Gaps in humanitarian WASH response: perspectives from people affected by crises, practitioners, global responders, and the literature.

Water, sanitation, and hygiene (WASH) interventions prevent and control disease in humanitarian response. To inform future funding and policy priorities, WASH 'gaps' were identified via 220 focus-group discussions with people affected by crises and WASH practitioners, 246 global survey respondents, and 614 documents. After extraction, 2,888 (48 per cent) gaps from direct feedback and 3,151 (52 per cent) from literature were categorised. People affected by crises primarily listed 'services gaps', including a need for water, sanitation, solid waste disposal, and hygiene items. Global survey respondents principally cited 'mechanism gaps' in providing services, including collaboration, WASH staffing expertise, and community engagement. Literature highlighted gaps in health (but not other) WASH intervention impacts. Overall, people affected by crises wanted the 'what' (services), responders wanted the 'how' (to supply), and researchers wanted the 'why' (health consequences). This study suggests a need for a renewed focus on basic WASH services, collaboration across stakeholders, and research on WASH outcomes beyond health.

Lessons learned from conducting six multi-country mixed-methods effectiveness research studies on water, sanitation, and hygiene (WASH) interventions in humanitarian response.

BACKGROUND: Provision of safe water, sanitation, and hygiene (WASH) to affected populations in humanitarian emergencies is necessary for dignity and communicable disease control. Additional evidence on WASH interventions is needed in humanitarian settings. Between 2008 and 2019, we completed six multi-country, mixed-methods effectiveness studies in humanitarian response on six different WASH interventions. In each evaluation, we conducted: key informant interviews; water point observations and water quality testing; household surveys with recipients, including survey and water quality testing; focus group discussions; and/or, secondary data analysis. The research questions were: "What is the effectiveness of [intervention] in reducing the risk of diarrhea/cholera transmission; and, what programmatic factors lead to higher effectiveness?" DISCUSSION: In all six multi-country, mixed-methods evaluations, policy-relevant outcomes were obtained. We found, in our individual research results, that: interventions could reduce the risk of disease in humanitarian contexts; this reduction of risk did not always occur, as there were large ranges in effectiveness; and, implementation factors were crucial to intervention effectiveness. When collaboratively reviewing our research process across evaluations, we found strategies for successfully conducting this research included: 1) working with partners to identify and evaluate programs; 2) rapidly obtaining approvals to deploy; and, 3) conducting research methodologies consistently. Personal connections, in-person communication, trust, and experience working together were key factors for success in identifying partners for evaluation. Successes in evaluation deployment occurred with flexibility, patience, commitment of adequate time, and understanding of processes; although we note access and security concerns in insecure contexts precluded deployment. Consistent and robust protocols, flexibility, and a consistent researcher on the ground in each context allowed for methodological consistency and high-quality results. CONCLUSIONS: In conclusion, we have found multi-country, mixed-methods results to be one crucial piece of the WASH evidence base in humanitarian contexts. This is particularly because evaluations of reductions in risk from real-world programming are policy-relevant, and are directly used to improve programming. In future, we need to flexibly work with donors, agencies, institutions, responders, local governments, local responders, and beneficiaries to design safe and ethical research protocols to answer questions related to WASH interventions effectiveness in humanitarian response, and, improve WASH programming.

Evidence summary of water, sanitation, and hygiene (WASH) coordination in humanitarian response.

Providing water, sanitation, and hygiene (WASH) to emergency-affected populations is necessary for reasons of dignity and disease control. Such a (humanitarian) response is coordinated via the 'cluster approach'. This study utilises a literature review, an appraisal of and analysis of Global WASH Cluster (GWC) documentation, and key informant interviews to summarise the outcomes and impacts of GWC coordination. Across these three datasets, consistent themes were identified, including: the cluster approach evolving into a cost-effective 'best-fit' model; cluster staff requiring technical and coordination skills; and cluster members facing participation-related trade-offs. Consistent intractable difficulties were pinpointed, too, such as: accountability to beneficiaries; cross-cluster and subnational cluster coordination; and working with national governments. Previous research was found to be largely subjective and not to address future cluster challenges. An analysis of cluster outcomes and impacts, including member and beneficiary perspectives, is needed. To facilitate this work, a theory of change for cluster coordination was also developed.

Molecular targeting of renal cell carcinoma by an oral combination.

The 5-year survival rate of patients with metastatic renal cell carcinoma (mRCC) is <12% due to treatment failure. Therapeutic strategies that overcome resistance to modestly effective drugs for mRCC, such as sorafenib (SF), could improve outcome in mRCC patients. SF is terminally biotransformed by UDP-glucuronosyltransferase-1A9 (A9) mediated glucuronidation, which inactivates SF. In a clinical-cohort and the TCGA-dataset, A9 transcript and/or protein levels were highly elevated in RCC specimens and predicted metastasis and overall-survival. This suggested that elevated A9 levels even in primary tumors of patients who eventually develop mRCC could be a mechanism for SF failure. 4-methylumbelliferone (MU), a choleretic and antispasmodic drug, downregulated A9 and inhibited SF-glucuronidation in RCC cells. Low-dose SF and MU combinations inhibited growth, motility, invasion and downregulated an invasive signature in RCC cells, patient-derived tumor explants and/or endothelial-RCC cell co-cultures; however, both agents individually were ineffective. A9 overexpression made RCC cells resistant to the combination, while its downregulation sensitized them to SF treatment alone. The combination inhibited kidney tumor growth, angiogenesis and distant metastasis, with no detectable toxicity; A9-overexpressing tumors were resistant to treatment. With effective primary tumor control and abrogation of metastasis in preclinical models, the low-dose SF and MU combinations could be an effective treatment option for mRCC patients. Broadly, our study highlights how targeting specific mechanisms that cause the failure of "old" modestly effective FDA-approved drugs could improve treatment response with minimal alteration in toxicity profile.

A Novel Splice Variant of HYAL-4 Drives Malignant Transformation and Predicts Outcome in Patients with Bladder Cancer.

PURPOSE: Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer. EXPERIMENTAL DESIGN: In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder. RESULTS: HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically. CONCLUSIONS: Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer.

RB constrains lineage fidelity and multiple stages of tumour progression and metastasis.

Mutations in the retinoblastoma (RB) tumour suppressor pathway are a hallmark of cancer and a prevalent feature of lung adenocarcinoma1-3. Although RB was the first tumour suppressor to be identified, the molecular and cellular basis that underlies selection for persistent RB loss in cancer remains unclear4-6. Methods that reactivate the RB pathway using inhibitors of cyclin-dependent kinases CDK4 and CDK6 are effective in some cancer types and are currently under evaluation for the treatment of lung adenocarcinoma7-9. Whether RB pathway reactivation will have therapeutic effects and whether targeting CDK4 and CDK6 is sufficient to reactivate RB pathway activity in lung cancer remains unknown. Here we model RB loss during lung adenocarcinoma progression and pathway reactivation in established oncogenic KRAS-driven tumours in mice. We show that RB loss enables cancer cells to bypass two distinct barriers during tumour progression. First, RB loss abrogates the requirement for amplification of the MAPK signal during malignant progression. We identify CDK2-dependent phosphorylation of RB as an effector of MAPK signalling and critical mediator of resistance to inhibition of CDK4 and CDK6. Second, RB inactivation deregulates the expression of cell-state-determining factors, facilitates lineage infidelity and accelerates the acquisition of metastatic competency. By contrast, reactivation of RB reprograms advanced tumours towards a less metastatic cell state, but is nevertheless unable to halt cancer cell proliferation and tumour growth due to adaptive rewiring of MAPK pathway signalling, which restores a CDK-dependent suppression of RB. Our study demonstrates the power of reversible gene perturbation approaches to identify molecular mechanisms of tumour progression, causal relationships between genes and the tumour suppressive programs that they control and critical determinants of successful cancer therapy.

Household Water Treatment and Cholera Control.

Water, sanitation, and hygiene are one part of a cholera control strategy. Household water treatment (HWT) in particular has been shown to improve the microbiological quality of stored water and reduce the disease burden. We conducted a systematic review of published and gray literature to determine the outcomes and impacts of HWT in preventing cholera specifically. Fourteen manuscripts with 18 evaluations of HWT interventions in cholera were identified. Overall, a moderate quality of evidence suggests that HWT interventions reduce the burden of disease in cholera outbreaks and the risk of disease transmission. Appropriate training for users and community health worker follow-up are necessary for use. Barriers to uptake include taste and odor concerns, and facilitators include prior exposure, ease of use, and links to preexisting development programming. Further research on local barriers and facilitators, HWT filters, scaling up existing development programs, program sustainability, integrating HWT and oral cholera vaccine, and monitoring in low-access emergencies is recommended.

A Systematic Review and Meta-Analysis of the Association between Water, Sanitation, and Hygiene Exposures and Cholera in Case-Control Studies.

Case-control studies are conducted to identify cholera transmission routes. Water, sanitation, and hygiene (WASH) exposures can facilitate cholera transmission (risk factors) or interrupt transmission (protective factors). To our knowledge, the association between WASH exposures and cholera from case-control studies has not been systematically analyzed. A systematic review was completed to close this gap, including describing the theory of risk and protection, developing inclusion criteria, searching and selecting studies, assessing quality of evidence, and summarizing associations between cholera and seven predicted WASH protective factors and eight predicted WASH risk factors using meta-analysis and sensitivity analysis. Overall, 47 articles describing 51 individual studies from 30 countries met the inclusion criteria. All eight predicted risk factors were associated with higher odds of cholera (odds ratio [OR] = 1.9-5.6), with heterogeneity (I2) of 0-92%. Of the predicted protective factors, five of seven were associated with lower odds of cholera (OR = 0.35-1.4), with heterogeneity of 57-91%; exceptions were insignificant associations for improved water source (OR = 1.1, heterogeneity 91%) and improved sanitation (OR = 1.4, heterogeneity 68%). Results were robust; 3/70 (5%) associations changed directionality or significance in sensitivity analysis. Meta-analysis results highlight that predicted risk factors are associated with cholera; however, predicted protective factors are not as consistently protective. This variable protection is attributed to 1) cholera transmission via multiple routes and 2) WASH intervention implementation quality variation. Water, sanitation, and hygiene interventions should address multiple transmission routes and be well implemented, according to international guidance, to ensure that field effectiveness matches theoretical efficacy. In addition, future case-control studies should detail WASH characteristics to contextualize results.

Molecular Characterization of Renal Cell Carcinoma: A Potential Three-MicroRNA Prognostic Signature.

Background: Aberrantly expressed miRNAs promote renal cell carcinoma (RCC) growth and metastasis and are potentially useful biomarkers for metastatic disease. However, a consensus clinically significant miRNA signature has not been identified. To identify an miRNA signature for predicting clinical outcome in RCC patients, we used a four-pronged interconnected approach.Methods: Differentially expressed miRNAs were identified and analyzed in 113 specimens (normal kidney: 59; tumor: 54). miRNA profiling was performed in matched normal and tumor specimens from 8 patients and extended to 32 specimens. Seven aberrantly expressed miRNAs were analyzed by qPCR, and their levels were correlated with RCC subtypes and clinical outcome. miRNA signature was confirmed in The Cancer Genome Atlas RCC dataset (n = 241).Results: Discovery phase identified miR-21, miR-142-3p, miR-142-5p, miR-150, and miR-155 as significantly upregulated (2-4-fold) and miR-192 and miR-194 as downregulated (3-60-fold) in RCC; miR-155 distinguished small tumors (<4 cm) from benign oncocytomas. In univariate and multivariate analyses, miRNA combinations (miR-21+194; miR-21+142-5p+194) significantly predicted metastasis and/or disease-specific mortality; miR-21+142-5p+194 (for metastasis): P = 0.0017; OR, 0.53; 95% confidence interval (CI), 0.75-0.33; 86.7% sensitivity; 82% specificity. In the TCGA dataset, combined biomarkers associated with metastasis and overall survival (miR-21+142-5p+194: P < 0.0001; OR, 0.37; 95% CI, 0.58-0.23).Conclusions: The interconnected discovery-validation approach identified a three-miRNA signature as a potential predictor of disease outcome in RCC patients.Impact: With 10% survival at 5 years, metastatic disease presents poor prognosis for RCC patients. The three-miRNA signature discovered and validated may potentially at an early stage detect and predict metastasis, to allow early intervention for improving patient prognosis. Cancer Epidemiol Biomarkers Prev; 27(4); 464-72. ©2018 AACR.

Hyaluronic acid family in bladder cancer: potential prognostic biomarkers and therapeutic targets.

BACKGROUND: Molecular markers of clinical outcome may aid in designing targeted treatments for bladder cancer. However, only a few bladder cancer biomarkers have been examined as therapeutic targets. METHODS: Data from The Cancer Genome Atlas (TCGA) and bladder specimens were evaluated to determine the biomarker potential of the hyaluronic acid (HA) family of molecules - HA synthases, HA receptors and hyaluronidase. The therapeutic efficacy of 4-methylumbelliferone (4MU), a HA synthesis inhibitor, was evaluated in vitro and in xenograft models. RESULTS: In clinical specimens and TCGA data sets, HA synthases and hyaluronidase-1 levels significantly predicted metastasis and poor survival. 4-Methylumbelliferone inhibited proliferation and motility/invasion and induced apoptosis in bladder cancer cells. Oral administration of 4MU both prevented and inhibited tumour growth, without dose-related toxicity. Effects of 4MU were mediated through the inhibition of CD44/RHAMM and phosphatidylinositol 3-kinase/AKT axis, and of epithelial-mesenchymal transition determinants. These were attenuated by HA, suggesting that 4MU targets oncogenic HA signalling. In tumour specimens and the TCGA data set, HA family expression correlated positively with ß-catenin, Twist and Snail expression, but negatively with E-cadherin expression. CONCLUSIONS: This study demonstrates that the HA family can be exploited for developing a biomarker-driven, targeted treatment for bladder cancer, and 4MU, a non-toxic oral HA synthesis inhibitor, is one such candidate.

Systematic In Vivo Inactivation of Chromatin-Regulating Enzymes Identifies Setd2 as a Potent Tumor Suppressor in Lung Adenocarcinoma.

Chromatin-modifying genes are frequently mutated in human lung adenocarcinoma, but the functional impact of these mutations on disease initiation and progression is not well understood. Using a CRISPR-based approach, we systematically inactivated three of the most commonly mutated chromatin regulatory genes in two KrasG12D-driven mouse models of lung adenocarcinoma to characterize the impact of their loss. Targeted inactivation of SWI/SNF nucleosome-remodeling complex members Smarca4 (Brg1) or Arid1a had complex effects on lung adenocarcinoma initiation and progression. Loss of either Brg1 or Arid1a were selected against in early-stage tumors, but Brg1 loss continued to limit disease progression over time, whereas loss of Arid1a eventually promoted development of higher grade lesions. In contrast to these stage-specific effects, loss of the histone methyltransferase Setd2 had robust tumor-promoting consequences. Despite disparate impacts of Setd2 and Arid1a loss on tumor development, each resulted in a gene expression profile with significant overlap. Setd2 inactivation and subsequent loss of H3K36me3 led to the swift expansion and accelerated progression of both early- and late-stage tumors. However, Setd2 loss per se was insufficient to overcome a p53-regulated barrier to malignant progression, nor establish the prometastatic cellular states that stochastically evolve during lung adenocarcinoma progression. Our study uncovers differential and context-dependent effects of SWI/SNF complex member loss, identifies Setd2 as a potent tumor suppressor in lung adenocarcinoma, and establishes model systems to facilitate further study of chromatin deregulation in lung cancer. Cancer Res; 77(7); 1719-29. ©2017 AACR.

Antitumor activity of sulfated hyaluronic acid fragments in pre-clinical models of bladder cancer.

Tumor cell-derived hyaluronidase HYAL-1 degrades hyaluronic acid (HA) into angiogenic fragments (AGF: 10-12 disaccharides). AGF support tumor growth and progression. Urine and tissue HAase/HYAL-1 levels are sensitive markers for high-grade bladder cancer (BCa) and its metastasis. In preclinical models of BCa, we evaluated whether o-sulfated AGF (sHA-F) inhibits HAase activity and has antitumor activity. At IC50 for HAase activity inhibition (5-20 µg/ml [0.4-1.7 µM]), sHA-F significantly inhibited proliferation, motility and invasion of HYAL-1 expressing BCa cells (253J-Lung, HT1376, UMUC-3), P<0.001. sHA-F did not affect the growth of HYAL-1 non-expressing BCa (5637, RT4, T24, TCCSUP) and normal urothelial (Urotsa, SV-HUC1) cells. sHA-F treatment induced apoptosis by death receptor pathway. sHA-F downregulated transcript and/or protein levels of HA receptors (CD44, RHAMM), p-AKT, ß-catenin, pß-Catenin(S552), Snail and Twist but increased levels of pß-Catenin(T41/S45), pGSK-3α/ß(S21/S9) and E-cadherin. sHA-F also inhibited CD44/Phosphoinositide 3-kinase (PI-3K) complex formation and PI-3K activity. AGF addition or myristoylated-AKT overexpression attenuated sHA-F effects. Contrarily, HYAL-1 expression sensitized RT4 cells to sHA-F treatment. In the 253J-L and HT1376 xenograft models, sHA-F treatment significantly inhibited tumor growth (P<0.001), plausibly by inhibiting angiogenesis and HA receptor-PI-3K/AKT signaling. This study delineates that sHA-F targets tumor-associated HA-HAase system and could be potentially useful in BCa treatment.

Recombinase-based conditional and reversible gene regulation via XTR alleles.

Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R).

Dietary supplement 4-methylumbelliferone: an effective chemopreventive and therapeutic agent for prostate cancer.

BACKGROUND: Prevention and treatment of advanced prostate cancer (PCa) by a nontoxic agent can improve outcome, while maintaining quality of life. 4-methylumbelliferone (4-MU) is a dietary supplement that inhibits hyaluronic acid (HA) synthesis. We evaluated the chemopreventive and therapeutic efficacy and mechanism of action of 4-MU. METHODS: TRAMP mice (7-28 per group) were gavaged with 4-MU (450mg/kg/day) in a stage-specific treatment design (8-28, 12-28, 22-28 weeks). Efficacy of 4-MU (200-450mg/kg/day) was also evaluated in the PC3-ML/Luc(+) intracardiac injection and DU145 subcutaneous models. PCa cells and tissues were analyzed for HA and Phosphoinositide 3-kinase (PI-3K)/Akt signaling and apoptosis effectors. HA add-back and myristoylated Akt (mAkt) overexpression studies evaluated the mechanism of action of 4-MU. Data were analyzed with one-way analysis of variance and unpaired t test or Tukey's multiple comparison test. All statistical tests were two-sided. RESULTS: While vehicle-treated transgenic adenocarcinoma of the prostate (TRAMP) mice developed prostate tumors and metastases at 28 weeks, both were abrogated in treatment groups, without serum/organ toxicity or weight loss; no tumors developed at one year, even after stopping the treatment at 28 weeks. 4-MU did not alter the transgene or neuroendocrine marker expression but downregulated HA levels. However, 4-MU decreased microvessel density and proliferative index (P < .0001,). 4-MU completely prevented/inhibited skeletal metastasis in the PC3-ML/Luc(+) model and DU145-tumor growth (85-90% inhibition, P = .002). 4-MU also statistically significantly downregulated HA receptors, PI-3K/CD44 complex and activity, Akt signaling, and ß-catenin levels/activation, but upregulated GSK-3 function, E-cadherin, and apoptosis effectors (P < .001); HA addition or mAkt overexpression rescued these effects. CONCLUSION: 4-MU is an effective nontoxic, oral chemopreventive, and therapeutic agent that targets PCa development, growth, and metastasis by abrogating HA signaling.

The impact of water, sanitation, and hygiene interventions on the health and well-being of people living with HIV: a systematic review.

BACKGROUND: Access to improved water supply and sanitation is poor in low-income and middle-income countries. Persons living with HIV/AIDS (PLHIV) experience more severe diarrhea, hospitalizations, and deaths from diarrhea because of waterborne pathogens than immunocompetent populations, even when on antiretroviral therapy (ART). METHODS: We examined the existing literature on the impact of water, sanitation, and hygiene (WASH) interventions on PLHIV for these outcomes: (1) mortality, (2) morbidity, (3) retention in HIV care, (4) quality of life, and (5) prevention of ongoing HIV transmission. Cost-effectiveness was also assessed. Relevant abstracts and articles were gathered, reviewed, and prioritized by thematic outcomes of interest. Articles meeting inclusion criteria were summarized in a grid for comparison. RESULTS: We reviewed 3355 citations, evaluated 132 abstracts, and read 33 articles. The majority of the 16 included articles focused on morbidity, with less emphasis on mortality. Contaminated water, lack of sanitation, and poor hygienic practices in homes of PLHIV increase the risk of diarrhea, which can result in increased viral load, decreased CD4 counts, and reduced absorption of nutrients and antiretroviral medication. We found WASH programming, particularly water supply, household water treatment, and hygiene interventions, reduced morbidity. Data were inconclusive on mortality. Research gaps remain in retention in care, quality of life, and prevention of ongoing HIV transmission. Compared with the standard threshold of 3 times GDP per capita, WASH interventions were cost-effective, particularly when incorporated into complementary programs. CONCLUSIONS: Although research is required to address behavioral aspects, evidence supports that WASH programming is beneficial for PLHIV.

Effectiveness of chlorine dispensers in emergencies: case study results from Haiti, Sierra Leone, Democratic Republic of Congo, and Senegal.

Dispensers are a source-based water quality intervention with promising uptake results in development contexts. Dispenser programs include a tank of chlorine with a dosing valve that is installed next to a water source, a local Promoter who conducts community education and refills the Dispenser, and chlorine refills. In collaboration with response organizations, we assessed the effectiveness of Dispensers in four emergency situations. In the three initial and four sustained response phase evaluations, 70 Dispenser sites were visited, 2057 household surveys were conducted, and 1676 water samples were analyzed. Across the evaluations, reported Dispenser use ranged from 9 to 97%, confirmed Dispenser use (as measured by free chlorine residual) ranged from 5 to 87%, and effective use (as measured by improvement in household water quality to meet international standards) ranged from 0 to 81%. More effective Dispenser interventions installed Dispensers at point-sources, maintained a high-quality chlorine solution manufacturing and distribution chain, maintained Dispenser hardware, integrated Dispensers projects within larger water programs, remunerated Promoters, had experienced project staff, worked with local partners to implement the project, conducted ongoing monitoring, and had a project sustainability plan. Our results indicate that Dispensers can be, but are not always, an appropriate strategy to reduce the risk of waterborne diseases in emergencies.

Enantioselective pharmacokinetics of primaquine in healthy human volunteers.

Primaquine (PQ), a racemic drug, is the only treatment available for radical cure of relapsing Plasmodium vivax malaria and blocking transmission of P. falciparum malaria. Recent studies have shown differential pharmacologic and toxicologic profiles of individual PQ enantiomers in rodent, dog, and primate animal models. This study was conducted in six healthy adult human volunteers to determine the plasma pharmacokinetic profile of enantiomers of PQ and carboxyprimaquine (cPQ), the major plasma metabolite. The individuals were orally administered PQ diphosphate, equivalent to 45-mg base, 30 minutes after a normal breakfast. Blood samples were collected at different time intervals, and plasma samples were analyzed for enantiomers of PQ and cPQ. Plasma PQ concentrations were low and variable for both parent enantiomers and peaked around 2-4 hours. Peak (-)-(R)-PQ concentrations ranged from 121 ng/ml to 221 ng/ml, and peak (+)-(S)-PQ concentrations ranged from 168 ng/ml to 299 ng/ml. The cPQ concentrations were much higher and were surprisingly consistent from subject to subject. Essentially all the cPQ detected in plasma was (-)-cPQ. The peak concentrations of (-)-cPQ were observed at 8 hours (range: 1104-1756 ng/ml); however, very high concentrations were sustained through 24 hours. (+)-cPQ was two orders of magnitude lower than (-)-cPQ, and in a few subjects it was detected but only under the limit of quantification. In vitro studies with primary human hepatocytes also suggested more rapid metabolism of (-)-PQ compared with (+)-PQ. The results suggest more rapid metabolism of (-)-PQ to (-) cPQ compared with (+)-PQ. Alternatively, (+)-PQ or (+)-cPQ could be rapidly converted to another metabolite(s) or distributed to tissues. This is the first clinical report on enantioselective pharmacokinetic profiles of PQ and cPQ and supports further clinical evaluation of individual PQ enantiomers.

Differential expression of SDF-1 isoforms in bladder cancer.

PURPOSE: SDF-1 is a ligand of the chemokine receptors CXCR4 and 7. The 6 known SDF-1 isoforms are generated by alternative mRNA splicing. While SDF-1 expression has been detected in various malignancies, only few groups have reported differential expression of SDF-1 isoforms and its clinical significance. We evaluated the expression of 3 SDF-1 isoforms (α, ß and γ) in bladder cancer. MATERIALS AND METHODS: Using quantitative polymerase chain reaction we measured SDF-1α, ß and γ mRNA levels in 25 normal and 44 bladder cancer tissues, and in 210 urine specimens (28 normal, 74 benign, 57 bladder cancer, 35 bladder cancer history, 8 other cancer history and 8 other cancer) from consecutive patients. Levels were correlated with clinical outcome. RESULTS: Of the SDF-1 isoforms only SDF-1ß mRNA was significantly over expressed 2.5-fold to sixfold in bladder cancer compared to normal bladder tissues. SDF-1α was expressed in bladder tissues but SDF-1γ was undetectable. On multivariate analysis SDF-1ß was an independent predictor of metastasis and disease specific mortality (p=0.017 and 0.043, respectively). In exfoliated urothelial cells only SDF-1ß mRNA levels were differentially expressed with 91.2% sensitivity and 73.8% specificity for detecting bladder cancer. In patients with a bladder cancer history increased SDF-1ß levels indicated a 4.3-fold increased risk of recurrence within 6 months (p=0.0001). CONCLUSIONS: SDF-1 isoforms are differentially expressed in bladder tissues and exfoliated urothelial cells. SDF-1ß mRNA levels in bladder cancer tissues predict a poor prognosis. Furthermore, SDF-1ß mRNA levels in exfoliated cells detect bladder cancer with high sensitivity and they are a potential predictor of future recurrence.

C-X-C chemokine receptor 7: a functionally associated molecular marker for bladder cancer.

BACKGROUND: C-X-C chemokine receptor 4 (CXCR4) and CXCR7 are 7-transmembrane chemokine receptors of the stroma-derived factor (SDF-1). CXCR4, but not CXCR7, has been examined in bladder cancer (BCa). This study examined the functional and clinical significance of CXCR7 in BCa. METHODS: CXCR4 and CXCR7 levels were measured in BCa cell lines, tissues (normal = 25; BCa = 44), and urine specimens (n = 186) by quantitative polymerase chain reaction and/or immunohistochemistry. CXCR7 function in BCa cells were examined by transient transfections using a CXCR7 expression vector or small interfering RNA. RESULTS: In BCa cell lines, CXCR7 messenger RNA levels were 5- to 37-fold higher than those for CXCR4. Transient overexpression of CXCR7 in BCa cell lines promoted growth and chemotactic motility. CXCR7 colocalized and formed a functional complex with epidermal growth factor receptor, phosphoinositide 3-kinase/Akt, Erk, and src and induced their phosphorylation. CXCR7 also induced up-regulation of cyclin-D1 and bcl-2. Suppression of CXCR7 expression reversed these effects and induced apoptosis. CXCR7 messenger RNA levels and CXCR7 staining scores were significantly (5- to 10-fold) higher in BCa tissues than in normal tissues (P < .001). CXCR7 expression independently associated with metastasis (P = .019) and disease-specific mortality (P = .03). CXCR7 was highly expressed in endothelial cells in high-grade BCa tissues when compared to low-grade BCa and normal bladder. CXCR7 levels were elevated in exfoliated urothelial cells from high-grade BCa patients (P = .0001; 90% sensitivity; 75% specificity); CXCR4 levels were unaltered. CONCLUSIONS: CXCR7 promotes BCa cell proliferation and motility plausibly through epidermal growth factor receptor receptor and Akt signaling. CXCR7 expression is elevated in BCa tissues and exfoliated cells and is associated with high-grade and metastasis.

Dietary supplement hymecromone and sorafenib: a novel combination for the control of renal cell carcinoma.

PURPOSE: Current treatments for metastatic renal cell carcinoma do not extend survival beyond a few months. Sorafenib is a targeted drug approved for metastatic renal cell carcinoma but it has modest efficacy. Hymecromone is a nontoxic dietary supplement with some antitumor activity at high doses of 450 to 3,000 mg per day. Hymecromone inhibits the synthesis of hyaluronic acid, which promotes tumor growth and metastasis. We recently noted that the hyaluronic acid receptors CD44 and RHAMM are potential predictors of metastatic renal cell carcinoma. In the current study we examined the antitumor properties of hymecromone, sorafenib and the combination in renal cell carcinoma models. MATERIALS AND METHODS: Using proliferation, clonogenic and apoptosis assays, we examined the effects of hymecromone (0 to 32 µg/ml), sorafenib (0 to 3.2 µg/ml) and hymecromone plus sorafenib in Caki-1, 786-O, ACHN and A498 renal cell carcinoma cells, and HMVEC-L and HUVEC endothelial cells. A Boyden chamber was used for motility and invasion assays. Apoptosis indicators, hyaluronic acid receptors, epidermal growth factor receptor and c-Met were evaluated by immunoblot. The efficacy of hymecromone, sorafenib and hymecromone plus sorafenib was assessed in the sorafenib resistant Caki-1 xenograft model. RESULTS: Hymecromone plus sorafenib synergistically inhibited proliferation (greater than 95%), motility/invasion (65%) and capillary formation (76%) in renal cell carcinoma and/or endothelial cells, and induced apoptosis eightfold (p <0.001). Hymecromone plus sorafenib inhibited hyaluronic acid synthesis and adding hyaluronic acid reversed the cytotoxicity of hymecromone plus sorafenib. Hymecromone plus sorafenib up-regulated pro-apoptotic indicators and down-regulated Mcl-1, CD44, RHAMM, phospho-epidermal growth factor receptor and phospho-cMet. In all assays hymecromone and sorafenib alone were ineffective. Oral administration of hymecromone (50 to 200 mg/kg) plus sorafenib (30 mg/kg) eradicated Caki-1 tumor growth without toxicity. Hymecromone and sorafenib alone were ineffective. CONCLUSIONS: To our knowledge this is the first study to show that the combination of sorafenib and the nontoxic dietary supplement hymecromone is highly effective for controlling renal cell carcinoma.