , Osteoclastos/metabolismo , Absorciometria de Fóton/métodos , Animais , Densidade Óssea/fisiologia , Reabsorção Óssea/genética , Diferenciação Celular/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismoRESUMO
Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.
Assuntos
Materiais Biocompatíveis/farmacologia , Células Gigantes de Corpo Estranho/ultraestrutura , Implantes Experimentais , Animais , Adesão Celular , Forma Celular , Reação a Corpo Estranho , Células Gigantes de Corpo Estranho/citologia , Camundongos , Mitocôndrias , Osteoclastos , OvinosRESUMO
BACKGROUND: There is a need for biomarkers to screen the effectiveness of (novel) therapeutic agents for psoriasis at an early stage. OBJECTIVE: We aimed to determine which of the changes in psoriatic skin correlates best with clinical improvement 4 weeks after effective adalimumab therapy. METHODS: Twenty-two psoriatic arthritis patients were randomized to receive adalimumab or placebo. T cell numbers and markers of innate immunity were estimated in lesional and nonlesional skin biopsies at baseline and after 4 weeks of treatment. RESULTS: CD161+ and elastase+ dermal cells in lesional skin were significantly reduced upon 4 weeks of successful adalimumab treatment compared with placebo. CONCLUSION: Early improvement of psoriasis lesions during adalimumab therapy is associated with a marked reduction of infiltrated dermal CD161+ T cells and elastase+ neutrophils, suggesting that these parameters could be used as biomarkers to monitor early changes after active treatment in small proof-of-concept studies of short duration.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Psoriásica/tratamento farmacológico , Psoríase/tratamento farmacológico , Pele/efeitos dos fármacos , Adalimumab , Adulto , Idoso , Artrite Psoriásica/metabolismo , Biomarcadores/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Psoríase/metabolismo , Linfócitos T/metabolismo , Adulto JovemRESUMO
Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work showed that a single, short co-treatment with a HSR (HSP90) inhibitor ganetespib prolongs and potentiates the effects of hyperthermia on DNA repair, enhances hyperthermic sensitization to radio- and chemotherapeutic agents, and reduces thermotolerance. In the current study, we first validated these results using an extended panel of cell lines and more robust methodology. Next, we examined the effects of hyperthermia and ganetespib on global proteome changes. Finally, we evaluated the potential of ganetespib to boost the efficacy of thermo-chemotherapy and thermo-radiotherapy in a xenograft murine model of cervix cancer. Our results revealed new insights into the effects of HSR inhibition on cellular responses to heat and show that ganetespib could be employed to increase the efficacy of hyperthermia when combined with radiation.
RESUMO
To investigate whether specific markers for innate immunity would diminish with successful treatment in psoriasis, we analyzed lesional and non-lesional skin biopsies taken from patients with moderate to severe psoriasis during 12 weeks of treatment with etanercept in correlation with the clinical response. In the clinical responders (PASI reduction >50%), all markers (CD3, CD68, CD161, elastase, BDCA-2, TNF-alpha) showed a decline during treatment, indicating a pivotal role for innate immunity in the pathogenesis of psoriasis.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Imunidade Inata , Imunoglobulina G/uso terapêutico , Psoríase/tratamento farmacológico , Psoríase/patologia , Receptores do Fator de Necrose Tumoral/uso terapêutico , Pele/patologia , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Biópsia , Complexo CD3/metabolismo , Etanercepte , Feminino , Humanos , Lectinas Tipo C/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Elastase Pancreática/metabolismo , Psoríase/metabolismo , Receptores Imunológicos/metabolismo , Pele/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Streptococcus suis is a porcine pathogen, causing severe invasive infections. S. suis serotype 9 is increasingly causing disease in Dutch and Chinese pig herds, but it is unknown whether all serotype 9 isolates are equally virulent and markers that can identify virulent strains are not available. Therefore, discrimination between virulent isolates and carriage isolates typically not associated with disease, is currently not possible. We collected tonsillar S. suis isolates from 6 herds not previously diagnosed with S. suis infections, and clinical S. suis isolates of previously diseased pigs. We confirmed the virulence of a virulent type strain and one representative clinical isolate, and the lack of virulence of two carriage isolates, in a pig infection model. Phylogenetic analysis of whole genome sequences of 124 isolates resulted in 10 groups, of which two were almost uniquely populated by clinical isolates. The population structure of S. suis serotype 9 appears highly diverse. However, analysis of the capsule loci sequences showed variation in a single region which fully correlated with a virulent genotype. Transmission electron microscopy suggested differences in capsule thickness between carriage and clinical genotypes. In conclusion, we found that that the S. suis serotype 9 population in the Netherlands is diverse. A distinct virulence-associated lineage was identified and could be discriminated based on the capsule locus sequence. Whilst the difference in virulence cannot be directly attributed to the DNA sequence, the correlation of capsule locus sequence with virulence could be used in the development of diagnostic tests to identify potential virulent S. suis serotype 9 in pigs.
Assuntos
DNA Bacteriano/genética , Filogenia , Sorogrupo , Infecções Estreptocócicas , Streptococcus suis , Doenças dos Suínos , Animais , Humanos , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus suis/genética , Streptococcus suis/patogenicidade , Streptococcus suis/ultraestrutura , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/microbiologiaRESUMO
Calcium bodies are internal epithelial sacs found in terrestrial isopods of the family Trichoniscidae that contain a mineralized extracellular matrix that is deposited and resorbed in relation to the molt cycle. Calcium bodies in several trichoniscids are filled with bacteria, the function of which is currently unknown. The woodlouse Hyloniscus riparius differs from other trichoniscids in that it possesses two different pairs of calcium bodies, the posterior pair being filled with bacteria and the anterior pair being devoid of bacteria. We explored the development of these organs and bacterial colonization of their lumen during the postmarsupial development with the use of optical clearing and whole-body confocal imaging of larval and juvenile stages. Our results show that calcium bodies are formed as invaginations of the epidermis in the region of intersegmental membranes during the postmarsupial development. The anterior pair of calcium bodies is generated during the first postmarsupial manca stage, whereas the posterior calcium bodies first appear in juveniles and are immediately colonized by bacteria, likely through a connection between the calcium body lumen and the body surface. Mineral is deposited in calcium bodies as soon as they are present.
Assuntos
Cálcio/metabolismo , Matriz Extracelular/fisiologia , Isópodes/crescimento & desenvolvimento , Muda , Animais , Epiderme/crescimento & desenvolvimento , Epiderme/ultraestrutura , Matriz Extracelular/ultraestrutura , Isópodes/ultraestrutura , Larva/crescimento & desenvolvimento , Larva/ultraestrutura , Microscopia Eletrônica de VarreduraRESUMO
Expression of the Autoimmune regulator (AIRE) outside of the thymus has long been suggested in both humans and mice, but the cellular source in humans has remained undefined. Here we identify AIRE expression in human tonsils and extensively analyzed these "extra-thymic AIRE expressing cells" (eTACs) using combinations of flow cytometry, CyTOF and single cell RNA-sequencing. We identified AIRE+ cells as dendritic cells (DCs) with a mature and migratory phenotype including high levels of antigen presenting molecules and costimulatory molecules, and specific expression of CD127, CCR7, and PDL1. These cells also possessed the ability to stimulate and re-stimulate T cells and displayed reduced responses to toll-like receptor (TLR) agonists compared to conventional DCs. While expression of AIRE was enriched within CCR7+CD127+ DCs, single-cell RNA sequencing revealed expression of AIRE to be transient, rather than stable, and associated with the differentiation to a mature phenotype. The role of AIRE in central tolerance induction within the thymus is well-established, however our study shows that AIRE expression within the periphery is not associated with an enriched expression of tissue-restricted antigens (TRAs). This unexpected finding, suggestive of wider functions of AIRE, may provide an explanation for the non-autoimmune symptoms of APECED patients who lack functional AIRE.
Assuntos
Células Dendríticas/imunologia , Tonsila Palatina/citologia , Fatores de Transcrição/metabolismo , Antígeno B7-H1/metabolismo , Comunicação Celular/imunologia , Células Cultivadas , Pré-Escolar , Células Dendríticas/metabolismo , Humanos , Lactente , Recém-Nascido , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Ativação Linfocitária , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Cultura Primária de Células , Receptores CCR7/metabolismo , Linfócitos T/imunologia , Fatores de Transcrição/imunologia , Proteína AIRERESUMO
The complement system is a key driver of neuroinflammation. Activation of complement by all pathways, results in the formation of the anaphylatoxin C5a and the membrane attack complex (MAC). Both initiate pro-inflammatory responses which can contribute to neurological disease. In this study, we delineate the specific roles of C5a receptor signaling and MAC formation during the progression of experimental autoimmune encephalomyelitis (EAE)-mediated neuroinflammation. MAC inhibition was achieved by subcutaneous administration of an antisense oligonucleotide specifically targeting murine C6 mRNA (5 mg/kg). The C5a receptor 1 (C5aR1) was inhibited with the C5a receptor antagonist PMX205 (1.5 mg/kg). Both treatments were administered systemically and started after disease onset, at the symptomatic phase when lymphocytes are activated. We found that antisense-mediated knockdown of C6 expression outside the central nervous system prevented relapse of disease by impeding the activation of parenchymal neuroinflammatory responses, including the Nod-like receptor protein 3 (NLRP3) inflammasome. Furthermore, C6 antisense-mediated MAC inhibition protected from relapse-induced axonal and synaptic damage. In contrast, inhibition of C5aR1-mediated inflammation diminished expression of major pro-inflammatory mediators, but unlike C6 inhibition, it did not stop progression of neurological disability completely. Our study suggests that MAC is a key driver of neuroinflammation in this model, thereby MAC inhibition might be a relevant treatment for chronic neuroinflammatory diseases.
Assuntos
Anti-Inflamatórios/uso terapêutico , Complexo de Ataque à Membrana do Sistema Complemento/antagonistas & inibidores , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Encefalite/tratamento farmacológico , Encefalite/etiologia , Encefalomielite Autoimune Experimental/complicações , Animais , Anti-Inflamatórios/química , Axônios/efeitos dos fármacos , Axônios/patologia , Axônios/ultraestrutura , Ativação do Complemento , Complexo de Ataque à Membrana do Sistema Complemento/química , Modelos Animais de Doenças , Exorribonucleases/uso terapêutico , Masculino , Camundongos , Microscopia Eletrônica , Modelos Biológicos , Peptídeos Cíclicos/uso terapêutico , RNA Mensageiro/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/química , Receptor da Anafilatoxina C5a/metabolismo , Sinaptofisina/metabolismo , Sinaptofisina/ultraestruturaRESUMO
Aims: The pathology of heart failure is characterized by poorly contracting and dilated ventricles. At the cellular level, this is associated with lengthening of individual cardiomyocytes and loss of sarcomeres. While it is known that the transcription factor myocyte enhancer factor-2 (MEF2) is involved in this cardiomyocyte remodelling, the underlying mechanism remains to be elucidated. Here, we aim to mechanistically link MEF2 target genes with loss of sarcomeres during cardiomyocyte remodelling. Methods and results: Neonatal rat cardiomyocytes overexpressing MEF2 elongated and lost their sarcomeric structure. We identified myotonic dystrophy protein kinase (DMPK) as direct MEF2 target gene involved in this process. Adenoviral overexpression of DMPK E, the isoform upregulated in heart failure, resulted in severe loss of sarcomeres in vitro, and transgenic mice overexpressing DMPK E displayed disruption of sarcomere structure and cardiomyopathy in vivo. Moreover, we found a decreased expression of sarcomeric genes following DMPK E gain-of-function. These genes are targets of the transcription factor serum response factor (SRF) and we found that DMPK E acts as inhibitor of SRF transcriptional activity. Conclusion: Our data indicate that MEF2-induced loss of sarcomeres is mediated by DMPK via a decrease in sarcomeric gene expression by interfering with SRF transcriptional activity. Together, these results demonstrate an unexpected role for DMPK as a direct mediator of adverse cardiomyocyte remodelling and heart failure.
Assuntos
Cardiomiopatias/enzimologia , Insuficiência Cardíaca/enzimologia , Fatores de Transcrição MEF2/metabolismo , Miócitos Cardíacos/enzimologia , Miotonina Proteína Quinase/metabolismo , Sarcômeros/enzimologia , Remodelação Ventricular , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Células HEK293 , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Fatores de Transcrição MEF2/genética , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/ultraestrutura , Miotonina Proteína Quinase/genética , Fosforilação , Ratos Wistar , Sarcômeros/genética , Sarcômeros/ultraestrutura , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 ± 7% as surface area and 24 ± 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease.
Assuntos
Vasos Sanguíneos/anatomia & histologia , Gengiva/anatomia & histologia , Histocitoquímica/métodos , Imageamento Tridimensional/métodos , Imagem Óptica/métodos , Células Endoteliais/química , Humanos , Microscopia , Microscopia Confocal , Microscopia de Fluorescência , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Coloração e Rotulagem/métodosRESUMO
The cause of lichen planus is still unknown. Previously we showed human herpes virus 7 (HHV-7) DNA and proteins in lesional lichen planus skin, and significantly less in non-lesional lichen planus, psoriasis or healthy skin. Remarkably, lesional lichen planus skin was infiltrated with plasmacytoid dendritic cells. If HHV-7 is associated with lichen planus, then HHV-7 replication would reduce upon lichen planus remission. HHV-7 DNA detection was performed by nested PCR and HHV-7 protein by immunohistochemistry on lesional skin biopsies from lichen planus patients before treatment and after remission. Biopsies were obtained from lichen planus lesions before treatment (n = 18 patients) and after remission (n = 13). Before treatment 61% biopsies contained HHV-7 DNA versus 8% after remission (P = 0.01). HHV-7-protein positive cell numbers diminished significantly after remission in both dermis and epidermis. Expression of HHV-7 was mainly detected in BDCA-2 positive plasmacytoid dendritic cells rather than CD-3 positive lymphocytes. HHV-7 replicates in plasmacytoid dendritic cells in lesional lichen planus skin and diminishes after remission. This study further supports our hypothesis that HHV-7 is associated with lichen planus pathogenesis.
Assuntos
Antígenos Virais/metabolismo , Células Dendríticas/metabolismo , Herpesvirus Humano 7/metabolismo , Líquen Plano/metabolismo , Líquen Plano/terapia , Acitretina/uso terapêutico , Corticosteroides/uso terapêutico , Adulto , Biópsia , Terapia Combinada , DNA Viral/efeitos dos fármacos , DNA Viral/metabolismo , Células Dendríticas/patologia , Células Dendríticas/virologia , Feminino , Herpesvirus Humano 7/genética , Herpesvirus Humano 7/patogenicidade , Humanos , Ceratolíticos/uso terapêutico , Líquen Plano/virologia , Masculino , Pessoa de Meia-Idade , Fototerapia/métodos , Indução de Remissão , Infecções por Roseolovirus/metabolismo , Infecções por Roseolovirus/patologia , Pele/metabolismo , Pele/patologia , Pele/virologia , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Several reports have indicated that the chemokine receptor CCR5 and its ligands, especially CCL5 (formerly known as RANTES), may play a role in the pathogenesis of psoriasis. The purpose of this investigation was to examine the expression of CCR5 and its ligands in chronic plaque psoriasis and to evaluate the clinical and immunohistochemical effect of a CCR5 receptor inhibitor. Immunohistochemical analysis showed low but significant increased total numbers of CCR5 positive cells in epidermis and dermis of lesional skin in comparison to non-lesional skin. However, relative expression of CCR5 proportional to the cells observed revealed that the difference between lesional and non-lesional skin was only statistically significant in the epidermis for CD3 positive cells and in the dermis for CD68 positive cells. Quantification of mRNA by reverse transcriptase-polymerase chain reaction only showed an increased expression of CCL5 (RANTES) in lesional skin. A randomized placebo-controlled clinical trial in 32 psoriasis patients revealed no significant clinical effect and no changes at the immunohistochemical level comparing patients treated with placebo or a CCR5 inhibitor SCH351125. We conclude that although CCR5 expression is increased in psoriatic lesions, this receptor does not play a crucial role in the pathogenesis of psoriasis.
Assuntos
Antagonistas dos Receptores CCR5 , Óxidos N-Cíclicos/uso terapêutico , Piperidinas/uso terapêutico , Psoríase/tratamento farmacológico , Piridinas/uso terapêutico , Adulto , Idoso , Método Duplo-Cego , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Oximas , Receptores CCR5/análise , Receptores CCR5/genética , Receptores CCR5/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/químicaRESUMO
Traditionally the evaluation of the cellular infiltrate and protein expression in skin tissue sections is done by manual quantification. However, for reliable evaluation of histology in the development of new anti-psoriatic treatments there is a need for a more time-efficient and reproducible method. To test the use of digital image analysis (DIA) in this situation we compared the assessment of immunohistochemically stained skin sections with the more traditional manual quantification (MQ) and semi-quantitative analysis (SQA). The number of CD3+ T cells and the expression of E-selectin were evaluated in stained paired skin biopsies from 11 patients with chronic plaque psoriasis before and after initiation of anti-psoriasis therapy. We observed significant correlations between MQ and DIA for the number of T cells (epidermis: r=0.88, P< or =0.01, dermis r=0.87, P< or =0.01). Both DIA and MQ were equally effective in detecting reductions of T-cell numbers in active-treated patients. MQ took 20 h, compared to 6 h for DIA. We also observed significant correlations between SQA and DIA for the expression of E-selectin (r=0.88, P< or =0.01), although DIA was more sensitive than SQA to detect (early) changes. SQA took 10 h, compared to 4 h for DIA. In conclusion, the quantification of the inflammatory infiltrate in psoriatic lesional skin by DIA generated similar results as MQ and SQA in a reliable, reproducible and higher time efficient fashion.
Assuntos
Processamento de Imagem Assistida por Computador , Psoríase/patologia , Pele/patologia , Linfócitos T/patologia , Complexo CD3/análise , Selectina E/análise , Humanos , Imuno-Histoquímica , Contagem de Linfócitos , Variações Dependentes do Observador , Psoríase/metabolismoRESUMO
Foreign body multinucleated giant cells (FBGCs) and osteoclasts share several characteristics, like a common myeloid precursor cell, multinuclearity, expression of tartrate-resistant acid phosphatase (TRAcP) and dendritic cell-specific transmembrane protein (DC-STAMP). However, there is an important difference: osteoclasts form and reside in the vicinity of bone, while FBGCs form only under pathological conditions or at the surface of foreign materials, like medical implants. Despite similarities, an important distinction between these cell types is that osteoclasts can resorb bone, but it is unknown whether FBGCs are capable of such an activity. To investigate this, we differentiated FBGCs and osteoclasts in vitro from their common CD14+ monocyte precursor cells, using different sets of cytokines. Both cell types were cultured on bovine bone slices and analyzed for typical osteoclast features, such as bone resorption, presence of actin rings, formation of a ruffled border, and characteristic gene expression over time. Additionally, both cell types were cultured on a biomimetic hydroxyapatite coating to discriminate between bone resorption and mineral dissolution independent of organic matrix proteolysis. Both cell types differentiated into multinucleated cells on bone, but FBGCs were larger and had a higher number of nuclei compared to osteoclasts. FBGCs were not able to resorb bone, yet they were able to dissolve the mineral fraction of bone at the surface. Remarkably, FBGCs also expressed actin rings, podosome belts and sealing zones--cytoskeletal organization that is considered to be osteoclast-specific. However, they did not form a ruffled border. At the gene expression level, FBGCs and osteoclasts expressed similar levels of mRNAs that are associated with the dissolution of mineral (e.g., anion exchange protein 2 (AE2), carbonic anhydrase 2 (CAII), chloride channel 7 (CIC7), and vacuolar-type H+-ATPase (v-ATPase)), in contrast the matrix degrading enzyme cathepsin K, which was hardly expressed by FBGCs. Functionally, the latter cells were able to dissolve a biomimetic hydroxyapatite coating in vitro, which was blocked by inhibiting v-ATPase enzyme activity. These results show that FBGCs have the capacity to dissolve the mineral phase of bone, similar to osteoclasts. However, they are not able to digest the matrix fraction of bone, likely due to the lack of a ruffled border and cathepsin K.
Assuntos
Reabsorção Óssea/patologia , Durapatita/metabolismo , Células Gigantes de Corpo Estranho/citologia , Monócitos/citologia , Osteoclastos/citologia , Animais , Reabsorção Óssea/metabolismo , Bovinos , Diferenciação Celular , Células Gigantes de Corpo Estranho/metabolismo , Técnicas Imunoenzimáticas , Microscopia Eletrônica de Transmissão , Monócitos/metabolismo , Osteoclastos/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Alefacept, a LFA-3/IgG1 fusion protein, interferes with the activation and proliferation of T cells by binding to the CD2 receptor on their surfaces. The clinical efficacy of this drug has been demonstrated in chronic plaque psoriasis. We performed a single-center, open-label study to investigate the immunohistochemical effects in psoriatic lesional skin. A group of 11 patients with plaque psoriasis all received 12 weekly doses of 7.5 mg alefacept intravenously. Skin biopsies were obtained at baseline and on days 8, 43 and 92, and were evaluated by digital image analysis after immunohistochemical staining. After completion of treatment, 8 out of the 11 patients experienced a reduction in PASI of 50% or more compared to baseline. Immunohistochemical analysis displayed a gradual decrease in the number of cutaneous T cells during therapy, with a significant reduction in epidermal CD8+ cells and dermal CD4+ cells on day 92. Patients with a reduction in PASI of 50% or more after therapy had a clearance of effector/memory T cells from the epidermis, in contrast to patients with a reduction in PASI of less than 50%. These findings support the hypothesis that effector/memory T cells play a prominent role in the pathogenesis of psoriasis, and that alefacept is capable of reducing these cells in lesional psoriatic skin.