Herpes Simplex Virus type 1 infects Langerhans cells and the novel epidermal dendritic cell,Epi-cDC2s,via different entry pathways

Skin mononuclear phagocytes (MNPs) provide the first interactions of invading viruses with the immune system. In addition to Langerhans cells (LCs), we recently described a second epidermal MNP population, Epi-cDC2s, in human anogenital epidermis that is closely related to dermal conventional dendritic cells type 2 (cDC2) and can be preferentially infected by HIV. Here we show that in epidermal explants topically infected with herpes simplex virus (HSV-1), both LCs and Epi-cDC2s interact with HSV-1 particles and infected keratinocytes. Isolated Epi-cDC2s support higher levels of infection than LCs in vitro, inhibited by acyclovir, but both MNP subtypes express similar levels of the HSV entry receptors nectin-1 and HVEM, and show similar levels of initial uptake. Using inhibitors of endosomal acidification, actin and cholesterol, we found that HSV-1 utilises different entry pathways in each cell type. HSV-1 predominantly infects LCs, and monocyte-derived MNPs, via a pH-dependent pathway. In contrast, Epi-cDC2s are mainly infected via a pH-independent pathway which may contribute to the enhanced infection of Epi-cDC2s. Both cells underwent apoptosis suggesting that Epi-cDC2s may follow the same dermal migration and uptake by dermal MNPs that we have previously shown for LCs. Thus, we hypothesize that the uptake of HSV and infection of Epi-cDC2s will stimulate immune responses via a different pathway to LCs, which in future may help guide HSV vaccine development and adjuvant targeting.     

Evaluation of the new serum-free medium (MDSS2) for the production of different biologicals: Use of various cell lines

The presence of serum in cell culture raises safety problems for the production of biologicals, thus a new serum-free medium (MDSS2) was developed. The evaluation of this medium for the growth of different cell lines (BHK-21 C13, BSR and Vero) has shown that cells grew in this medium similarly to standard serum-containing medium, independently of the culture system used: in static (as monolayer) as well as in agitated systems (in suspension in spinner and perfusion reactors). BHK-21 and BSR cells grew as aggregate cultures and could proliferate in both static and agitated culture systems. Vero cells stayed attached to a substrate and proliferated equally in static and in agitated microcarrier-culture systems. The cell densities obtained with BHK-21 cells depended only on the culture system used. They ranged from 2–3×10⁶ to 6–12×10⁶ cells per ml for static batch and perfusion reactor cultures respectively. The cell concentration was 3 to 6 times higher than in classical cultures performed in serum-containing medium. The cell densities obtained with Vero cells were indistinguishable from those obtained in serum-containing medium, whatever the cell culture system used. These cell lines have been used for the production of rabies virus. With respect to BHK-21 and BSR, similar production rates of rabies glycoprotein have been found as in the standard roller bottle process. The production of rabies virus and of viral glycoprotein by Vero cells cultivated in serum-free medium was augmented 1.5-fold and 2.5-fold, respectively, when compared to serum-containing medium.A recombinant BHK-21 cell line, producing human IL-2, can also proliferate in MDSS2, after addition of insulin. The specific IL-2 production rate was augmented 3–4 fold in comparison to serum-containing medium.For the cells tested, the MDSS2 serum-free medium is a good growth and production medium. Its use for cultivating other cell lines and/or for the production of other biologicals is discussed.     

Communication at the Garden Fence – Context Dependent Vocalization in Female House Mice

House mice (Mus musculus) live in social groups where they frequently interact with conspecifics, thus communication (e.g. chemical and/or auditory) is essential. It is commonly known that male and female mice produce complex vocalizations in the ultrasonic range (USV) that remind of high-pitched birdsong (so called mouse song) which is mainly used in social interactions. Earlier studies suggest that mice use their USVs for mate attraction and mate choice, but they could also be used as signal during hierarchy establishment and familiarization, or other communication purposes. In this study we elucidated the vocalization behaviour of interacting female mice over an extended period of time under semi-natural conditions. We asked, if the rate or structure of female vocalization differs between different social and non-social contexts. We found that female USV is mainly used in social contexts, driven by direct communication to an unknown individual, the rate of which is decreased over time by a familiarization process. In addition we could show that female mice use two distinct types of USVs, differing in their frequency, which they use differently depending on whether they directly or indirectly communicate with another female. This supports the notion that vocalization in mice is context dependent, driven by a reasonable and yet underestimated amount of complexity that also involves the interplay between different sensory signals, like chemical and auditory cues.     

Comparison of Buprestidae collected by Cerceris fumipennis (Hymenoptera: Crabronidae) with those collected by purple prism traps

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Solution and solid state conformational preferences of a family of cyclic disulphide bridged tetrapeptides

A set of cyclic tetrapeptides of the general form cyclo (Boc‐Cys‐Pro‐ X ‐Cys‐OMe) with X being L‐ / D‐Ala , L‐ / D‐Val , and L‐ / D‐Trp was synthesized. These peptides serve as model systems for structure elucidation in solution and feature a variety of structural motifs — namely a β‐turn with intramolecular hydrogen bonding interactions, cis/trans isomerism, and a disulphide bond. In this work, we performed a comprehensive structural analysis focussing on their β‐turn conformational preferences using NMR, VCD, and Raman spectroscopy. Our results provide evidence for a strong influence of a single stereocenter on the structures of the peptides whereas solvent polarity does not significantly affect them. Additionally, the solid state conformational preferences were studied by crystal structure analysis. Overall, a general trend for the conformational preferences of this set of peptides can be concluded from the results of the complementary investigations.     

Cobalamin potentiates vinblastine cytotoxicity through downregulation of mdr-1 gene expression in HepG2 cells.

BACKGROUND: P-glycoprotein (Pgp), produced by multidrug resistance-1 gene (mdr-1), is a main mechanism developed by cancer cells to guard against anti-cancer drugs. Alterations of DNA methylation of the mdr-1 gene promoter are known to be linked to mdr-1 gene expression and are probably related to intracellular S-adenosyl-methionine. We here used HepG2 cells to determine the role of the methionine cycle (through the use of the Methionine-Synthase (MS) cofactor, cobalamin) on mdr-1 gene expression. METHODS: Semiquantitative RT-PCR of mdr-1 gene, cellular retention of rhodamine-123, and vinblastine cytotoxicity were carried out on cells cultivated with and without cobalamin. Methylation status of the mdr-1 gene promoter was determined by methylation-specific PCR. RESULTS: Addition of cobalamin to the cells led to an increase in MS activity, to a significant decrease in mdr-1 gene expression which is correlated to an increase in retention of the Pgp substrate Rhodamine 123. Furthermore, cobalamin potentiated cell sensitivity to vinblastine to the same range as that of the Pgp blocker verapamil and prevented methotrexate-induced up-regulation of mdr-1 gene expression. However, no modification in methylation of the mdr-1 gene promoter was observed. CONCLUSION: Cobalamin downregulates mdr-1 gene expression, as well as Pgp expression and function, and significantly increases cytotoxicity of vinblastine. The identification of this novel way of diminishing cellular resistance to the chemotherapeutic agent vinblastine holds promises of leading to better treatments for cancer patients.