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Additional supportive data is provided as Fig S1 (Generation and validation of Tg(Tff1-CreERT2)mice), Fig. S2 (Tff1-CreERT2 transgene, endogenous Tff1, and Lgr5 expression in the stomach), Fig. S3 (Tff1-CreERT2-mediated I2-galactosidase reporter activity in various organs), Fig. S4 (KrasG12D-induced gastric tumorigenesis is associated with aberrant mucous production, ectopic expression of intestinal genes and immune cell infiltration), Fig. S5 (Gastric-specific activation of oncogenic BrafV600E triggers tumorigenesis associated with increased Erk and Stat3 phosphorylation), Fig. S6 (Tff1-CreERT2 activity in established gastric tumors of gp130F/F mice), Fig. S7 (Co-activation of Kras and gp130 signalling induces intestinalization associated with severe atrophy) and Table S1 (Incidence and onset of gastric tumor formation).

About 5% to 10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli, including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre recombinase–mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg), comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre recombinase (CreERT2)–coding sequence. The resulting Tg(Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf. The administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);KrasLSL-G12D/+ and Tg(Tff1-CreERT2);BrafLSL-V600E/+ mice resulted in gastric metaplasia, inflammation, and adenoma development, characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130F/F mice, which carry a knockin mutation in the Il6 signal transducer (Il6st), we generated Tg(Tff1-CreERT2);Stat3fl/fl;gp130F/F mice that also harbor a conditional Stat3 knockout allele and found that tamoxifen administration conferred a significant reduction in their tumor burden. Conversely, excessive Kras activity in Tg(Tff1-CreERT2);KrasLSL-G12D/+;gp130F/F mice promoted more extensive gastric inflammation, metaplastic transformation, and tumorigenesis than observed in Tg(Tff1-CreERT2);KrasLSL-G12D/+ mice. Collectively, our findings demonstrate that advanced gastric tumorigenesis requires oncogenic KRAS or BRAF in concert with aberrant STAT3 activation in epithelial precursor cells of the glandular stomach, providing a new conditional model of gastric cancer in which to investigate candidate therapeutic targets and treatment strategies. Cancer Res; 76(8); 2277–87. ©2016 AACR.

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Background In most settings, Female Sex Workers (FSW) bear a disproportionate burden of Human Immunodeficiency Virus (HIV) disease worldwide. Representative data to inform the development of behavioral and biomedical interventions for FSW in Namibia have not been published. Objectives Our objectives were to measure HIV prevalence, identify risk factors for infection, and describe uptake of prevention, testing, and treatment among FSW in Namibia. Methods We conducted cross-sectional surveys using Respondent-driven Sampling (RDS) in the Namibian cities of Katima Mulilo, Oshikango, Swakopmund/Walvis Bay, and Windhoek. Participating FSW completed behavioral questionnaires and rapid HIV testing. Results City-specific ranges of key indicators were: HIV prevalence (31.0-52.3%), reached by prevention programs in the past 12 months (46.9-73.6%), condom use at last sex with commercial (82.1-91.1%) and non-commercial (87.0-94.2%) partners, and tested for HIV within past 12 months or already aware of HIV-positive serostatus (56.9-82.1%). Factors associated with HIV infection varied by site and included: older age, having multiple commercial or non-commercial sex partners, unemployment, being currently out of school, and lower education level. Among HIV-positive FSW, 57.1% were aware of their HIV-positive serostatus and 33.7% were on antiretroviral treatment. Discussion Our results indicate extremely high HIV prevalence and low levels of case identification and treatment among FSW in Namibia. Our results, which are the first representative community-based estimates among FSW in Namibia, can inform the scale-up of interventions to reduce the risk for HIV acquisition and onward transmission, including treatment as prevention and pre-exposure prophylaxis.

Background In 2016, Namibia had ~ 230,000 people living with HIV (PLHIV) and 9154 new tuberculosis (TB) cases, including 3410 (38%) co-infected cases. TB preventative therapy (TPT), consisting of intensive case finding and isoniazid preventative therapy, is critical to reducing TB disease and mortality. Methods Between November 2014 and February 2015, data was abstracted from charts of PLHIV enrolled in HIV treatment. Fifty-five facilities were purposively selected based on patient volume, type and location. Charts were randomly sampled. The primary outcome was to estimate baseline TPT in PLHIV, using nationally weighted proportions. Qualitative surveys were conducted and summarized to evaluate TPT practices and quantify challenges encountered by health care workers (HCW). Results Among 861 PLHIV sampled, 96% were eligible for TPT services, of which 87.1% were screened for TB at least once. For PLHIV eligible for preventative therapy (646/810; 82.6%), 45.4% (294/646) initiated therapy and 45.7% (139/294) of those completed therapy. The proportion of eligible PLHIV completing TB screening, initiating preventative therapy and then completing preventative therapy was 20.7%. Qualitative surveys with 271 HCW identified barriers to TPT implementation including: lack of training (61.3% reported receiving training on TPT); misunderstandings about timing of TPT initiation (46.7% correctly reported TPT should be started with antiretroviral therapy); and variable screening practices and responsibilities (66.1% of HCWs screened for TB at every encounter). Though barriers were evident, 72.2% HCWs surveyed described their clinical performance as very good, often placing responsibility of difficulties on patients and downplaying challenges like staff shortages and medication stock outs. Conclusions In this study, only 1 in 5 eligible PLHIV completed the TPT cascade in Namibia. Lack of training, irregularities with TB screening and timing of TPT, unclear prescribing and recording responsibilities, and a clinical misperception may have contributed to suboptimal programmatic implementation. Addressing these challenges will be critical with continued TPT scale-up.

Background Direct measures of HIV incidence are needed to assess the population-level impact of prevention programs but are scarcely available in the subnational epidemic hotspots of sub-Saharan Africa. We created a sentinel HIV incidence cohort within a community-based program that provided home-based HIV testing to all residents of Namibia’s Zambezi region, where approximately 24% of the adult population was estimated to be living with HIV. Objective The aim of this study was to estimate HIV incidence, detect correlates of HIV acquisition, and assess the feasibility of the sentinel, community-based approach to HIV incidence surveillance in a subnational epidemic hotspot. Methods Following the program’s initial home-based testing (December 2014-July 2015), we purposefully selected 10 clusters of 60 to 70 households each and invited residents who were HIV negative and aged ≥15 years to participate in the cohort. Consenting participants completed behavioral interviews and a second HIV test approximately 1 year later (March-September 2016). We used Poisson models to calculate HIV incidence rates between baseline and follow-up and multivariable Cox proportional hazard models to assess the correlates of seroconversion. Results Among 1742 HIV-negative participants, 1624 (93.23%) completed follow-up. We observed 26 seroconversions in 1954 person-years (PY) of follow-up, equating to an overall incidence rate of 1.33 per 100 PY (95% CI 0.91-1.95). Among women, the incidence was 1.55 per 100 PY (95% CI 1.12-2.17) and significantly higher among those aged 15 to 24 years and residing in rural areas (adjusted hazard ratio [aHR] 4.26, 95% CI 1.39-13.13; P=.01), residing in the Ngweze suburb of Katima Mulilo city (aHR 2.34, 95% CI 1.25-4.40; P=.01), who had no prior HIV testing in the year before cohort enrollment (aHR 3.38, 95% CI 1.04-10.95; P=.05), and who had engaged in transactional sex (aHR 17.64, 95% CI 2.88-108.14; P=.02). Among men, HIV incidence was 1.05 per 100 PY (95% CI 0.54-2.31) and significantly higher among those aged 40 to 44 years (aHR 13.04, 95% CI 5.98-28.41; P Conclusions Nearly three decades into Namibia’s generalized HIV epidemic, these are the first estimates of HIV incidence for its highest prevalence region. By creating a sentinel incidence cohort from the infrastructure of an existing community-based testing program, we were able to characterize current transmission patterns, corroborate known risk factors for HIV acquisition, and provide insight into the efficacy of prevention interventions in a subnational epidemic hotspot. This study demonstrates an efficient and scalable framework for longitudinal HIV incidence surveillance that can be implemented in diverse sentinel sites and populations.

BACKGROUND Direct measures of HIV incidence are needed to assess the population-level impact of prevention programs but are scarcely available in the subnational epidemic hotspots of sub-Saharan Africa. We created a sentinel HIV incidence cohort within a community-based program that provided home-based HIV testing to all residents of Namibia’s Zambezi region, where approximately 24% of the adult population was estimated to be living with HIV. OBJECTIVE The aim of this study was to estimate HIV incidence, detect correlates of HIV acquisition, and assess the feasibility of the sentinel, community-based approach to HIV incidence surveillance in a subnational epidemic hotspot. METHODS Following the program’s initial home-based testing (December 2014-July 2015), we purposefully selected 10 clusters of 60 to 70 households each and invited residents who were HIV negative and aged ≥15 years to participate in the cohort. Consenting participants completed behavioral interviews and a second HIV test approximately 1 year later (March-September 2016). We used Poisson models to calculate HIV incidence rates between baseline and follow-up and multivariable Cox proportional hazard models to assess the correlates of seroconversion. RESULTS Among 1742 HIV-negative participants, 1624 (93.23%) completed follow-up. We observed 26 seroconversions in 1954 person-years (PY) of follow-up, equating to an overall incidence rate of 1.33 per 100 PY (95% CI 0.91-1.95). Among women, the incidence was 1.55 per 100 PY (95% CI 1.12-2.17) and significantly higher among those aged 15 to 24 years and residing in rural areas (adjusted hazard ratio [aHR] 4.26, 95% CI 1.39-13.13; P=.01), residing in the Ngweze suburb of Katima Mulilo city (aHR 2.34, 95% CI 1.25-4.40; P=.01), who had no prior HIV testing in the year before cohort enrollment (aHR 3.38, 95% CI 1.04-10.95; P=.05), and who had engaged in transactional sex (aHR 17.64, 95% CI 2.88-108.14; P=.02). Among men, HIV incidence was 1.05 per 100 PY (95% CI 0.54-2.31) and significantly higher among those aged 40 to 44 years (aHR 13.04, 95% CI 5.98-28.41; PP=.02). No seroconversions occurred among persons with HIV-positive partners on antiretroviral treatment. CONCLUSIONS Nearly three decades into Namibia’s generalized HIV epidemic, these are the first estimates of HIV incidence for its highest prevalence region. By creating a sentinel incidence cohort from the infrastructure of an existing community-based testing program, we were able to characterize current transmission patterns, corroborate known risk factors for HIV acquisition, and provide insight into the efficacy of prevention interventions in a subnational epidemic hotspot. This study demonstrates an efficient and scalable framework for longitudinal HIV incidence surveillance that can be implemented in diverse sentinel sites and populations.

Temporal and spatial regulation of genes mediated by tissue-specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium-specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain confers tamoxifen-inducible irreversible somatic recombination and allows simultaneous doxycycline-dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1-CreERT2 and Tff2-rtTA transgene activity in a partially overlapping subset of long-term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626-635, 2016. © 2016 Wiley Periodicals, Inc.

About 5% to 10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli, including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre recombinase–mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg), comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre recombinase (CreERT2)–coding sequence. The resulting Tg(Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf. The administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);KrasLSL-G12D/+ and Tg(Tff1-CreERT2);BrafLSL-V600E/+ mice resulted in gastric metaplasia, inflammation, and adenoma development, characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130F/F mice, which carry a knockin mutation in the Il6 signal transducer (Il6st), we generated Tg(Tff1-CreERT2);Stat3fl/fl;gp130F/F mice that also harbor a conditional Stat3 knockout allele and found that tamoxifen administration conferred a significant reduction in their tumor burden. Conversely, excessive Kras activity in Tg(Tff1-CreERT2);KrasLSL-G12D/+;gp130F/F mice promoted more extensive gastric inflammation, metaplastic transformation, and tumorigenesis than observed in Tg(Tff1-CreERT2);KrasLSL-G12D/+ mice. Collectively, our findings demonstrate that advanced gastric tumorigenesis requires oncogenic KRAS or BRAF in concert with aberrant STAT3 activation in epithelial precursor cells of the glandular stomach, providing a new conditional model of gastric cancer in which to investigate candidate therapeutic targets and treatment strategies. Cancer Res; 76(8); 2277–87. ©2016 AACR.

Background: Sativex® (THC:CBD oromucosal spray) is indicated as add-on treatment for patients with moderate to severe multiple sclerosis (MS) spasticity. We aimed to determine whether antispasticity treatment history influenced the efficacy and safety of add-on THC:CBD oromucosal spray in MS spasticity patients. Methods: Post hoc analysis of an enriched-design clinical trial of THC:CBD oromucosal spray versus placebo, using records of patients under previous and current ineffective antispasticity therapies. Subgroups were patients with at least 1 failed therapy attempt with either baclofen or tizanidine (Group 1) or at least 2 failed therapy attempts with both baclofen and tizanidine (Group 2). Summary: Of 241 patients in the intent-to-treat population, 162 and 57 patients met the criteria for Groups 1 and 2, respectively. In all groups, response on the spasticity 0-10 Numerical Rating Scale was significantly greater with THC:CBD oromucosal spray versus placebo, for minimal clinically important difference (MCID ≥18% improvement vs. baseline) and clinically important difference (CID, ≥30% improvement vs. baseline). THC:CBD oromucosal spray improved spasticity-related symptoms such as sleep quality and timed 10-meter walk independent of the number of prior failed therapy attempts. Tolerability was not influenced by pre-treatment history. Conclusions: THC:CBD oromucosal spray provided consistent relief with good tolerability in MS spasticity patients irrespective of their antispasticity pre-treatment history.

Background Key populations, including female sex workers (FSWs), are at a disproportionately high risk for HIV infection. Estimates of the size of these populations serve as denominator data to inform HIV prevention and treatment programming and are necessary for the equitable allocation of limited public health resources. Objective This study aimed to present the respondent-driven sampling (RDS) adjusted reverse tracking method (RTM; RadR), a novel population size estimation approach that combines venue mapping data with RDS data to estimate the population size, adjusted for double counting and nonattendance biases. Methods We used data from a 2014 RDS survey of FSWs in Windhoek and Katima Mulilo, Namibia, to demonstrate the RadR method. Information from venue mapping and enumeration from the survey formative assessment phase were combined with survey-based venue-inquiry questions to estimate population size, adjusting for double counting, and FSWs who do not attend venues. RadR estimates were compared with the official population size estimates, published by the Namibian Ministry of Health and Social Services (MoHSS), and with the unadjusted RTM. Results Using the RadR method, we estimated 1552 (95% simulation interval, SI, 1101-2387) FSWs in Windhoek and 453 (95% SI: 336-656) FSWs in Katima Mulilo. These estimates were slightly more conservative than the MoHSS estimates-Windhoek: 3000 (1800-3400); Katima Mulilo: 800 (380-2000)-though not statistically different. We also found 75 additional venues in Windhoek and 59 additional venues in Katima Mulilo identified by RDS participants' responses that were not detected during the initial mapping exercise. Conclusions The RadR estimates were comparable with official estimates from the MoHSS. The RadR method is easily integrated into RDS studies, producing plausible population size estimates, and can also validate and update key population maps for outreach and venue-based sampling.

BACKGROUND Key populations, including female sex workers (FSWs), are at a disproportionately high risk for HIV infection. Estimates of the size of these populations serve as denominator data to inform HIV prevention and treatment programming and are necessary for the equitable allocation of limited public health resources. OBJECTIVE This study aimed to present the respondent-driven sampling (RDS) adjusted reverse tracking method (RTM; RadR), a novel population size estimation approach that combines venue mapping data with RDS data to estimate the population size, adjusted for double counting and nonattendance biases. METHODS We used data from a 2014 RDS survey of FSWs in Windhoek and Katima Mulilo, Namibia, to demonstrate the RadR method. Information from venue mapping and enumeration from the survey formative assessment phase were combined with survey-based venue-inquiry questions to estimate population size, adjusting for double counting, and FSWs who do not attend venues. RadR estimates were compared with the official population size estimates, published by the Namibian Ministry of Health and Social Services (MoHSS), and with the unadjusted RTM. RESULTS Using the RadR method, we estimated 1552 (95% simulation interval, SI, 1101-2387) FSWs in Windhoek and 453 (95% SI: 336-656) FSWs in Katima Mulilo. These estimates were slightly more conservative than the MoHSS estimates—Windhoek: 3000 (1800-3400); Katima Mulilo: 800 (380-2000)—though not statistically different. We also found 75 additional venues in Windhoek and 59 additional venues in Katima Mulilo identified by RDS participants’ responses that were not detected during the initial mapping exercise. CONCLUSIONS The RadR estimates were comparable with official estimates from the MoHSS. The RadR method is easily integrated into RDS studies, producing plausible population size estimates, and can also validate and update key population maps for outreach and venue-based sampling.

Background In 2015, Namibia implemented an Acceleration Plan to address the high burden of HIV (13.0% adult prevalence and 216 311 people living with HIV [PLHIV]) and achieve the UNAIDS 90-90-90 targets by 2020. We provide an update on Namibia’s overall progress toward achieving these targets and estimate the percent reduction in HIV incidence since 2010. Methods Data sources include the 2013 Namibia Demographic and Health Survey (2013 NDHS), the national electronic patient monitoring system, and laboratory data from the Namibian Institute of Pathology. These sources were used to estimate (1) the percentage of PLHIV who know their HIV status, (2) the percentage of PLHIV on antiretroviral therapy (ART), (3) the percentage of patients on ART with suppressed viral loads, and (4) the percent reduction in HIV incidence. Results In the 2013 NDHS, knowledge of HIV status was higher among HIV-positive women 91.8% (95% confidence interval [CI], 89.4%–93.7%) than HIV-positive men 82.5% (95% CI, 78.1%–86.1%). At the end of 2016, an estimated 88.3% (95% CI, 86.3%–90.1%) of PLHIV knew their status, and 165 939 (76.7%) PLHIV were active on ART. The viral load suppression rate among those on ART was 87%, and it was highest among ≥20-year-olds (90%) and lowest among 15–19-year-olds (68%). HIV incidence has declined by 21% since 2010. Conclusions With 76.7% of PLHIV on ART and 87% of those on ART virally suppressed, Namibia is on track to achieve UNAIDS 90-90-90 targets by 2020. Innovative strategies are needed to improve HIV case identification among men and adherence to ART among youth.

In order to further investigate the molecular mechanisms that regulate oligodendrocyte (OC) survival, we utilized microarrays to characterize changes in OC gene expression after exposure to the cytokines neurotrophin3, insulin, or leukemia inhibitory factor (LIF) in vitro. We identified and validated the induction and secretion of the neuropeptide galanin in OCs, specifically in response to LIF. We next established that galanin is an OC survival factor and showed that autocrine or paracrine galanin secretion mediates LIF-induced OC survival in vitro. We also revealed that galanin is up-regulated in OCs in the cuprizone model of central demyelination, and that oligodendroglial galanin expression is significantly regulated by endogenous LIF in this context. We also showed that knock-out of galanin reduces OC survival and exacerbates callosal demyelination in the cuprizone model. These findings suggest a potential role for the use of galanin agonists in the treatment of human demyelinating diseases.

BACKGROUND. Poliovirus (PV) antibody seroprevalence studies assess population immunity, verify an immunization program’s performance and vaccine efficacy, and guide polio eradication strategy. Namibia experienced a polio outbreak among adults in 2006, yet population seroimmunity was unknown. METHODS. We tested 2061 specimens from Namibian pregnant females aged 15–44 years for neutralizing antibody to PV types 1–3 (PV1–3); all females were sampled during the 2010 National HIV Sentinel Survey. We determined the proportion of females seropositive for PV antibody by 5-year age strata, and analyzed factors associated with seropositivity, including age, gravidity, human immunodeficiency virus (HIV) infection status, residence, and antiretroviral treatment, by log-binomial regression. RESULTS. The seroprevalence was 94.6% for PV1, 97.0% for PV2, and 85.1% for PV3. HIV-positive females had significantly lower seroprevalence than HIV-negative females for PV1 (91.8% vs 95.3%; P < .01) and PV3 (80.0% vs 86.1%; P < .01) but not for PV2 (96.4% vs 97.1%; P = .3). The prevalence ratio of seropositivity for HIV-positive females versus HIV-negative females was 0.95 (95% confidence interval [CI], .92–.98) for PV1, 0.99 (95% CI, .97–1.01) for PV2, and 0.92 (95% CI, .87–.96) for PV3. CONCLUSIONS. Despite relatively high PV seroprevalence, Namibia might remain at risk for a PV outbreak, particularly in lower-seroprevalence populations, such as HIV-positive females. Namibia should continue to maintain high routine polio vaccination coverage.

We conducted a microarray study to identify genes that are differentially regulated in the spinal cords of mice with the inflammatory disease experimental autoimmune encephalomyelitis (EAE) relative to healthy mice. In total 181 genes with at least a two-fold increase in expression were identified, and most of these genes were associated with immune function. Unexpectedly, ceruloplasmin (Cp), a ferroxidase that converts toxic ferrous iron to its nontoxic ferric form and also promotes the efflux of iron from astrocytes in the CNS, was shown to be highly upregulated (13.2-fold increase) in EAE spinal cord. Expression of Cp protein is known to be increased in several neurological conditions, but the role of Cp regulation in CNS autoimmune disease is not known. To investigate this, we induced EAE in Cp gene knockout, heterozygous, and wild-type mice. Cp knockout mice were found to have slower disease evolution than wild-type mice (EAE days 13–17; P = 0.05). Interestingly, Cp knockout mice also exhibited a significant increase in the number of astrocytes with reactive morphology in early EAE compared with wild-type mice at the same stage of disease. CNS iron levels were not increased with EAE in these mice. Based on these observations, we propose that an increase in Cp expression could contribute to tissue damage in early EAE. In addition, endogenous CP either directly or indirectly inhibits astrocyte reactivity during early disease, which could also worsen early disease evolution.

Temporal and spatial regulation of genes mediated by tissue-specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium-specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain confers tamoxifen-inducible irreversible somatic recombination and allows simultaneous doxycycline-dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1-CreERT2 and Tff2-rtTA transgene activity in a partially overlapping subset of long-term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626-635, 2016. © 2016 Wiley Periodicals, Inc.

HIV drug resistance (HIVDR) testing is not routinely available in many resource-limited settings, therefore, antiretroviral therapy (ART) program and site factors known to be associated with HIVDR should be monitored to optimize the quality of patient care and minimize the emergence of preventable HIVDR.In 2009, Namibia selected 5 World Health Organization Early Warning Indicators (EWIs) and piloted abstraction at 9 ART sites: "ART prescribing practices, patients lost to follow-up at 12 months, patient retention on first-line ART at 12 months, on-time antiretroviral drug pick-up, and antiretroviral drug-supply continuity".Records supported monitoring of 3 of 5 selected EWIs. Nine of 9 (100%) sites met the target of 100% initiated on appropriate first-line regimens. Eight of 9 (89%) sites met the target of ≤20% lost to follow-up, although 20.8% of ART starters (range: 4.6%-44.6%) had a period of absence without documented ART coverage of 2.3 months (range: 1.5-3.9 months). Six of 9 (67%) sites met the target of 0% switched to a second-line regimen.EWI monitoring directly resulted in public health action which will optimize the quality of care, specifically the strengthening of ART record systems permitting monitoring of 5 EWIs in future years and protocols for improved ART patient defaulter tracing.

The World Health Organization (WHO) prospective surveys of acquired HIV drug resistance (HIVDR) evaluate HIVDR emerging after the first year of antiretroviral therapy (ART) and associated factors.Consecutive ART starters in 2009 were enrolled at 3 sentinel sites in Namibia. Genotyping was performed at start and after 12 months in patients with HIV viral load (VL)1000 copies per mL. HIVDR outcomes were: HIVDR prevention (VL ≤1000 copies/mL), possible HIVDR (VL1000 copies/mL without detectable HIVDR or loss to follow-up or ART stop), and HIVDR (VL1000 copies/mL with detectable HIVDR). Adherence was assessed using medication possession ratio (MPR).Of 394 starters, at 12 months, 80% were on first-line ART, 1% died, 4% transferred out, 1% stopped ART,1% switched to second-line, and 15% were lost to follow-up. Among patients on first-line, 77% had VL testing, and 94% achieved VL ≤1000 copies per mL. At baseline, 7% had HIVDR. After 12 months, among patients with VL testing, 5% had HIVDR. A majority of patients failing therapy had high-level resistance to nonnucleoside reverse transcriptase inhibitors but none to protease inhibitors. All sites achieved the WHO target of ≥70% HIVDR prevention. Factors associated with not achieving HIVDR prevention were: baseline resistance to nonnucleoside reverse transcriptase inhibitors [odds ratio (OR) 3.0, P = 0.023], WHO stage 3 or 4 at baseline (OR 2.0, P = 0.012), and MPR75% (OR 4.9, P = 0.021).Earlier ART initiation and removal of barriers to on-time drug pickups may help to prevent HIVDR. These data inform decisions at national and global levels on the effectiveness of first- and second-line regimens.

OBJECTIVE: Food insecurity is emerging as an important barrier to antiretroviral therapy (ART) adherence. The objective of this study was to determine if food insecurity is associated with poor ART adherence among HIV-positive adults in a resource-limited setting that uses the public health model of delivery. DESIGN: A cross-sectional study using a 1-time questionnaire and routinely collected pharmacy data. METHODS: Participants were HIV-infected adults on ART at the public ART clinics in Windhoek Namibia: Katutura State Hospital Katutura Health Centre and Windhoek Central Hospital. Food insecurity was measured by the Household Food Insecurity Access Scale (HFIAS). Adherence was assessed by the pharmacy adherence measure medication possession ratio (MPR). Multivariate regression was used to assess whether food insecurity was associated with ART adherence. RESULTS: Among 390 participants 7% were food secure 25% were mildly or moderately food insecure and 67% were severely food insecure. In adjusted analyses severe household food insecurity was associated with MPR

In order to further investigate the molecular mechanisms that regulate oligodendrocyte (OC) survival, we utilized microarrays to characterize changes in OC gene expression after exposure to the cytokines neurotrophin3, insulin, or leukemia inhibitory factor (LIF) in vitro. We identified and validated the induction and secretion of the neuropeptide galanin in OCs, specifically in response to LIF. We next established that galanin is an OC survival factor and showed that autocrine or paracrine galanin secretion mediates LIF-induced OC survival in vitro. We also revealed that galanin is up-regulated in OCs in the cuprizone model of central demyelination, and that oligodendroglial galanin expression is significantly regulated by endogenous LIF in this context. We also showed that knock-out of galanin reduces OC survival and exacerbates callosal demyelination in the cuprizone model. These findings suggest a potential role for the use of galanin agonists in the treatment of human demyelinating diseases.

We conducted a microarray study to identify genes that are differentially regulated in the spinal cords of mice with the inflammatory disease experimental autoimmune encephalomyelitis (EAE) relative to healthy mice. In total 181 genes with at least a two-fold increase in expression were identified, and most of these genes were associated with immune function. Unexpectedly, ceruloplasmin (Cp), a ferroxidase that converts toxic ferrous iron to its nontoxic ferric form and also promotes the efflux of iron from astrocytes in the CNS, was shown to be highly upregulated (13.2-fold increase) in EAE spinal cord. Expression of Cp protein is known to be increased in several neurological conditions, but the role of Cp regulation in CNS autoimmune disease is not known. To investigate this, we induced EAE in Cp gene knockout, heterozygous, and wild-type mice. Cp knockout mice were found to have slower disease evolution than wild-type mice (EAE days 13-17; P = 0.05). Interestingly, Cp knockout mice also exhibited a significant increase in the number of astrocytes with reactive morphology in early EAE compared with wild-type mice at the same stage of disease. CNS iron levels were not increased with EAE in these mice. Based on these observations, we propose that an increase in Cp expression could contribute to tissue damage in early EAE. In addition, endogenous CP either directly or indirectly inhibits astrocyte reactivity during early disease, which could also worsen early disease evolution.

Background HIV drug resistance (HIVDR) testing is not routinely available in many resource-limited settings, therefore antiretroviral therapy (ART) program and site factors known to be associated with emergence of HIVDR should be monitored to optimize the quality of patient care and minimize the emergence of preventable HIVDR. Methods In 2010, Namibia selected five World Health Organization Early Warning Indicators (EWIs) and scaled-up monitoring from 9 to 33 ART sites: ART prescribing practices, Patients lost to follow-up (LTFU) at 12 months, Patients switched to a second-line regimen at 12 months, On-time antiretroviral (ARV) drug pick-up, and ARV drug-supply continuity. Results Records allowed reporting on three of the five selected EWIs. 22 of 33 (67%) sites met the target of 100% initiated on appropriate first-line regimens. 17 of 33 (52%) sites met the target of ≤20% LTFU. 15 of 33 (45%) sites met the target of 0% switched to a second-line regimen. Conclusions EWI monitoring directly resulted in public health action which will optimize the quality of care, specifically the strengthening of ART record systems, engagement of ART sites, and operational research for improved adherence assessment and ART patient defaulter tracing.

The EphA4 receptor tyrosine kinase is a major regulator of axonal growth and astrocyte reactivity and is a possible inflammatory mediator. Given that multiple sclerosis (MS) is primarily an inflammatory demyelinating disease and in mouse models of MS, such as experimental autoimmune encephalomyelitis (EAE), axonal degeneration and reactive gliosis are prominent clinical features, we hypothesised that endogenous EphA4 could play a role in modulating EAE. EAE was induced in EphA4 knockout and wildtype mice using MOG peptide immunisation and clinical severity and histological features of the disease were then compared in lumbar spinal cord sections. EphA4 knockout mice exhibited a markedly less severe clinical course than wildtype mice, with a lower maximum disease grade and a slightly later onset of clinical symptoms. Numbers of infiltrating T cells and macrophages, the number and size of the lesions, and the extent of astrocytic gliosis were similar in both genotypes; however, EphA4 knockout mice appeared to have decreased axonal pathology. Blocking of EphA4 in wildtype mice by administration of soluble EphA4 (EphA4-Fc) as a decoy receptor following induction of EAE produced a delay in onset of clinical symptoms; however, most mice had clinical symptoms of similar severity by 22 days, indicating that EphA4 blocking treatment slowed early EAE disease evolution. Again there were no apparent differences in histopathology. To determine whether the role of EphA4 in modulating EAE was CNS mediated or due to an altered immune response, MOG primed T cells from wildtype and EphA4 knockout mice were passively transferred into naive recipient mice and both were shown to induce disease of equivalent severity. These results are consistent with a non-inflammatory, CNS specific, deleterious effect of EphA4 during neuroinflammation that results in axonal pathology.

Presentation of AML antigens by dendritic cells (DC) could potentially induce a T cell-mediated anti-leukemic immune response. In the present study, we generated DC from adherent (AD-DC) and non-adherent (NAD-DC) myeloblasts obtained from bone marrows of AML patients. Both cell populations displayed morphological, phenotypic and functional properties of DC. The functions of NAD-DC were compared to AD-DC that had been fused with autologous AML blasts (FC/AML). The FC/AML induced greater T cell proliferation and CTL activity against autologous AML blasts (9/10 cases) as compared to NAD-DC. FC/AML may thus represent a promising strategy for DC-based immunotherapy of patients with AML.

Background In response to concerns about the emergence of HIV drug resistance (HIVDR), the World Health Organization (WHO) has developed a comprehensive set of early warning indicators (EWIs) to monitor HIV drug resistance and good programme practice at antiretroviral therapy (ART) sites. Methods In 2012, Namibia utilized the updated WHO EWI guidance and abstracted data from adult and pediatric patients from 50 ART sites for the following EWIs: 1. On-time Pill Pick-up, 2. Retention in Care, 3. Pharmacy Stock-outs, 4. Dispensing Practices, and 5. Virological Suppression. Results Data for EWIs one through four were abstracted and validated. EWI 5 – Virological Suppression was not included due to poor data entry at many sites. On-time Pill Pick-up national estimate was 87.9% (87.2–88.7) of patients picking up pills on time for adults and 90.0% (88.9–90.9) picking up pills on time for pediatrics. Retention in Care national estimate was 82% of patients retained on ART after 12 months for adults and 83% for pediatrics. Pharmacy Stock-outs national estimate was 99% of months without a stock-out for adults and 97% for pediatrics. Dispensing Practices national estimate was 0.01% (0.003–0.064) of patients dispensed mono- or dual-therapy for adults and 0.25% (0.092–0.653) for pediatrics. Conclusions The successful 2012 EWI exercise provides Namibia a solid evidence base, which can be used to make national statements about programmatic functioning and possible HIVDR. This evidence base will serve to contextualize results from Namibia's surveys of HIVDR, which involves genotype testing. EWI abstraction has prompted the national program and its counterparts to engage sites in dialogue regarding the need to strengthen adherence and retention of patients on ART. The EWI collection process and EWI results will serve to optimize patient care and support Namibia in making evidence-based recommendations and take action to minimize the emergence of preventable HIVDR.

The visual-analogue scale (VAS), Likert item (rating scale), pills identification test (PIT), and medication possession ratio (MPR) provide estimates of antiretroviral therapy (ART) adherence which correlate with HIV viral suppression. These simple adherence measures are inexpensive and easy to administer; however, require validation and adjustment prior to implementation. The objective of this study was to define the optimal adherence assessment measure in Namibia to identify patients at risk for sub-optimal adherence and poor virologic response 6 months after ART initiation. We conducted a cross-sectional survey in HIV-infected adults receiving ART for 6–12 months prior to the adherence assessment. Adherence measures included 30-day VAS, 30-day Likert item, self-reported treatment interruptions, PIT, and MPR. Association of adherence measures with 6-month HIV-1 RNA level was assessed using two thresholds (1000 copies/mL and 5000 copies/mL). Adherence was assessed in 236 patients, mean age 37.3 years, 54% female. Mean adherence was 98.1% by 30-day VAS, 84.7% by 30-day Likert item, 97.0% by self-reported treatment interruptions, 90.6% by PIT, and 98.8% by MPR. Agreement between adherence measures was poor using kappa statistic. 76% had HIV-1 RNA