RESUMO
BACKGROUND: Stringent control of proteolytic activity represents a major therapeutic approach for wound-bed preparation. OBJECTIVES: We tested whether a protease-modulating polyacrylate- (PA-) containing hydrogel resulted in a more efficient wound-bed preparation of venous leg ulcers when compared to an amorphous hydrogel without known protease-modulating properties. METHODS: Patients were randomized to the polyacrylate-based hydrogel (n = 34) or to an amorphous hydrogel (n = 41). Wound beds were evaluated by three blinded experts using photographs taken on days 0, 7 and 14. RESULTS: After 14 days of treatment there was an absolute decrease in fibrin and necrotic tissue of 37.6 ± 29.9 percentage points in the PA-based hydrogel group and by 16.8 ± 23.0 percentage points in the amorphous hydrogel group. The absolute increase in the proportion of ulcer area covered by granulation tissue was 36.0 ± 27.4 percentage points in the PA-based hydrogel group and 14.5 ± 22.0 percentage points in the control group. The differences between the groups were significant (decrease in fibrin and necrotic tissue P = 0.004 and increase in granulation tissue P = 0.0005, respectively). CONCLUSION: In particular, long-standing wounds profited from the treatment with the PA-based hydrogel. These data suggest that PA-based hydrogel dressings can stimulate normalization of the wound environment, particularly in hard-to-heal ulcers.
Assuntos
Resinas Acrílicas , Hidrogéis , Úlcera da Perna/terapia , Peptídeo Hidrolases/administração & dosagem , Úlcera Varicosa/terapia , Ferimentos e Lesões/terapia , Resinas Acrílicas/efeitos adversos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The histoarchitecture and function of the epidermis depend on a well-controlled balance between keratinocyte proliferation and differentiation. This balance is perturbed after skin injury, and imbalance is a characteristic feature of major human skin diseases such as psoriasis and epidermal cancers. Recent studies have highlighted the importance of fibroblast-derived soluble factors for the regulation of keratinocyte proliferation and differentiation. Therefore, identification of these paracrine-acting factors and the elucidation of their mechanisms of action are necessary for understanding epidermal homeostasis, repair and disease, and these approaches will offer new potential targets for drug therapy. Here, we review exciting recent findings on the identification, regulation and function of paracrine-acting cytokines in the skin. In particular, we describe the role of fibroblast-derived mitogens as regulators of keratinocyte proliferation and differentiation, and we summarize the regulation of these factors by keratinocyte-derived interleukin 1 that involves the transcription factors c-Jun and JunB.
Assuntos
Queratinócitos/citologia , Comunicação Parácrina , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Substâncias de Crescimento/metabolismo , Substâncias de Crescimento/farmacologia , Substâncias de Crescimento/fisiologia , Humanos , Comunicação Parácrina/fisiologiaRESUMO
Epithelial-mesenchymal interactions control epidermal growth and differentiation, but little is known about the mechanisms of this interaction. We have examined the effects of human dermal microvascular endothelial cells (DMEC) and fibroblasts on keratinocytes in conventional (feeder layer) and organotypic cocultures (lifted collagen gels) and demonstrated the induction of paracrine growth factor gene expression. Clonal keratinocyte growth was similarly stimulated in cocultures with irradiated DMEC and fibroblasts as feeder cells. This effect is most probably caused by induction of growth factor expression in cocultured dermal cells. Keratinocytes stimulated mRNA levels for KGF and IL-6 in both mesenchymal cell types and GM-CSF in fibroblasts. The feeder effect could not be replaced by conditioned media or addition of isolated growth factors. In organotypic cocultures with keratinocytes growing on collagen gels (repopulated with dermal cells), a virtually normal epidermis was formed within 7 to 10 d. Keratinocyte proliferation was drastically stimulated by dermal cells (histone 3 mRNA expression and BrdU labeling) which continued to proliferate as well in the gel. Expression of all typical differentiation markers was provoked in the reconstituted epithelium, though with different localization as compared to normal epidermis. Keratins K1 and K10 appeared coexpressed but delayed, reflecting conditions in epidermal hyperplasia. Keratin localization and proliferation were normalized under in vivo conditions, i.e., in surface transplants on nude mice. From these data it is concluded that epidermal homeostasis is in part controlled by complex reciprocally induced paracrine acting factors in concert with cell-cell interactions and extracellular matrix influences.
Assuntos
Comunicação Celular , Endotélio Vascular/citologia , Fatores de Crescimento de Fibroblastos , Fibroblastos/fisiologia , Substâncias de Crescimento/genética , Queratinócitos/fisiologia , Pele/irrigação sanguínea , Sequência de Bases , Diferenciação Celular , Divisão Celular , Células Cultivadas , Colagenases/biossíntese , Colagenases/genética , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Substâncias de Crescimento/biossíntese , Humanos , Interleucinas/biossíntese , Interleucinas/genética , Queratinócitos/citologia , Queratinócitos/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Técnicas de Cultura de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The function of keratinocyte growth factor (KGF) in normal and wounded skin was assessed by expression of a dominant-negative KGF receptor transgene in basal keratinocytes. The skin of transgenic mice was characterized by epidermal atrophy, abnormalities in the hair follicles, and dermal hyperthickening. Upon skin injury, inhibition of KGF receptor signaling reduced the proliferation rate of epidermal keratinocytes at the wound edge, resulting in substantially delayed reepithelialization of the wound.
Assuntos
Fatores de Crescimento de Fibroblastos , Substâncias de Crescimento/fisiologia , Queratinócitos/citologia , Receptores de Fatores de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento/fisiologia , Pele/citologia , Cicatrização/fisiologia , Envelhecimento , Animais , Divisão Celular , Movimento Celular , Epiderme/patologia , Células Epiteliais , Fator 10 de Crescimento de Fibroblastos , Fator 7 de Crescimento de Fibroblastos , Cabelo/citologia , Cabelo/crescimento & desenvolvimento , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Fenótipo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento/genética , Transdução de SinaisRESUMO
OBJECTIVE: This prospective, multicentre application study was conducted to assess the clinical performance of Hydrosorb comfort hydrogel dressing. METHOD: Eighty-one patients (average age 67 years) with acute or chronic wounds received three dressing changes. The condition of the wound and patient-reported pain were assessed at the beginning and end of the study period. RESULTS: The mean proportion of the wound surface covered with slough fell from 63% to 34%, and the mean area of new granulation and epithelial tissue increased from 25% to 37% and 13% to 28%, respectively. The average wound size decreased from 4.7 x 2.9cm to 3.7 x 2.3cm; 29.6% of the patients reported no pain at baseline, increasing to 56.3% at the final assessment. CONCLUSION: Both acute and chronic wounds can be effectively treated with the Hydrosorb comfort hydrogel dressing.
Assuntos
Curativos Hidrocoloides/normas , Ferimentos e Lesões/terapia , Doença Aguda , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Documentação , Feminino , Tecido de Granulação , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação em Enfermagem , Dor/etiologia , Atenção Primária à Saúde , Estudos Prospectivos , Higiene da Pele/instrumentação , Higiene da Pele/métodos , Higiene da Pele/enfermagem , Resultado do Tratamento , Cicatrização , Ferimentos e Lesões/complicações , Ferimentos e Lesões/patologiaRESUMO
OBJECTIVE: To assess the clinical efficacy, tolerance and acceptance of a novel, hydroactive-impregnated dressing (Hydrotul) in the local treatment of acute and chronic wounds. METHOD: In a prospective observational study 24 centres in France, Belgium, Germany and Austria recruited 74 patients. At each dressing change the investigators evaluated the condition of the wound, perilesional skin and patient-reported pain. Overall, five dressing changes were documented, or until complete healing occurred. The hydroactive properties of the dressing were assessed in laboratory tests by measuring fluid absorption capacity and kinetics. RESULTS: Patients were treated for an average of 17 days. The wound condition improved markedly during the observation period. The wound area covered with fibrinous slough decreased from 29% to 14%, epithelialisation increased from 19% to 54% and 22 wounds were completely healed by the end of the study. The number of patients reporting severe and moderate wound pain decreased from 35% to 19% and the proportion of patients without wound pain doubled from 27% to 60%. In laboratory tests, Hydrotul absorbed two to three times the amount of fluid compared with other impregnated wound dressings and the kinetics of absorption was much faster. CONCLUSION: The novel hydroactive impregnated dressing supports the healing process in patients with acute and chronic wounds and reduces wound pain. The dressing absorbs excess wound exudate while keeping the wound surface moist and protecting perilesional skin.
Assuntos
Bandagens , Pomadas/uso terapêutico , Cicatrização , Absorção , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
The structure of a new segment of the gene locus encoding the variable regions of human immunoglobulins of the Kappa type (VK) has been elucidated. This segment (cluster B) encompasses six VK sequences, which belong to three different subgroups and which are arranged in the same transcriptional orientation. Part of cluster B was found to be very similar to another region of the VK gene locus, which was cloned previously (cluster A). Sequence differences between the homologous region of clusters A and B range from 0.2% to 3.7% depending on the position of the VK sequences. The divergence is in the same range for genes and pseudogenes. Hybridization experiments with DNAs from different individuals clearly demonstrate that the two segments are located at different positions within the VK locus and do not represent allelic variants. The sequence homology between clusters A and B is higher than the homology of both clusters to an allelic variant, which is represented by a DNA segment that had been isolated from another individual. These results, together with a report in the literature of two other homologous regions in the VK locus, make it very likely that a major part of even the whole locus is duplicated. In this case, VK gene numbers would be higher than previously estimated on the basis of hybridization studies. An inverse orientation of VK gene clusters would explain published data on rearrangement products in B-cells if an inversion-deletion mechanism is assumed.
Assuntos
Mapeamento Cromossômico , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Alelos , Autorradiografia , Sequência de Bases , DNA , Genes , Humanos , Hibridização de Ácido NucleicoRESUMO
Epidermodysplasia verruciformis-associated human papillomaviruses and in particular human papillomavirus type 5 were recently shown to be highly prevalent in psoriatic skin. We have analyzed lesional skin from 54 psoriasis patients for infections with genital-specific and epidermodysplasia verruciformis-specific human papillomaviruses to define the spectrum of involved human papillomavirus types and to test if it is influenced by psoralen ultraviolet A therapy. Using polymerase chain reaction analysis we could detect human papillomavirus sequences in skin lesions of 83% of the tested patients. In contrast, human papillomavirus-DNA was only demonstrated in 19% of skin samples from 42 dermatologically healthy, immunocompetent individuals. Sequence analysis of the polymerase chain reaction amplimers revealed 14 human papillomavirus types, all belonging to the epidermodysplasia verruciformis or epidermodysplasia verruciformis-related papillomaviruses. Only in one case we identified sequences related to those of genital viruses, which, however, represented a putatively new human papillomavirus type. The most prevalent human papillomavirus type in our patient series was human papillomavirus type 36, found in 62% of the patients positive for human papillomavirus-DNA, followed by human papillomavirus type 5 (38%) and human papillomavirus type 38 (24%). Multiple infections with two to five different human papillomavirus types could be detected in skin samples of 63% of the analyzed patients. The overall human papillomavirus detection rate did not differ significantly between patients which have been subjected to psoralen ultraviolet A photochemotherapy or solely treated with topical preparations (77 vs 89%). Human papillomavirus type 5, however, could be detected significantly more frequent in lesions of psoralen ultraviolet A-treated patients (p < 0.001). Our data strongly argue for infections with epidermodysplasia verruciformis-specific papillomaviruses being an almost consistent feature of the lesional psoriatic skin and substantiate the importance of further studies to elucidate a possible involvement of human papillomaviruses in psoriasis pathology.
Assuntos
Terapia PUVA , Papillomaviridae , Infecções por Papillomavirus/tratamento farmacológico , Psoríase/tratamento farmacológico , Infecções Tumorais por Vírus/tratamento farmacológico , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Biópsia , DNA Viral/genética , Epidermodisplasia Verruciforme/virologia , Humanos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Papillomaviridae/classificação , Papillomaviridae/genética , Prevalência , Psoríase/epidemiologia , Psoríase/virologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Pele/patologia , Pele/virologia , Dermatopatias Virais/tratamento farmacológico , Dermatopatias Virais/patologiaRESUMO
In a patient with metastatic melanoma transmitted by the renal allograft, HLA serves as an alloantigen per se and is associated with tumor antigens at the same time. The influence of this antigeneic pattern on the Vbeta T-cell repertoire in an allogeneic melanoma, allograft, and peripheral blood mononuclear cells (PBMC) was assessed by polymerase chain reaction. Vbeta13.1 and 19 were found in both the melanoma and the graft. Vbeta14 was detected only in the melanoma and Vbeta6 was detected only in the kidney. PBMC revealed an unrestricted Vbeta pattern. Markers for cytotoxic activity of T cells--granzyme B and perforin--were not expressed during immunosuppressive therapy as clinically reflected in a nonrejecting allograft and in a progressing melanoma. In vitro PBMC proliferated to recombinant interleukin-2, whereas recombinant interferon-gamma did not augment this response. Initiation of immune therapy, in addition to discontinuation of immunosuppression, might support the rejection of the allogeneic tumor by dominant Vbeta T cells.
Assuntos
Transplante de Rim/efeitos adversos , Melanoma/etiologia , Linfócitos T/imunologia , Imunologia de Transplantes , Idoso , Feminino , Granzimas , Teste de Histocompatibilidade , Humanos , Transplante de Rim/imunologia , Transplante de Rim/patologia , Melanoma/patologia , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Estudos Retrospectivos , Serina Endopeptidases/análiseRESUMO
Epidermal-dermal cell-cell interactions are recognized to influence keratinocyte proliferation in vivo as well as in vitro. To study the underlying molecular mechanisms of epithelial-mesenchymal interactions epidermal-dermal cell co-cultures and organotypic cultures were used. Steady-state mRNA levels are described for transforming growth factors (TGF) alpha, beta1 and beta2, factors known to stimulate or inhibit the epidermal proliferation rate. In epidermal-dermal monolayer co-cultures TGF alpha hybridization signals were absent. TGF beta1 mRNA was expressed in all cell types (keratinocytes, fibroblasts and microvascular endothelial cells), yet not regulated. In contrast, TGF beta2 mRNA was significantly induced in mesenchymal cells when they were co-cultured with keratinocytes. In organotypic cultures epidermal proliferation is dependent on the presence of fibroblasts within the gel. TGF beta1 was expressed at low levels in all cell types whereas TGF beta2 transcripts were not detectable at all. TGF alpha mRNA was present in keratinocytes at high levels, independent of epidermal cell proliferation or added epidermal growth factor. These results indicate complex regulative mechanisms for TGF alpha, beta1 and beta2 at the mRNA level. However, post-transcriptional steps are involved in the activation of TGF beta1 and 2 and also have to be considered.
RESUMO
Human papillomaviruses (HPV) infect keratinocytes of skin and mucosa. Persistent infection can lead to the formation of benign tumors. In cases of high-risk HPV, such as HPV16 or 18, these may further progress to cancer. In order to support viral replication in suprabasal keratinocytes, the HPV E7 protein employs various strategies to keep keratinocytes in cycle and counteracts anti-proliferative signals from outside. HPV16 E7 can directly interfere with transforming growth factor-beta (TGF-beta) signalling by binding to Smad proteins mediating growth arrest. It has been speculated that this property of HPV16 E7 contributes to HPV-associated carcinogenesis. Here, we show that E7 proteins from different low- and high-risk HPV types bind to Smad 1 to 4. The E7 protein from HPV1, a low-risk HPV causing plantar warts, efficiently inhibited Smad 3-induced transcription. Our data strongly indicate that the Smad-binding capacity of E7 proteins from different HPVs may preserve keratinocyte proliferation required for the productive viral life cycle rather than promoting carcinogenesis.
Assuntos
Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Smad/metabolismo , Ativação Transcricional , Proliferação de Células , Queratinócitos/citologia , Queratinócitos/virologia , Papillomaviridae/química , Verrugas/virologiaRESUMO
Bacterial colonisation of wounds may delay wound healing. Modern silver-containing dressings are antimicrobial, yet cellular toxicity is a serious side-effect. We provide data for a newly formulated silver-containing ointment dressing, Atrauman Ag, for antimicrobial activity and cytotoxicity. Atrauman Ag effectively killed a panel of commensal skin as well as pathogenic bacterial strains while cytotoxicity for HaCaT keratinocytes was only around 10%. With these favourable in vitro tests, Atrauman Ag was analysed in 86 patients with traumatic and non-healing wounds of different aetiologies. The wound state was evaluated for 3 subsequent dressing changes. The slough score was reduced from 59.2 to 35.8%, granulation tissue increased from 27 to 40% and epithelialisation went up from 12.1 to 24%. We conclude that Atrauman Ag has a superior profile of antimicrobial activity over cellular toxicity and the low silver ion release rate may prevent interference with wound-healing mechanisms.
Assuntos
Antibacterianos/uso terapêutico , Curativos Oclusivos , Prata/administração & dosagem , Cicatrização/efeitos dos fármacos , Idoso , Bactérias/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pomadas/uso terapêutico , Pomadas/toxicidade , Prata/uso terapêutico , Prata/toxicidadeRESUMO
Activins are members of the transforming growth factor beta (TGF beta) superfamily, which comprises a growing group of dimeric proteins. TGF beta and several other members of this superfamily are known to play an important role in wound healing. However, expression of activin during wound healing has not been demonstrated so far. In this study we have analyzed the expression pattern of activin and activin receptors in normal and wounded skin. We found a large induction of activin A and a minor induction of activin B mRNA expression 1 day after skin injury and high expression levels of activin A and B were found within the first 7 days after wounding. At 13 days after injury, expression of activin A mRNA had returned to the basal level, whereas high levels of activin B persisted. In situ hybridization studies revealed expression of activin A in the granulation tissue below the wound and activin B in the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue. All known types of activin receptors as well as the activin binding protein follistatin were expressed in normal and wounded skin. However, no significant induction of receptor gene expression was seen during the repair process. The distribution of activins and activin receptors in the wound suggests multiple autocrine and paracrine activities of the ligands during wound healing. Our data provide evidence for a novel function of activin and indicate that--besides TGF beta s themselves--other members of this superfamily might also play an important role in tissue repair.
Assuntos
Expressão Gênica/fisiologia , Inibinas/genética , Oligopeptídeos , Peptídeos/genética , Cicatrização/genética , Receptores de Ativinas , Ativinas , Animais , Epiderme/química , Epitélio/química , Feminino , Folistatina , Glicoproteínas/genética , Tecido de Granulação/química , Inibinas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/fisiologia , RNA Mensageiro/análise , Receptores de Fatores de Crescimento/genética , PeleRESUMO
Gene transfer into the skin is a promising approach to treat inherited or acquired dermatological diseases and systemic monogenic deficiencies. For this purpose, the efficient and sustained gene delivery into keratinocytes is of critical importance. Recombinant adeno-associated virus (rAAV) vectors hold the potential to achieve a long-term gene transfer into various human organs. In order to evaluate this potential for skin gene therapy, human keratinocytes were transduced in vitro with rAAV vectors encoding the reporter genes beta-galactosidase (rAAV/LacZ) or green fluorescent protein (rAAV/GFP). Using rAAV/LacZ at a multiplicity of infection (MOI) of five transducing particles per cell, up to 70% of human keratinocytes were transduced within 48 h. This effect was independent of individual skin donors and different body areas serving as the source for keratinocyte isolation. rAAV had no significant influence on cell viability, but induced a growth arrest in transduced keratinocytes. This growth arrest was overcome by replating cells in fresh media. rAAV/GFP-transduced keratinocytes could be passaged several times, expressed GFP for up to 50 days, and passed the transgene to their daughter cells, suggesting that keratinocyto precursor cells were also transduced. Taken together, the results suggest that rAAV is a promising gene transfer vehicle for skin gene therapy.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Queratinócitos/metabolismo , Transfecção/métodos , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Imuno-Histoquímica , Proteínas Luminescentes/genética , beta-Galactosidase/genéticaRESUMO
The VK gene segments that have been transposed from the kappa locus on the short arm of chromosome 2 at 2p11-12 to other chromosomal sites are called orphons. The 18 VK orphons sequenced up to now carry defects and are to be considered pseudogenes. We now describe the VKI gene segment V108 whose sequence is without any defects and which was localized to the long arm of chromosome 2 at 2q12-14 by in situ hybridization. The V108 region may have been transposed from the short to the long arm of chromosome 2 by a pericentric inversion. Possible reasons for the conservation of its sequence are discussed. In spite of its bona fide sequence V108 is considered to be an unlikely candidate for a VK-JK rearrangement and subsequent functional expression.
Assuntos
Rearranjo Gênico , Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Humanos Par 2 , Clonagem Molecular , Cosmídeos , Humanos , Células Híbridas , Dados de Sequência Molecular , Pseudogenes , Mapeamento por RestriçãoRESUMO
The cutaneous basement membrane zone, composed of numerous macromolecules, plays a multifunctional role in tissue regeneration and maintenance. To elucidate the cellular origin and dynamics of basement membrane formation, de novo synthesis, deposition, and ultrastructural assembly of its components were analyzed in organotypic cultures of adult skin keratinocytes on collagen gels with or without collagen-embedded dermal cells. Collagen IV and laminin-1 deposition occurred only in the presence of mesenchymal cells: patchy at day 4 and continuous after 1 week. Chain-specific mRNA expression started at day 2 in both keratinocytes and fibroblasts. It steadily increased up to day 10, however, with a reciprocal induction pattern, mRNA abundance shifting from keratinocytes to fibroblasts. On the other hand, laminin-5 staining was first observed at day 4, but in keratinocyte both mono- and cocultures. This was followed by nidogen, which was detected in cocultures but also in dermal monocultures. Laminin-5 protein persisted throughout day 21, whereas nidogen steadily increased in intensity. Expression kinetics revealed high levels of laminin-5 transcripts early and in keratinocytes only, whereas nidogen was expressed later and predominantly in fibroblasts. Although basement membrane protein deposition was continuous at day 14, the ultrastructural organization was still fragmentary, eventually normalizing at 3 weeks. These data demonstrate a dynamic interaction and cooperation of epithelial and mesenchymal skin cells in basement membrane formation. This interaction is supposedly mediated via diffusible factors. Our findings further extend the scope of epithelial-mesenchymal interactions stressing that both cell compartments are essential to constitute a tissue-specific extracellular matrix structure.
Assuntos
Membrana Basal/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Pele/citologia , Comunicação Celular , Técnicas de Cocultura , Colágeno/biossíntese , Colágeno/genética , Técnicas de Cultura/métodos , Regulação da Expressão Gênica , Humanos , Laminina/biossíntese , Laminina/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , RNA Mensageiro/biossíntese , Fatores de TempoRESUMO
It has long been speculated that pro-inflammatory cytokines play an important role in wound repair. However, little is known about the temporal and spatial expression pattern of these cytokines during normal and impaired wound healing. In this study we show a strong and early induction of interleukins 1 alpha and beta (IL-alpha and beta) and of tumour necrosis factor alpha (TNF-alpha) expression after cutaneous injury. Highest levels of these cytokines were seen as early as 12-24 h after wounding. After completion of the proliferative phase of wound healing, mRNA levels of these cytokines returned to the basal level. During the early phase of wound repair, proinflammatory cytokines were predominantly expressed in polymorphonuclear leukocytes, suggesting a novel function of these cells in the initiation of wound healing. At later stages of the repair process, expression of IL-1 alpha, IL-1 beta and TNF-alpha was also seen in macrophages. Furthermore, TNF-alpha was detected in the hyperproliferative epithelium at the wound edge and IL-1 alpha was found in keratinocytes of the hair follicles. Induction of these cytokines after injury was significantly reduced during wound repair in healing-impaired glucocorticoid-treated mice. This finding demonstrates that wound healing defects are associated with impaired cytokine expression and suggests that the early induction of these genes is important for normal repair.
Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mediadores da Inflamação/metabolismo , Interleucina-1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Cicatrização/fisiologia , Animais , Feminino , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
Cellular immunity plays a major role in controlling human papilloma virus infection and development of cervical carcinoma. Mononuclear cell infiltration possibly due to the action of chemokines becomes prominent in the tumor tissue. In fact, the macrophage chemoattractant protein-1, MCP-1, was detected in cervical squamous cell carcinoma in situ, whereas absent in cultured cells. From this, unknown environmental factors were postulated regulating chemokine expression in vivo. In this study, we show high CD40 expression on cervical carcinoma cells and CD40 ligand (CD40L) staining on attracted T cells in tumor tissue, suggesting a paracrine stimulation mechanism via CD40L-CD40 interactions. We therefore investigated chemokine synthesis in nonmalignant and malignant human papilloma virus-positive cell lines after CD40L exposure. Constitutive expression of MCP-1, MCP-3, RANTES, and IFN-gamma-inducible protein-10 was almost undetectable in all cell lines tested. CD40L was able to induce MCP-1 production; however, despite much higher CD40 expression in malignant cells, MCP-1 induction was significantly lower compared with nontumorigenic cells. After sensitization with IFN-gamma, another T cell-derived cytokine showing minimal effects on CD40 expression levels, CD40 ligation led to a more than 20-fold MCP-1 induction in carcinoma cell lines. An even stronger effect was observed for IFN-gamma-inducible protein-10. Our study highlights the synergism of T cell-derived mediators such as CD40L and IFN-gamma for chemokine responses in cervical carcinoma cells, helping to understand the chemokine expression patterns observed in vivo.
Assuntos
Antígenos CD40/metabolismo , Carcinoma de Células Pequenas/imunologia , Quimiocinas/biossíntese , Interferon gama/farmacologia , Glicoproteínas de Membrana/metabolismo , Neoplasias do Colo do Útero/imunologia , Ligante de CD40 , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/virologia , Linhagem Celular Transformada , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Quimiocina CCL2/biossíntese , Quimiocina CXCL10 , Quimiocinas CXC/biossíntese , Sinergismo Farmacológico , Feminino , Humanos , Interleucina-8/biossíntese , Queratinócitos/metabolismo , Ligantes , NF-kappa B/metabolismo , Papillomaviridae , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , Ligação Proteica/imunologia , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/virologiaRESUMO
CD40, a member of the TNF receptor family, has been characterized as an important T-B cell interaction molecule. In B cells it co-stimulates isotype switching, proliferation, adhesion and is involved in cell death regulation. In addition to B cells, CD40 expression was found on transformed cells and carcinomas. However, little is known about its functions in these cell types. Recent studies show that CD40 mediates the production of pro-inflammatory cytokines in non-hematopoietic cells, inhibits proliferation or induces cell death. In some cell types the apoptotic program triggered by CD40 is only executed when protein synthesis is blocked, suggesting the existence of constitutively expressed resistance proteins. Here we demonstrate that CD40, similar to the 55-kDa TNF receptor (p55TNFR), has a dual role in the regulation of apoptosis in such cells. In the fibroblast cell line SV80 both CD40 and the p55TNFR trigger apoptosis when protein synthesis is blocked with cycloheximide (CHX). Simultaneous activation of both receptors results in markedly enhanced cell death. However, CD40 activation more than 4 h prior to a challenge with TNF/CHX paradoxically conferred resistance to TNF-induced cell death. Protection correlated with NF-kappaB induction and up-regulation of the anti-apoptotic zinc finger protein A20. Overexpression of A20 in turn rendered SV80 cells resistant to TNF cytotoxicity. In conclusion, our data provide evidence that CD40 may regulate cell death in non-hematopoietic cells in a dual fashion: the decision upon apoptosis or survival of a CD40-activated cell seems to depend on its ability to up-regulate resistance factors.
Assuntos
Apoptose/efeitos dos fármacos , Antígenos CD40/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antígenos CD/fisiologia , Linhagem Celular , Cricetinae , Cicloeximida/farmacologia , Fibroblastos/fisiologia , Humanos , NF-kappa B/fisiologia , RNA Mensageiro/análise , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Dedos de ZincoRESUMO
Contact of various cells with extracellular matrix molecules modulates their cellular functions and phenotype. Most investigations have employed dishes coated with purified matrix constituents or plain collagen I lattices omitting the effects of other important matrix components such as proteoglycans. In this study we analyze the effect of purified glycosaminoglycans (GAGs) on human fibroblasts and human umbilical vein endothelial cells (HUVEC) embedded within collagen I/III lattices. HUVEC contracted collagen I/III gels far less efficiently than fibroblasts and addition of heparan sulfate and heparin almost completely inhibited contraction. In collagen gels HUVEC down-regulated collagenase mRNA while increasing collagen I, IV mRNA expression. Addition of heparin and heparan sulfate reversed the collagen IV mRNA induction whereas hyaluronic acid and chondroitin sulfate enhanced fibronectin and collagenase transcripts. Fibroblasts readily contracted collagen gels, and mRNA levels for fibronectin, collagenase and interleukin-6 were stimulated. Gel contraction was mostly unaffected by the different glycosaminoglycans. Fibroblasts responded to the addition of dermatan sulfate, heparan sulfate and heparin with a decrease in fibronectin, collagenase and interleukin-6 mRNA. Binding studies revealed saturable binding sites on fibroblasts and HUVEC for 35S-labelled heparin, demonstrating specificity for heparin and heparan sulfate over other GAGs in competition experiments. This study implies that glycosaminoglycans participate in cell-matrix interactions by effectively modulating the cellular phenotype via high affinity binding sites.