Site-directed in vitro immunization leads to a complete human monoclonal IgG4λ that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands

<p>Abstract</p> <p>Background</p> <p>The ability to acquire fully human monoclonal antibodies (mAbs) with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG) molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags) such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4) because proper hosts are lacking.</p> <p>Results</p> <p>Here we developed an optimized protocol for site-directed <it>in vitro </it>immunizing peripheral blood mononuclear cells (PBMC) by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (γ4λhuCD152), which contains variable (V) regions of the heavy chain and the light chain derived from the VH3 and Vλ human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, γ4λhuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4λs as revealed by the isoelectric focusing (IEF) analysis. Furthermore, γ4λhuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2) of CD152, whereby it can react to activated CD3⁺cells.</p> <p>Conclusion</p> <p>In a context of specific cell depletion and conditioned medium,<it>in vitro </it>induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, γ4λhuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4λ mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both <it>in situ </it>and <it>ex situ</it>, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may otherwise prevent the access of whole IgG1 molecules.</p

Aerosolized Colistin for the Treatment of Multidrug-resistant Acinetobacter baumannii Pneumonia: Experience in a Tertiary Care Hospital in Northern Taiwan

Background/PurposeVentilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) Acinetobacter baumannii in critically ill patients presents an emerging challenge to clinicians. Administration of aerosolized colistin as an adjunctive therapy is one therapeutic option mentioned in limited evidence-based studies. This study aimed to evaluate the effectiveness of adjunctive aerosolized colistin treatment for VAP due to MDR pathogens.MethodsWe retrospectively reviewed the medical records of patients who had received aerosolized colistin for treatment of VAP due to MDR A. baumannii in our hospital from August to December 2008.ResultsForty-five patients were enrolled in our study. The mean age was 71 ± 15 years. The mean Acute Physiological and Chronic Health Evaluation II (APACHE II) scores on the day of intensive care unit admission and on the first day of aerosolized colistin administration were 22.5 ± 6.7 and 18.9 ± 5.7, respectively. The mean duration of intensive care unit stay was 34 ± 16 days. The mean daily dosage of aerosolized colistin was 4.29 ± 0.82 million IU, and the mean duration of administration was 10.29 days. Seventeen patients (37.8%) had a favorable microbiological outcome and 26 (57.8%) showed a clinical response. Mortality due to all causes was 42.2%. No adverse effects related to inhaled colistin were recorded.ConclusionAerosolized colistin may be considered as an adjunct to intravenous treatments in patients with VAP due to colistin-susceptible MDR A. baumannii in critically ill patients

Clinical characteristics and treatment outcomes of patients with tubo-ovarian abscess at a tertiary care hospital in Northern Taiwan

Background/PurposeControversy exists regarding the need for surgical intervention in patients with tubo-ovarian abscess (TOA). This study was aimed at investigating the clinical characteristics and treatment outcomes in patients with TOA at a tertiary care hospital in Taiwan.MethodsThe medical records of 83 patients who presented at the hospital with TOA between January 1, 2006, and December 31, 2007, were retrospectively reviewed. Outcomes of patients who received medical treatment alone or underwent surgical intervention were analyzed using univariate and logistic regression analyses.ResultsAmong the 83 patients with TOA, 13 patients (15.7%) underwent surgical intervention, and 70 patients (84.3%) received medical treatment alone. Significant variables related to surgical treatment in the univariate analysis were length of stay (short vs. long; t = −2.267, p = 0.026), department of admission (emergency room vs. outpatient department; χ2 = 7.459, p = 0.006), number of live births (nulliparous vs. multiparous; χ2 = 18.202, p = 0.001), and C-reactive protein (CRP) level (high vs. low; t = −2.250, p = 0.028). Logistic regression analysis performed to determine influential factors for surgical treatment showed that the operation odds ratio of three to four live births versus no live births was 33.995 (p = 0.043) and that of two live births versus no live births was 13.598 (p = 0.026).ConclusionPatients with TOA who underwent surgery had a longer duration of hospitalization. Among the patients who underwent surgical intervention, those admitted to the emergency room had higher CRP levels and were more likely to be multiparous

Phenyl radical + propene: a prototypical reaction surface for aromatic-catalyzed 1,2-hydrogen-migration and subsequent resonance-stabilized radical formation

The C[subscript 9]H[subscript 11] potential energy surface (PES) was experimentally and theoretically explored because it is a relatively simple, prototypical alkylaromatic radical system. Although the C[subscript 9]H[subscript 11] PES has already been extensively studied both experimentally (under single-collision and thermal conditions) and theoretically, new insights were made in this work by taking a new experimental approach: flash photolysis combined with time-resolved molecular beam mass spectrometry (MBMS) and visible laser absorbance. The C[subscript 9]H[subscript 11] PES was experimentally accessed by photolytic generation of the phenyl radical and subsequent reaction with excess propene (C[subscript 6]H[subscript 5] + C[subscript 3]H[subscript 6]). The overall kinetics of C[subscript 6]H[subscript 5] + C[subscript 3]H[subscript 6] was measured using laser absorbance with high time-resolution from 300 to 700 K and was found to be in agreement with earlier measurements over a lower temperature range. Five major product channels of C[subscript 6]H[subscript 5] + C[subscript 3]H[subscript 6] were observed with MBMS at 600 and 700 K, four of which were expected: hydrogen (H)-abstraction (measured by the stable benzene, C[subscript 6]H[subscript 6], product), methyl radical (CH[subscript 3])-loss (styrene detected), H-loss (phenylpropene isomers detected) and radical adduct stabilization. The fifth, unexpected product observed was the benzyl radical, which was rationalized by the inclusion of a previously unreported pathway on the C[subscript 9]H[subscript 11] PES: aromatic-catalysed 1,2-H-migration and subsequent resonance stabilized radical (RSR, benzyl radical in this case) formation. The current theoretical understanding of the C[subscript 9]H[subscript 11] PES was supported (including the aromatic-catalyzed pathway) by quantitative comparisons between modelled and experimental MBMS results. At 700 K, the branching to styrene + CH[subscript 3] was 2-4 times greater than that of any other product channel, while benzyl radical + C[subscript 2]H[subscript 4] from the aromatic-catalyzed pathway accounted for ∼10% of the branching. Single-collision conditions were also simulated on the updated PES to explain why previous crossed molecular beam experiments did not see evidence of the aromatic-catalyzed pathway. This experimentally validated knowledge of the C[subscript 9]H[subscript 11] PES was added to the database of the open-source Reaction Mechanism Generator (RMG), which was then used to generalize the findings on the C[subscript 9]H[subscript 11] PES to a slightly more complicated alkylaromatic system.Think Global Education Trus

Managing cardiac arrest with refractory ventricular fibrillation in the emergency department: Conventional cardiopulmonary resuscitation versus extracorporeal cardiopulmonary resuscitation

AbstractAimRefractory ventricular fibrillation, resistant to conventional cardiopulmonary resuscitation (CPR), is a life threatening rhythm encountered in the emergency department. Although previous reports suggest the use of extracorporeal CPR can improve the clinical outcomes in patients with prolonged cardiac arrest, the effectiveness of this novel strategy for refractory ventricular fibrillation is not known. We aimed to compare the clinical outcomes of patients with refractory ventricular fibrillation managed with conventional CPR or extracorporeal CPR in our institution.MethodThis is a retrospective chart review study from an emergency department in a tertiary referral medical center. We identified 209 patients presenting with cardiac arrest due to ventricular fibrillation between September 2011 and September 2013. Of these, 60 patients were enrolled with ventricular fibrillation refractory to resuscitation for more than 10min. The clinical outcome of patients with ventricular fibrillation received either conventional CPR, including defibrillation, chest compression, and resuscitative medication (C-CPR, n=40) or CPR plus extracorporeal CPR (E-CPR, n=20) were compared.ResultsThe overall survival rate was 35%, and 18.3% of patients were discharged with good neurological function. The mean duration of CPR was longer in the E-CPR group than in the C-CPR group (69.90±49.6min vs 34.3±17.7min, p=0.0001). Patients receiving E-CPR had significantly higher rates of sustained return of spontaneous circulation (95.0% vs 47.5%, p=0.0009), and good neurological function at discharge (40.0% vs 7.5%, p=0.0067). The survival rate in the E-CPR group was higher (50% vs 27.5%, p=0.1512) at discharge and (50% vs 20%, p=0. 0998) at 1 year after discharge.ConclusionsThe management of refractory ventricular fibrillation in the emergency department remains challenging, as evidenced by an overall survival rate of 35% in this study. Patients with refractory ventricular fibrillation receiving E-CPR had a trend toward higher survival rates and significantly improved neurological outcomes than those receiving C-CPR

Pervasive brain monitoring and data sharing based on multi-tier distributed computing and linked data technology

EEG-based Brain-computer interfaces (BCI) are facing grant challenges in their real-world applications. The technical difficulties in developing truly wearable multi-modal BCI systems that are capable of making reliable real-time prediction of users’ cognitive states under dynamic real-life situations may appear at times almost insurmountable. Fortunately, recent advances in miniature sensors, wireless communication and distributed computing technologies offered promising ways to bridge these chasms. In this paper, we report our attempt to develop a pervasive on-line BCI system by employing state-of-art technologies such as multi-tier fog and cloud computing, semantic Linked Data search and adaptive prediction/classification models. To verify our approach, we implement a pilot system using wireless dry-electrode EEG headsets and MEMS motion sensors as the front-end devices, Android mobile phones as the personal user interfaces, compact personal computers as the near-end fog servers and the computer clusters hosted by the Taiwan National Center for High-performance Computing (NCHC) as the far-end cloud servers. We succeeded in conducting synchronous multi-modal global data streaming in March and then running a multi-player on-line BCI game in September, 2013. We are currently working with the ARL Translational Neuroscience Branch and the UCSD Movement Disorder Center to use our system in real-life personal stress and in-home Parkinson’s disease patient monitoring experiments. We shall proceed to develop a necessary BCI ontology and add automatic semantic annotation and progressive model refinement capability to our system

SIRT1 Activation by a c-MYC Oncogenic Network Promotes the Maintenance and Drug Resistance of Human FLT3-ITD Acute Myeloid Leukemia Stem Cells

SummaryThe FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes

Anodization of nanoporous alumina on impurity-induced hemisphere curved surface of aluminum at room temperature

Nanoporous alumina which was produced by a conventional direct current anodization [DCA] process at low temperatures has received much attention in various applications such as nanomaterial synthesis, sensors, and photonics. In this article, we employed a newly developed hybrid pulse anodization [HPA] method to fabricate the nanoporous alumina on a flat and curved surface of an aluminum [Al] foil at room temperature [RT]. We fabricate the nanopores to grow on a hemisphere curved surface and characterize their behavior along the normal vectors of the hemisphere curve. In a conventional DCA approach, the structures of branched nanopores were grown on a photolithography-and-etched low-curvature curved surface with large interpore distances. However, a high-curvature hemisphere curved surface can be obtained by the HPA technique. Such a curved surface by HPA is intrinsically induced by the high-resistivity impurities in the aluminum foil and leads to branching and bending of nanopore growth via the electric field mechanism rather than the interpore distance in conventional approaches. It is noted that by the HPA technique, the Joule heat during the RT process has been significantly suppressed globally on the material, and nanopores have been grown along the normal vectors of a hemisphere curve. The curvature is much larger than that in other literatures due to different fabrication methods. In theory, the number of nanopores on the hemisphere surface is two times of the conventional flat plane, which is potentially useful for photocatalyst or other applications

Functional characterization of cellulases identified from the cow rumen fungus Neocallimastix patriciarum W5 by transcriptomic and secretomic analyses

<p>Abstract</p> <p>Background</p> <p><it>Neocallimastix patriciarum</it> is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (<it>GHs</it>) produced by this anaerobic fungus.</p> <p>Results</p> <p>We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to <it>N. patriciarum </it>to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative <it>GH </it>contigs and classified them into 25 <it>GH</it> families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the <it>GH1</it>, <it>GH3</it>, <it>GH5</it>, <it>GH6</it>, <it>GH9</it>, <it>GH18</it>, <it>GH43 </it>and <it>GH48 </it>gene families, which were highly expressed in <it>N. patriciarum </it>cultures grown on different feedstocks.</p> <p>Conclusions</p> <p>These <it>GH </it>genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.</p