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Abstract Background The impact of periodontitis on large populations of people living with HIV (PLHIV) in resource-constrained settings remains largely un-investigated. This study aims to address this knowledge gap by providing a comprehensive description of the periodontal health status among a sizable cohort of Ugandans living with HIV. Methods This was a cross-sectional study with 4,449 participants who were over 18-years old with data captured on their reported age, gender, tobacco use, length of time on HAART and alcohol use. Periodontal health was assessed using the WHO periodontal probe and the modified CPI data entry form. Descriptive statistics were reported using frequencies for the affected number of sextants in the surveyed participants. This was followed by additional regression analysis using the R statistical computing environment, with the periodontal health outcomes (bleeding on probing, pocket depth and clinical attachment loss) individually as the dependent, recorded as binary outcomes. A multilevel model was run with clinical attachment loss as the dependant variable controlling for all the other factors. The 95% confidence intervals were used to report the level of significance for each test. Results There were 3,103/4,449 (69.7%) female participants. The mean age was 44.3 years (SD 10.1 years) with a range of 18 to 89 years. About 66% of the participants had bleeding on probing at one or more of the examined sites/tooth surfaces. The odds for bleeding on probing were significantly higher for female participants (adjusted Odds ratio: 1.49, 95% CI 1.19 to 1.86), and higher in individuals who reported tobacco use (adjusted odds ratio 1.62, 95% CI 1.09 to 2.41). Slightly under half of our participants (48.2%) had moderate to severe clinical attachment loss. Conclusions This study found that among Ugandans living with HIV, periodontal disease is a significant public health concern. The majority of study participants had bleeding on probing and almost half of them recording moderate to severe clinical attachment loss, worsened by age and time on HAART. This highlights the need for comprehensive oral health care and targeted interventions for this population.

BackgroundDental caries is a multifactorial disease that affects many people. Even though microorganisms play a crucial role in causing dental caries, diagnosis is routinely macroscopic. In order to improve early detection especially in HIV patients who are disproportionately affected, there is need to reconcile the macroscopic and microscopic characteristics of dental caries. Therefore, the aim of this study was to characterize the oral microbiota profile along the decayed, missing, filled teeth (DMFT) index using amplicon sequencing data.MethodsAmplicon sequencing of the V6-V8 region of the 16S rRNA gene was done on DNA recovered from whole unstimulated saliva of 59 HIV positive and 29 HIV negative individuals. The microbial structure, composition and co-occurrence networks were characterized using QIIME-2, Phyloseq, Microbiome-1.9.2 and Metacoder in R.ResultsWe characterized the oral microbiota into 2,093 operational taxonomic units (OTUs), 21 phyla and 239 genera from 2.6 million high quality sequence reads. While oral microbiota did not cluster participants into distinct groups that track with the DMFT index, we observed the following: (a) The proportion of accessory microbiota was highest in the high DMFT category while the core size (∼50% of richness) remained relatively stable across all categories. (b) The abundance of core genera such as Stomatobaculum, Peptostreptococcus and Campylobacter was high at onset of dental caries, (c) A general difference in oral microbial biomass. (d) The onset of dental caries (low DMFT) was associated with significantly lower oral microbial entropy.ConclusionsAlthough oral microbial shifts along the DMFT index were not distinct, we demonstrated the potential utility of microbiota dynamics to characterize oral disease. Therefore, we propose a microbial framework using the DMFT index to better understand dental caries among HIV positive people in resource limited settings.

Abstract Background Chronic Human Immunodeficiency Virus (HIV) infection is associated with reduced saliva flow rate due to infiltration of HIV and proliferation of CD8 lymphocytes in salivary glands. It is unclear whether HIV infection and antiretroviral therapy (ART) increase caries risk. This study aimed to determine the prevalence and factors associated with dental caries in HIV infected adults attending the Mulago Immune Suppression Syndrome (ISS) clinic in Uganda. Methods A cross-sectional study was conducted among HIV infected persons. Dental examinations were performed by two calibrated dentists using the WHO Radke’s caries classification criteria and reported using the decayed (D), missing (M), filled (F), teeth (DMFT) index. The prevalence and factors associated with dental caries was examined through linear regression analyses. Results Overall, 748 participants (females = 491, 65.6%) with a mean age of 39 ± 9.4 years were included in the final analysis; of whom 83.7% had caries (DMFT> 0), with a significantly (p 2 years but 5 years 6.6 ± 6.0 p

Abstract Background The estimated number of people living with human immunodeficiency virus (HIV) (PLHIV) in Uganda is 1.5 million (7.3%). As of June 2016, 60% (898,197) of PLHIV were enrolled and receiving antiretroviral therapy (ART). In scientific literature, the effect of HIV and ART on dental caries remains equivocal. At the Prosthetics Clinic of the Department of Dentistry, Makerere University College of Health Sciences, we have seen a number of PLHIV who require replacement of missing teeth with partial or complete dentures as a result of extensive caries. Here we report a case of an HIV positive female patient with extensive dental caries resulting in complete edentulous jaws, associated with psychological stress and stigmatization. Case presentation A 52-year-old patient, HIV positive for fourteen (14) years and receiving antiretroviral therapy (ART) for the last four years wanted to replace her missing teeth for effective feeding and cosmetic reasons. A diagnosis of partially edentulous maxillary and mandibular arches, cervical caries of tooth # 12, 15, 25, 34 and retained roots of tooth # 11, 13, 22 and 35 was made. Following oral health education and mouth preparation, this patient received a set of removable acrylic full upper and lower dentures. Conclusion This case may represent the long-term effects of HIV and ART on oral health status especially tooth surfaces in some PLHIV. Further evaluation is required to ascertain if this was an isolated case or it is a common finding among HIV positive adult patients receiving long-term ART in sub-Saharan Africa. Information emerging from these studies would establish the magnitude of dental caries among PLHIV and guide the development of appropriate oral health care guidelines in the management of people living with HIV.

Objective: The use of fasting plasma glucose (FPG) level > or =7.0 mmol/l leads to underdiagnosis of type 2 diabetes compared with the oral glucose tolerance test (OGTT). The OGTT is of limited use for population screening. Most of the increase in cardiovascular risk in relation to increasing blood glucose occurs before the threshold at which the diagnosis of type 2 diabetes is made. The aim of this study was to evaluate the use of HbA(1c) and FPG as predictors of type 2 diabetes and cardiovascular risk and, accordingly, to develop a rational approach to screening for abnormalities of glucose tolerance.Research Design and Methods: OGTT and measurement of HbA(1c) and FPG levels were performed in 505 subjects screened for type 2 diabetes. Anthropomorphic measurements were obtained. A cardiovascular risk factor questionnaire was completed.Results: The subjects were aged 19-88 years (mean 53.8). The incidence of type 2 diabetes was 10.4% based on the OGTT and 4% based on an FPG level > or =7.0 mmol/l. Using high-performance liquid chromatography (HPLC), HbA(1c) of <4.7 and > or =6.2% predicted with certainty the absence or presence of type 2 diabetes as defined by the OGTT. The corresponding cutoffs were <5.0 and > or =6.8% for HbA(1c) (DCA2000 HPLC device; Bayer Diagnostics, Mulgrave, Australia) and <4.7 and > or =6.4 mmol/l for FPG. However, 75-85% of subjects in each case had intermediate values, which were therefore nondiagnostic. Cardiovascular risk increased at least 2.2 times at an HbA(1c) level > or =6.2% (by HPLC), 1.8-2.2 times at an HbA(1c) level of 5.6-6.1% (by HPLC), 2 times at an FPG level > or =6.4 mmol/l, and 1.7-1.9 times at an FPG level of 5.6-6.3 mmol/l.Conclusions: Measurement of FPG and HbA(1c) levels will diagnose or exclude type 2 diabetes with certainty in a minority (15%) of people. There is a continuous relationship between FPG and HbA(1c) and cardiovascular risk. Accordingly, we propose that there is a rational basis for using either FPG and HbA(1c) for purposes of screening and assigning risk. Individuals with an HbA(1c) level of 5.6-6.1% and an FPG level of 5.6-6.3 mmol/l are at greatest risk for cardiovascular disease and should be targeted for further evaluation. An algorithm outlining a cost-effective approach is presented. [ABSTRACT FROM AUTHOR]

Purpose: Health promotion and prevention interventions to support children's early language and literacy development are essential, however little is known about how social media can be used in this context. This study evaluated an intervention which aimed to build capacity of parents in supporting children's language and literacy skills, implemented using Facebook., Method: A cross-sector partnership of Australian speech-language pathologists developed a suite of universal messages for parents to support their child's language development. Messages were delivered across 12 weeks via Facebook. A convergent mixed methods study design was used to evaluate survey responses from parents of children aged 0-5 years, before ( n  = 136) and after the intervention ( n  = 61). Social media analytics were collected., Result: Participants identified social media was a helpful way to receive information about supporting their child's language and literacy development, reporting an increase in knowledge following the intervention. Participants reported they trusted information provided in Facebook posts as it was from professionals and evidence-based sources. The usability (i.e. look and layout) was generally rated highly., Conclusion: Social media is a valuable tool for speech-language pathologists to deliver universal messages. Future social media initiatives should be co-designed with families and social media professionals to ensure delivery of trusted, consistent, and user-friendly information.

Background: Evidence supports the effectiveness of shared book reading for promoting language and literacy development, but it is known that families experiencing vulnerability may have reduced access to books and are less likely to share books regularly at home. Community hubs often provide support to families experiencing vulnerability and may provide an opportunity to create environments that support families to engage in shared book reading, especially if families are invited into the creation of these environments through co-design. However, there is currently little evidence regarding the impact of co-designing shared book reading environments with families in community settings., Aims: The current study is part of a broader project which used co-design to develop a shared book reading environment in collaboration with stakeholders in a community hub. This small-scale study aimed to provide a preliminary evaluation of the impact of this co-designed shared book reading environment at the community hub on (1) the frequency of shared book reading at the hub; (2) participants' confidence and enjoyment of reading with children at the hub; and (3) participants' experiences of shared book reading at the hub., Method & Procedures: The co-design project was carried out in four phases with families, staff and community partners at the community hub. A convergent mixed-methods approach was used to collect and analyse quantitative and qualitative data in the first and last phase of the project to evaluate project outcomes. Quantitative data (environmental observations of reading at the hub and participant ratings of confidence and enjoyment of reading with children at the hub) were analysed using descriptive statistics and nonparametric statistical tests. Qualitative data (participants' responses regarding their experiences of changes implemented in the community hub) were analysed using inductive content analysis., Outcome & Results: An increased frequency of shared book reading was observed within the community hub after changes were implemented, and positive changes in enjoyment of book reading were recorded. Qualitative responses from participants following conclusion of the project were organised into three main categories, which showed that (1) children and families were reading more and enjoying reading activities that were happening; (2) children and families were borrowing more books; and (3) families had changed the way they were reading at home with their children., Conclusions & Implications: A co-designed shared book reading environment within a community hub had a positive impact on shared book reading experiences for families in areas with high levels of vulnerability., What This Paper Adds: What is already known on this subject Community hubs provide a unique opportunity to support early language and literacy development in partnership with families in areas of vulnerability; however, there is limited literature investigating the impacts of these supports. A previous study utilised co-design to develop a shared book reading environment with stakeholders at a community hub in an area where a high proportion of children are considered to be 'vulnerable' in terms of language and literacy development. It was found that co-design enabled the development of collaborative changes to support shared book reading that were valued and owned by families, staff and community partners at the community hub. What this study adds The current study focused on evaluating the impact of a co-design project on the frequency of shared book reading at the hub and participants' experiences, confidence and enjoyment of reading with children at the hub. Analysis of pre- and post-data provide preliminary evidence that a co-designed shared book reading environment in a community hub can have a positive impact on increasing families' awareness and engagement with book borrowing, engagement in shared book reading activities in the community and at home, and participants' confidence and enjoyment of reading with children. What are the clinical implications of this work? Speech pathologists have an important role to play in prevention and promotion. This study has shown that collaboration with families experiencing vulnerability can support the implementation of strategies to promote shared reading in a community hub and demonstrate increased quality and frequency of shared book reading. The utilisation of a community hub also highlights the opportunities for the speech pathology profession to deliver community-based promotion and prevention initiatives as a strategy to address equitable language outcomes., (© 2024 Royal College of Speech and Language Therapists.)

Background: The impact of periodontitis on large populations of people living with HIV (PLHIV) in resource-constrained settings remains largely un-investigated. This study aims to address this knowledge gap by providing a comprehensive description of the periodontal health status among a sizable cohort of Ugandans living with HIV., Methods: This was a cross-sectional study with 4,449 participants who were over 18-years old and captured their reported age, gender, tobacco use, length of time on HAART and alcohol use. Periodontal health was assessed using the WHO periodontal probe and the modified CPI data entry form. Descriptive statistics were reported using frequencies for the affected number of sextants in the surveyed participants. This was followed by additional regression analysis using the R statistical computing environment, with the periodontal health outcomes (bleeding on probing, pocket depth and clinical attachment loss) individually as the dependant, recoded as binary outcomes. A multilevel model was run with clinical attachment loss as the dependant variable controlling for all the other factors. The 95% confidence intervals were used to report the level of significance for each test., Results: There were 3,103/4,449 (69.7%) female participants. The mean age was 44.3 years (SD 10.1 years) with a range of 18 to 89 years. About 66% of the participants had bleeding on probing at one or more of the examined sites/tooth surfaces. The odds for bleeding on probing were significantly higher for female participants (adjusted Odds ratio: 1.49, 95% CI 1.19 to 1.86), and higher in individuals who reported tobacco use (adjusted odds ratio 1.62, 95% CI 1.09 to 2.41). Slightly under half of our participants (48.2%) had moderate to severe clinical attachment loss., Conclusions: This study found that among Ugandans living with HIV, periodontal disease is a significant public health concern. The majority (66%) had bleeding on probing, with a sizeable number (48.2%) of participants recording moderate to severe clinical attachment loss, worsened by age and time on HAART. This highlight the need for comprehensive oral health care and targeted interventions for this population., Competing Interests: Competing interests None to declare

This study investigated the effects of Lacticaseibacillus rhamnosus HN001 supplementation on the architecture and gene expression in small intestinal tissues of piglets used as an animal model for infant humans. Twenty-four 10-d-old entire male piglets (4·3 (sd 0·59) kg body weight) were fed an infant formula (IF) (control) or IF supplemented with 1·3 × 10 5 (low dose) or 7·9 × 10 6 (high dose) colony-forming units HN001 per ml of reconstituted formula ( n 8 piglets/treatment). After 24 d, piglets were euthanised. Samples were collected to analyse the histology and gene expression (RNAseq and qPCR) in the jejunal and ileal tissues, blood cytokine concentrations, and blood and faecal calprotectin concentrations. HN001 consumption altered (false discovery rate < 0·05) gene expression (RNAseq) in jejunal tissues but not in ileal tissues. The number of ileal goblet cells and crypt surface area increased quadratically ( P < 0·05) as dietary HN001 levels increased, but no increase was observed in the jejunal tissues. Similarly, blood plasma concentrations of IL-10 and calprotectin increased linearly ( P < 0·05) as dietary HN001 levels increased. In conclusion, supplementation of IF with HN001 affected the architecture and gene expression of small intestine tissue, blood cytokine concentration and frequencies, and blood calprotectin concentrations, indicating that HN001 modulated small intestinal tissue maturation and immunity in the piglet model.

Issue Addressed: Queensland children have a higher level of developmental vulnerability compared to the Australian average. This paper reports on Healthy Kids-a capacity building strategy for the early childhood education and care (ECEC) sector targeting communities experiencing socio-economic and child development vulnerabilities. These communities may face additional barriers when engaging and participating in health promotion models. This paper reports on the development, key components and principles of a capacity building model referred to as Healthy Kids, that strategically responds to these barriers and supports these communities., Methods: The development of the Healthy Kids model emerged through a quality improvement process that included an environmental scan, and review of existing capacity building, health promotion, and workforce development approaches. It also involved consultation and engagement with the ECEC sector., Results: Evidence indicates Healthy Kids to be an innovative health promotion model focussed on building capacity through a workforce development strategy for the ECEC sector in a way that is accessible, low cost, and sustainable. SO WHAT?: This paper shares a model for building capacity through the establishment of localised cross-sector communities of practice across a large geographic region with a centralised coordinating hub. The hub and spoke model has facilitated community ownership to grow and be sustained over time. This model offers opportunities for partnerships, transferability, and contextualisation for those interested in contemporary health promotion, capacity building, and workforce development. The model offers an approach for those willing to step outside traditional boundaries to work across sectors and settings to achieve sustainable knowledge and skills, processes and resources that enables a collective commitment to improving health outcomes., (© 2023 The Authors. Health Promotion Journal of Australia published by John Wiley & Sons Australia, Ltd on behalf of Australian Health Promotion Association.)

Purpose: Community hubs often provide support to families in areas of high vulnerability and can provide unique opportunities for delivering early literacy programs. This study used a co-design process to engage families, staff, and community partners within a community hub to design an environment that supported shared book reading., Method: Co-design was enacted in four phases: 1) interviews to understand user experiences relating to shared book reading; 2) focus groups to refine ideas into actions to support shared book reading and prioritise these actions; 3) implementation of changes; and 4) understanding of participants' experiences of involvement., Result: Participant identified changes were implemented within four categories: 1) changing how books are organised, 2) showing families how to share books, 3) giving families information about how books can be borrowed, and 4) running more activities about books. Participants indicated they enjoyed being a part of a co-design process to affect change at the community hub., Conclusion: Co-design enabled the development of collaborative changes to support book reading that were valued and owned by families, staff, and community partners. Community hubs can provide unique opportunities to engage with families in areas of vulnerability to support the development of early language and literacy skills.

The probiotic Lacticaseibacillus rhamnosus strain HN001 has been shown to have several beneficial health effects for both pediatric and maternal groups, including reduced risk of eczema in infants and gestational diabetes and postnatal depression in mothers. While L. rhamnosus HN001 appears to modify immune and gut barrier biomarkers, its mode of action remains to be fully elucidated. To gain insights into the role of HN001 on the infant microbiome, the impacts of L. rhamnosus HN001 supplementation was studied in 10-day old male piglets that were fed either infant formula, or infant formula with L. rhamnosus HN001 at a low (1.3 × 10 5 CFU/ml) or high dose (7.9 × 10 6 CFU/ml) daily for 24 days. The cecal and fecal microbial communities were assessed by shotgun metagenome sequencing and host gene expression in the cecum and colon tissue was assessed by RNA-seq. Piglet fecal samples showed only modest differences between controls and those receiving dietary L. rhamnosus HN001. However, striking differences between the three groups were observed for cecal samples. While total lactobacilli were significantly increased only in the high dose L. rhamnosus HN001 group, both high and low dose groups showed an up to twofold reduction across the Firmicutes phylum and up to fourfold increase in Prevotella compared to controls. Methanobrevibacter was also decreased in HN001 fed piglets. Microbial genes involved in carbohydrate and vitamin metabolism were among those that differed in relative abundance between those with and without L. rhamnosus HN001. Changes in the cecal microbiome were accompanied by increased expression of tight junction pathway genes and decreased autophagy pathway genes in the cecal tissue of piglets fed the higher dose of L. rhamnosus HN001. Our findings showed supplementation with L. rhamnosus HN001 caused substantial changes in the cecal microbiome with likely consequences for key microbial metabolic pathways. Host gene expression changes in the cecum support previous research showing L. rhamnosus HN001 beneficially impacts intestinal barrier function. We show that fecal samples may not adequately reflect microbiome composition higher in the gastrointestinal tract, with the implication that effects of probiotic consumption may be missed by examining only the fecal microbiome., Competing Interests: Authors NH and JD were former and employed by the Fonterra Co-operative Group Ltd., respectively. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Young, Maclean, Dunstan, Ryan, Peters, Armstrong, Anderson, Dewhurst, van Gendt, Dilger, Dekker, Haggarty and Roy.)

The milk fat globule membrane (MFGM) appears to play an important role in infant neurocognitive development; however, its mechanism(s) of action remains unclear. This study aimed to investigate the role of a dietary MFGM supplement on the lipid profiles of different neonatal brain regions. Ten-day-old male piglets (4−5 kg) were fed unsupplemented infant formula (control, n = 7) or an infant formula supplemented with low (4%) or high (8%) levels of MFGM (n = 8 each) daily for 21 days. Piglets were then euthanized, and brain tissues were sectioned. Untargeted liquid chromatography-mass spectrometry lipidomics was performed on the cerebellum, hippocampus, prefrontal cortex, and the rest of the brain. The analyses identified 271 and 171 lipids using positive and negative ionization modes, respectively, spanning 16 different lipid classes. MFGM consumption did not significantly alter the lipidome in most brain regions, regardless of dose, compared to the control infant formula. However, 16 triacylglyceride species were increased in the hippocampus (t-test, p-value < 0.05) of the high-supplemented piglets. Most lipids (262 (96.7%) and 160 (93.6%), respectively) differed significantly between different brain regions (ANOVA, false discovery rate corrected p-value < 0.05) independent of diet. Thus, this study highlighted that dietary MFGM altered lipid abundance in the hippocampus and detected large differences in lipid profiles between neonatal piglet brain regions.

Gastrointestinal conditions in which the transit of contents is altered may benefit from nutritional approaches to influencing health outcomes. Milk proteins modulate the transit of contents along different regions, suggesting that they have varying effects on neuromuscular function to alter gastrointestinal motility. We tested the hypothesis that bovine whey and casein milk protein hydrolysates could have direct modulatory effects on colonic motility patterns in isolated rat large intestine. Casein protein hydrolysate (CPH), whey protein concentrate (WPC), whey protein hydrolysate (WPH), and a milk protein hydrolysate (MPH; a hydrolyzed blend of 60% whey to 40% casein) were compared for their effects on spontaneous contractile waves. These contractions propagate along the length of the isolated intact large intestine (22 cm) between the proximal colon and rectum and were detected by measuring activity at 4 locations. Milk proteins were perfused through the tissue bath, and differences in contraction amplitude and frequency were quantified relative to pretreatment controls. Propagation frequency was decreased by CPH, increased by MPH, and unaffected by intact whey proteins. The reduced motility with CPH and increased motility with MPH indicate a direct action of these milk proteins on colon tissue and provide evidence for differential modulation by hydrolysate type. These findings mirror actions on lower gastrointestinal transit reported in vivo, with the exception of WPH, suggesting that other factors are required., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objective: Prognostication is important in clinical decision-making, especially for older people. The aim was to present estimates of life expectancy for older people in New Zealand., Methods: Statistics New Zealand age-sex-specific death rates were used to derive quartiles of expected years of life remaining in people aged over 65 years., Results: Given current patterns and trends in New Zealand death rates, 50% of women reaching age 80 years in 2016 can expect to live at least another 10.5 years, 25% will live over 14.7 years, and 25% will die within 6.2 years. Comparable results for men reaching age 80 years in 2016 are 8.5 years, 12.7 years and 4.6 years, respectively. Of those reaching age 90 years in 2016, median expected years of life left is 4.2 years for women and 3.4 years for men., Conclusion: Demographic norms are useful as a guide when specific predictive tools are unavailable., (© 2016 AJA Inc.)

Beta-casomorphin-5 (βCM-5) is a milk-derived bioactive peptide that slows gastro-intestinal transit (GIT) in vivo and blocks the peristaltic reflex in the guinea pig colon in vitro. We wanted to establish an in vitro model system in which effects of dairy-derived substances containing opioid peptides on intestinal motility can be assessed and used to predict in vivo outcomes. Because βCM-5 is an opioid agonist that acts on enteric neurons, we used this substance to compare two different isolated colonic tissue preparations to determine which would more closely mimic the in vivo response previously reported in the literature. We compared and characterized the effects of βCM-5 on spontaneous contractions in isolated segments of distal colon (1 cm length) compared with propagating contractions along the isolated intact large intestine (22 cm length). In short segments of distal colon, βCM-5 increased the tension and frequency of spontaneous contractions in a concentration-dependent manner. At 20 μM βCM-5 tension increased by 71 ± 17% and the frequency doubled (n = 9), effects inhibited by naloxone (n = 7) and therefore mediated by opioid receptors. In contrast 20 μM βCM-5 disrupted propagating contractions in the large intestine preparation. At 20 μM βCM-5 reduced the proportion of contractions initiated in the proximal colon reaching the rectum by 83 ± 11% (n = 5) and this effect was also inhibited by naloxone, consistent with altered GIT reported in vivo. Our results demonstrate that the isolated whole large intestine provides an ideal preparation that mimics the reduced propagation of GIT in vivo in response to an opioid agonist, whereas short colon segments did not. The findings of the current study reveal that preserving large segments of intact large intestine, and hence intact enteric neural circuitry provides an ideal in vitro model to investigate the effect of opioid receptor modulators on intestinal transit.

CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.