Real-time cytotoxicity assay for rapid and sensitive detection of ricin from complex matrices

Background

In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits.

Methodology/Findings

This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index–time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material.

Conclusions/Significance

The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices.     

Abbott RealTime HBV Assay Is More Sensitive in Detection of Low Viral Load and Little Impacted by Drug Resistant Mutation in Chronic Hepatitis B Patients under Nucleot(s)ide Analogues Therapy

Background

Selection of drug-resistant strains may lead to failure of HBV antiviral therapy. There is little information whether there is detection difference in drug resistant mutations between different viral load assays of HBV.

Objectives

This study is aimed to investigate whether there is drug-resistant strains related detection difference between Abbott RealTime HBV (RealTime) and CobasAmpliPrep/CobasTaqMan HBV assays 2.0 (TaqMan).

Study Design

One hundred and thirty-four CHB patients who received HBV anti-viral therapy were enrolled. HBV virological markers were tested 3 months apart regularly. Serum HBV DNA levels were determined using the TaqMan and RealTime. YMDD (rt180M and rt204V) mutation was checked in patients who experienced virologic breakthrough (VBT).

Results

The correlation of HBV DNA observed between the RealTime and TaqMan was good for all 571 samples (R² = 0.797; P<0.001). However, the correlation in the 434 samples with HBV DNA level <3 log₁₀ IU/ml was not as good as in all samples (R² = 0.457). Overall, 21.5% of samples had a detection difference of ≥1 log₁₀ IU/ml with 91.9% of these having HBV DNA level <3 log₁₀ IU/ml. Twenty-four patients experiencedVBT. Three of these patients had acquired the YMDD mutation and exhibited discordant viral load results between the two methods tested. In each case, persistent HBV DNA was detected by RealTime and undetectable with TaqMan. Of the patients who experienced a VBT and had acquired YMDD mutation, 4.7% had undetectable HBV DNA by TaqMan while all were detectable with RealTime.

Conclusions

RealTime assay is more sensitive and is little impacted by the development of drug resistant mutation.     

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection,Identification and Genotyping in Clinical Specimens

Objective

Early diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.

Methods

To this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.

Results

A new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.

Conclusions

The first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.     

7 Tesla Magnetic Resonance Imaging to Detect Cortical Pathology in Multiple Sclerosis

Background

Neocortical lesions (NLs) are an important pathological component of multiple sclerosis (MS), but their visualization by magnetic resonance imaging (MRI) remains challenging.

Objectives

We aimed at assessing the sensitivity of multi echo gradient echo (ME-GRE) T₂ ^*-weighted MRI at 7.0 Tesla in depicting NLs compared to myelin and iron staining.

Methods

Samples from two MS patients were imaged post mortem using a whole body 7T MRI scanner with a 24-channel receive-only array. Isotropic 200 micron resolution images with varying T₂ ^* weighting were reconstructed from the ME-GRE data and converted into R₂ ^* maps. Immunohistochemical staining for myelin (proteolipid protein, PLP) and diaminobenzidine-enhanced Turnbull blue staining for iron were performed.

Results

Prospective and retrospective sensitivities of MRI for the detection of NLs were 48% and 67% respectively. We observed MRI maps detecting only a small portion of 20 subpial NLs extending over large cortical areas on PLP stainings. No MRI signal changes suggestive of iron accumulation in NLs were observed. Conversely, R₂ ^* maps indicated iron loss in NLs, which was confirmed by histological quantification.

Conclusions

High-resolution post mortem imaging using R₂ ^* and magnitude maps permits detection of focal NLs. However, disclosing extensive subpial demyelination with MRI remains challenging.     

Analytical Performance of the Roche LightCycler? Mycobacterium Detection Kit for the Diagnosis of Clinically Important Mycobacterial Species

Background

The LightCycler® Mycobacterium Detection Kit based on real-time PCR technology for the detection of Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii was recently developed. This study evaluated its analytical sensitivity, specificity and reproducibility.

Methodology/Principal Findings

Plasmid standards were prepared and used to determine the limit of detection. The assay was also performed against organisms other than mycobacteria, other mycobacterial strains and interfering substances to exclude cross-reactivity and interference. Reference standards were prepared and tested to assess the assay''s reproducibility. All PCR assays were performed using the LightCycler® 2.0 Instrument. The detection limit for M. tuberculosis was 28 copies per microlitre. Neither cross-reactivity nor interference occurred with non-mycobacterial organisms and substances tested. Overall reproducibility for consecutive measurements, run-to-run, lot-to-lot, day-to-day and laboratory-to-laboratory achieved a coefficient of variance of less than two percent.

Significance

The LightCycler® Mycobacterium Detection kit has shown to be a robust and accurate assay with the potential to be used as a rapid TB diagnostic test.     

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

Background

The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.

Methodology/Principal Findings

Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH₂) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH₂ supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.

Conclusions/Significance

The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.     

Micro-Arrayed Human Embryonic Stem Cells-Derived Cardiomyocytes for In Vitro Functional Assay

Introduction

The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality.

Methods

hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling.

Results

Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H₂O₂. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H₂O₂, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay.

Conclusions

Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.     

13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

Background

Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers.

Methodology/Principal Findings

We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [¹³C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of δ¹³CO₂ formation were determined. Samples obtained prior to inoculation served as control samples for background ¹³CO₂ conversion in the rabbit model. ¹³CO₂, from metabolic conversion of [¹³C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of ¹³CO₂ formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of ¹³CO₂ formation.

Conclusions/Significance

Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the δ¹³CO₂ signal from urease-positive gastrointestinal organisms.     

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background

DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response.

Aim

To investigate the effect of red blood cells on H₂O₂-induced DNA damage and repair in human peripheral blood mononuclear cells.

Methods

DNA breaks were induced in peripheral blood mononuclear cells by H₂O₂ in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by ³H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation.

Results

Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA.

Conclusion

Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.     

Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

Background  

Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.     

Evaluation of Rapid Blood Sample Collection in the Detection of Circulating Filarial Antigens for Epidemiological Survey by rWbSXP-1 Capture Assay

Background

Lymphatic filariasis is a neglected tropical disease leading to profound disfiguring causing socio economic burden in the tropics. Current diagnosis strategies available during field surveys and epidemics are based on traditional microscopic detections and a few antigen/antibody assays. We have compared different sampling methodologies and standardized the highly sensitive and reliable rWbSXP-1 antigen detection assay to our new sampling methodology.

Methodology

Samples collected as serum, whole blood, whole blood on filter paper and whole blood on microscopic slides from patients belonging to various clinical groups of filariasis [endemic normal(EN), chronic pathology(CP), microfilaraemic(MF) and non-endemic normal(NEN)] were collected and standardized the rWbSXP-1 antigen detection assay using monoclonal antibody raised against rWbSXP-1 protein. The whole blood collected on microscopic slide based sampling method was employed in the field and the presence of circulating filarial antigen (CFA) was assessed using the rWbSXP-1 assay.

Principal Findings

The sampling methods were compared and no significant difference was observed for the detection of CFA (MF, P = 0.304, EN, P = 0.675, CP, P = 0.5698, NEN, P = 0.4494). Further the optimized sampling method was utilized to collect the 1106 samples from Polur, Tiruvannamalai. The rWbSXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17.

Conclusions

Four sampling methodologies were analyzed and the new sampling methodology of whole blood collected on microscopic slide was found to be convenient for the detection of CFA using rWbSXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The rWbSXP-1 antigen assay perceived a 7.32% increased result which was read as false negatives on the conventional microscopic staining method. This new sampling methodology coupled with the rWbSXP-1 antigen assay can be used in epidemiological surveys for lymphatic filariasis and the same sampling methodology can be expanded to other antigen based high affinity assays.     

Effects of karanjin on cell cycle arrest and apoptosis in human A549, HepG2 and HL-60 cancer cells

Background

We have investigated the potential anticancer effects of karanjin, a principal furanoflavonol constituent of the Chinese medicine Fordia cauliflora, using cytotoxic assay, cell cycle arrest, and induction of apoptosis in three human cancer cell lines (A549, HepG2 and HL-60 cells).

Results

MTT cytotoxic assay showed that karanjin could inhibit the proliferation and viability of all three cancer cells. The induction of cell cycle arrest was observed via a PI (propidium iodide)/RNase Staining Buffer detection kit and analyzed by flow cytometry: karanjin could dose-dependently induce cell cycle arrest at G2/M phase in the three cell lines. Cell apoptosis was assessed by Annexin V-FITC/PI staining: all three cancer cells treated with karanjin exhibited significantly increased apoptotic rates, especially in the percentage of late apoptosis cells.

Conclusion

Karanjin can induce cancer cell death through cell cycle arrest and enhance apoptosis. This compound may be effective clinically for cancer pharmacotherapy.     

Inhibitory Effect of Tanshinone IIA on Rat Hepatic Stellate Cells

Background

Anti-inflammation via inhibition of NF-κB pathways in hepatic stellate cells (HSCs) is one therapeutic approach to hepatic fibrosis. Tanshinone IIA (C₁₉H₁₈O₃, Tan IIA) is a lipophilic diterpene isolated from Salvia miltiorrhiza Bunge, with reported anti-inflammatory activity. We tested whether Tan IIA could inhibit HSC activation.

Materials and Methods

The cell line of rat hepatic stellate cells (HSC-T6) was stimulated with lipopolysaccharide (LPS) (100 ng/ml). Cytotoxicity was assessed by MTT assay. HSC-T6 cells were pretreated with Tan IIA (1, 3 and 10 µM), then induced by LPS (100 ng/ml). NF-κB activity was evaluated by the luciferase reporter gene assay. Western blotting analysis was performed to measure NF-κB-p65, and phosphorylations of MAPKs (ERK, JNK, p38). Cell chemotaxis was assessed by both wound-healing assay and trans-well invasion assay. Quantitative real-time PCR was used to detect gene expression in HSC-T6 cells.

Results

All concentrations of drugs showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, all of which were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5, IL-1β, TNF-α, IL-6, ICAM-1, iNOS, and α-SMA in HSC-T6 cells.

Conclusion

Our results demonstrated that Tan IIA decreased LPS-induced HSC activation.     

A Fiber-Optic Fluorescence Microscope Using a Consumer-Grade Digital Camera for In Vivo Cellular Imaging

Background

Early detection is an essential component of cancer management. Unfortunately, visual examination can often be unreliable, and many settings lack the financial capital and infrastructure to operate PET, CT, and MRI systems. Moreover, the infrastructure and expense associated with surgical biopsy and microscopy are a challenge to establishing cancer screening/early detection programs in low-resource settings. Improvements in performance and declining costs have led to the availability of optoelectronic components, which can be used to develop low-cost diagnostic imaging devices for use at the point-of-care. Here, we demonstrate a fiber-optic fluorescence microscope using a consumer-grade camera for in vivo cellular imaging.

Methods

The fiber-optic fluorescence microscope includes an LED light, an objective lens, a fiber-optic bundle, and a consumer-grade digital camera. The system was used to image an oral cancer cell line labeled with 0.01% proflavine. A human tissue specimen was imaged following surgical resection, enabling dysplastic and cancerous regions to be evaluated. The oral mucosa of a healthy human subject was imaged in vivo, following topical application of 0.01% proflavine.

Findings

The fiber-optic microscope resolved individual nuclei in all specimens and tissues imaged. This capability allowed qualitative and quantitative differences between normal and precancerous or cancerous tissues to be identified. The optical efficiency of the system permitted imaging of the human oral mucosa in real time.

Conclusion

Our results indicate this device as a useful tool to assist in the identification of early neoplastic changes in epithelial tissues. This portable, inexpensive unit may be particularly appropriate for use at the point-of-care in low-resource settings.     

Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol

Background

Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.

Methodology/Principal Findings

Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.

Conclusions/Significance

This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.     

The value of plasma paraquat concentration in predicting therapeutic effects of haemoperfusion in patients with acute paraquat poisoning

Objective

This study was aimed to analyze the scavenging effect of haemoperfusion on plasma paraquat (PQ) and to evaluate the clinical significance of PQ examination in the treatment of patients with acute paraquat poisoning.

Methods

85 patients with acute paraquat intoxication by oral ingestion were admitted in West China Hospital from Jun, 2010 to Mar, 2011. A standardized therapeutic regimen including emergency haemoperfusion was given on all subjects. A total of 91 whole blood samples were taken before (0h), underway (1h after haemoperfusion beginning) and at the end (2h) of the haemoperfusion therapy. The clearance rate was calculated and related factors were analyzed.

Results

As heamoperfusion was going on, the plasma paraquat concentration of the patients kept falling down. After 1 hour of haemoperfusion, the average clearance rate (R₁) was 37.06±21.81%. After 2 hours of haemoperfusion, the average clearance rate (R₂) was 45.99±23.13%. The average of R₁/R₂ ratio was 76.61±22.80%. In the high paraquat concentration group (plasma paraquat concentration (C₀) >300 ng/mL), both the averages of R₁ and R₂ were significantly higher than those of the low paraquat concentration group (C₀≤200 ng/mL) (p<0.05), and there was no significant difference of R₁/R₂ between these two groups (p>0.05).

Conclusions

The dynamic monitoring of plasma PQ concentration was not only critical in the clinical evaluation but also helpful in guiding the treatment of patients with acute PQ intoxication. Haemoperfusion can effectively eliminate paraquat from the plasma in patients with high initial plasma PQ concentration, while in patients with low initial plasma PQ concentration (<200 ng/ml), the clearance effect of harmoperfusion was very limited. Increasing HP time might improve the overall clearance rate of HP on plasma PQ yet decrease the elimination efficiency of HP, while repeated HP treatment was helpful against the rebound phenomena.