The role played by alkaline phosphatase in lipid droplet formation in different lipid-storing cell types

Thesis (Ph.D. (Chemical Pathology))--University of the Witwatersrand, Faculty of Health Sciences, 2012Alkaline phosphatases (ALPs) are a group of membrane-bound glycoproteins that occur in many species of animals and have a wide tissue distribution. ALPs have been shown to play a role in cell differentiation and organogenesis. In humans, the physiological role of ALP in skeletal mineralization is well documented. In routine clinical practice, ALP measurement is frequently used in the differential diagnosis of liver and bone diseases. Studies have shown the presence of tissue non-specific ALP (TNSALP) activity in rat adipocytes, human preadipocytes and in a murine preadipocytic cell line, 3T3-L1. ALP has also been shown to play a role in adipogenesis in 3T3-L1 cells and human preadipocytes. The purpose of the present study was to determine whether the ALP that is expressed in a human hepatocarcinoma cell line, HepG2 has a role in intracellular lipid accumulation. Intracellular lipid droplet accumulation in HepG2 cells was induced by addition of oleic acid coupled to albumin (Sigma-Aldrich, UK) to culture medium (Earle’s Minimum Essential Medium [EMEM]) and used at a final concentration of 400M. Tissue non-specific ALP inhibitors (levamisole and histidine) inhibited ALP activity and intracellular lipid accumulation in both the 3T3-L1 and HepG2 cells. Post-transcriptional silencing of the tissue non-specific alkaline phosphatase (TNSALP) gene using siRNA oligos inhibited intracellular lipid accumulation in both 3T3-L1 and HepG2 cells. In both cell lines, the ALP mRNA levels decreased in cells transfected with the anti-ALP siRNA compared to untransfected cells. This decrease in gene expression was mirrored by a corresponding fall in ALP activity in both cell lines. Quantification of the expression levels of the peroxisome proliferator activated receptor gamma (PPAR) gene (an important regulator of adipogenesis) using real-time quantitative polymerase chain reaction (real-time qPCR) showed an upward regulation of its expression four days after induction of intracellular lipid droplet accumulation in both cell types after which the levels declined. Neither levamisole nor histidine affected the expression of PPAR. Immunostaining of HepG2 cells with monoclonal antibodies against adipophilin and staining for ALP using the ELF 97 kit (Molecular Probes, Holland) demonstrated that ALP activity was localized to the surface of the lipid droplet membrane. A previous investigation has shown that ALP activity is higher in preadipocytes isolated from black compared to white females. Investigation of single nucleotide polymorphisms in the promoter region of the human TNSALP gene shows that genetic variation in the ALP promoter is not responsible for the ethnic differences in ALP activity observed in black and white South Africans. In conclusion, the close association of ALP activity with the lipid droplet membrane in HepG2 and 3T3-L1 cells and the ability to block intracellular lipid accumulation using sequence specific oligonucleotides for ALP and pharmacological agents (histidine & levamisole) strongly indicates that ALP is involved in intracellular lipid accumulation in HepG2 cells and 3T3-L1 cells. This study also shows that PPAR gene expression increases during lipid accumulation in HepG2 cells but that inhibition of ALP with histidine or levamisole does not affect the expression of this gene. Thus, ALP must act downstream of PPAR during intra-cellular lipid accumulatio

How useful are malaria risk maps at the country level? Perceptions of decision-makers in Kenya, Malawi and the Democratic Republic of Congo

BackgroundDeclining malaria prevalence and pressure on external funding have increased the need for efficiency in malaria control in sub-Saharan Africa (SSA). Modelled Plasmodium falciparum parasite rate (PfPR) maps are increasingly becoming available and provide information on the epidemiological situation of countries. However, how these maps are understood or used for national malaria planning is rarely explored. In this study, the practices and perceptions of national decision-makers on the utility of malaria risk maps, showing prevalence of parasitaemia or incidence of illness, was investigated.MethodsA document review of recent National Malaria Strategic Plans was combined with 64 in-depth interviews with stakeholders in Kenya, Malawi and the Democratic Republic of Congo (DRC). The document review focused on the type of epidemiological maps included and their use in prioritising and targeting interventions. Interviews (14 Kenya, 17 Malawi, 27 DRC, 6 global level) explored drivers of stakeholder perceptions of the utility, value and limitations of malaria risk maps.ResultsThree different types of maps were used to show malaria epidemiological strata: malaria prevalence using a PfPR modelled map (Kenya); malaria incidence using routine health system data (Malawi); and malaria prevalence using data from the most recent Demographic and Health Survey (DRC). In Kenya the map was used to target preventative interventions, including long-lasting insecticide-treated nets (LLINs) and intermittent preventive treatment in pregnancy (IPTp), whilst in Malawi and DRC the maps were used to target in-door residual spraying (IRS) and LLINs distributions in schools. Maps were also used for operational planning, supply quantification, financial justification and advocacy. Findings from the interviews suggested that decision-makers lacked trust in the modelled PfPR maps when based on only a few empirical data points (Malawi and DRC).ConclusionsMaps were generally used to identify areas with high prevalence in order to implement specific interventions. Despite the availability of national level modelled PfPR maps in all three countries, they were only used in one country. Perceived utility of malaria risk maps was associated with the epidemiological structure of the country and use was driven by perceived need, understanding (quality and relevance), ownership and trust in the data used to develop the maps

Task sharing within a managed clinical network to improve child health in Malawi

The Ministry of Health, Malawi, for its full support and collaboration during the development and implementation of this programme, including providing salaries for MOH staff during training. Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH for scholarships for BSc for CO PCH 2013–2014 intake and the salary of a paediatrician to provide training to this cadre. University of St Andrews Global Health Implementation programme for funding BSc PCH 2013–2017. University of Edinburgh for funding BSc PCH 2013–2016. The Scottish Government for funding the virtual learning environment for this cadre. Nchima Trust for funding BSc PCH 2014 intake. ELMA Philanthropies for funding MMED scholarships for specialist paediatricians. ELMA Philanthropies for funding BSc 2015–2018 intake, funding for supervision and mentoring visitsBackground Eighty per cent of Malawi’s 8 million children live in rural areas, and there is an extensive tiered health system infrastructure from village health clinics to district hospitals which refers patients to one of the four central hospitals. The clinics and district hospitals are staffed by nurses, non-physician clinicians and recently qualified doctors. There are 16 paediatric specialists working in two of the four central hospitals which serve the urban population as well as accepting referrals from district hospitals. In order to provide expert paediatric care as close to home as possible, we describe our plan to task share within a managed clinical network and our hypothesis that this will improve paediatric care and child health. Presentation of the hypothesis Managed clinical networks have been found to improve equity of care in rural districts and to ensure that the correct care is provided as close to home as possible. A network for paediatric care in Malawi with mentoring of non-physician clinicians based in a district hospital by paediatricians based at the central hospitals will establish and sustain clinical referral pathways in both directions. Ultimately, the plan envisages four managed paediatric clinical networks, each radiating from one of Malawi’s four central hospitals and covering the entire country. This model of task sharing within four hub-and-spoke networks may facilitate wider dissemination of scarce expertise and improve child healthcare in Malawi close to the child’s home. Testing the hypothesis Funding has been secured to train sufficient personnel to staff all central and district hospitals in Malawi with teams of paediatric specialists in the central hospitals and specialist non-physician clinicians in each government district hospital. The hypothesis will be tested using a natural experiment model. Data routinely collected by the Ministry of Health will be corroborated at the district. This will include case fatality rates for common childhood illness, perinatal mortality and process indicators. Data from different districts will be compared at baseline and annually until 2020 as the specialists of both cadres take up posts. Implications of the hypothesis If a managed clinical network improves child healthcare in Malawi, it may be a potential model for the other countries in sub-Saharan Africa with similar cadres in their healthcare system and face similar challenges in terms of scarcity of specialists.Publisher PDFPeer reviewe