Sensitive RNase A detection and intracellular imaging using a natural compound-assisted tetrahedral DNA nanoprobe.

We report an intracellular imaging and assay nanoplatform for RNase A using a DNA tetrahedron-based fluorescent probe as a substrate. Importantly, a natural compound was used as an RNase A activity stimulator to improve the sensitivity. This platform provides an alternative for the diagnosis and prognosis of RNase A-related diseases and drug screening.

Homogeneous Electrochemiluminescence Biosensor for the Detection of RNase A Activity and Its Inhibitor.

Ribonuclease A (RNase A) is increasingly considered as a biomarker for tumor diagnosis, and it is of great significance to develop an ultrasensitive, cost-effective assay for RNase A detection. Electrochemiluminescence (ECL) technology has distinctive advantages in the development of biosensors for diverse targets. However, most of the ECL biosensors require the complex process of electrode modification, which is laborious and time consuming. In this work, an immobilization-free homogeneous ECL assay was developed for the highly sensitive detection of RNase A activity for the first time. On the basis of the fact that RNase A can specifically hydrolyze RNA at the site of ribonucleotide uracil (rU), a rU-containing chimeric DNA probe is designed and labeled with Ru(bpy)32+ (act as ECL indicator). The chimeric DNA probe hardly diffuses to the surface of negatively charged indium tin oxide (ITO) electrode due to the strong electrostatic repulsion between the negatively charged DNA and ITO electrode, resulting in a weak ECL signal detected. When the RNase A is present, the chimeric DNA probe is hydrolyzed into small fragments, which contains little negative charge and can diffuse easily to the ITO electrode surface due to the decreased electrostatic repulsion. In this case, an enhanced ECL signal can be detected. Under the optimal conditions, there is a linear relationship between the ECL signal and the concentration of RNase A in the range of 0.001-0.10 ng/mL, and the detection limit is 0.2 pg/mL. In addition, the proposed ECL sensing system is also applied to detect the RNase A inhibitor, taking As3+ as an example. The proposed homogeneous ECL sensing system provides a new approach for the highly sensitive and convenient detection of RNase A as well as other ribonucleases only by redesigning a responding chimeric DNA probe.

Analysis of the Ribonuclease A Superfamily of Antimicrobial Peptides in Patients Undergoing Chronic Peritoneal Dialysis.

Infectious peritonitis is a common complication in patients undergoing chronic peritoneal dialysis (PD), limiting the duration of PD as a modality for renal replacement therapy and increasing patient morbidity and mortality. Antimicrobial peptides (AMPs) serve critical roles in mucosal defense, but their expression and activity during peritonitis are poorly understood. We hypothesized that AMPs belonging to the Ribonuclease (RNase) A Superfamily are present in peritoneal fluid and increase during peritonitis in patients undergoing chronic PD. In the absence of peritonitis, we detected RNase 3, RNase 6, and RNase 7 in cell-free supernatants and viable cells obtained from peritoneal fluid of chronic PD patients. The cellular sources of these RNases were eosinophils (RNase 3), macrophages (RNase 6), and mesothelial cells (RNase 7). During peritonitis, RNase 3 increased 55-fold and RNase 7 levels increased 3-fold on average, whereas RNase 6 levels were unchanged. The areas under the receiver-operating characteristic curves for RNase 3 and RNase 7 were 0.99 (95% confidence interval (CI): 0.96-1.0) and 0.79 (95% CI: 0.64-0.93), respectively, indicating their potential as biomarkers of peritonitis. Discrete omental reservoirs of these RNases were evident in patients with end stage kidney disease prior to PD initiation, and omental RNase 3 reactive cells increased in patients undergoing PD with a history of peritonitis. We propose that constitutive and inducible pools of antimicrobial RNases form a network to shield the peritoneal cavity from microbial invasion in patients undergoing chronic PD.

Repeated arctigenin treatment produces antidepressant- and anxiolytic-like effects in mice.

Depression is the root of various diseases. It is one of the most debilitating conditions globally. Antidepressant drugs are usually the first-line of depression treatment. Arctigenin (ARC), one of active ingredient of Arctium lappa L, has been found to exert neuroprotective, anti-decrepitude, and anti-inflammatory activities. Thus, the aim of this study was to investigate the potential antidepressant- and anxiolytic-like effects of ARC using acute and chronic mild stress (CMS) mice model. ICR mice model received acute stress or chronic mild stress assessed by open field test (OFT), novelty suppressed feeding (NSF), sucrose preference test (SPT), forced-swimming test (FST), and tail suspension test (TST). After the final test, blood was collected to detect the serum levels of angiogenin (ANG), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay (ELISA). The behavioral results showed that repeated ARC (10, 30 mg/kg) administration significantly relieved the antidepressant- and anxiolytic-like effects. And repeated ARC administration at the dose of 10 and 30 mg/kg could significantly block depressive- and anxiety-like behaviors caused by CMS. Finally, ELISA results showed that ARC administration increased the serum levels of angiogenin (ANG), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF). Results showed that chronic ARC administration produces antidepressant- and anxiolytic-like effects, which provides direct evidence for the first time that ARC may be a novel strategy for the treatment of depression and even stress-related disorders. The present data supports further exploration for developing ARC administration as a novel therapeutic strategy for depression and even stress-related disorders.

Using ZIF-8 as stationary phase for capillary electrophoresis separation of proteins.

Recently, the separation of proteins has received much attention, although many techniques require expensive instrumentation and trained analysts. In this work, a low-cost, effective, and environmental friendship capillary electrophoresis (CE) for proteins separation was first time introduced. The ZIF-8 with outstanding properties of large surface area, and accessible tunnels and cages were coated the inner surface of silica capillary as a separation media by electrostatic interaction. The fast baseline separation of Lys, CC, BSA and RNase A can be obtained within 10 min using the ZIF-8 nanocrystals coated capillary column under the optimum separation conditions. Meanwhile, this system showed good reproducibility and stability. Using L-glutamic acid as the selector ligand, the D- and L-phenylalanine were successfully separated by the ZIF-8 nanocrystals coated capillary column. Furthermore, the method was also applied to separate egg white proteins, and three main proteins were separated in a single run.

Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe.

In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba2+, Co2+, Pb2+, As3+, and Cu2+ inhibited RNase A activity with IC50 values of 93.7 µM (Ba2+), 90.9 µM (Co2+), 110.6 µM (Pb2+), 171.5 µM (As3+), and 165.1 µM (Cu2+), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.

Streptavidin-triggered signal amplified fluorescence polarization for analysis of DNA-protein interactions.

Fluorescence polarization (FP) is a sensitive, robust, and homogeneous assay format, able to probe a diversity of biological molecules and their interactions. Herein, we describe a new FP strategy based on the use of streptavidin as a signal amplifier. Such signal amplified fluorescence polarization (SAFP) was used to monitor the binding affinity of human angiogenin and a single-stranded DNA aptamer. Streptavidin was bound to a biotinylated single-stranded DNA aptamer and the interaction between this complex and Alexa Fluor 488 labelled human angiogenin was measured. A dissociation constant of 135.3 ± 32.9 nM and a limit of detection of 6.3 nM were successfully extracted only when the FP signal was increased (without binding hindrance) via streptavidin. Moreover, the demonstrated approach was specific to target molecules without any non-specific binding. The streptavidin-triggered SAFP method unlike amplification strategies that utilize nanomaterials (such as graphene oxides, carbon nanotubes, and metal nanoparticles) is not compromised by fluorescence quenching, and it is able to operate within nanomolar concentration regimes. Furthermore, unlike the other FP signal amplification strategies that use dual binding DNA probes, the presented method is simple to implement with signal amplification only requiring the binding of streptavidin with biotinylated DNA. This method could be expanded to analyze molecular interactions and it may be a useful tool for FP measurement by reducing the concentration of rare and expensive protein samples.

Photosensitive diazotized poly(ethylene glycol) covalent capillary coatings for analysis of proteins by capillary electrophoresis.

A new method for the fabrication of covalently cross-linked capillary coatings of poly(ethylene glycol) (PEG) is described using diazotized PEG (diazo-PEG) as a new photosensitive coating agent. The film of diazo-PEG depends on ionic bonding and was first prepared on the inner surface of capillary by self-assembly, and ionic bonding was converted into covalent bonding after reaction of ultraviolet light with diazo groups through unique photochemical reaction. The covalently bonded coating impedance adsorption of protein on the central surface of capillary and hence the four proteins ribonuclease A, cytochrome c, bovine serum albumin, and lysosome can be baseline separated by using capillary electrophoresis (CE). The covalently cross-linked diazo-PEG capillary column coatings not only improved the CE separation performance for proteins compared to non-covalently cross-linked coatings or bare capillary but also showed a remarkable chemical solidity and repeatability. Because photosensitive diazo-PEG took the place of the highly noxious and silane moisture-sensitive coating reagents in the fabrication of covalent coating, this technique shows the advantage of being environment-friendly and having a high efficiency for CE to make the covalently bonded capillaries.

Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering.

The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.

Online Hydrophobic Interaction Chromatography-Mass Spectrometry for Top-Down Proteomics.

Recent progress in top-down proteomics has led to a demand for mass spectrometry (MS)-compatible chromatography techniques to separate intact proteins using volatile mobile phases. Conventional hydrophobic interaction chromatography (HIC) provides high-resolution separation of proteins under nondenaturing conditions but requires high concentrations of nonvolatile salts. Herein, we introduce a series of more-hydrophobic HIC materials that can retain proteins using MS-compatible concentrations of ammonium acetate. The new HIC materials appear to function as a hybrid form of conventional HIC and reverse phase chromatography. The function of the salt seems to be preserving protein structure rather than promoting retention. Online HIC-MS is feasible for both qualitative and quantitative analysis. This is demonstrated with standard proteins and a complex cell lysate. The mass spectra of proteins from the online HIC-MS exhibit low charge-state distributions, consistent with those commonly observed in native MS. Furthermore, HIC-MS can chromatographically separate proteoforms differing by minor modifications. Hence, this new HIC-MS combination is promising for top-down proteomics.

Ribonuclease 5 coordinates signals for the regulation of intraocular pressure and inhibits neural apoptosis as a novel multi-functional anti-glaucomatous strategy.

Glaucoma is a vision-threatening disorder characterized by progressive death of retinal ganglion cells (RGCs), although little is known about therapeutic milestones. Due to its complex and multifactorial pathogenesis, multipronged therapeutic approach is needed. Angiogenin (ANG), now called ribonuclease (RNase) 5, has been previously known as angiogenic factor and more recently its biologic activity is extended to promoting cell survival via its ribonucleolytic activity. Here, we revealed the defect of ANG in human glaucomatous trabecular meshwork (TM) cells and identified novel multiple functions of ANG as an anti-glaucomatous strategy. ANG was highly expressed in normal eyes and normal TM cells compared to glaucomatous TM cells. ANG induced intraocular pressure (IOP) lowering in rat models of both normal and elevated IOP, and as a possible mechanism, activated Akt-mediated signals for nitric oxide (NO) production, an important regulator of IOP in glaucomatous TM cell. Moreover, we demonstrated ANG-induced production of matrix metalloproteinase (MMP)-1 and -3 and rho-kinase inhibition for TM remodeling. For anti-glaucomatous defense optimization, ANG not only elicited immune-modulative pathways via indolamine 2,3-dioxygenase (IDO) activation in TM cells and suppression of Jurkat T cells, but also rescued neural stem cells (NSCs) from apoptosis induced by glaucomatous stress. These results demonstrate that novel multi-functional effects of ANG may have benefits against glaucoma in ocular tissues.

Evaluation of a Porcine Gastric Mucin and RNase A Assay for the Discrimination of Infectious and Non-infectious GI.1 and GII.4 Norovirus Following Thermal, Ethanol, or Levulinic Acid Plus Sodium Dodecyl Sulfate Treatments.

Human noroviruses (NoVs) are a major source of foodborne illnesses worldwide. Since human NoVs cannot be cultured in vitro, methods that discriminate infectious from non-infectious NoVs are needed. The purpose of this study was to evaluate binding of NoV genotypes GI.1 and GII.4 to histo-blood group antigens expressed in porcine gastric mucin (PGM) as a surrogate for detecting infectious virus following thermal (99 °C/5 min), 70% ethanol or 0.5% levulinic acid (LV) plus 0.01 or 0.1% sodium dodecyl sulfate (SDS) sanitizer treatments and to determine the limit of detection of GI.1 and GII.4 binding to PGM. Treated and control virus samples were applied to 96-well plates coated with 1 µg/ml PGM followed by RNase A (5 ng/µl) treatment for degradation of exposed RNA. Average log genome copies per ml (gc/ml) reductions and relative differences (RD) in quantification cycle (Cq) values after thermal treatment were 1.77/5.62 and 1.71/7.25 (RNase A) and 1.73/5.50 and 1.56/6.58 (no RNase A) for GI.1 and GII.4, respectively. Treatment of NoVs with 70% EtOH resulted in 0.05/0.16 (GI.1) and 3.54/10.19 (GII.4) log reductions in gc/ml and average RD in Cq value, respectively. LV (0.5%) combined with 0.1 % SDS provided a greater decrease of GI.1 and GII.4 NoVs with 8.97 and 8.13 average RD in Cq values obtained, respectively than 0.5% LV/0.01 % SDS. Virus recovery after PGM binding was variable with GII.4 > GI.1. PGM binding is a promising surrogate for identifying infectious and non-infectious NoVs after capsid destruction, however, results vary depending on virus strain and inactivation method.

[Angiogenin, bFGF and VEGF: angiogenic markers in breath condensate of patients with pulmonary hypertension]. / Die angiogenetischen Faktoren VEGF, Angiogenin und bFGF im Atemkondensat von Patienten mit pulmonaler Hypertonie.

Pulmonary arterial hypertension (PAH) is associated with a change in vascular architecture. A characteristic histological feature is the plexiform lesion. Similar alterations are observed in the pulmonary vascular bed of patients with chronic thromboembolic pulmonary hypertension (CTEPH). Cytokines involved in angiogenesis were found in both serum and lung tissue of patients with PAH and CTEPH, although their role in the formation of plexiform lesions remains unclear. The examination of breath condensate is a noninvasive technique to analyse proteins possibly associated with the pathogenesis of various lung diseases.Breath condensate of 22 patients with pulmonary hypertension (PAH: n = 12; CTEPH: n = 10) and 7 healthy volunteers was examined using a multiplex fluorescent bead immunoassay to determine the concentrations of the biomarkers angiogenin, bFGF, VEGF, IL-8, and TNF-α. Significantly higher levels of angiogenin, bFGF and TNF-α were observed in breath condensate of patients with pulmonary hypertension in comparison to healthy controls. Similarly, breath condensate levels of VEGF were elevated in patients with PAH as against healthy volunteers. However, IL-8 levels in breath condensate did not differ between the two groups. The data suggest that breath condensate of patients with pulmonary hypertension is characterized by increased levels of the angiogenic factors angiogenin, VEGF and bFGF as well as TNF-α, but not IL-8. A larger study is needed to confirm these results and to determine the prognostic and therapeutic implications of these findings.

Aptasensor for electrochemical sensing of angiogenin based on electrode modified by cationic polyelectrolyte-functionalized graphene/gold nanoparticles composites.

Herein, a label-free and highly sensitive electrochemical aptasensor for the detection of angiogenin was proposed based on a conformational change of aptamer and amplification by poly(diallyldimethyl ammonium chloride) (PDDA)-functionalized graphene/gold nanoparticles (AuNPs) composites-modified electrode. PDDA-functionalized graphene (P-GR) nanosheets as the building block in the self-assembly of GR nanosheets/AuNPs heterostructure enhanced the electrochemical detection performance. The electrochemical aptasensor has an extraordinarily sensitive response to angiogenin in a linear range from 0.1pM to 5nM with a detection limit of 0.064pM. The developed sensor provides a promising strategy for the cancer diagnosis in medical application in the future.

Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells.

Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses.

Separation of PEGylated variants of ribonuclease A and apo-α-lactalbumin via reversed phase chromatography.

The covalent attachment of polyethylene glycol (PEG) molecules to pharmaceutical proteins, "PEGylation", often results in a population of conjugate species that includes differing numbers and locations of attached PEG chains. As some portion of this population may be biologically inactive, a challenging separation problem arises. An interesting alternative to the size-based resolution of these conjugates involves the use of reversed phase chromatography (RPC), treating the PEG moieties as hydrophobic purification tags. The use of RPC raises concerns about protein denaturation in the mobile and on the stationary phase. Here, the potential dual role of conjugated PEG chains as both group-specific separation tags and as steric or structural stabilizers in RPC was explored. In this work, RPC with C18-based media was used to resolve PEGylation number variants of ribonuclease A (RNase A) and apo-α-lactalbumin (apo-αLac) in a neutral pH mobile phase. While the attachment of 20kDa PEG molecules did not modify the structures of RNase A and apo-αLac, as confirmed by structural analysis using circular dichroism, exposure to the mobile phase modifier, acetonitrile, and to the C18 media during separation resulted in perturbations to both the secondary and tertiary structures of all species studied. RNase A experienced small perturbations that were mediated to some extent by PEGylation; these results were consistent with activity assays which showed that PEGylated RNase A species retained native-like activity after RPC separation. Apo-αLac, a more hydrophobic and less stable protein than RNase A, experienced extensive structural perturbations regardless of PEGylation state. The temperature of the mobile phase was found to strongly influence chromatographic separation of PEG-conjugates with conjugate species becoming more strongly retained with increasing temperature. This work shows that it is feasible to employ RPC with neutral pH mobile phases to resolve PEG conjugate number heterogeneity.

Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens: Procedures and Mechanisms.

We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.

Top-down tandem mass spectrometry on RNase A and B using a Qh/FT-ICR hybrid mass spectrometer.

Protein characterization using top-down approaches emerged with advances in high-resolution mass spectrometers and increased diversity of available activation modes: collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD) electron capture dissociation (ECD), and electron transfer dissociation (ETD). Nevertheless, top-down approaches are still rarely used for glycoproteins. Hence, this work summarized the capacity of top-down approaches to improve sequence coverage and glycosylation site assignment on the glycoprotein Ribonuclease B (RNase B). The glycan effect on the protein fragmentation pattern was also investigated by comparing the fragmentation patterns of RNase B and its nonglycosylated analog RNase A. The experiments were performed on a Bruker 12-T Qh/FT-ICR SolariX mass spectrometer using vibrational (CID/IRMPD) and radical activation (ECD/ETD) with/without pre- or post-activation (IRMPD or CID, respectively). The several activation modes yielded complementary sequence information. The radical activation modes yielded the most extensive sequence coverage that was slightly improved after a CID predissociation activation event. The combination of the data made it possible to obtain 90% final sequence coverage for RNase A and 86% for RNase B. Vibrational and radical activation modes showed high retention of the complete glycan moiety (>98% for ETD and ECD) facilitating unambiguous assignment of the high-mannose glycosylation site. Moreover, the presence of the high-mannose glycan enhanced fragmentation around the glycosylation site.

PPARγ agonists improve survival and neurocognitive outcomes in experimental cerebral malaria and induce neuroprotective pathways in human malaria.

Cerebral malaria (CM) is associated with a high mortality rate, and long-term neurocognitive impairment in approximately one third of survivors. Adjunctive therapies that modify the pathophysiological processes involved in CM may improve outcome over anti-malarial therapy alone. PPARγ agonists have been reported to have immunomodulatory effects in a variety of disease models. Here we report that adjunctive therapy with PPARγ agonists improved survival and long-term neurocognitive outcomes in the Plasmodium berghei ANKA experimental model of CM. Compared to anti-malarial therapy alone, PPARγ adjunctive therapy administered to mice at the onset of CM signs, was associated with reduced endothelial activation, and enhanced expression of the anti-oxidant enzymes SOD-1 and catalase and the neurotrophic factors brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the brains of infected mice. Two months following infection, mice that were treated with anti-malarials alone demonstrated cognitive dysfunction, while mice that received PPARγ adjunctive therapy were completely protected from neurocognitive impairment and from PbA-infection induced brain atrophy. In humans with P. falciparum malaria, PPARγ therapy was associated with reduced endothelial activation and with induction of neuroprotective pathways, such as BDNF. These findings provide insight into mechanisms conferring improved survival and preventing neurocognitive injury in CM, and support the evaluation of PPARγ agonists in human CM.

A clinicopathological correlation of the expression of the angiopoietin-Tie-2 receptor pathway in the brain of adults with Plasmodium falciparum malaria.

BACKGROUND: Plasma angiopoietin (Ang)-2 is associated with disease severity and mortality in adults and children with falciparum malaria. However the mechanism of action of the angiopoietins in fatal malaria is unclear. This study aimed to determine whether the expression of Ang-1 and Ang-2 and their receptor Tie-2 in cerebral endothelial or parenchymal cells was specific to cerebral malaria (CM), correlated with coma or other severe clinical features, and whether plasma and CSF levels of these markers correlated with the clinical and neuropathological features of severe and fatal malaria in Vietnamese adults. METHODS: Immunohistochemistry was performed for Ang-1, Ang-2 and Tie-2 on post-mortem brain tissue from fatal malaria cases and controls. Quantitative ELISA for plasma and cerebrospinal fluid levels of Ang-1, Ang-2 and Tie-2 was done to compare fatal cases with surviving patients from the same study. RESULTS: Immunohistochemistry revealed significant differences in expression in endothelial and parenchymal cells compared to controls. However there was no significant difference in expression of these markers on endothelial cells, astroglial cells or neurons between CM and non-cerebral malaria cases. Immunostaining of Ang-1, Ang-2 and Tie-2 was also not associated with Plasmodium falciparum-infected erythrocyte sequestration in the brain. However Ang-1 and Ang-2 expression in neurons was significantly correlated with the incidence of microscopic haemorrhages. Plasma levels of Ang-2 and Ang-2/Ang-1 ratio were associated with the number of severe malaria complications and were significant and independent predictors of metabolic acidosis and fatal outcome. CONCLUSIONS: The independent prognostic significance of Ang-2 and the Ang-2/Ang-1 ratio in severe malaria was confirmed, although immunohistochemistry in fatal cases did not reveal increased expression on brain endothelium in cerebral versus non-cerebral cases. Activation of the Ang-Tie-2 pathway in severe malaria is therefore related to acidosis, number of severity criteria and outcome, but is not a specific event in the brain during cerebral malaria.