RESUMEN
Laser scar revision was studied to measure the effects of targeting extracellular matrix protein versus tissue water on scar revision. We compared the free electron laser used at 7.7 microm (the amide III protein absorption band) to the carbon dioxide (CO2) laser and dermabrasion.Nude mice (n = 40) that had rejected skin grafts on their dorsal surface and developed mature scars were used as a model for scar revision. One-half of each scar was revised with either the free electron laser at 7.7 microm (32 to 38 mJ, nonoverlapping pulses delivered with a computerized adjustable pattern generator at 30 Hz, and two to three passes), a 100-microsec CO2 resurfacing laser (500 mJ, 5.0 Hz, and two to five passes), or dermabrasion. The untreated portion of each scar served as an internal control. Evaluation was by measurement of the clinical size of the scar using photography with quantitative computer image analysis to compare the data and histology to evaluate the quality and depth of the scars. The free electron laser at 7.7 microm was significantly better than the CO2 laser and dermabrasion for scar size reduction (p < 0.046 and p < 0.018). The CO2 laser and a highly skilled dermabrader were not statistically significantly different (p < 0.44). The result seen with less skilled dermabraders was significantly worse than all other methods (p < 0.009). The free electron laser at 7.7 microm, which is preferentially absorbed by the proteins of the extracellular matrix, provided better scar reduction than the CO2 resurfacing laser and dermabrasion. Dermabrasion by a skilled operator resulted in improvement similar to the results obtained with the CO2 resurfacing laser, but less skilled operators had significantly poorer results.
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Cicatriz/cirugía , Terapia por Láser , Animales , Dermabrasión , Terapia por Láser/métodos , Ratones , Ratones DesnudosRESUMEN
BACKGROUND AND OBJECTIVE: Short pulsed and scanned CO(2) lasers that target water molecules are currently used for cutaneous resurfacing. These CO(2) resurfacing lasers produce acute cutaneous contraction, which can be quantitated as a measure of the laser's effect. We postulated that targeting the vibrational and rotational modes of proteins with specific infrared laser wavelengths might be more effective at inducing cutaneous contraction than the CO(2) resurfacing lasers. STUDY DESIGN/MATERIALS AND METHODS: The Vanderbilt University Free Electron Laser (FEL) was used at wavelengths between 6.0-8.6 microm. The cutaneous contraction and histologic thermal damage observed was compared to that seen with a scanned CO(2) resurfacing laser. RESULTS: Peaks of cutaneous contraction at 7.2-7.4 and 7.6-7.7 microm were found, which were three-fold more efficient at producing cutaneous contraction than the 10.6 microm CO(2) laser. The 7.2 microm wavelength is associated with the CH bend of C-CH(3), 7.4 microm to the CH bend of O=C-CH(3), 7.6 microm to the C-C-C stretch, and 7.7 microm to the amide III (C-N-H) absorption band for proteins. Using light microscopy, an approximately 40 microm denaturation zone of dermal collagen was found at all FEL wavelengths tested, regardless of the effectiveness of cutaneous contraction. CONCLUSION: The mechanism of action of these infrared wavelengths on cutaneous contraction is unknown, but appears to be independent of the amount of collagen denatured as observed by light microscopy. Infrared lasers such as the FEL that target vibrational and rotational modes of proteins therefore hold promise for cutaneous application at selected wavelengths.
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Procedimientos Quirúrgicos Dermatologicos , Terapia por Láser/métodos , Piel/patología , Colágeno/análisis , Técnicas de Cultivo , Humanos , Rayos Infrarrojos/uso terapéutico , Dosis de Radiación , Sensibilidad y Especificidad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Photopheresis or extracorporeal photochemotherapy (ECP) is a novel immunomodulatory therapy based upon pheresis of light-sensitive cells. Whole blood is removed from patients who have previously ingested the photosensitizing agent 8-methoxypsoralen (8-MOP) followed by leukapheresis and exposure of the 8-MOP containing white blood cells (WBCs) extracorporeally to an ultraviolet A (UVA) light source prior to their return to the patient. In 1988, the Food and Drug Administration (FDA) approved photopheresis for the treatment of cutaneous T-cell lymphoma (CTCL). Treatment of CTCL with photopheresis has been reported in over 300 patients worldwide. Photopheresis has also demonstrated encouraging results in the treatment of solid organ transplant rejection, graft versus host disease, scleroderma, and other autoimmune diseases although fewer patients have been studied. This review will focus on the North American experience with photopheresis.
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Fotoféresis , Artritis Reumatoide/terapia , Enfermedad Injerto contra Huésped/terapia , Humanos , Linfoma Cutáneo de Células T/terapia , Metoxaleno , América del Norte , Fotoféresis/métodos , Fármacos Fotosensibilizantes , Esclerodermia Sistémica/terapiaRESUMEN
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes implicated in the invasion and metastasis of many cancers. In situ hybridization techniques were used to reveal sites of expression of collagenase (MMP-1), gelatinase 72 kd (MMP-2), gelatinase 92 kd (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in head and neck carcinomas (N = 21). Both TIMP-1 and gelatinase 72 kd were expressed in nearly all tumors, whereas the expression of collagenase and gelatinase 92 kd showed variability. Tumor-associated expression of MMPs was strongest in stromal cells near advancing margins. No differences in expression levels were detected between primary and metastatic sites. This paper reviews the literature and discusses the significance and possible implications of MMPs in head and neck squamous cell carcinoma.
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Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/enzimología , Metaloendopeptidasas/metabolismo , Adulto , Anciano , Carcinoma de Células Escamosas/metabolismo , Colagenasas/metabolismo , Cartilla de ADN , Gelatinasas/metabolismo , Amplificación de Genes , Expresión Génica , Glicoproteínas/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Histocitoquímica , Humanos , Hibridación in Situ , Metaloproteinasa 1 de la Matriz , Metaloproteinasa 2 de la Matriz , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Metaloendopeptidasas/biosíntesis , Persona de Mediana Edad , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Inhibidores Tisulares de MetaloproteinasasRESUMEN
The present study was designed to characterize similarities and differences among three wounding modalities in partial-thickness porcine wounds. We hypothesized that inherent differences, such as endogenous cytokine delivery into excisional wounds or ablation of eschar during laser vaporization, should accelerate the magnitude and sequence of reparative events above the delayed repair that is frequently observed in patients with burns. A constant mid-dermal depth of injury was created by a Padgett dermatome, a computer-controlled pulsed CO(2) laser, or a temperature-controlled metal template. Wounds were harvested after 5, 10, or 15 days. After 5 days, significant resurfacing differences were apparent with values of 54% in excisions, 29% in lasers, and 12% in standard thermal burns. Sequences of fibroblastic proliferation were measurably different among the three wound modalities. At day 5 the bromodeoxyuridine labeling index for fibroblasts showed laser wound levels greater than excision wound levels, which were greater than burn wound levels; but by day 10, the proliferative profiles indicated that burn wound levels were greater than excision wound levels, which were greater than laser wound levels. Capillary areas (an assessment of angiogenesis) differed among the three wound types throughout the study. Peak values were observed at day 5 in both excisional and laser injuries; however, standard thermal burns did not peak until day 10. Both the magnitude and sequence of expression of three matrix metalloproteinases (-1, -2, and -9) differed among the three types of injuries. Laser wounds showed the earliest peak in matrix metalloproteinase-1 expression, whereas burns showed the least expression at day 5. In conclusion, although the three types of wounds undergo similar reparative processes such as reepithelialization, fibroblastic proliferation, angiogenesis, and expression of matrix metalloproteinases, the magnitude and temporal sequences are measurably altered among the three wound modalities. A greater understanding of specific differences within wound environments may lead to more insightful design of interventional wound therapies.
RESUMEN
BACKGROUND: Few studies have assessed the long-term outcome of patients with cutaneous T-cell lymphoma (CTCL) treated with extracorporeal photochemotherapy (ECP). OBJECTIVE: Our objective was to assess the efficacy, safety, and survival of a cohort of patients with refractory T-cell lymphoma in various stages of cutaneous involvement who were treated with ECP. METHODS: Twenty patients who had received at least 6 months of ECP between September 1988 and April 1991 were reevaluated and the data analyzed statistically to obtain outcome data through December 1995. RESULTS: A complete response (disappearance of all lesions) was obtained in five patients (25%) and a partial response (disappearance of at least 50% of lesions) in five patients (25%). Of the 10 responders, seven (70%) were weaned from ECP. Two of seven patients had a relapse. Ten patients (50%) showed no response to ECP. No statistically significant differences between responders and nonresponders were found with respect to demographic, clinical, or laboratory variables. Seven patients died of causes directly related to CTCL and two patients died of unrelated causes. Median survival time for the entire cohort was 96 months (range, 16 to 152 months). An assessment of early response after 6 to 8 months of ECP had a sensitivity of 100% and a specificity of 90% for predicting long-term (> 4 years) outcome. Adverse effects were minimal. CONCLUSION: ECP is a safe effective alternative therapy for CTCL that is refractory to other therapies; it can induce a long-term, disease-free remission in a minority of patients. Response in the first 6 to 8 months of treatment predicts long-term outcome.
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Linfoma Cutáneo de Células T/tratamiento farmacológico , Fotoféresis , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Linfoma Cutáneo de Células T/mortalidad , Masculino , Metoxaleno/efectos adversos , Metoxaleno/uso terapéutico , Persona de Mediana Edad , Fotoféresis/efectos adversos , Fármacos Fotosensibilizantes/efectos adversos , Fármacos Fotosensibilizantes/uso terapéutico , Neoplasias Cutáneas/mortalidad , Tasa de SupervivenciaRESUMEN
BACKGROUND AND OBJECTIVE: We evaluated the physical changes in human skin following CO2 laser cutaneous resurfacing with either the Sharplan SilkTouch handpiece or the Coherent UltraPulse laser. STUDY DESIGN/MATERIALS AND METHODS: Three-hundred five human tissue samples and matched controls were used. Up to five laser passes were performed per specimen. Parameters evaluated included: lateral skin shrinkage, transient temperature change, isometric tension development, elasticity change, and histologic change. RESULTS: Skin shrinkage increased in direct proportion to laser pass number. Isometric tension exponentially increased and elasticity exponentially decreased with successive laser passes. The zone of thermal denaturation for the SilkTouch handpiece was 115 +/- 15 microns, and was independent of laser pass number. The zone of thermal denaturation was patchy for the UltraPulse laser treatments, regardless of pass number. A greater temperature increase was also measured for SilkTouch irradiation than with the UltraPulse laser. CONCLUSION: The observed alterations in tissue length, tension development, and elasticity obtained with SilkTouch or UltraPulse treatment may contribute to the changes in clinical appearance associated with laser cutaneous resurfacing. Our findings support a role for extracellular matrix contraction in the mechanism of action for CO2 lasers in cutaneous resurfacing.
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Rayos Láser , Ritidoplastia/métodos , Piel/efectos de la radiación , Elasticidad , Femenino , Humanos , Técnicas In Vitro , Piel/patología , Temperatura Cutánea/efectos de la radiaciónRESUMEN
The role of matrix metalloproteinases in parathyroid hormone (PTH)-induced bone resorption was assayed using a fetal rat limb bone culture system. Cotreatment of bones with PTH and recombinant inhibitor of metalloproteinases, TIMP-1, in vitro, inhibited the PTH-stimulated 45Ca release from the limb bones without affecting beta-glucuronidase release. TIMP-1 was fully effective when added during only the final 24 h of a 72 h culture with PTH but was ineffective when added for only the first 24 h of the 72 h culture. In contrast, calcitonin (CT) was effective when added for either the first 24 or the final 24 h of the culture. Using in situ hybridization, the mRNA for collagenase was detected in mononuclear cells of cultured bone. Treatment of the bones with PTH resulted in an increase in the number of cells producing collagenase mRNA, some of which had osteoclastic morphology, PTH also caused a dramatic induction of the mRNA for the 92-kD gelatinase B metalloproteinase in both mononuclear and osteoclastic cells. There was no detectable mRNA for the metalloproteinases stromelysin-1, stromelysin-2, or matrilysin in PTH-treated or control cultures. These results suggest that PTH-induced bone resorption is mediated, at least in part, by the induction of collagenase and gelatinase B mRNA in bone cells.
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Resorción Ósea/inducido químicamente , Resorción Ósea/enzimología , Colagenasas/biosíntesis , Hormona Paratiroidea/farmacología , Animales , Resorción Ósea/metabolismo , Calcio/metabolismo , Colagenasas/genética , ADN Complementario , Inhibidores Enzimáticos/farmacología , Glicoproteínas/farmacología , Hibridación in Situ , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Técnicas de Cultivo de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Inhibidores Tisulares de MetaloproteinasasRESUMEN
We report a patient with severe epidermolysis bullosa acquisita (EBA) whose disease was refractory to conventional treatments. New bullae continued to develop over greater than 50% of his body surface area despite therapy. His course was complicated by hyperglycaemia, sepsis, hypoxia caused by pulmonary Aspergillus infection and an idiopathic cardiomyopathy. His EBA resolved after treatment with extracorporeal photochemotherapy (ECP). Hence, ECP may be effective in the treatment of severe EBA which has failed to respond to standard treatment regimens.
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Epidermólisis Ampollosa Adquirida/terapia , Fotoféresis/métodos , Enfermedad Aguda , Adulto , Epidermólisis Ampollosa Adquirida/patología , Humanos , MasculinoRESUMEN
Singlet oxygen has been postulated to be generated by Ultraviolet (UV) A irradiation of mammalian cells. We studied the role of singlet oxygen in the downstream signaling of the complex UV response leading to the induction of matrix-metalloproteinase-1 (interstitial collagenase/MMP-1). Exposure of cultured human fibroblasts to singlet oxygen, generated in a dark reaction by thermodissociation of the endoperoxide of the disodium salt of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) induced collagenase mRNA steady state levels in a dose dependent manner. The increase in collagenase expression after singlet-oxygen exposure generated with 3 mM NDPO2 was equivalent to that observed with UVA at a dose rate of 200-300 kJ/m2 and developed in a similar time course. In contrast, mRNA levels of TIMP-1, the specific tissue inhibitor of metalloproteinases, remained unchanged. Indirect evidence for the role of singlet oxygen in the UVA induction of collagenase comes from studies using singlet oxygen enhancer or quencher. Accordingly, incubation in deuterium oxide, an enhancer of singlet-oxygen lifetime, led to an additional increase in steady-state levels of collagenase mRNA after exposure to NDPO2 or to UVA irradiation. In contrast, sodium azide, a potent quencher of singlet oxygen, almost totally abrogated the induction of collagenase after exposure of fibroblasts to NDPO2 or to UVA irradiation. Similar results were obtained in studies of the proteins by radioimmunoprecipitation of MMP-1 and TIMP-1 using specific antibodies. Collectively, our data provide circumstantial evidence that singlet oxygen mediates the UVA induction of collagenase in vitro, whereas it does not exert any effect on TIMP-1 synthesis. The unbalanced synthesis of interstitial collagenase may contribute to the connective tissue damage in vivo related to photoaging and other photocutaneous disorders.
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Colagenasas/biosíntesis , Oxígeno/farmacología , Piel/efectos de la radiación , Rayos Ultravioleta , Azidas/farmacología , Supervivencia Celular/efectos de los fármacos , Colagenasas/metabolismo , Colagenasas/efectos de la radiación , Óxido de Deuterio/farmacología , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Masculino , Metaloproteinasa 1 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Mutágenos/farmacología , Naftoles/farmacología , ARN Mensajero/análisis , Oxígeno Singlete , Azida Sódica , Inhibidores Tisulares de MetaloproteinasasRESUMEN
Degradative events in remodeling connective tissues are mediated through the actions of one or more members of the matrix metalloproteinase family. Conversely, members of the tissue inhibitors of metalloproteinase (TIMP) family act to attenuate proteolysis. Because collagenase and TIMP are rapidly secreted into the extracellular matrix following their biosynthesis and may not remain near their cell of origin, we undertook an immunohistochemical examination of human burn injuries to establish the distribution of these proteins during acute wound repair. Immunostaining for collagenase and TIMP was markedly increased within the wound bed but not in adjacent regions of histologically normal skin. Immunoreactive collagenase was first noted at the eschar-dermal interface by day 3 after injury and became very prominent in the dermis from day 5 to day 17. By day 5, focal patches of immunoreactive collagenase were found at the epidermal-dermal junctions at the wound margins. Within the wound bed, intense staining for collagenase was noted in the connective tissue surrounding the surviving epithelial appendages and around blood vessels. Immunoreactive TIMP was detected by day 2 both in the dermis and the overlying eschar but rapidly assumed the same interfacial pattern as described for collagenase. Staining for TIMP was only sporadically found at the dermal-epidermal margins and surrounding surviving epithelial appendages. Like collagenase, TIMP was prominently localized about vascular structures. These studies demonstrate that, in acute wounds, immunoreactive collagenase and TIMP are generally increased throughout the area of injury but particularly so at interface zones including eschar-dermis, epidermis-dermis, appendages-dermis, and around vascular structures.
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Quemaduras/enzimología , Colagenasas/análisis , Glicoproteínas/análisis , Colagenasas/farmacocinética , Glicoproteínas/farmacocinética , Humanos , Inmunohistoquímica , Inhibidores de la Metaloproteinasa de la Matriz , Distribución Tisular , Inhibidores Tisulares de Metaloproteinasas , Cicatrización de Heridas/fisiologíaRESUMEN
The process of wound healing sets in motion a complex and dynamic series of events, which includes the remodeling of the extracellular matrix. Degradation of matrix macromolecules is mediated through the actions of the matrix metalloproteinase family. Conversely, the actions of this enzyme family are regulated by tissue inhibitors of metalloproteinases (TIMPs). In this study, we have developed riboprobes derived from human cDNAs representing collagenase, 72-kDa gelatinase, and TIMP and have found them to be sufficiently specific and sensitive for use in in situ hybridization studies of porcine burn wounds. Expression of these mRNAs, although not seen in uninjured skin, was found to be a predictable and locally distinct event in wound repair. Transcripts for collagenase and TIMP but not 72-kDa gelatinase were detected at the resurfacing epithelial margin; label was also detected in and around follicular epithelium within the wound bed. Transcripts for both metalloenzymes and TIMP were found throughout the viable dermis and subcutaneous tissues underlying the wound bed. However, expression of 72-kDa gelatinase was most prominent in the superficial dermis adjacent to the resurfacing epidermis at the wound margin. Collagenase and TIMP transcripts were particularly prominent in a perivascular pattern in the dermis and in the connective tissue network surrounding adipocytes in the subcutaneous zone. Numerous cell types appeared to be involved, including keratinocytes, fibroblasts, macrophages, and endothelial cells. Future exploitation of this porcine thermal injury model is likely to provide information about the spatial and temporal patterns of matrix metalloproteinase and TIMP expression in cutaneous wound healing.
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Quemaduras/metabolismo , Colagenasas/genética , Gelatinasas/genética , Glicoproteínas/genética , ARN Mensajero/metabolismo , Cicatrización de Heridas/fisiología , Animales , Secuencia de Bases , Colagenasas/metabolismo , Espacio Extracelular/metabolismo , Gelatinasas/química , Gelatinasas/metabolismo , Metaloendopeptidasas/antagonistas & inhibidores , Sondas Moleculares/genética , Datos de Secuencia Molecular , Peso Molecular , Piel/metabolismo , Porcinos , Distribución Tisular , Inhibidores Tisulares de MetaloproteinasasRESUMEN
Interstitial collagenase, a matrix metalloproteinase, is known to be actively involved in remodeling of cutaneous tissues including those affected by trauma, neoplasia, and inflammation. Conversely, collagenase activity is blocked by tissue inhibitor of metalloproteinases (TIMP). Because both collagenase and TIMP are rapidly secreted into the extracellular matrix, their sites of synthesis remain ambiguous. To determine the site and sequence of collagenase and TIMP expression in cutaneous wound repair, we examined partial and full thickness excisions of human burn wounds representing days 2 to 34 postinjury. Prominent labeling for collagenase and TIMP was detected in epithelial cells at the burn margin and at the edges of surviving hair follicles and eccrine sweat structures in the wound bed. Within the dermis, cells expressing collagenase and TIMP were at first perivascular in location and later appeared at the interface zone between viable and nonviable dermis. A diversity of cell types including macrophages, fibroblasts, endothelial cells, and keratinocytes appeared to express mRNAs for collagenase and TIMP. Little if any labeling was detected in necrotic regions, in adjacent nonwounded dermis, or epidermis. Our data indicate that collagenase and TIMP are temporally and spatially regulated during cutaneous wound repair.
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Quemaduras/enzimología , Quemaduras/fisiopatología , Colagenasas/genética , Glicoproteínas/genética , ARN Mensajero/análisis , Cicatrización de Heridas/genética , Adolescente , Adulto , Anciano , Secuencia de Bases , Quemaduras/patología , Niño , Preescolar , Colagenasas/análisis , Femenino , Fibroblastos/química , Fibroblastos/enzimología , Fibroblastos/patología , Glicoproteínas/análisis , Humanos , Hibridación in Situ , Lactante , Macrófagos/química , Macrófagos/enzimología , Macrófagos/patología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Mensajero/genética , Piel/química , Piel/enzimología , Piel/patología , Factores de Tiempo , Inhibidores Tisulares de Metaloproteinasas , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: Many regimens are used for cutaneous T-cell lymphoma (CTCL), but with advanced disease response rates and patient survival are not adequate with any current therapy. Recently extracorporeal photochemotherapy (ECP) was proposed as an alternative therapy. OBJECTIVE: Our purpose is to present the results of ECP in patients with CTCL refractory to other treatments. METHODS: Patients with CTCL received ECP at 3- to 5-week intervals for at least 6 months. All patients except one were in stage T2 (patch/plaque) or higher. Eight patients had extracutaneous disease involving lymph nodes (six patients), bone marrow (five), or Sézary cells (six). The interval between initial symptoms and diagnosis was 5.9 +/- 1.9 years (mean +/- standard error of the mean) and the interval between diagnosis and ECP was 2.2 +/- 0.4 years. RESULTS: A complete response (disappearance of all lesions) was obtained in five patients (25%) and a partial response (disappearance of at least 50% of lesions) in six patients (30%). Four patients (20%) showed stabilization of their disease and five progressed (25%). The only variable that predicted responders versus nonresponders was the number of ECP sessions (p < 0.05 by multivariate logistic regression). In contrast, no separate beneficial effect of adjunctive chemotherapy (p > 0.5) or electron beam therapy (p > 0.1) was found. CONCLUSION: Long-term ECP may be an effective alternative treatment for CTCL refractory to other therapies and is likely to be even more useful when combined with other modalities.
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Linfoma Cutáneo de Células T/tratamiento farmacológico , Fotoquimioterapia/métodos , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Terapia Combinada , Circulación Extracorporea , Femenino , Humanos , Linfoma Cutáneo de Células T/patología , Masculino , Metoxaleno/uso terapéutico , Persona de Mediana Edad , Estadificación de Neoplasias , Fotoquimioterapia/efectos adversos , Inducción de Remisión , Neoplasias Cutáneas/patologíaRESUMEN
We have recently demonstrated that human keratinocytes synthesize and secrete procollagenase and tissue inhibitor of metalloproteinases (TIMP) in culture. We have examined the response of keratinocyte collagenase production to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), interleukin-1, extracellular matrix proteins and phagocytosis. Collagenase production in keratinocytes was markedly stimulated by TPA and paralleled the morphologic changes induced by the phorbol ester. Synthesis of collagenase increased six- to 34-fold with TPA, whereas the level of TIMP rose only three-fold. Interleukin-1 did not stimulate collagenase production by the keratinocytes, in contrast to its effect on cultured fibroblasts. When keratinocytes were plated on type I or type IV collagen, they synthesized increased amounts of collagenase compared with cells cultured on laminin or in the absence of matrix. TIMP synthesis was not increased by collagen. Finally, phagocytosis of latex beads did not augment collagenase production by the keratinocytes.
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Colagenasas/biosíntesis , Queratinocitos/enzimología , Células Cultivadas , Colagenasas/metabolismo , Inducción Enzimática/efectos de los fármacos , Proteínas de la Matriz Extracelular/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Glicoproteínas/biosíntesis , Glicoproteínas/metabolismo , Humanos , Interleucina-1/farmacología , Queratinocitos/efectos de los fármacos , Fagocitosis , Proteínas Recombinantes/farmacología , Acetato de Tetradecanoilforbol/farmacología , Inhibidores Tisulares de MetaloproteinasasRESUMEN
Remodeling of the extracellular matrix by matrix-degrading metalloproteinases (MMPs) has been implicated in the early morphogenesis of branched organs. Growth factors such as EGF and TGF alpha are known to regulate the expression of MMPs in a variety of systems. We therefore examined the effects of EGF, TGF alpha, and collagenase upon in vitro branching of the embryonic lung. Lung rudiments from 11.5 day post coitum mice underwent extensive growth and repetitive branching during a 3-day period in organ culture. Lungs treated with EGF or TGF alpha were larger than controls, yet displayed fewer branches along with markedly dilated end buds which lacked clefts, indicating that these growth factors inhibit normal lung branching. Addition of purified mammalian collagenase to lung cultures similarly inhibited epithelial branching and produced end bud enlargement. In addition, gelatin-substrate enzymography of the conditioned medium from EGF- and TGF alpha-treated lungs revealed a marked induction of a metalloproteinase activity which most likely corresponds to the 72kDa type IV collagenase/gelatinase which degrades basement membrane collagens. Lungs maintained in the presence of both TGF alpha and TIMP, a specific inhibitor of MMPs, branched repeatedly and displayed normal, narrow end buds as seen with controls, suggesting that TIMP is capable of preventing or reversing the observed growth factor mediated effects upon lung branching. Taken together, these results provide evidence that the growth factors EGF and TGF alpha guide lung development, at least in part, by inducing the expression of matrix-degrading metalloproteinases.
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Factor de Crecimiento Epidérmico/farmacología , Matriz Extracelular/efectos de los fármacos , Pulmón/embriología , Metaloendopeptidasas/biosíntesis , Factor de Crecimiento Transformador alfa/farmacología , Animales , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Glicoproteínas/farmacología , Pulmón/enzimología , Metaloendopeptidasas/antagonistas & inhibidores , Morfogénesis , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas , Inhibidores Tisulares de MetaloproteinasasRESUMEN
To investigate the mechanism of cyclosporine (CS)-induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non-inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 micrograms/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity. Two of the fibroblasts strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased. This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS-induced gingival enlargement.
Asunto(s)
Ciclosporina/farmacología , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Colagenasa Microbiana/metabolismo , Células Cultivadas , Colágeno , Fibroblastos/citología , Fibroblastos/enzimología , Encía/citología , Encía/enzimología , Hiperplasia Gingival/inducido químicamente , Glicoproteínas/biosíntesis , Humanos , Colagenasa Microbiana/antagonistas & inhibidores , Colagenasa Microbiana/biosíntesis , Radioinmunoensayo , Inhibidores Tisulares de MetaloproteinasasRESUMEN
Human keratinocytes in culture are known to produce collagenase. As part of studies to ascertain the physiologic stimuli for collagenase production by keratinocytes, we wanted to determine whether extracellular matrix could modulate the production of collagenase in vitro. Immunoprecipitable collagenase from the conditioned medium of cells grown on different types of matrix was measured. Metabolically labeled human keratinocytes were cultured in 0.1 mM calcium in serum-free medium on colloidal gold-coated coverslips plus type IV collagen, type I collagen, or laminin or in the absence of matrix. Immunoprecipitation of the conditioned medium with anti-collagenase antiserum was performed and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, fluorography, and densitometry. The keratinocytes cultured on type IV or type I collagen produced more collagenase than did those cultured on laminin or in the absence of matrix. This effect did not reflect a general increase in secreted proteins, because the production of tissue inhibitor of metalloproteinase, or TIMP, did not increase under the same conditions. Phagocytosis of the gold salts by the keratinocytes migrating on types I or IV collagen did not account for the increased collagenase produced by these cells since the effect persisted in the absence of the colloidal gold and phagocytosis of latex beads did not augment collagenase production.