Harvesting pre-polarized macrophages using thermo-responsive substrates.

In the cell culture environment macrophages are highly adherent cells. Currently used methods to harvest macrophages have the disadvantage of reducing cell viability and their ability to re-attach after seeding. Although thermo-responsive surfaces have been employed to harvest cell sheets no reports are available to use these to harvest (pre-polarized) macrophages. We show that this method significantly improves the yield of living macrophages and percentage of subsequent cell reattachment, whilst having a minimal effect on the cell phenotype.

Tracking immune-related cell responses to drug delivery microparticles in 3D dense collagen matrix.

Beyond the therapeutic purpose, the impact of drug delivery microparticles on the local tissue and inflammatory responses remains to be further elucidated specifically for reactions mediated by the host immune cells. Such immediate and prolonged reactions may adversely influence the release efficacy and intended therapeutic pathway. The lack of suitable in vitro platforms limits our ability to gain insight into the nature of immune responses at a single cell level. In order to establish an in vitro 3D system mimicking the connective host tissue counterpart, we utilized reproducible, compressed, rat-tail collagen polymerized matrices. THP1 cells (human acute monocytic leukaemia cells) differentiated into macrophage-like cells were chosen as cell model and their functionality was retained in the dense rat-tail collagen matrix. Placebo microparticles were later combined in the immune cell seeded system during collagen polymerization and secreted pro-inflammatory factors: TNFα and IL-8 were used as immune response readout (ELISA). Our data showed an elevated TNFα and IL-8 secretion by macrophage THP1 cells indicating that Placebo microparticles trigger certain immune cell responses under 3D in vivo like conditions. Furthermore, we have shown that the system is sensitive to measure the differences in THP1 macrophage pro-inflammatory responses to Active Pharmaceutical Ingredient (API) microparticles with different API release kinetics. We have successfully developed a tissue-like, advanced, in vitro system enabling selective "readouts" of specific responses of immune-related cells. Such system may provide the basis of an advanced toolbox enabling systemic evaluation and prediction of in vivo microparticle reactions on human immune-related cells.

From implantation to degradation - are poly (l-lactide)/multiwall carbon nanotube composite materials really cytocompatible?

Poly (l-lactide)'s (PLLA) biodegradable properties are of special value in orthopaedic applications, but its mechanical strength limits its usage. To overcome this PLLA can be reinforced by multiwall carbon nanotubes (MWCNT). In this study the PLLA and MWCNT were combined to prepare nanostructured composites (nanocomposite) at 0, 0.1, 0.5 and 1wt.% reinforcement. The in vitro biocompatibility of these PLLA/MWCNT nanocomposites was evaluated taking into account the various stages of implantation including nanocomposite degradation. PLLA/MWCNT nanocomposites were highly biocompatible with human bone marrow stromal cells (HBMC). The potential surface degradation product, MWCNT, did not induce toxic responses on HBMC. However, the combination of MWCNT with lactic acid, resembling release after bulk degradation, significantly inhibited HBMC proliferation and activity. This study demonstrates the importance of comprehensive evaluations of novel materials for medical applications in predicting possible adverse effects during nanocomposite degradation. FROM THE CLINICAL EDITOR: This study scrutinizes the cytocompatibility of poly-L-lactide reinforced by multiwall carbon nanotubes, and concludes that the combination of MWCNT with lactic acid significantly inhibited human bone marrow stromal cell proliferation and activity, highlighting the importance of comprehensive evaluations of novel materials.

Homeodomain-only protein HOP is a novel modulator of late differentiation in keratinocytes.

The homeodomain-only protein (HOP) contains an atypical homeodomain which is unable to bind to DNA due to mutations in residues important for DNA binding. Recently, HOP was reported to regulate proliferation/differentiation homeostasis in different cell types. In the present study, we performed transcriptional profiling of cultured primary human keratinocytes and noted a robust induction of HOP upon calcium-induced cell differentiation. Immunohistochemistry of human skin localized HOP to the granular layer in the epidermis. Overexpression of HOP using a lentiviral vector up-regulated FLG and LOR expression during keratinocyte differentiation. Conversely, decreasing HOP expression using small interfering RNA markedly reduced the calcium-induced expression of late markers of differentiation in vitro, with the most prominent effect on profilaggrin (FLG) mRNA. Moreover, mRNA levels of profilaggrin and loricrin were downregulated in the epidermis of HOP knockout mice. Analysis of skin disorders revealed altered HOP expression in lichen planus, psoriasis and squamous cell carcinoma (SCC). Our data indicate that HOP is a novel modulator of late terminal differentiation in keratinocytes.

The TRAF-interacting protein (TRIP) is a regulator of keratinocyte proliferation.

The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.