Rejection of dendritic-cell-depleted epithelial-cultured allografts.
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Animals↗
Biomedical subjects
F A Auger
Publications and source records attributed to F A Auger.
Surgical model for in vivo evaluation of cultured epidermal sheets in mice.
The objective of this study was to establish a novel surgical model for the grafting of cultured epidermal sheets in mice in order to optimize studies on the behavior of these grafts. Graft-related skin immunology and wound-healing studies also would benefit from this adapted surgical approach. Adapted tie-over surgical procedures were established for mice and promptly applied. Early-stage observation of grafts was made possible by replacing the cotton dressing with a saddle-like plastic tube dressing with a screw cap. We grafted normal Balb/c mice and athymic nude (nu/nu) mice with cultured human epidermis. Evaluation of graft rejection was carried out with the first group, whereas the second provided information on epidermal stratification and terminal differentiation. This innovation permitted direct evaluation of the grafted tissue at any time. Advances in applied transplantation research will certainly provide additional tools for human applications.
Animals↗
Influence of initial collagen and cellular concentrations on the final surface area of dermal and skin equivalents: a Box-Behnken analysis.
Our laboratory has been involved in finding optimal conditions for producing dermal and skin equivalents. As an original approach, a Box-Behnken experimental design was used to study the effects of the initial collagen and fibroblast concentrations and the initial gel thickness on the contraction of dermal and skin equivalents. The final surface area of dermal equivalent varied significantly with the initial concentration of collagen and fibroblast, whereas the initial thickness of gel had no appreciable effect on the contraction of the dermal equivalent. When keratinocytes were grown on these dermal equivalents they produced a very severe contraction, to an extent that all skin equivalents had a similar final surface area. This severe contraction was independent of collagen and fibroblast concentrations. Models for the prediction of the final percentage contraction of dermal and skin equivalents as a function of the initial concentration of collagen, the logarithm of fibroblast concentration, and the initial gel thickness were obtained and analyzed. Keratinocytes grown at the lowest seeding density did not contract the equivalents. However, histologic analysis has shown an incomplete coverage by these cells of the equivalents. The extensive contraction of the skin equivalent presenting adequate morphology is a major drawback toward its clinical utilization for burn wound coverage.
Cells, Cultured↗
Impaired antibody-dependent cell-mediated cytotoxic activity in patients with the acquired immunodeficiency syndrome.
Since the acquired immunodeficiency syndrome (AIDS) is characterized by opportunistic infections and malignancies indicative of a profound suppression in cell-mediated immunity, we investigated the antibody-dependent cell-mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells of patients with AIDS against chicken red blood cells (CRBC). A marked decrease in ADCC-CRBC activity was observed from patients with AIDS as compared to healthy controls. Furthermore, suppression in ADCC activity was seen when mononuclear cells from healthy subjects were assayed using media containing 25% or 40% sera from AIDS patients. Two of two patients with AIDS and impaired ADCC-CRBC activity were also found to have in vivo impaired reticuloendothelial system Fc-specific clearance of 51Cr-labelled, anti-Rho (D) IgG-sensitized autologous erythrocytes. These data provide further evidence of monocyte-macrophage dysfunction in AIDS and help explain the widespread occurrence of opportunistic pathogens in AIDS.
Acquired Immunodeficiency Syndrome↗
Improved micromethod for mezlocillin quantitation in serum and urine by high-pressure liquid chromatography.
A rapid, sensitive, and specific method of analysis for mezlocillin in serum and urine by high-pressure liquid chromatography is described. A solid-phase extraction column was used to remove interfering substances from samples before chromatography. Quantitation included the use of an internal standard, nafcillin. Mezlocillin was chromatographed with a phosphate buffer-acetonitrile (73:27) mobile phase and a C-18 reverse-phase column and detected at a wavelength of 220 nm. The assay had a sensitivity of 1.6 micrograms/ml and a linearity of up to 600 micrograms/ml and 16 mg/ml in serum and urine, respectively, with only 0.1 ml of sample. The interday and intraday coefficients of variation for replicate analyses of spiked serum and urine specimens were less than 6.5%.
Chromatography, High Pressure Liquid↗
Permanent grafting of living skin substitutes: surgical parameters to control for successful results.
Autologous mesh grafting, widely used in the treatment of severe burns, remains the most conventional approach for permanent skin replacement. However, during the last decade several types of skin substitutes were reported as suitable alternatives for full-thickness burn wound coverage. The clinical use of such dressings requires new surgical skills to maintain the integrity of the grafts and favor their permanent implantation in vivo. This article reports observations made on nude mice grafted with cultured human skin equivalents. Some parameters such as the quality of adhesion between the implant and the graft bed, the size, the stability and the thickness of the graft, the humidity of the chamber, and the protocol of antibiotic administration were identified as crucial for the success of the surgery. The grafting procedures are described in this paper. These results should be taken into consideration in all transplantations of skin grafts in vivo.
Animals↗
Cyclic traction machine for long-term culture of fibroblast-populated collagen gels.
Our research group has been investigating the effect of cyclic deformations on the evolution of fibroblast populated collagen gels (FPCG). Since existing traction machines are not designed for such an application, we had to design a cyclic traction machine adapted to tissue culture inside an incubator over an extended period of time. Biocompatible materials were used for fabrication to allow for easy sterilization and to prevent any adverse reaction from the tissue. The traction machine is based on a computer-controlled stepping motor system for easy adjustment of the deformation amplitude and frequency. The maximum stretching speed achieved is around 1 mm/s. The traction machine can measure FPCG mechanical properties and perform rupture tests to determine its ultimate strength. Several FPCGs have been successfully cultured with the machine for up to four weeks without any adverse reaction.
Biocompatible Materials↗
Reconstructed human cornea produced in vitro by tissue engineering.
The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson's trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as beta1, alpha3, alpha4, alpha5, and alpha6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4-5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the alpha4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.
Cell Division↗
CD36(+)-dendritic epidermal cells: a putative actor in the cutaneous immune system.
UNLABELLED: In the present study we have investigated by indirect immunofluorescence staining and mixed lymphocyte reaction methods, the localization, distribution, percentage, and the immunological involvement of CD36(+)-dendritic epidermal cells (CD36(+)-DECs) in normal human skin. Human epidermal cell suspensions were obtained from skin specimen of healthy persons. First, an indirect immunofluorescent staining method was performed on frozen skin sections, freshly isolated cells, nonadherent and adherent cells and second, the allogeneic mixed epidermal cell-lymphocyte reaction (ELR) method was performed with human peripheral blood mononuclear cells and irradiated CD36(+)-DECs plus CD-1a+ (Langerhans cells) and/ConA (at 10 micrograms/mL). We found that CD36(+)-DECs were localized in the epidermis mainly in the basal layer. They were non adherent cells. The percentage of these CD36(+)-DECs was of about 2%. These CD36(+)-DECs were AE3 (which recognizes keratin normally expressed by keratinocytes) positive cells. Our immunoreactivity study using allogeneic mixed ELR, showed that CD36(+)-DECs stimulated allogeneic lymphocyte proliferation. Their stimulatory effects were important when Langerhans cells and ConA were added separately or together to the PBMCs culture. The above results suggest that CD36(+)-DECs may contribute to the immunological role of skin and could be involved in cutaneous allograft recognition and rejection. ABBREVIATIONS: DECs: dendritic epidermal cells; ConA: concanavalin A; DPM: disintegrations per minute; ELR: epidermal cell-lymphocyte reaction; LC: Langerhans cells; PBMCs: peripheral blood mononuclear cells.
Adult↗
Involvement of male-specific minor histocompatibility antigen H-Y in epidermal equivalent allograft rejection.
This study describes the involvement of male-specific minor histocompatibility antigen H-Y in vitro cultured epidermal equivalent (EE) rejection. Male and female Balb/c or C3H/HeN keratinocytes were isolated and cultured separately. Male EE were grafted onto adult male (isografts) and adult female (H-Y allografts) mice. As controls, Balb/c EE were grafted onto adult C3H/HeN (complete allografts) mice. Fourteen, 21, and 30 days postgrafting, histological studies showed well-organized cutaneous tissues with complete basement membranes (laminin and type IV collagen deposition) in H-Y allografts compared to the isografts. This cutaneous organization was altered 150 days postgrafting, which is a sign of the H-Y EE allograft rejection. Complete allografts were totally rejected 21 days postgrafting. Immunological studies revealed leucocyte infiltration of H-Y allografts. Significant infiltration was detected even 150 days postgrafting. Leucocyte phenotyping revealed the presence of Mac-I+, CD8+ and CD4+ cells in the H-Y allografts. Humoral immune analysis revealed the presence of circulating anti-H-Y allogeneic keratinocyte cytotoxic antibodies in female recipient sera. Our data suggest that male-specific minor histocompatibility antigen H-Y induces cellular and humoral activation of the recipient immune system even after grafting EE free of cutaneous active immune cells such as T lymphocytes and Langerhans cells.
Animals↗
Production of tissue-engineered three-dimensional human bronchial models.
We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.
Bronchi↗
Three-dimensional production of bronchi in vitro.
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Bronchi↗