Studies on feline calicivirus with particular reference to persistence.

The molecular evolution of feline calicivirus (FCV) was studied in cell culture and in persistently infected cats. Sequence analysis of the 5' hypervariable region of the FCV capsid (5'HVR; located at the 5' end of variable region E), a region known to contain linear neutralising B cell epitopes, showed FCV existed as a quasispecies which evolved at the nucleotide and amino acid level during persistent infection. Quasispecies heterogeneity tended to decrease the course of persistence. Sequential isolates from a cat showed marked antigenic variation during the course of persistent infection. Sequential passage of FCV in cell culture was also associated with sequence evolution of the 5'HVR. However, these isolates showed no change in antigenicity suggesting that individual substitutions observed in viruses from cats, but not in viruses from cell culture, may be responsible for changes in antigenicity. Alternatively, the observed antigenic changes may be associated with mutations elsewhere in the genome. In order to identify regions of the FCV capsid protein containing liner B-cell epitopes, two approaches were used. Firstly, an expression library containing random, short (100-300bp) fragments of an FCV capsid gene was constructed. This library was screened using polyclonal antisera from a cat that had been challenged experimentally with FCV to identify immunoreactive clones containing B-cell epitopes. Initial screening identified five clones that reacted positively to feline antisera in immunoblots. FCV derived sequence from these clones all mapped to the 5'HVR, suggesting this region contains the immunodominant linear epitopes of the capsid. The second approach used to identify B-cell epitopes was to map more accurately the epitope of a neutralising monoclonal antibody (IG9) which had already been shown to lie in a 37 amino acid region of the 5'HVR (Milton et al. (1992), Journal of General Virology 73, 2435-2439). Replication of plaque purified IG9-sensitive parent virus in sub-neutralising concentrations of IG9 led to the generation of a neutralisation resistant escape mutant. Sequence analysis of this mutant and the parent virus revealed a single non-synonymous nucleotide substitution within the 5'HVR, suggesting this residue is critical to the correct formation of the IG9 epitope

Combinatorial biological complexity: a study of amino acid side chains and alternative splicing

Both, laymen and experts have always been intrigued by nature’s vast complexity and variety. Often, these phenomena arise from combination of parts, as for example, cell types of the human body, or the diverse proteins of a cell. In this thesis I investigate three instances of combinatorial complexity: combinations of aliphatic amino acid side chains, alternative mRNA splicing in fungi, and mutually exclusively spliced exons in human and mouse. In the first part the number of aliphatic amino acid side chains is studied. Structural combinations yield a vast theoretical number, yet we find that only a fraction of them is realized in nature. Reasons especially with respect to restrictions by the genetic code are discussed. Moreover, strategies for the need for increased diversity are examined. In the second part, the extent of alternative splicing (AS) in fungi is investigated. A genome-wide, comparative multi-species study is conducted. I find that AS is common in fungi, but with lower frequency compared to plants and animals. AS is more common in more complex fungi, and is over-represented in pathogens. It is hypothesized that AS contributes to multi-cellular complexity in fungi. In the third part, mutually exclusive exons (MXEs) of mouse and human are detected and characterized. Rather unexpected patterns arose: the majority of MXEs originate from non-adjacent exons and frequently appear in clusters. Known regulatory mechanisms of MXE splicing are unsuitable for these MXEs, and thus, new mechanisms have to be sought. Summarizing it is hypothesized that complexity from combinations constitutes a universal principle in biology. However, there seems to be a need to restrict the combinatorial potential. This is highlighted by the interdependence of MXEs and the low number of realized amino acids in the genetic code. Combinatorial complexity and its restriction are discussed with respect to other biological systems to further substantiate the hypotheses

Investigation into the changing colonisation of skin bacteria during isotretinoin treatment for acne vulgaris

Poster presentatio

Advances in Parvovirus Research 2020

Viruses of the Parvoviridae family constitute a most diverse and intriguing field of research. Parvoviruses can differ widely in their structure, genome organization and expression, virus–cell interactions, and impact on hosts. The translational implication of research on parvoviruses is relevant, since many viruses are important human and veterinary pathogens, while other viruses can be engineered as tools for oncolytic therapy or as sophisticated gene delivery vectors. Exploring the diversity and inherent complexity in the biology of these apparently simple viruses is a still challenging topic for the scientific community. The Special Issue of Viruses is a collection of recent contributions in the field of parvovirus research, encompassing many aspects of basic and translational research on viruses of the family Parvoviridae, including on their structure, replication, and gene expression in addition to virus–host interactions and the development of vaccines and viral vectors

Biology of interleukin-6 production by human natural killer cells

NK cells secrete a variety of immune-regulatory cytokines and chemokines such as interferon - γ (IFN-γ) and tumour necrosis factor- α (TNF-α). However, in some of infections where NK cells have an important role in the defence mechanism, IL-6 appears to play a significant anti-viral role. Considering the role of both IL-6 and NK cells in these infections raises the possibility that IL-6 secretion by NK cells could be a main defence mechanism against them. Morover, NK cell-mediated IL-6 secretion may provide a critical link between the innate and adaptive immune response of the host. Furthermore, changes in this pathway in various autoimmune diseases, for example rheumatoid arthritis (RA) may be relevant for the pathogenesis of these disorders. The results presented in this thesis demonstrated that peripheral blood NK cells from healthy individuals have the ability to secrete IL-6 after co-culture with target cells against which these cells are known to exhibit direct cytotoxicity (either K562 or HeLa cells). The described secretory response was rapid, with IL-6 being significantly higher as early as 1 hour in HeLa co-cultures compared to 6 hours in case of K562 co-cultures. These findings were further confirmed when NK cells were activated alone with high doses of IL-2 or with non specific chemical activators (PMA+ ionomycin). These experiments clearly showed that NK cells have the potential to secrete IL-6 following activation. To analyse whether IL-6 secretion in the co-culture experiments was the result of direct cell-cell interactions between NK cells and the target cells or was induced by the presence of soluble mediators, co-culture experiments were set up where the target cells were separated from NK cells using a 0.4 µm pore size inserts. Separating NK cells and target cells abolished increases in cytokine production proving that direct interaction between NK cells and target cells is necessary for triggering IL-6 production by NK cells, as the semi-permeable membrane of Transwell chambers allows for the free passage of soluble factors but prevents direct cell-cell contact. Investigating the activating pathway which triggers the secretion of IL-6 by NK cells was the next step. This aim was achieved by inducing NK cell activation with immobilized antibodies against NK cell activating receptors and assessing the effect of the engagement of these receptors on peripheral blood NK IL-6 gene expression and protein secretion by quantitative real time PCR and ELISA.The results demonstrated that NKG2D and NKp46 were the two main receptors involved in the IL-6 mRNA expression and secretion by NK cells. The final aim of this thesis was to evaluate the biological significance of this secretion through an in vitro experimental model. We hypothesized that IL-6 secreted by NK cells could contribute to the migration of other inflammatory and immune cells to the site of inflammation. This hypothesis was based on the observations of others that IL-6 could induce direct CD4+ T cell migration. To test this hypothesis, an in vitro transmigration assay using Transwell inserts with 8 and 3 µm pore size were used. Our results demonstrated that CD4+ T cell migration in response to NK cells was inhibited by about 30% in the presence of neutralizing antibody to IL-6. These results signify the relative biological importance of IL-6 induced secretion by NK cells. In conclusion NK cells can contribute to IL-6 secretion. Given that NK cells appear at inflammation sites at the earliest stages of the process, where the number of other cells with a potential to secrete IL-6 is low, it is possible that NK cell-mediated IL-6 secretion is essential in orchestrating and potentiating the later stages of the adaptive immune response

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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