196 research outputs found
Use of A Collagen/Elastin Matrix As Transport Carrier System to Transfer Proliferating Epidermal Cells to Human Dermis in Vitro
This in vitro study describes a novel cell culture, transport, and transfer protocol that may be highly suitable for delivering cultured proliferating keratinocytes and melanocytes to large open skin wounds (e.g., burns). We have taken into account previous limitations identified using other keratinocyte transfer techniques, such as regulatory issues, stability of keratinocytes during transport (single cell suspensions undergo terminal differentiation), ease of handling during application, and the degree of epidermal blistering resulting after transplantation (both related to transplanting keratinocyte sheets). Large numbers of proliferating epidermal cells (EC) (keratinocytes and melanocytes) were generated within 10-14 days and seeded onto a three-dimensional matrix composed of elastin and collagen types I, III, and V (Matriderm®), which enabled easy and stable transport of the EC for up to 24 h under ambient conditions. All culture conditions were in accordance with the regulations set by the Dutch Central Committee on Research Involving Human Subjects (CCMO). As an in vitro model system for clinical in vivo transfer, the EC were then transferred from Matriderm onto human acellular dermis during a period of 3 days. After transfer the EC maintained the ability to regenerate into a fully differentiated epidermis containing melanocytes on the human dermis. Proliferating keratinocytes were located in the basal layer and keratin-10 expression was located in differentiating suprabasal layers similar to that found in human epidermis. No blistering was observed (separation of the epidermis from the basement membrane). Keratin-6 expression was strongly upregulated in the regenerating epidermis similar to normal wound healing. In summary, we show that EC-Matriderm contains viable, metabolically active keratinocytes and melanocytes cultured in a manner that permits easy transportation and contains epidermal cells with the potential to form a pigmented reconstructed epidermis. This in vitro study has produced a robust protocol that is ready for clinical studies in the future
The formation of vault-tubes: a dynamic interaction between vaults and vault PARP
Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles that are
composed of a major vault protein (MVP), two minor vault proteins
[telomerase-associated protein 1 (TEP1), vault poly(ADP-ribose) polymerase
(VPARP)] and small untranslated RNA molecules. Not all expressed TEP1 and
VPARP in cells is bound to vaults. TEP1 is known to associate with the
telomerase complex, whereas VPARP is also present in the nuclear matrix
and in cytoplasmic clusters (VPARP-rods). We examined the subcellular
localization and the dynamics of the vault complex in a non-small cell
lung cancer cell line expressing MVP tagged with green fluorescent
protein. Using quantitative fluorescence recovery after photobleaching
(FRAP) it was shown that vaults move temperature independently by
diffusion. However, incubation at room temperature (21 degrees C) resulted
in the formation of distinct tube-like structures in the cytoplasm.
Raising the temperature could reverse this process. When the vault-tubes
were formed, there were fewer or no VPARP-rods present in the cytoplasm,
suggesting an incorporation of the VPARP into the vault-tubes. MVP
molecules have to interact with each other via their coiled-coil domain in
order to form vault-tubes. Furthermore, the stability of microtubules
influenced the efficiency of vault-tube formation at 21 degrees C. The
dynamics and structure of the tubes were examined using confocal
microscopy. Our data indicate a direct and dynamic relationship between
vaults and VPARP, providing further clues to unravel the function of
vaults
IL-21 promotes the expansion of CD27+CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells
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118572.pdf (publisher's version ) (Open Access)BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-gamma receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT
Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics
Vaults are ribonucleoprotein particles with a distinct structure and a
high degree of conservation between species. Although no function has been
assigned to the complex yet, there is some evidence for a role of vaults
in multidrug resistance. To confirm a direct relation between vaults and
multidrug resistance, and to investigate other possible functions of
vaults, we have generated a major vault protein (MVP/lung
resistance-related protein) knockout mouse model. The MVP(-/-) mice are
viable, healthy, and show no obvious abnormalities. We investigated the
sensitivity of MVP(-/-) embryonic stem cells and bone marrow cells derived
from the MVP-deficient mice to various cytostatic agents with different
mechanisms of action. Neither the MVP(-/-) embryonic stem cells nor the
MVP(-/-) bone marrow cells showed an increased sensitivity to any of the
drugs examined, as compared with wild-type cells. Furthermore, the
activities of the ABC-transporters P-glycoprotein, multidrug
resistance-associated protein and breast cancer resistance protein were
unaltered on MVP deletion in these cells. In addition, MVP wild-type and
deficient mice were treated with the anthracycline doxorubicin. Both
groups of mice responded similarly to the doxorubicin treatment. Our
results suggest that MVP/vaults are not directly involved in the
resistance to cytostatic agents
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