Development of a Glycocluster Adjuvant Mimicking Natural Yeast beta-(1,2)-Structures

Allergy is characterized by T helper (Th) 2-type immune response after encounter with an allergen leading to subsequent immunoglobulin (Ig) E-mediated hypersensitivity reaction and further allergic inflammation. Allergen-specific immunotherapy (SIT) balances the Th2-biased immunity towards Th1 and T regulatory responses. Adjuvants are used in allergen preparations to intensify and modify SIT. β-(1,2)-oligomannoside constituents present in Candida albicans (C. albicans) cell wall possess Th1-type immunostimulatory properties. The aim of this thesis was to develop a β-(1,2)-linked carbohydrate compound with known structure and anti-allergic properties to be applied as an adjuvant in SIT. First the immunostimulatory properties of various fungal extracts were studied. C. albicans appeared to be the most promising Th1-inducing extract, which led to the synthesis of various mono- or divalent oligomannosides designed on the basis of C. albicans. These carbohydrates did not induce strong cytokine production in human peripheral blood mononuclear cell (PBMC) cultures. In contrast to earlier reports using native oligosaccharides from C. albicans, synthetic -(1,2)-linked mannotetraose did not induce any tumor necrosis factor production in murine macrophages. Next, similarities with synthesized divalent mannosides and the antigenic epitopes of β-(1,2)-linked C. albicans mannan were investigated. Two divalent compounds inhibited specific IgG antibodies binding to below 3 kDa hydrolyzed mannan down to the level of 30–50% showing similar antigenicity to C. albicans. Immunomodulatory properties of synthesized carbohydrate assemblies ranging from mono- to pentavalent were evaluated. A trivalent acetylated dimannose (TADM) induced interleukin-10 (IL-10) and interferon-γ responses. TADM also suppressed birch pollen induced IL-4 and IL-5 responses in allergen (Bet v) stimulated PBMCs of birch pollen allergic subjects. This suppression was stronger with TADM than with other used adjuvants, immunostimulatory oligonucleotides and monophosphoryl lipid A. In a murine model of asthma, the allergen induced inflammatory responses could also be suppressed by TADM on cytokine and antibody levels.

Studies on membrane properties of cholesterol and 3-beta modified sterol analogs

Cholesterol (Chol) is an important lipid in cellular membranes functioning both as a membrane fluidity regulator, permeability regulator and co-factor for some membrane proteins, e.g. G-protein coupled receptors. It also participates in the formation of signaling platforms and gives the membrane more mechanical strenght to prevent osmotic lysis of the cell. The sterol structure is very conserved and already minor structural modifications can completely abolish its membrane functions. The right interaction with adjacent lipids and the preference of certain lipid structures over others are also key factors in determining the membrane properties of cholesterol. Because of the many important properties of cholesterol it is of value to understand the forces and structural properties that govern the membrane behavior of this sterol. In this thesis we have used established fluorescence spectroscopy methods to study the membrane behavior of both cholesterol and some of its 3β-modified analogs. Using several fluorescent probes we have established how the acyl chain order of the two main lipid species, sphingomyelin (SM) and phosphatidylcholine (PC) affect sterol partitioning as well as characterized the membrane properties of 3β-aminocholesterol and cholesteryl phosphocholine. We concluded that cholesterol prefers SM over PC at equal acyl chain order, indicating that other structural properties besides the acyl chain order are important for sphingomyelin-sterol interactions. A positive charge at the 3β position only caused minor changes in the sterol membrane behavior compared to cholesterol. A large phosphocholine head group caused a disruption in membrane packing together with other membrane lipids with large head groups, but was also able to form stable fluid bilayers together with ceramide and cholesterol. The Ability of the large head group sterol to form bilayers together with ceramide was further explored in the last paper where cholesteryl phosphocholine/ceramide (Chol-PC/Cer) complexes were successfully used to transfer ceramide into cultured cells.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

The Role of Integrins in Enterovirus Infections and in Metastasis of Cancer

Integrins are a family of transmembrane glycoproteins, composed of two different subunits (alpha and beta). Altered expression of integrins in tumor cells contributes to metastasis tendency by influencing on the cells‟ attachment to adjacent cells and their migration. Viral pathogens, including certain enteroviruses, use integrins as receptors. Enteroviruses have also been suggested to be involved in the etiopathogenesis of type 1 diabetes. The study focuses on the role of integrins in the pathogenesis of metastasis to cortical bone and on type 1 diabetes (T1D) and echovirus 1 infection. In the first part of the thesis, the role of different integrins in the initial attachment of MDA-MD-231 breast cancer cells to bovine cortical bone disks was studied. A close correlation between alpha2beta1 and alpha3beta1 integrin receptor expression and the capability of the tumor to attach to bone were observed. In the second part, a possible correlation between susceptibility to enterovirus infections in diabetic children and differences in enterovirus receptor genes, including certain integrins, was investigated. In parallel, virus-specific neutralizing antibodies and diabetic risk alleles were studied. In the diabetic group, an amino acid change was detected in the polio virus receptor and the neutralizing antibody titers against echovirus 30 were lower. However, to obtain statistically sustainable results, a larger number of individuals should be analyzed. Echovirus 1 (EV1) enters cells by attaching to the alpha2I domain of the alpha2beta1 integrin. In the third part EV1 was shown to attach to a chimeric receptor construct of the transferrin receptor and the alpha2I domain and to enter cells through clathrin-mediated endocytosis that is normally not used by the virus. The chimeric receptor was recycled to the plasma membrane, whereas the virus remained in intracellular vesicles. The virus replication cycle was initiated in these cells, suggesting that evolution pressure could possibly cause the virus to evolve to use a different entry mechanism. Moreover, a cDNA microarray analysis of host gene expression during EV1 replication showed that 0.53% of the total genes, including several immediate early genes, were differently expressed.

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.