Stability of inflammation markers in human blood collected using volumetric absorptive microsampling (VAMS) under typical laboratory storage temperatures.

Dried blood spots (DBS) collected on filter paper such as Guthrie cards are stored for years at room temperature. The assumption is that once dried, the samples remain stable and quantifiable indefinitely since the metabolites these were initially designed to measure, are known for their extended stability. The concentration of other blood proteins such as cytokines, however, are known to vary with storage even in liquid samples stored at -80 °C for extended periods of time. We sought to determine if cytokines are stable for up to 5 months when stored as a dried blood sample using volumetric absorptive microsampling (VAMS) devices. To test this, blood was collected from 4 healthy participants, spiked with recombinant cytokines, and collected into 30 µL VAMS devices. These prepared VAMS devices were stored at room temperature, 4 °C, or -20 °C for up to 5 months and matching VAMS liquid extracts were stored at -80 °C for the same period of time. At each timepoint, the samples were extracted from the VAMS devices and the extracts were analysed by Luminex® for quantification of up to 31 cytokines. These methods were also tested in a remote clinical study over a period of up to 8 months. Cytokine analysis revealed that room temperature, the current standard for DBS and VAMS storage, performed the poorest out of all storage temperatures with significant losses in 13/21 analytes compared to 4 °C at 5 months. Storage at 4 °C or colder performed well for the majority of analytes tested, however out of those, the optimal storage temperature differed for each analyte. There were a small number of analytes that performed poorly regardless of storage conditions and for fractalkine, this was found to be caused by inefficient recovery during extraction. Cytokine concentrations from finger-prick samples were also found to be much more variable that those in venous blood samples. Our results highlight the need to understand the stability of analytes of interest before committing to longitudinal collection and storage of samples in VAMS devices. These data give confidence that storage at 4 °C or colder was beneficial for cytokine stability. Wherein 25/31 cytokines were quantifiably stable at -20 °C when stored for 3 months and 17/21 were quantifiably stable after 5 months when stored at 4 °C.

Critical illness and multi-organ failure following topical application of skin-lightening preparation.

Topical application of skin-lightening cream is increasingly undertaken in many non-Caucasian populations for cultural and social reasons. It is a rare cause of poisoning that has potential to lead to significant harm due to skin damage and systemic absorption of cream following application over prolonged periods of time. This case report describes for the development of multi-organ failure in an adult due to salicylate toxicity after whole-body application of a skin-lightening cream for 24 h. It highlights the need for vigilance and awareness of the toxic potential of topical salicylates.

Phylogenetic characterisation of circulating, clinical influenza isolates from Bali, Indonesia: preliminary report from the BaliMEI project.

BACKGROUND: Human influenza represents a major public health concern, especially in south-east Asia where the risk of emergence and spread of novel influenza viruses is particularly high. The BaliMEI study aims to conduct a five year active surveillance and characterisation of influenza viruses in Bali using an extensive network of participating healthcare facilities. METHODS: Samples were collected during routine diagnostic treatment in healthcare facilities. In addition to standard clinical and molecular methods for influenza typing, next generation sequencing and subsequent de novo genome assembly were performed to investigate the phylogeny of the collected patient samples. RESULTS: The samples collected are characteristic of the seasonally circulating influenza viruses with indications of phylogenetic links to other samples characterised in neighbouring countries during the same time period. CONCLUSIONS: There were some strong phylogenetic links with sequences from samples collected in geographically proximal regions, with some of the samples from the same time-period resulting to small clusters at the tree-end points. However this work, which is the first of its kind completely performed within Indonesia, supports the view that the circulating seasonal influenza in Bali reflects the strains circulating in geographically neighbouring areas as would be expected to occur within a busy regional transit centre.

Best practices to prevent transmission and control outbreaks of hand, foot, and mouth disease in childcare facilities: a systematic review.

INTRODUCTION: Hand, foot, and mouth disease continues to cause seasonal epidemics in the Asia-Pacific Region. Since the current Enterovirus 71 vaccines do not provide cross-protection for all Enterovirus species that cause hand, foot, and mouth disease, there is an urgent need to identify appropriate detection tools and best practice to prevent its transmission and to effectively control its outbreaks. This systematic review aimed to identify characteristics of outbreak and assess the impact and effectiveness of detection tools and public health preventive measures to interrupt transmission. The findings will be used to recommend policy on the most effective responses and interventions in Hong Kong to effectively minimise and contain the spread of the disease within childcare facilities. METHODS: We searched the following databases for primary studies written in Chinese or English: MEDLINE, EMBASE, Global Health, WHO Western Pacific Region Index Medicus database, China National Knowledge Infrastructure Databases, and Chinese Scientific Journals Database. Studies conducted during or retrospective to outbreaks of hand, foot, and mouth disease caused by Enterovirus 71 from 1980 to 2012 within childcare facilities and with a study population of 0 to 6 years old were included. RESULTS: Sixteen studies conducted on outbreaks in China showed that hand, foot, and mouth disease spread rapidly within the facility, with an outbreak length of 4 to 46 days, especially in those with delayed notification (after 24 hours) of clustered outbreak (with five or more cases discovered within the facility) to the local Center for Disease Control and Prevention and delayed implementation of a control response. The number of classes affected ranged from 1 to 13, and the attack rate for children ranged from 0.97% to 28.18%. CONCLUSIONS: Communication between key stakeholders about outbreak confirmation, risk assessment, and surveillance should be improved. Effective communication facilitates timely notification (within 24 hours) of clustered outbreaks to a local Center for Disease Control and Prevention. Timely implementation of a control response is effective in minimising incidence and length of an outbreak in childcare facilities. The government should provide incentives for childcare facilities to train infection control specialists who can serve as the first contact, knowledge, and communication points, as well as facilitate exchange of information and provision of support across stakeholders during a communicable disease epidemic.

Sub-nanosecond time-resolved near-field scanning magneto-optical microscope.

We report on the development of a new magnetic microscope, time-resolved near-field scanning magneto-optical microscope, which combines a near-field scanning optical microscope and magneto-optical contrast. By taking advantage of the high temporal resolution of time-resolved Kerr microscope and the sub-wavelength spatial resolution of a near-field microscope, we achieved a temporal resolution of ∼50 ps and a spatial resolution of <100 nm. In order to demonstrate the spatiotemporal magnetic imaging capability of this microscope, the magnetic field pulse induced gyrotropic vortex dynamics occurring in 1 µm diameter, 20 nm thick CoFeB circular disks has been investigated. The microscope provides sub-wavelength resolution magnetic images of the gyrotropic motion of the vortex core at a resonance frequency of ∼240 MHz.

Assessing the effects of HIV/AIDS and TB disease control programmes on health systems in low- and middle-income countries of Southeast Asia: a semi-systematic review of the literature.

OBJECTIVE: To systematically review the literature on if and how HIV/AIDS and TB programmes have impacted on general healthcare systems in Association of Southeast Asian Nations (ASEAN) countries. METHODS: Medline, Embase, Global Health and CINHAL were searched for English language literature published between 1st January 2003 and 31st March 2011. Papers included had to focus on: HIV and/or TB control programmes; the low- and-middle-income ASEAN countries; and factors related to any health systems functions. The effects were examined along six system functions: Stewardship and Governance; Financing; Planning; Service Delivery; Monitoring and Evaluation; and Demand Generation. A comprehensive thematic analytical tool aligned with the above six health systems functions was developed to support data extraction and analysis. FINDINGS: 88 papers met the inclusion criteria. Most programme effects highlighted were related with health service delivery. The other five health system functions were seldom scrutinized, and each covered by less than a quarter of papers. Overall 69% of effects highlighted were positive effects whereas 31% were negative. CONCLUSION: There was a paucity of robust evidence. Effects on health systems were rarely a focus of research protocols but more often a minor component in the Results/Discussion sections. Particular attention should be paid by Global Health Initiatives to the negative effects that emerged from this study, such as the development of parallel systems, specific incentives not available to the general health systems, and lack of integration of services with private healthcare providers.

Health service resource needs for pandemic influenza in developing countries: a linked transmission dynamics, interventions and resource demand model.

We used a mathematical model to describe a regional outbreak and extrapolate the underlying health-service resource needs. This model was designed to (i) estimate resource gaps and quantities of resources needed, (ii) show the effect of resource gaps, and (iii) highlight which particular resources should be improved. We ran the model, parameterized with data from the 2009 H1N1v pandemic, for two provinces in Thailand. The predicted number of preventable deaths due to resource shortcomings and the actual resource needs are presented for two provinces and for Thailand as a whole. The model highlights the potentially huge impact of health-system resource availability and of resource gaps on health outcomes during a pandemic and provides a means to indicate where efforts should be concentrated to effectively improve pandemic response programmes.

Hyperalgesia, synovitis and multiple biomarkers of inflammation are suppressed by interleukin 1 inhibition in a novel animal model of gouty arthritis.

BACKGROUND: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis. OBJECTIVE: To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers. METHODS: MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation. RESULTS: Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling. CONCLUSIONS: IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.

Vascular endothelial growth factor is up-regulated after status epilepticus and protects against seizure-induced neuronal loss in hippocampus.

Vascular endothelial growth factor (VEGF) is a protein factor which has been found to play a significant role in both normal and pathological states. Its role as an angiogenic factor is well-established. More recently, VEGF has been shown to protect neurons from cell death both in vivo and in vitro. While VEGF's potential as a protective factor has been demonstrated in hypoxia-ischemia, in vitro excitotoxicity, and motor neuron degeneration, its role in seizure-induced cell loss has received little attention. A potential role in seizures is suggested by Newton et al.'s [Newton SS, Collier EF, Hunsberger J, Adams D, Terwilliger R, Selvanayagam E, Duman RS (2003) Gene profile of electroconvulsive seizures: Induction of neurotrophic and angiogenic factors. J Neurosci 23:10841-10851] finding that VEGF mRNA increases in areas of the brain that are susceptible to cell loss after electroconvulsive-shock induced seizures. Because a linear relationship does not always exist between expression of mRNA and protein, we investigated whether VEGF protein expression increased after pilocarpine-induced status epilepticus. In addition, we administered exogenous VEGF in one experiment and blocked endogenous VEGF in another to determine whether VEGF exerts a neuroprotective effect against status epilepticus-induced cell loss in one vulnerable brain region, the rat hippocampus. Our data revealed that VEGF is dramatically up-regulated in neurons and glia in hippocampus, thalamus, amygdala, and neocortex 24 h after status epilepticus. VEGF induced significant preservation of hippocampal neurons, suggesting that VEGF may play a neuroprotective role following status epilepticus.

VEGF trap as a novel antiangiogenic treatment currently in clinical trials for cancer and eye diseases, and VelociGene- based discovery of the next generation of angiogenesis targets.

The concept that tumors can be controlled by directly targeting their vascular supply has finally come of age, because clinical trials using a humanized monoclonal antibody that blocks VEGF have demonstrated exciting efficacy in cancer patients, as well as in vascular eye diseases that can lead to blindness. However, data suggest that these current regimens may not provide complete VEGF inhibition and, thus, that the maximum therapeutic potential of VEGF blockade has not yet been achieved. We describe the status of a very potent and high-affinity VEGF blocker, termed the VEGF Trap, that may provide the opportunity to maximize the potential of VEGF blockade in cancer as well as in vascular eye diseases. We also describe use of the VEGF Trap as a research tool, when coupled to high-throughput mouse genetics approaches such as VelociGene that can be exploited in strategies to discover and validate the next generation of angiogenesis targets.

Angiopoietic factors and retinopathy in pregnancies complicated with Type 1 diabetes.

AIMS: To evaluate the role of systemic angiopoietic factors in the progression of diabetic retinopathy during pregnancy. METHODS: In a prospective study of 26 pregnant women with diabetes and eight non-diabetic pregnant women, retinopathy was graded from fundus photographs. Plasma levels of angiopoietin-1, angiopoietin-2, human vascular endothelial growth factor A (hVEGF-A), and total soluble receptor of vascular endothelial growth factor (sVEGF) receptor-1 were measured during the first and third trimester and 3 months postpartum. RESULTS: In diabetic women, levels of angiopoietin-2 were 26.5 ng/ml (12.1-47.7) (median and range) during the first trimester, 2.9 ng/ml (0.6-3.5) during the third trimester, and 0.5 ng/ml (0.3-0.7) 3 months postpartum, compared with 44.3 (38.3-61.9), 5.7 (3.1-8.4) and 0.9 (0.6-4.9) ng/ml, respectively, in non-diabetic women (P = 0.002 between groups). Levels of angiopoietin-1 and sVEGF receptor-1 did not differ between the groups. Postpartum hVEGF-A levels were lowest in women with progression of retinopathy. In logistic regression analyses, progression of retinopathy during pregnancy was not explained by the levels of the angiopoietic factors. CONCLUSIONS: The circulating levels of angiopoietic factors in pregnant diabetic women were either lower than (Ang-2) or similar to (Ang-1, hVEGF-A, VEGFR-1) those levels observed in non-diabetic pregnant women. The levels of angiopoietic factors measured here appear not to be connected with the progression of retinopathy during pregnancy.

VEGF-initiated blood-retinal barrier breakdown in early diabetes.

PURPOSE: The objectives of this study were to (1) determine whether endogenous vascular endothelial growth factor (VEGF) triggers diabetic blood-retinal barrier breakdown, and (2) identify the site as well as phenotype of the hyperpermeable diabetic retinal vessels. METHODS: Retinal VEGF mRNA levels were quantified in 1-week diabetic rats using the RNase protection assay. VEGF bioactivity was blocked via the systemic administration of a highly specific VEGF-neutralizing soluble Flt/F(c) construct (VEGF TrapA(40)). An inactive IL6 receptor/F(c) construct (IL6R Trap) was used as an isotype control. Blood-retinal barrier breakdown was quantified using the Evans blue technique and was spatially localized with fluorescent microspheres. RESULTS: Retinal VEGF mRNA levels in 1-week diabetic animals were 3.2-fold higher than in nondiabetic controls (P < 0.0001). Similarly, retinal vascular permeability in 8-day diabetic animals was 1.8-fold higher than in normal nondiabetic controls (P < 0.05). Diabetes-induced blood-retinal barrier breakdown was dose-dependently inhibited with VEGF TrapA(40), with 25 mg/kg producing complete inhibition of the diabetes-induced increases (P < 0.05). Blood-retinal barrier breakdown in diabetic animals treated with solvent alone or IL6R Trap did not differ significantly from untreated diabetic animals (P > 0.05). Spatially, early blood-retinal barrier breakdown was localized to the retinal venules and capillaries of the superficial retinal vasculature. CONCLUSIONS: Early blood-retinal barrier breakdown in experimental diabetes is VEGF dependent and is restricted, in part, to the venules and capillaries of the superficial inner retinal vasculature. VEGF inhibition should prove a useful therapeutic approach in the treatment of early diabetic blood-retinal barrier breakdown.

Luteal angiogenesis: prevention and intervention by treatment with vascular endothelial growth factor trap(A40).

The possibility of stimulating or inhibiting paracrine factors regulating angiogenesis may lead to new approaches for the treatment of pathological conditions of the female reproductive tract. We examined the effects of a clinical candidate, a soluble truncated form of the Flt-1 receptor, vascular endothelial growth factor trap(A40) (VEGF trap), in a primate model to determine its ability to prevent the onset of luteal angiogenesis or intervene with the on-going process. Marmosets were treated from the day of ovulation until luteal day 3 (prevention regimen) or on luteal day 3 for 1 day (intervention regimen). Effects of VEGF inhibition were studied by obtaining a proliferation index using bromodeoxyuridine incorporation, quantifying endothelial cell area using CD31, and assessing luteal function by plasma progesterone. After both treatments, intense luteal endothelial proliferation was suppressed, a concomitant decrease in endothelial cell area confirmed the inhibition of vascular development, and a marked fall in plasma progesterone levels showed that luteal function was compromised. In situ hybridization was used to localize and quantify compensatory effects on the expression of angiogenic genes. VEGF messenger ribonucleic acid (mRNA) expression in luteal cells was increased, whereas expression of its receptor, Flt, was decreased. Inhibition of VEGF resulted in localized increased expression of angiopoietin-2 mRNA and its receptor, Tie-2. The results show that the VEGF trap can prevent luteal angiogenesis and inhibit the established process with resultant suppression of luteal function. Luteal Flt mRNA expression is dependent upon VEGF, and VEGF inhibition results in abortive increases in expression of VEGF, angiopoietin-2, and Tie-2.

Vascular-specific growth factors and blood vessel formation.

A recent explosion in newly discovered vascular growth factors has coincided with exploitation of powerful new genetic approaches for studying vascular development. An emerging rule is that all of these factors must be used in perfect harmony to form functional vessels. These new findings also demand re-evaluation of therapeutic efforts aimed at regulating blood vessel growth in ischaemia, cancer and other pathological settings.

Angiopoietin-1 is an antipermeability and anti-inflammatory agent in vitro and targets cell junctions.

Inflammation is a basic pathological mechanism that underlies many diseases. An important component of the inflammatory response is the passage of plasma components and leukocytes from the blood vessel into the tissues. The endothelial monolayer lining blood vessels reacts to stimuli such as thrombin or vascular endothelial growth factor by changes in cell-cell junctions, an increase in permeability, and the leakage of plasma components into tissues. Other stimuli, such as tumor necrosis factor-alpha (TNF-alpha), are responsible for stimulating the transmigration of leukocytes. Here we show that angiopoietin-1, a cytokine essential in fetal angiogenesis, not only supports the localization of proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) into junctions between endothelial cells and decreases the phosphorylation of PECAM-1 and vascular endothelial cadherin, but it also strengthens these junctions, as evidenced by a decrease in basal permeability and inhibition of permeability responses to thrombin and vascular endothelial growth factor. Furthermore, angiopoietin-1 inhibits TNF-alpha-stimulated leukocyte transmigration. Angiopoietin-1 may thus have a major role in maintaining the integrity of endothelial monolayers.

Angiopoietin-1 protects the adult vasculature against plasma leakage.

Pathological increases in vascular leakage lead to edema and swelling, causing serious problems in brain tumors, in diabetic retinopathy, after strokes, during sepsis and also in inflammatory conditions such as rheumatoid arthritis and asthma. Although many agents and disease processes increase vascular leakage, no known agent specifically makes vessels resistant to leaking. Vascular endothelial growth factor (VEGF) and the angiopoietins function together during vascular development, with VEGF acting early during vessel formation, and angiopoietin-1 acting later during vessel remodeling, maturation and stabilization. Although VEGF was initially called vascular permeability factor, there has been less focus on its permeability actions and more effort devoted to its involvement in vessel growth and applications in ischemia and cancer. Recent transgenic approaches have confirmed the profound permeability effects of VEGF (refs. 12-14), and have shown that transgenic angiopoietin-1 acts reciprocally as an anti-permeability factor when provided chronically during vessel formation, although it also profoundly affects vascular morphology when thus delivered. To be useful clinically, angiopoietin-1 would have to inhibit leakage when acutely administered to adult vessels, and this action would have to be uncoupled from its profound angiogenic capabilities. Here we show that acute administration of angiopoietin-1 does indeed protect adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents.

Brain-derived neurotrophic factor transgenic mice exhibit passive avoidance deficits, increased seizure severity and in vitro hyperexcitability in the hippocampus and entorhinal cortex.

Transgenic mice overexpressing brain-derived neurotrophic factor from the beta-actin promoter were tested for behavioral, gross anatomical and physiological abnormalities. Brain-derived neurotrophic factor messenger RNA overexpression was widespread throughout brain. Overexpression declined with age, such that levels of overexpression decreased sharply by nine months. Brain-derived neurotrophic factor transgenic mice had no gross deformities or behavioral abnormalities. However, they showed a significant passive avoidance deficit. This deficit was dependent on continued overexpression, and resolved with age as brain-derived neurotrophic factor transcripts decreased. In addition, the brain-derived neurotrophic factor transgenic mice showed increased seizure severity in response to kainic acid. Hippocampal slices from brain-derived neurotrophic factor transgenic mice showed hyperexcitability in area CA3 and entorhinal cortex, but not in dentate gyrus. Finally, area CA1 long-term potentiation was disrupted, indicating abnormal plasticity. Our data suggest that overexpression of brain-derived neurotrophic factor in the brain can interfere with normal brain function by causing learning impairments and increased excitability. The results also support the hypothesis that excess brain-derived neurotrophic factor could be pro-convulsant in the limbic system.

Co-infusion with a TrkB-Fc receptor body carrier enhances BDNF distribution in the adult rat brain.

Fusion proteins comprising the Fc domain of human IgG and extracellular domains of receptor tyrosine kinases can neutralize the activity of their cognate ligands when administered in molar excess. We have generated a fusion protein using the ectodomain of TrkB (TrkB-Fc). Although the ability of TrkB-Fc to neutralize the activity of brain-derived neurotrophic factor (BDNF) in vitro has been demonstrated, there have been no conclusive demonstrations of its ability to neutralize the activity of BDNF in vivo. We co-infused TrkB-Fc with BDNF into the cortex and hippocampus of adult rats to determine whether TrkB-Fc would interfere with the ability of BDNF to upregulate neuropeptide Y (NPY). We report here that rather than neutralizing the activity of exogenous BDNF, co-infusion with the TrkB-Fc fusion protein greatly increased the volume of tissue in which neuropeptide Y immunostaining was upregulated. In addition, TrkB-Fc greatly enhanced BDNF's distribution through adult brain parenchyma. TrkB-Fc also markedly increased the otherwise limited diffusion of BDNF into brain parenchyma following intraventricular infusion. These results show that rather than neutralizing or sequestering BDNF, the TrkB-Fc, at close to molar equivalence to BDNF, can function as a carrier for BDNF and thus enhance the delivery or penetration of this polypeptide into the brain.