Mahmoud Rouabhia

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.

Porphyromonas gingivalis-Epithelial Cell Interactions in Periodontitis

Emerging data on the consequences of the interactions between invasive oral bacteria and host cells have provided new insights into the pathogenesis of periodontal disease. Indeed, modulation of the mucosal epithelial barrier by pathogenic bacteria appears to be a critical step in the initiation and progression of periodontal disease. Periodontopathogens such as Porphyromonas gingivalis have developed different strategies to perturb the structural and functional integrity of the gingival epithelium. P. gingivalis adheres to, invades, and replicates within human epithelial cells. Adhesion of P. gingivalis to host cells is multimodal and involves the interaction of bacterial cell-surface adhesins with receptors expressed on the surfaces of epithelial cells. Internalization of P. gingivalis within host cells is rapid and requires both bacterial contact-dependent components and host-induced signaling pathways. P. gingivalis also subverts host responses to bacterial challenges by inactivating immune cells and molecules and by activating host processes leading to tissue destruction. The adaptive ability of these pathogens that allows them to survive within host cells and degrade periodontal tissue constituents may contribute to the initiation and progression of periodontitis. In this paper, we review current knowledge on the molecular cross-talk between P. gingivalis and gingival epithelial cells in the development of periodontitis.

Tissue-engineered human skin substitutes developed from collagen- populated hydrated gels: clinical and fundamental applications

The field of tissue engineering has opened several avenues in biomedical sciences, through ongoing progress. Skin substitutes are currently optimised for clinical as well as fundamental applications. The paper reviews the development of collagen-populated hydrated gels for their eventual use as a therapeutic option for the treatment of burn patients or chronic wounds: tools for pharmacological and toxicological studies, and cutaneous models for in vitro studies. These skin substitutes are produced by culturing keratinocytes on a matured dermal equivalent composed of fibroblasts included in a collagen gel. New biotechnological approaches have been developed to prevent contraction (anchoring devices) and promote epithelial cell differentiation. The impact of dermo-epidermal interactions on the differentiation and organisation of bio-engineered skin tissues has been demonstrated with human skin cells. Human skin substitutes have been adapted for percutaneous absorption studies and toxicity assessment. The evolution of these human skin substitutes has been monitored in vivo in preclinical studies showing promising results. These substitutes could also serve as in vitro models for better understanding of the immunological response and healing mechanism in human skin. Thus, such human skin substitutes present various advantages and are leading to the development of other bio-engineered tissues, such as blood vessels, ligaments and bronchi.

UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.

Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity by expression of a dominant-negative mutant of p38 or with SB203580 impaired cell viability and led to an increase in UVB-induced apoptosis. This sensitization to apoptosis was independent of caspase activities. Inhibition of p38 did not sensitize transformed HaCaT keratinocytes to UVB-induced apoptosis. In normal keratinocytes, expression of a dominant-negative mutant of p53 increased UVB-induced cell death, pointing to a role for p53. In these cells, UVB triggered a p38-dependent phosphorylation of p53 on Ser-15. This phosphorylation was associated with an SB203580-sensitive accumulation of p53, even in the presence of a serine phosphatase inhibitor. Accumulated p53 was localized mainly in the cytoplasm, independently of CRM1 nuclear export. In HaCaT cells, p53 was localized exclusively in the nucleus and its distribution and level were not affected by UVB or p38 inhibition. However, UVB induced an SB203580-insensitive phosphorylation on Ser-15 of mutated p53. Overall, our results suggest that, in normal human keratinocytes, protection against UVB depends on p38-mediated phosphorylation and stabilization of p53 and is tightly associated with the cytoplasmic sequestration of wild-type p53. We conclude that the p38/p53 pathway plays a key role in the adaptive response of normal human keratinocytes against UV stress.

Kinetics of the breakdown of cross-linked soy protein films for drug delivery

The aim of the present work was to investigate the potential of soy protein isolate (SPI) films as controlled release systems for active compounds. Mechanical properties, dissolution and compound release kinetics of SPI films prepared with different concentrations of formaldehyde were measured over time in the absence or presence of digestive enzymes at gastric or intestinal pH. The effect of formaldehyde on tensile strength, elastic modulus, % elongation and swelling suggested that increasing its concentration increased film cross-linking density. Film bulk erosion in the presence of digestive enzymes followed first-order kinetics. Methylene blue or rifampicin release followed variable kinetics depending on compound solubility during a 1-2h initial phase, followed by zero-order release. Cross-linking density appears to provide effective means of regulating the erosion and release rate of SPI films. SPI film networks displayed excellent compound binding capacity, especially for hydrophobic molecules, and hence potential for use in controlled release systems based on matrix erosion.

Alcohol Dehydrogenase Restricts the Ability of the Pathogen Candida albicans To Form a Biofilm on Catheter Surfaces through an Ethanol-Based Mechanism

ABSTRACT Candida biofilms formed on indwelling medical devices are increasingly associated with severe infections. In this study, we used proteomics and Western and Northern blotting analyses to demonstrate that alcohol dehydrogenase (ADH) is downregulated in Candida biofilms. Disruption of ADH1 significantly (P = 0.0046) enhanced the ability of Candida albicans to form biofilm. Confocal scanning laser microscopy showed that the adh1 mutant formed thicker biofilm than the parent strain (210 μm and 140 μm, respectively). These observations were extended to an engineered human oral mucosa and an in vivo rat model of catheter-associated biofilm. Inhibition of Candida ADH enzyme using disulfiram and 4-methylpyrazole resulted in thicker biofilm (P < 0.05). Moreover, biofilms formed by the adh1 mutant strain produced significantly smaller amounts of ethanol, but larger amounts of acetaldehyde, than biofilms formed by the parent and revertant strains (P < 0.0001), demonstrating that the effect of Adh1p on biofilm formation is mediated by its enzymatic activity. Furthermore, we found that 10% ethanol significantly inhibited biofilm formation in vitro, with complete inhibition of biofilm formation at ethanol concentrations of ≥20%. Similarly, using a clinically relevant rabbit model of catheter-associated biofilm, we found that ethanol treatment inhibited biofilm formation by C. albicans in vivo (P < 0.05) but not by Staphylococcus spp. (P > 0.05), indicating that ethanol specifically inhibits Candida biofilm formation. Taken together, our studies revealed that Adh1p contributes to the ability of C. albicans to form biofilms in vitro and in vivo and that the protein restricts biofilm formation through an ethanol-dependent mechanism. These results are clinically relevant and may suggest novel antibiofilm treatment strategies.

In vitro models of tissue penetration and destruction by Porphyromonas gingivalis.

ABSTRACT Porphyromonas gingivalis is a gram-negative anaerobic bacterium that is considered the key etiologic agent of chronic periodontitis. Arg- and Lys-gingipain cysteine proteinases produced by P. gingivalis are key virulence factors and are believed to be essential for significant tissue component degradation, leading to host tissue invasion by periodontopathogens. Two in vitro models were used to determine the extent to which P. gingivalis can reach connective tissue. The tissue penetration potential of P. gingivalis was first investigated by using an engineered human oral mucosa model composed of normal human epithelial cells and fibroblasts. Internalized bacteria were assessed by transmission electron microscopy. Bacteria were observed within multilayered gingival epithelial cells and in the space between the stratified epithelium and the lamina propria. A gingipain-null mutant strain of P. gingivalis was found to be less potent in penetrating tissue than the wild-type strain. Proinflammatory responses to P. gingivalis infection were evaluated. P. gingivalis increased the secretion of interleukin-1β, interleukin-6, interleukin-8, and tumor necrosis factor alpha. In the second part of the study, the contribution of P. gingivalis gingipains to tissue penetration was investigated by using a reconstituted basement membrane model (Matrigel). The penetration of 14C-labeled P. gingivalis cells through Matrigel was significantly reduced when leupeptin, a specific inhibitor of Arg-gingipain activity, was added or when a gingipain-null mutant was used. The results obtained with these two relevant models support the capacities of P. gingivalis to infiltrate periodontal tissue and to modulate the proinflammatory response and suggest a critical role of gingipains in tissue destruction.

The regulation of cell functions electrically using biodegradable polypyrrole-polylactide conductors.

Electricity has a long history of being used as an alternative clinical treatment and as an effective approach to modifying cellular behaviours in vitro. It has been difficult, however, to take advantage of this modality in tissue generation because of the lack of suitable conductive, biocompatible scaffolding materials. In this study, in order to electrically regulate cell activities, a largely biodegradable conductor made of 5% conductive polypyrrole and 95% biodegradable poly(L-lactide) (PPy/PLLA) was prepared. Human cutaneous fibroblasts were cultured on the conductors in the presence or absence of a direct current (DC) electrical field (EF) of 50 mV/mm. The growth of the cells was characterized using fluorescent staining, SEM, and a MTT assay. The RNA expressions of interleukin-6 (IL-6) and interleukin-8 (IL-8) were assayed by RT-PCR. The amounts of IL-6 and IL-8 secreted by the fibroblasts were quantified by ELISA. The results showed that the PPy/PLLA conductors supported cell adhesion, spreading, and proliferation in both the presence and absence of the EF. Electrical stimulation (ES) applied through PPy/PLLA conductors dramatically enhanced cytokine secretion approximately 10-fold when compared to the non-ES controls. This effect lasted several days after the end of the ES. These findings highlight for the first time the possibility of a potent, effective approach to regulating tissue regeneration in conductive scaffolds through ES-modulated cytokine secretion, and to increasing cytokine productivity for biotechnological applications.

Interleukin-18 and Gamma Interferon Production by Oral Epithelial Cells in Response to Exposure to Candida albicans or Lipopolysaccharide Stimulation

ABSTRACT Oral candidiasis is a collective name for a group of disorders caused by the dimorphic fungus Candida albicans. Host defenses against C. albicans essentially fall into two categories: specific immune mechanisms and local oral mucosal epithelial cell defenses. Since oral epithelial cells secrete a variety of cytokines and chemokines in response to oral microorganisms and since C. albicans is closely associated with oral epithelial cells as a commensal organism, we wanted to determine whether interleukin-18 (IL-18) and gamma interferon (IFN-γ) were produced by oral epithelial cells in response to C. albicans infection and lipopolysaccharide (LPS) stimulation. Our results showed that IL-18 mRNA and protein were constitutively expressed by oral epithelial cells and were down-regulated by Candida infections but increased following LPS stimulation. Both C. albicans and LPS significantly decreased pro-IL-18 (24 kDa) levels and increased active IL-18 (18 kDa) levels. This effect was IL-1β-converting-enzyme dependent. The increase in active IL-18 protein levels promoted the production of IFN-γ by infected cells. No effect was obtained with LPS. Although produced only at an early stage, secreted IFN-γ seemed to be a preferential response by oral epithelial cells to C. albicans growth. These results provide additional evidence for the contribution of oral epithelial cells to local (direct contact) and systemic (IL-18 and IFN-γ production) defense against exogenous stimulation such as C. albicans infection or LPS stimulation.

Virulence of the Fungal Pathogen Candida albicans Requires the Five Isoforms of Protein Mannosyltransferases

ABSTRACT The PMT gene family in Candida albicans encodes five isoforms of protein mannosyltransferases (Pmt proteins Pmt1p, Pmt2p, Pmt4p, Pmt5p, and Pmt6p) that initiate O mannosylation of secretory proteins. We compared virulence characteristics of pmt mutants in two complex, three-dimensional models of localized candidiasis, using reconstituted human epithelium (RHE) and engineered human oral mucosa (EHOM); in addition, mutants were tested in a mouse model of hematogenously disseminated candidiasis (HDC). All pmt mutants showed attenuated virulence in the HDC model and at least one model of localized candidiasis. The pmt5 mutant, which lacks in vitro growth phenotypes, was less virulent in the EHOM and HDC assays but had no consistent phenotype in the RHE assay. In contrast, the pmt4 and pmt6 mutants were less virulent in the RHE and HDC assays but not in the EHOM assay. The results stress the contribution of all Pmt isoforms to the virulence of C. albicans and suggest that the importance of individual Pmt isoforms may differ in specific host niches. We propose that Pmt proteins may be suitable targets for future novel classes of antifungal agents.