Mutational profile of previously treated chronic lymphocytic leukemia patients progressing on acalabrutinib or ibrutinib.

Chronic lymphocytic leukemia (CLL) progression during Bruton tyrosine kinase (BTK) inhibitor treatment is typically characterized by emergent B-cell receptor pathway mutations. Using peripheral blood samples from relapsed/refractory CLL patients in ELEVATE-RR (NCT02477696) (median 2 prior therapies), we report clonal evolution data for patients progressing on acalabrutinib or ibrutinib (median follow-up 41 months). Paired (baseline and progression) samples were available for 47 (excluding 1 Richter) acalabrutinib-treated and 30 (excluding 6 Richter) ibrutinib-treated patients. At progression, emergent BTK mutations were observed in 31 (66%) acalabrutinib-treated and 11 (37%) ibrutinib-treated patients (median variant allele fraction [VAF]: 16.1% vs 15.6%). BTK C481S mutations were most common in both groups; T474I (n = 9; 8 co-occurring with C481) and the novel E41V mutation within the pleckstrin homology domain of BTK (n = 1) occurred with acalabrutinib, while neither mutation occurred with ibrutinib. L528W and A428D co-mutations presented in one ibrutinib-treated patient. Pre-existing TP53 mutations were present in 25 (53.2%) acalabrutinib-treated and 16 (53.3%) ibrutinib-treated patients at screening. Emergent TP53 mutations occurred with acalabrutinib and ibrutinib (13% vs 7%; median VAF: 6.0% vs 37.3%, respectively). Six acalabrutinib-treated patients and one ibrutinib-treated patient had emergent TP53/BTK co-mutations. Emergent PLCG2 mutations occurred in 3 (6%) acalabrutinib-treated and 6 (20%) ibrutinib-treated patients. One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 co-mutations. While common BTK C481 mutations were observed with both treatments, patterns of mutation and co-mutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W, A428D) in this patient population.

Acalabrutinib CYP3A-mediated drug-drug interactions: Clinical evaluations and physiologically based pharmacokinetic modelling to inform dose adjustment strategy.

AIMS: Clinical drug interaction studies with itraconazole and rifampicin have demonstrated that acalabrutinib is a sensitive substrate of CYP3A. A physiologically based pharmacokinetic (PBPK) model was developed based on the data of these studies. One of the active CYP3A metabolites, ACP-5862, was identified but never studied in a drug interaction scenario. This study aims to evaluate both parent and metabolite exposure change with coadministration of moderate CYP3A inhibitors and its impact on safety and efficacy. METHODS: In an open label, randomized, 2-period study, we investigated the effect of coadministration of fluconazole or isavuconazole on the pharmacokinetics of acalabrutinib. Bruton tyrosine kinase receptor occupancy and safety were compared between different treatments. Experimental data were compared to PBPK simulation results. RESULTS: Least square means of acalabrutinib maximum plasma concentration and area under the curve increased 1.37 (1.14-1.64) and 1.60 (1.45-1.77)-fold in the presence of isavuconazole and 1.48 (1.10-1.98) and 2.16 (1.94-2.40)-fold in the presence of fluconazole, respectively. For ACP-5862, these values are 0.72 (0.63-0.82) and 0.91 (0.86-0.97) fold for isavuconazole and 0.65 (0.49-0.87) and 0.95 (0.91-0.99) fold for fluconazole coadministration. The PBPK model was able to recover acalabrutinib and ACP-5862 PK profiles in the study. Bruton tyrosine kinase receptor occupancy change was minimal in the presence of isavuconazole. There were no deaths, serious adverse events (AEs), or subject discontinuation due to AEs in this study. Only mild (Grade 1) AEs were reported during the study, by 17% of the study population. CONCLUSION: Our results demonstrate the impact of fluconazole and isavuconazole on the pharmacokinetics of acalabrutinib and ACP-5862, and suggest that no dose adjustment is needed for concomitant administration with moderate CYP3A inhibitors. the current PBPK model can be used to propose dose adjustment for drug interactions via CYP3A.

Comprehensive behavioural intervention for tics: a neurophysiological intervention.

BACKGROUND: Gilles de la Tourette Syndrome (GTS) is a childhood-onset neuropsychiatric disorder characterised by motor and vocal tics. While Comprehensive Behavioural Intervention for Tics (CBIT) is an effective, non-pharmacological treatment for patients with GTS, the underlying neurophysiological basis of this intervention has not been investigated. METHODS: To investigate the clinical effectiveness of CBIT in reducing tic severity in young people with GTS and explore neurophysiological mechanisms associated with clinical change. RESULTS: There was a significant overall improvement in tic severity of large effect size. The Cortical Silent Period (CSP) to motor evoked potential (MEP) ratio (CSP/MEP ratio) increased after the intervention with a small effect size. Other neurophysiological measures of inhibition were not significantly related to the change in tic severity. CONCLUSIONS: Alongside significant clinical improvements, these results suggest a role for motor cortical Gamma-aminobutyric acid (GABA)-ergic inhibitory circuitry in the neurophysiological changes underlying CBIT treatment. These findings need to be replicated in larger studies using control samples.

Modulation of chemotherapy-induced cytotoxicity in SH-SY5Y neuroblastoma cells by caffeine and chlorogenic acid.

Chemotherapy is an important treatment modality for malignancy but is limited by significant toxicity and it susceptibility to numerous drug interactions. While the interacting effects with medications are well known, there is limited evidence on the interaction with commonly consumed food and natural products. The aim of this study was to evaluate the bioactive constituents of coffee (caffeine and chlorogenic acid) on the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in vitro. Pretreatment with caffeine (100 nM and 10 µM) sensitized SH-SY5Y cells to doxorubicin-induced toxicity and increased apoptosis and sensitized PC3 cells to gemcitabine-induced toxicity. Pretreatment with 10 µM caffeine decreased total cell reactive oxygen species (ROS) production but increased mitochondrial ROS production. In contrast, caffeine (10 nM and 10 µM) protected cells against gemcitabine-induced toxicity and apoptosis. Similarly, 1 µM and 10 µM caffeine protected cells against paclitaxel-induced toxicity and mitochondrial ROS production. Chlorogenic acid had no effect on chemotherapy-induced toxicity in SH-SY5Y cells. In conclusion, this study provides preliminary evidence that caffeine, not chlorogenic acid, modulates the cytotoxicity of doxorubicin, gemcitabine, and paclitaxel in SH-SY5Y cells via different mechanisms.

Outside-In Hemofiltration for Prolonged Operation without Clogging.

Hemofiltration (HF) is used extensively for continuous renal replacement therapy, but long-term treatment is limited by thrombosis leading to fiber clogging. Maximum filter life is typically less than 20 hours. We have achieved for the first time continuous and consistent hemofiltration for more than 100 hours using outside-in hemofiltration with the blood flow into the inter-fiber space (IFS). Although thrombi do deposit in the IFS, they have minimal affect on the blood flow and filtrate flux due to the three-dimensional system of interconnected hydrodynamic flow channels in the IFS. Microscopic examination of sections of the fiber bundle showed that deposited thrombi have dimensions about the size of the gaps between the hollow fibers and remain isolated from each other. A simple mathematical model is developed to describe the effect of thrombus deposition on the fluid flow that accounts for the enhanced performance arising from the interconnected flow. The hydrodynamic advantage of outside-in HF decreases at low anticoagulant concentration due to the instability in the blood and the very high volume fraction of thrombi that deposit in the entrance zone of the filter. These results clearly demonstrate the significant potential advantages of using outside-in hemofiltration for long-term renal replacement therapy.

Multiplex-microsphere-quantitative polymerase chain reaction: nucleic acid amplification and detection on microspheres.

We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.

Liraglutide modulates adhesion molecules and enhances cell properties in three-dimensional cultures of olfactory ensheathing cells.

Cell transplantation using olfactory ensheathing cells (OECs) is a promising approach for nerve repair but there are numerous limitations with their delivery method. Three-dimensional (3D) cell culture systems potentially offer a powerful approach for cell production and delivery options. To further optimise the use of OECs, strategies to promote cell viability and maintain cell behaviours in 3D cultures become important. We previously demonstrated an anti-diabetic drug, liraglutide, could modulate OEC migration and re-model extracellular matrix in two-dimensional (2D) cultures. In the present study, we further investigated its beneficial effects in our 3D culture system using primary OECs. OECs treated with liraglutide at 100 nM showed improved cell viability and had modulated expression of N-cadherin and ß1-integrin (two important cell adhesion molecules). When formed into 3D spheroids, the pre-treated OECs generated spheroids with an increased volume and a decreased cell density compared to control spheroids. OECs that subsequently migrated out of the liraglutide pre-treated spheroids had higher capacity for migration with increased duration and length, which was attributed to a reduction in the pauses during the migration. Moreover, OECs that migrated out from liraglutide spheroids had a more bipolar morphology consistent with higher migratory capacity. In summary, liraglutide improved the viability of OECs, modulated cell adhesion molecules, and resulted in stable 3D cell constructs which conferred enhanced migratory capacity on the OECs. Overall, liraglutide may potentially improve the therapeutic use of OECs for neural repair by enhancing the generation of stable 3D constructs and increasing the migratory behaviour of OECs.

PrimRglo: a multiplexable quantitative real-time polymerase chain reaction system for nucleic acid detection.

We report the development of a new real-time polymerase chain reaction (PCR) detection system that uses oligonucleotide "tagged" PCR primers, a fluorophore-labeled "universal" detection oligonucleotides, and a complementary quenching oligonucleotide. The fluorescence signal decreases as PCR product accumulates due to the increase in detection/quencher hybrid formation as the tagged primer is consumed. We use plasmids containing the influenza A matrix gene and the porA and ctrA genes of Neisseria meningitidis as targets for developing the system. Cycle threshold (Ct) values were generated, and the sensitivity of the new system (dubbed "PrimRglo") compared favorably with the commonly used SYBR green and Taqman detection systems and, unlike the latter system, does not require the design of a new dual-labeled detection oligonucleotide for each new target sequence.

Liraglutide modulates olfactory ensheathing cell migration with activation of ERK and alteration of the extracellular matrix.

Transplantation of olfactory ensheathing cells (OECs) is a promising approach for repairing the injured nervous system that has been extensively trialed for nervous system repair. However, the method still needs improvement and optimization. One avenue of improving outcomes is to stimulate OEC migration into the injury site. Liraglutide is a glucagon-like peptide-1 receptor agonist used for management of diabetes and obesity. It has been shown to be neuroprotective and to promote cell migration, but whether it can stimulate glial cells remains unknown. In the current study, we investigated the effects of liraglutide on OEC migration and explored the involved mechanisms. We showed that liraglutide at low concentration (100 nM) overall promoted OEC migration over time. Liraglutide modulated the migratory behavior of OECs by reducing time in arrest, and promoted random rather than straight migration. Liraglutide also induced a morphological change of primary OECs towards a bipolar shape consistent with improved migration. We found that liraglutide activated extracellular signal-regulated kinase (ERK), which has key roles in cell migration; the timing of ERK activation correlated with stimulation of migration. Furthermore, liraglutide also modulated the extracellular matrix by upregulating laminin-1 and down-regulating collagen IV. In summary, we found that liraglutide can stimulate OEC migration and re-model the extracellular matrix to better promote cell migration, and possibly also to become more conducive for axonal regeneration. Thus, liraglutide may improve OEC transplantation outcomes.

Turbulent fluid flow is a novel closed-system sample extraction method for flexible endoscope channels of various inner diameters.

OVERVIEW: Effective sample extraction from endoscope channels is crucial for monitoring manual cleaning adequacy as well as for ensuring optimal sensitivity for culture after disinfection. The objective of this study was to compare the efficacy of Turbulent Fluid Flow (TFF) to Flush (F) or Flush-Brush-Flush (FBF) methods. MATERIALS & METHODS: Pseudomonas aeruginosa and Enterococcus faecalis in artificial test soil-2015 (ATS2015) were used as bacterial markers while protein and carbohydrate were the organic markers for biofilm formed inside 3.2-mm and 1.37-mm polytetrafluoroethylene (PTFE) channels. TFF was generated using compressed air and sterile water to provide friction for sample extraction. Extraction for biofilm coated PTFE channels as well as for colonoscope channels perfused with ATS2015 containing 108 CFU/mL P. aeruginosa, E. faecalis and Candida albicans was determined using TFF compared to FBF and F. RESULTS: The extraction ratio for P. aeruginosa and E. faecalis from biofilm extracted by TFF compared to the positive control was significantly better than F for 1.37-mm channels (≥0.94 for both bacteria by TFF versus 0.69 to 0.72 by F for P. aeruginosa and E. faecalis, respectively) but not significantly different between TFF and FBF for 3.2-mm channels. F was also ineffective for extraction of protein and carbohydrate from 1.37-mm channels. Extraction efficacy by TFF from inoculated colonoscope channels was >98% for all test markers. CONCLUSIONS: The novel TFF method for extraction of samples from colonoscope channels is a more effective method than the existing FBF and F methods.

A microfluidic device for in situ fixation and super-resolved mechanosensation studies of primary cilia.

The primary cilium plays an important role in mechanosensation in mammalian cells. To understand mechanosensation in the primary cilium, we combined a microfluidic device with super-resolution microscopy to study the primary cilium phenotypes. The microfluidic system enabled the precise control of the flow shear within a well-confined cell-culture environment. In addition, in situ cilia fixation was possible by switching from the culture medium to the fixation buffer instantaneously, which preserved the real-time cilium phenotype under the flow shear. After fixation, multiple cilium-specific proteins were immunostained to quantify the cilia bending behavior. We found that >50% of the primary cilia of mouse inner medullary collecting duct cells were highly aligned with the direction of flow under 11 Pa shear stress. Finally, we used super-resolution microscopy to observe the redistribution of two major cilium-specific proteins under flow shear, acetylated alpha-tubulin, and intraflagellar transport protein 88. To the best of our knowledge, this is the first platform to combine a microfluidic device with super-resolution microscopy to enable flow stimulation and in situ fixation for the observation of ciliary protein. This system can potentially be applied to the future development of a stimulation-enabled organ-on-a-chip to observe the intercellular signaling of primary cilia or for the analysis of disease mechanisms associated with ciliary mutations at the organ level.

A broad spectrum, one-step reverse-transcription PCR amplification of the neuraminidase gene from multiple subtypes of influenza A virus.

BACKGROUND: The emergence of high pathogenicity strains of Influenza A virus in a variety of human and animal hosts, with wide geographic distribution, has highlighted the importance of rapid identification and subtyping of the virus for outbreak management and treatment. Type A virus can be classified into subtypes according to the viral envelope glycoproteins, hemagglutinin and neuraminidase. Here we review the existing specificity and amplification of published primers to subtype neuraminidase genes and describe a new broad spectrum primer pair that can detect all 9 neuraminidase subtypes. RESULTS: Bioinformatic analysis of 3,337 full-length influenza A neuraminidase segments in the NCBI database revealed semi-conserved regions not previously targeted by primers. Two degenerate primers with M13 tags, NA8F-M13 and NA10R-M13 were designed from these regions and used to generate a 253 bp cDNA product. One-step RT-PCR testing was successful in 31/32 (97%) cases using a touchdown protocol with RNA from over 32 different cultured influenza A virus strains representing the 9 neuraminidase subtypes. Frozen blinded clinical nasopharyngeal aspirates were also assayed and were mostly of subtype N2. The region amplified was direct sequenced and then used in database searches to confirm the identity of the template RNA. The RT-PCR fragment generated includes one of the mutation sites related to oseltamivir resistance, H274Y. CONCLUSION: Our one-step RT-PCR assay followed by sequencing is a rapid, accurate, and specific method for detection and subtyping of different neuraminidase subtypes from a range of host species and from different geographical locations.

A case of central nervous system nocardiosis in a patient with lupus treated with belimumab.

Belimumab was approved by the United States Food and Drug Administration in March 2011 as the first biological agent for treating active systemic lupus erythematosus (SLE). To the best of our knowledge, this is the first case report regarding a patient with SLE treated with belimumab who was diagnosed with central nervous system nocardiosis.

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step.

BACKGROUND: Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. RESULTS: The strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants. CONCLUSION: This strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.

Application of the PrimRglo assay chemistry to multiplexed bead assays.

In this unit, we describe a multiplex microsphere quantitative PCR. The system is based on the use of two additional oligonucleotides within a single tube PCR reaction. The first oligonucleotide is modified with a single base pair mismatch and is otherwise equivalent to a universal sequence added to the forward PCR primer. Further, this first extra oligonucleotide is coupled to Luminex microspheres. The second additional oligonucleotide is designed to be complementary to the universal sequence, and is modified with the fluorescent dye Cy3. As the PCR reaction proceeds, the second oligonucleotide is able to bind to the microspheres. Thus, quantitative monitoring of PCR progress takes place. The microsphere-mediated Cy3-detection is measured using flow cytometry directly after the PCR reaction. This allows a flow cytometer analysis from up to 150 different spheres and, therefore, multiple genes in one reaction. The multiplex microsphere qPCR is demonstrated using three target genes from Influenza A and Neisseria meningitidis. The multiplex microsphere system will enable a higher degree of multiplexing than is possible with currently available qPCR systems.

Synthesis and reactivity of a cyclopentadienyl-indenyl ligand ring-coupled by a chiral bridge derived from ethyl (S)-(-) lactate.

In order to prepare a new unsymmetrical chiral ligand, C9H7CH(CH3)CH2C5H5, with an indenyl moiety connected to a cyclopentadienyl unit by a chiral ethylene bridge, we reacted the optically active tosylate 4, derived from ethyl (S)-(-) lactate, with LiCp. The only product resulting from this reaction was an optically active spirocyclopropane 6 obtained with a high diastereoselectivity (81.8% de). We also observed that, when the reduction of the ethyl indene lactate 2 was realized with an excess of LiAlH4 in Et2O under reflux, spirocyclopropane 6 was obtained in high yield with the same diastereoselectivity. When tosylate 4 was reacted with MgCp2, in place of LiCp, ligand 5 was obtained in good yield as a mixture of two double-bond isomers of the Cp unit. Ligand 5 was fully structurally characterized after conversion into transition monometallic complexes such as C9H7CH(CH3)CH2η(5)-C5H4Mo(CO)3Me 7 and C9H7CH(CH3)CH2η(5)-C5H4Rh(COD) 9, in which the indene moiety was kept intact due to the difference in reactivity of the indenyl moiety with respect to the cyclopentadienyl unit. An attempt to prepare a heterobimetallic complex from the sodium indenide salt of 7 and [RhCl(COD)]2, afforded a rhodium cyclopentadienyl complex 9, resulting from a metal exchange reaction with loss of the molybdenum part initially coordinated to the Cp unit, and conservation of the optical purity. An homobimetallic rhodium complex 10 could be prepared by deprotonation of ligand 5 with TlOEt, followed by quenching with [RhCl(COD)]2. The bis-rhodium complex 10 is obtained as a mixture of two diastereoisomers 10a and 10b with respect to the planar chirality of the indenyl ring system, with a good diastereoisomeric excess de = 70%. The structures of both complexes 9 and major (pS)-diastereoisomer 10a were determined by single crystal X-ray diffraction.

A new, multiplex, quantitative real-time polymerase chain reaction system for nucleic acid detection and quantification.

Quantitative real-time polymerase chain reaction (qPCR) has emerged as a powerful investigative and diagnostic tool with potential to generate accurate and reproducible results. qPCR can be designed to fulfil the four key aspects required for the detection of nucleic acids: simplicity, speed, sensitivity, and specificity. This chapter reports the development of a novel real-time multiplex quantitative PCR technology, dubbed PrimRglo™, with a potential for high-degree multiplexing. It combines the capacity to simultaneously detect many viruses, bacteria, or nucleic acids, in a single reaction tube, with the ability to quantitate viral or bacterial load. The system utilizes oligonucleotide-tagged PCR primers, along with complementary fluorophore-labelled and quencher-labelled oligonucleotides. The analytic sensitivity of PrimRglo technology was compared with the widely used Taqman(®) and SYBR green detection systems.