Rik J. Scheper

Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts.

Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin–eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely from human cell lines, provides an excellent opportunity to study in vitro skin biology and can also be used for drug targeting and testing new therapeutics, and ultimately, for incorporating into skin-on-a chip in the future.

Intermittent exposure to doxorubicin in vitro selects for multifactorial non‐P‐glycoprotein‐associated multidrug resistance in RPMI 8226 human myeloma cells

The purpose of the present study was to evaluate whether intermittent exposure to a constant dose of doxorubicin selects for multidrug resistance (MDR) in RPMI 8226 human myeloma cells and, if so, to determine the molecular mechanism. In an attempt to approximate clinical doxorubicin treatment in vitro, cells were exposed to a fixed dose of doxorubicin for 4 d alternating with growth in drug‐free medium for 17 d. An MDR subline emerged, termed 8226/DOXint5, which was 3–4‐fold resistant to doxorubicin, etoposide and m‐AMSA, and 1.6‐fold resistant to vincristine. Sensitivity to docetaxel, melphalan and cisplatin was normal. Verapamil normalized vincristine sensitivity but had little effect on resistance to the other agents. Cellular uptake and retention of daunorubicin and vincristine were reduced by approximately 10%. The 8226/DOXint5 cells showed diminished DNA topoisomerase IIα expression and increased expression of the multidrug resistance protein MRP. Expression of MDR1/P‐glycoprotein was not detected. Immunostaining showed 70% of the cells to over‐express the lung‐resistance protein LRP. This new MDR myeloma cell line may prove to be a useful model for the development of strategies to overcome low‐level, multifactorial MDR, which might be a common phenomenon in clinical myeloma treated with doxorubicin.

Localisation of the multidrug resistance-associated protein, MRP, in resistant large-cell lung tumour cells

The drug transport protein, P-glycoprotein, confers multidrug resistance (MDR) by expelling drugs across the cell surface. The structurally similar multidrug resistance-associated protein, or MRP, is also involved with drug efflux. In MDR variants of the human lung tumour cell line COR-L23 that overexpress MRP, there are also changes in intracellular drug distribution. To ascertain whether MRP could be involved in either process, experiments were performed to identify where MRP was located in these cells. Following separation of membranes by sucrose gradient centrifugation, MRP was found predominantly in the lighter membrane fractions containing plasma membrane enzyme activity. Immunofluorescent staining with a monoclonal antibody raised against MRP confirmed that MRP is present at the cell surface of these MDR lung tumour cells.

Restrictions to the use of 111indium-oxine as a radiolabel in lymphocyte migration studies

Guinea pig T lymphocytes were labelled with various concentrations of 111In-oxine. The capacity of these cells to migrate into a skin inflammatory site was compared with that of 51Cr-labelled cells. The results show that even with low dosages of 111In-oxine which did not impair lymph node localization (1-10 microCi/10(8) cells/ml), migration into an inflammatory site was markedly reduced. Moreover, using this isotope, a significant contribution to the radioactivity recovered from an inflammatory site is made by the local accumulation of non-cell-bound label. These results stress the limited use of 111 In-oxine as a radiolabel in lymphocyte migration studies.

Comparative migration of guinea pig T and B lymphocytes from capillary tubes

Abstract Using a capillary tube migration technique, a comparison was made between the random mobility of separated guinea pig T- and B-lymph node lymphocyte subpopulations, and macrophage-rich peritoneal exudate cells. A decrease in random movement was found in that order, which would fit with the hypothesis that migration on a substrate is an adherence-dependent phenomenon. In order to characterize a possible dichotomy between T-and B-cell locomotion, the effects of several factors which might interfere with their migration were studied. These factors included drugs which affect cell metabolism, cell surface ligands, and some factors which may play a role in inflammatory foci (acidity, immune complexes, and lymphokines). The results emphasize the similarity in the mode of locomotion of T and B lymphocytes. However, a remarkable difference was found in the stronger inhibition of B-cell migration by pH values below pH 7.0. The relevance of these findings to the migration of T and B lymphocytes into inflammatory foci is discussed.

Effector functions of CD8-positive guinea pig T lymphocytes

The response of guinea pig T lymphocytes to different stimuli was analysed with focus on the functions of CD8-positive T cells, which so far had been poorly defined in this animal model. For identification and purification of guinea pig cytotoxic T lymphocytes, three monoclonal antibodies, directed against the CD8 differentiation antigen were characterized and compared with respect to expression pattern and biochemical characteristics of the corresponding cell surface antigen. The antibodies were used for the identification of the cytotoxic T lymphocyte subpopulation within alloreactive T cell lines, and for the depletion of CD8-positive cells in in vitro assays. Purified CD4- and CD8-positive cells were tested for their ability to proliferate in response to antigen, mitogen or anti-guinea pig Thy-1 monoclonal antibodies. Both, CD4- and CD8-positive cells showed IL-2 release and subsequent proliferation after polyclonal stimulation. Cytotoxic activity in CD8-positive alloreactive T cells was expressed in vitro only after repeated stimulation.

E and EAC Rosette-Forming Cells in Healthy Donors and Cancer Patients

The influence of age and sex on the lymphocyte count and numbers of E and EAC rosette-forming cells (RFC) was investigated using peripheral blood of healthy donors. In the female donors we found a lower total lymphocyte count and percentage of EAC RFC than in male donors. The percentage and total number of E RFC appeared to be low in younger donors. Total lymphocyte count and numbers of E and EAC RFC were investigated in untreated and treated patients with advanced solid tumors and in patients with untreated malignant lymphomas. The lymphocyte count in all these groups was decreased, but the E/EAC RFC ratio was normal, except in the group with untreated solid tumors, where we found an increased percentage of E RFC. It is suggested that in these patients a nonspecific impairment of bone marrow function might be expressed in numbers of peripheral T and B cells.