RESUMEN

Percutaneous medical devices remain susceptible to infection and failure. We hypothesize that healing of the skin into the percutaneous device will provide a seal, preventing bacterial attachment, biofilm formation, and subsequent device failure. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] with sphere-templated pores (40 microm) and interconnecting throats (16 microm) were implanted in normal C57BL/6 mice for 7, 14, and 28 days. Poly(HEMA) was either untreated, keeping the surface nonadhesive for cells and proteins, or modified with carbonyldiimidazole (CDI) or CDI reacted with laminin 332 to enhance adhesion. No clinical signs of infection were observed. Epidermal and dermal response within the poly(HEMA) pores was evaluated using light and transmission electron microscopy. Cells (keratinocytes, fibroblasts, endothelial cells, inflammatory cells) and basement membrane proteins (laminin 332, beta4 integrin, type VII collagen) could be demonstrated within the poly(HEMA) pores of all implants. Blood vessels and dermal collagen bundles were evident in all of the 14- and 28-day implants. Fibrous capsule formation and permigration were not observed. Sphere-templated polymers with 40 microm pores demonstrate an ability to recapitulate key elements of both the dermal and the epidermal layers of skin. Our morphological findings indicate that the implant model can be used to study the effects of biomaterial pore size, pore interconnect (throat) size, and surface treatments on cutaneous biointegration. Further, this model may be used for bacterial challenge studies.

Asunto(s)

Dermis/efectos de los fármacos , Dermis/fisiología , Epidermis/efectos de los fármacos , Epidermis/fisiología , Implantes Experimentales , Metacrilatos/química , Metacrilatos/farmacología , Animales , Dermis/citología , Dermis/ultraestructura , Células Epidérmicas , Epidermis/ultraestructura , Inmunohistoquímica , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Ratones , Ratones Endogámicos C57BL , Fenotipo , Porosidad/efectos de los fármacos , Fijación del Tejido

RESUMEN

Percutaneous devices are critical for health care. Access to tissue, vessels and internal organs afforded by these devices provides the means to treat and monitor many diseases. Unfortunately, such access is not restricted, and infection may compromise the usefulness of the device and even the life of the patient. New biomaterials offer the possibility of maintaining internal access while limiting microbial access, but understanding of the cutaneous/biomaterial interface and models to study this area are limited. This paper focuses on models useful for studying the morphology and biology of the intersection of skin and percutaneous biomaterials. An organ culture and a mouse model are described that offer promising possibilities for improved understanding of this critical interface.

Asunto(s)

Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/química , Modelos Animales de Enfermedad , Infecciones Relacionadas con Prótesis/inducido químicamente , Infecciones Relacionadas con Prótesis/patología , Piel/efectos de los fármacos , Piel/patología , Animales , Procedimientos Quirúrgicos Dermatologicos , Humanos

RESUMEN

Percutaneous medical devices are integral in the management and treatment of disease. The space created between the skin and the device becomes a haven for bacterial invasion and biofilm formation and results in infection. We hypothesize that sealing this space via integration of the skin into the device will create a barrier against bacterial invasion. The purpose of this study was to develop an animal model in which the interaction between skin and biomaterials can be evaluated. Porous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] rods were implanted for 7 days in the dorsal skin of C57 BL/6 mice. The porous poly(HEMA) rods were surface-modified with carbonyldiimidazole (CDI) or CDI plus laminin 5; unmodified rods served as control. Implant sites were sealed with 2-octyl cyanoacrylate; corn pads and adhesive dressings were tested for stabilization of implants. All rods remained intact for the duration of the study. There was histological evidence of both epidermal and dermal integration into all poly(HEMA) rods regardless of treatment. This in vivo model permits examination of the implant/skin interface and will be useful for future studies designed to facilitate skin cell attachment where percutaneous devices penetrate the skin.

Asunto(s)

Materiales Biocompatibles , Modelos Animales , Piel , Animales , Ratones , Polihidroxietil Metacrilato , Absorción Cutánea

RESUMEN

Immunohistochemistry (IHC) is a valuable tool for labeling structures in tissue samples. Quantification of immunolabeled structures using traditional approaches has proved to be difficult. Manual counts of IHC-stained structures are inherently biased, require multiple observers, and generate qualitative data. Stereological methods provide accurate quantification but are complex and labor-intensive when staining must be compared among large numbers of samples. In an effort to quickly, objectively, and reproducibly quantify cutaneous innervation in a large number of counterstained tissue sections, we developed a color subtractive-computer-assisted image analysis (CS-CAIA) system. To develop and test the CS-CAIA method, tissue sections of diabetic (db/db) mouse skin and their wild-type (db/-) littermates were stained by IHC for the neural marker PGP 9.5. The brown-red PGP 9.5 peroxidase stain was colorimetrically isolated through a scripted process of color background removal. The remaining stain was thresholded and binarized for computer determination of nerve profile counts (number of stained regions), area fraction (total area of nerve profiles per unit area of tissue), and area density (total number of nerve profiles per unit area of tissue). Using CS-CAIA, epidermal nerve profile counts, area fraction, and area density were significantly lower in db/db compared to db/- mice.

Asunto(s)

Diabetes Mellitus/patología , Piel/inervación , Animales , Diabetes Mellitus/genética , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes

RESUMEN

Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Dermatitis Alérgica por Contacto/patología , Dermatitis Alérgica por Contacto/prevención & control , Neprilisina/fisiología , Sustancia P/toxicidad , Administración Cutánea , Animales , Antiinflamatorios no Esteroideos/antagonistas & inhibidores , Antiinflamatorios no Esteroideos/metabolismo , Permeabilidad Capilar/genética , Permeabilidad Capilar/inmunología , Capsaicina/administración & dosificación , Aceite de Crotón/toxicidad , Dermatitis Alérgica por Contacto/enzimología , Dermatitis Alérgica por Contacto/genética , Dermatitis Irritante/enzimología , Dermatitis Irritante/genética , Dermatitis Irritante/patología , Dermatitis Irritante/prevención & control , Inhibidores Enzimáticos/administración & dosificación , Femenino , Glicopéptidos/administración & dosificación , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neprilisina/antagonistas & inhibidores , Neprilisina/deficiencia , Neprilisina/metabolismo , Antagonistas del Receptor de Neuroquinina-1 , Piperidinas/administración & dosificación , Quinuclidinas/administración & dosificación , Piel/irrigación sanguínea , Piel/enzimología , Piel/patología

RESUMEN

The neurological system plays an important role in modulating some inflammatory skin diseases. Neuro-cutaneous interactions may be mediated by the release of neuropeptides such as substance P (SP) which activate immunocompetent cells in the skin by binding to high affinity neurokinin receptors (NKR). Since epidermal keratinocytes produce a variety of cytokines and are intimately associated with cutaneous sensory fibers, we tested the ability of these cells to participate in the cutaneous neuroimmune system by the secretion of potent cytokines such as interleukin 1 (IL-1) in response to released SP. RT-PCR studies demonstrated that cultured PAM 212 murine keratinocytes expressed mRNA for NK-2R but not NK-1R. Correspondingly, the addition of SP to these cells resulted in a rapid increase in intracellular Ca2+ levels that could be specifically blocked by an NK-2R antagonist. NK-2R was also shown in normal mouse epidermis by immunohistochemistry. SP augmented the expression of PAM 212 keratinocyte IL-1alpha mRNA in a dose and time dependent manner and this induction was inhibited by an NK-2R antagonist. Secretion of bioactive IL-1alpha by the PAM 212 keratinocytes was likewise stimulated by SP in a dose dependent manner. These data support the hypothesis that SP released from cutaneous sensory nerves contributes to neuroimmune inflammatory responses in the skin by modulating the expression and release of cytokines from epidermal keratinocytes.

Asunto(s)

Interleucina-1/biosíntesis , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Receptores de Neuroquinina-2/metabolismo , Sustancia P/farmacología , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular Transformada , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Interleucina-1/genética , Líquido Intracelular/efectos de los fármacos , Líquido Intracelular/metabolismo , Queratinocitos/metabolismo , Cinética , Ratones , Neuroinmunomodulación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/antagonistas & inhibidores , Receptores de Neuroquinina-2/genética , Piel/inervación , Piel/metabolismo

RESUMEN

There is increasing evidence that the cutaneous neurosensory system can directly modulate inflammatory responses in the skin by the release of neuropeptides such as substance P (SP). Dermal microvascular endothelial cell (DMEC) cellular adhesion molecule (CAM) expression plays a key role in directing leukocyte trafficking during cutaneous inflammatory responses. In recent studies, our laboratory examined the direct effect of SP on DMEC CAM expression and function in vitro and in vivo. Our studies indicate that DMEC express high affinity functional receptors for SP. After exposure to SP, DMEC expressed significant levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), which was accompanied by increased binding to leukocytes expressing the appropriate integrin counter receptors for these CAM. We then determined the in vivo effect of released neuropeptides on DMEC CAM expression. Our results indicate that the topical cutaneous application of the neuropeptide-releasing agent capsaicin resulted in increased ICAM-1 and VCAM-1 immunostaining of microvascular cells in the skin of human volunteers. Little is known regarding the cellular regulatory events by which SP modulates DMEC CAM expression. Our studies indicate that SP-induced cellular Ca+2 signals led to the activation of the NF-kappaB pathway, resulting in nuclear translocation of p65/p50 heterodimers that bind to high-affinity tandem kappaB sites on the VCAM-1 promoter, whereas SP activation induced NF-AT activation and ICAM-1 DNA binding. Thus, these studies further support the role of the cutaneous neurologic system in modulating inflammatory processes in the skin.

Asunto(s)

Dermatitis/inmunología , Endotelio Vascular/inmunología , Endotelio Vascular/inervación , Piel , Animales , Dermatitis/fisiopatología , Humanos , Neuroinmunomodulación/fisiología , Piel/irrigación sanguínea , Piel/inmunología , Piel/inervación

RESUMEN

Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.

Asunto(s)

Neprilisina/biosíntesis , Piel/enzimología , Heridas y Lesiones/enzimología , Anciano , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Colorantes , Contaminación de Medicamentos , Endotelio Vascular/citología , Femenino , Fibroblastos/enzimología , Humanos , Inmunohistoquímica , Queratinocitos/enzimología , Queratinocitos/metabolismo , Queratinas/inmunología , Masculino , Microcirculación , Persona de Mediana Edad , Neprilisina/genética , Neprilisina/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/química

RESUMEN

BACKGROUND: Evaluation of silicone-induced morbidity in skin has been hampered by the difficulty of detecting silicone in tissue because conventional methods are nonquantitative and insensitive. OBJECTIVE: We attempted to determine whether silicone could be identified and quantitated in skin by means of electron spectroscopy for chemical analysis (ESCA). METHODS: Skin biopsy specimens were obtained from the nose, chin, malar region, and inner arm of a patient who had received injections of silicone gel in his nose and chin. Frozen sections were dried under vacuum and examined by means of ESCA. Contiguous sections were examined by light microscopy. RESULTS: The surface concentrations of silicone were as follows: chin, 20.6% +/- 3.6%; nose, 19.0%; malar region, 2.6% +/- 1.6%; inner arm, 0.0% +/- 0.0%. Light microscopy revealed homogeneous "globules" consistent with silicone in the chin and nose sections only; the malar region and inner arm sections showed no evidence of silicone. CONCLUSION: ESCA can be used to detect silicone in skin in a specific, highly sensitive, and quantitative manner. This is the first report of quantification of silicone in skin by means of ESCA.

Asunto(s)

Geles de Silicona/análisis , Piel/patología , Brazo/patología , Mejilla/patología , Mentón/patología , Dermis/patología , Microanálisis por Sonda Electrónica/instrumentación , Microanálisis por Sonda Electrónica/métodos , Dermatosis Facial/inducido químicamente , Dermatosis Facial/patología , Secciones por Congelación , Histiocitos/patología , Humanos , Linfocitos/patología , Masculino , Persona de Mediana Edad , Nariz/patología , Geles de Silicona/efectos adversos , Piel/química , Vacio

RESUMEN

Sensory nerves in skin are capable of releasing multiple neuropeptides, which modulate inflammatory responses by activating specific cutaneous target cells. Extravasation of particular subsets of leukocytes depends upon the regulated expression of cellular adhesion molecules such as VCAM-1 on microvascular endothelial cells. We examined the direct effect of cutaneous neuropeptides on the expression and function of human dermal microvascular endothelial cell (HDMEC) VCAM-1. A significant increase in VCAM-1 immunostaining of microvascular endothelium was observed in vivo following capsaicin application to human skin. Multiple cutaneous sensory C-fiber-released neuropeptides were evaluated for their ability to induce VCAM-1 cell surface expression on HDMEC. Only substance P (SP) was found to be capable of inducing HDMEC VCAM-1 expression. This SP-mediated VCAM-1 induction appeared to be a direct effect that did not require the release of other HDMEC-derived soluble factors. Increased HDMEC VCAM-1 mRNA expression was detected 1 h after the addition of SP, with peak mRNA increase at 6-9 h postinduction. FACS studies demonstrated a 6.5-fold increase in endothelial cell surface VCAM-1 expression detectable 16 h after addition of SP, which was specifically blocked by a neurokinin-1 receptor antagonist. Increased VCAM-1 cell surface expression on SP-treated HDMEC resulted in a 4-fold increase in the functional binding of 51Cr-labeled MOLT-4 T cells. These data indicate that SP is capable of directly and specifically up-regulating functional endothelial VCAM-1 expression and thus may play a key role in modulating certain inflammatory responses in the skin.

Asunto(s)

Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Piel/irrigación sanguínea , Piel/efectos de los fármacos , Sustancia P/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Capsaicina/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Humanos , Inflamación/etiología , Neuroinmunomodulación/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sustancia P/fisiología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

There is increasing evidence that sensory nerves may participate in cutaneous inflammatory responses by the release of neuropeptides such as substance P (SP). We examined the direct effect of SP on human dermal microvascular endothelial cell (HDMEC) intercellular adhesion molecule 1 (ICAM-1) expression and function. Our results indicated that, although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMEC intracellular Ca2+ levels, primarily by the activation of NK-1R. Immunohistochemistry studies likewise demonstrated that HDMEC predominantly expressed NK-1R. The addition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1 mRNA levels, followed by a fivefold increase in ICAM-1 cell surface expression. This functionally resulted in a threefold increase in 51Cr-labeled binding of J-Y lymphoblastoid cells to HDMEC. In vivo studies demonstrated a marked increase in microvascular ICAM-1 immunostaining 24 and 48 h after application of capsaicin to the skin. These results indicate that neuropeptides such as SP are capable of directly activating HDMEC to express increased levels of functional ICAM-1 and further support the role of the cutaneous neurological system in modulating inflammatory processes in the skin.

Asunto(s)

Endotelio Vascular/metabolismo , Molécula 1 de Adhesión Intercelular/fisiología , Neuropéptidos/fisiología , Piel/irrigación sanguínea , Capsaicina/farmacología , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Senescencia Celular/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/fisiología , Microcirculación/fisiología , ARN Mensajero/metabolismo , Receptores de Neuroquinina-1/metabolismo , Receptores de Taquicininas/genética , Piel/efectos de los fármacos , Sustancia P/farmacología

RESUMEN

In a study initially designed to evaluate reinnervation of human cutaneous wounds using an antibody to the neuroneal marker protein gene product (PGP) 9.5, we observed marked immunostaining of cells with morphologic features of fibroblasts in the wounds. PGP 9.5 has recently been shown to be an important enzyme in the highly conserved ubiquitin system of proteolysis. Because the ubiquitin system is known to play an important role in regulating the cell cycle, the presence of PGP 9.5 in cells at a wound site was of considerable interest. Our objectives were to clarify the time frame for the appearance of PGP 9.5 and ubiquitin in wounds, to verify that PGP 9.5 is produced by wound fibroblasts, and to evaluate a potential role for these proteins in the tissue repair process. Standard incisional human wounds were stained with antibodies specific for PGP 9.5 and ubiquitin. At 7 d, stellate cells with morphologic features of fibroblasts stained for PGP 9.5, whereas earlier wounds were generally negative. In 14 and 21 d incised wounds and in chronic granulation tissue from nonhealing ulcers there was strong cellular staining for PGP 9.5 and for ubiquitin. These stellate cells also showed expression of mRNA for PGP 9.5 by reverse transcriptase-polymerase chain reaction in situ hybridization. PGP 9.5 was detected in cultured fibroblasts both by reverse transcriptase-polymerase chain reaction and by northern blot analysis. Confocal microscopy showed colocalization of antibodies to PGP 9.5 and prolyl-4-hydroxylase (a fibroblast marker) as well as colocalization of PGP 9.5 and the platelet derived growth factor beta receptor. We conclude that ubiquitin and PGP 9.5 were expressed by fibroblasts during the granulation tissue and remodeling phases wound healing. The mRNA for PGP 9.5 was demonstrated in stellate cells in chronic wounds and in fibroblasts in culture. The appearance of these degradative proteins in later wounds suggests a downregulation function in the wound healing response.

Asunto(s)

Fibroblastos/química , Tioléster Hidrolasas/biosíntesis , Heridas y Lesiones/patología , Apoptosis , Northern Blotting , ADN Complementario/análisis , Fibroblastos/ultraestructura , Expresión Génica , Técnicas Genéticas , Humanos , Hibridación in Situ , Microscopía Confocal , Microscopía Electrónica , Proteínas del Tejido Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Receptores del Factor de Crecimiento Derivado de Plaquetas , Tioléster Hidrolasas/genética , Factores de Tiempo , Ubiquitina Tiolesterasa , Ubiquitinas/análisis

RESUMEN

The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. It has been observed that certain skin diseases such as psoriasis and atopic dermatitis have a neurogenic component. Neuropeptides released by sensory nerves that innervate the skin and often contact epidermal and dermal cells can directly modulate functions of keratinocytes, Langerhans cells (LC), mast cells, dermal microvascular endothelial cells and infiltrating immune cells. Among these neuropeptides the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and somatostatin (SOM) have been reported to effectively modulate skin and immune cell functions such as cell proliferation, cytokine production or antigen presentation under physiological or pathophysiological conditions. Expression and regulation of their corresponding receptors that are expressed on a variety of skin cells as well as the presence of neuropeptide-specific peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE) determine the final biological response mediated by these peptides on the target cell or tissue. Likewise, skin cells like keratinocytes or fibroblasts are a source for neurotrophins such as nerve growth factor that are required not only for survival and regeneration of sensory neurons but also to control responsiveness of these neurons to external stimuli. Therefore, neuropeptides, neuropeptide receptors, neuropeptide-degrading enzymes and neurotrophins participate in a complex, interdependent network of mediators that modulate skin inflammation, wound healing and the skin immune system. This review will focus on recent studies demonstrating the role of tachykinins, CGRP, SOM and VIP and their receptors and neuropeptide-degrading enzymes in mediating neurogenic inflammation in the skin.

Asunto(s)

Neuropéptidos/fisiología , Sistemas Neurosecretores/fisiología , Fenómenos Fisiológicos de la Piel , Piel/inmunología , Animales , Humanos

RESUMEN

OBJECTIVE: To determine the prognostic value of analyzing lymph node (LN) DNA from patients with mycosis fungoides for the presence of a monoclonal T-cell population. DESIGN: Inception cohort study. SETTING: A tertiary care referral center in Seattle, Wash. PATIENTS: Fifty-five uniformly staged patients with the diagnosis of mycosis fungoides and who had a lymph node biopsy, 21 with clinically abnormal nodes and 34 with normal nodes. MAIN OUTCOME MEASURES: Lymph nodes were evaluated by Southern blot analysis for T-cell receptor beta-chain (TCRB) gene rearrangement and by histopathologic examination for the LN classification using the National Cancer Institute system. Patients were observed clinically for a mean (+/- SD) of 4.7 +/- 3.4 years. RESULTS: Patients with detectable TCRB gene rearrangement in lymph node DNA had an increased likelihood of a poor clinical outcome and a decreased probability of survival (P < .001 for both) compared with patients with the TCRB germline. Although patients with clinically enlarged nodes were more likely to have the TCRB gene rearranged, those with normal nodes and the TCRB gene rearranged also had a poor clinical outcome and a decreased probability of survival. Similar to those with the TCRB gene rearranged, most patients with advanced histopathologic changes (LN3 and LN4) had a poor prognosis. The presence of a rearranged TCRB gene, however, correctly predicted some patients with intermediate LN scores (LN2) who had a poor clinical outcome. CONCLUSIONS: Detection of a monoclonal T-cell population, as demonstrated by a rearranged TCRB gene on Southern blot analysis, in LNs of patients with mycosis fungoides is predictive of a poor clinical outcome and a reduced probability of survival. Lymph node TCRB gene analysis provides additional prognostic information for patients with mycosis fungoides with intermediate LN histopathology.

Asunto(s)

Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Ganglios Linfáticos/metabolismo , Micosis Fungoide/genética , Neoplasias Cutáneas/genética , Antineoplásicos/uso terapéutico , Biopsia , Southern Blotting , Causas de Muerte , Estudios de Cohortes , Intervalos de Confianza , ADN de Neoplasias/análisis , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Ganglios Linfáticos/patología , Linfoma Cutáneo de Células T/patología , Masculino , Persona de Mediana Edad , Micosis Fungoide/patología , Estadificación de Neoplasias , Probabilidad , Pronóstico , Estudios Prospectivos , Inducción de Remisión , Reproducibilidad de los Resultados , Neoplasias Cutáneas/patología , Tasa de Supervivencia , Linfocitos T/metabolismo , Linfocitos T/patología , Resultado del Tratamiento

RESUMEN

There is increasing experimental evidence that the neurologic system can directly participate in cutaneous inflammation and wound healing. Recent studies indicate that neuropeptides released by cutaneous nerves such as c-fibers can activate a number of target cells including keratinocytes, Langerhans cells, mast cells, and endothelial cells. One such neuropeptide, substance P (SP), is able to specifically bind to murine and human keratinocytes and induce the release of cytokines such as interleukin 1 (IL-1). Other studies demonstrate that SP can also activate mast cells to produce the potent pro-inflammatory cytokine tumor necrosis factor alpha (TNF alpha). More recently, we examined the effect of cutaneous neuropeptides on human dermal microvascular endothelial cell (HDMEC) activities. Our studies indicate that the c-fiber-derived calcitonin gene-related peptide (CGRP) is capable of stimulating HDMEC to secrete the neutrophil chemotactic factor interleukin 8 (IL-8). In addition, SP is able to directly activate HDMEC to express high levels of the important cellular adhesion molecule vascular cellular adhesion molecule 1 (VCAM-1). Thus, these studies support the role that the neurologic system may play in mediating the biologic processes that occur during inflammation and wound healing in the skin.

Asunto(s)

Dermatitis/fisiopatología , Fenómenos Fisiológicos del Sistema Nervioso , Piel/inervación , Cicatrización de Heridas/fisiología , Animales , Endopeptidasas/metabolismo , Humanos , Neuropéptidos/farmacología , Receptores de Neuropéptido/fisiología , Piel/efectos de los fármacos

RESUMEN

The purpose of the study was to test the hypothesis that significant delays in cutaneous wound healing could be demonstrated using standard wounds and high quality histological methods in patients with severe peripheral vascular disease (PVD) and/or diabetes mellitus (DM) compared to healthy elderly controls. Additionally, we proposed that standard wounds on the arms of elderly controls would heal more rapidly than comparable wounds on the legs. In order to test these hypotheses we developed and characterized a partial thickness wound model which could be used safely in human subjects. The study population consisted of 25 elderly normal volunteers, 17 patients with PVD, and 24 patients with DM. Standard wounds were created using a Simplate II bleeding-time device. A total of 309 wounds ranging in age from 1 to 25 days were determined to be suitable for analysis. A global index of wound maturity was developed based on selected epidermal and dermal events of repair which could be scored histologically. The superficial component (within 0.1 mm of the epidermis) and deep components of dermal wounds were analyzed separately. Simultaneously created arm and leg wounds were studied in 15 of the elderly controls. Transcutaneous partial pressure of oxygen (TcPO2) measurements were used to estimate the severity of cutaneous ischemia. Data analysis revealed that the most striking differences observed were in dermal events of repair. Control wounds were more mature than dermal wounds from patients with PVD (P < 0.05). A significant reduction in the number of neutrophils and macrophages (P < 0.05) was demonstrated in 7-day-old wounds of patients with PVD compared to controls. Patients with DM showed a similar trend of reduced wound maturity but it did not reach statistical significance. Wounds created in skin with TcPO2 > 20 were more mature than wounds with TcPO2 < or = 20 (P < 0.05) and arm wounds were more mature than leg wounds (P < 0.01). The most significant difference noted in this wound model was that the superficial compartment of dermal wounds was significantly more mature than the deep compartment (P < 0.001). Good agreement was observed between two independent scorers of wound histology and no complications were noted in either patients or controls when using this human wound model. We conclude that the model described allows evaluation of both epidermal and dermal events of repair with relative safety even in patients with PVD and DM.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Complicaciones de la Diabetes , Enfermedades Vasculares Periféricas/complicaciones , Cicatrización de Heridas/fisiología , Anciano , Traumatismos del Brazo/fisiopatología , Traumatismos del Brazo/cirugía , Monitoreo de Gas Sanguíneo Transcutáneo , Recuento de Células , Angiopatías Diabéticas , Células Epidérmicas , Epidermis/lesiones , Fibroblastos/citología , Humanos , Traumatismos de la Pierna/fisiopatología , Traumatismos de la Pierna/cirugía , Persona de Mediana Edad , Monocitos/citología , Neutrófilos/citología , Factores de Tiempo

RESUMEN

We reported that interstitial collagenase is produced by keratinocytes at the edge of ulcers in pyogenic granuloma, and in this report, we assessed if production of this metalloproteinase is a common feature of the epidermal response in a variety of wounds. In all samples of chronic ulcers, regardless of etiology, and in incision wounds, collagenase mRNA, localized by in situ hybridization, was prominently expressed by basal keratinocytes bordering the sites of active re-epithelialization indicating that collagenolytic activity is a characteristic response of the epidermis to wounding. No expression of mRNAs for 72- and 92-kD gelatinases or matrilysin was seen in keratinocytes, and no signal for any metalloproteinase was detected in normal epidermis. Immunostaining for type IV collagen showed that collagenase-positive keratinocytes were not in contact with an intact basement membrane and, unlike normal keratinocytes, expressed alpha 5 beta 1 receptors. These observations suggest that cell-matrix interactions influence collagenase expression by epidermal cells. Indeed, as determined by ELISA, primary cultures of human keratinocytes grown on basement membrane proteins (Matrigel; Collaborative Research Inc., Bedford, MA) did not express significant levels of collagenase, whereas cells grown on type I collagen produced markedly increased levels. These results suggest that migrating keratinocytes actively involved in re-epithelialization acquire a collagenolytic phenotype upon contact with the dermal matrix.

Asunto(s)

Colagenasas/biosíntesis , Matriz Extracelular/fisiología , Granuloma/fisiopatología , Queratinocitos/enzimología , Queratinocitos/fisiología , Úlcera/fisiopatología , Cicatrización de Heridas/fisiología , Membrana Basal/fisiología , Células Cultivadas , Expresión Génica , Granuloma/metabolismo , Granuloma/patología , Humanos , Inmunohistoquímica , Integrinas/biosíntesis , Sondas ARN , Úlcera/metabolismo , Úlcera/patología

RESUMEN

PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

Asunto(s)

Queratinocitos/metabolismo , Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Piel/metabolismo , Heridas y Lesiones/fisiopatología , Anticuerpos Monoclonales , Biopsia , Células Cultivadas , Medios de Cultivo Condicionados , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Recién Nacido , Cinética , Sustancias Macromoleculares , Masculino , Factor de Crecimiento Derivado de Plaquetas/análisis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores del Factor de Crecimiento Derivado de Plaquetas/análisis , Receptores del Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Heridas y Lesiones/patología

RESUMEN

Pyoderma gangrenosum is a rare cutaneous disorder that may complicate recovery after a cardiac operation. The lesions appear to represent a necrotizing infection; however, they do not respond to surgical debridement and antibiotic therapy. The treatment is based on high-dose corticosteroids and concomitant treatment of any underlying systemic disease.

Asunto(s)

Puente de Arteria Coronaria , Complicaciones Posoperatorias/diagnóstico , Piodermia Gangrenosa/diagnóstico , Infección de la Herida Quirúrgica/diagnóstico , Anciano , Diagnóstico Diferencial , Esquema de Medicación , Humanos , Masculino , Complicaciones Posoperatorias/tratamiento farmacológico , Complicaciones Posoperatorias/patología , Prednisolona/administración & dosificación , Piodermia Gangrenosa/tratamiento farmacológico , Piodermia Gangrenosa/patología

RESUMEN

The percutaneous absorption and metabolism of lonapalene (6-chloro-2,3-dimethoxynaphthalene-1,4-diol-diacetate; RS-43179), a topically effective 5-lipoxygenase inhibitor, has been measured in six subjects with stable plaque-type psoriasis of the lower extremities. Lonapalene readily penetrates psoriatic skin, is rapidly and completely metabolized, and is almost entirely excreted in the urine. Unexpectedly we observed a trend for thigh (T) plaque skin to be more permeable than lower leg (LL) plaque skin as measured by total absorption (T, 44.8 +/- 13.4%; LL, 24.9 +/- 12.6% applied dose excreted), peak plasma levels (T, 209 +/- 107; LL, 146 +/- 81 ng Eq/ml), and peak rate of urinary excretion (T, 591.7 +/- 112.2; LL, 318.4 +/- 143.9 micrograms Eq/hr). There were also differences in the metabolic profiles between the two sites as measured by the quantity and proportion of dealkylated and conjugated products excreted in the urine.

Asunto(s)

Inhibidores de la Lipooxigenasa/farmacocinética , Naftalenos/farmacocinética , Psoriasis/metabolismo , Absorción Cutánea , Administración Cutánea , Cromatografía Líquida de Alta Presión , Humanos , Técnicas In Vitro , Inhibidores de la Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/uso terapéutico , Masculino , Naftalenos/metabolismo , Naftalenos/uso terapéutico , Psoriasis/tratamiento farmacológico