Detection of Pathogen-Specific Antibodies by Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) is a method for enzymatically replicating DNA that has great utility for clinical diagnosis at the point of care (POC), given its high sensitivity, specificity, speed, and technical requirements (isothermal conditions). Here, we adapted LAMP for measuring protein analytes by creating a protein-DNA fusion (referred to here as a “LAMPole”) that attaches oligonucleotides (LAMP templates) to IgG antibodies. This fusion consists of a DNA element covalently bonded to an IgG-binding polypeptide (protein L/G domain). In our platform, LAMP is expected to provide the most suitable means for amplifying LAMPoles for clinical diagnosis at the POC, while quantitative PCR is more suitable for laboratory-based quantification of antigen-specific IgG abundance. As proof of concept, we measured serological responses to a protozoan parasite by quantifying changes in solution turbidity in real time. We observed a >6-log fold difference in signal between sera from vaccinated versus control mice and in a clinical patient sample versus a control. We assert that LAMPoles will be useful for increasing the sensitivity of measuring proteins, whether it be in a clinical laboratory or in a field setting, thereby improving acute diagnosis of a variety of infections.     

Complex karyotype in mantle cell lymphoma is a strong prognostic factor for the time to treatment and overall survival,independent of the MCL international prognostic index

Mantle cell lymphoma (MCL) is usually an aggressive disease. However, a few patients do have an “indolent” evolution (iMCL) defined by a long survival time without intensive therapy. Many studies highlight the prognostic role of additional genetic abnormalities, but these abnormalities are not routinely tested for and do not yet influence the treatment decision. We aimed to evaluate the prognostic impact of these additional abnormalities detected by conventional cytogenetic testing, as well as their relationships with the clinical characteristics and their value in identifying iMCL. All consecutive MCL cases diagnosed between 1995 and 2011 at four institutions were retrospectively selected on the basis of an informative karyotype with a t(11;14) translocation at the time of diagnosis. A total of 125 patients were included and followed for an actual median time of 35 months. The median overall survival (OS) and survival without treatment (TFS) were 73.7 and 1.3 months, respectively. In multivariable Cox models, a high mantle cell lymphoma international prognostic index score, a complex karyotype, and blastoid morphology were independently associated with a shortened OS. Spleen enlargement, nodal presentation, extra‐hematological involvement, and complex karyotypes were associated with shorter TFS. A score based on these factors allowed for the identification of “indolent” patients (median TFS 107 months) from other patients (median TFS: 1 month). In conclusion, in this multicentric cohort of MCL patients, a complex karyotype was associated with a shorter survival time and allowed for the identification of iMCL at the time of diagnosis. © 2013 Wiley Periodicals, Inc.     

Design of donecopride,a dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer's disease treatment

RS67333 is a partial serotonin subtype 4 receptor (5-HT₄R) agonist that has been widely studied for its procognitive effect. More recently, it has been shown that its ability to promote the nonamyloidogenic cleavage of the precursor of the neurotoxic amyloid-β peptide leads to the secretion of the neurotrophic protein sAPPα. This effect has generated great interest in RS67333 as a potential treatment for Alzheimer’s disease (AD). We show herein that RS67333 is also a submicromolar acetylcholinesterase (AChE) inhibitor and therefore, could contribute, through this effect, to the restoration of the cholinergic neurotransmission that becomes altered in AD. We planned to pharmacomodulate RS67333 to enhance its AChE inhibitory activity to take advantage of this pleiotropic pharmacological profile in the design of a novel multitarget-directed ligand that is able to exert not only a symptomatic but also, a disease-modifying effect against AD. These efforts allowed us to select donecopride as a valuable dual (h)5-HT₄R partial agonist (K_i = 10.4 nM; 48.3% of control agonist response)/(h)AChEI (IC₅₀ = 16 nM) that further promotes sAPPα release (EC₅₀ = 11.3 nM). Donecopride, as a druggable lead, was assessed for its in vivo procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improvement of memory performances observed at 0.3 and 1 mg/kg on the object recognition test. On the basis of these in vitro and in vivo activities, donecopride seems to be a promising drug candidate for AD treatment.Among the large family of serotonin receptors (5-HTR), some of them, such as the subtype 4 (5-HT₄R), are of particular interest in improving memory performance and therefore, decreasing memory deficits, such as those that occur in Alzheimer''s disease (AD). In the CNS, they are located in structures that are primarily involved in cognitive functions, like the olfactory tubercles, basal ganglia, septum, substantia nigra, superior colliculi, hippocampus, and cortex. Several compounds act as agonists to 5-HT₄R (BIMU1, BIMU8, RS17017, SL65.0155, VRX-03011, prucalopride, RS67333, and RS67506). One of the most affine (pK_i = 7.88) and selective vs. other receptors is RS67333, which acts as a partial agonist (1). With respect to the potential therapeutic modulation of 5-HT₄R with RS67333 and excluding its putative antidepressant-like activity (2–4), most studies focused on the promnesic or antiamnesic actions of this compound. These effects on cognitive functions that concern learning and memory are probably, in part, because of the fact that the pharmacological stimulation of these receptors increases the release of ACh in the hippocampus and cortex, and it also increases serotonin, dopamine, and GABA release (5–13). Concerning the selective aspects of memory functions, RS67333 has been shown to improve object recognition in adult (14, 15) and aged animals (16, 17) and place recognition (10) in rodents. It also increases spatial learning on the Morris water maze task in rodents, and it even reverses the deleterious effect of atropine (18) or scopolamine during this same task.Based on the structural analogy existing between RS67333 and donepezil (Fig. 1), we postulated that RS67333 could improve learning and memory by not only activating 5-HT₄R but also, inhibiting acetylcholinesterase (AChE) activity. Indeed, several early studies reported that the inhibition of AChE improved cognition and that this effect is the main reason for the initial use of donepezil, galantamine, and rivastigmine as cognitive enhancers in AD (19, 20). AChE inhibition has also been reported to improve performances in healthy rodents or animal models of memory deficiency (21). The hypothesis of an involvement of AChE inhibition by RS67333 in memory improvement is an issue that has never been tested.Open in a separate windowFig. 1.RS67333 and donepezil are chemically close.Furthermore, such a pharmacological profile could be also exploited to lead to pleiotropic compounds that are theoretically useful in AD treatment. Indeed, today, it is well-established that 5-HT₄R activation not only favors ACh release but also, is involved in the nonamyloidogenic cleavage of amyloid precursor protein (APP) in the neurotrophic sAPPα fragment, with secretion that is detrimental to amyloid-β peptide (Aβ) production (22–24). However, inhibiting the catalytic activity of AChE is widely used to restore cholinergic neurotransmission in AD, and interacting with the peripheral anionic site (PAS) of this enzyme could also reduce amyloid aggregation, for which AChE would be responsible (25). These activities seem to be synergistic when they are associated in an AD animal model, which we have recently shown in mice (26). A second pharmacological approach based on the fact that a single compound may be able to hit multiple targets is now emerging. This concept, called multitarget-directed ligands (MTDLs) (27, 28), would have inherent advantages over a combination of drugs called multiple medication therapy. It would specially obviate the problems linked to the complexity of the pharmacokinetic profile of the combined drugs and the risk of drug–drug interactions. Moreover, MTDL could also alleviate compliance difficulties associated with multiple medication therapy. It has also been shown that MTDLs generally show a higher synergistic effect than that observed with a combination of drugs. Numerous examples of MTDL against AD have been recently described (27). Most of them associate an AChE inhibitory effect with another activity hitting another molecular target of AD, such as antioxidant effect, monoamine oxidase inhibition, calcium channel blocking effect, metal chelating activity, etc. However, no MTDLs associating an inhibition of AChE and 5-HT₄R agonist effect have been hitherto described. Among the different ways to synthesize such MTDLs, one of them is to merge the frameworks of two selective starting compounds, each one exerting an activity toward a sole target (28). This goal is even more easily reached if the starting compounds are structurally close, and it is the reason why we considered the structural analogy between donepezil and RS67333 as a good starting point to design MTDLs displaying both activities (AChE inhibition and 5-HT₄R agonist activities). We will, thus, provide proof of this concept with the synthesis and biological evaluation of MR31147 (donecopride) as conceived from the pharmacomodulation of RS67333.     

Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knockout mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.The subthalamic nucleus (STN) has long been a structure of interest for researchers and clinicians alike. There is ample evidence that high-frequency stimulation of the STN improves symptoms such as tremor, rigidity, and slowness of movement, so called bradykinesia, in patients with Parkinson disease (see ref. 1 for review), but the mechanism through which this is achieved is still unknown. Some studies suggest that electrical stimulation causes a hyperexcitation of this structure (2), whereas others find evidence that the opposite is true (3–5). Other possible interpretations include the activation of the zona incerta, a neighboring white-matter structure (6) or of fibers coming from the motor cortex (7). Bilateral lesions of the STN improve locomotion (8), a result that is consistent with the inactivation hypothesis. However, previous studies have also found cognitive side effects when using high-frequency stimulation of the STN (9), findings supported by lesion studies in experimental animals, which led to abnormalities in operant tasks involving attention and impulsivity (10, 11). The projections of the STN to other regions help explain the multiple roles of this structure: It sends projections to other targets in the basal ganglia, such as the internal segment of the globus pallidus [also termed the entopeduncular nucleus (EP) in rodents] and the substantia nigra pars reticulata (SNr) (12, 13). The STN is also part of a circuit that includes the prefrontal cortex and the nucleus accumbens (14). It is currently unknown, however, whether these different roles reflect a heterogeneous population of cells, characterized by distinct gene expression. If that is the case, it would allow direct control over each cell population, facilitating the investigation of their respective roles. In rodents, the STN is believed to be composed solely of glutamatergic neurons, characterized by expression of the subtype 2 Vesicular glutamate transporter (Vglut2), whereas the other two subtypes (Vglut1 and Vglut3) have not been detected (15, 16). Selective targeted deletion of Vglut2 expression in this nucleus would therefore provide a specific loss-of-function model that would bypass a common problem presented by traditional lesions with pharmacological agents, which have patterns of diffusion that likely affect surrounding structures (17). It is known, however, that Vglut2 is expressed in many other parts of the brain (18), and a complete knockout in the mouse is not viable (19, 20). There is also evidence that the promoter driving expression of the Paired-like homeodomain 2 (Pitx2) gene is strong in the mouse STN (21) but is also not specific to this structure and a full knockout of Pitx2 expression results in premature death (22). To achieve the desired level of specificity, using a conditional knockout technique previously used to eliminate glutamatergic transmission in other cell types (23), we crossed Pitx2-Cre and Vglut2-lox mice, producing Vglut2^{f/f;Pitx2-Cre} conditional knockout (cKO) mice in which Vglut2 expression in the STN was strongly reduced in comparison with expression levels in littermate control mice. To understand the physiological contribution of the selected subpopulation of STN cells, we characterized these cKO mice with regard to anatomical, electrophysiological, and molecular properties, as well as their performance in a range of behavioral tasks.     

Securing sustainable funding for viral hepatitis elimination plans

The majority of people infected with chronic hepatitis C virus (HCV) in the European Union (EU) remain undiagnosed and untreated. During recent years, immigration to EU has further increased HCV prevalence. It has been estimated that, out of the 4.2 million adults affected by HCV infection in the 31 EU/ European Economic Area (EEA) countries, as many as 580 000 are migrants. Additionally, HCV is highly prevalent and under addressed in Eastern Europe. In 2013, the introduction of highly effective treatments for HCV with direct‐acting antivirals created an unprecedented opportunity to cure almost all patients, reduce HCV transmission and eliminate the disease. However, in many settings, HCV elimination poses a serious challenge for countries’ health spending. On 6 June 2018, the Hepatitis B and C Public Policy Association held the 2nd EU HCV Policy summit. It was emphasized that key stakeholders should work collaboratively since only a few countries in the EU are on track to achieve HCV elimination by 2030. In particular, more effort is needed for universal screening. The micro‐elimination approach in specific populations is less complex and less costly than country‐wide elimination programmes and is an important first step in many settings. Preliminary data suggest that implementation of the World Health Organization (WHO) Global Health Sector Strategy on Viral Hepatitis can be cost saving. However, innovative financing mechanisms are needed to raise funds upfront for scaling up screening, treatment and harm reduction interventions that can lead to HCV elimination by 2030, the stated goal of the WHO.