Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.

Theory-driven formative research on on-site, shared sanitation quality improvement among landlords and tenants in peri-urban Lusaka, Zambia.

Rapid, unplanned urbanization in low-income countries is leading to increasing problems of dealing with human waste. On-site sanitation systems are often rudimentary, unhygienic, and poorly maintained. In-depth, on-site interactive interviews were conducted with 33 landlords and 33 tenants in a neighborhood in peri-urban Lusaka to understand on-site, shared sanitation quality improvement behaviors and preferences. Respondents were asked about housing characteristics, toilet histories, and financial decision-making. Improved, shared toilets were common (79%), but many were of low quality and poorly cleaned. Poor coordination among tenants, barriers to communication between landlords and tenants, and landlords viewing sanitation as a required basic service to provide instead of something for which tenants will pay more rent all limit the quality of sanitation in this setting. Landlord-directed interventions targeting non-health motivations for sanitation improvement and introducing effective cleaning systems may increase peri-urban sanitation quality.

Infection-avoidance behaviour in humans and other animals.

Compared with living free, the parasitic way of life has many attractions. Parasites create problems for all animals. Potential hosts can respond by learning to live with parasites (tolerance), actively fighting them (resistance), or they can avoid becoming infected in the first place (avoidance). I propose here a new classification of avoidance behaviour according to the epidemiology of infection risk, where animals must avoid (i) conspecifics, (ii) parasites and their vectors, (iii) parasite-rich environments, and (iv) niche infestation. I further explore how the disgust adaptive system, which coordinates avoidance behaviour, may form a continuum with the immune system through the sharing of signalling pathways, sites of action, and evolutionary history.

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.

Theory-based formative research on oral rehydration salts and zinc use in Lusaka, Zambia.

BACKGROUND: A theoretically grounded formative research study was carried out to investigate behaviour related to the use of Oral Rehydration Salts (ORS) and zinc tablets. The purpose was to inform the design of the behaviour change component of the Programme for Awareness and Elimination of Diarrhoea in Lusaka Province, Zambia, which aims to reduce childhood morbidity and mortality from diarrhoeal disease. METHODS: Fourteen behaviour trials were conducted among caregivers of children under-five with diarrhoea. Caregivers were recruited from two clinics situated in rural and peri-urban Lusaka. Trials took ten days and data were captured using video, observation and repeated interviews. Additional data were collected through focus group discussions with mothers, observations in clinics and pharmacies and interviews with clinic and pharmacy staff. Findings were organised according to categories of behavioural determinants from Evo-Eco theory. RESULTS: Participants were all familiar with ORS and most knew its purpose. ORS use was motivated by symptoms of dehydration, rather than the start of a diarrhoea episode, and was stopped when the child had visibly recovered energy. Only four of 14 behaviour trial participants were observed to correctly prepare ORS. Errors were mainly associated with measurement, resulting in a solution that was too concentrated. ORS was not observed to be given to children at clinics. Although zinc was unknown in this population, it was positively received by mothers keen to learn whether zinc would work better than alternative treatments to stop diarrhoea. CONCLUSIONS: ORS was sub-optimally prepared and used at home. It was not used while waiting to be seen at a clinic. In homes, the behaviour change intervention should promote early and continued use of correctly prepared ORS. In the longer-term, these behaviours may best be encouraged by changing the product design or sachet size. Despite its unfamiliarity, this population was well disposed to the use of zinc as a treatment for diarrhoea; when zinc is new to a population, promoting zinc as a solution to stopping diarrhoea, which mothers seek, may drive initial trial. Ensuring the availability of zinc in public clinics and private pharmacies prior to commencement of any promotion activities is crucial.

Design of an Intervention to Minimize Ingestion of Fecal Microbes by Young Children in Rural Zimbabwe.

We sought to develop a water, sanitation, and hygiene (WASH) intervention to minimize fecal-oral transmission among children aged 0-18 months in the Sanitation Hygiene Infant Nutrition Efficacy (SHINE) trial. We undertook 4 phases of formative research, comprising in-depth interviews, focus group discussions, behavior trials, and a combination of observations and microbiological sampling methods. The resulting WASH intervention comprises material inputs and behavior change communication to promote stool disposal, handwashing with soap, water treatment, protected exploratory play, and hygienic infant feeding. Nurture and disgust were found to be key motivators, and are used as emotional triggers. The concept of a safe play space for young children was particularly novel, and families were eager to implement this after learning about the risks of unprotected exploratory play. An iterative process of formative research was essential to create a sequenced and integrated longitudinal intervention for a SHINE household as it expects (during pregnancy) and then cares for a new child.

HIF-dependent regulation of AKAP12 (gravin) in the control of human vascular endothelial function.

Hypoxia has been widely implicated in many pathological conditions, including those associated with inflammation and tumorigenesis. A number of recent studies have implicated hypoxia in the control of vasculogenesis and permeability, the basis for which is not fully understood. Here we examine the transcriptional regulation of angiogenesis and permeability by hypoxia in endothelial cells. Guided by a global profiling approach in cultured endothelial cells, these studies revealed the selective induction of human gravin (protein kinase A anchoring protein 12) by hypoxia. Analysis of the cloned gravin promoter identified a functional hypoxia-responsive region including 2 binding sites for hypoxia-inducible factor (HIF). Site-directed mutagenesis identified the most distal HIF-binding site as essential for the induction of gravin by hypoxia. Further studies examining gravin gain and loss of function confirmed strong dependence of gravin in control of microvascular endothelial tube formation, wherein gravin functions as a "braking" system for angiogenesis. Additional studies in confluent endothelia revealed that gravin functionally couples to control endothelial barrier function in response to protein kinase A (PKA) agonists. Taken together, these results demonstrate transcriptional coordination of gravin by HIF-1α and amplified PKA-dependent endothelial responses. These findings provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.

Hygiene and health: systematic review of handwashing practices worldwide and update of health effects.

OBJECTIVE: To estimate the global prevalence of handwashing with soap and derive a pooled estimate of the effect of hygiene on diarrhoeal diseases, based on a systematic search of the literature. METHODS: Studies with data on observed rates of handwashing with soap published between 1990 and August 2013 were identified from a systematic search of PubMed, Embase and ISI Web of Knowledge. A separate search was conducted for studies on the effect of hygiene on diarrhoeal disease that included randomised controlled trials, quasi-randomised trials with control group, observational studies using matching techniques and observational studies with a control group where the intervention was well defined. The search used Cochrane Library, Global Health, BIOSIS, PubMed, and Embase databases supplemented with reference lists from previously published systematic reviews to identify studies published between 1970 and August 2013. Results were combined using multilevel modelling for handwashing prevalence and meta-regression for risk estimates. RESULTS: From the 42 studies reporting handwashing prevalence we estimate that approximately 19% of the world population washes hands with soap after contact with excreta (i.e. use of a sanitation facility or contact with children's excreta). Meta-regression of risk estimates suggests that handwashing reduces the risk of diarrhoeal disease by 40% (risk ratio 0.60, 95% CI 0.53-0.68); however, when we included an adjustment for unblinded studies, the effect estimate was reduced to 23% (risk ratio 0.77, 95% CI 0.32-1.86). CONCLUSIONS: Our results show that handwashing after contact with excreta is poorly practiced globally, despite the likely positive health benefits.

Assessing the impact of drinking water and sanitation on diarrhoeal disease in low- and middle-income settings: systematic review and meta-regression.

OBJECTIVE: To assess the impact of inadequate water and sanitation on diarrhoeal disease in low- and middle-income settings. METHODS: The search strategy used Cochrane Library, MEDLINE & PubMed, Global Health, Embase and BIOSIS supplemented by screening of reference lists from previously published systematic reviews, to identify studies reporting on interventions examining the effect of drinking water and sanitation improvements in low- and middle-income settings published between 1970 and May 2013. Studies including randomised controlled trials, quasi-randomised trials with control group, observational studies using matching techniques and observational studies with a control group where the intervention was well defined were eligible. Risk of bias was assessed using a modified Ottawa-Newcastle scale. Study results were combined using meta-analysis and meta-regression to derive overall and intervention-specific risk estimates. RESULTS: Of 6819 records identified for drinking water, 61 studies met the inclusion criteria, and of 12,515 records identified for sanitation, 11 studies were included. Overall, improvements in drinking water and sanitation were associated with decreased risks of diarrhoea. Specific improvements, such as the use of water filters, provision of high-quality piped water and sewer connections, were associated with greater reductions in diarrhoea compared with other interventions. CONCLUSIONS: The results show that inadequate water and sanitation are associated with considerable risks of diarrhoeal disease and that there are notable differences in illness reduction according to the type of improved water and sanitation implemented.

Burden of disease from inadequate water, sanitation and hygiene in low- and middle-income settings: a retrospective analysis of data from 145 countries.

OBJECTIVE: To estimate the burden of diarrhoeal diseases from exposure to inadequate water, sanitation and hand hygiene in low- and middle-income settings and provide an overview of the impact on other diseases. METHODS: For estimating the impact of water, sanitation and hygiene on diarrhoea, we selected exposure levels with both sufficient global exposure data and a matching exposure-risk relationship. Global exposure data were estimated for the year 2012, and risk estimates were taken from the most recent systematic analyses. We estimated attributable deaths and disability-adjusted life years (DALYs) by country, age and sex for inadequate water, sanitation and hand hygiene separately, and as a cluster of risk factors. Uncertainty estimates were computed on the basis of uncertainty surrounding exposure estimates and relative risks. RESULTS: In 2012, 502,000 diarrhoea deaths were estimated to be caused by inadequate drinking water and 280,000 deaths by inadequate sanitation. The most likely estimate of disease burden from inadequate hand hygiene amounts to 297,000 deaths. In total, 842,000 diarrhoea deaths are estimated to be caused by this cluster of risk factors, which amounts to 1.5% of the total disease burden and 58% of diarrhoeal diseases. In children under 5 years old, 361,000 deaths could be prevented, representing 5.5% of deaths in that age group. CONCLUSIONS: This estimate confirms the importance of improving water and sanitation in low- and middle-income settings for the prevention of diarrhoeal disease burden. It also underscores the need for better data on exposure and risk reductions that can be achieved with provision of reliable piped water, community sewage with treatment and hand hygiene.

Unintentional behaviour change.

We argue that the authors ignore a broad range of possible means of changing behaviour: unintentional change. Most of the behaviours that people seek to change - either in themselves or that are the subject of public health campaigns-are habitual, and hence not necessarily responsive to intentions. An evolutionary approach should take into account all kinds of evolved behavioural responses.

Hygiene intervention reduces contamination of weaning food in Bangladesh.

OBJECTIVE: This study was conducted to measure the impact of a hygiene intervention on the contamination of weaning food in Bangladesh. METHODS: Sixty households were selected: 30 study and 30 control households. Samples of weaning food were collected from all the 60 households at baseline and examined for faecal coliforms (FC), faecal streptococci (FS) and Clostridium perfringens (CP) following standard procedures. After cooking, food samples were collected on three occasions before feeding. Following Hazard Analysis Critical Control Point (HACCP) procedures, critical control points were determined. The mothers in the 30 study households were then trained for 4 weeks in how to attain the control point conditions. Then, again the food samples were collected and analysed. RESULTS: At baseline, weaning foods from study and control households were heavily contaminated with FC and FS. The FC and FS counts were 1.84 log(10) and 1.92 log(10) colony-forming unit (cfu)/g, respectively, in the study households, and 0.86 log(10) and 1.33 log(10)  cfu/g, respectively, in the control households in the first feeding. After the intervention, the FC and FS counts in study households had dropped to 0.10 log(10) and 0.09 log(10)  cfu/g, respectively, a statistically significant reduction (P < 0.001). Monitoring the sustainability of the behaviour change after 3 months showed that the mothers were maintaining food hygiene. CONCLUSIONS: A hygiene intervention following the HACCP approach reduced the weaning food contamination significantly. Awareness building among mothers about weaning food hygiene could be an important intervention for preventing weaning food-related diarrhoea in Bangladesh.

Behaviour change for better health: nutrition, hygiene and sustainability.

As the global population grows there is a clear challenge to address the needs of consumers, without depleting natural resources and whilst helping to improve nutrition and hygiene to reduce the growth of noncommunicable diseases. For fast-moving consumer goods companies, like Unilever, this challenge provides a clear opportunity to reshape its business to a model that decouples growth from a negative impact on natural resources and health. However, this change in the business model also requires a change in consumer behaviour. In acknowledgement of this challenge Unilever organised a symposium entitled 'Behaviour Change for Better Health: Nutrition, Hygiene and Sustainability'. The intention was to discuss how consumers can be motivated to live a more healthy and sustainable lifestlye in today's environment. This article summarises the main conclusions of the presentations given at the symposium. Three main topics were discussed. In the first session, key experts discussed how demographic changes - particularly in developing and emerging countries - imply the need for consumer behaviour change. The second session focused on the use of behaviour change theory to design, implement and evaluate interventions, and the potential role of (new or reformulated) products as agents of change. In the final session, key issues were discussed regarding the use of collaborations to increase the impact and reach, and to decrease the costs, of interventions. The symposium highlighted a number of key scientific challenges for Unilever and other parties that have set nutrition, hygiene and sustainability as key priorities. The key challenges include: adapting behaviour change approaches to cultures in developing and emerging economies; designing evidence-based behaviour change interventions, in which products can play a key role as agents of change; and scaling up behaviour change activities in cost-effective ways, which requires a new mindset involving public-private partnerships.

Male commuters in north and south England: risk factors for the presence of faecal bacteria on hands.

BACKGROUND: A previous study found that the prevalence of contamination with bacteria of faecal-origin on the hands of men differed across UK cities, with a general trend of increased contamination in northern cities. The aim of this study was to (1) confirm the north-south trend (2) identify causes for the trend. METHODS: Hand swabs from commuters (n = 308) at train stations in 4 cities were tested for the presence of faecal bacteria. RESULTS: The prevalence of hand contamination with faecal bacteria was again higher in cities in the north compared to the south (5% in London, 4% in Birmingham, 10% in Liverpool and 19% in Newcastle). Contamination risk decreased with age and better personal hygiene (self-reported). Soil contact and shaking hands increased contamination with faecal bacteria. However, in multivariable analysis, none of these factors fully explained the variation in contamination across cities. CONCLUSION: The study confirmed the north-south differences in faecal contamination of hands without finding a clear cause for the trend. Faecal contamination of hands was associated with personal hygiene indicators suggesting that microbiological testing may contribute to evaluating hygiene promotion campaigns.

Food Hygiene Practices of Rural Women and Microbial Risk for Children: Formative Research in Nepal.

Formative research was conducted in a rural hill setting in Nepal during April-June 2012 to inform the design of an intervention to promote safe food hygiene practices. A variety of methods underpinned by Behavior Centered Design theory and Hazard Analysis Critical Control Points was used to pinpoint key risk behaviors and their environmental and psychological determinants in 68 households with a mother having a child aged 6-59 months. These included video recordings, observation of daily routine, teach-the-researcher sessions, in-depth interviews, observations of actual behaviors, focus group discussions, motive mapping, microbiological assessment, and identification of critical control points. Physical settings, especially the kitchen, form a challenging environment for mothers, including family members in rural hill settings of Nepal to practice adequate food hygiene behaviors. Prevalent food hygiene practices of mothers were inadequate, leading to frequent exposure of young children to highly contaminated food, water, and milk. We identified six critical control points; of these, five needed improving. Determinants of these behaviors included physical and social environment as well as psychological brief and individual motives. Five key food hygiene risk behaviors are suggested for prioritization. While designing a food hygiene intervention package, consideration should be given to the physical, biological, and social environment, and immediate motives behind each practice should be taken into consideration while framing key messages. Creative and engaging activities should be designed around the motives of nurture, disgust, affiliation, and social status/respect.

Evaluation of a behavior-centered design strategy for creating demand for oral PrEP among young women in Cape Town, South Africa.

BACKGROUND: There is an urgent need to find effective interventions that reduce young South African women's vulnerability to HIV, and pre-exposure prophylaxis (PrEP) is highly effective when taken consistently. As national programs in Africa launch PrEP programs for young women, it is critical to understand how to effectively create awareness, stimulate interest, and increase uptake of PrEP. METHODS: Behavior-centered design (BCD) guided the development of a PrEP social marketing campaign for young women. Ethnographic observations, in-depth interviews, and focus-group discussions with young South African women informed the content and design of a 90-second PrEP demand creation video and two informational brochures. A short survey was administered to young women at their homes after watching a video to evaluate PrEP interest. Of 800 households with a 16-25-year-old female identified from a Cape Town township census, 320 women in these households viewed the video and completed a survey about the video and their interest in PrEP. RESULTS: In focus groups, young women from the township preferred local characters and messaging that was empowering, simple, and motivational. From the household survey of young women who viewed the video, most reported interest in learning more about PrEP (67.7% 'definitely interested' and 9.4% 'somewhat interested') and taking PrEP (56.4% 'definitely interested' and 12.5% 'somewhat interested'). Factors significantly associated with interest in taking PrEP were having a primary partner with whom they regularly have sex (80.0% vs. 65.2% without a primary partner; adjusted odds ratio (AOR)=3.1, 95% CI: 1.3, 7.0) and being in a sexual partnership for <6 months (86.8% vs. 68.5% for >12 months; AOR=3.0, 95% CI: 1.2, 7.3). CONCLUSIONS: A positively framed PrEP demand creation video generated high interest in PrEP among young South African women, particularly among women with a primary partner and a shorter-term relationship. Registration: NCT03142256; registered on 5 May 2017.

Adaptation to inflammatory acidity through neutrophil-derived adenosine regulation of SLC26A3.

Acute intestinal inflammation includes the early accumulation of neutrophils (PMN). Based on recent evidence that PMN infiltration "imprints" changes in the local tissue environment through local oxygen depletion and the release of adenine nucleotides, we hypothesized that the interaction between transmigrating PMN and intestinal epithelial cells (IECs) results in inflammatory acidification of the tissue. Using newly developed tools, we revealed that active PMN transepithelial migration (TEM) significantly acidifies the local microenvironment, a decrease of nearly 2 pH units. Using unbiased approaches, we sought to define acid-adaptive pathways elicited by PMN TEM. Given the significant amount of adenosine (Ado) generated during PMN TEM, we profiled the influence of Ado on IECs gene expression by microarray and identified the induction of SLC26A3, the major apical Cl-/HCO3- exchanger in IECs. Utilizing loss- and gain-of-function approaches, as well as murine and human colonoids, we demonstrate that Ado-induced SLC26A3 promotes an adaptive IECs phenotype that buffers local pH during active inflammation. Extending these studies, chronic murine colitis models were used to demonstrate that SLC26A3 expression rebounds during chronic DSS-induced inflammation. In conclusion, Ado signaling during PMN TEM induces an adaptive tissue response to inflammatory acidification through the induction of SLC26A3 expression, thereby promoting pH homeostasis.

Neutrophils as sources of dinucleotide polyphosphates and metabolism by epithelial ENPP1 to influence barrier function via adenosine signaling.

Extracellular adenosine signaling is established as a protective component in mucosal inflammatory responses. The sources of extracellular adenosine include enzymatic processing from nucleotides, such as ATP and AMP, that can be liberated from a variety of cell types, including infiltrating leukocytes. Here we demonstrate that activated human neutrophils are a source of diadenosine triphosphate (Ap3A), providing an additional source of nucleotides during inflammation. Profiling murine enteroids and intestinal epithelial cell lines revealed that intestinal epithelia prominently express apical and lateral ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1), a member of the ENPP family of enzymes that metabolize diadenosine phosphates, especially Ap3A. Extensions of these studies demonstrated that intestinal epithelia metabolize Ap3A to ADP and AMP, which are further metabolized to adenosine and made available to activate surface adenosine receptors. Using loss and gain of ENPP1 approaches, we revealed that ENPP1 coordinates epithelial barrier formation and promotes epithelial wound healing responses. These studies demonstrate the cooperative metabolism between Ap3A and ENPP1 function to provide a significant source of adenosine, subserving its role in inflammatory resolution.