Selective activation of tumour necrosis factor receptor-mediated intracellular signalling pathways.

Tumour necrosis factor (TNF) is a pleiotropic cytokine that has been shown to play a major role in defence against infections and malignancy, and regulation of the innate and adaptive immune responses. Despite its beneficial role, the cytokine has been implicated in the pathophysiology of a range of diseases including sepsis, cerebral malaria and autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. While blocking the activity of excessive TNF has become a therapeutic approach to managing patients with these diseases, there are concerns since this also decreases resistance against infection and cancer. Attempts to target intracellular signalling pathways used by TNF, such as the p38 mitogen activated protein kinase (MAPK) have also met with limitations and studies have been discontinued due to toxicity. Since most proteins exert their biological activity through the interaction between very small regions of their folded surfaces to their cognate receptors, smaller peptides which mimic the shape of the proteins at these points of contact with the receptors can be used to mimic and/or block the actions of these proteins. We have previously demonstrated that the TNF mimetic peptides TNF₇₀₋₈₀ and TNF₁₃₂₋₁₅₀ exhibited distinct biological activities, which in combination represented the spectrum of biological activities displayed by TNF. Research in this thesis sought to use these properties to develop new targets for development of anti-inflammatory agents. The mimetic TNF₇₀₋₈₀ was shown to bind and act as a ligand for the TNF receptor I (TNFRI) and selectively activated the p38 MAPK pathway, and not the c-Jun NH2-terminal kinase (JNK) and extracellular-signal-regulated kinase 1 and 2 (ERK1/ERK2) pathways. In contrast TNF₁₃₂₋₁₅₀ selectively activated the JNK and ERK1/ERK2 pathways. This is consistent with the biological properties of these peptides. The basis for the activation of a restricted signalling pathway by TNF₇₀₋₈₀ was related to a reduced capability to recruit adapter proteins. The peptide mimetic ligated TNFR was able to functionally couple TNF receptor associated factor 2 (TRAF2) to the p38 and NF-kB pathway but was unable to effect the coupling of germinal centre kinase (GCK) and apoptosis signal-regulating kinase (ASK1) to TRAF2, probably explaining the lack of activation of JNK and ERK1/ERK2 pathways. Using the ability of TNF₇₀₋₈₀ to activate p38, we identified the region to which TNF₇₀₋₈₀ binds to the TNFRI. Synthetic peptides representing the 206-211 amino acid residues of the TNFRI were made and examined for anti-TNF effects in vitro and in vivo. These TNFR mimetic peptides were found to selectively block TNF induced p38 activation and associated functions of neutrophil superoxide production, CD11b upregulation and cytokine production. Similar results were found with the monocytic cell line, Mono Mac 6. These TNFRI-derived peptides were found to inhibit leukocyte infiltration into inflammatory sites in acute and chronic inflammation models. Our findings open new opportunities for the development of therapeutics which selectively target the TNFR-p38 signalling pathway in chronic inflammatory diseases.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 200

Independent and combined effects of improved water, sanitation, and hygiene, and improved complementary feeding, on child stunting and anaemia in rural Zimbabwe: a cluster-randomised trial.

BACKGROUND: Child stunting reduces survival and impairs neurodevelopment. We tested the independent and combined effects of improved water, sanitation, and hygiene (WASH), and improved infant and young child feeding (IYCF) on stunting and anaemia in in Zimbabwe. METHODS: We did a cluster-randomised, community-based, 2 × 2 factorial trial in two rural districts in Zimbabwe. Clusters were defined as the catchment area of between one and four village health workers employed by the Zimbabwe Ministry of Health and Child Care. Women were eligible for inclusion if they permanently lived in clusters and were confirmed pregnant. Clusters were randomly assigned (1:1:1:1) to standard of care (52 clusters), IYCF (20 g of a small-quantity lipid-based nutrient supplement per day from age 6 to 18 months plus complementary feeding counselling; 53 clusters), WASH (construction of a ventilated improved pit latrine, provision of two handwashing stations, liquid soap, chlorine, and play space plus hygiene counselling; 53 clusters), or IYCF plus WASH (53 clusters). A constrained randomisation technique was used to achieve balance across the groups for 14 variables related to geography, demography, water access, and community-level sanitation coverage. Masking of participants and fieldworkers was not possible. The primary outcomes were infant length-for-age Z score and haemoglobin concentrations at 18 months of age among children born to mothers who were HIV negative during pregnancy. These outcomes were analysed in the intention-to-treat population. We estimated the effects of the interventions by comparing the two IYCF groups with the two non-IYCF groups and the two WASH groups with the two non-WASH groups, except for outcomes that had an important statistical interaction between the interventions. This trial is registered with ClinicalTrials.gov, number NCT01824940. FINDINGS: Between Nov 22, 2012, and March 27, 2015, 5280 pregnant women were enrolled from 211 clusters. 3686 children born to HIV-negative mothers were assessed at age 18 months (884 in the standard of care group from 52 clusters, 893 in the IYCF group from 53 clusters, 918 in the WASH group from 53 clusters, and 991 in the IYCF plus WASH group from 51 clusters). In the IYCF intervention groups, the mean length-for-age Z score was 0·16 (95% CI 0·08-0·23) higher and the mean haemoglobin concentration was 2·03 g/L (1·28-2·79) higher than those in the non-IYCF intervention groups. The IYCF intervention reduced the number of stunted children from 620 (35%) of 1792 to 514 (27%) of 1879, and the number of children with anaemia from 245 (13·9%) of 1759 to 193 (10·5%) of 1845. The WASH intervention had no effect on either primary outcome. Neither intervention reduced the prevalence of diarrhoea at 12 or 18 months. No trial-related serious adverse events, and only three trial-related adverse events, were reported. INTERPRETATION: Household-level elementary WASH interventions implemented in rural areas in low-income countries are unlikely to reduce stunting or anaemia and might not reduce diarrhoea. Implementation of these WASH interventions in combination with IYCF interventions is unlikely to reduce stunting or anaemia more than implementation of IYCF alone. FUNDING: Bill & Melinda Gates Foundation, UK Department for International Development, Wellcome Trust, Swiss Development Cooperation, UNICEF, and US National Institutes of Health.The SHINE trial is funded by the Bill & Melinda Gates Foundation (OPP1021542 and OPP113707); UK Department for International Development; Wellcome Trust, UK (093768/Z/10/Z, 108065/Z/15/Z and 203905/Z/16/Z); Swiss Agency for Development and Cooperation; US National Institutes of Health (2R01HD060338-06); and UNICEF (PCA-2017-0002)