Generation and characterization of a functional human adipose-derived multipotent mesenchymal stromal cell line

Multipotent mesenchymal stromal cells (MSC) and MSC-derived products have emerged as promising therapeutic tools. To fully exploit their potential, further mechanistic studies are still necessary and bioprocessing needs to be optimized, which requires an abundant supply of functional MSC for basic research. To address this need, here we used a novel technology to establish a human adipose-derived MSC line with functional characteristics representative of primary MSC. Primary MSC were isolated and subjected to lentiviral transduction with a library of expansion genes. Clonal cell lines were generated and evaluated on the basis of their morphology, immunophenotype, and proliferation potential. One clone (K5 iMSC) was then selected for further characterization. This clone had integrated a specific transgene combination including genes involved in stemness and maintenance of adult stem cells. Favorably, the K5 iMSC showed cell characteristics resembling juvenile MSC, as they displayed a shorter cell length and enhanced migration and proliferation compared with the non-immortalized original primary MSC (p < 0.05). Still, their immunophenotype and differentiation potential corresponded to the original primary MSC and the MSC definition criteria, and cytogenetic analyses revealed no clonal aberrations. We conclude that the technology used is applicable to generate functional MSC lines for basic research and possible future bioprocessing applications.     

Opportunistic predation on birds trapped in mist nets in two areas in the Atlantic Forest of southeastern Brazil

Mist netting is the most popular method for capturing birds, but it can increase the predation rates of individuals trapped in the nets. From 2008 to 2017, we recorded eight instances of opportunistic bird predation from mist nets (MNs) in a matrix mixing restored forest and fragments of semideciduous seasonal forest in southeastern Brazil, three times (37.5%) by exotic primates and five times (62.5%) by birds of prey. Overall predation rates (1.17–1.20%) at these two sites were considered high but were lower than in other Brazilian studies. Placing MNs near the edges of forest fragments may have allowed attacks by either forest predators or marmosets, which are exotic edge species. Some previously described precautions may decrease the predation rates of birds in MNs, such as shorter observation intervals, greater attention to given site selection and maintaining a safe distance between the MNs and the ground.     

Dichloro(6-aminoethylaminopurine)platinum(II) and its hydroxy analogues: Synthesis and preliminary evaluation

The synthesis, chemical characterization and functional evaluation are reported for dichloro(6-aminoethylaminopurine)platinum(II) and dichloro(6-hydroxyethylaminopurine)platinum(II) and dichloro(6-hydroxyethylamethylaminopurine)platinum(II) (i.e. Pt(6-AEAP), Pt(6-HEAP) and Pt(6-MHEAP) new complexes of platinum(II). Certain reaction conditions favored the formation of the tripurine platinum complex, but the monopurine complex could be obtained either by hydrolysis of the tripurine or by reacting at reduced temperature and concentration. Although neither compound was as effective as cis-diamminedichloroplatinum(II) (i.e. DDP) at reducing tumor cell viability or proliferation, both were associated with much less renal toxicity than DDP in the mouse kidney (i.e. Pt(6-AEAP):～20 × less; Pt(6-MHEAP): ～100 × less).     

Pathology background of targeted therapy; quality control in pathology

The administration of targeted therapy of gastric carcinoma is a very important recent improvement of its treatment and prognosis. The basis of the successful treatment is the excellent quality of pathology, now including HER2 testing: the use of validated methods and strict criteria. This is especially important if we consider that many gastric cancers are diagnosed in small biopsy material, in which HER2 testing is challenging. This requires standardized, validated methods and experienced pathologists. Being of diagnostic and predictive significance, high quality of both the technique and the interpretation of the test is mandatory. In order to achieve general high quality in this field, technical and interpretation external quality control of HER2 testing is necessary. Hungarian pathologists with the help of Roche Hungary Ltd. completed an external quality control round which showed that most of the participating laboratories are able. Kulka J, Szirtes I, Szász AM, Kupcsulik P, Kenessey I, Lotz G, Tímár J. Pathology background of targeted therapy; quality control in pathology.     

Vault nanocapsules as adjuvants favor cell-mediated over antibody-mediated immune responses following immunization of mice

Background

Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier.

Methodology and Principal Findings

We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8⁺ and CD4⁺ memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8⁺ memory T cells and production of IFNγ plus CD4⁺ memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes.

Conclusions/Significance

These experiments show that vault nanocapsules induced strong anti-OVA CD8⁺ and CD4⁺ T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8⁺ and CD4⁺ T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.     

Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research

Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight emetic or taste aversive compounds were employed to investigate the acute (10 min) effect of compounds on Dictyostelium cell behaviour (shape, speed and direction of movement) in a shallow chemotaxic gradient (Dunn chamber). Compound concentrations were chosen based on those previously reported to be emetic or aversive in in vivo studies and results were recorded and quantified by automated image analysis. Dictyostelium cell motility was rapidly and strongly inhibited by four structurally distinct tastants (three bitter tasting compounds--denatonium benzoate, quinine hydrochloride, phenylthiourea, and the pungent constituent of chilli peppers--capsaicin). In addition, stomach irritants (copper chloride and copper sulphate), and a phosphodiesterase IV inhibitor also rapidly blocked movement. A concentration-dependant relationship was established for five of these compounds, showing potency of inhibition as capsaicin (IC(50) = 11.9 ± 4.0 μM) > quinine hydrochloride (IC(50) = 44.3 ± 6.8 μM) > denatonium benzoate (IC(50) = 129 ± 4 μM) > phenylthiourea (IC(50) = 366 ± 5 μM) > copper sulphate (IC(50) = 1433 ± 3 μM). In contrast, 21 compounds within the cytotoxic and receptor agonist/antagonist classes did not affect cell behaviour. Further analysis of bitter and pungent compounds showed that the effect on cell behaviour was reversible and not cytotoxic, suggesting an uncharacterised molecular mechanism of action for these compounds. These results therefore demonstrate that Dictyostelium has potential as a non-sentient model in the analysis of the molecular effects of tastants, although it has limited utility in identification of emetic agents in general.     

The use of supramolecular structures as protein ligands

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.