A simple optical fiber device for quantitative fluorescence microscopy of single living cells

A simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultiplier are converted to voltage, amplified, and displayed on a recorder. In the excitation pathway a shutter control unit is mounted. With this control unit the period that the excitation pathway is `opened¿ and `closed¿ can be adjusted, to reduce cell damage and/or bleaching of the probe. This option allows time-lapse recording of experiments up to 1 h. We have used this set-up with a single and dual emission fluorescent probe to determine intracellular calcium concentrations and pH, respectively. In Fluo-3-loaded K562 target cells bound to natural killer cells, a temporary rise in [Ca2+]i was accompanied by bleb formation. The simple construction of this set-up is interchangeable between different types of fluorescence microscopes and can easily be combined with other microscopy techniques, e.g., patch clamp

Flow Cytometric Measurement of [Ca2+]i and pHi in Conjugated Natural Killer Cells and K562 Target Cells during the Cytotoxic Process1,2

We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly compared to the nonconjugated cells. This is achieved by labeling one cell type with the Ca2+-specific dye Fluo-3, while the other cell type is labeled with the pH-sensitive dye SNARF-1. As these fluorochromes have different emission spectra, events positive for both fluorochromes are identified as conjugates. The results show that the conjugates can be clearly distinguished from single cytotoxic cells [natural killer (NK) cells] and target cells [K562 cells, (TC)]. Upon binding, [Ca2+]i is increased in the NK cells as well as in the TC. In conjugated NK cells this increase of [Ca2+]i is temperature dependent and is followed by a decrease to a normal [Ca2+]i value later on. The [Ca2+]i in NK cells increases in 2 steps, which may be related to the binding-and lethal hit phase. Upon conjugate formation, NK cells show a slight increase in pHi (0.2-0.3 pH units). TC do not reveal a significant change in pHi

Chronic Diseases in North-West Tanzania and Southern Uganda. Public Perceptions of Terminologies, Aetiologies, Symptoms and Preferred Management

Research outputs produced to support a quantitative population survey, quantitative health facility survey, focus groups and in-depth interviews performed by the projec

Heavy burden of non-communicable diseases at early age and gender disparities in an adult population of Burkina Faso: world health survey

<p>Abstract</p> <p>Background</p> <p>WHO estimates suggest that age-specific death rates from non-communicable diseases are higher in sub-Saharan Africa than in high-income countries. The objectives of this study were to examine, in Burkina Faso, the prevalence of non-communicable disease symptoms by age, gender, socioeconomic group and setting (rural/urban), and to assess gender and socioeconomic inequalities in the prevalence of these symptoms.</p> <p>Methods</p> <p>We obtained data from the Burkina Faso World Health Survey, which was conducted in an adult population (18 years and over) with a high response rate (4822/4880 selected individuals). The survey used a multi-stage stratified random cluster sampling strategy to identify participants. The survey collected information on socio-demographic and economic characteristics, as well as data on symptoms of a variety of health conditions. Our study focused on joint disease, back pain, angina pectoris, and asthma. We estimated prevalence correcting for the sampling design. We used multiple Poisson regression to estimate associations between non-communicable disease symptoms, gender, socioeconomic status and setting.</p> <p>Results</p> <p>The overall crude prevalence and 95% confidence intervals (CI) were: 16.2% [13.5; 19.2] for joint disease, 24% [21.5; 26.6] for back pain, 17.9% [15.8; 20.2] for angina pectoris, and 11.6% [9.5; 14.2] for asthma. Consistent relationships between age and the prevalence of non-communicable disease symptoms were observed in both men and women from rural and urban settings. There was markedly high prevalence in all conditions studied, starting with young adults. Women presented higher prevalence rates of symptoms than men for all conditions: prevalence ratios and 95% CIs were 1.20 [1.01; 1.43] for joint disease, 1.42 [1.21; 1.66] for back pain, 1.68 [1.39; 2.04] for angina pectoris, and 1.28 [0.99; 1.65] for asthma. Housewives and unemployed women had the highest prevalence rates of non-communicable disease symptoms.</p> <p>Conclusions</p> <p>Our work suggests that social inequality extends into the distribution of non-communicable diseases among social groups and supports the thesis of a differential vulnerability in Burkinabè women. It raises the possibility of an abnormally high rate of premature morbidity that could manifest as a form of premature aging in the adult population. Increased prevention, screening and treatment are needed in Burkina Faso to address high prevalence and gender inequalities in non-communicable diseases.</p

Rationale and cross-sectional study design of the Research on Obesity and type 2 Diabetes among African Migrants: the RODAM study.

INTRODUCTION: Obesity and type 2 diabetes (T2D) are highly prevalent among African migrants compared with European descent populations. The underlying reasons still remain a puzzle. Gene-environmental interaction is now seen as a potential plausible factor contributing to the high prevalence of obesity and T2D, but has not yet been investigated. The overall aim of the Research on Obesity and Diabetes among African Migrants (RODAM) project is to understand the reasons for the high prevalence of obesity and T2D among sub-Saharan Africans in diaspora by (1) studying the complex interplay between environment (eg, lifestyle), healthcare, biochemical and (epi)genetic factors, and their relative contributions to the high prevalence of obesity and T2D; (2) to identify specific risk factors within these broad categories to guide intervention programmes and (3) to provide a basic knowledge for improving diagnosis and treatment. METHODS AND ANALYSIS: RODAM is a multicentre cross-sectional study among homogenous sub-Saharan African participants (ie, Ghanaians) aged >25 years living in rural and urban Ghana, the Netherlands, Germany and the UK (http://rod-am.eu/). Standardised data on the main outcomes, genetic and non-genetic factors are collected in all locations. The aim is to recruit 6250 individuals comprising five subgroups of 1250 individuals from each site. In Ghana, Kumasi and Obuasi (urban stratum) and villages in the Ashanti region (rural stratum) are served as recruitment sites. In Europe, Ghanaian migrants are selected through the municipality or Ghanaian organisations registers. ETHICS AND DISSEMINATION: Ethical approval has been obtained in all sites. This paper gives an overview of the rationale, conceptual framework and methods of the study. The differences across locations will allow us to gain insight into genetic and non-genetic factors contributing to the occurrence of obesity and T2D and will inform targeted intervention and prevention programmes, and provide the basis for improving diagnosis and treatment in these populations and beyond

Setting research priorities to improve global newborn health and prevent stillbirths by 2025.

BACKGROUND: In 2013, an estimated 2.8 million newborns died and 2.7 million were stillborn. A much greater number suffer from long term impairment associated with preterm birth, intrauterine growth restriction, congenital anomalies, and perinatal or infectious causes. With the approaching deadline for the achievement of the Millennium Development Goals (MDGs) in 2015, there was a need to set the new research priorities on newborns and stillbirth with a focus not only on survival but also on health, growth and development. We therefore carried out a systematic exercise to set newborn health research priorities for 2013-2025. METHODS: We used adapted Child Health and Nutrition Research Initiative (CHNRI) methods for this prioritization exercise. We identified and approached the 200 most productive researchers and 400 program experts, and 132 of them submitted research questions online. These were collated into a set of 205 research questions, sent for scoring to the 600 identified experts, and were assessed and scored by 91 experts. RESULTS: Nine out of top ten identified priorities were in the domain of research on improving delivery of known interventions, with simplified neonatal resuscitation program and clinical algorithms and improved skills of community health workers leading the list. The top 10 priorities in the domain of development were led by ideas on improved Kangaroo Mother Care at community level, how to improve the accuracy of diagnosis by community health workers, and perinatal audits. The 10 leading priorities for discovery research focused on stable surfactant with novel modes of administration for preterm babies, ability to diagnose fetal distress and novel tocolytic agents to delay or stop preterm labour. CONCLUSION: These findings will assist both donors and researchers in supporting and conducting research to close the knowledge gaps for reducing neonatal mortality, morbidity and long term impairment. WHO, SNL and other partners will work to generate interest among key national stakeholders, governments, NGOs, and research institutes in these priorities, while encouraging research funders to support them. We will track research funding, relevant requests for proposals and trial registers to monitor if the priorities identified by this exercise are being addressed

Setting research priorities to improve global newborn health and prevent stillbirths by 2025

Background In 2013, an estimated 2.8 million newborns died and 2.7 million were stillborn. A much greater number suffer from long term impairment associated with preterm birth, intrauterine growth restriction, congenital anomalies, and perinatal or infectious causes. With the approaching deadline for the achievement of the Millennium Development Goals (MDGs) in 2015, there was a need to set the new research priorities on newborns and stillbirth with a focus not only on survival but also on health, growth and development. We therefore carried out a systematic exercise to set newborn health research priorities for 2013-2025. Methods We used adapted Child Health and Nutrition Research Initiative (CHNRI) methods for this prioritization exercise. We identified and approached the 200 most productive researchers and 400 program experts, and 132 of them submitted research questions online. These were collated into a set of 205 research questions, sent for scoring to the 600 identified experts, and were assessed and scored by 91 experts. Results Nine out of top ten identified priorities were in the domain of research on improving delivery of known interventions, with simplified neonatal resuscitation program and clinical algorithms and improved skills of community health workers leading the list. The top 10 priorities in the domain of development were led by ideas on improved Kangaroo Mother Care at community level, how to improve the accuracy of diagnosis by community health workers, and perinatal audits. The 10 leading priorities for discovery research focused on stable surfactant with novel modes of administration for preterm babies, ability to diagnose fetal distress and novel tocolytic agents to delay or stop preterm labour. Conclusion These findings will assist both donors and researchers in supporting and conducting research to close the knowledge gaps for reducing neonatal mortality, morbidity and long term impairment. WHO, SNL and other partners will work to generate interest among key national stakeholders, governments, NGOs, and research institutes in these priorities, while encouraging research funders to support them. We will track research funding, relevant requests for proposals and trial registers to monitor if the priorities identified by this exercise are being addressed