Immunization with a Trypanosoma cruzi cyclophilin-19 deletion mutant protects against acute Chagas disease in mice.

Human infection with the protozoan parasite Trypanosoma cruzi causes Chagas disease for which there are no prophylactic vaccines. Cyclophilin 19 is a secreted cis-trans peptidyl isomerase expressed in all life stages of Trypanosoma cruzi. This protein in the insect stage leads to the inactivation of insect anti-parasitic peptides and parasite transformation whereas in the intracellular amastigotes it participates in generating ROS promoting the growth of parasites. We have generated a parasite mutant with depleted expression of Cyp19 by removal of 2 of 3 genes encoding this protein using double allelic homologous recombination. The mutant parasite line failed to replicate when inoculated into host cells in vitro or in mice indicating that Cyp19 is critical for infectivity. The mutant parasite line also fails to replicate in or cause clinical disease in immuno-deficient mice further validating their lack of virulence. Repeated inoculation of mutant parasites into immuno-competent mice elicits parasite-specific trypanolytic antibodies and a Th-1 biased immune response and challenge of mutant immunized mice with virulent wild-type parasites is 100% effective at preventing death from acute disease. These results suggest that parasite Cyp19 may be candidate for small molecule drug targeting and that the mutant parasite line may warrant further immunization studies for prevention of Chagas disease.

MicroRNA-21 Deficiency Promotes the Early Th1 Immune Response and Resistance toward Visceral Leishmaniasis.

MicroRNA-21 (miR-21) inhibits IL-12 expression and impairs the Th1 response necessary for control of Leishmania infection. Recent studies have shown that Leishmania infection induces miR-21 expression in dendritic cells and macrophages, and inhibition of miR-21 restores IL-12 expression. Because miR-21 is known to be expressed due to inflammatory stimuli in a wide range of hematopoietic cells, we investigated the role of miR-21 in regulating immune responses during visceral leishmaniasis (VL) caused by Leishmania donovani infection. We found that miR-21 expression was significantly elevated in dendritic cells, macrophages, inflammatory monocytes, polymorphonuclear neutrophils, and in the spleen and liver tissues after L. donovani infection, concomitant with an increased expression of disease exacerbating IL-6 and STAT3. Bone marrow dendritic cells from miR-21 knockout (miR-21KO) mice showed increased IL-12 production and decreased production of IL-10. On L. donovani infection, miR-21KO mice exhibited significantly greater numbers of IFN-γ- and TNF-α-producing CD4+ and CD8+ T cells in their organs that was associated with increased production of Th1-associated IFN-γ, TNF-α, and NO from the splenocytes. Finally, miR-21KO mice displayed significantly more developing and mature hepatic granulomas leading to reduction in organ parasitic loads compared with wild type counterparts. Similar results were noted in L. donovani-infected wild type mice after transient miR-21 depletion. These observations indicate that miR-21 plays a critical role in pathogenesis of VL by suppressing IL-12- and Th1-associated IFN-γ and also inducing disease-promoting induction of the IL-6 and STAT-3 signaling pathway. miR-21 could therefore be used as a potential target for developing host-directed treatment for VL.

Miconazole induces autophagic death in glioblastoma cells via reactive oxygen species-mediated endoplasmic reticulum stress.

Miconazole is an antifungal agent that is used for the treatment of superficial mycosis. However, recent studies have indicated that miconazole also exhibits potent anticancer effects in various types of cancer via the activation of apoptosis. The main aim of the present study was to observe the effect of miconazole on autophagic cell death of cancer cells. Cytotoxicity was measured by viable cell counting after miconazole treatment in glioblastoma cell lines (U343MG, U87MG and U251MG). Induction of autophagy was analyzed by examining microtubule-associated protein light chain 3 (LC3)-II expression levels using western blotting and by detecting GFP-LC3 translocation using a fluorescence microscope. Intracellular ROS production was measured using a fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate. It was found that miconazole induced autophagic cell death in the U251MG glioblastoma cell line via the generation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress response. An association between miconazole-induced ROS production and autophagy was also identified; in particular, pretreatment of the cells with a ROS scavenger resulted in a reduction in the levels of LC3-II. Miconazole-induced ER stress was associated with increases in binding immunoglobulin protein (BiP), inositol-requiring enzyme 1α (IRE1α) and CHOP expression, and phospho-eIF2α levels. The inhibition of ER stress via treatment with 4-phenylbutyric acid or BiP knockdown reduced miconazole-induced autophagy and cell death. These findings suggest that miconazole induces autophagic cell death by inducing an ROS-dependent ER stress response in U251MG glioma cancer cells and provide new insights into the potential antiproliferative effects of miconazole.

Assessment of the validity of the measurement of newborn and maternal health-care coverage in hospitals (EN-BIRTH): an observational study.

BACKGROUND: Progress in reducing maternal and neonatal deaths and stillbirths is impeded by data gaps, especially regarding coverage and quality of care in hospitals. We aimed to assess the validity of indicators of maternal and newborn health-care coverage around the time of birth in survey data and routine facility register data. METHODS: Every Newborn-BIRTH Indicators Research Tracking in Hospitals was an observational study in five hospitals in Bangladesh, Nepal, and Tanzania. We included women and their newborn babies who consented on admission to hospital. Exclusion critiera at admission were no fetal heartbeat heard or imminent birth. For coverage of uterotonics to prevent post-partum haemorrhage, early initiation of breastfeeding (within 1 h), neonatal bag-mask ventilation, kangaroo mother care (KMC), and antibiotics for clinically defined neonatal infection (sepsis, pneumonia, or meningitis), we collected time-stamped, direct observation or case note verification data as gold standard. We compared data reported via hospital exit surveys and via hospital registers to the gold standard, pooled using random effects meta-analysis. We calculated population-level validity ratios (measured coverage to observed coverage) plus individual-level validity metrics. FINDINGS: We observed 23 471 births and 840 mother-baby KMC pairs, and verified the case notes of 1015 admitted newborn babies regarding antibiotic treatment. Exit-survey-reported coverage for KMC was 99·9% (95% CI 98·3-100) compared with observed coverage of 100% (99·9-100), but exit surveys underestimated coverage for uterotonics (84·7% [79·1-89·5]) vs 99·4% [98·7-99·8] observed), bag-mask ventilation (0·8% [0·4-1·4]) vs 4·4% [1·9-8·1]), and antibiotics for neonatal infection (74·7% [55·3-90·1] vs 96·4% [94·0-98·6] observed). Early breastfeeding coverage was overestimated in exit surveys (53·2% [39·4-66·8) vs 10·9% [3·8-21·0] observed). "Don't know" responses concerning clinical interventions were more common in the exit survey after caesarean birth. Register data underestimated coverage of uterotonics (77·9% [37·8-99·5] vs 99·2% [98·6-99·7] observed), bag-mask ventilation (4·3% [2·1-7·3] vs 5·1% [2·0-9·6] observed), KMC (92·9% [84·2-98·5] vs 100% [99·9-100] observed), and overestimated early breastfeeding (85·9% (58·1-99·6) vs 12·5% [4·6-23·6] observed). Inter-hospital heterogeneity was higher for register-recorded coverage than for exit survey report. Even with the same register design, accuracy varied between hospitals. INTERPRETATION: Coverage indicators for newborn and maternal health care in exit surveys had low accuracy for specific clinical interventions, except for self-report of KMC, which had high sensitivity after admission to a KMC ward or corner and could be considered for further assessment. Hospital register design and completion are less standardised than surveys, resulting in variable data quality, with good validity for the best performing sites. Because approximately 80% of births worldwide take place in facilities, standardising register design and information systems has the potential to sustainably improve the quality of data on care at birth. FUNDING: Children's Investment Fund Foundation and Swedish Research Council.

Cyclophilin 19 secreted in the host cell cytosol by Trypanosoma cruzi promotes ROS production required for parasite growth.

Infection by Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, depends on reactive oxygen species (ROS), which has been described to induce parasite proliferation in mammalian host cells. It is unknown how the parasite manages to increase host ROS levels. Here, we found that intracellular T. cruzi forms release in the host cytosol its major cyclophilin of 19 kDa (TcCyp19). Parasites depleted of TcCyp19 by using CRISPR/Cas9 gene replacement proliferate inefficiently and fail to increase ROS, compared to wild type parasites or parasites with restored TcCyp19 gene expression. Expression of TcCyp19 in L6 rat myoblast increased ROS levels and restored the proliferation of TcCyp19 depleted parasites. These events could also be inhibited by cyclosporin A, (a cyclophilin inhibitor), and by polyethylene glycol-linked to antioxidant enzymes. TcCyp19 was found more concentrated in the membrane leading edges of the host cells in regions that also accumulate phosphorylated p47phox , as observed to the endogenous cyclophilin A, suggesting some mechanisms involved with the translocation process of the regulatory subunit p47phox in the activation of the NADPH oxidase enzymatic complex. We concluded that cyclophilin released in the host cell cytosol by T. cruzi mediates the increase of ROS, required to boost parasite proliferation in mammalian hosts.

The role of vascular endothelium and exosomes in human protozoan parasitic diseases.

The vascular endothelium is a vital component in maintaining the structure and function of blood vessels. The endothelial cells (ECs) mediate vital regulatory functions such as the proliferation of cells, permeability of various tissue membranes, and exchange of gases, thrombolysis, blood flow, and homeostasis. The vascular endothelium also regulates inflammation and immune cell trafficking, and ECs serve as a replicative niche for many bacterial, viral, and protozoan infectious diseases. Endothelial dysfunction can lead to vasodilation and pro-inflammation, which are the hallmarks of many severe diseases. Exosomes are nanoscale membrane-bound vesicles that emerge from cells and serve as important extracellular components, which facilitate communication between cells and maintain homeostasis during normal and pathophysiological states. Exosomes are also involved in gene transfer, inflammation and antigen presentation, and mediation of the immune response during pathogenic states. Protozoa are a diverse group of unicellular organisms that cause many infectious diseases in humans. In this regard, it is becoming increasingly evident that many protozoan parasites (such as Plasmodium, Trypanosoma, Leishmania, and Toxoplasma) utilize exosomes for the transfer of their virulence factors and effector molecules into the host cells, which manipulate the host gene expression, immune responses, and other biological activities to establish and modulate infection. In this review, we discuss the role of the vascular endothelium and exosomes in and their contribution to pathogenesis in malaria, African sleeping sickness, Chagas disease, and leishmaniasis and toxoplasmosis with an emphasis on their actions on the innate and adaptive immune mechanisms of resistance.

MicroRNA-155 Deficiency Exacerbates Trypanosoma cruzi Infection.

Chagas disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a public health problem affecting 6 to 8 million people, mainly in Latin America. The role of microRNAs in the pathogenesis of Chagas disease has not been well described. Here, we investigate the role of microRNA-155 (miR-155), a proinflammatory host innate immune regulator responsible for T helper type 1 and type 17 (Th1 and Th17) development and macrophage responses during T. cruzi infection. For this, we compared the survival and parasite growth and distribution in miR-155-/- and wild-type (WT) C57BL/6 mice. The lack of miR-155 caused robust parasite infection and diminished survival of infected mice, while WT mice were resistant to infection. Immunological analysis of infected mice indicated that, in the absence of miR-155, there was decreased interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) production. In addition, we found that there was a significant reduction of CD8-positive (CD8+) T cells, natural killer (NK) cells, and NK-T cells and increased accumulation of neutrophils and inflammatory monocytes in miR-155-/- mice. Collectively, these data indicate that miR-155 is an important immune regulatory molecule critical for the control of T. cruzi infection.

Ibrutinib treatment inhibits breast cancer progression and metastasis by inducing conversion of myeloid-derived suppressor cells to dendritic cells.

BACKGROUND: Ibrutinib is a Bruton's tyrosine kinase (BTK) and interleukin-2-inducible kinase (ITK) inhibitor used for treating chronic lymphocytic leukaemia (CLL) and other cancers. Although ibrutinib is known to inhibit the growth of breast cancer cell growth in vitro, its impact on the treatment and metastasis of breast cancer is unclear. METHODS: Using an orthotopic mouse breast cancer model, we show that ibrutinib inhibits the progression and metastasis of breast cancer. RESULTS: Ibrutinib inhibited proliferation of cancer cells in vitro, and Ibrutinib-treated mice displayed significantly lower tumour burdens and metastasis compared to controls. Furthermore, the spleens and tumours from Ibrutinib-treated mice contained more mature DCs and lower numbers of myeloid-derived suppressor cells (MDSCs), which promote disease progression and are linked to poor prognosis. We also confirmed that ex vivo treatment of MDSCs with ibrutinib switched their phenotype to mature DCs and significantly enhanced MHCII expression. Further, ibrutinib treatment promoted T cell proliferation and effector functions leading to the induction of antitumour TH1 and CTL immune responses. CONCLUSIONS: Ibrutinib inhibits tumour development and metastasis in breast cancer by promoting the development of mature DCs from MDSCs and hence could be a novel therapeutic agent for the treatment of breast cancer.

Equity and Coverage in the Continuum of Reproductive, Maternal, Newborn and Child Health Services in Nepal-Projecting the Estimates on Death Averted Using the LiST Tool.

INTRODUCTION: The third Sustainable Development Goal, focused on health, includes two targets related to the reduction in maternal, newborn and under-five childhood mortality. We found it imperative to examine the equity and coverage of reproductive, maternal, newborn and child health (RMNCH) interventions from 2001 to 2016 in Nepal; and the death aversion that will take place during the SDG period. METHODS: We used the datasets from the Nepal Demographic Health Surveys (NDHS) 2001, 2006, 2011 and 2016. We calculated the coverage and equity for RMNCH interventions and the composite coverage index (CCI). Based on the Annualized Rate of Change (ARC) in the coverage for selected RMNCH indicators, we projected the trend for the RMNCH interventions by 2030. We used the Lives Saved Tools (LiST) tool to estimate the maternal, newborn, under-five childhood deaths and stillbirths averted. We categorised the interventions into four different patterns based on coverage and inequity gap. RESULTS: Between 2001 and 2016, a significant improvement is seen in the overall RMNCH intervention coverage-CCI increasing from 46 to 75%. The ARC was highest for skilled attendance at birth (11.7%) followed by care seeking for pneumonia (8.2%) between the same period. In 2016, the highest inequity existed for utilization of the skilled birth attendance services (51%), followed by antenatal care (18%). The inequity gap for basic immunization services reduced significantly from 27.4% in 2001 to 5% in 2016. If the current ARC continues, then an additional 3783 maternal deaths, 36,443 neonatal deaths, 66,883 under-five childhood deaths and 24,024 stillbirths is expected to be averted by the year 2030. CONCLUSION: Nepal has experienced an improvement in the coverage and equity in RMNCH interventions. Reducing inequities will improve coverage for skilled birth attendants and antenatal care. The current annual rate of change in RMNCH coverage will further reduce the maternal, neonatal, under-five childhood deaths and stillbirths.

Effect of a scaled-up neonatal resuscitation quality improvement package on intrapartum-related mortality in Nepal: A stepped-wedge cluster randomized controlled trial.

BACKGROUND: Improving quality of intrapartum care will reduce intrapartum stillbirth and neonatal mortality, especially in resource-poor settings. Basic neonatal resuscitation can reduce intrapartum stillbirth and early neonatal mortality, if delivered in a high-quality health system, but there is a dearth of evidence on how to scale up such evidence-based interventions. We evaluated the scaling up of a quality improvement (QI) package for neonatal resuscitation on intrapartum-related mortality (intrapartum stillbirth and first day mortality) at hospitals in Nepal. METHODS AND FINDINGS: We conducted a stepped-wedge cluster randomized controlled trial in 12 hospitals over a period of 18 months from April 14, 2017, to October 17, 2018. The hospitals were assigned to one of four wedges through random allocation. The QI package was implemented in a stepped-wedge manner with a delay of three months for each step. The QI package included improving hospital leadership on intrapartum care, building health workers' competency on neonatal resuscitation, and continuous facilitated QI processes in clinical units. An independent data collection system was set up at each hospital to gather data on mortality through patient case note review and demographic characteristics of women using semi-structured exit interviews. The generalized linear mixed model (GLMM) and multivariate logistic regression were used for analyses. During this study period, a total of 89,014 women-infant pairs were enrolled. The mean age of the mother in the study period was 24.0 ± 4.3 years, with 54.9% from disadvantaged ethnic groups and 4.0% of them illiterate. Of the total birth cohort, 54.4% were boys, 16.7% had gestational age less than 37 weeks, and 17.1% had birth weight less than 2,500 grams. The incidence of intrapartum-related mortality was 11.0 per 1,000 births during the control period and 8.0 per 1,000 births during the intervention period (adjusted odds ratio [aOR], 0.79; 95% CI, 0.69-0.92; p = 0.002; intra-cluster correlation coefficient [ICC], 0.0286). The incidence of early neonatal mortality was 12.7 per 1,000 live births during the control period and 10.1 per 1,000 live births during the intervention period (aOR, 0.89; 95% CI, 0.78-1.02; p = 0.09; ICC, 0.1538). The use of bag-and-mask ventilation for babies with low Apgar score (<7 at 1 minute) increased from 3.2% in the control period to 4.0% in the intervention period (aOR, 1.52; 95% CI, 1.32-1.77, p = 0.003). There were two major limitations to the study; although a large sample of women-infant pairs were enrolled in the study, the clustering reduced the power of the study. Secondly, the study was not sufficiently powered to detect reduction in early neonatal mortality with the number of clusters provided. CONCLUSION: These results suggest scaled-up implementation of a QI package for neonatal resuscitation can reduce intrapartum-related mortality and improve clinical care. The QI intervention package is likely to be effective in similar settings. More implementation research is required to assess the sustainability of QI interventions and quality of care. TRIAL REGISTRATION: ISRCTN30829654.

Scaling Up Safer Birth Bundle Through Quality Improvement in Nepal (SUSTAIN)-a stepped wedge cluster randomized controlled trial in public hospitals.

BACKGROUND: Each year, 2.2 million intrapartum-related deaths (intrapartum stillbirths and first day neonatal deaths) occur worldwide with 99% of them taking place in low- and middle-income countries. Despite the accelerated increase in the proportion of deliveries taking place in health facilities in these settings, the stillborn and neonatal mortality rates have not reduced proportionately. Poor quality of care in health facilities is attributed to two-thirds of these deaths. Improving quality of care during the intrapartum period needs investments in evidence-based interventions. We aim to evaluate the quality improvement package-Scaling Up Safer Bundle Through Quality Improvement in Nepal (SUSTAIN)-on intrapartum care and intrapartum-related mortality in public hospitals of Nepal. METHODS: We will conduct a stepped wedge cluster randomized controlled trial in eight public hospitals with each having least 3000 deliveries a year. Each hospital will represent a cluster with an intervention transition period of 2 months in each. With a level of significance of 95%, the statistical power of 90% and an intra-cluster correlation of 0.00015, a study period of 19 months should detect at least a 15% change in intrapartum-related mortality. Quality improvement training, mentoring, systematic feedback, and a continuous improvement cycle will be instituted based on bottleneck analyses in each hospital. All concerned health workers will be trained on standard basic neonatal resuscitation and essential newborn care. Portable fetal heart monitors (Moyo®) and neonatal heart rate monitors (Neobeat®) will be introduced in the hospitals to identify fetal distress during labor and to improve neonatal resuscitation. Independent research teams will collect data in each hospital on intervention inputs, processes, and outcomes by reviewing records and carrying out observations and interviews. The dose-response effect will be evaluated through process evaluations. DISCUSSION: With the global momentum to improve quality of intrapartum care, better understanding of QI package within a health facility context is important. The proposed package is based on experiences from a similar previous scale-up trial carried out in Nepal. The proposed evaluation will provide evidence on QI package and technology for implementation and scale up in similar settings. TRIAL REGISTRATION NUMBER: ISRCTN16741720 . Registered on 2 March 2019.

"Every Newborn-BIRTH" protocol: observational study validating indicators for coverage and quality of maternal and newborn health care in Bangladesh, Nepal and Tanzania.

BACKGROUND: To achieve Sustainable Development Goals and Universal Health Coverage, programmatic data are essential. The Every Newborn Action Plan, agreed by all United Nations member states and >80 development partners, includes an ambitious Measurement Improvement Roadmap. Quality of care at birth is prioritised by both Every Newborn and Ending Preventable Maternal Mortality strategies, hence metrics need to advance from health service contact alone, to content of care. As facility births increase, monitoring using routine facility data in DHIS2 has potential, yet validation research has mainly focussed on maternal recall surveys. The Every Newborn - Birth Indicators Research Tracking in Hospitals (EN-BIRTH) study aims to validate selected newborn and maternal indicators for routine tracking of coverage and quality of facility-based care for use at district, national and global levels. METHODS: EN-BIRTH is an observational study including >20 000 facility births in three countries (Tanzania, Bangladesh and Nepal) to validate selected indicators. Direct clinical observation will be compared with facility register data and a pre-discharge maternal recall survey for indicators including: uterotonic administration, immediate newborn care, neonatal resuscitation and Kangaroo mother care. Indicators including neonatal infection management and antenatal corticosteroid administration, which cannot be easily observed, will be validated using inpatient records. Trained clinical observers in Labour/Delivery ward, Operation theatre, and Kangaroo mother care ward/areas will collect data using a tablet-based customised data capturing application. Sensitivity will be calculated for numerators of all indicators and specificity for those numerators with adequate information. Other objectives include comparison of denominator options (ie, true target population or surrogates) and quality of care analyses, especially regarding intervention timing. Barriers and enablers to routine recording and data usage will be assessed by data flow assessments, quantitative and qualitative analyses. CONCLUSIONS: To our knowledge, this is the first large, multi-country study validating facility-based routine data compared to direct observation for maternal and newborn care, designed to provide evidence to inform selection of a core list of indicators recommended for inclusion in national DHIS2. Availability and use of such data are fundamental to drive progress towards ending the annual 5.5 million preventable stillbirths, maternal and newborn deaths.

Host-Directed Drug Therapies for Neglected Tropical Diseases Caused by Protozoan Parasites.

The neglected tropical diseases (NTDs) caused by protozoan parasites are responsible for significant morbidity and mortality worldwide. Current treatments using anti-parasitic drugs are toxic and prolonged with poor patient compliance. In addition, emergence of drug-resistant parasites is increasing worldwide. Hence, there is a need for safer and better therapeutics for these infections. Host-directed therapy using drugs that target host pathways required for pathogen survival or its clearance is a promising approach for treating infections. This review will give a summary of the current status and advances of host-targeted therapies for treating NTDs caused by protozoa.

Oral hygiene practices and their socio-demographic correlates among Nepalese adult: evidence from non communicable diseases risk factors STEPS survey Nepal 2013.

BACKGROUND: Oral diseases remain a significant public health problem in Nepal, as do oral health behaviours. Socio-demographic factors play a crucial role in driving oral hygiene practices. This study aims to identify oral hygiene practices and associated socio-demographic factors in Nepalese population. METHODS: This descriptive, cross-sectional study recruited 4200 adults (15-69 years) through multistage cluster sampling. Data obtained from the WHO NCD STEPS instrument version 2.2 were analysed in STATA 13.0 using complex sample weighted analysis. RESULTS: Prevalence of cleaning teeth at least once a day was 94.9 % (95 % CI: 93.7-95.9), while that of cleaning teeth at least twice a day was 9.9 % (95 % CI: 8.2-11.9). Use of fluoridated toothpaste was seen among 71.4 % (95 % CI: 67.9-74.7) respondents. A 3.9 % (95 % CI: 3.1-5.0) made a dental visit in the last 6 months. The 45-69 years age group had lesser odds of cleaning teeth at least once a day (AOR: 0.4; 95 % CI: 0.2-0.8), in comparison to 15-29 years age group. Women had greater odds of cleaning teeth at least twice a day (AOR: 1.7; 95 % CI: 1.1-2.4) and having visited a dentist in the last 6 months (AOR: 2.2; 95 % CI: 1.2-3.8) compared to men. With reference to rural residents, urban population had higher odds of using fluoridated toothpaste (AOR: 2.3; 95 % CI: 1.4-3.4) and making a dental visit within the last 6 months (AOR: 1.9; 95 % CI:1.1-3.6). Inhabitants of the Terai had five-fold (AOR: 4.9; 95 % CI: 3.1-7.8) greater odds of cleaning teeth once per day than did hill residents. Those with higher education had greater odds than non-formal education holders of cleaning teeth at least once a day (AOR: 9.0; 95 % CI: 2.9-27.7), cleaning teeth at least twice a day (AOR: 5.6; 95 % CI: 2.9-10.6), using fluoridated toothpaste (AOR: 13.9; 95 % CI: 8.4-23.1), and having visited a dentist in the last 6 months (AOR: 2.8; 95 % CI: 1.4-5.4). CONCLUSIONS: Cleaning teeth at least once a day is widely prevalent in Nepal and a substantial number of population use fluoridated toothpaste. However, cleaning teeth twice a day and visiting a dentist is less common. Being women, Terai residents, urban residents, and educated were significantly associated with oral hygiene practices assessed in this study.

Juglone induces cell death of Acanthamoeba through increased production of reactive oxygen species.

Juglone (5-hydroxy-1,4-naphthoquinone) is a major chemical constituent of Juglans mandshruica Maxim. Recent studies have demonstrated that juglone exhibits anti-cancer, anti-bacterial, anti-viral, and anti-parasitic properties. However, its effect against Acanthamoeba has not been defined yet. The aim of this study was to investigate the effect of juglone on Acanthamoeba. We demonstrate that juglone significantly inhibits the growth of Acanthamoeba castellanii at 3-5 µM concentrations. Juglone increased the production of reactive oxygen species (ROS) and caused cell death of A. castellanii. Inhibition of ROS by antioxidant N-acetyl-l-cysteine (NAC) restored the cell viability. Furthermore, our results show that juglone increased the uptake of mitochondrial specific dye. Collectively, these results indicate that ROS played a significant role in the juglone-induced cell death of Acanthamoeba.

Tigecycline inhibits proliferation of Acanthamoeba castellanii.

Acanthamoeba is an opportunistic protozoan parasite responsible for different diseases in humans, such as granulomatous amoebic encephalitis and amoebic keratitis. Tigecycline, a third-generation tetracycline antibiotic, has potential activity to treat most of the antibiotic resistant bacterial infections. The effects of tigecycline in eukaryotic cells as well as parasites are less well studied. In the present study, we tested the effects of tigecycline on trophozoites of Acanthamoeba castellanii. The inhibitory effect of tigecycline on Acanthamoeba was determined by resazurin reduction and trypan blue exclusion assays. We found that tigecycline significantly inhibited the growth of Acanthamoeba (46.4 % inhibition at the concentration of 100 µM) without affecting cell viability and induction of encystation, whereas other tetracycline groups of antibiotics such as tetracycline and doxycycline showed no inhibitory effects. Furthermore, tigecycline decreased cellular adenosine triphosphate (ATP) level by 26 % than the control and increased mitochondrial mass, suggesting mitochondrial dysfunction in tigecycline-treated cells. These findings suggest that mitochondrial dysfunction with decreased ATP production might play an important mechanism of tigecycline in suppression of Acanthamoeba proliferation.

Chloroquine has a cytotoxic effect on Acanthamoeba encystation through modulation of autophagy.

Encystation of Acanthamoeba castellanii is associated with resistance to chemotherapeutic agents. Blocking the encystation process could potentiate the efficacy of chemotherapeutic agents and biocides. During encystation, autophagy is highly stimulated and required for proper encystation of Acanthamoeba. In this study, the cytotoxic effect of chloroquine, a well-known autophagy-inhibitory drug, was tested in A. castellanii. Chloroquine was able to selectively reduce cell survival during the encystation of A. castellanii. However, A. castellanii trophozoites and mature cysts were resistant to chloroquine. Chloroquine treatment led to an increase in the number and size of lysosomes in encysting cells. Moreover, chloroquine inhibited the degradation of long-lived proteins in the encysting cells. Decreased autophagic flux, indicated by an increased number of lysosomes and decreased degradation of long-lived proteins, may be the mechanism by which cell death is induced by chloroquine in encysting Acanthamoeba. These results suggest a potential novel therapeutic application of chloroquine as an anti-Acanthamoeba drug. Our findings also suggest that targeting autophagy could be a therapeutic strategy against Acanthamoeba infection.

Translational suppression of HIF-1α by miconazole through the mTOR signaling pathway.

BACKGROUND: Miconazole is an imidazole antifungal agent that has amply been used in the treatment of superficial mycosis. Preliminary data indicate that miconazole may also induce anticancer effects. As yet, however, little is known about the therapeutic efficacy of miconazole on cancer and the putative mechanism(s) involved. Here, we show that miconazole suppresses hypoxia inducible factor-1α (HIF-1α) protein translation in different cancer-derived cells. METHODS: The effect of miconazole on HIF-1α expression was examined by Western blotting and reverse transcriptase polymerase chain reaction assays in human U87MG and MCF-7 glioma and breast cancer-derived cell lines, respectively. The transcriptional activity of the HIF-1 complex was confirmed using a luciferase assay. To assess whether angiogenic factors are increased under hypoxic conditions in these cells, vascular endothelial growth factor (VEGF) levels were measured by ELISA. Metabolic labeling was performed to examine HIF-1α protein translation and global protein synthesis. The role of the mammalian target of rapamycin (mTOR) signaling pathway was examined to determine translation regulation of HIF-1α after miconazole treatment. RESULTS: Miconazole was found to suppress HIF-1α protein expression through post-transcriptional regulation in U87MG and MCF-7 cells. The suppressive effect of HIF-1α protein synthesis was found to be due to inhibition of mTOR. Miconazole significantly inhibited the transcriptional activity of the HIF-1 complex and the expression of its target VEGF. Moreover, miconazole was found to suppress global protein synthesis by inducing phosphorylation of the translation initiation factor 2α (eIF2α). CONCLUSION: Our data indicate that miconazole plays a role in translational suppression of HIF-1α. We suggest that miconazole may represent a novel therapeutic option for the treatment of cancer.

Identification of lysosomotropic compounds based on the distribution and size of lysosomes.

Lysosomal accumulation of drugs with their specific physicochemical properties is of key importance to drug distribution in the body. Several attempts have been made to treat various human diseases by employing the accumulation of lysosomal drugs, and many methods to identify lysosomal accumulation of drugs have been proposed. Among those, the use of high-content screening has increased tremendously because of improved efficiency and accuracy as well as the development of automatic image acquisition and analytical techniques. Conventional methods to identify lysosomal accumulation of drugs by evaluating changes in the lysosomal area are unable to maximize the advantages of phenotypic high-content screening. Lysosomal distribution and the size of lysosomes are affected by lysosomal accumulating drugs. Therefore, we present image acquisition conditions and analytical methods to utilize lysosomal distribution and size as parameters for identifying lysosomal accumulating drugs. These two parameters will help to improve the reliability of the screening methods for identifying lysosomal accumulation of drugs by maximizing usage of information from image-based screening.

Minocycline inhibits angiogenesis in vitro through the translational suppression of HIF-1α.

Minocycline was recently found to be effective against cancer. However, the precise molecular mechanisms of minocycline in cancer are poorly understood. Hypoxia-inducible factor-1 (HIF-1, a heterodimeric transcription factor composed of HIF-1α and ß) activates the transcription of genes that are involved in angiogenesis in cancer. In this study, we found that minocycline significantly inhibits HIF-1α protein expression and suppresses HIF-1 transcriptional activity. The tube formation assay showed that minocycline has anti-angiogenic activity and suppresses hypoxia-induced vascular endothelial growth factor (VEGF) expression. The metabolic labeling assay showed that minocycline reduces HIF-1α protein translation and global protein synthesis. In addition, minocycline suppresses mTOR signaling and increases the phosphorylation of eIF2α, which is known to be related to the translational regulation of HIF-1α expression. These findings collectively indicate that minocycline is a potential inhibitor of HIF-1α and provide new insight into the discovery of drugs for cancer treatment.