Evaluation of the toxicity of eugenol at anesthetic doses in African clawed frogs (Xenopus laevis).

Eugenol has been shown to induce anesthesia in African clawed frogs (Xenopus laevis). The toxicity of eugenol, administered at anesthetic doses, was evaluated in Xenopus frogs with an average body weight of 28.2 ± 13.7 g. Frogs were immersed in 250 mL of an aqueous solution containing 350 µl/L of eugenol for ten minutes and received a single administration (group 1, twelve animals) or three consecutive daily administrations (group 2, twelve animals). In each group, six frogs were scheduled to be euthanized the following day (subgroup A) and the other six were scheduled to be euthanized after a one-week recovery period (subgroup B). Morphologic changes consistent with renal tubular apoptosis affecting distal tubules in the medulla were observed in all subgroup A animals, ranging from mild to moderate in group 1, and from mild to severe in group 2. In subgroup B, renal tubular regeneration was present in all but one animal examined. These findings suggest that eugenol toxicity in amphibians is first manifested by renal tubular apoptosis. Other eugenol-related lesions were massive hepatic necrosis in group 2 (n = 6), hyaline membranes in the lung (n = 5), and adipose tissue hemorrhages in group/subgroup 2B (n = 4).

The use of dual-energy X-ray absorptiometry to estimate the dissected composition of lamb carcasses.

A total of 140 male and female Dorset and Suffolk lambs were slaughtered according to four live weight classes (36-39kg, 41-44kg, 46-49kg and 51-54kg). Total tissue, fat and lean masses, and bone mineral content measured by dual-energy X-ray absorptiometry (DXA) were used to predict dissected tissue weights. The DXA total weights accurately predict half-carcasses and primal cuts weights (shoulder, leg, loin and flank) (R(2)>0.99, CVe<1.3%). The prediction of the half-carcass dissected fat percentage is weaker (R(2)=0.77, CVe=10.4%). Fatness prediction accuracy is equivalent for the shoulder, leg and loin (R(2) between 0.68 and 0.78, CVe between 10% and 13%). The R(2) obtained when predicting dissected lean content from DXA variables is 0.93 for the half-carcass and higher than 0.83 for all cuts other than flank (CVe are between 3.5% and 6.5%, except for the flank, which is 9.1%). The prediction of bone weight using the bone mineral content is not very accurate for the half-carcass, shoulder and leg (R(2): 0.48, 0.47 and 0.43; CVe: 10.2%, 12.0% and 11.6%, respectively). The situation improves, however, for the loin (R(2)=0.70, CVe=10.7%). In conclusion, DXA is an effective technology for predicting total weight and the amount of lean and fat in lamb carcasses and their primal cuts.

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.

Production of bioengineered cancer tissue constructs in vitro: epithelium-mesenchyme heterotypic interactions.

A few models have been established to study cancer cells in vitro. However, the cellular interactions have rarely been studied specifically using bioengineered cancer constructs combining human carcinoma cells and tumor-associated fibroblasts. We developed an in vitro model of tridimensional bioengineered cancer tissue constructs (bCTC) by seeding mammary epithelial cancer cells or normal keratinocytes over a mesenchymal layer containing tumor-derived fibroblastic cells or normal skin fibroblasts. After the introduction of epithelial cells, each construct was cultured for another 10 d. Histologic analyses showed that carcinoma cell lines could invade the subjacent mesenchymal layer and that the capacity to migrate was related to the invasive potential of cancer cells and the type of fibroblasts used, while noninvasive populations did not. Of the tested epithelial cells, MDA-MB-231 and, to a lesser degree, HDQ-P1 cell lines were invasive, and the invasion was deeper into the mesenchymal component containing tumor-derived fibroblasts. However, with normal skin fibroblasts, the mesenchymal layer was degraded twice faster than with tumor-derived fibroblastic cells. MDA-MB-231 cells and normal keratinocytes induced the highest level of gelatinase B, and the level was lowest with the MCF-7 cell line. The activated form of gelatinase B was, however, induced to the highest levels in the keratinocyte-seeded bCTC containing tumor-derived but not normal fibroblasts. MDA-MB-231 was the only epithelial cancer cell line whose activity of gelatinase A was reduced when cocultured with tumor-derived fibroblasts but not under normal fibroblast stimulation. Finally, a 50/48-kDa gelatinase band has been observed in bCTCs with noninvasive epithelial cells only. Our study demonstrates the selective secretion of gelatinases according to the phenotype of the cells seeded in the various bCTCs.

Selective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer.

The main drawback of the selective culture of human mammary epithelial cells from primary breast cancer is the overgrowth of tumor-associated stromal fibroblasts. This drawback may be overcome by using, in primary culture, lethally irradiated 3T3 cells which act as a feeder layer to maintain tumor-derived epithelial cell proliferation. These 3T3 cells, exposed to 60 Gy at confluence, form a specific cellular substrate which constitutes an obstacle to fibroblast attachment. Enzyme-disaggregated breast cells from six primary breast carcinomas were cocultured over lethally irradiated but living 3T3 cells. The method led to the purification of tumor-derived epithelial cells from all six cancer samples, and long-term culture was obtained in one. The epithelial nature of these purified tumor-derived epithelial cells was demonstrated by their general morphology and by the expression of cytokeratins and Epithelial Membrane Antigen. These results confirm the stimulatory effect of a this stromal feeder layer on breast epithelial cell growth and show that this stromal feeder layer can also control the fibroblast overgrowth. Our results provide an alternative approach in the selective culture of epithelial cells from primary breast carcinoma.

Establishment and characterization of a new cell line derived from a human primary breast carcinoma.

A new cell line, designated HDQ-P1, was successfully established from a primary ductal infiltrating mammary carcinoma by using a 3T3 feeder layer lethally irradiated to 60 Gy. The HDQ-P1 cells have been grown in culture for over 115 passages and have a doubling time of 60 hours. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, telomerase expression, tumor antigen expression, xenograft implantation into nude mice, colony formation in soft agar, TP53 sequencing, and gene copy number of C-MYC, C-ERBB-2, and C-H-RAS oncogenes. The epithelial nature of this cell line was confirmed by ultrastructural analysis, expression of cytokeratins, and epithelial membrane antigen. The HDQ-P1 cells possess an extensively rearranged and polyploid karyotype, with an average of 20 recurrent marker chromosomes. Scatchard analysis demonstrated that both primary tumor and HDQ-P1 cells were estrogen- and progesterone-receptor negative. The HDQ-P1 cells had the same expression of human telomerase reverse transcriptase as other established breast cancer cell lines such as MDA-MB-231, SK-BR-3, and MCF-7. Direct DNA sequencing showed a point mutation which yielded to a stop codon at the amino acid 213 in exon 6 of the TP53 gene. A five-fold amplification of C-MYC was observed in HDQ-P1 cells. No amplification of C-ERBB-2 and C-H-RAS genes were observed. This cell line presents unique characteristics and may prove to be a good experimental model for investigating breast cancer biology.

Multistep production of bioengineered skin substitutes: sequential modulation of culture conditions.

Many studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboidal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.

The Script Concordance test: a tool to assess the reflective clinician.

BACKGROUND: The Script Concordance (SC) test is a new assessment tool. It is designed to probe whether knowledge of examinees is efficiently organized for clinical actions. That kind of organization of knowledge is named a script. The SC test places examinees in written, but authentic, clinical situations in which they must interpret data to make decisions. PURPOSE: The SC test is designed to measure the degree of concordance that exists between examinees' scripts and scripts of a panel of experts. The objective of this article is to provide interested educators with the practical "how to" information needed to build and use an SC test. METHODS: The theoretical background of the SC test is described. The principles of construction of an SC test are presented, including the writing of clinical cases, the choice of item format, the validation of the test, and the elaboration of the scoring system. RESULTS: A series of studies have shown that the SC test has interesting psychometric properties, in terms of reliability, face validity, and construct validity. Results from these studies are succinctly presented and commented. CONCLUSION: The SC test is a simple and direct approach to testing organization and use of knowledge. It has the strong advantage for a testing method of being relatively easy to construct and use and to be machine-scorable. It can be either paper- or computer-based and can be used in undergraduate, postgraduate, or continuing medical education.

Educational skills and knowledge needed and problems encountered by continuing medical education providers.

BACKGROUND: The objective of this study was to identify the training needs and difficulties encountered by continuing medical education (CME) providers in Quebec. METHODS: A questionnaire comprised of open-ended and closed questions was sent to 224 general practitioners across Quebec who organize CME meetings. To complement and validate the data, interviews were conducted with 18 physicians selected from this group, based on their years of experience with CME, and with the managers of two organizations involved in CME. RESULTS: The questionnaire response rate was 54%. Quantitative analysis was used to identify the main training needs expressed by CME providers affiliated with the Quebec Federation of General Practitioners, namely, methods for identifying needs (74%), group leadership techniques (69%), basic principles in adult education (69%), and organization of CME activities (66%). The main problems encountered by respondents in their duties are stimulating and maintaining the interest and participation of physicians in formal CME activities (52%), identifying and meeting physicians' educational needs (32%), and motivating physicians to get involved in any kind of CME initiative (18%). The interviews highlighted the wide disparity in the approaches used by CME providers when planning activities and the failure of providers to pass on relevant information to their successors. IMPLICATIONS: Based on the difficulties and the training needs identified, we were able to develop tools (structured training program, biannual newsletter, reference books, and resources) suited to the needs of general practitioners who organize CME activities.

Isolation and characterization of human airway fibroblasts in culture.

Asthma is considered an airway inflammatory disorder characterized by variable airflow obstruction and airway hyperresponsiveness (1). The inflammatory component of asthma has been studied extensively over the past few years, but, more recently, the potential contribution of airway wall remodeling to functional and clinical changes has been emphasized (2,3). Although the methods of sampling of bronchial tissue were previously limited, being obtained mostly from autopsic or surgical specimens, they have improved recently.

Participation in CME activities.

OBJECTIVE: To compare the continuing medical education (CME) activities of family physicians in the province of Quebec with more than 25 years in practice with those with less than 25 years in practice. DESIGN: Mailed questionnaire survey. SETTING: Family practices in the province of Quebec. PARTICIPANTS: All physicians (n = 722) with more than 25 years in practice (expressed as older) and a matched sample of 721 physicians with less than 25 years in practice (expressed as younger). MAIN OUTCOME MEASURES: Types of CME activities and time spent on them, participant characteristics. RESULTS: Older physicians spent more time in individual CME activities than younger ones (21 hours vs 18 hours monthly). Younger physicians, however, spent more time in group CME activities than older ones did (100 hours vs 80 hours yearly). Excluding physicians who devoted no time to CME activities, only two activities differentiated between the two groups: older physicians spent more time than their younger colleagues reading and listening to audiocassettes. CONCLUSIONS: Older physicians maintained their clinical competence by participating in different CME activities from younger physicians. They participated in as many CME activities as their younger colleagues.

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.

Attitudes toward obstetrics training. Residents surveyed at McGill University and University of Montreal.

OBJECTIVE: To determine family medicine residents' attitudes toward family practice training in obstetrics and neonatology before and after implementation of a modified obstetrics curriculum at McGill University (MG). DESIGN: Two-group pretest and posttest. Fifty-seven respondents, 31 at MG, 26 at University of Montreal (UM), were case matched as first-year and second-year residents. SETTING: Departments of Family Medicine at MG and UM. PARTICIPANTS: Family medicine residents at MG and UM. INTERVENTION: A modified obstetrics curriculum was introduced at MG (study group); no modifications were introduced at UM (control group). First- and second-year residents' attitudes toward the adequacy of training were assessed through responses to a questionnaire administered in July 1992 and July 1994. MAIN OUTCOME MEASURES: Changes in response scores before and after implementation of the modified curriculum. RESULTS: Repeated multivariate analysis of variance (MANOVA) showed respondents believed family practice obstetrics training was adequate in general, but that family practitioners were inadequately trained in emergency obstetric skills. Scores for items assessing neonatology skills increased significantly in the MG group after the intervention. CONCLUSIONS: Residents' overall confidence in their obstetrics training did not appear to improve, but this might be due to a time lag between curriculum modification and attitudinal change. McGill residents' confidence in neonatology skills improved significantly after curriculum modification.

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.

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.

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.

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.