Functional Characterization of Cultured Keratinocytes after Acute Cutaneous Burn Injury

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable--keratinocyte-derived--cytokine and growth factor milieu. In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters. Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß. Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients

Detectie van antilichamen gericht tegen polymeren (APA) in serum van vrouwen met siliconen borstimplantaten. Introductie en reproduceerbaarheid van de test in het RIVM

Siliconen bevattende borst implantaten (SBI) worden veelvuldig gebruikt om medische en kosmetische redenen. De laatste tijd is een discussie op gang gekomen dat het aanwezig zijn van een siliconen borstimplantaat mogelijk kan leiden tot gezondheidsklachten. Recentelijk is een laboratoriumtest beschreven, die de APA-test genoemd wordt (Antipolymeer Antilichaam), die antilichamen in het serum gericht tegen polymeren detecteert (Tenenbaum et al, Lancet 1997; 349: 449-454). De hoogte van het antistofgehalte bleek gecorreleerd te zijn aan de ernst van de klachten bij vrouwen met een siliconen borstimplantaat. In het rapport wordt de introductie en reproduceerbaarheid van de APA-test in het RIVM beschreven. De resultaten verkregen in het RIVM werden vergeleken met de resultaten behaald door 'Autoimmune Technologies, LLC' (New Orleans, USA) (producent en patenthouder van de test). Referentie en test monsters werden verkregen van Autoimmune Technologies LCC. Vervanging van de door 'Autoimmune Technologies' bijgeleverde chemicalien door chemicalien van lokale leveranciers gaf dezelfde resultaten in de APA-test. Concluderend kan gezegd worden dat de APA-test succesvol geintroduceerd is in het RIVM. De variatie binnen de test, in zesvoud uitgevoerd op een dag en tussen verschillende testen uitgevoerd op zes verschillende dagen, lagen binnen een acceptabele marge. Om deze reden kan worden geconcludeerd dat de APA-test reproduceerbare uitslagen geeft en geschikt is om te gebruiken voor het onderzoek naar de aanwezigheid van antipolymeer antistoffen in serum van vrouwen met een siliconen borstimplantaat.Silicone breast implants (SBI) are widely used in women both for medical and cosmetic reasons. During the last decade there has been much debate whether the use of SBI is related to health complaints. Recently a laboratory assay has been described, designated the APA-assay ( Anti-Polymer Antibody), which detects antibodies directed to a polymer in serum of patients with SBI (Tenenbaum et al, 1997). The level of antipolymer antibodies was described to be associated to the severity of the clinical symptoms of SBI recipients. The report describes the introduction and performance of the APA-assay at the RIVM. The results obtained at the RIVM were compared to the results obtained by Autoimmune Technologies LLC, (New Orleans, USA) (manufacturer and patent holder of the assay). Reference and test samples were provided by Autoimmune Technologies LCC. Substitution of the chemicals supplied by Autoimmune Technologies, by chemicals obtained from local suppliers had no impact, simmilar results were observed using both sets of chemicals. In conclusion, the APA-assay was successfully introduced in our laboratory. Intra-assay variability and inter-assay variability were found to be within an acceptable range. Hence, the APA-assay gives reproducible results, and can be used for the evaluation of the presence of antipolymer antibodies in serum of women with a silicone breast implant.VWS/IG

Ranking of allergenic potency of rubber chemicals in a modified local lymph node assay

Ranking of allergenic potency of rubber chemicals in a modified local lymph node assay. De Jong WH, Van Och FM, Den Hartog Jager CF, Spiekstra SW, Slob W, Vandebriel RJ, Van Loveren H. Laboratory for Pathology and Immunobiology, National Institute for Public Health and the Environment, RIVM, P.O.Box 1, 3720 BA Bilthoven, The Netherlands. [email protected] A modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5-95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy inductio

Onderzoek naar de aanwezigheid van antipolymeer antistoffen in een groep Nederlandse vrouwen met een siliconenborstimplantaat

Het doel van het hier beschreven onderzoek is vast te stellen of er een populatie Nederlandse vrouwen is met een hoge prevalentie van antipolymeer antistoffen (APA) en ernstige klachten/ziekte-verschijnselen en blootstelling aan een siliconenborstimplantaat. Omdat de antigeenspecificiteit van de antipolymeer antistoffen (APA) nog niet is aangetoond, gebruiken wij de term polymeer bindende immunoglobulinen. De studiepopulatie werd geselecteerd uit een bestand van de Consumentenbond, Den Haag, waar vrouwen met een SBI zich op vrijwillige basis hadden laten registreren. De definitieve selectie werd uitgevoerd middels een vragenlijst met zelf gerapporteerde klachten. In totaal werden 42 vrouwen in de studie opgenomen. Bij deze vrouwen werd klinisch onderzoek uitgevoerd en bloed afgenomen. In de onderzoeksgroep werd bij een gering aantal vrouwen een verhoogd gehalte aan polymeer bindende immunoglobulines waargenomen. Ook bij een groep vrouwen zonder siliconenborstimplantaat, waarvan het serum onderzocht werd als controle op de uitvoering van de laboratorium test, werd bij een aantal vrouwen een verhoogd gehalte aan polymeer bindende immunoglobulines waargenomen. Met een gemiddelde implantatietijd van 17 jaar kan geconcludeerd worden dat blootstelling aan siliconen op zich geen inductie geeft van polymeer bindende immunoglobulinen. De studiepopulatie werd wat betreft ziekteverschijnselen in subgroepen (minimaal, gering, matig, ernstig) onderverdeeld op basis van afname van functionele capaciteit (zelfredzaamheid, wat kan men nog wel en niet meer doen in het dagelijkse leven) en algemene beoordeling door een arts wat betreft de ziekte-activiteit en een globale schatting van de pijn. De meeste vrouwen met SBI (34 van de 42) werden ingedeeld in de groep met minimale ziekteverschijnselen. De onderzoekspopulatie bleek wat betreft de ernst van de ziekteverschijnselen niet overeen te komen met de groep vrouwen waarbij in de literatuur een verhoogde prevalentie van polymeer bindende immunoglobulines was gevonden.The purpose of the study described in this report was to determine whether there exists a population of Dutch women with high prevalence of antipolymer antibodies (APA) and severe complaints, and exposure to a SBI. As the antigen specific nature of the antipolymer antibody has not yet been established, we refer to the term polymer binding immunoglobulins. The study population was selected from a voluntary registry of SBI recipients of a Dutch consumers (Consumentenbond, The Hague, The Netherlands) organisation. The final selection was based on self reported complaints in a questionnaire which was used to determine the complaints at the time of the study. A total of 42 SBI recipients was included in the study, clinically examined and blood samples were obtained. Only in a few SBI recipients an increase in the level of polymer binding immunoglobulins was detected. Also in a few blood samples of non SBI recipients, included for control on the performance of the APA assay, an increase in the level of polymer binding immunoglobulins was demonstrated. As the mean duration of the SBI implant was 17 years it can be concluded that exposure to silicones does not lead to the induction of polymer binding immunoglobulins. The study population of SBI recipients was categorized in severity subgroups based on the functional capacity, and the physicians global assessment of pain and disease activity. Most (34 of 42) SBI recipients belonged to the limited severity subgroup on a increasing scale of limited, mild, moderate and advanced. Conclusively, our selection method failed to include a large proportion of SBI recipients which could be categorized in the moderate or advanced severity subgroups. In this respect our study population is not comparable to the severity subgroups of SBI recipients or severe fibromyalgia patients reported to have a high prevalence of antipolymer antibodies.VWS IG

Skin Sensitization Tests: The LLNA and the RhE IL-18 Potency Assay

Contact allergy is of considerable importance to the toxicologist, and regulatory authorities worldwide require testing for skin sensitization potential and appropriate hazard labeling to enable management of the risk to human health. Although traditionally the identification of skin-sensitizing chemicals has been carried out using animal models, in Europe legislative changes have promoted, and now require, the use of non-animal methods (i.e., Cosmetic Directive, REACH). Several in vitro alternatives for hazard identification have now been validated, but do not provide information on the potency of a skin sensitizer. Here, we describe an animal model, the local lymph node assay (LLNA), and an in vitro model, the RhE IL-18 potency assay, in the context of the identification and potency classification of skin sensitizers. These two assays have been chosen among the different available tests as representative of an alternative in vivo model (the LLNA) and a promising in vitro method with the potential of both hazard identification and potency classification