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Biomedical subjects

L Germain

Publications and source records attributed to L Germain.

At least 55 records · Page 3Linked to original sources
PubMed1997

Differential expression of collagens XII and XIV in human skin and in reconstructed skin.

Collagens XII and XIV localize near the surface of collagen fibrils and may be involved in epithelial-mesenchymal interactions as well as in the modulation of tissue biomechanical properties. Moreover, human skin fibroblasts cultured in monolayer are known to lose their ability to produce collagen XIV and to switch the transcription of collagen XII from the small splice variant (220 kDa) to the large (320 kDa), whereas the small form is the main form found in human skin. We have investigated the expression patterns of these two molecules in human skin as a function of donor age and anatomic site, by using immunohistology with specific monoclonal antibodies. We demonstrated changes in the expression patterns of collagens XII and XIV in human skin after birth. Moreover, in adult scalp skin, very strong staining of collagen XII fibril bundles was observed around hair follicles, in association with very low expression of collagen XIV. We also investigated the expression of collagens XII and XIV by fibroblasts and keratinocytes cultured in a reconstructed skin. In these culture conditions, fibroblasts recovered their ability to produce collagen XIV and re-expressed the small splice variant of collagen XII. These results could be explained by the deposition of large amounts of collagen fibrils by fibroblasts in this culture system. Thus, the re-expression of these collagens suggests that the deposition of banded collagen fibrils is a pre-requisite for the expression of collagen XIV and small variant of collagen XII.

Adult↗
PubMed1997

Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide.

Many reports have cited coexpression of platelet-derived growth factor (PDGF) and its receptors by tumor cells or cells supporting tumor growth, suggesting both autocrine and paracrine mechanisms for PDGF-mediated tumor growth. We found that a small organic molecule, N-[4-(trifluoromethyl)phenyl] 5-methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation. SU101 inhibited PDGF-stimulated tyrosine phosphorylation of PDGF receptor (PDGFR) beta in C6 (rat glioma) and NIH3T3 cells engineered to overexpress human PDGFRbeta (3T3-PDGFRbeta). SU101 blocked both PDGF- and epidermal growth factor (EGF)-stimulated DNA synthesis. Previously, this compound was shown to inhibit pyrimidine biosynthesis by interfering with the enzymatic activity of dihydroorotate dehydrogenase. In the current study, EGF-stimulated DNA synthesis was restored by the addition of saturating quantities of uridine, whereas PDGF-induced DNA synthesis was not, suggesting that the compound demonstrated some selectivity for the PDGFR pathway that was independent of pyrimidine biosynthesis. Selectivity was further demonstrated by the ability of the compound to block the entry of PDGF-stimulated cells into the S phase of the cell cycle, without affecting cell cycle progression of EGF-stimulated cells. In cell growth assays, SU101 selectively inhibited the growth of PDGFRbeta-expressing cell lines more efficiently than it inhibited the growth of PDGFRbeta-negative cell lines. SU101 inhibited the s.c., i.p., and intracerebral growth of a panel of cell lines including cells from glioma, ovarian, and prostate origin. In contrast, SU101 failed to inhibit the in vitro or s.c. growth of A431 and KB tumor cells, both of which express EGF receptor but not PDGFRbeta. SU101 also inhibited the growth of D1B and L1210 (murine leukemia) cells in syngeneic immunocompetent mice, without causing adverse effects on the immune response of the animals. In an i.p. model of tumor growth in syngeneic immunocompetent mice, SU101 prevented tumor growth and induced long-term survivors in animals implanted with 7TD1 (murine B-cell hybridoma) tumor cells. Because PDGFRbeta was detected on most of the tumor cell lines in which in vivo growth was inhibited by SU101, these data suggest that SU101 is an effective inhibitor of PDGF-driven tumor growth in vivo.

3T3 Cells↗
PubMed1996

Grafting on nude mice of living skin equivalents produced using human collagens.

Autologous epidermal transplantation for human burn management is an example of a significant breakthrough in tissue engineering. However, the main drawback with this treatment remains the fragility of these grafts during and after surgery. A new human bilayered skin equivalent (hSE) was produced in our laboratory to overcome this problem. The aim of the present work was to study skin regeneration after hSE grafting onto nude mice. A comparative study was carried out over a period of 90 days, between anchored bovine skin equivalent, hSE and hSE+, the latter containing additional matrix components included at concentrations similar to those in human skin in vivo. The addition of a dermal layer to the epidermal sheet led to successful graft take, enhanced healing, and provided mechanical resistance to the grafts after transplantation. In situ analysis of the grafts showed good ultrastructural organization, including the deposition of a continuous basement membrane 1 week after surgery.

Animals↗
PubMed1996

Use of human vessels and human vascular smooth muscle cells in pharmacology.

Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, but these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.

Calcium↗
PubMed1996

Stimulation of human keratinocyte proliferation through growth factor exchanges with dermal fibroblasts in vitro.

Progress in biotechnology has led to new therapeutic approaches in various fields of human health care, such as the autologous grafting of cultured epidermal cell sheets on burned patients. These cultures depend on various parameters but growth factors are of paramount importance. Cutaneous cells are known to secrete various growth factors in vivo, although only a few have been identified. The aim of this study was to determine if such factors are secreted from human cutaneous cells in culture, to evaluate their effects on epidermal cell proliferation in vitro and to analyse them on SDS-PAGE. Human skin fibroblasts and keratinocytes were co-cultured for 8-10 days using a Costar trans-filter system. Dermo-epidermal cooperation was observed in such a co-culture system through the exchange of secretion products in the culture medium. Epidermal cell growth and metabolic activities were highly stimulated in co-culture (2-fold and 1.5-fold, respectively, P < 0.02) compared to the control. The de novo synthesis of secretion products, notably of a protein of about 40 kDa, was specifically induced in co-culture. The identification of new keratinocyte growth factors could accelerate graftable epidermal sheet production in vitro for human wound coverage and possibly enhance wound healing in vivo.

Cell Division↗
PubMed1996

Cultured epidermal sheet grafting with Hemaseel HMN fibrin sealant on nude mice.

Grafting of cultured epidermal sheets is a promising technique for skin restoration in extensive burns, but the technique has some limitations, resulting in variable graft takes. These experiments were designed to evaluate the innocuity of Hemaseel HMN fibrin sealant in the grafting process and in vivo evolution of cultured epidermis. A total of 30 mice were grafted, 15 were controls, 15 received tissue sealant application before the deposition of the cultured human epidermal sheets. Seven days after transplantation, compared to controls, the percentage of graft take over the total surface area grafted was greater in animals that had received the tissue sealant application. No difference was found 14 and 21 days postgrafting. In contrast, the percentage of graft take over the bony area (spinal) was significantly increased in animals grafted with previous application of sealant compared to controls at 7, 14 and 21 days postgrafting. Immunohistological and ultrastructural analysis showed that the evolution of the cultured human epidermis after transplantation was similar in both groups. The basement membrane was well structured 21 days after transplantation. The sealant was present at 4 days but not at 21 days postgrafting. Therefore, we conclude that the application of fibrin sealant before cultured epidermal sheet deposition on nude mouse graft bed is innocuous and enhances their mechanical stability. Since in this nude mouse system Hemaseel HMN fibrin sealant increased the percentage of graft take over areas difficult to engraft, we think that it may be advantageous in cultured epidermal sheet grafting on burn patients.

Animals↗
PubMed1996

In vitro models to study wound healing fibroblasts.

Phenotypic and contractile properties of human fibroblasts from dermis and from an experimental wound model were studied in vitro. When cultured in monolayer, dermal fibroblasts had an elongated spindle shape, were small in diameter and grew at a high rate. Wound fibroblasts grew slowly and were large, star shaped and had cytoplasmic stress fibres. Smooth muscle alpha actin was detected in 10 percent of dermal cells, whereas 20-80 per cent of wound fibroblasts contained this protein in their cytoplasm. The contractile property of cells was evaluated using a three-dimensional cell culture model. Our results show that wound fibroblasts contract collagen gels during the first days more strongly than dermal fibroblasts. These results show that, in vitro, wound fibroblasts have greater contractile capacity than dermal cells. The significant proportion of wound fibroblasts containing alpha-smooth muscle actin suggests that alpha-smooth muscle actin ratio may be related to wound contraction.

Cells, Cultured↗
PubMed1996

Morphologic and functional properties of bronchial cells isolated from normal and asthmatic subjects.

Recent advances in biomedical sciences have led to the development of various methods for the evaluation of the physiopathology of respiratory diseases. This study reports morphologic and functional features of cells isolated by a new method from bronchial biopsies of normal and asthmatic subjects. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. The cells were cultured for several passages and stored frozen. Two selective culture media were used in order to obtain pure epithelial and fibroblastic cell populations. Immunofluorescence analysis of intermediate filaments, keratins, and vimentin confirmed the type of the isolated cells. The proportions of alpha-actin-expressing cells varied among the fibroblastic cell populations isolated from normal and asthmatic subjects. Interestingly, the population containing high numbers of alpha-actin-expressing cells and presenting the fastest collagen contraction kinetic was isolated from bronchial biopsies of an asthmatic subject. Moreover, the fibroblastic cells that showed the best contractile properties 24 h after their seeding in floating collagen gels were isolated from bronchial biopsies of asthmatic patients having PC20 values below 1 mg/ml. On the basis of these data, we propose a new approach to isolate, culture and characterize human bronchial cells in vitro.

Adult↗
PubMed1996

Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage.

This study was undertaken to evaluate keratin 19 (K19) as a biochemical marker for skin stem cells in order to address some long standing questions concerning these cells in the field of cutaneous biology. We first used the well-established mouse model enabling us to identify skin stem cells as [3H]thymidine-label-retaining cells. A site directed antibody was raised against a synthetic peptide of K19. It reacted specifically with a 40 kDa protein (K19) on immunoblotting. It labelled the bulge area of the outer root sheath on mouse skin by immunohistochemistry. Double-labelling revealed that K19-positive-cells were also [3H]thymidine-label-retaining cells, suggesting that K19 is a marker for skin stem cells of hair follicles. K19-expression was then used to investigate the variation in mouse and human skin stem cells as a function of body site, donor age and culture time. K19 was expressed in the hair follicle and absent from the interfollicular epidermis at hairy sites (except for some K18 coexpressing Merkel cells). In contrast, at glabrous sites, K19-positive-cells were in deep epidermal rete ridges. K19 expressing cells also contained high levels of alpha 3 beta 1 integrin. The proportion of K19-positive-cells was greater in newborn than older foreskins. This correlated with keratinocyte culture lifespan variation with donor age. Moreover, it could explain clinical observations that children heal faster than adults. In conclusion, K19 expression in skin provides an additional tool to allow further characterization of skin stem cells under normal and pathological conditions in situ and in vitro.

3T3 Cells↗
PubMed1995

Suppression of tumor growth in vivo by local and systemic 90K level increase.

Expression levels of the immunostimulatory 90K antigen in mammary carcinoma, glioblastoma, and other tumor-derived cell lines inversely correlate with their tumorigenicity in athymic mice. Engineered enhancement of 90K expression results in significant (> 80%) tumor growth inhibition, not by direct action on the tumor cell, but by stimulation of the residual cell-mediated immune defense of the nude mouse. Enhanced 90K level effects are both localized and systemic and involve induction of ICAM-1 and VCAM-1 in the tumor endothelium. The findings presented suggest a role for 90K as a molecular alarm signal for the body's cellular defense against pathogens, which in a subset of tumors is suppressed to allow cancer progression.

Animals↗
PubMed1995

Allogeneic-syngeneic cultured epithelia. A successful therapeutic option for skin regeneration.

Organ transplantation has progressed rapidly during the last decades. Furthermore, tissue engineering has and will continue to enlarge the scope of organ grafting. Thus, severe skin wounds, as observed in large burn trauma patients, can now be permanently treated with cultured autologous epithelial sheets. However, the time required for autologous cell growth is a major limitation. We propose to alleviate this pitfall through a novel chimeric (allogeneic-syngeneic) epithelial cell culture technique. These chimeric epidermal grafts implanted in an animal model have been shown to allow the reappearance of a histologically normal epidermal coverage similar to simultaneously performed isografts. The regenerated epidermis contained only syngeneic keratinocytes. Thus, allogeneic cells were eliminated passively. This new culture technology could find multiple applications, notably in various skin disease therapies.

Animals↗
PubMed1995

Skin equivalent produced with human collagen.

Several studies have recently been conducted on cultured skin equivalent (SE), prepared using human keratinocytes seeded on various types of dermal equivalents (DE). We previously showed the advantages of our anchorage method in preventing the severe surface reduction of DE due to fibroblast contractile properties in vitro. A new anchored human SE was established in our laboratory in order to obtain a bioengineered tissue that would possess the appropriate histological and biological properties. In order to compare the effects of different collagen origins on the evolution of SE in vitro, human keratinocytes were seeded on three types of anchored DE. A comparative study was carried out between bovine SE (bSE), human SE (hSE), and human skin equivalent containing additional dermal matrix components (hSE+). Immunohistological analysis showed that hSE and hSE+ presented good structural organization, including the deposition of several basement membrane constituents. Higher amounts of transglutaminase, ceramides, and keratin 1 were detected in the epidermal layers of all SE when cultured at the air-liquid interface. However, a 92 kDa gelatinase activity was higher in bovine skin equivalent (bSE) compared to hSE cultures. The use of human collagens comparatively to bovine collagen as SE matricial component delayed the degradation of the dermal layer in culture.

Animals↗
PubMed1995

Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or Dispase.

The basement membrane zone is important for graft adhesion and stability. The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets. A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin. After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20-30 min) or Dispase (for 45-60 min), and deposited on a muscular graft bed of athymic mice. Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting. Bullous pemphigoid antigens and laminin were detected at the dermo-epidermal junction, showing an almost continuous line 2 days postgrafting. Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting. Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting. Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting. The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase. However, it differed from that of other wound healing models. Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models.

Animals↗
PubMed1995

A method for the evaluation of tensile properties of skin equivalents.

In vitro production of anchored skin equivalent is a new therapeutical option for burn patients. A skin equivalent is a combined culture of dermal and epidermal layers. The dermal layer provides important mechanical properties, such as tensile resistance and nonlinear elasticity, to the skin equivalent during its development. Prior to any in vivo human transplantation, the tensile properties of cutaneous equivalents have to be evaluated as a function of its structural components, in view of establishing the culture conditions leading to the best mechanical resistance and stretchability characteristics. However, the handling and clamping of skin equivalents are frequent causes of tearing and lack of repeatability in the measuring of tensile properties. A new indentation method involving a specially designed culture dish has been developed to minimize the risk of damage. Using this new culture dish, cutaneous equivalents were installed on an indentation apparatus. The central loading of a spherical tip was transmitted to the central area of a circular anchored cutaneous equivalent and was recorded with tip position. The tests were achieved at a constant low deflection rate of the tip. This new and accurate method gave repeatability in three central load-deflection characteristics of anchored dermal equivalent: the high-modulus (0.15 g mm-1), the central load of rupture (1.49 g), the rupture deflection (0.470 mm). This indentation test is expected to be an efficient tool in the evaluation of various skin equivalent models tensile properties.

Biological Dressings↗
PubMed1995

Study of the tensile properties of living skin equivalents.

The living skin equivalent is one of the more advanced clinical applications in the field of tissue engineering. It is a promising therapeutic option for burn victims and a strong potential for manifold in vitro experiments. However, researchers have encountered major drawbacks in the reconstruction of the dermal layer. Peripheral anchorage of the dermal equivalent component has been a valuable solution to many of these problems. In this work, we have carried out the mechanical analysis of skin equivalent models, based on this dermal anchoring technique, with a study of their biaxial tensile properties. Differences between models were related to the origin of collagen, either bovine or human, and on the culture techniques: immersion or at the air-liquid interface. The study was accomplished in vitro using 25.4-mm-diameter disk-shaped specimens with an indentation test. In appropriate wet condition, the specimens were punctured with a spherical tip at a quasi-static rate. We measured the load applied against the tip vs deflection up to the breaking point. Our results show that skin equivalents presented a typical exponential load-deflection relationship. All skin equivalents presented large extensibility up to 1.41 expressed in a ratio of deflection vs specimen's radius. The maximum tensile strength (0.871-1.169 Newton) and energy calculations (3.75-6.432 N.mm) was offered by living skin equivalent, made with human types I and III collagens, cultured at the air-liquid interface. In these conditions, our results suggest the tensile properties of living skin equivalents were enhanced due to the development of well stratified stratum corneum.

Adolescent↗
PubMed1994

Insulin binding to human cultured lymphocytes measured by flow cytometry using three ligands.

The binding of insulin to cultured IM-9 human lymphocytes was studied by flow cytometry using FITC-insulin and biotinylated insulins coupled to streptavidin-phycoerythrin (N alpha beta 1-biotinylinsulin (B-insulin) and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin (NBC-insulin)). The reference methods were 125I-insulin binding and the insulin-antiinsulin antibody complexes for flow cytometry. There was a close correlation between 125I-insulin binding and increase in fluorescence for B-insulin, NBC-insulin, and insulin-anti-insulin antibody complexes, but not for FITC-insulin. NBC-insulin gave the largest increase in fluorescence (79 +/- 9 channels) and the the insulin-antiinsulin antibody complexes the smallest (34 +/- 2 channels) (P < 0.05). FITC-insulin and B-insulin gave similar results: 47 +/- 6 and 59 +/- 6 channels. The concentration reducing 125I-insulin binding by 50% was 1.1 x 10(-9) M for native insulin, 2.7 x 10(-9) M for B-insulin, 3.3 x 10(-9) M for NBC-insulin, and 6.6 x 10(-9) M for FITC-insulin (P < 0.05). Nonspecific binding was low for B-insulin and NBC-insulin but reached 75% for 10(-6) M FITC-insulin. These results suggest that B-insulin and NBC-insulin are suitable ligands for insulin binding studies using flow cytometry. This two-step procedure is easier than the insulin-antiinsulin antibody complex technique. Its poor affinity, specificity, and sensitivity make FITC-insulin less suitable.

Animals↗
PubMed1994

Human wound healing fibroblasts have greater contractile properties than dermal fibroblasts.

Contractile and phenotypic properties of human fibroblasts from healing wounds were compared to those of dermal fibroblasts using in vitro models. Wound fibroblasts were recovered from implants, made of a polyvinyl alcohol sponge threaded into a perforated silicone tube, 12 days after their subcutaneous implantation in human volunteers. Dermal fibroblasts were isolated from the skin of healthy subjects. Two morphologically different fibroblast populations were observed in cells cultured from implants. In order to characterize these fibroblast populations, intracellular alpha-actin expression was studied by immunofluorescence labeling of cells cultured in monolayer. This protein was detected in less than 1% of the dermal fibroblasts. By contrast, 30 to 40% of wound fibroblasts were labeled and contained fiber networks of alpha-actin. These results confirm the presence of myofibroblasts in human wound healing tissues. The contractile property of fibroblasts and myofibroblasts was evaluated using a three-dimensional cell culture model (fibroblast populated collagen gels). Cells were incorporated in a collagen matrix and cultured for 14 days. The surface area of collagen gels was measured every day. Our results show that wound fibroblasts strongly contract collagen gels during the first 24 hr (surface area at 24 hr = 20-55% of initial surface area) in comparison to dermal fibroblasts (surface area at 24 hr = 70-75% of initial surface area). This superior level of contraction was observed until the fifth day of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗