bluuebunny/crossref_metadata_2025 huggingface dataset:
Intravenous immune globulin–related hemolysis: comparing two different methods for case assessment
Authors: Taylor Elaine , Vu Duc , Legare Carole , Keene Daniel
Published: July 2015

BACKGROUND

Hemolysis is a rare adverse event associated with the use of intravenous immune globulin (IVIG). Using the Canadian experience, this article compares two methods used for case ascertainment of hemolysis after IVIG.

STUDY DESIGN AND METHODS

This is a retrospective, descriptive analysis of case reports of hemolytic reaction after exposure to IVIG. The cases were ascertained from the Canada Vigilance database using the standardized MedDRA query for hemolytic disorders between the years 2006 and 2012 inclusively. The presence of a causal relationship was determined by using the Transfusion Transmitted Injuries Surveillance System (TTISS) algorithm. Cases were then reviewed against the standardized case definition of IVIG‐associated hemolysis proposed by the IVIG Hemolysis Pharmacovigilance Group.

RESULTS

A total of 942 adverse event reports after exposure to IVIG had been received by Canada Vigilance during the study period. A search for the adverse event of hemolytic disorder retrieved 313 reports (33%). Using a modified TTISS definition of hemolysis and the TTISS causality assessment algorithm, 226 cases were found to be at least possibly related to administration of IVIG, whereas 69 cases met the definition of the IVIG Hemolysis Pharmacovigilance Group. The amount of IVIG distributed increased over the study period; this trend was not reflected in the number of adverse reaction reports for “hemolytic disorder” received.

CONCLUSION

The number of case reports per year received for the adverse event “hemolytic disorder” after exposure to IVIG fluctuated. The number of confirmed case reports varied depending on the case definition being used.
The role of isoagglutinins in intravenous immunoglobulin–related hemolysis
Authors: Bellac Caroline L. , Hottiger Thomas , Jutzi Markus P. , Bögli‐Stuber Katja , Sänger Michael , Hanschmann Kay‐Martin , Keller‐Stanislawski Brigitte , Funk Markus B.
Published: July 2015

BACKGROUND

Increased reporting of intravenous immunoglobulin (IVIG)‐related hemolytic reactions (HRs) triggered an investigation by the German and Swiss health authorities to identify potential risk factors.

STUDY DESIGN AND METHODS

From the EudraVigilance database HRs reported between 2008 and 2013 were retrieved for seven IVIG preparations. HRs were classified as mild to moderate (hemoglobin [Hb] decline < 2 g/dL)] or severe (Hb decline > 2 g/dL) and separately analyzed for IVIG doses of less than 2 g/kg body weight and 2 g/kg body weight or more. It was assessed whether HR reporting rates correlate with the isoagglutinin content of the different preparations.

RESULTS

Of 569 HR cases retrieved, 103 cases were excluded due to insufficient data, leaving 466 for analysis. Ninety‐three cases were classified as mild to moderate and 373 as severe. Approximately 80% of the severe HRs concerned patients with blood group A and only three patients with blood group O. Testing of isoagglutinin titers revealed substantial differences between the seven preparations. IVIG products with high anti‐A/anti‐B titers (≥32) had elevated HR reporting rates, particularly when cumulative doses at least 2 g/kg were administered.

CONCLUSION

The isoagglutinin content of IVIGs correlates with the risk for HRs. Exclusion of high‐titer donations and manufacturing steps that deplete isoagglutinins should be considered for risk mitigation. In patients with blood groups A or AB receiving doses of at least 2 g/kg, the use of IVIG batches with low isoagglutinin titers should be considered to prevent HRs.
Complementary use of passive surveillance and Mini‐Sentinel to better characterize hemolysis after immune globulin
Authors: Winiecki Scott , Baer Bethany , Chege Wambui , Jankosky Christopher , Mintz Paul , Baker Meghan , Woodworth Tiffany , Nguyen Michael
Published: July 2015

BACKGROUND

Hemolysis after intravenous immune globulins (IGIVs) is a known complication, but expanding indications and recent manufacturing changes warrant ongoing postmarketing surveillance. Characterization of post‐IGIV hemolysis to date has been limited to small case series.

STUDY DESIGN AND METHODS

We queried the Food and Drug Administration's (FDA) Adverse Event Reporting System (FAERS) from 2007 to 2014. All reported post‐IGIV hemolysis cases were classified using a prespecified case definition and a case series analysis performed. We also conducted two assessments using FDA's Mini‐Sentinel (MS) system to quantify the risk of hemolysis by six product indications and by IGIV formulation and evaluate the onset interval.

RESULTS

A total of 109 FAERS cases met our definition. For cases with available information, 83% (66/80) received IGIV doses of at least 2 g/kg, 98.1% (51/52) had non‐O blood group, and 75% (64/85) of events occurred within 4 days of IGIV exposure. We identified 313,045 treatment episodes and 337 post‐IGIV hemolytic events in MS from 2006 to 2014, with 72% occurring within 2 days. Rates of hemolysis were highest among patients with Kawasaki disease (KD) and immune thrombocytopenia (ITP). The risk among patients receiving nonlyophilized products was 2.3 times higher than that in patients receiving lyophilized products.

CONCLUSION

With the largest case series to date, FAERS data support that higher doses and non‐O blood group are key risk factors. The incident rate of post‐IGIV hemolysis is estimated at one per 1000 IGIV treatment episodes, with most occurring within 2 days of exposure. The risk is higher in patients with KD and ITP and after receipt of nonlyophilized IGIV.
Do immune complexes play a role in hemolytic sequelae of intravenous immune globulin?
Authors: Zimring James C.
Published: July 2015

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one‐to‐one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two‐hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis.

IVIG‐mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop.
Survival of Pseudomonas aeruginosa in modified Romanowsky staining solutions
Authors: Duffield Richard , Wong Hui‐San , Trott Darren J. , Hill Peter B.
Published: July 2015

Background

Anecdotal reports suggest that rapid staining solutions can become contaminated with micro‐organisms, especially Pseudomonas aeruginosa.

Hypothesis/Objectives

To determine whether inoculation of rapid Romanowsky‐type stains with P. aeruginosa results in viable bacterial contamination, which could lead to cross‐contamination of slides during cytological staining.

Methods

Pseudomonas aeruginosa was inoculated into clean and organically contaminated staining solutions (fixative, eosin and methylene blue) and positive (broth) and negative (bleach) control solutions. Subsequent viability and survival were detected by measuring colony‐forming units per millilitre at various time points up to 2 weeks. Each sample was stained and microscopically examined to determine whether bacteria were visible.

Results

No bacteria could be cultured at any time point from the bleach or fixative solution. In clean eosin and methylene blue staining solutions, viable bacteria were recovered for up to 1 h, but by 24 h all bacteria were dead. In staining solutions contaminated with hair and dead skin cells, bacteria survived in methylene blue for up to 1 h, and viable bacteria persisted in the eosin stain for 2 weeks. In solutions containing viable organisms, the bacteria could be observed by microscopic examination; no bacteria were visible when the solutions contained no viable organisms.

Conclusions and clinical importance

Pseudomonas aeruginosa can survive in commonly used staining solutions for variable periods of time, but is unable to proliferate. Although theoretically this might complicate cytological interpretation and subsequent diagnosis, the likelihood of this in clinical practice appears remote when the correct staining technique is used.
Comparison of the stability and pharmacokinetics in dogs of modified ciclosporin capsules stored at −20°C and room temperature
Authors: Bachtel Jeremy C. , Pendergraft Jennifer S. , Rosychuk Rodney A. W. , Gustafson Daniel L. , Hansen Ryan J. , Lunghofer Paul J.
Published: July 2015

Background

Placement of ciclosporin (Atopica®; Novartis Animal Health, Greensboro, NC, USA) capsules in a freezer prior to administration may reduce the incidence of vomiting in dogs. However, its impact on ciclosporin stability and pharmacokinetics is unknown.

Hypothesis/Objectives

The purpose of this study was to evaluate the stability of Atopica® capsules and pharmacokinetics of ciclosporin in dogs after storage at −20°C in comparison with storage of capsules at 15–25°C. We hypothesized that there would be no difference in stability or pharmacokinetic parameters between freezer‐stored and room‐temperature Atopica® capsules.

Animals

Eight healthy research beagle dogs received one 5.0 mg/kg oral dose each of freezer‐stored and room‐temperature Atopica® capsules with a 1 week washout period between.

Methods

Ciclosporin concentrations of all available Atopica® capsule strengths were assessed for stability after −20°C storage at five time points over 30 days and at room temperature (15–25°C). A blinded, randomized cross‐over study was also performed to compare blood concentrations of ciclosporin after capsule storage for 28 days at −20 versus 15–25°C. Blood samples were obtained over a 24 h period after administration. Capsule and whole‐blood ciclosporin concentrations were assessed via high‐performance liquid chromatography–tandem mass spectrometry.

Results

There was no significant difference in stability between freezer‐stored and room‐temperature Atopica® capsules at any time point. In the cross‐over study, there were no significant differences in pharmacokinetic parameters assessed.

Conclusions and clinical importance

Placing Atopica® capsules in a −20°C freezer for 28 days does not affect stability or absorption in the dog.
Effect of topical latanoprost 0.005% on intraocular pressure and pupil diameter in normal and glaucomatous cats
Authors: McDonald Jessica E. , Kiland Julie A. , Kaufman Paul L. , Bentley Ellison , Ellinwood N. Matthew , McLellan Gillian J.
Published: July 2015

Objective

To determine the effects of latanoprost on intraocular pressure (IOP) and pupil diameter (PD) in cats with inherited primary congenital glaucoma (PCG) and normal cats.

Animals studied and procedures

IOPandPDwere measured in both eyes (OU) of 12 adult cats (six normal, sixPCG), three times per week for 3 weeks prior to, for 3 weeks during, and for 2 weeks following twice‐daily treatment with 0.005% latanoprost to the right eye (OD) and vehicle to the left (control) eye (OS).IOPandPDwere measured hourly, for 8 h, 1 day prior to, and on the first and last days of treatment. Aqueous humor flow rate (AHF) was determined at baseline and at the end of the treatment phase in six normal cats.

Results

MeanIOPwas significantly lower in treated vs. control eyes ofPCGcats, for up to 8 h following a single latanoprost treatment, and a maximalIOPreduction of 63% occurred in treated eyes at 3 h. Latanoprost acutely loweredIOPin cats withPCG, but this effect appeared to diminish over 3 weeks of treatment.AHFwas modestly increased in the treated eyes of normal cats after 3 weeks of latanoprost treatment, althoughIOPwas not significantly affected. Latanoprost caused miosis, with rebound mydriasis at 24 h posttreatment, in the treated eyes of all cats.

Conclusions

Further research is needed to determine the suitability and efficacy of latanoprost treatment for long‐termIOP‐lowering in cats withPCGor other forms of glaucoma.
Atomic and electronic structure of exfoliated black phosphorus
Authors: Wu Ryan J. , Topsakal Mehmet , Low Tony , Robbins Matthew C. , Haratipour Nazila , Jeong Jong Seok , Wentzcovitch Renata M. , Koester Steven J. , Mkhoyan K. Andre
Published: July 2015

Black phosphorus, a layered two-dimensional crystal with tunable electronic properties and high hole mobility, is quickly emerging as a promising candidate for future electronic and photonic devices. Although theoretical studies using ab initio calculations have tried to predict its atomic and electronic structure, uncertainty in its fundamental properties due to a lack of clear experimental evidence continues to stymie our full understanding and application of this novel material. In this work, aberration-corrected scanning transmission electron microscopy and ab initio calculations are used to study the crystal structure of few-layer black phosphorus. Directly interpretable annular dark-field images provide a three-dimensional atomic-resolution view of this layered material in which its stacking order and all three lattice parameters can be unambiguously identified. In addition, electron energy-loss spectroscopy (EELS) is used to measure the conduction band density of states of black phosphorus, which agrees well with the results of density functional theory calculations performed for the experimentally determined crystal. Furthermore, experimental EELS measurements of interband transitions and surface plasmon excitations are also consistent with simulated results. Finally, the effects of oxidation on both the atomic and electronic structure of black phosphorus are analyzed to explain observed device degradation. The transformation of black phosphorus into amorphous PO3 or H3PO3 during oxidation may ultimately be responsible for the degradation of devices exposed to atmosphere over time.
Investigation of blister formation in sputtered Cu2ZnSnS4 absorbers for thin film solar cells
Authors: Bras Patrice , Sterner Jan , Platzer-Björkman Charlotte
Published: July 2015

Blister formation in Cu2ZnSnS4 (CZTS) thin films sputtered from a quaternary compound target is investigated. While the thin film structure, composition, and substrate material are not correlated to the blister formation, a strong link between sputtering gas entrapment, in this case argon, and blistering effect is found. It is shown that argon is trapped in the film during sputtering and migrates to locally form blisters during the high temperature annealing. Blister formation in CZTS absorbers is detrimental for thin film solar cell fabrication causing partial peeling of the absorber layer and potential shunt paths in the complete device. Reduced sputtering gas entrapment, and blister formation, is seen for higher sputtering pressure, higher substrate temperature, and change of sputtering gas to larger atoms. This is all in accordance with previous publications on blister formation caused by sputtering gas entrapment in other materials.
Calibration of GafChromic EBT3 for absorbed dose measurements in 5 MeV proton beam and 60Co γ‐rays
Authors: Vadrucci M. , Esposito G. , Ronsivalle C. , Cherubini R. , Marracino F. , Montereali R. M. , Picardi L. , Piccinini M. , Pimpinella M. , Vincenti M. A. , De Angelis C.
Published: July 2015

Purpose:

To study EBT3 GafChromic film in low‐energy protons, and for comparison purposes, in a reference 60Co beam in order to use it as a calibrated dosimetry system in the proton irradiation facility under construction within the framework of the Oncological Therapy with Protons (TOP)‐Intensity Modulated Proton Linear Accelerator for RadioTherapy (IMPLART) Project at ENEA‐Frascati, Italy.

Methods:

EBT3 film samples were irradiated at the Istituto Nazionale di Fisica Nucleare—Laboratori Nazionali di Legnaro, Italy, with a 5 MeV proton beam generated by a 7 MV Van de Graaff CN accelerator. The nominal dose rates used were 2.1 Gy/min and 40 Gy/min. The delivered dose was determined by measuring the particle fluence and the energy spectrum in air with silicon surface barrier detector monitors. A preliminary study of the EBT3 film beam quality dependence in low‐energy protons was conducted by passively degrading the beam energy. EBT3 films were also irradiated at ENEA‐National Institute of Ionizing Radiation Metrology with gamma radiation produced by a 60Co source characterized by an absorbed dose to water rate of 0.26 Gy/min as measured by a calibrated Farmer type ionization chamber. EBT3 film calibration curves were determined by means of a set of 40 film pieces irradiated to various doses ranging from 0.5 Gy to 30 Gy absorbed dose to water. An EPSON Expression 11000XL color scanner in transmission mode was used for film analysis. Scanner response stability, intrafilm uniformity, and interfilm reproducibility were verified. Optical absorption spectra measurements were performed on unirradiated and irradiated EBT3 films to choose the most sensitive color channel to the dose range used.

Results:

EBT3 GafChromic films show an under response up to about 33% for low‐energy protons with respect to 60Co gamma radiation, which is consistent with the linear energy transfer dependence already observed with higher energy protons, and a negligible dose‐rate dependence in the 2–40 Gy/min range. Short‐ and long‐term scanner stabilities were 0.5% and 1.5%, respectively; film uniformity and reproducibility were better than 0.5%.

Conclusions:

The main purpose of this study was to implement EBT3 dosimetry in the proton low‐energy radiobiology line of the TOP‐IMPLART accelerator, having a maximum energy of 7 MeV. Low‐energy proton and 60Co calibrated sources were used to investigate the behavior of film response vs to be written in italicum dose. The calibration in 5 MeV protons is currently used for dose assessment in the radiobiological experiments at the TOP‐IMPLART accelerator carried out at that energy value.
Perception of acoustic cues to Tokyo Japanese pitch-accent contrasts in native Japanese and naive English listeners
Authors: Shport Irina A.
Published: July 2015

This study examines how native language shapes the perception of a prosodic contrast. In Tokyo Japanese, a high-low pitch accent is a lexical property of a word, and the F0 fall after the peak associated with the accented syllable is the fundamental cue to accent perception. In English, pitch accents do not create lexically contrastive F0 patterns. A hypothesis that English listeners naive to Japanese use the F0 fall cue less than Japanese listeners was tested in two experiments. The alignment of F0 peak, the presence and magnitude of F0 fall were manipulated in a trisyllabic nonword to resynthesize Japanese 1st-syllable accented, 2nd-syllable accented, and unaccented patterns. In an AX-discrimination experiment, both listener groups showed sensitivity to the presence of F0 fall at every peak location. In a categorization experiment, the English group did not use the F0 fall cue in decisions about whether the 1st or the 2nd syllable sounded more prominent. The Japanese group relied on the F0 fall information, some listeners much heavily than others. These findings suggest that one's native language constrains how much attention the prosodic dimension of F0 change receives and that individual listeners may have qualitatively different perceptual strategies.
Ultrasonic wave properties of human bone marrow in the femur and tibia
Authors: Kawasaki Satoshi , Ueda Ryohei , Hasegawa Akihiko , Fujita Akifumi , Mihata Teruhisa , Matsukawa Mami , Neo Masashi
Published: July 2015

Ultrasonic wave properties of human bone marrow obtained in the femur and tibia were measured using an ultrasound pulse technique. The measured frequency range was 4–10 MHz, and the temperature range was 30 °C–40 °C. The sound velocity was 1410 m/s, and the attenuation coefficient was 4.4 dB/cm at 36 °C (10 MHz). These values decreased with temperature. Site dependence and individual differences in elderly human bone marrow were negligible. The slopes of the attenuation coefficient were estimated by a power law. The values of the exponent n were 2.0 (30 °C–38 °C) and 2.3 (40 °C).
Poisson's ratio from polarization of acoustic zero-group velocity Lamb mode
Authors: Baggens Oskar , Ryden Nils
Published: July 2015

Poisson's ratio of an isotropic and free elastic plate is estimated from the polarization of the first symmetric acoustic zero-group velocity Lamb mode. This polarization is interpreted as the ratio of the absolute amplitudes of the surface normal and surface in-plane components of the acoustic mode. Results from the evaluation of simulated datasets indicate that the presented relation, which links the polarization and Poisson's ratio, can be extended to incorporate plates with material damping. Furthermore, the proposed application of the polarization is demonstrated in a practical field case, where an increased accuracy of estimated nominal thickness is obtained.
Summer declines in activity and body temperature offer polar bears limited energy savings
Authors: Whiteman J. P. , Harlow H. J. , Durner G. M. , Anderson-Sprecher R. , Albeke S. E. , Regehr E. V. , Amstrup S. C. , Ben-David M.
Published: July 2015

Not that unusual after all

As polar ice recedes, polar bears are facing a changed habitat with reduced summer foraging opportunities. It has been hypothesized that they might be able to resist summer food shortages by reducing their metabolic needs in a sort of “walking hibernation.” Whiteman
et al.
monitored energy expenditure in polar bears both on and off the ice and found energy reductions, but that these were more akin to normal mammalian fasting levels. Thus, it appears that polar bears have no energetic protections against reduced summer food supplies and will face increasing starvation threats if summer foraging habitats continue to decline.

Science
, this issue p.
295
Advances in natural language processing
Authors: Hirschberg Julia , Manning Christopher D.
Published: July 2015

Natural language processing employs computational techniques for the purpose of learning, understanding, and producing human language content. Early computational approaches to language research focused on automating the analysis of the linguistic structure of language and developing basic technologies such as machine translation, speech recognition, and speech synthesis. Today’s researchers refine and make use of such tools in real-world applications, creating spoken dialogue systems and speech-to-speech translation engines, mining social media for information about health or finance, and identifying sentiment and emotion toward products and services. We describe successes and challenges in this rapidly advancing area.
Response to Comment on “Sedimentary DNA from a submerged site reveals wheat in the British Isles 8000 years ago”
Authors: Smith Oliver , Momber Garry , Bates Richard , Garwood Paul , Fitch Simon , Pallen Mark , Gaffney Vincent , Allaby Robin G.
Published: July 2015

Bennett questions the rigor of the dating of our sample from which sedimentary ancient DNA was obtained and the reliability of the taxonomic identification of wheat. We present a further radiocarbon date from S308 that confirms the lateral consistency of the palaeosol age. The suggestion of taxonomic false positives in our data illustrates a misinterpretation of the phylogenetic intersection analysis.
Living supramolecular polymerization
Authors: Mukhopadhyay Rahul Dev , Ajayaghosh Ayyappanpillai
Published: July 2015

Greater control is achieved over the chain growth and properties of dynamic materials
Epidemiology, Clinical Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections
Authors: Crump John A. , Sjölund-Karlsson Maria , Gordon Melita A. , Parry Christopher M.
Published: July 2015

Salmonella entericainfections are common causes of bloodstream infection in low-resource areas, where they may be difficult to distinguish from other febrile illnesses and may be associated with a high case fatality ratio. Microbiologic culture of blood or bone marrow remains the mainstay of laboratory diagnosis. Antimicrobial resistance has emerged inSalmonella enterica, initially to the traditional first-line drugs chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole. Decreased fluoroquinolone susceptibility and then fluoroquinolone resistance have developed in association with chromosomal mutations in the quinolone resistance-determining region of genes encoding DNA gyrase and topoisomerase IV and also by plasmid-mediated resistance mechanisms. Resistance to extended-spectrum cephalosporins has occurred more often in nontyphoidal than in typhoidalSalmonellastrains. Azithromycin is effective for the management of uncomplicated typhoid fever and may serve as an alternative oral drug in areas where fluoroquinolone resistance is common. In 2013, CLSI lowered the ciprofloxacin susceptibility breakpoints to account for accumulating clinical, microbiologic, and pharmacokinetic-pharmacodynamic data suggesting that revision was needed for contemporary invasiveSalmonellainfections. Newly established CLSI guidelines for azithromycin andSalmonella entericaserovar Typhi were published in CLSI document M100 in 2015.
Diagnostic Performance of the New Version (v2.0) of GenoType MTBDR
sl
Assay for Detection of Resistance to Fluoroquinolones and Second-Line Injectable Drugs: a Multicenter Study
Authors: Tagliani Elisa , Cabibbe Andrea M. , Miotto Paolo , Borroni Emanuele , Toro Juan Carlos , Mansjö Mikael , Hoffner Sven , Hillemann Doris , Zalutskaya Aksana , Skrahina Alena , Cirillo Daniela M.
Published: July 2015

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDR
sl
assay compared to phenotypic DST and sequencing on a panel of 228
Mycobacterium tuberculosis
isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the e
is
promoter region in the MTBDR
sl
v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDR
sl
v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDR
sl
v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.
Buffer AVL Alone Does Not Inactivate Ebola Virus in a Representative Clinical Sample Type
Authors: Smither Sophie J. , Weller Simon A. , Phelps Amanda , Eastaugh Lin , Ngugi Sarah , O'Brien Lyn M. , Steward Jackie , Lonsdale Steve G. , Lever Mark S.
Published: July 2015

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-resource, outbreak scenarios. The EBOV inactivation efficacy of Buffer AVL (Qiagen) was tested against marmoset serum (EBOV concentration of 1 × 10
8
50% tissue culture infective dose per milliliter [TCID
50
· ml
−1
]) and murine blood (EBOV concentration of 1 × 10
7
TCID
50
· ml
−1
) at 4:1 vol/vol buffer/sample ratios. Posttreatment cell culture and enzyme-linked immunosorbent assay (ELISA) analysis indicated that treatment with Buffer AVL did not inactivate EBOV in 67% of samples, indicating that Buffer AVL, which is designed for RNA extraction and not virus inactivation, cannot be guaranteed to inactivate EBOV in diagnostic samples. Murine blood samples treated with ethanol (4:1 [vol/vol] ethanol/sample) or heat (60°C for 15 min) also showed no viral inactivation in 67% or 100% of samples, respectively. However, combined Buffer AVL and ethanol or Buffer AVL and heat treatments showed total viral inactivation in 100% of samples tested. The Buffer AVL plus ethanol and Buffer AVL plus heat treatments were also shown not to affect the extraction of PCR quality RNA from EBOV-spiked murine blood samples.
Expansion of Simian Immunodeficiency Virus (SIV)-Specific CD8 T Cell Lines from SIV-Naive Mauritian Cynomolgus Macaques for Adoptive Transfer
Authors: Mohns Mariel S. , Greene Justin M. , Cain Brian T. , Pham Ngoc H. , Gostick Emma , Price David A. , O'Connor David H.
Published: July 2015

CD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). However, the specific qualities and characteristics of an effective CD8 T cell response remain unclear. Although targeting breadth, cross-reactivity, polyfunctionality, avidity, and specificity are correlated with HIV control, further investigation is needed to determine the precise contributions of these various attributes to CD8 T cell efficacy. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques (MCM). These cells exhibited an effector memory phenotype, produced cytokines in response to cognate antigen, and suppressed viral replicationin vitro. We further cultured cell lines specific for four SIV-derived epitopes, Nef103–111RM9, Gag389–394GW9, Env338–346RF9, and Nef254–262LT9. These cell lines were up to 94.4% pure, as determined by major histocompatibility complex (MHC) tetramer analysis. After autologous transfer into two MCM recipients, expanded CD8 T cells persisted in peripheral blood and lung tissue for at least 24 weeks and trafficked to multiple extralymphoid tissues. However, these cells did not impact the acute-phase SIV load after challenge compared to historic controls. The expansion and autologous transfer of SIV-specific T cells into naive animals provide a unique model for exploring cellular immunity and the control of SIV infection and facilitate a systematic evaluation of therapeutic adoptive transfer strategies for eradication of the latent reservoir.

IMPORTANCECD8 T cells play a crucial role in the control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Autologous adoptive transfer studies followed by SIV challenge may help define the critical elements of an effective T cell response to HIV and SIV infection. We developed protocols for isolating and expanding SIV-specific CD8 T cells from SIV-naive Mauritian cynomolgus macaques. This is an important first step toward the development of autologous transfer strategies to explore cellular immunity and potential therapeutic applications in the SIV model.
Similarities between Human Immunodeficiency Virus Type 1 and Hepatitis C Virus Genetic and Phenotypic Protease Quasispecies Diversity
Authors: Martinez Miguel Angel , Nevot Maria , Jordan-Paiz Ana , Franco Sandra
Published: July 2015

Human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) are two highly variable RNA viruses that cause chronic infections in humans. Although HCV likely preceded the AIDS epidemic by some decades, the global spread of both viruses is a relatively recent event. Nevertheless, HCV global diversity is higher than that of HIV-1. To identify differences in mutant diversity, we compared the HIV-1 protease and HCV NS3 protease quasispecies. Three protease gene quasispecies samples per virus, isolated from a total of six infected patients, were genetically and phenotypically analyzed at high resolution (HIV-1, 308 individual clones; HCV, 299 clones). Single-nucleotide variant frequency did not differ between quasispecies from the two viruses (HIV-1, 2.4 × 10
−3
± 0.4 × 10
−3
; HCV, 2.1 × 10
−3
± 0.5 × 10
−3
) (
P
= 0.1680). The proportion of synonymous substitutions to potential synonymous sites was similar (3.667 ± 0.6667 and 2.183 ± 0.9048, respectively) (
P
= 0.2573), and Shannon's entropy values did not differ between HIV-1 and HCV (0.84 ± 0.02 and 0.83 ± 0.12, respectively) (
P
= 0.9408). Of note, 65% (HIV-1) and 67% (HCV) of the analyzed enzymes displayed detectable protease activity, suggesting that both proteases have a similar mutational robustness. In both viruses, there was a rugged protease enzymatic activity landscape characterized by a sharp peak, representing the master sequence, surrounded by a collection of diverse variants present at lower frequencies. These results indicate that nucleotide quasispecies diversification during chronic infection is not responsible for the higher worldwide genetic diversity observed in HCV.

IMPORTANCE
HCV global diversity is higher than that of HIV-1. We asked whether HCV genetic diversification during infection is responsible for the higher worldwide genetic diversity observed in HCV. To this end, we analyzed and compared the genotype and enzymatic activities of HIV-1 and HCV protease quasispecies existing in infected individuals. Our results indicate that HIV-1 and HCV protease quasispecies have very similar genetic diversity and comparable rugged enzymatic activity landscapes. Therapy for HCV has expanded, with new therapeutic agents such as the direct-acting antivirals (DAAs). DAAs, which target HCV NS3 protease and other virus proteins, have improved cure rates. However, major questions remain to be elucidated regarding the virologic correlates of HCV eradication. The findings shown here may help our understanding of the different therapeutic responses observed during chronic HCV infection.
pUL69 of Human Cytomegalovirus Recruits the Cellular Protein Arginine Methyltransferase 6 via a Domain That Is Crucial for mRNA Export and Efficient Viral Replication
Authors: Thomas Marco , Sonntag Eric , Müller Regina , Schmidt Stefanie , Zielke Barbara , Fossen Torgils , Stamminger Thomas
Published: July 2015

The regulatory protein pUL69 of human cytomegalovirus acts as a viral mRNA export factor, facilitating the cytoplasmic accumulation of unspliced RNA via interaction with the cellular mRNA export factor UAP56. Here we provide evidence for a posttranslational modification of pUL69 via arginine methylation within the functionally important N terminus. First, we demonstrated a specific immunoprecipitation of full-length pUL69 as well as pUL69aa1-146 by a mono/dimethylarginine-specific antibody. Second, we observed a specific electrophoretic mobility shift upon overexpression of the catalytically active protein arginine methyltransferase 6 (PRMT6). Third, a direct interaction of pUL69 and PRMT6 was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. We mapped the PRMT6 interaction motif to the pUL69 N terminus and identified critical amino acids within the arginine-rich R1 box of pUL69 that were crucial for PRMT6 and/or UAP56 recruitment. In order to test the impact of putative methylation substrates on the functions of pUL69, we constructed various pUL69 derivatives harboring arginine-to-alanine substitutions and tested them for RNA export activity. Thus, we were able to discriminate between arginines within the R1 box of pUL69 that were crucial for UAP56/PRMT6-interaction and/or mRNA export activity. Remarkably, nuclear magnetic resonance (NMR) analyses revealed the same α-helical structures for pUL69 sequences encoding either the wild type R1/R2 boxes or a UAP56/PRMT6 binding-deficient derivative, thereby excluding the possibility that R/A amino acid substitutions within R1 affected the secondary structure of pUL69. We therefore conclude that the pUL69 N terminus is methylated by PRMT6 and that this critically affects the functions of pUL69 for efficient mRNA export and replication of human cytomegalovirus.

IMPORTANCE
The UL69 protein of human cytomegalovirus is a multifunctional regulatory protein that acts as a viral RNA export factor with a critical role for efficient replication. Here, we demonstrate that pUL69 is posttranslationally modified via arginine methylation and that the protein methyltransferase PRMT6 mediates this modification. Furthermore, arginine residues with a crucial function for RNA export and for binding of the cellular RNA export factor UAP56 as well as PRMT6 were mapped within the arginine-rich R1 motif of pUL69. Importantly, we demonstrated that mutation of those arginines did not alter the secondary structure of R1, suggesting that they may serve as critical methylation substrates. In summary, our study reveals a novel posttranslational modification of pUL69 which has a significant impact on the function of this important viral regulatory protein. Since PRMTs appear to be amenable to selective inhibition by small molecules, this may constitute a novel target for antiviral therapy.
Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k
Authors: Condezo Gabriela N. , Marabini Roberto , Ayora Silvia , Carazo José M. , Alba Raúl , Chillón Miguel , San Martín Carmen
Published: July 2015

Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating.

IMPORTANCE
Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.
Differential Effects of Variable Frequencies of Manual Acupuncture at St36 in Rats with Atropine-Induced Inhibition of Gastric Motility
Authors: Gao Li-Li , Guo Yi , Sha Tao , Liu Yang-Yang , Tang Jia-Bei , Yuan Fang , Zhou Tao , Hong Shou-Hai , Ming Dong
Published: July 2015

Objective

The ‘intensity-response’ relationship between acupuncture stimulation and therapeutic effect is currently the focus of much research interest. The same needling manipulation with different frequencies can generate differential levels of stimulus. This study aimed to examine the effects on gastric motility induced by four twirling frequencies based on relatively standardised manual acupuncture (MA) manipulations.

Methods

Twirling manipulations at 1, 2, 3, and 4 Hz were practised before the experiments by a single operator using an MA parameter measurement device and stability was evaluated through time-frequency analysis. Forty-eight Sprague-Dawley rats were randomly divided into six groups (n=8 each): Control, Model, Model+MA (1, 2, 3, and 4 Hz). Rats in the five Model groups received injections of atropine into the tail vein to inhibit gastric motility, which was continuously recorded by a balloon in the gastric antrum. Rats in the four Model+MA groups received MA at 1, 2, 3 and 4 Hz, respectively, for 70 s and needles were retained for a further 5 min.

Results

The amplitude of waveforms produced by the four twirling frequencies was relatively consistent and reproducible. The gastric motility amplitude in all groups decreased after modelling (injections of atropine) (p<0.01). Twirling manipulation at 1, 2, and 3 Hz (but not 4 Hz) increased gastric motility amplitude (p<0.05). The increase in gastric motility amplitude induced by MA at 2 Hz was greater than for all other frequencies (p<0.05).

Conclusions

Acupuncture at ST36 helped recover gastric motility amplitude in rats with atropine-induced gastric inhibition and the effects induced by 1–3 Hz frequency were greater than those induced by 4 Hz.
Inhibition of Spinal Microglia and Astrocytes Contributes to the Anti-Allodynic Effect of Electroacupuncture in Neuropathic Pain Induced by Spinal Nerve Ligation
Authors: Liang Yi , Qiu Yujie , Du Junying , Liu Jin , Fang Junfan , Zhu Ji , Fang Jianqiao
Published: July 2015

Objective

Besides neurons, activated microglia and astrocytes in the spinal cord dorsal horn (SCDH) contribute to the pathogenesis of chronic pain. Electroacupuncture (EA) has been used widely to treat various chronic pain diseases, however, the underlying mechanisms of EA are still not fully understood.

Methods

Male Sprague-Dawley rats were randomly divided into four groups, including an untreated healthy Control group (n=14), a True-spinal nerve ligation (SNL) group that underwent SNL and remained untreated (n=25), a True-SNL+EA group that underwent SNL followed by EA treatment (n=25), and a Sham-SNL group that underwent sham surgery and remained untreated (n=15). SNL was performed unilaterally at L5 and EA was applied to ST36 and BL60 bilaterally once per day. Paw withdrawal thresholds (PWTs) were measured ipsilaterally at baseline and 1, 3, 7, and 14 days after ligation. Activation of microglia and astrocytes in the SCDH were examined bilaterally by immunofluorescence staining, and concentrations of interleukin-1β (IL-1β) and interleukin (IL-6) were measured in the ipsilateral SCDH by ELISA.

Results

SNL significantly decreased PWTs and activated glial cells in the superficial laminae of the ipsilateral SCDH. In rats with SNL, glial fibrillary acidic protein (GFAP) immunoreactivity peaked at 7 days and was maintained until 14 days post-ligation, while anti-integrin alphaM (OX-42) immunoreactivity peaked at 3 days and declined gradually. EA significantly alleviated SNL-induced mechanical allodynia. Furthermore, EA reduced microglial activation (OX-42 positive ratios) in the lumbar SCDH at 3 days post-ligation and suppressed astrocyte activation (GFAP positive ratios) at all time points observed.

Conclusions

EA stimulation alleviates SNL-induced neuropathic pain, at least in part through inhibition of spinal glial activation. Moreover, inhibition of spinal microglia and astrocyte activation may contribute to the immediate effects and maintenance of EA analgesia, respectively.
Recurrent presumed herpes simplex keratitis and episcleritis in keratosis follicularis (Darier's disease)
Authors: Radia Meera , Gilhooley Michael James , Panos Chris , Claoué Charles
Published: July 2015

Keratosis follicularis (Darier's disease) is an autosomal dominant dermatological disorder characterised by abnormal epidermal differentiation and loss of normal cell-to-cell adhesion. Cardinal features include diffuse hyperkeratotic warty papules with scaly plaques in seborrhoeic regions with associated mucous membrane changes. Darier's disease is rare (prevalence 2.7 in 100 000), with few ocular sequelae reported: commonly dry eye with or without Sjögren's syndrome. This is the first report, to the best of our knowledge, to describe a case of recurrent herpes simplex virus (HSV) keratitis and episcleritis in a 47-year-old man suffering from Darier's disease. The patient's condition predisposed him towards developing ocular complications due to several factors: impaired desmosome function leading to poor cell-to-cell adhesion in the corneal epithelium, dry eye and HSV invasion of inflamed periocular skin presumably combining to allow viral colonisation of a poorly protected cornea.
Pitfalls in diagnosis and treatment of alveolar echinococcosis: a sentinel case series
Authors: Stojkovic M , Mickan C , Weber TF , Junghanss T
Published: July 2015

Background

Alveolar echinococcosis (AE) is a neglected zoonosis presenting with focal liver lesions (FLL) with a wide range of imaging patterns resembling benign as well as malignant FLLs. Complementary serology and histopathology may be misleading.

Objective

The objective of our study is to highlight pitfalls leading to wrong diagnoses and harmful interventions in patients with AE.

Design

This retrospective sentinel case series analyses diagnostic and treatment data of patients with confirmed AE.

Results

80 patients treated between 1999 and 2014 were included in the study. In 26/80 patients treatment decisions were based on a wrong diagnosis. AE was mistaken for cystic echinococcosis (CE) in 12/26 patients followed by cholangiocellular carcinoma (CCA) in 5/26 patients; 61/80 patients had predominantly infiltrative liver lesions and 19/80 patients had a predominantly pseudocystic radiological presentation. Serology correctly differentiated betweenEchinococcus multilocularisandEchinococcus granulosusin 53/80 patients. Histopathology reports attributed the rightEchinococcus speciesin 25/58 patients but failed to differentiateE. multilocularisfromE. granulosusin 25/58 patients. Although contraindicated in AE 8/25 patients treated surgically had instillation of a protoscolicidal agent intraoperatively. One of the eight patients developed toxic cholangitis and liver failure and died 1 year after liver transplantation.

Conclusions

Misclassification of AE leads to a critical delay in growth inhibiting benzimidazole treatment, surgical overtreatment and bares the risk of liver failure if protoscolicidal agents are instilled in AE pseudocysts.