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M Rouabhia

Publications and source records attributed to M Rouabhia.

At least 37 records · Page 2Linked to original sources

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.

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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.

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Zinc (Zn2+) and fish immune response effect on carp IL2-like production and activity.

Supernatants obtained from PHA-activated pronephric carp lymphocytes were able to provoke the growth of carp blasts but not from fresh lymphocytes without mitogens. These supernatants contain certainly soluble growth factor(s) such as IL2. The addition of Zn2+ (1 mM) to lymphocyte cultures could not enhance supernatants' ability to promote blast proliferation. When Zn2+ and PHA (50 micrograms/ml) were added, respectively, supernatants obtained could enhance significant tritiated thymidine incorporation in blasts. Zn2+ by itself is not able to provoke the growth of blasts in vitro. In contrast, the addition of Zn2+ and PHA-stimulated lymphocyte supernatants in vitro to blasts or nonstimulated lymphocyte cultures enhances their mitotic capacity.

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Improvement of human keratinocyte isolation and culture using thermolysin.

We propose a modification of the conventional keratinocyte isolation method which has shown a significant improvement in the purity, colony forming efficiency (c.f.e.) and growth capacity of the isolated epidermal cell population. This method utilized thermolysin since it selectively digests the dermo-epidermal junction. Following separation from the dermis, the epidermis was digested with trypsin to obtain a single cell suspension. Compared with the conventional procedure, this isolation method was shorter and resulted in (i) cells displaying a higher colony forming efficiency, (ii) cells reaching confluence 1-3 days earlier, (iii) cells not contaminated by fibroblasts, (iv) a cell population containing all the basal layer keratinocytes. These cells were suitable for the establishment of primary cultures and could be subcultured. Such cell populations should be advantageous in studies of epithelial-mesenchymal interactions in which keratinocyte populations, free of fibroblasts, are desirable. In the treatment of extensively burned patients using cultured epidermal sheets, the main problem remains the time required for their production. Thus, the absence of fibroblast overgrowth of the keratinocyte cultures and the significantly reduced time to obtain confluent cultures and epidermal sheets with our method have very important implications for the treatment of large burn wounds.

Adolescent↗

Cultured epithelium allografts: Langerhans cell and Thy-1+ dendritic epidermal cell depletion effects on allograft rejection.

The effects of in vitro dendritic cell (DC) depletion on the survival of epidermal sheet allografts were studied in a murine model. Newborn (1-3 days old) mouse skin was used. Langerhans cell (LC) and Thy-1+ dendritic epidermal cell (Thy-1+ DEC) depletion was achieved using: (1) a prolonged culture period (7 days), or (2) the anti-IA and anti-Thy-1.2 mAbs followed by complement treatment. DC (LC and Thy-1+ DEC) depletion was assessed on sheets and cultured cell suspensions by an indirect immunofluorescence procedure. They showed that, after 7 days of culture or after the antibody-complement treatment, epidermal cultures were depleted of LC and Thy-1+ DEC. Cultured sheets were grafted onto the muscle of H-2-incompatible recipients. The control experiments were: (1) full epidermis and DC undepleted sheet allografts, and (2) DC depleted and undepleted sheet isografts. The full epidermis was totally rejected after 9 days. However, no rejection sign was ever seen in any of the isografts. The in vitro produced and allografted epithelia did not show any necrotic sign until the 11th day postgrafting. However, on days 12-13, the DC depleted allograft's color changed from pink to brown. On days 14-16, degradation of the allografts resulted in a complete denudation of the underlying muscle. Immunohistological analysis of the allografts revealed the presence of a monocyte and lymphocyte infiltration starting from the 11th day postgrafting, with the presence of polymorphonuclear leukocytes on day 14. These results suggest that LC and Thy-1+ DEC depletions were not sufficient to prevent allograft rejection.

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Optimization of murine keratinocyte culture for the production of graftable epidermal sheets.

The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1-3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (greater than 1 mM) Ca2+ levels. After a 3 day culture at the 10(4) cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium. Under these conditions confluence was not obtained. At the 10(5) cells/cm2 seeding inoculum, the percentage of confluence increased to 95-100% during the first 72 h of culture in both high and low Ca2+ culture media. Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 10(5) cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.

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Effect of luteotrophic hormone (LH) and thymosin on mitogen-induced blast transformation of Balb/c mouse lymphoid cells.

The present study investigates the effect of a pituitary hormone (LH) and a thymic factor (Thymosin Fraction 5 = TF5) on in vitro and in vivo spleen cell proliferative response of 2, 6 and 8-month-old Balb/c mice. In vitro experiments showed that the addition of LH at various concentrations (0.5, 5, 50 or 500 ng/ml) to cultures increased significantly the proliferative response to some known mitogens (ConA, PHA, PWM). The LH stimulation was further enhanced by subsequent addition of TF5 to these cultures. However, the addition of LH to LPS cultures resulted in a blockage of the cell growth which persisted after the addition of TF5. In vivo experiments showed that injection of LH (5 or 50 ng/ml) to 2, 6 and 8-month-old Balb/c mice had a significant increase on blast transformation of lymphoid cells following their incubation with ConA, PHA and PWM, and a significant decrease when they were incubated with LPS. The physiological significance of these findings which point out an intimate connection between immune and endocrine functions is discussed.

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Modulatory effect of metal ions on the immune response of fish: in vivo and in vitro influence of MnCl2 on NK activity of carp pronephros cells.

The in vivo and in vitro influence of MnCl2 on carp pronephros cells was investigated. Increased cytotoxicity against both YAC-1 and P 815 target cells was observed following an intraperitoneal injection of 40, 80, or 120 micrograms MnCl2/g body wt administrated 24 hr prior to the in vitro 51Cr release assay. Similarly, in vitro treatment of carp pronephros cells, at a final concentration of 60 micrograms/culture, resulted in an increase of NK cell activity in both YAC-1 and P 815 target cell lines. However, a significant decrease in this activity was shown with lower doses of MnCl2 (40 and 20 micrograms/culture).

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Effects of metal ions on cyprinid fish immune response: in vitro effects of Zn2+ and Mn2+ on the mitogenic response of carp pronephros lymphocytes.

Lymphocytes from the pronephros of carp (Cyprinus carpio L) have been subjected to transformation by mitogens, concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharides (LPS), with Zn or Mn at varying concentrations. Addition of Zn2+ (10(-7) to 10(-3) M) to mitogen-stimulated T and B cells enhanced [3H]thymidine incorporation. Addition of 10(-5) M Zn2+ inhibited the response to Con A, PHA, and LPS. At this concentration, Zn was toxic. Addition of Mn2+ (10(-7) to 10(-3) M) to mitogen-stimulated lymphocytes enhanced [3H]thymidine incorporation. This effect was observed with Con A- and PHA-stimulated lymphocytes, but not with LPS-stimulated lymphocytes. In contrast, addition of 10(-1) M Mn2+ to lymphocyte cultures exerted an inhibitor on the response to Con A or to PHA, while the response to LPS was unaffected. Addition of 10(-1) M Mn2+ to Con A- or PHA-stimulated cultures at different times after initiation of stimulation indicated that Mn2+ was inhibitory only when it was added before the first 16 hr of cultures. The inhibition induced by 10(-1) M Mn2+ could be reversed by adding 2 mM CaCl2 to the culture.

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Interaction between luteotrophic hormone (LH) and thymosin on natural killer cell activity in BALB/c mice.

The present study investigates the effect of a pituitary hormone (luteotrophic hormone, LH) and a thymic factor (thymosin fraction V, TVF) on in vitro and in vivo natural killer (NK) cell activity of BALB/c mice (2, 6 and 8 months old). In vitro it was found that LH at various concentrations (0.5, 5, 50 and 500 ng/ml) significantly reduced the NK cell activity. This inhibition was not blocked by TFV. In vivo it was observed that there was a great inhibition of NK cell activity by LH (5 and 50 ng/ml) on 2- and 6-month-old mice, but a significant increase of NK cell activity on 8-month-old mice. The physiological significance of these observations points to an intimate connection between immune and endocrine system functions.

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The thymus. Key organ between endocrinologic and immunologic systems.

Our data permitted us to postulate an immunoregulatory circuit linking the neuroendocrine structure and the immune system via hormones secreted by the thymus which permit differentiation of T cells. In our first approach we have considered the thymus as a key organ; it exerts its function upon the two systems. We can also consider the thymus a key organ because a major part of the lymphoid cells responsible for the secretion of immunotransmitters or described as target cells for immunohormones or opioids are mature T cells; these T cells have had contact--either cellular (nurse cells) or humoral (thymic hormones)--with thymus during ontogeny. We hypothesize that during this differentiation the properties of secreting hormones or opioids and of receiving an endocrine message appear. Concerning the effect of TF V, the different thymosin properties led us to postulate that among the TF V polypeptides some could exert their effects on the immune system and some on the endocrine system directly or indirectly, either in synergism or in antagonism with classical hormones. There is no doubt that neurotransmitters and hormones can modulate in vivo ontogenesis, differentiation, and expression of the immune response. We believe that the thymus via its hormonal function is one factor in this modulation and one of the main channels of this bidirectional action between the immune and the endocrine systems. We have summarized our hypothesis in Figure 2.

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[Neuroimmunomodulation of the immune response by the endocrine system: effect of luteinizing hormone (LH) on the proliferative response of lymphocytes].

Proliferative response of splenic lymphocytes from adult (2 months old) male mice to human luteotrophic hormone (hLH) was studied in vitro. hLH induced lymphoblastic transformation at a concentration of 0.1 micrograms/ml or higher. This hormone potentiated the mitogen activities of concanavalin A (ConA) and phytohemagglutinin (PHA) for T cells and of pokweed mitogen for B and T cells. The data suggest that peripherally circulating LH may regulate a certain function of lymphoid cells.

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In vitro effect of methionine-enkephalin and thymosin on murine expression of Thy1-2 antigen.

The present study investigates the in vitro effect of methionine-enkephalin (Met-Enk) on Thy1-2 antigen density and the interaction between this opioid peptide and a thymic hormone (thymosin fraction V (TFV]. In vitro, it was found that Met-Enk at various concentrations (28.10(-11), 28.10(-10), 28.10(-9) and 28.10(-8) M) had an increased positive effect on the Thy1-2 antigen density of Balb/C mouse thymocytes. This action was observed at all concentrations of Met-Enk. The addition of TFV to Met-Enk thymocyte cultures showed an additive effect of these two hormones. The physiological significance of these observations is that they indicate an interaction between immune and endocrine system functions.

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