Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) a-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions

Measurement of event background fluctuations for charged particle jet reconstruction in Pb-Pb collisions at sNN=2.76\sqrt{s_{\rm NN}} = 2.76 TeV

The effect of event background fluctuations on charged particle jet reconstruction in Pb-Pb collisions at $\sqrt{s_{\rm NN}} = 2.76$ TeV has been measured with the ALICE experiment. The main sources of non-statistical fluctuations are characterized based purely on experimental data with an unbiased method, as well as by using single high $p_{\rm T}$ particles and simulated jets embedded into real Pb-Pb events and reconstructed with the anti-$k_{\rm T}$ jet finder. The influence of a low transverse momentum cut-off on particles used in the jet reconstruction is quantified by varying the minimum track $p_{\rm T}$ between 0.15 GeV/$c$ and 2 GeV/$c$. For embedded jets reconstructed from charged particles with $p_{\rm T} > 0.15$ GeV/$c$, the uncertainty in the reconstructed jet transverse momentum due to the heavy-ion background is measured to be 11.3 GeV/$c$ (standard deviation) for the 10% most central Pb-Pb collisions, slightly larger than the value of 11.0 GeV/$c$ measured using the unbiased method. For a higher particle transverse momentum threshold of 2 GeV/$c$, which will generate a stronger bias towards hard fragmentation in the jet finding process, the standard deviation of the fluctuations in the reconstructed jet transverse momentum is reduced to 4.8-5.0 GeV/$c$ for the 10% most central events. A non-Gaussian tail of the momentum uncertainty is observed and its impact on the reconstructed jet spectrum is evaluated for varying particle momentum thresholds, by folding the measured fluctuations with steeply falling spectra.Comment: 21 pages, 5 captioned figures, 3 tables, authors from page 16, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/350

Neutral pion and η\eta meson production in proton-proton collisions at s=0.9\sqrt{s}=0.9 TeV and s=7\sqrt{s}=7 TeV

The first measurements of the invariant differential cross sections of inclusive $\pi^0$ and $\eta$ meson production at mid-rapidity in proton-proton collisions at $\sqrt{s}=0.9$ TeV and $\sqrt{s}=7$ TeV are reported. The $\pi^0$ measurement covers the ranges $0.4<p_T<7$ GeV/$c$ and $0.3<p_T<25$ GeV/$c$ for these two energies, respectively. The production of $\eta$ mesons was measured at $\sqrt{s}=7$ TeV in the range $0.4<p_T<15$ GeV/$c$. Next-to-Leading Order perturbative QCD calculations, which are consistent with the $\pi^0$ spectrum at $\sqrt{s}=0.9$ TeV, overestimate those of $\pi^0$ and $\eta$ mesons at $\sqrt{s}=7$ TeV, but agree with the measured $\eta/\pi^0$ ratio at $\sqrt{s}=7$ TeV.Comment: 17 pages, 5 captioned figures, 2 tables, authors from page 12, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/310

Pallidothalamic-projecting neurons in Macaca fascicularis co-express GABAergic and glutamatergic markers as seen in control, MPTP-treated and dyskinetic monkeys

GABAergic neurons within the internal division of the globus pallidus (GPi) are the main source of basal ganglia output reaching the thalamic ventral nuclei in monkeys. Following dopaminergic denervation, pallidothalamic-projecting neurons are known to be hyperactive, whereas a reduction in GPi activity is typically observed in lesioned animals showing levodopa-induced dyskinesia. Besides the mRNAs coding for GABAergic markers (GAD65 and GAD67), we show that all GPi neurons innervating thalamic targets also express transcripts for the isoforms 1 and 2 of the vesicular glutamate transporter (vGlut1 and vGlut2 mRNA). Indeed, dual immunofluorescent detection of GAD67 and vGlut1/2 confirmed the data gathered from in situ hybridization experiments, therefore demonstrating that the detected mRNAs are translated into the related proteins. Furthermore, the dopaminergic lesion resulted in an up-regulation of expression levels for both GAD65 and GAD67 mRNA within identified pallidothalamic-projecting neurons. This was coupled with a down-regulation of GAD65/67 mRNA expression levels in GPi neurons innervating thalamic targets in monkeys showing levodopa-induced dyskinesia. By contrast, the patterns of gene expression for both vGlut1 and vGlut2 mRNAs remained unchanged across GPi projection neurons in control, MPTP-treated and dyskinetic monkeys. In summary, both GABAergic and glutamatergic markers were co-expressed by GPi efferent neurons in primates. Although the status of the dopaminergic system directly modulates the expression levels of GAD65/67 mRNA, the observed expression of vGlut1/2 mRNA is not regulated by either dopaminergic removal or by continuous stimulation with dopaminergic agonists

Glutamatergic and cholinergic pedunculopontine neurons innervate the thalamic parafascicular nucleus in rats: changes following experimental parkinsonism

The tegmental pedunculopontine nucleus (PPN) is a basal ganglia-related structure that has recently gained renewed interest as a potential surgical target for the treatment of several aspects of Parkinson's disease. However, the underlying anatomical substrates sustaining the choice of the PPN nucleus as a surgical candidate remain poorly understood. Here, we characterized the chemical phenotypes of different subtypes of PPN efferent neurons innervating the rat parafascicular (PF) nucleus. Emphasis was placed on elucidating the impact of unilateral nigrostriatal denervation on the expression patterns of the mRNA coding the vesicular glutamate transporter type 2 (vGlut2 mRNA). We found a bilateral projection from the PPN nucleus to the PF nucleus arising from cholinergic and glutamatergic efferent neurons, with a small fraction of projection neurons co-expressing both cholinergic and glutamatergic markers. Furthermore, the unilateral nigrostriatal depletion induced a bilateral twofold increase in the expression levels of vGlut2 mRNA within the PPN nucleus. Our results support the view that heterogeneous chemical profiles account for PPN efferent neurons innervating thalamic targets. Moreover, a bilateral enhancement of glutamatergic transmission arising from the PPN nucleus occurs following unilateral dopaminergic denervation, therefore sustaining the well-known hyperactivity of the PF nucleus in parkinsonian-like conditions. In conclusion, our data suggest that the ascending projections from the PPN that reach basal ganglia-related targets could play an important role in the pathophysiology of Parkinson's disease

A direct projection from the subthalamic nucleus to the ventral thalamus in monkeys

The current basal ganglia model considers the internal division of the globus pallidus and the substantia nigra pars reticulata as the sole sources of basal ganglia output to the thalamus. However, following the delivery of retrograde tracers into the ventral anterior/ventral lateral thalamic nuclei, a moderate number of labeled neurons were found within the subthalamic nucleus (STN) in control cases, MPTP-treated monkeys and animals with levodopa-induced dyskinesias. Furthermore, dual tracing experiments showed that subthalamo-thalamic and subthalamo-pallidal projections arise from different subpopulations of STN efferent neurons. Moreover, upregulated expression of the mRNA coding the vesicular glutamate transporter 2 (vGlut2) was found in retrogradely-labeled STN neurons in MPTP-treated monkeys. By contrast, there is a reduction in vGlut2 mRNA expression in subthalamo-thalamic neurons in dyskinetic monkeys. In conclusion, our findings support the presence of a direct projection from the STN to the ventral thalamus that appears to be functionally modulated by dopaminergic activity

Neuroanatomical tracing combined with in situ hybridization: analysis of gene expression patterns within brain circuits of interest

Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols. The subsequent detection at the single-cell level of the expression of a given mRNA within retrograde-labeled neurons provides information regarding cellular function. This may be of particular interest when trying to elucidate the performance of brain circuits of interest in animal models of brain diseases. Several combinations of retrograde tracing with either single- and double-ISH are presented here, together with some criteria that influence the selection of the tracer to be used in conjunction with the strong demands of the ISH

Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis

The putative presence of the cannabinoid receptor type 2 (CB(2)-R) in the central nervous system is still a matter of debate. Although first described in peripheral and immune tissues, evidence suggesting the existence of CB(2)-Rs in glial cells and even neurons has been made available more recently. By taking advantage of newly designed CB(2)-R mRNA riboprobes, we have demonstrated by in situ hybridization and PCR the existence of CB2-R transcripts in a variety of brain areas of the primate Macaca fascicularis, including the cerebral cortex and the hippocampus, as well as in the external and internal divisions of the globus pallidus, both pallidal segments showing the highest abundance of CB(2)-R transcripts. In this regard, the presence of the messenger coding CB(2)-Rs within the pallidal complex highlights their consideration as potential targets for the treatment of movement disorders of basal ganglia origin

The diagnosis and management of allergic reactions in patients sensitized to non-specific lipid transfer proteins

Sensitization to one or more non-specific lipid transfer proteins (nsLTPs), initially thought to exist mainly in southern Europe, is becoming accepted as a cause of allergic reactions to plant foods across Europe and beyond. The peach nsLTP allergen Pru p 3 is a dominant sensitizing allergen and peaches a common food trigger, although multiple foods can be involved. A frequent feature of reactions is the requirement for a cofactor (exercise, alcohol, non-steroidal anti-inflammatory drugs, Cannabis sativa) to be present for a food to elicit a reaction. The variability in the food and cofactor triggers makes it essential to include an allergy-focused diet and clinical history in the diagnostic workup. Testing on suspected food triggers should also establish whether sensitization to nsLTP is present, using purified or recombinant nsLTP allergens such as Pru p 3. The avoidance of known trigger foods and advice on cofactors is currently the main management for this condition. Studies on immunotherapy are promising, but it is unknown whether such treatments will be useful in populations where Pru p 3 is not the primary sensitizing allergen. Future research should focus on the mechanisms of cofactors, improving diagnostic accuracy and establishing the efficacy of immunotherapy. © 2021 European Academy of Allergy and Clinical Immunology and John Wiley &amp; Sons Ltd

Time to Relapse and the Patterns of Relapse Are Prognostic for Post-Relapse Survival after CD34+-Selected Allogeneic Hematopoietic Stem Cell Transplantation in Multiple Myeloma

Allogeneic hematopoietic stem cell transplantation (HCT), defined by its underlying graft-versus-multiple myeloma (MM) effect, is a potentially curative approach for high-risk pts. However, relapse remains the main cause of treatment failure. Predictors for post-relapse survival after HCT are not well characterized, and we hypothesized that the time to relapse and the patterns of relapse are prognostic for post-relapse survival. Methods: All pts with MM who underwent a CD34+-selected alloHCT at Memorial Sloan Kettering Cancer Center on protocol (NCT 01131169/01119066) or off study from 01/2010 to 12/2017 and progressed after alloHCT were included. Pts were conditioned with busulfan (0.8 mg/kg x 10), melphalan (70 mg/m2 × 2), and fludarabine (25mg/m2 × 5). The K-M method was used to estimate OS post-HCT relapse. Significant predictors were considered in a Cox model. Results: Of the 115 transplanted pts, 60 (52%) relapsed and were analyzed. The median age was 56 yrs (35- 66), 62% male and 82% with high-risk cytogenetics (CG). KPS was ≥ 80 in 100% and ≥ 90 in 52%. Pts received a median of 4 lines of therapy pre-HCT (1-10), with 88% achieving at least a partial response (PR) prior to alloHCT. All pts had received at least one autoHCT. Of the 38% who received a planned or preemptive post-HCT therapy, 13 received DLI and 10 other strategies. Relapse was characterized as very early (24 mo, 22%). At relapse, 27% presented with extramedullary disease (EMD). Median post-relapse OS was significantly different for pts who relapsed very early, early, or late; 4 mo, 17 mo, and 72 mo, respectively (p = 0.002). Age ≥55, relapse with EMD, lower KPS, <PR prior to HCT, <PR by day 100, and no maintenance were also prognostic for worse post-relapse OS in univariate analysis. There was no significant difference for post-relapse OS for number of lines, high-risk CG, or era of alloHCT. In multivariate analysis adjusting for sex and age, risk factors for worse post-relapse OS included very early (HR 4.4, 95%CI 1.4-13.5) or early relapse (HR 2.5, 95%CI 1.4-13.5), relapse with EMD (HR 5.2, 95%CI 2.1-12.9), and lack of DLI as planned post-HCT therapy (HR for DLI compared with no DLI = 0.11, 95%CI 0.01-0.9). Conclusion: For this very high-risk group of MM pts, differences in time to relapse after alloHCT define very distinct post-relapse survival patterns. Very early relapses were associated with greater chemoresistance and dismal prognosis. In contrast, the median post-relapse OS for late relapses was 6 yrs, suggesting different underlying disease biology and/or host/donor characteristics. Similar to the post-auto setting, relapse with EMD conferred poor prognosis. Of note, post-relapse OS was superior for pts who received a DLI as planned post-HCT therapy prior to relapse. Efforts to characterize optimal post-relapse therapeutic strategies and mechanism(s) driving relapses are ongoing