Use of biochemical markers to monitor changes in bone turnover in cynomolgus monkeys

The ovariectomized old cynomolgus monkey is a recognized model of human osteoporosis, and the same species can be used for the assessment of the efficacy and potential toxicity of agents intended to prevent or treat osteoporosis. Several assays have been developed that can measure the same biochemical markers of bone turnover as are used in human patients for the diagnosis and treatment follow-up of bone-related diseases, including osteoporosis. The aim of the present study was to describe the results obtained with these assays in normal control monkeys, their variations with age and sex, and their sensitivity in monitoring the bone turnover induced by ovariectomy in old skeletally mature cynomolgus monkeys. Seven old cynomolgus monkeys were bilaterally ovariectomized and 13 age-matched monkeys were sham-operated. Bone mineral density and biochemical markers were measured before and at regular intervals after surgery for up to 20 months. Total alkaline phosphatase (total ALP), bone-specific alkaline phosphatase isoenzyme (bone ALP) and osteocalcin (OC) were highly correlated to the decrease in bone mineral density (BMD) induced by ovariectomy. Deoxypyridinoline (DPD) measured by enzyme-linked immunoassay was insensitive to the bone resorption induced by ovariectomy, but cross-linked N-telopeptide (NTX-I) was higher in ovariectomized monkeys than in control monkeys. These results demonstrate that reliable biochemical parameters are available to adequately monitor and provide insight into osteoclastic bone resorption and osteoblastic bone formation, the two components of bone turnover in this animal model, and can thus be used to assess the efficacy and toxicity of potential therapeutic agents.     

A Novel Assay for Measurement of Membrane‐Protein Surface Expression using a β‐lactamase Reporter

The trafficking of membrane proteins is dynamic and contributes to the homeostatic control of their cell surface localization and their function in signal transduction. Therefore, it is important to have sensitive techniques that allow measurement of surface expression. The current assays for such measurement are time consuming and low throughput. Here, we describe a quantitative, one‐step and potentially high‐throughput assay, using the β‐lactamase enzyme (βlac) as a reporter, for measurement of surface expression of proteins. In this assay, the βlac is fused to the extracellular portion of the plasma membrane protein of interest. To selectively measure surface expression, a cell‐impermeable substrate of βlac, nitrocefin, is used. We demonstrate the utility of the βlac assay using well‐established paradigms of internalization and molecular chaperoning, applied to two G‐protein‐coupled receptors and a monoamine transporter. Considering its simplicity and low cost, this assay could become a standard technique in the measurement of protein surface expression .      

Pre-Eclampsia Increases the Risk of Postpartum Haemorrhage: A Nationwide Cohort Study in The Netherlands

Background

Postpartum haemorrhage is a leading cause of maternal morbidity and mortality worldwide. Identifying risk indicators for postpartum haemorrhage is crucial to predict this life threatening condition. Another major contributor to maternal morbidity and mortality is pre-eclampsia. Previous studies show conflicting results in the association between pre-eclampsia and postpartum haemorrhage. The primary objective of this study was to investigate the association between pre-eclampsia and postpartum haemorrhage. Our secondary objective was to identify other risk indicators for postpartum haemorrhage in the Netherlands.

Methods

A nationwide cohort was used, containing prospectively collected data of women giving birth after 19 completed weeks of gestation from January 2000 until January 2008 (n =  1 457 576). Data were extracted from the Netherlands Perinatal Registry, covering 96% of all deliveries in the Netherlands. The main outcome measure, postpartum haemorrhage, was defined as blood loss of ≥1000 ml in the 24 hours following delivery. The association between pre-eclampsia and postpartum haemorrhage was investigated with uni- and multivariable logistic regression analyses.

Results

Overall prevalence of postpartum haemorrhage was 4.3% and of pre-eclampsia 2.2%. From the 31 560 women with pre-eclampsia 2 347 (7.4%) developed postpartum haemorrhage, compared to 60 517 (4.2%) from the 1 426 016 women without pre-eclampsia (odds ratio 1.81; 95% CI 1.74 to 1.89). Risk of postpartum haemorrhage in women with pre-eclampsia remained increased after adjusting for confounders (adjusted odds ratio 1.53; 95% CI 1.46 to 1.60).

Conclusion

Women with pre-eclampsia have a 1.53 fold increased risk for postpartum haemorrhage. Clinicians should be aware of this and use this knowledge in the management of pre-eclampsia and the third stage of labour in order to reach the fifth Millenium Developmental Goal of reducing maternal mortality ratios with 75% by 2015.     

Malaria Burden and Artemisinin Resistance in the Mobile and Migrant Population on the Thai–Myanmar Border, 1999–2011: An Observational Study

Background

The Shoklo Malaria Research Unit has been working on the Thai–Myanmar border for 25 y providing early diagnosis and treatment (EDT) of malaria. Transmission of Plasmodium falciparum has declined, but resistance to artesunate has emerged. We expanded malaria activities through EDT and evaluated the impact over a 12-y period.

Methods and Findings

Between 1 October 1999 and 30 September 2011, the Shoklo Malaria Research Unit increased the number of cross-border (Myanmar side) health facilities from two to 11 and recorded the number of malaria consultations. Changes in malaria incidence were estimated from a cohort of pregnant women, and prevalence from cross-sectional surveys. In vivo and in vitro antimalarial drug efficacy were monitored. Over this period, the number of malaria cases detected increased initially, but then declined rapidly. In children under 5 y, the percentage of consultations due to malaria declined from 78% (95% CI 76–80) (1,048/1,344 consultations) to 7% (95% CI 6.2–7.1) (767/11,542 consultations), p<0.001. The ratio of P. falciparum/P. vivax declined from 1.4 (95% CI 1.3–1.4) to 0.7 (95% CI 0.7–0.8). The case fatality rate was low (39/75,126; 0.05% [95% CI 0.04–0.07]). The incidence of malaria declined from 1.1 to 0.1 episodes per pregnant women-year. The cumulative proportion of P. falciparum decreased significantly from 24.3% (95% CI 21.0–28.0) (143/588 pregnant women) to 3.4% (95% CI 2.8–4.3) (76/2,207 pregnant women), p<0.001. The in vivo efficacy of mefloquine-artesunate declined steadily, with a sharp drop in 2011 (day-42 PCR-adjusted cure rate 42% [95% CI 20–62]). The proportion of patients still slide positive for malaria at day 3 rose from 0% in 2000 to reach 28% (95% CI 13–45) (8/29 patients) in 2011.

Conclusions

Despite the emergence of resistance to artesunate in P. falciparum, the strategy of EDT with artemisinin-based combination treatments has been associated with a reduction in malaria in the migrant population living on the Thai–Myanmar border. Although limited by its observational nature, this study provides useful data on malaria burden in a strategically crucial geographical area. Alternative fixed combination treatments are needed urgently to replace the failing first-line regimen of mefloquine and artesunate. Please see later in the article for the Editors'' Summary     

1H, 15N and 13C Backbone resonance assignments of a conformational mutant of the adhesion protein ∆-Bd37 from Babesia divergens

We report here the resonance assignment of EDK-?-Bd37, conformational mutant potentially displaying the “open” conformation of Bd37, a 25 kDa surface protein from the Apicomplexa parasite Babesia divergens that could undergo drastic conformational changes during erythrocyte invasion.     

Monocyte Chemoattractant Protein-1/CCL2 Produced by Stromal Cells Promotes Lung Metastasis of 4T1 Murine Breast Cancer Cells

MCP-1/CCL2 plays an important role in the initiation and progression of cancer. Since tumor cells produce MCP-1, they are considered to be the main source of this chemokine. Here, we examined whether MCP-1 produced by non-tumor cells affects the growth and lung metastasis of 4T1 breast cancer cells by transplanting them into the mammary pad of WT or MCP-1^−/− mice. Primary tumors at the injected site grew similarly in both mice; however, lung metastases were markedly reduced in MCP-1^−/− mice, with significantly longer mouse survival. High levels of MCP-1 mRNA were detected in tumors growing in WT, but not MCP-1^−/− mice. Serum MCP-1 levels were increased in tumor-bearing WT, but not MCP-1^−/− mice. Transplantation of MCP-1^−/− bone marrow cells into WT mice did not alter the incidence of lung metastasis, whereas transplantation of WT bone marrow cells into MCP-1^−/− mice increased lung metastasis. The primary tumors of MCP-1^−/− mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of MCP-1, and intravenous injection of 4T1 cells producing a high level of MCP-1 resulted in increased tumor foci in the lung of WT and MCP-1^−/− mice. Thus, stromal cell-derived MCP-1 in the primary tumors promotes lung metastasis of 4T1 cells, but tumor cell-derived MCP-1 can also contribute once tumor cells enter the circulation. A greater understanding of the source and role of this chemokine may lead to novel strategies for cancer treatment.     

Adding parasites to the guppy-predation story: insights from field surveys

Studies of phenotypic variation in nature often consider only a single potential selective agent. In such cases, it remains an open question as to whether variation attributed to that single measured agent might be influenced by some other unmeasured agent. Previous research has shown that phenotypic variation in the Trinidadian guppy (Poecilia reticulata) is strongly influenced by predation regime, and we here ask whether parasitism might represent an additional important selective agent shaping this variation. We performed a field survey of 26 natural guppy populations of known predation regime in northern Trinidad. We quantified levels of parasitism of guppies by the monogenean ecotoparasite, Gyrodactylus, and examined whether this parasite was associated with guppy body size or male colour. Spatial variation in Gyrodactylus parasitism was consistent between years, and parasite prevalence was generally, but not always, higher at high-predation sites than at low-predation sites. Consistent with previous work, predation regime was related to guppy size and some aspects of male colour, whereas parasitism showed few and only minor associations with the same traits. Moreover, a consideration of parasitism did not alter any interpretations regarding associations between guppy traits and predation regimes. These results suggest that parasitism, at least as quantified in the present study, does not play a major role in shaping variation in guppy body size or colour. Nevertheless, considerable variation in these traits, even within a predation regime, suggests the likely importance of other selective agents beyond just predation regime.