The tumour microenvironment shapes dendritic cell plasticity in a human organotypic melanoma culture.

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14+ DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system.

Health-related quality of life analysis in stage III melanoma patients treated with adjuvant dendritic cell therapy.

BACKGROUND: Health-related quality of life (HRQoL) is an important issue in the rapidly evolving field of adjuvant treatment for stage III melanoma. Dendritic cell vaccination is one of the adjuvant forms of therapy currently investigated. METHODS: We enrolled adults with stage III melanoma to receive adjuvant dendritic cell vaccination after a complete radical lymph node dissection. HRQoL assessment was one of the secondary endpoints of this trial and investigated with the EORTC-QLQ-C30 questionnaire at baseline and week 26. RESULTS: Fifteen patients with a median age of 50 years were included in the study, with twelve evaluable patients on study at time of the second questionnaire. Global health status and role functioning improved clinically relevant with a mean difference of 15 (p = 0.010) and 26 points (p = 0.005), respectively. DISCUSSION: Despite the small number of patients, we found a clinically relevant improved global health status. Besides, compared to the other investigated therapies, toxicity of dendritic cell vaccination is low, which supports our finding. CONCLUSION: This is the first description of HRQoL in melanoma patients receiving dendritic cell vaccination. We show the expected improvement in global health status after surgical treatment of stage III melanoma. Thus, adjuvant dendritic cell vaccination does not seem to hamper this improvement, as shown in our small explorative study.

Bovine lens crystallins do contain helical structure: a circular dichroism study.

In order to settle a recent discussion on the secondary structure of lens crystallins, we have measured the circular dichroism (CD) spectra of alpha-, beta(H)-, and beta(L)-crystallin from 178 to 250 nm and of gamma-crystallin from 168 to 250 nm. The results were analysed by means of a newly developed algorithm that almost doubles the reliability of secondary structure prediction and that allows discrimination between alpha- and 3(10)-helical, and between extended and polyproline beta-type structure. The results indicate that the crystallins studied contain a non-negligible amount of alpha-helical structure, although at least 50% of it is in the form of single and/or distorted loops. In alpha-crystallin, which is related to the chaperones, the helical content is lower than in beta- and gamma-crystallin. In some cases, the helices may play a role in DNA binding by the crystallins.

Transient electric birefringence study of intermediate filament formation from vimentin and glial fibrillary acidic protein.

Mg2+-induced polymerization of type III intermediate filament proteins vimentin and glial fibrillary acidic protein was studied by transient electric birefringence. In the absence of MgCl2 we found a net permanent dipole moment, approximately 45-nm-long dimers for vimentin, approximately 65-nm-long tetramers, hexamers, and possibly octamers for both proteins, and 100-nm aggregates for glial fibrillary acidic protein. Controlled oligomerization occurred after the addition of MgCl2. Although the solutions contained (small) aggregates of different sizes, more or less discrete steps in polymer formation were observed, and it was possible to discriminate between an increase in width and length. At the first stage of polymerization (in 0.3 mM MgCl2 for vimentin and 0.2 mM MgCl2 for glial fibrillary acidic protein), the permanent dipole moment disappeared without a change in length of the particles. At higher MgCl2 concentrations, structures of approximately 100 nm were formed, which strongly tended to laterally assemble into full-width intermediate filament structures consisting of about 32 monomers. This contrasts with previous models where first full-width (approximately 10-nm) aggregates are formed, which then increase in length. Subsequently, two discrete elongation steps of 35 nm are observed that increase the length to 135 and 170 nm, respectively. Possible structural models are suggested for the polymerization.

Characterization of multiple oligomeric vimentin intermediate filament units by transient electric birefringence measurements.

In this work we have studied the structure of soluble vimentin units from which intermediate filaments (IFs) are built. Several oligomeric forms have been presented in the literature as IF "building blocks", but there is still no agreement on this matter. By comparing our data with various models as proposed in the literature we can favour certain models and reject others. Transient electric birefringence (TEB) measurements were performed from which information is obtained concerning electric and hydrodynamic properties of the particles under investigation. TEB decay analysis at pH 6.8 after 70 microseconds pulses (at 20 degrees C in aqueous solution) yielded three decay times: 1.1(+/- 0.3) microseconds, 4.0(+/- 1.0) microseconds and 20.0(+/- 5.0) microseconds, with amplitudes of 45% to 60%, 30% to 45%, and less than 10%, respectively. At pH 8.5 after 70 microseconds pulses, more than 90% of the TEB signal with the second decay time is obtained, while the remainder had a decay time of 15.0(+/- 4.0) microseconds. Only when the pulse duration was decreased, the fast decay time around 1 microsecond was observed, suggesting that only a minor fraction of the particles at this pH value causes such a short decay time. At both pH values, the TEB measurements indicated that, at least in part, the molecules are oriented by a permanent dipole moment. It will be shown that the shortest decay time originates from bent or flexible dimers, and the second decay time from particles with a length of 54 to 65 nm containing, at least in part, a relatively large overall dipole moment. The longest decay time is probably due to larger aggregates. These results are consistent with a model in which single dimers, antiparallel staggered tetramers and hexamers coexist. Alternatively, but less likely on the basis of literature data, a model of parallel in-register tetramers with a considerable length contribution of the head groups would fit our research.

Determination of oxidized, reduced and protein-bound glutathione in eye lenses by high-performance liquid chromatography and electrochemical detection.

A method for the detection of oxidized, reduced and protein-bound glutathione in eye lenses has been developed. Homogenized lens samples are deproteinated with acetonitrile and perchloric acid. Protein-bound glutathione is reduced by 1,4-dithiothreitol. Separation of the different forms of glutathione and dithiothreitol is performed by ion-pair reversed-phase high-performance liquid chromatography with sodium octylsulphate as the ion-pairing agent. The compounds are detected amperometrically using on-line-generated bromine, which oxidizes thiols and disulphides. In this way two samples can be analysed in triplicate in a single day. The lower detection limits are 80 and 48 nmol per gram wet lens for reduced and oxidized glutathione, respectively. The amounts of free reduced and protein-bound glutathione in calf lenses, determined with this method, are 6.8 +/- 0.4 and 0.96 +/- 0.03 mumol per gram wet lens, respectively. That of oxidized glutathione is less than 0.048 mumol per gram wet lens.