RESUMEN
The aim of this study was to investigate the local changes in the ultrastructure of human skin after iontophoresis, using cryo-scanning, transmission and freeze fracture electron microscopy in human skin in vitro and in vivo. Human dermatomed skin was subjected to passive diffusion for 6 hours followed by nine hours of iontophoresis at 0.5 mA/cm2. The skin was processed and examined using both cryo-scanning electron microscopy (Cryo-SEM) and transmission electron microscopy (TEM). In addition, iontophoresis patches were applied to healthy volunteers for 3.5h with 0.5h of passive delivery followed by 3h of iontophoresis at a current density of 0.25 mA/cm2. Subsequently, a series of tape stripping were performed, which were visualized by freeze fracture transmission electron microscopy (FFTEM). In vitro, the cryo-scanning electron microscopy study revealed that electric current induced changes in the water distribution in the stratum corneum. Transmission electron microscopy showed no local changes in the ultrastructure of the stratum corneum; however, layers of detached corneocytes were frequently observed especially at the anodal site. In vivo, there was no evidence of perturbation of the stratum corneum lipid organization; however, changes in the fracture were noticed deeper in the stratum corneum at the anodal side, indicating a weakening of the desmosomal structure. The in vitro/in vivo studies suggest that iontophoresis results in the formation of intercellular water pools (in vitro observation) and a weakening of the desmosomal structure (in vivo observation) only in the upper part of the stratum corneum. However, no changes in the lipid organization were observed in vitro and in vivo at the current densities of 0.5 and 0.25 mA/cm2, respectively. Therefore, even at relatively high current densities, no drastic changes in the ultrastructure of the stratum corneum are observed. As far as structural changes in stratum corneum are concerned iontophoresis is therefore a safe method at the experimental conditions we used.
Asunto(s)
Iontoforesis , Piel/ultraestructura , Adulto , Agua Corporal/metabolismo , Microscopía por Crioelectrón , Epidermis/ultraestructura , Femenino , Humanos , Piel/metabolismoRESUMEN
In many multi-disciplinary fields of science, such as tissue engineering, where material and biological sciences are combined, there is a need for a tool that combines ultrastructural and chemical data analysis in a non-destructive manner at high resolution. We show that a combination of confocal Raman spectroscopy (CRS) and scanning electron microscopy (SEM) can be used for such analysis. Studies of atomic composition can be done by X-ray microanalysis in SEM, but this is only possible for atomic numbers greater than five and does not reveal molecular identity. Raman spectroscopy, however, can provide information on molecular composition and identity by detection of wavelength shifts caused by molecular vibrations. In this study, CRS-SEM revealed that early in vitro-formed bone extracellular matrix (ECM) produced by rat osteoprogenitor cells resembles mature bone chemically. We gained insight into the structure and chemical composition of the ECM, which was composed of mainly mineralized collagen type I fibres and areas of dense carbonated calcium phosphate related to the collagen fibre density, as revealed by Raman imaging of SEM samples. We found that CRS-SEM allows the study of specimens in a non-destructive manner and provides high-resolution structural and chemical information about inorganic and organic constituents by parallel measurements on the same sample.
Asunto(s)
Matriz Ósea/química , Matriz Ósea/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Espectrometría Raman/métodos , Animales , Células Cultivadas , Masculino , Osteocitos/metabolismo , Osteocitos/ultraestructura , RatasRESUMEN
We here present TYSON, a new program for automatic and semi-automatic particle selection from electron micrographs. TYSON employs a three-step strategy of searching, sorting and selecting single particles. In the first step, TYSON finds the positions of potential particles by one of three different methods: local averaging, template matching or local variance. The practical merits and drawbacks of these methods are discussed. In the second step, these potential particles are automatically sorted according to their probability of being true positives. Many criteria are provided for this sort. In the final -interactive- step, whole categories of poorly fitting false positives can be removed with a single mouse-click. We present results obtained using cryo-EM micrographs of both spherical virus particles and asymmetric particles. The procedures are fast and use of TYSON allowed, for example, some 20,000 particles to be selected in a single working day.
Asunto(s)
Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Algoritmos , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/ultraestructura , Procesamiento Automatizado de Datos , Escherichia coli/química , Escherichia coli/enzimología , Análisis de Fourier , Levivirus/química , Levivirus/ultraestructura , Reconocimiento de Normas Patrones Automatizadas , Ribosomas/química , Ribosomas/ultraestructura , Interfaz Usuario-Computador , Virión/química , Virión/ultraestructuraRESUMEN
In this study, we investigated to what extent the stability and transduction capacity of polyplexed DNA can be improved by optimizing the condensing peptide sequence. We have synthesized a small library of cationic peptides, at which the lysine/arginine ratio and the cation charge were varied. All peptides were able to compact DNA, at which polyplexes of short lysine-rich sequences were considerably larger than those of elongated or arginine-rich peptides (GM102 and GM202). In addition, the arginine-rich peptides GM102 and GM202 rendered the polyplexes resistant to plasma incubation or DNase I-mediated digestion. While all peptides were found to improve the transfection efficiency in HepG2 cells, only the GM102- and GM202-derived polyplexes could be specifically targeted to HepG2 cells by incorporation of a ligand-derivatized YKAK(8)WK peptide. We propose that GM102 and GM202 combine the advantage of small condensing peptides to give small-sized polyplexes with the superior stability of condensing polymers, which makes GM102 and GM202 excellent candidates for future in vivo gene therapy studies.
Asunto(s)
Arginina/química , Técnicas de Transferencia de Gen , Vectores Genéticos/química , Oligopéptidos/química , Secuencia de Aminoácidos , Animales , Fenómenos Químicos , Química Física , Desoxirribonucleasa I/química , Estabilidad de Medicamentos , Marcación de Gen/métodos , Vectores Genéticos/farmacocinética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oligopéptidos/genética , Oligopéptidos/farmacocinética , Fragmentos de Péptidos/química , Distribución Tisular , Transducción Genética , TransfecciónRESUMEN
Uptake of particulate antigen carrier systems by specialized M-cells of the gut-associated lymphoid tissue is still a limiting step in inducing efficient immune responses after oral vaccination. Although transport of soluble drugs over the epithelial barrier of the gut is extensively studied in vitro by using the Caco-2 cell model, this was for long time not possible for particles due to the absence of M-cells. By co-culturing Caco-2 cells with cultured human B-lymphocytes (Raji-cells), cells which are morphologically and functionally similar to M-cells can be induced. This human M-cells model makes it possible to study the uptake of microparticles for oral vaccine delivery. In this way, chitosan microparticles, which have demonstrated to target the Peyer's patches efficiently in vivo, could be tested in vitro. The development of this M-cells model facilitates the optimization of the microparticles in order to target them even more efficiently to the M-cells in the gut. In this study, the integrity of the human M-cell model was investigated by determining the transepithelial electrical resistance (TEER), 14C-mannitol transport and morphology using scanning electron microscopy. The uptake of particles was investigated by measuring transport of both fluorescently labeled microspheres (Fluospheres) and chitosan microparticles using flowcytometry. No discontinuities or abnormalities could be found in the co-culture. Scanning electron microscopy showed that morphologically different cells were present in the human M-cell model. Both commercially available Fluospheres (size 0.2 microm) and chitosan microparticles (size 1.7 microm) for oral vaccine delivery were transported at a significantly higher amount by the human M-cell model compared to the transport by the Caco-2 cell monoculture. Since chitosan microparticles were proven to be taken up by Peyer's patches in mice as well, this human M-cell model is able to predict the M-cell uptake of microparticles for oral vaccine delivery. This M-cell model is a new tool, which can be used to scan, develop and optimize microparticles for oral vaccine delivery. Since the M-cell uptake can now be studied in vitro, the targeting of these cells can be studied more efficiently and can now be done in cells from human origin.
Asunto(s)
Quitina/análogos & derivados , Quitina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Mucosa Intestinal/metabolismo , Tejido Linfoide/metabolismo , Vacunas/administración & dosificación , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/ultraestructura , Transporte Biológico/fisiología , Células CACO-2 , Quitina/farmacocinética , Quitosano , Técnicas de Cocultivo/métodos , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/ultraestructura , Tejido Linfoide/efectos de los fármacos , Tejido Linfoide/ultraestructura , Microesferas , Células Tumorales CultivadasRESUMEN
There are several skin diseases in which the lipid composition in the intercellular matrix of the stratum corneum is different from that of healthy human skin. It has been shown that patients suffering from atopic dermatitis have a reduced ceramide content in the stratum corneum, whereas in the stratum corneum of lamellar ichthyosis patients, the amount of free fatty acids is decreased and the ceramide profile is altered. Both patient groups also show elevated levels of transepidermal water loss indicative of an impaired barrier function. As ceramides and free fatty acids are essential for a proper barrier function, we hypothesized that changes in the composition of these lipids would be reflected in the lipid organization in stratum corneum of atopic dermatitis and lamellar ichthyosis patients. We investigated the lateral lipid packing using electron diffraction and the lamellar organization using freeze fracture electron microscopy. In atopic dermatitis stratum corneum, we found that, in comparison with healthy stratum corneum, the presence of the hexagonal lattice (gel phase) is increased with respect to the orthorhombic packing (crystalline phase). In lamellar ichthyosis stratum corneum, the hexagonal packing was predominantly present, whereas the orthorhombic packing was observed only occasionally. This is in good agreement with studies on stratum corneum lipid models that show that the presence of long-chain free fatty acids is involved in the formation of the orthorhombic packing. The results of this study also suggest that the ceramide composition is important for the lateral lipid packing. Finally, using freeze fracture electron microscopy, changes in the lamellar organization in stratum corneum of both patient groups could be observed.
Asunto(s)
Dermatitis Atópica/metabolismo , Epidermis/metabolismo , Ictiosis Lamelar/metabolismo , Metabolismo de los Lípidos , Dermatitis Atópica/patología , Epidermis/patología , Humanos , Ictiosis Lamelar/patología , Lípidos/químicaRESUMEN
PURPOSE: To study at the ultrastructural level which part of the skin is associated with percutaneous iodide transport by passive diffusion and iontophoresis. METHODS: Following passive diffusion or iontophoresis of iodide, the morphology and the ion distribution of the skin was preserved by rapid freezing. The skin was kept frozen until and during examination by transmission electron microscopy (TEM) and X-ray microanalysis (XRMA). The intrinsic electron absorbing characteristics of cryopreserved skin allow direct TEM examination without additional staining. XRMA can be used to obtain in a relatively nondestructive way in situ information on ion distributions across the skin. RESULTS: After passive diffusion, iodide was mainly found in the stratum corneum (SC), whereas there was little iodide in the viable epidermis. Iontophoresis up to 300 microA/cm2 did not significantly affect this distribution. With iontophoresis at 1,000 microA/cm2, the amount of iodide increased dramatically and was equally distributed over the SC and viable epidermis. The presence of iodide in the SC suggests that iodide is present inside corneocytes. CONCLUSIONS: Iontophoresis up to 300 microA/cm2 does not significantly perturb skin structures in contrast to iontophoresis at 1,000 microA/cm2. The presence of iodide inside corneocytes suggests the possibility of transcellular percutaneous iodide transport.
Asunto(s)
Criopreservación , Transporte Iónico/fisiología , Iontoforesis , Piel/metabolismo , Piel/ultraestructura , Administración Cutánea , Agar/ultraestructura , Microanálisis por Sonda Electrónica/métodos , Geles , Humanos , Iontoforesis/métodos , Microscopía Electrónica , Yoduro de Potasio/farmacocinéticaRESUMEN
Subcellular regulation mechanisms of calcium concentrations related to oxygen sensing in the carotid body are unclear. In the present study, we investigated the ultrastructural distribution patterns of calcium in carotid body cells and its changes evoked by hypoxia. Carotid bodies were dissected from anesthetized cats exposed in vivo to normoxic or acute hypoxic conditions. We used the oxalate-pyroantimonate technique that yields an electron-opaque calcium precipitate. X-ray microanalysis and appropriate controls confirmed the presence of calcium in the precipitate. Calcium precipitates were found in all types of cells in carotid body parenchyma: chemoreceptor cells, sustentacular cells, and nerve endings. In normoxic chemoreceptor cells, the precipitate was localized in dense core vesicles, mitochondria, and nuclei, but rarely in the cytoplasm. The most apparent effect of hypoxia was disappearance of the precipitate from dense core vesicles and was associated with its appearance in the cytoplasm. The amount of precipitate throughout the carotid body parenchyma was decreased overall due to hypoxia. These results indicate the involvement of subcellular calcium trafficking in hypoxia-sensing in the carotid body. The redistribution pattern of granular calcium deposits from organelles to the cytoplasm of chemoreceptor cells agrees with biochemical data of calcium release from intracellular stores during hypoxia.
Asunto(s)
Antimonio/metabolismo , Calcio/metabolismo , Cuerpo Carotídeo/metabolismo , Hipoxia de la Célula/fisiología , Animales , Cuerpo Carotídeo/ultraestructura , Gatos , Quelantes/metabolismo , Células Quimiorreceptoras/metabolismo , Células Quimiorreceptoras/ultraestructuraRESUMEN
Although ultraviolet (UV) B radiation is known to be immunosuppressive, there is little information regarding a relevant immunological endpoint to assess human subjects in vivo. Therefore, we have examined the effect of in vivo UV radiation on the ability of human epidermal cells (EC) to present herpes simplex virus (HSV) antigens to memory T cells. Human volunteers, who were seropositive for HSV, were exposed to one minimal erythemal dose (MED) for four consecutive days. EC, prepared from suction blister roofs, were co-cultured with autologous T cells in the presence of HSV. HSV antigen presentation by UV-exposed EC was increased compared with control, nonexposed EC. This up-regulation correlated with an influx of macrophages into the epidermis, which are considered to be associated with UV-induced tolerance. Altering the UV protocol to a sub-erythemal UV dose for four consecutive days or to a single high dose of 2 MED, resulted in suppressed HSV antigen presentation, without the influx of the UV-macrophages. One of the goals of the present study was to eventually use this HSV system to investigate sunscreen immunoprotection. A pilot study with a TiO2-containing sunscreen suggested that the endpoint for UV-induced immunosuppression presented here is promising to be used for human in vivo sunscreen immunoprotection studies.
Asunto(s)
Presentación de Antígeno/efectos de la radiación , Antígenos Virales/inmunología , Epidermis/efectos de la radiación , Herpesvirus Humano 1/inmunología , Adulto , Presentación de Antígeno/inmunología , Antígenos CD1/inmunología , División Celular , Relación Dosis-Respuesta en la Radiación , Células Epidérmicas , Epidermis/efectos de los fármacos , Epidermis/inmunología , Antígenos HLA-DR/inmunología , Humanos , Células de Langerhans/citología , Macrófagos/inmunología , Persona de Mediana Edad , Protectores Solares/farmacología , Linfocitos T/citología , Linfocitos T/inmunología , Factores de Tiempo , Titanio/farmacología , Rayos Ultravioleta , VoluntariosRESUMEN
We used scanning electron microscopy in combination with X-ray microanalysis to evaluate Co-, Cr-, and Mo-based human femoral hip prostheses. In total, 23 retrieved implants and four new implants were included in this study. Scanning electron microscopy of the polished surface of all arthroplasties showed, in addition to the polishing marks, small round and angular holes or pits. Other types of surface irregularities were interpreted as wear or corrosion of the metal compound. In all cases studied, corrosion propagated from holes at the surface of the polished prosthesis heads, in some cases also along phase boundaries. X-ray microanalysis of the intact prosthetic surface showed a relative composition of the elements Co, Cr, and Mo, which was in agreement with the manufacturer's information (63:33:4%). However, X-ray microanalysis spot analysis of the surface holes showed deviation in the relative composition of the elements Co, Cr, and Mo and also the presence of Si, Ti, and Al. Furthermore, Ti and Al could be traced back at an artificially made fracture plane of a new prosthesis. Therefore, Ti and Al have to be present during the manufacturing process. These impurities in the metal prosthesis alloy may create a galvanic element with the Co, Cr, Mo alloy of the implant. If this is the case, such a galvanic element in combination with the electrolyte environment formed by body fluids, can induce galvanic corrosion with release of metal particles.
Asunto(s)
Cartílago Articular , Prótesis de Cadera , Falla de Prótesis , Artroplastia de Reemplazo de Cadera , Cromo/análisis , Cobalto/análisis , Microanálisis por Sonda Electrónica , Humanos , Microscopía Electrónica de Rastreo , Molibdeno/análisis , Reoperación , Propiedades de SuperficieRESUMEN
The main problem with topical application of compounds to administer drugs to and regulate drug levels in a human body, is the barrier formed by the intercellular lipid matrix of the stratum corneum (SC). In a search for possibilities to overcome this barrier function, a good understanding of the organization and phase behavior of these lipids is required. SC lipid model studies especially provide a wealth of information with respect to the lipid organization and the importance of certain subclasses of lipids for the structure. Previously, we have shown that electron diffraction (ED) provides detailed information on the lateral lipid packing in both intact SC (G.S.K. Pilgram et al., J. Invest. Dermatol. 113 (1999) 403) and SC lipid models (G.S.K. Pilgram et al., J. Lipid Res. 39 (1998) 1669). In the present study, we used ED to examine the influence of two azones and sebaceous lipids on the lateral phase behavior of lipids isolated from human SC. We established that human SC lipids are arranged in an orthorhombic packing pattern. Upon mixing with the two enhancers the orthorhombic packing pattern was still observed; however, an additional fluid phase became more apparent. In mixtures with sebaceous lipids, the presence of the hexagonal lattice increased. These findings provide a basis for the mechanism by which these enhancers and sebaceous lipids interact with human SC lipids.
Asunto(s)
Azepinas/farmacología , Lípidos/química , Sebo/química , Piel/metabolismo , Femenino , Humanos , Lípidos/aislamiento & purificación , Sustancias Macromoleculares , Microscopía Electrónica/métodos , Permeabilidad/efectos de los fármacos , Glándulas Sebáceas/metabolismo , Sebo/metabolismo , Piel/químicaRESUMEN
The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Streptomyces/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , División Celular , Eliminación de Gen , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutagénesis , Plásmidos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Esporas Bacterianas/fisiología , Esporas Bacterianas/ultraestructura , Streptomyces/citología , Streptomyces/genéticaRESUMEN
HYPOTHESIS/BACKGROUND: Endotoxin can induce morphologic changes to middle ear epithelium, which can disturb the mucociliary clearance system (MCS) and lead to otitis media with effusion (OME). The bactericidal/permeability-increasing (BPI) protein is a major component of neutrophil granules and binds with high affinity to endotoxin. In this study, the capacity to inhibit the effects of endotoxin by rBPI21, a recombinant amino-terminal analog derived from BPI, was investigated on cultured human middle ear epithelium using light microscopy and scanning- and transmission electron microscopy. METHODS: Human middle ear epithelium was air-exposed cultured on a collagenous underlayer with different additions of endotoxin and rBPI21 to the culture medium. The tissue specimens were inspected after 4 weeks for the number of ciliated and secretory cells, thickness of the mucosal layer, and cell size. RESULTS: The morphologic changes induced by endotoxin were increased thickness of the mucosal layer and increased number of secretory cells. These changes were significantly diminished or even absent when endotoxin was added with rBPI21 to the culture medium. CONCLUSION: rBPI21 can inhibit morphologic changes in the middle ear epithelium due to endotoxin. Hence, the authors believe that rBPI21 can be a new therapeutic agent in the treatment of OME.
Asunto(s)
Antibacterianos/farmacología , Actividad Bactericida de la Sangre/efectos de los fármacos , Oído Medio/efectos de los fármacos , Oído Medio/metabolismo , Endotoxinas/metabolismo , Haemophilus influenzae/metabolismo , Salmonella typhimurium/metabolismo , Células Cultivadas , Oído Medio/ultraestructura , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Epitelio/ultraestructura , Humanos , Depuración Mucociliar/efectos de los fármacos , Membrana Mucosa/ultraestructuraRESUMEN
PURPOSE: Investigation of the relationship between changes in human SC lipid organization induced by N-alkyl-azocycloheptane-2-one and SC permeability to the model compound HgCl2. METHODS: Human dermatomed skin was treated with propylene glycol (PG), oleyl-Azone (OAz) or dodecyl-Azone (DAz) in 0.15 M PG. Untreated skin served as control. The lateral lipid organization was studied by electron diffraction. Hg was measured on tape-strips by X-ray microanalysis and in the acceptor phase by atom absorption spectrometry. RESULTS: In control and PG treated samples, the lipid packing was mainly orthorhombic, while a small fraction was hexagonal. In OAz and DAz treated samples, the orthorhombic lipid organization remained, however, the hexagonal packing was recorded less frequently. The amount of Hg decreased as a function of depth in all SC samples, however, the penetration profile increased significantly upon OAz treatment. The cumulative amount of Hg in the acceptor phase of OAz treated samples also increased significantly compared to control and PG treated samples. CONCLUSIONS: The increased penetration of Hg into OAz treated skin could not be related to an orthorhombic-hexagonal phase transition. Alternatively, phase separation of OAz and/or formation of grain boundaries might affect SC permeability, hereby increasing Hg penetration. A similar mechanism is proposed for DAz.
Asunto(s)
Azepinas/farmacocinética , Desinfectantes/farmacocinética , Membrana Dobles de Lípidos/metabolismo , Cloruro de Mercurio/farmacocinética , Propilenglicol/farmacocinética , Humanos , Membrana Dobles de Lípidos/química , Permeabilidad , Absorción Cutánea/efectos de los fármacos , Absorción Cutánea/fisiologíaRESUMEN
BACKGROUND: Melanocytic dendrites consist of a central core of microtubules and a subcortical actin network. Several studies provide arguments supporting the hypothesis that actin-based and microtubule-based motor proteins co-operate in melanosome transport towards the dendrite tips. Melanosomes can move bidirectionally along microtubules in vitro, and in murine melanocytes, they move towards the cell periphery and back again. Microtubules have a fast-growing plus end and a slow-growing minus end. Microtubule-associated motor proteins move unidirectionally either towards the plus or towards the minus end. However, it is not known which motor protein is responsible for minus end-directed movement of melanosomes. OBJECTIVES: We aimed to investigate the in vitro expression of the minus end-directed motor protein cytoplasmic dynein in normal human epidermal melanocytes, keratinocytes and dermal fibroblasts. METHODS: Reverse transcription-polymerase chain reaction and Northern blot analysis were used. In addition, an attempt to obtain insight into the subcellular localization of cytoplasmic dynein, immunofluorescence studies and immunogold electron microscopic studies were performed. RESULTS: The three different forms of cytoplasmic dynein heavy chain were expressed in all studied skin cells. Immunofluorescence staining showed similar punctate distributions for dynein heavy chain 1 and dynein heavy chain 2 in melanocytes, with accentuation in the perinuclear area and dendrite tips. Double labelling with a melanosome marker showed apparent co-localization of both dynein heavy chains 1 and 2 with melanosomes in the perinuclear area and dendrite tips. For the dynein intermediate chain of 74 kDa, again a punctate staining pattern was seen with intense centrosomal staining. A close association of dynein intermediate chain 74 and alpha-tubulin with the melanosome surface was detected using immunogold electron microscopy. CONCLUSIONS: The colocalization of different subunits of the cytoplasmic dynein complex with melanosomes is consistent with the hypothesis that this motor protein supports minus end-directed melanosome movement along microtubules.
Asunto(s)
Citoplasma/metabolismo , Dineínas/metabolismo , Melanosomas/metabolismo , Adulto , Técnicas de Cultivo de Célula , Dendritas/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Recién Nacido , Queratinocitos/metabolismo , Microscopía Inmunoelectrónica , Microtúbulos/metabolismo , Piel/metabolismo , Células Tumorales CultivadasRESUMEN
Cis-urocanic acid (UCA) has been indicated as an important mediator of ultraviolet (UV)-induced immunosuppression. In this study we describe a rapid, noninvasive method for the determination of the protective capacity of various sunscreens against the UV-induced isomerization of trans-UCA into its cis form. For this purpose we applied sunscreens prior to in vivo exposure of human volunteers with single or repeated broadband UVB irradiations of 100 mJ per cm2. We found significant but different levels of protection against UCA photoisomerization by all sunscreens that correlated with the sun protection factor. A comparison of various sunscreens with a sun protection factor of 10, showed that the best protection was offered by the sunscreens (containing organic UV filters or TiO2) with broad absorption spectra. The ability to inhibit cis-UCA formation was not influenced by the penetration characteristics of sunscreens, as determined by application of the sunscreen on quartz glass that was placed on the skin, preventing penetration of sunscreen in the skin. In addition ex vivo UV exposure of human skin was employed to permit other tests of immunomodulation, in this case the mixed epidermal cell lymphocyte reaction. The advantage of this ex vivo method is that there is no need to take biopsies from volunteers. Ex vivo irradiation of human skin with a single dose of 200 mJ per cm2 resulted in similar protection by the sunscreens against cis-UCA formation as in the in vivo system. Furthermore, the mixed epidermal cell lymphocyte reaction data correlated with the cis-UCA findings. We conclude that UCA isomerization is an excellent method to determine sunscreen efficacy and that broad-spectrum sunscreens offer good immunoprotection.
Asunto(s)
Piel/efectos de la radiación , Protectores Solares/farmacología , Rayos Ultravioleta , Relación Dosis-Respuesta en la Radiación , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Tolerancia Inmunológica/efectos de la radiación , Isomerismo , Prueba de Cultivo Mixto de Linfocitos , Dosis de Radiación , Titanio/farmacología , Ácido Urocánico/químicaRESUMEN
In this study, the efficacy of bactericidal/permeability-increasing protein (BPI) was assessed in a rat model of chronic otitis media with effusion. BPI injection prevented disturbance of the mucociliary clearance system of the middle ear. Hence, it is postulated that BPI can be a new therapy for chronic otitis media with effusion.
Asunto(s)
Antiinfecciosos/uso terapéutico , Proteínas Sanguíneas/uso terapéutico , Proteínas de la Membrana , Otitis Media con Derrame/tratamiento farmacológico , Animales , Péptidos Catiónicos Antimicrobianos , Enfermedad Crónica , Modelos Animales de Enfermedad , Oído Medio/inmunología , Oído Medio/patología , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Membrana Mucosa/lesiones , Membrana Mucosa/patología , Otitis Media con Derrame/inmunología , Otitis Media con Derrame/patología , Ratas , Proteínas Recombinantes/uso terapéuticoRESUMEN
Microtubuli play an important role in the organization of organelles and membrane traffic. They are present in melanocytic dendrites through which melanosomes are transported towards keratinocytes. Besides the actin-based motility systems, microtubuli-associated motor proteins also play a critical role in melanosome movement, as has recently been confirmed in mouse melanocytes. We investigated the in vitro expression of two forms of human conventional kinesin and its receptor kinectin in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription polymerase chain reaction and northern blot analysis. In an attempt to gain insight into the subcellular distribution of kinesin and kinectin in melanocytes, double immunofluorescent staining and immunogold electron microscopy were performed. In all studied skin cells ubiquitous and neuronal kinesin are expressed, as well as the kinectin receptor. Immunofluorescent staining shows distinct but partially overlapping distributions for kinesin heavy chain and melanosomes, suggesting that kinesin is associated with some but not all of the melanosomes. Similar observations for kinectin indicate that this receptor can colocalize with melanosomes, which was confirmed by immunoelectron microscopy. The latter technique allowed us to demonstrate a close association between kinesin heavy chain, microtubuli, and melanosomes. The combined data from reverse transcription polymerase chain reaction, northern blot analysis, double immunofluorescent staining, and immunogold electron microscopy suggest that kinesins and kinectin have an important role in microtubuli-based melanosome transport in human melanocytes.
Asunto(s)
Cinesinas/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Reactivos de Enlaces Cruzados , Citoplasma/química , Humanos , Inmunohistoquímica , Melanocitos/metabolismo , Melanocitos/ultraestructura , Melanosomas/química , Microtúbulos/químicaRESUMEN
Melanocytic dendrites consist of a central core of microtubules (MT) and a subcortical actin network. In previous reports we showed the presence of MT-associated motor proteins kinesin and cytoplasmic dynein on the melanosomal surface, forming a link with MT (Vancoillie et al. J Invest Dermatol 2000;114:421-429; Vancoillie et al. Br J Dermatol 2000;143:258-306). We could also demonstrate the association of kinectin, the kinesin receptor, with melanosomes. The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. Therefore, in this study, we investigated the in vitro expression of dynactin subunits P150Glued and P50 in normal human epidermal melanocytes, keratinocytes, and dermal fibroblasts by reverse transcription-polymerase chain reaction and northern blot analysis. In an attempt to gain an insight into the subcellular localization of dynactin, immunofluorescence and immunoelectron microscopy (IEM) studies were performed. The two isoforms of P150Glued and P50 are expressed in all studied skin cells. Immunofluorescence staining shows punctate distributions for P150Glued and P50 in melanocytes. P150Glued shows a clear centrosomal staining and accentuation in the dendrite tips. P50 is also accentuated in the perinuclear area and dendrite tips. Immunofluorescence double-labeling with a melanosome marker showed apparent colocalization of both P150Glued and P50 with melanosomes. By IEM, P50 is detected on the surface of the majority of melanosomes in melanocytes. The colocalization of different subunits of the dynactin complex with melanosomes is consistent with the earlier finding of cytoplasmic dynein association with melanosomes and supports the hypothesis that this complex could form a link between cytoplasmic dynein and the melanosomal membrane.
Asunto(s)
Melanocitos/metabolismo , Melanosomas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Northern Blotting , Membrana Celular/metabolismo , Células Cultivadas , Centrosoma/metabolismo , Citoplasma/metabolismo , Complejo Dinactina , Dineínas/química , Dineínas/metabolismo , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Microscopía Confocal , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Células Tumorales CultivadasRESUMEN
The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.