Marta Aymerich

Abstract Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale 1–3 . Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter 4 ; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation 5,6 ; analyses timings and patterns of tumour evolution 7 ; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity 8,9 ; and evaluates a range of more-specialized features of cancer genomes 8,10–18 .

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

Abstract Mantle cell lymphoma (MCL) is typically a very aggressive disease with poor outcomes, but some cases display an indolent behavior that might not necessitate treatment at diagnosis. To define molecular criteria that might permit recognition of such cases, we compared the clinicopathologic features, gene expression, and genomic profile of patients who had indolent or conventional disease (iMCL or cMCL). Patients with iMCL displayed nonnodal leukemic disease with predominantly hypermutated IGVH and noncomplex karyotypes. iMCL and cMCL shared a common gene expression profile that differed from other leukemic lymphoid neoplasms. However, we identified a signature of 13 genes that was highly expressed in cMCL but underexpressed in iMCL. SOX11 was notable in this signature and we confirmed a restriction of SOX11 protein expression to cMCL. To validate the potential use of SOX11 as a biomarker for cMCL, we evaluated SOX11 protein expression in an independent series of 112 cases of MCL. Fifteen patients with SOX11-negative tumors exhibited more frequent nonnodal presentation and better survival compared with 97 patients with SOX11-positive MCL (5-year overall survival of 78% versus 36%, respectively; P = 0.001). In conclusion, we defined nonnodal presentation, predominantly hypermutated IGVH, lack of genomic complexity, and absence of SOX11 expression as qualities of a specific subtype of iMCL with excellent outcomes that might be managed more conservatively than cMCL. Cancer Res; 70(4); 1408–18

Abstract Mitochondria are essential cellular organelles that play critical roles in cancer. Here, as part of the International Cancer Genome Consortium/The Cancer Genome Atlas Pan-Cancer Analysis of Whole Genomes Consortium, which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumor types, we performed a multidimensional, integrated characterization of mitochondrial genomes and related RNA sequencing data. Our analysis presents the most definitive mutational landscape of mitochondrial genomes and identifies several hypermutated cases. Truncating mutations are markedly enriched in kidney, colorectal and thyroid cancers, suggesting oncogenic effects with the activation of signaling pathways. We find frequent somatic nuclear transfers of mitochondrial DNA, some of which disrupt therapeutic target genes. Mitochondrial copy number varies greatly within and across cancers and correlates with clinical variables. Co-expression analysis highlights the function of mitochondrial genes in oxidative phosphorylation, DNA repair and the cell cycle, and shows their connections with clinically actionable genes. Our study lays a foundation for translating mitochondrial biology into clinical applications.

Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.

Chronic lymphocytic leukemia (CLL) is a frequent hematological neoplasm in which underlying epigenetic alterations are only partially understood. Here, we analyze the reference epigenome of seven primary CLLs and the regulatory chromatin landscape of 107 primary cases in the context of normal B cell differentiation. We identify that the CLL chromatin landscape is largely influenced by distinct dynamics during normal B cell maturation. Beyond this, we define extensive catalogues of regulatory elements de novo reprogrammed in CLL as a whole and in its major clinico-biological subtypes classified by IGHV somatic hypermutation levels. We uncover that IGHV-unmutated CLLs harbor more active and open chromatin than IGHV-mutated cases. Furthermore, we show that de novo active regions in CLL are enriched for NFAT, FOX and TCF/LEF transcription factor family binding sites. Although most genetic alterations are not associated with consistent epigenetic profiles, CLLs with MYD88 mutations and trisomy 12 show distinct chromatin configurations. Furthermore, we observe that non-coding mutations in IGHV-mutated CLLs are enriched in H3K27ac-associated regulatory elements outside accessible chromatin. Overall, this study provides an integrative portrait of the CLL epigenome, identifies extensive networks of altered regulatory elements and sheds light on the relationship between the genetic and epigenetic architecture of the disease.

Abstract Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS) high –B cell receptor (BCR) low -signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT.

NOTCH1 has been found recurrently mutated in a subset of patients with chronic lymphocytic leukemia (CLL). To analyze biological features and clinical impact of NOTCH1 mutations in CLL, we sequenced this gene in 565 patients. NOTCH1 mutations, found in 63 patients (11%), were associated with unmutated IGHV, high expression of CD38 and ZAP-70, trisomy 12, advanced stage and elevated lactate dehydrogenase. Sequential analysis in 200 patients demonstrated acquisition of mutation in one case (0.5%) and disappearance after treatment in two. Binet A and B patients with NOTCH1-mutated had a shorter time to treatment. NOTCH1-mutated patients were more frequently refractory to therapy and showed shorter progression-free and overall survival after complete remission. Overall survival was shorter in NOTCH1-mutated patients, although not independently from IGHV. NOTCH1 mutation increased the risk of transformation to diffuse large B-cell lymphoma independently from IGHV, with this being validated in resampling tests of replicability. In summary, NOTCH1 mutational status, that was rarely acquired during the course of the disease, identify a genetic subgroup with high risk of transformation and poor outcome. This recently identified genetic subgroup of CLL patients deserves prospective studies to define their best management.

Pigment epithelial-derived factor (PEDF), an angiogenesis inhibitor with neurotrophic properties, balances angiogenesis in the eye and blocks tumor progression. Its neurotrophic function and the ability to block vascular leakage is replicated by the PEDF 44-mer peptide (residues 58-101). We analyzed PEDFs' three-dimensional structure and identified a potential receptor-binding surface. Seeking PEDF-based antiangiogenic agents we generated and tested peptides representing the middle and lower regions of this surface. We identified previously unknown antiangiogenic epitopes consisting of the 34-mer (residues 24-57) and a shorter proximal peptide (TGA, residues 16-26) with the critical stretch L19VEEED24 and a fragment within the 44-mer (ERT, residues 78-94), which retained neurotrophic activity. The 34-mer and TGA, but not the 44-mer reproduced PEDF angioinhibitory signals hinged on c-jun-NH2-kinase-dependent nuclear factor of activated T cell deactivation and caused apoptosis. Conversely, the ERT, but not the 34-mer/TGA induced neuronal differentiation. For the 44-mer/ERT, we showed a novel ability to cause neuroendocrine differentiation in prostate cancer cells. PEDF and the peptides bound endothelial and PC-3 prostate cancer cells. Bound peptides were displaced by PEDF, but not by each other, suggesting multiple receptors. PEDF and its active fragments blocked tumor formation when conditionally expressed by PC-3 cells. The 34- and 44-mer used distinct mechanisms: the 34-mer acted on endothelial cells, blocked angiogenesis, and induced apoptosis whereas 44-mer prompted neuroendocrine differentiation in cancer cells. Our results map active regions for the two PEDF functions, signaling via distinct receptors, identify candidate peptides, and provide their mechanism of action for future development of PEDF-based tumor therapies.

It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D(1) receptors (D(1)Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D(1)R and σ(1)R were found to heteromerize in transfected cells, where cocaine robustly potentiated D(1)R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D(1)R stimulation in a dopamine transporter-independent and σ(1)R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ(1)R KO mice, providing evidence for the existence of σ(1)R-D(1)R heteromers in the brain. Therefore, these results provide a molecular explanation for which D(1)R plays a more significant role in the behavioral effects of cocaine, through σ(1)R-D(1)R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction.

Glial cell line-derived neurotrophic factor (GDNF) has shown promise in the treatment of neurodegenerative disorders of basal ganglia origin such us Parkinson's disease (PD). In this study, we investigated the neurorestorative effect of controlled GDNF delivery using biodegradable microspheres in an animal model with partial dopaminergic lesion. Microspheres were loaded with N-glycosylated recombinant GDNF and prepared using the Total Recirculation One-Machine System (TROMS). GDNF-loaded microparticles were unilaterally injected into the rat striatum by stereotaxic surgery two weeks after a unilateral partial 6-OHDA nigrostriatal lesion. Animals were tested for amphetamine-induced rotational asymmetry at different times and were sacrificed two months after microsphere implantation for immunohistochemical analysis. The putative presence of serum IgG antibodies against rat glycosylated GDNF was analyzed for addressing safety issues. The results demonstrated that GDNF-loaded microspheres, improved the rotational behavior induced by amphetamine of the GDNF-treated animals together with an increase in the density of TH positive fibers at the striatal level. The developed GDNF-loaded microparticles proved to be suitable to release biologically active GDNF over up to 5 weeks in vivo. Furthermore, none of the animals developed antibodies against GDNF demonstrating the safety of glycosylated GDNF use.

Abstract Whether advances in treatment are prolonging survival of patients with chronic lymphocytic leukemia (CLL) is unclear. We analyzed presentation patterns and survival over time in 929 patients followed from 1980 to 2008 at the Hospital Clinic of Barcelona. The 5- and 10-year relative survival (adjusted for the expected survival in the general population) was estimated in patients seen in 2 periods of time: 1980-1994 (n = 451) and 1995-2004 (n = 365). We found that CLL shortens life expectancy in all age groups independently of clinical features at diagnosis. Nevertheless, survival is improving, particularly in some groups of patients. Thus, relative survival was significantly higher in the 1995-2004 cohort than in the 1980-1994 group both at 5 years (incidence rate ratio [IRR] = 0.46; P = .004) and 10 years (IRR = 0.65; P = .007) from diagnosis. The improved survival was largely due to a decrease in CLL-attributable mortality in patients younger than 70 years in Binet stage B or C at diagnosis (IRR = 0.40; P = .001 at 5 years; IRR = 0.33; P < .001 at 10 years). These results suggest that newer treatments are changing the prognosis of CLL, particularly in younger patients with advanced disease, whereas no improvement is yet observed in older subjects or those with lower-risk disease.

Fludarabine, cyclophosphamide, and mitoxantrone (FCM) results in a high response rate in previously treated patients with chronic lymphocytic leukemia (CLL). The aim of this study was to investigate FCM as frontline therapy in CLL.Sixty-nine patients under the age of 65 years with active CLL were treated. Patients received six cycles of fludarabine 25 mg/m(2) i.v. x 3 days, cyclophosphamide 200 mg/m(2) i.v. x 3 days, and mitoxantrone 6 mg/m(2) i.v. x 1 day. Treatment outcome was correlated with clinical and biological variables. The clinical significance of eradicating minimal residual disease (MRD) was also analyzed.The overall response, MRD-negative complete response (CR), MRD-positive CR, nodular partial response (PR), and PR rates were 90%, 26%, 38%, 14%, and 12%, respectively. Severe (grades 3 or 4) neutropenia developed in 10% of the patients. Major and minor infections were reported in 1% and 8% of cases, respectively. Median response duration was 37 months. Patients with del(17p) failed to attain CR. Patients achieving MRD-negative CR had a longer response duration and overall survival than patients with an inferior response. Low serum lactate dehydrogenase levels, low ZAP-70 expression, and mutated IgV(H) genes predicted longer response duration. Finally, both low ZAP-70 and CD38 expression in leukemic cells correlated with MRD-negativity achievement.FCM induces a high response rate, including MRD-negative CRs in untreated patients with active CLL. Treatment toxicity is acceptable. Both high ZAP-70 and increased CD38 expression predict failure to obtain MRD-negative response. Patients in whom MRD can be eradicated have longer response duration and overall survival than those with inferior response. These results indicate that FCM can be an ideal companion for chemoimmunotherapy of patients with CLL.

Key Points Mutations in the TLR/MYD88 pathway occur in 4% of patients with CLL, and they are the most frequent in young patients. TLR/MYD88 mutations in CLL patients confer a good outcome, which is similar to that of the age- and gender-matched healthy population.

Genome studies of chronic lymphocytic leukemia (CLL) have revealed the remarkable subclonal heterogeneity of the tumors, but the clinical implications of this phenomenon are not well known. We assessed the mutational status of 28 CLL driver genes by deep-targeted next-generation sequencing and copy number alterations (CNA) in 406 previously untreated patients and 48 sequential samples. We detected small subclonal mutations (0.6–25% of cells) in nearly all genes (26/28), and they were the sole alteration in 22% of the mutated cases. CNA tended to be acquired early in the evolution of the disease and remained stable, whereas the mutational heterogeneity increased in a subset of tumors. The prognostic impact of different genes was related to the size of the mutated clone. Combining mutations and CNA, we observed that the accumulation of driver alterations (mutational complexity) gradually shortened the time to first treatment independently of the clonal architecture, IGHV status and Binet stage. Conversely, the overall survival was associated with the increasing subclonal diversity of the tumors but it was related to the age of patients, IGHV and TP53 status of the tumors. In conclusion, our study reveals that both the mutational complexity and subclonal diversity influence the evolution of CLL.

Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer.

The endocannabinoid system (ECS) exerts a modulatory effect of important functions such as neurotransmission, glial activation, oxidative stress, or protein homeostasis. Dysregulation of these cellular processes is a common neuropathological hallmark in aging and in neurodegenerative diseases of the central nervous system (CNS). The broad spectrum of actions of cannabinoids allows targeting different aspects of these multifactorial diseases. In this review, we examine the therapeutic potential of the ECS for the treatment of chronic neurodegenerative diseases of the CNS focusing on Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. First, we describe the localization of the molecular components of the ECS and how they are altered under neurodegenerative conditions, either contributing to or protecting cells from degeneration. Second, we address recent advances in the modulation of the ECS using experimental models through different strategies including the direct targeting of cannabinoid receptors with agonists or antagonists, increasing the endocannabinoid tone by the inhibition of endocannabinoid hydrolysis, and activation of cannabinoid receptor-independent effects. Preclinical evidence indicates that cannabinoid pharmacology is complex but supports the therapeutic potential of targeting the ECS. Third, we review the clinical evidence and discuss the future perspectives on how to bridge human and animal studies to develop cannabinoid-based therapies for each neurodegenerative disorder. Finally, we summarize the most relevant opportunities of cannabinoid pharmacology related to each disease and the multiple unexplored pathways in cannabinoid pharmacology that could be useful for the treatment of neurodegenerative diseases.

Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research.

Pigment epithelium-derived factor (PEDF) has neuronal differentiation and survival activity on retinoblastoma and cerebellar granule (CG) cells. Here, we investigated the presence of PEDF receptors on retinoblastoma Y-79 and CG cells. PEDF radiolabeled with ^l25I remained biologically active and was used for radioligand binding analysis. The binding was saturable and specific to a single class of receptors on both cells and with similar affinities (<i>K</i> _d = 1.7–3.6 nm, <i>B</i> _max = 0.5–2.7 × 10⁵ sites/Y-79 cell; and <i>K</i> _d = 3.2 nm, <i>B</i> _max = 1.1 × 10³ sites/CG cell). A polyclonal antiserum to PEDF, previously shown to block the PEDF neurotrophic activity, prevented the¹²⁵I-PEDF binding. We designed two peptides from a region previously shown to confer the neurotrophic property to human PEDF, synthetic peptides 34-mer (positions 44–77) and 44-mer (positions 78–121). Only peptide 44-mer competed for the binding to Y-79 cell receptors (EC₅₀ = 5 nm) and exhibited neuronal differentiating activity. PEDF affinity column chromatography of membrane proteins from both cell types revealed a PEDF-binding protein of ∼80 kDa. These results are the first demonstration of a PEDF-binding protein with characteristics of a PEDF receptor and suggest that the region comprising amino acid positions 78–121 of PEDF might be involved in ligand-receptor interactions.

Abstract Vesicular glutamate transporters (VGLUTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Three isoforms of the VGLUT have been identified, now known as VGLUT1, VGLUT2, and VGLUT3. Both VGLUT1 and VGLUT2 have been considered definitive markers of glutamatergic neurons, whereas VGLUT3 is expressed in nonglutamatergic neurons such as cholinergic striatal interneurons. It is widely believed that VGLUT1 and VGLUT2 are expressed in a complementary manner at the cortical and thalamic levels, suggesting that these glutamatergic neurons fulfill different physiological functions. In the present work, we analyzed the pattern of VGLUT1 and VGLUT2 mRNA expression at the thalamic level by using single and dual in situ hybridization. In accordance with current beliefs, we found significant expression of VGLUT2 mRNA in all the thalamic nuclei, while moderate expression of VGLUT1 mRNA was consistently found in both the principal relay and the association thalamic nuclei. Interestingly, individual neurons within these nuclei coexpressed both VGLUT1 and VGLUT2 mRNAs, suggesting that these individual thalamic neurons may have different ways of trafficking glutamate. These results call for a reappraisal of the previously held concept regarding the mutually exclusive distribution of VGLUT transporters in the central nervous system. J. Comp. Neurol. 501:703–715, 2007. © 2007 Wiley‐Liss, Inc.

Changes in cannabinoid receptor expression and concentration of endocannabinoids have been described in Parkinson's disease; however, it remains unclear whether they contribute to, or result from, the disease process. To evaluate whether targeting the endocannabinoid system could provide potential benefits in the treatment of the disease, the effect of a monoacylglycerol lipase inhibitor that prevents degradation of 2-arachidonyl-glycerol was tested in mice treated chronically with probenecid and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTPp). Chronic administration of the compound, JZL184 (8 mg/kg), prevented MPTPp-induced motor impairment and preserved the nigrostriatal pathway. Furthermore, none of the hypokinetic effects associated with cannabinoid receptor agonism were observed. In the striatum and substantia nigra pars compacta, MPTPp animals treated with JZL184 exhibited astroglial and microglial phenotypic changes that were accompanied by increases in TGFβ messenger RNA expression and in glial cell-derived neurotrophic factor messenger RNA and protein levels. JZL184 induced an increase in β-catenin translocation to the nucleus, implicating the Wnt/catenin pathway. Together, these results demonstrate a potent neuroprotective effect of JZL184 on the nigrostriatal pathway of parkinsonian animals, likely involving restorative astroglia and microglia activation and the release of neuroprotective and antiinflammatory molecules.

The GPR55 receptor is expressed abundantly in the brain, especially in the striatum, suggesting it might fulfill a role in motor function. Indeed, motor behavior is impaired in mice lacking GPR55, which also display dampened inflammatory responses. Abnormal-cannabidiol (Abn-CBD), a synthetic cannabidiol (CBD) isomer, is a GPR55 agonist that may serve as a therapeutic agent in the treatment of inflammatory diseases. In this study, we explored whether modulating GPR55 could also represent a therapeutic approach for the treatment of Parkinson's disease (PD). The distribution of GPR55 mRNA was first analyzed by in situ hybridization, localizing GPR55 transcripts to neurons in brain nuclei related to movement control, striatum, globus pallidus, subthalamic nucleus, substantia nigra and cortex. Striatal expression of GPR55 was downregulated in parkinsonian conditions. When Abn-CBD and CBD (5 mg/kg) were chronically administered to mice treated over 5 weeks with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp), Abn-CBD but not CBD prevented MPTPp induced motor impairment. Although Abn-CBD protected dopaminergic cell bodies, it failed to prevent degeneration of the terminals or preserve dopamine levels in the striatum. Both compounds induced morphological changes in microglia that were compatible with an anti-inflammatory phenotype that did not correlate with a neuroprotective activity. The symptomatic relief of Abn-CBD was further studied in the haloperidol-induced catalepsy mouse model. Abn-CBD had an anti-cataleptic effect that was reversed by CBD and PSB1216, a newly synthesized GPR55 antagonist, and indeed, two other GPR55 agonists also displayed anti-cataleptic effects (CID1792197 and CID2440433). These results demonstrate for the first time that activation of GPR55 might be beneficial in combating PD.

T-cell prolymphocytic leukaemia (T-PLL) is an aggressive leukaemia. The primary genetic alteration in T-PLL are the inv(14)(q11q32)/t(14;14)(q11;q32) leading to TRD/TRA-TCL1A fusion, or the t(X;14)(q28;q11) associated with TRD/TRA-MTCP1 fusion. However, additional cooperating abnormalities are necessary for emergence of the full neoplastic phenotype. Though the pattern of secondary chromosomal aberrations is remarkably conserved, targets of the changes are largely unknown. We analysed a cohort of 43 well-characterized T-PLL for hotspot mutations in the genes JAK3, STAT5B and RHOA. Additionally, we selected a subset of 23 T-PLL cases for mutational screening of 54 genes known to be recurrently mutated in T-cell and other haematological neoplasms. Activating mutations in the investigated regions of the JAK3 and STAT5B genes were detected in 30% (13/43) and 21% (8/39) of the cases, respectively, and were mutually exclusive. Further, we identified mutations in the genes encoding the epigenetic regulators EZH2 in 13% (3/23), TET2 in 17% (4/23) and BCOR in 9% (2/23) of the cases. We confirmed that the JAK-STAT pathway is a major mutational target, and identified epigenetic regulators recurrently mutated in T-PLL. These findings complement the mutational spectrum of secondary aberrations in T-PLL and underscore the potential therapeutical relevance of epigenetic regulators in T-PLL.

G-Protein-coupled receptors (GPCRs) were classically described as monomers. We now appreciate that they also function as homo- and hetero-oligomers, for which structural information is lacking. Here, we use available 3D structures and biochemical considerations to present and evaluate experimentally testable structural models for GPCR oligomers and associated G proteins. G-Protein-coupled receptors (GPCRs) were classically described as monomers. We now appreciate that they also function as homo- and hetero-oligomers, for which structural information is lacking. Here, we use available 3D structures and biochemical considerations to present and evaluate experimentally testable structural models for GPCR oligomers and associated G proteins. GPCRs comprise the largest family of proteins in mammals. They mediate diverse important physiological functions in mammalian body systems and are implicated in the pathophysiology of serious diseases. GPCRs have been classically described as monomeric transmembrane (TM) receptors that form a ternary complex together with the ligand and the associated G protein. It is now accepted that GPCRs may interact with additional proteins to selectively modulate distinct intracellular signal transduction pathways. A particular case is the formation of GPCR homo- and heterodimers, or higher-order oligomers. High-resolution crystal structures available for GPCRs (mostly belonging to rhodopsin-like/class A receptors) often reveal intimate association between monomers through the TM domains. These associations are compatible with the spatial restrictions imposed by the membrane and with previous experiments investigating the topology of such complexes [1Guo W. et al.Dopamine D2 receptors form higher order oligomers at physiological expression levels.EMBO J. 2008; 27: 2293-2304Crossref PubMed Scopus (290) Google Scholar]. With only a few exceptions (see Table S1 in the supplemental information online), these receptor pairs have twofold rotational symmetry; that is, they form head-to-head interfaces that occur mainly through TMs 1, 4, 4/5, and 5/6 (Figure 1A) [2Gonzalez A. et al.Modeling of G protein-coupled receptors using crystal structures: from monomers to signaling complexes.Adv. Exp. Med. Biol. 2014; 796: 15-33Crossref PubMed Google Scholar]. Remarkably, two crystal structures revealed not only dimers, but also homo-oligomeric strands of receptors with two different interfaces: TM1 and TM5/6 for the μ-opioid receptor (μOR) [3Manglik A. et al.Crystal structure of the μ-opioid receptor bound to a morphinan antagonist.Nature. 2012; 485: 321-326Crossref PubMed Scopus (1083) Google Scholar] and TM1 and TM4/5 for the β1-adrenergic receptor (β1-AR) [4Huang J. et al.Crystal structure of oligomeric β-adrenergic G protein-coupled receptors in ligand-free basal state.Nat. Struct. Mol. Biol. 2013; 4: 419-425Crossref Scopus (220) Google Scholar]. Regardless of whether dimers and oligomers adopt these structural arrangements in vivo, there is no doubt that they constitute high-resolution structural information that can facilitate the task of modeling GPCR dimers and/or oligomers [5Johnston J.M. et al.Assessing the relative stability of dimer interfaces in G protein-coupled receptors.PLoS Comput. Biol. 2012; 8: e1002649Crossref PubMed Scopus (90) Google Scholar]. In this forum, we show how tetramers, emerging from the combination of crystallographic interfaces, may fit into the current understanding of GPCR activation and G-protein-coupling. Let us first consider the simplest case of head-to-head dimers interacting with a G protein. The crystal structures of the β2AR-Gs complex [6Rasmussen S.G. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2363) Google Scholar] and of opsin with a Gα C-terminal (Ct)-derived peptide [7Scheerer P. et al.Crystal structure of opsin in its G-protein-interacting conformation.Nature. 2008; 455: 497-502Crossref PubMed Scopus (936) Google Scholar] revealed the precise changes (mainly in TMs 5 and 6) required for GαCt binding (Figure 1B). Such conformational changes are possible in most dimer models (e.g., the TM1 dimer in Figure 1C). An exception is the TM5/6 dimer, in which a compact four-helix bundle would impede the conformational shift (Figure 1D). Thus, the prediction is that the TM5/6 dimer is an unlikely GPCR functional entity, because it would not be able to activate and engage G-protein-mediated signaling. The now-assumed 2:1 (receptor:G protein) stoichiometry [8Jastrzebska B. et al.Asymmetry of the rhodopsin dimer in complex with transducin.FASEB J. 2013; 27: 1572-1584Crossref PubMed Scopus (57) Google Scholar] fits with a cross-sectional area (in the plane of the plasma membrane) of a receptor being approximately half of the maximum cross-sectional area of a heterotrimeric G protein. This implies that the protomer that binds GαCt, but not the partner protomer, undergoes the conformational changes needed in TMs 5 and 6 (Figure 1C). Still, not all dimeric structures are compatible with the binding of the G protein. For instance, in a TM4 dimer, the N terminus of the Gα would clash with the partner protomer (itself not interacting with GαCt) (Figure 1E). Thus, TM1 and TM4/5 dimers, which appear more frequently in the crystals, are the most plausible dimeric models. Unlike α subunits, which directly interact with one of the protomers, βγ subunits do not necessarily establish interactions with the partner protomer in these dimers (e.g., see Figure 1F, illustrating the TM1 dimer). Another key element is the ‘clam shell-like’ opening of the small globular domain in the Gα subunit, known as the α-helical (AH) domain, which occurs in every GTP–GDP exchange [9Chung K.Y. et al.Conformational changes in the G protein Gs induced by the β2 adrenergic receptor.Nature. 2011; 477: 611-615Crossref PubMed Scopus (299) Google Scholar, 10Westfield G.H. et al.Structural flexibility of the G α s α-helical domain in the β2-adrenoceptor Gs complex.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 16086-16091Crossref PubMed Scopus (181) Google Scholar]. When the AH domain is modeled in the ‘closed’ conformation [11Sunahara R.K. et al.Crystal structure of the adenylyl cyclase activator Gsα.Science. 1997; 278: 1943-1947Crossref PubMed Scopus (273) Google Scholar], both TM1 and TM4/5 models can accommodate the G protein. However, the open conformation of the AH domain observed in the crystal structure of the β2AR-Gs complex would sterically clash with the second protomer of a TM4/5 dimer (Figure 1G). One may then wonder whether the conformation of AH observed in the β2AR-Gs crystal may be physiological or if its movement should be smaller in magnitude. Remarkably, the closed conformation of the AH domain is not attainable in the reported crystal lattice due to steric clash with the Ras domain of a neighboring crystal unit (not shown). As additional evidence suggests [9Chung K.Y. et al.Conformational changes in the G protein Gs induced by the β2 adrenergic receptor.Nature. 2011; 477: 611-615Crossref PubMed Scopus (299) Google Scholar, 10Westfield G.H. et al.Structural flexibility of the G α s α-helical domain in the β2-adrenoceptor Gs complex.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 16086-16091Crossref PubMed Scopus (181) Google Scholar], a slightly less-opened conformation than that observed in the crystal structure would make the steric hindrance disappear in TM4/5 dimers. The simplest way to form a tetramer is to combine two head-to-head dimers using an additional interface. As an example, Figure 2A combines two TM4/5 dimers (in gray and red) using the TM1 interface to give a TM4/5–TM1 heterotetramer (see Figure 2 for nomenclature) modeled using the crystal structure of β1-AR (Protein Data Bank id: 4GPO [4Huang J. et al.Crystal structure of oligomeric β-adrenergic G protein-coupled receptors in ligand-free basal state.Nat. Struct. Mol. Biol. 2013; 4: 419-425Crossref Scopus (220) Google Scholar]). Two G proteins may potentially bind either to the inner or outer protomers in a GPCR tetramer giving rise to ‘in-in’, ‘in-out’, or ‘out-out’ possibilities (Figure 2A). In addition to the TM1 and TM4/5 interfaces described for homodimers, TM5/6 (Figure 2B) and TM4 (Figure 2C) are possible interdimer interfaces for a tetramer. For example, the TM1–TM5/6 tetramer, which is equivalent to the arrangement observed in the crystal structure of μ-OR (Protein Data Bank id: 4DKL [3Manglik A. et al.Crystal structure of the μ-opioid receptor bound to a morphinan antagonist.Nature. 2012; 485: 321-326Crossref PubMed Scopus (1083) Google Scholar]), may bind two Gα in the two external protomers in the out-out mode. The in-in and in-out modes are forbidden because TMs 5 and 6 in the internal protomers are blocked and, therefore, cannot bind Gα. The combination of the above-described basic dimer models interacting through TM1 or TM4/5 using TM1, TM4, TM4/5, or TM5/6 as interdimer interfaces gives rise to only six possible tetramers. The relative distance between inter- and intradimer interfaces is useful to divide tetramers into ‘linear’ tetramers (distant interdimer interfaces, as in TM1–TM4/5, Figure 2A), ‘zigzagged’ tetramers (intermediate situation, as in TM1–TM5/6, Figure 2B) and ‘compact’ tetramers (close interdimer interfaces, as in TM4/5–TM4, Figure 2C). Compact tetramers encounter difficulties in accommodating two G proteins due to excessive crowding at the cytoplasmic region. Still, the TM4/5–TM4 (Figure 2C) and the TM4/5–TM5/6 tetramer (not shown) are feasible in the out-out mode. By contrast, linear and zigzagged tetramers are more permissive, except in the in-in mode, which also suffers from steric hindrance between the two G proteins in all but TM4/5–TM1 tetramers (Figure 2A). Overall, there are only ten likely combinations of tetramers with two G proteins: one following the in-in mode (TM4/5–TM1), three in the in-out mode (TM1–TM4, TM1–TM4/5, and TM4/5–TM1) and six in the out-out mode (TM1–TM4, TM1–TM4/5, TM1–TM5/6, TM4/5–TM1, TM4/5–TM4, and TM4/5–TM5/6). It should be noted that a stoichiometry with more than two G proteins per tetramer is forbidden due to steric issues. The above-described hypothetical tetrameric models of GPCRs are compatible with available structural data and current knowledge of signal transduction. Complexes are symmetrical in the GPCR arrangement (both dimer and tetrameric models display twofold rotational symmetry), but G-protein-binding may be asymmetrical. Furthermore, the proposed structures may be helpful for understanding GPCR oligomerization and for designing novel approaches to understand GPCR function. For instance, the structures may be useful to reveal, at the residue level, how one receptor may allosterically modulate another receptor, or how two G proteins can communicate with each other and affect heteromer-mediated signaling. This work was supported by grant SAF2012/39875-C02-01 and SAF2013-48271-C2-2-R from the Spanish Ministry of Economia y competitividad MINECO. Download .docx (.05 MB) Help with docx files

Elements of the endocannabinoid system are strongly expressed in the basal ganglia where they suffer profound rearrangements after dopamine depletion. Modulation of the levels of the endocannabinoid 2-arachidonoyl-glycerol by inhibiting monoacylglycerol lipase alters glial phenotypes and provides neuroprotection in a mouse model of Parkinson's disease. In this study, we assessed whether inhibiting fatty acid amide hydrolase could also provide beneficial effects on the time course of this disease. The fatty acid amide hydrolase inhibitor, URB597, was administered chronically to mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) over 5weeks. URB597 (1mg/kg) prevented MPTPp induced motor impairment but it did not preserve the dopamine levels in the nigrostriatal pathway or regulate glial cell activation. The symptomatic relief of URB597 was confirmed in haloperidol-induced catalepsy assays, where its anti-cataleptic effects were both blocked by antagonists of the two cannabinoid receptors (CB1 and CB2), and abolished in animals deficient in these receptors. Other fatty acid amide hydrolase inhibitors, JNJ1661010 and TCF2, also had anti-cataleptic properties. Together, these results demonstrate an effect of fatty acid amide hydrolase inhibition on the motor symptoms of Parkinson's disease in two distinct experimental models that is mediated by cannabinoid receptors.

Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) family, is a survival factor for various types of neurons. We studied the mechanisms by which human PEDF protects motor neurons from degeneration, with the goal of eventually conducting human clinical trials. We first searched for a molecular region of human PEDF essential to motor neuron protection. Using a spinal cord culture model of chronic glutamate toxicity, we show herein that a synthetic 44 mer peptide from an N-terminal region of the human PEDF molecule that lacks the homologous serpin-reactive region contains its full neuroprotective activity. We also investigated the presence and distribution of PEDF receptors in the spinal cord. Using a fluoresceinated PEDF probe, we show that spinal motor neurons contain specific binding sites for PEDF. Kinetics analyses using a radiolabeled PEDF probe demonstrate that purified rat motor neurons contain a single class of saturable and specific binding sites. This study indicates that a small peptide fragment of the human PEDF molecule could be engineered to contain all of its motor neuron protective activity, and that the neuroprotective action is likely to be mediated directly on motor neurons via a single class of PEDF receptors. The data support the pharmacotherapeutic potential of PEDF as a neuroprotectant in human motor neuron degeneration.

The present study describes the thalamic innervation coming from the rat parafascicular nucleus (PF) onto striatal and subthalamic efferent neurons projecting either to the globus pallidus (GP) or to the substantia nigra pars reticulata (SNr) by using a protocol for multiple neuroanatomical tracing. Both striatofugal neurons targeting the ipsilateral SNr (direct pathway) as well as striatal efferent neurons projecting to the ipsilateral GP (indirect pathway) were located within the terminal fields of the thalamostriatal afferents. In the subthalamic nucleus (STN), both neurons projecting to ipsilateral GP as well as neurons projecting to ipsilateral SNr also appear to receive thalamic afferents. Although the projections linking the caudal intralaminar nuclei with the ipsilateral striatum and STN are far more prominent, we also noticed that thalamic axons could gain access to the contralateral STN. Furthermore, a small number of STN neurons were seen to project to both the contralateral GP and PF nuclei. These ipsi- and contralateral projections enable the caudal intralaminar nuclei to modulate the activity of both the direct and the indirect pathway.

Abstract Glial cell line‐derived neurotrophic factor (GDNF) is a member of the transforming growth factor‐β superfamily that when exogenously administrated exerts a potent trophic action on dopaminergic (DA) cells. Although we know a lot about its signalling mechanisms and pharmacological effects, physiological actions of GDNF on the adult brain remain unclear. Here, we have used morphological and molecular techniques, and an experimental model of Parkinson's disease in rats, to investigate whether GDNF constitutively expressed in the adult mesostriatal system plays a neuroprotective role on midbrain DA cells. We found that although all midbrain DA cells express both receptor components of GDNF (GFRα1 and Ret), those in the ventral tegmental area (VTA) and rostromedial substantia nigra (SNrm) also contain GDNF but not GDNFmRNA. The levels of GDNFmRNA are significantly higher in the ventral striatum (vSt), the target region of VTA and SNrm cells, than in the dorsal striatum (dSt), the target region of DA cells in the caudoventral substantia nigra (SNcv). After fluoro‐gold injection in striatum, VTA and SNrm DA cells show triple labelling for tyrosine hydroxylase, GDNF and fluoro‐gold, and after colchicine injection in the lateral ventricle, they become GDNF‐immunonegative, suggesting that GDNF in DA somata comes from their striatal target. As DA cells in VTA and SNrm are more resistant than those in SNcv to intracerebroventricular injection of 6‐OHDA, as occurs in Parkinson's disease, we can suggest that the fact that they project to vSt, where GDNF expression is significantly higher than in the dSt, is a neuroprotective factor involved in the differential vulnerability of midbrain DA neurons.

The position of the caudal intralaminar nuclei within basal ganglia circuitry has largely been neglected in most studies dealing with basal ganglia function. During the past few years, there has been a growing body of evidence suggesting that the thalamic parafascicular nucleus in rodents (PF) exerts a multifaceted modulation of basal ganglia nuclei, at different levels. Our aim was to study the activity of the thalamostriatal pathway in rats with unilateral dopaminergic depletion. The experimental approach comprised first unilateral delivery of 6-OHDA in the medial forebrain bundle. Thirty days post-lesioning, animals showing a clear asymmetry were then subjected to bilateral injection of Fluoro-Gold (FG) within the striatum. Subsequently, expression of the mRNA encoding the vesicular glutamate transporter 2 (vGLUT2) was detected within thalamostriatal-projecting neurons (FG-labeled) by in situ hybridization and the results were confirmed by laser-guided capture microdissection microscopy followed by real-time PCR. The data showed that there was a marked neuronal loss restricted to PF neurons projecting to the dopamine-depleted striatum. Moreover, PF neurons innervating the dopamine-depleted striatum were intensely hyperactive. These neurons showed a marked increase on the expression of vGLUT2 mRNA as well as for the mRNA encoding the subunit I of cytochrome oxidase as compared with those neurons projecting to the striatum with normal dopamine content. Thus, the selective neurodegeneration of PF neurons innervating the striatum together with the increased activity of the thalamostriatal pathway coexist after nigrostriatal denervation.

Abstract Objective Chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal‐onset multisystem inflammatory disease (NOMID), is a severe, early‐onset autoinflammatory disease characterized by an urticaria‐like rash, arthritis/arthropathy, variable neurologic involvement, and dysmorphic features, which usually respond to interleukin‐1 blockade. CINCA/NOMID has been associated with dominant Mendelian inherited NLRP3 mutations. However, conventional sequencing analyses detect true disease‐causing mutations in only ∼55–60% of patients, which suggests the presence of genetic heterogeneity. We undertook the current study to assess the presence of somatic, nongermline NLRP3 mutations in a sporadic case of CINCA/NOMID. Methods Clinical data, laboratory results, and information on treatment outcomes were gathered through direct interviews. Exhaustive genetic studies, including Sanger method sequencing, subcloning, restriction fragment length polymorphism assay, and pyrosequencing, were performed. Results The patient's CINCA/NOMID was diagnosed based on clinical features (early onset of the disease, urticaria‐like rash, knee arthropathy, and dysmorphic features). The patient has exhibited a successful response to anakinra within the last 28 months. Analysis of NLRP3 identified a novel heterozygous variant (p.D303H) that was detected in ∼30–38% of circulating leukocytes. The absence of this variant in healthy controls and in the patient's parents suggested a de novo true disease‐causing mutation. Additional analyses showed that this novel mutation was present in both leukocyte subpopulations and epithelial cells. Conclusion Our findings identify the novel p.D303H NLRP3 variant in a Spanish patient with CINCA/NOMID as a new disease‐causing mutation, which was detected as a somatic, nongermline mutation in hematopoietic and nonhematopoietic cell lineages. Our data provide new insight into the role of low‐level mosaicism in NLRP3 as the pathophysiologic mechanism underlying cryopyrin‐associated periodic syndrome.

ABSTRACT GPR40, the free fatty acid receptor 1, is expressed strongly in the primate pancreas and brain. While the role of pancreatic GPR40 in glucose homeostasis has been extensively studied, the absence of this G‐protein‐coupled receptor from the brain of rodents has hampered studies into its role in the central nervous system. However, we found intense GPR40 mRNA expression by in situ hybridization in mouse hippocampal and motor cortex neurons. Furthermore, in a neuroblastoma cell GPR40 was activated by docosahexaenoic acid and selective agonists, yet not by palmitic acid. Significantly, the activation of GPR40 provoked the phosphorylation of the cAMP response element‐binding protein, CREB. The receptor was also functional in primary cultures of murine neurons, in which its activation by a selective agonist produced the phosphorylation of CREB and of extracellular signal‐regulated kinases, ERK1/2. These results suggest that mice represent a suitable model for elucidating the role of GPR40 in brain function. © 2014 Wiley Periodicals, Inc.

Neuroinflammation involves the activation of glial cells, which is associated to the progression of neurodegeneration in Parkinson's disease. Recently, we and other researchers demonstrated that dopamine receptor D3 (D3R)-deficient mice are completely refractory to neuroinflammation and consequent neurodegeneration associated to the acute intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we examined the therapeutic potential and underlying mechanism of a D3R-selective antagonist, PG01037, in mice intoxicated with a chronic regime of administration of MPTP and probenecid (MPTPp). Biodistribution analysis indicated that intraperitoneally administered PG01037 crosses the blood-brain barrier and reaches the highest concentration in the brain 40 min after the injection. Furthermore, the drug was preferentially distributed to the brain in comparison to the plasma. Treatment of MPTPp-intoxicated mice with PG01037 (30 mg/kg, administrated twice a week for five weeks) attenuated the loss of dopaminergic neurons in the substantia nigra pars compacta, as evaluated by stereological analysis, and the loss of striatal dopaminergic terminals, as determined by densitometric analyses of tyrosine hydroxylase and dopamine transporter immunoreactivities. Accordingly, the treatment resulted in significant improvement of motor performance of injured animals. Interestingly, the therapeutic dose of PG01037 exacerbated astrogliosis and resulted in increased ramification density of microglial cells in the striatum of MPTPp-intoxicated mice. Further analyses suggested that D3R expressed in astrocytes favours a beneficial astrogliosis with anti-inflammatory consequences on microglia. Our findings indicate that D3R-antagonism exerts a therapeutic effect in parkinsonian animals by reducing the loss of dopaminergic neurons in the nigrostriatal pathway, alleviating motor impairments and modifying the pro-inflammatory phenotype of glial cells.

Mutations in genes of the RAS-BRAF-MAPK-ERK pathway have not been fully explored in patients with chronic lymphocytic leukemia. We, therefore, analyzed the clinical and biological characteristics of chronic lymphocytic leukemia patients with mutations in this pathway and investigated the in vitro response of primary cells to BRAF and ERK inhibitors. Putative damaging mutations were found in 25 of 452 patients (5.5%). Among these, BRAF was mutated in nine patients (2.0%), genes upstream of BRAF (KITLG, KIT, PTPN11, GNB1, KRAS and NRAS) were mutated in 12 patients (2.6%), and genes downstream of BRAF (MAPK2K1, MAPK2K2, and MAPK1) were mutated in five patients (1.1%). The most frequent mutations were missense, subclonal and mutually exclusive. Patients with these mutations more frequently had increased lactate dehydrogenase levels, high expression of ZAP-70, CD49d, CD38, trisomy 12 and unmutated immunoglobulin heavy-chain variable region genes and had a worse 5-year time to first treatment (hazard ratio 1.8, P=0.025). Gene expression analysis showed upregulation of genes of the MAPK pathway in the group carrying RAS-BRAF-MAPK-ERK pathway mutations. The BRAF inhibitors vemurafenib and dabrafenib were not able to inhibit phosphorylation of ERK, the downstream effector of the pathway, in primary cells. In contrast, ulixertinib, a pan-ERK inhibitor, decreased phospho-ERK levels. In conclusion, although larger series of patients are needed to corroborate these findings, our results suggest that the RAS-BRAF-MAPK-ERK pathway is one of the core cellular processes affected by novel mutations in chronic lymphocytic leukemia, is associated with adverse clinical features and could be pharmacologically inhibited.

Neuroinflammation constitutes a fundamental process involved in Parkinson's disease (PD). Microglial cells play a central role in the outcome of neuroinflammation and consequent neurodegeneration of dopaminergic neurons in the substantia nigra. Current evidence indicates that CD4+ T-cells infiltrate the brain in PD, where they play a critical role determining the functional phenotype of microglia, thus regulating the progression of the disease. We previously demonstrated that mice bearing dopamine receptor D3 (DRD3)-deficient CD4+ T-cells are completely refractory to neuroinflammation and consequent neurodegeneration induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we aimed to determine whether DRD3-signalling is altered in peripheral blood CD4+ T-cells obtained from PD patients in comparison to healthy controls (HC). Furthermore, we evaluated the therapeutic potential of targeting DRD3 confined to CD4+ T-cells by inducing the pharmacologic antagonism or the transcriptional inhibition of DRD3-signalling in a mouse model of PD induced by the chronic administration of MPTP and probenecid (MPTPp). In vitro analyses performed in human cells showed that the frequency of peripheral blood Th1 and Th17 cells, two phenotypes favoured by DRD3-signalling, were significantly increased in PD patients. Moreover, naïve CD4+ T-cells obtained from PD patients displayed a significant higher Th1-biased differentiation in comparison with those naïve CD4+ T-cells obtained from HC. Nevertheless, DRD3 expression was selectively reduced in CD4+ T-cells obtained from PD patients. The results obtained from in vivo experiments performed in mice show that the transference of CD4+ T-cells treated ex vivo with the DRD3-selective antagonist PG01037 into MPTPp-mice resulted in a significant reduction of motor impairment, although without significant effect in neurodegeneration. Conversely, the transference of CD4+ T-cells transduced ex vivo with retroviral particles codifying for an shRNA for DRD3 into MPTPp-mice had no effects neither in motor impairment nor in neurodegeneration. Notably, the systemic antagonism of DRD3 significantly reduced both motor impairment and neurodegeneration in MPTPp mice. Our findings show a selective alteration of DRD3-signalling in CD4+ T-cells from PD patients and indicate that the selective DRD3-antagonism in this subset of lymphocytes exerts a therapeutic effect in parkinsonian animals dampening motor impairment.

Abstract Background Inflammation is a critical process for the progression of neuronal death in neurodegenerative disorders. Microglia play a central role in neuroinflammation and may affect neuron vulnerability. Next generation sequencing has shown the molecular heterogeneity of microglial cells; however, the variability in their response to pathological inputs remains unknown. Methods To determine the effect of an inflammatory stimulus on microglial cells, lipopolysaccharide (LPS) was administered peripherally to mice and the inflammatory status of the cortex, hippocampus, midbrain, and striatum was assessed. Microglial activation and interaction with the immune system were analyzed in single cell suspensions obtained from the different brain regions by fluorescence-activated cell sorting, next generation RNA sequencing, real-time PCR, and immunohistochemical techniques. Antigen-presenting properties of microglia were evaluated by the ability of isolated cells to induce a clonal expansion of CD4 + T cells purified from OT-II transgenic mice. Results Under steady-state conditions, the midbrain presented a high immune-alert state characterized by the presence of two unique microglial subpopulations, one expressing the major histocompatibility complex class II (MHC-II) and acting as antigen-presenting cells and another expressing the toll-like receptor 4 (TLR4), and by the presence of a higher proportion of infiltrating CD4 + T cells. This state was not detected in the cortex, hippocampus, or striatum. Systemic LPS administration induced a general increase in classic pro-inflammatory cytokines, in co-inhibitory programmed death ligand 1 (PD-L1), and in cytotoxic T lymphocyte antigen 4 (CTLA-4) receptors, as well as a decrease in infiltrating effector T cells in all brain regions. Interestingly, a specific immune-suppressive response was observed in the midbrain which was characterized by the downregulation of MHC-II microglial expression, the upregulation of the anti-inflammatory cytokines IL10 and TGFβ, and the increase in infiltrating regulatory T cells. Conclusions These data show that the midbrain presents a high immune-alert state under steady-state conditions that elicits a specific immune-suppressive response when exposed to an inflammatory stimulus. This specific inflammatory tone and response may have an impact in neuronal viability.

The present study analyses the anatomical arrangement of the projections linking the Wistar rat parafascicular thalamic nucleus (PF) and basal ganglia structures, such as the striatum and the subthalamic nucleus (STN), by using neuroanatomical tract-tracing techniques. Both the thalamostriatal and the striato-entopeduncular projections were topographically organized, and several areas of overlap between identified circuits were noticed, sustaining the existence of up to three separated channels within the Nauta-Mehler loop. Thalamic afferents arising from dorsolateral PF territories are in register with striatofugal neurons located in dorsolateral striatal areas, which in turn project to dorsolateral regions of the entopeduncular nucleus (ENT). Medial ENT regions are innervated by striatal neurons located within medial striatal territories, these neurons being the target for thalamic afferents coming from medial PF areas. Finally, afferents from neurons located in ventrolateral PF areas approached striatal neurons in ventral and lateral striatal territories, which in turn project towards ventral and lateral ENT regions. Efferent STN neurons projecting to ENT were found to be the apparent postsynaptic target for thalamo-subthalamic axons. The thalamo-subthalamic projection was also topographically organized. Medial, central and lateral STN territories are innervated by thalamic neurons located within medial, ventrolateral and dorsolateral PF areas, respectively. Thus, each individual PF subregion projects in a segregated fashion to specific parts of the striato-entopeduncular and subthalamo-entopeduncular systems. These circuits enabled the caudal intralaminar nuclei to modulate basal ganglia output.

The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.

The pedunculopontine tegmental nucleus (PPN) and laterodorsal tegmental nucleus (LDT) are functionally associated brainstem structures implicated in behavioral state control and sensorimotor integration. The PPN is also involved in gait and posture, while the LDT plays a role in reward. Both nuclei comprise characteristic cholinergic neurons intermingled with glutamatergic and GABAergic cells whose absolute numbers in the rat have been only partly established. Here we sought to determine the complete phenotypical profile of each nucleus to investigate potential differences between them. Counts were obtained using stereological methods after the simultaneous visualization of cholinergic and either glutamatergic or GABAergic cells. The two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, were separately analyzed. Dual in situ hybridization revealed coexpression of GAD65 and GAD67 mRNAs in ∼90% of GAD-positive cells in both nuclei; thus, the estimated mean numbers of (1) cholinergic, (2) glutamatergic, and (3) GABAergic cells in PPN and LDT, respectively, were (1) 3,360 and 3,650; (2) 5,910 and 5,190; and (3) 4,439 and 7,599. These data reveal significant differences between PPN and LDT in their relative phenotypical composition, which may underlie some of the functional differences observed between them. The estimation of glutamatergic cells was significantly higher in the caudal PPN, supporting the reported functional rostrocaudal segregation in this nucleus. Finally, a small subset of cholinergic neurons (8% in PPN and 5% in LDT) also expressed the glutamatergic marker Vglut2, providing anatomical evidence for a potential corelease of transmitters at specific target areas.

Abstract Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88 , a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88- mutated and -unmutated cases. In the Eµ -TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Abstract Immunoglobulin (Ig) gene rearrangements and oncogenic translocations are routinely assessed during the characterization of B cell neoplasms and stratification of patients with distinct clinical and biological features, with the assessment done using Sanger sequencing, targeted next-generation sequencing, or fluorescence in situ hybridization (FISH). Currently, a complete Ig characterization cannot be extracted from whole-genome sequencing (WGS) data due to the inherent complexity of the Ig loci. Here, we introduce IgCaller, an algorithm designed to fully characterize Ig gene rearrangements and oncogenic translocations from short-read WGS data. Using a cohort of 404 patients comprising different subtypes of B cell neoplasms, we demonstrate that IgCaller identifies both heavy and light chain rearrangements to provide additional information on their functionality, somatic mutational status, class switch recombination, and oncogenic Ig translocations. Our data thus support IgCaller to be a reliable alternative to Sanger sequencing and FISH for studying the genetic properties of the Ig loci.

The present study is focused on the analysis of the vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) used by thalamic neurons giving rise to the thalamostriatal system. Instead of studying the distribution of VGLUT proteins at the level of thalamostriatal terminals, this report is focused on identifying the expression of the VGLUT mRNAs within the parent cell bodies of thalamic neurons innervating the striatum. For this purpose, we have combined dual in situ hybridization to detect both VGLUT1 and VGLUT2 mRNAs together with retrograde tracing with cholera toxin. Our results show that VGLUT2 is the only vesicular glutamate transporter expressed in thalamostriatal-projecting neurons located in the midline and intralaminar nuclei, whereas all neurons from the ventral thalamic nuclei innervating the striatum express both VGLUTs, at least at the mRNA level. Indeed, the mRNAs encoding for VGLUT1 and VGLUT2 displayed a sharp complementary subcellular distribution within neurons from the ventral thalamic nuclei giving rise to thalamostriatal projections. The differential distribution of VGLUT mRNAs lead us to conclude that the thalamostriatal pathway is a dual system, composed by a preponderant projection arising from the midline and intralaminar nuclei using VGLUT2 as the glutamate transporter, together with another important source of striatal afferents arising from neurons in the ventral thalamic relay nuclei containing both kinds of vesicular glutamate transporters.

Glial cell-line derived neurotrophic factor (GDNF), a potent neurotrophic factor for dopaminergic neurons, appeared as a promising candidate for treating Parkinson’s disease. GDNF microencapsulation could ensure protection against degradation due to the fragile nature of the protein. Poly(lactide-co-glycolide) (PLGA) microparticles loaded with recombinant glycosylated GDNF obtained in a mammalian cell line were prepared by TROMS, a semi-industrial technique capable of encapsulating fragile molecules maintaining their native properties. The effects of several parameters as PLGA copolymer type, PEG 400 quantity co-encapsulated with GDNF or drug loading, on the properties of the particles were investigated. Microparticles showed a mean diameter between 8 and 30 μm, compatible with their stereotaxic implantation. The drug entrapment efficiency ranged from 50.6% to 100% depending on the microsphere composition. GDNF was better encapsulated using hydrophilic polymers with high molecular weight such as RG 503H. In vitro drug release was influenced by the polymer type as well as by the amount of PEG 400 co-encapsulated with GDNF. Microparticles prepared using PLGA RG 503H released 67% of the total protein content within 40 days. Moreover, very low concentrations of poly(vinyl alcohol) were detected after microparticles washing and freeze-drying. Finally, a PC-12 bioassay demonstrated that the in vitro GDNF released was bioactive.

Abstract Background: Glial cell‐derived neurotrophic factor is a survival factor for dopaminergic neurons and a promising candidate for the treatment of Parkinson's disease. However, the delivery issue of the protein to the brain still remains unsolved. Our aim was to investigate the effect of long‐term delivery of encapsulated glial cell‐derived neurotrophic factor within microspheres. Methods: A single dose of microspheres containing 2.5 μg of glial cell‐derived neurotrophic factor was implanted intrastriatally in animals 2 weeks after a 6‐hydroxydopamine lesion. Results: The amphetamine test showed a complete behavioral recovery after 16 weeks of treatment, which was maintained until the end of the study (week 30). This effect was accompanied by an increase in dopaminergic striatal terminals and neuroprotection of dopaminergic neurons. Conclusions: The main achievement was the long‐term neurorestoration in parkinsonian animals induced by encapsulated glial cell‐derived neurotrophic factor, suggesting that microspheres may be considered as a means to deliver glial cell‐derived neurotrophic factor for Parkinson's disease treatment. © 2011 Movement Disorder Society

The clinical course of chronic lymphocytic leukemia (CLL) is extremely heterogeneous and while some patients achieve a normal lifespan, others succumb to the disease shortly after diagnosis. Recurrent chromosomal aberrations as detected by chromosome banding analysis (CBA) or fluorescent in situ hybridization (FISH) have a reproducible prognostic power in terms of response to therapy and survival.1–3 In particular, patients whose tumor cells harbor 17p deletions (17p-) are considered to have a shorter survival and, hence, high-risk CLL. This poor prognosis is, however, not universally true for all patients with 17p- CLL. Indeed, we and others have observed that some clinical-biological features, such as presence of B symptoms, advanced clinical stage, size of the 17p- clone, β2-microglobulin (β2M) concentration and IGH mutational status have a significant impact on the outcome of this subgroup of patients.4,5 Novel molecular studies have helped in the understanding of 17p- CLL. On one hand, TP53 mutations are present in more than 80% of cases with 17p deletion and in around 5% of patients without 17p deletion.6,7 On the other hand, next generation sequencing studies have revealed novel genetic aberrations such as NOTCH1 and SF3B1 mutations that have a negative impact on survival.8–10 Finally, genomic complexity, as defined by karyotyping1 or copy number (CN) arrays, has also been independently associated with disease transformation and poor outcome in patients with CLL.11,12 The aim of this study was to evaluate the prognostic value of concomitant molecular abnormalities in patients with CLL and TP53 aberrations as diagnosed by FISH, CBA or DNA sequencing. From our database, we identified 55 of 763 (7%) CLL patients with TP53 disruption detected at any time over the course of the disease. Importantly, CBA/FISH and molecular studies were performed on samples drawn on the same date. Other clinical and biological characteristics, such as Binet stage, CD38 and ZAP70 expression or β2M serum concentration, were also analyzed at the time of detection of TP53 disruption using conventional methods. All patients from this study signed an informed consent and were recruited into the International Cancer Genome Consortium Chronic Lymphocytic Leukemia project, which was reviewed by the Institutional Review Board. CBA was performed on Giemsa-banded chromosomes obtained after a 72-h culture and stimulation with tetradecanoyl-phorbol-acetate. A complex karyotype was defined as the presence of 3 or more clonal chromosomal aberrations. FISH studies for 11q, 13q and 17p deletions and gains of chromosome 12 were performed using the Vysis CLL probe kit (Abbott, Des Plaines, IL, USA). IGHV-IGHD-IGHJ rearrangements and mutational status and TP53 mutation analysis were analyzed following ERIC recommendations.13,14 NOTCH1 and SF3B1 mutations were evaluated as previously described.8,9 CN analysis was performed using two different platforms: a custom Agilent 8×60K oligonucleotide array (I Salaverria et al., manuscript in preparation) and the Genome-Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA). Nexus 6.0 Discovery Edition (Biodiscovery, El Segundo, CA, USA) was used for global analysis and visualization. All data have been up-loaded to the GEO database ({type:entrez-geo,attrs:{text:GSE56277,term_id:56277}}GSE56277). Comparison between groups was performed using Fisher’s exact and Mann-Whitney tests. Overall survival (OS) and time to first treatment (TTFT) were calculated from the date of sampling to the date of death or front-line treatment, respectively, or last follow up. Appropriate cutoff points for copy number alterations (CNAs) were calculated using maximally selected rank statistics. Cox regression multivariate models were fitted in order to assess the independent prognostic value of those covariates that were significant by univariate analysis. Data were collected from 55 patients with TP53 disruption (Figure 1). Thirty-four patients had 17p- diagnosed by interphase FISH and 6 by CBA before the availability of FISH. The remaining 15 patients were identified to have TP53 mutations by Sanger sequencing but had no identifiable 17p deletion by CBA and/or FISH. Thirty (55%) patients had de novo aberrations (i.e. detected within 6 months of CLL diagnosis) and 25 (45%) acquired them at a median of 58 months from diagnosis (range 8–194 months), 23 of these patients (92%) after CLL-specific therapy. Median age of the entire population was 67 years (range 30–98 years) when the TP53 disruption was detected. Patients with acquired aberrations had a higher incidence of Binet stage B–C disease and elevated β2M concentration at the time of detection of TP53 disruption, consistent with a more advanced disease (Online Supplementary Table S1). CBA was performed in 48 patients and yielded adequate metaphases in 45 of them (Figure 1). Chromosome abnormalities involving 17p were observed in 27 of 45 (60%) patients (Online Supplementary Table S1), and 23 of 45 (52%) patients had a complex karyotype. Figure 1. Graphical representation of the whole cohort of patients according to TP53 alterations. The percentage of cells with 17p deletion by FISH is illustrated in the vertical bar plots. The squares below indicate the presence of 17p losses by copy number arrays ... In the 34 patients with a positive FISH test, the median percentage of 17p- cells was 40% (range 10–98%). Forty-two cases were studied by CN-arrays: 27 using SNP arrays 6.0 and 15 using a customized 8×60K array (Figure 2 and Online Supplementary Table S2). CN-arrays revealed 17p losses in 27 of 42 (64%) patients. Furthermore, 17p losses were more frequent in patients with 25% or more cells with deletion by FISH compared to patients with a lower allelic burden (93% vs. 24%; P<0.001). Indeed, CN-arrays only failed to detect a 17p loss in one out of 15 patients with 25% or more 17p- cells by FISH, which is considered the limit of detection of CN-arrays.11 In addition, CN-arrays were able to detect a copy number neutral loss of heterozygosity (CNN-LOH) of the 17p region in 2 of 14 (14%) patients who had TP53 mutations without 17p deletion by FISH (Online Supplementary Figure S1). The presence of other genomic aberrations that have been reported to confer prognostic value independently of TP53 disruption, such as gains at 2p and 8q or losses at 8p,15 was too low in our series (6%, 9% and 6%, respectively) to have any prognostic impact. Figure 2. Copy number profiles of cases with de novo (A) and acquired (B) TP53 disruption. In the x-axis the chromosomes are represented horizontally from 1 to 22, in the y-axis the percentage of cases showing the copy number alterations. Gains are represented ... The number of CNAs was equally distributed among patients with de novo and acquired TP53 disruption (Online Supplementary Table S1). The maxstat analysis revealed two possible cut offs for CNAs with a high prognostic power: three and nine CNAs per case (Online Supplementary Figure S2). For the validation of the cut offs, prediction error curves for their different values were estimated using a .632+ bootstrap strategy. The integrated Brier score between time 0 and time 54 of each estimated curve was used as a performance measure of the corresponding cut offs (Online Supplementary Figure S3). There was a significant correlation between the presence or absence of a complex karyotype by CBA and the number of CNAs by CN-array (P=0.011, Mann-Whitney test). TP53 mutations were detected in 43 of 55 (78%) patients. NOTCH1 and SF3B1 mutations were identified in 10 of 54 (19%) and 8 of 49 (16%) patients, respectively, but no clear association was evident between the presence of these mutations and any other genomic aberration and/or prognostic marker. The great majority (86%) of patients with 17p disruption by CN-arrays (either 17p loss or CNN-LOH) also had concurrent TP53 mutations. Among patients with de novo TP53 disruption, 21 of 30 (70%) required CLL-specific therapy (Online Supplementary Table S4). Median TTFT was nine months, and covariates predictive of a shorter TTFT were unmutated IGHV genes (P=0.011) and high ZAP70 expression (P=0.011). Multivariate analysis revealed that IGHV mutational status was the only variable with independent prognostic value in terms of TTFT (hazard ratio [HR] 13.8, 95% confidence interval [CI] 1.7–112.9; P=0.014) (Figure 3A). Median overall survival for the entire cohort was 37 (95%CI: 34–41) months from the time of sampling. We tested both possible cut offs for CNAs (three and nine alterations) and found that both were equally significant (P=0.024). By multivariate analysis, the only factor with independent prognostic value was the number of CNAs (0–3 vs. 4–9 vs. >9; P=0.024) (Figure 3B). Hazard ratios for CNAs were 7.63 (95%CI: 1.6–37.0; P=0.011) for the 0–3 versus 4–9 comparison and 7.35 (95%CI: 1.62–33.3; P=0.010) for the 4–9 versus >9 comparison (Online Supplementary Table S5). Figure 3. (A) Time to first treatment in patients with de novo TP53 disruption according to IGHV mutational status [(mutated (n = 6) vs. unmutated (n = 20)]; (B) overall survival in patients with TP53 disruption according to the number of copy number alterations ... In conclusion, the prognosis of CLL patients with a TP53 disruption is modulated by their genomic complexity as assessed by CN-arrays but also by additional molecular features such as IGHV mutations. Genomic complexity as determined by CN-arrays was predictive of OS and IGHV mutational status was predictive of TTFT, which is in keeping with previous results.11 Finally, SNP-arrays were very helpful in the detection of 17p CNN-LOH. These results require validation but provide further evidence of the expanding role of CN-arrays and molecular testing in the prognostic workup of patients with CLL.

Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.

Public opinion surrounding the recreational use and therapeutic potential of cannabis is shifting. This review describes new work examining the behavioural and neural effects of cannabis and the endocannabinoid system, highlighting key regions within corticolimbic brain circuits. First, we consider the role of human genetic factors and cannabis strain chemotypic differences in contributing to interindividual variation in the response to cannabinoids, such as THC, and review studies demonstrating that THC-induced impairments in decision-making processes are mediated by actions at prefrontal CB1 receptors. We further describe evidence that signalling through prefrontal or ventral hippocampal CB1 receptors modulates mesolimbic dopamine activity, aberrations of which may contribute to emotional processing deficits in schizophrenia. Lastly, we review studies suggesting that endocannabinoid tone in the amygdala is a critical regulator of anxiety, and report new data showing that FAAH activity is integral to this response. Together, these findings underscore the importance of cannabinoid signalling in the regulation of cognitive and affective behaviours, and encourage further research given their social, political, and therapeutic implications.

The neurotrophic activity of pigment epithelium-derived factor (PEDF), an extracellular factor present in the retina, is mediated by binding to cell-surface receptors in responsive cell cultures. In the present study, the expression of PEDF receptors in native neural retinas from adult steers was examined.Binding reactions were performed with (125)I-PEDF and fluoresceinated PEDF using plasma membranes, detergent-soluble membrane proteins, or cryosections of retina from adult bovine eyes. Radioligand-binding and competition analyses were performed with a computer-assisted program. Ligand blot analysis of detergent-soluble membrane proteins was performed with (125)I-PEDF followed by autoradiography. Ligand-affinity column chromatography of detergent-soluble membrane proteins was performed with PEDF-coupled resin followed by SDS-PAGE. Binding of fluoresceinated PEDF to retina cryosections was detected by confocal microscopy.Radioligand-binding assays showed that (125)I-PEDF bound in a specific and saturable fashion to one class of sites on retina membranes (K(d) = 2.5-6.5 nM; maximum binding [B(max)] = 1-48 x 10(10) sites/retina). A peptide of 44 amino acids (44-mer), identified as the receptor-binding region of PEDF, competed efficiently for (125)I-PEDF binding to retina membranes with kinetics similar to the full-length PEDF. Ligand blot analysis and ligand-affinity chromatography revealed a specific and high-affinity PEDF-binding protein of approximately 85 kDa in retina plasma membranes. Confocal microscopy showed that fluorescein-conjugated PEDF stained exclusively the inner segments of photoreceptors and cells of the ganglion cell layer in retinal cryosections.Altogether, these data conclusively demonstrate the existence of PEDF receptors discretely distributed on the surface of cells from the adult neural retina of bovine eyes. Furthermore, they provide evidence for the direct action of PEDF on photoreceptor and ganglion cell neurons and an anatomic basis for studies to assess PEDF neurotrophic effects on the adult retina.

To compare T-cell reconstitution in two groups of patients submitted to allogeneic stem cell transplantation (SCT): those receiving reduced-intensity conditioning (RIC, n = 24) and those receiving myeloablative conditioning (MA, n = 27).Fifty-one consecutive patients undergoing SCT were evaluated. Serial assessments of lymphocyte subsets and T cell receptor excision circles (TRECs) levels were performed using multiparametric flow cytometry and real-time PCR, respectively.During the first 6 months posttransplant, total and naïve CD4(+) T cell counts were higher after RIC-SCT than after MA-SCT (total CD4(+): p = 0.04, p = 0.08, and p = 0.058; naïve CD4(+): p = 0.14, p = 0.05, and p = 0.01 at 1, 3, and 6 months, respectively). In both groups of patients, TRECs levels were low or undetectable in the first 3 months after SCT and progressively increased during the study. However, a higher proportion of patients with detectable levels of TRECs was observed in RIC-SCT at 1 and 3 months and more patients in this group reached normal levels of TRECs at 6 months post-SCT. In the multivariate analysis, including factors such as type of donor (sibling vs unrelated), dose of CD34(+) cells infused with the graft, patient age, and graft-vs-host disease (GVHD), the most important factor influencing TRECs recovery in the early period after SCT was the type of conditioning regimen.In this study, the pattern of immune reconstitution after RIC-SCT was different from that of MA-SCT and was characterized by higher posttransplant naïve CD4(+) T cell counts and TRECs levels in the early period after transplant.

Chronic lymphocytic leukemia (CLL) is a common disease with highly variable clinical course. Several recurrent chromosomal alterations are associated with prognosis and may guide risk-adapted therapy. We have developed a targeted genome-wide array to provide a robust tool for ascertaining abnormalities in CLL and to overcome limitations of the 4-marker fluorescence in situ hybridization (FISH). DNA from 180 CLL patients were hybridized to the qChip®Hemo array with a high density of probes covering commonly altered loci in CLL (11q22-q23, 13q14, and 17p13), nine focal regions (2p15-p16.1, 2p24.3, 2q13, 2q36.3-q37.1, 3p21.31, 8q24.21, 9p21.3, 10q24.32, and 18q21.32-q21.33) and two larger regions (6q14.1-q22.31 and 7q31.33-q33). Overall, 86% of the cases presented copy number alterations (CNA) by array. There was a high concordance of array findings with FISH (84% sensitivity, 100% specificity); all discrepancies corresponded to subclonal alterations detected only by FISH. A chromothripsis-like pattern was detected in eight cases. Three showed concomitant shattered 5p with gain of TERT along with isochromosome 17q. Presence of 11q loss was associated with shorter time to first treatment (P = 0.003), whereas 17p loss, increased genomic complexity, and chromothripsis were associated with shorter overall survival (P < 0.001, P = 0.001, and P = 0.02, respectively). In conclusion, we have validated a targeted array for the diagnosis of CLL that accurately detects, in a single experiment, all relevant CNAs, genomic complexity, chromothripsis, copy number neutral loss of heterozygosity, and CNAs not covered by the FISH panel. This test may be used as a practical tool to stratify CLL patients for routine diagnostics or clinical trials.

Diet is a modifiable risk factor for several neoplasms but evidence for chronic lymphocytic leukemia (CLL) is sparse. Previous studies examining the association between single-food items and CLL risk have yielded mixed results, while few studies have been conducted on overall diet, reporting inconclusive findings. This study aimed to evaluate the association between adherence to three dietary patterns and CLL in the multicase-control study (MCC-Spain) study. Anthropometric, sociodemographic, medical and dietary information was collected for 369 CLL cases and 1605 controls. Three validated dietary patterns, Western, Prudent and Mediterranean, were reconstructed in the MCC-Spain data. The association between adherence to each dietary pattern and CLL was assessed, overall and by Rai stage, using mixed logistic regression models adjusted for potential confounders. High adherence to a Western dietary pattern (i.e. high intake of high-fat dairy products, processed meat, refined grains, sweets, caloric drinks, and convenience food) was associated with CLL [ORQ4 vs. Q1=1.63 (95%CI 1.11; 2.39); P-trend=0.02; OR 1-SD increase=1.19 (95%CI: 1.03; 1.37)], independently of Rai stages. No differences in the association were observed according to sex, Body Mass Index, energy intake, tobacco, physical activity, working on a farm, or family history of hematologic malignancies. No associations were observed for Mediterranean and Prudent dietary patterns and CLL. This study provides the first evidence for an association between a Western dietary pattern and CLL, suggesting that a proportion of CLL cases could be prevented by modifying dietary habits. Further research, especially with a prospective design, is warranted to confirm these findings.

Abstract Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1 -mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1 -mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1 -mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1 , and NPM1 gene expression as well as cell proliferation in NOTCH1 -mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4 , NRARP , and VEGFA , together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1 -mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

The t(14;19)(q32;q13) often juxtaposes BCL3 with immunoglobulin heavy chain (IGH) resulting in overexpression of the gene. In contrast to other oncogenic translocations, BCL3 rearrangement (BCL3-R) has been associated with a broad spectrum of lymphoid neoplasms. Here we report an integrative whole-genome sequence, transcriptomic, and DNA methylation analysis of 13 lymphoid neoplasms with BCL3-R. The resolution of the breakpoints at single base-pair revealed that they occur in two clusters at 5’ (n=9) and 3’ (n=4) regions of BCL3 associated with two different biological and clinical entities. Both breakpoints were mediated by aberrant class switch recombination of the IGH locus. However, the 5’ breakpoints (upstream) juxtaposed BCL3 next to an IGH enhancer leading to overexpression of the gene whereas the 3’ breakpoints (downstream) positioned BCL3 outside the influence of the IGH and were not associated with its expression. Upstream BCL3-R tumors had unmutated IGHV, trisomy 12, and mutated genes frequently seen in chronic lymphocytic leukemia (CLL) but had an atypical CLL morphology, immunophenotype, DNA methylome, and expression profile that differ from conventional CLL. In contrast, downstream BCL3-R neoplasms were atypical splenic or nodal marginal zone lymphomas (MZL) with mutated IGHV, complex karyotypes and mutated genes typical of MZL. Two of the latter four tumors transformed to a large B-cell lymphoma. We designed a novel fluorescence in situ hybridization assay that recognizes the two different breakpoints and validated these findings in 17 independent tumors. Overall, upstream or downstream breakpoints of BCL3-R are mainly associated with two subtypes of lymphoid neoplasms with different (epi)genomic, expression, and clinicopathological features resembling atypical CLL and MZL, respectively.

We have compared the performance of two methods designed to simultaneously detect two different mRNAs within a single brain section by dual ISH. Specific mRNA riboprobes labeled with biotin and digoxigenin were simultaneously hybridized and visualized using either brightfield or fluorescence microscopy. For brightfield visualization, the biotin-labeled riboprobe was detected with a peroxidase chromogen, whereas, an alkaline phosphatase substrate was used for the detection of the digoxigenin-labeled riboprobe. Dual fluorescent ISH involved the detection of the biotin-labeled riboprobe with an Alexa®488-conjugated streptavidin followed by the visualization of the digoxigenin-labeled riboprobe with the red fluorescent substrate HNPP. The dual ISH protocols presented here offer sensitive methods to detect the expression of two mRNAs of interest, with both colorimetric and fluorescent ISH each having its strengths and limitations. For example, dual colorimetric ISH has proven to be particularly useful to study the distribution of two mRNAs in different brain nuclei, whereas, dual fluorescent ISH has provided better results when studying the co-localization of two different mRNAs in single neurons. The comprehensive step-by-step procedure is presented, together with a troubleshooting section in which the advantages and limitations of these procedures are reviewed in depth. Moreover, alternative protocols for dual ISH were also compared to those presented here.

Chronic lymphocytic leukemia (CLL) is frequently associated with autoimmune hemolytic anemia (AIHA). However, the mechanisms governing the association between CLL and AIHA are poorly understood. MicroRNAs (miRNAs) have been associated with different clinico-biological forms of CLL and are also known to play a substantial role in autoimmunity. However, there are no studies correlating miRNA expression with the likelihood that patients with CLL will develop AIHA. In this study, we found that malignant B-cells from patients with CLL subsequently developing AIHA present nine down-regulated (i.e. miR-19a, miR-20a, miR-29c, miR-146b-5p, miR-186, miR-223, miR-324-3p, miR-484 and miR-660) miRNAs. Interestingly, two of these miRNAs (i.e. miR-20a and miR-146b-5p) are involved in autoimmune phenomena, and one (i.e. miR-146b-5p) in both autoimmunity and CLL. Furthermore, we demonstrated that miR-146b-5p modulates CD80, a molecule associated with the B–T-cell synapse and in restoration of the antigen presenting cell capacity of CLL cells.

Clinical responses to bendamustine in chronic lymphocytic leukemia (CLL) are highly heterogeneous and no specific markers to predict sensitivity to this drug have been reported.In order to identify biomarkers of response, we analyzed the in vitro activity of bendamustine and the gene expression profile in primary CLL cells.We observed that mRNA expression of CD69 (CD69) and ITGAM (CD11b) constitute the most powerful predictor of response to bendamustine.When we interrogated the predictive value of the corresponding cell surface proteins, the expression of the activation marker CD69 was the most reliable predictor of sensitivity to bendamustine.Importantly, a multivariate analysis revealed that the predictive value of CD69 expression was independent from other clinico-biological CLL features.We also showed that when CLL cells were co-cultured with distinct subtypes of stromal cells, an upregulation of CD69 was accompanied by a reduced sensitivity to bendamustine.In agreement with this, tumor cells derived from lymphoid tumor niches harbored higher CD69 expression and were less sensitive to bendamustine than their peripheral blood counterparts.Furthermore, pretreatment of CD69 high CLL cases with the B-cell receptor (BCR) pathway inhibitors ibrutinib and idelalisib decreased CD69 levels and enhanced bendamustine cytotoxic effect.Collectively, our findings indicate that CD69 could be a predictor of bendamustine response in CLL patients and the combination of clinically-tested BCR signaling inhibitors with bendamustine may represent a promising strategy for bendamustine low responsive CLL cases.

Management of levodopa-induced dyskinesias (LID) is one of the main challenges in the treatment of Parkinson's disease patients. Mechanisms involved in the appearance of these involuntary movements are not well known but modifications in the activity of different neurotransmitter pathways seem to play an important role. The objective of this study was to determine differences in the expression levels of the endocannabinoid system (ECS) elements that would support a role in LID. The basal ganglia nuclei, putamen, external segment of the globus pallidus (GPe), internal segment of the globus pallidus (GPi), subthalamic nucleus (STN) and substantia nigra (SN) were dissected out from cryostat sections obtained from two groups of parkinsonian monkeys treated with levodopa to induce dyskinesias. One group of dyskinetic animals was sacrificed under the effect of levodopa, during the active phase of LID, and the other group 24 h after the last levodopa dose (OFF levodopa). Biochemical analysis by real-time PCR for ECS elements was performed. CB1 receptor expression was upregulated in the putamen, GPe and STN during the active phase of dyskinesia and downregulated in the same nuclei and in the SN when dyskinetic animals were OFF levodopa. Changes in the 2-arachidonoyl glycerol (2-AG) synthesizing/degrading enzymes affecting the pallidal-subthalamic projections in dyskinetic animals OFF levodopa would suggest that 2-AG may play a role in LID. Anandamide (AEA) synthesizing/degrading enzymes were altered specifically in the GPe of untreated parkinsonian monkeys, suggesting that increased AEA levels may be a compensatory mechanism. These results indicate that the expression of the ECS elements is influenced by alterations in dopaminergic neurotransmission. On one hand, changes in CB1 receptor expression and in the 2-AG synthesizing/degrading enzymes suggest that they could be a therapeutic target for the active phase of LID. On the other hand, AEA metabolism could provide a non-dopaminergic target for symptomatic relief. However, further research is needed to unravel the mechanism of action of the ECS and how they could be modulated for a therapeutic purpose.

The situation of the caudal intralaminar thalamic nuclei within basal ganglia circuits has gained increased attention over the past few years. Although initially considered as a "non-specific" thalamic nuclei, tract-tracing studies carried out over the past two decades have demonstrated that the centromedian-parafascicular thalamic complex (CM-Pf) is connected to virtually all basal ganglia components and related nuclei. Although the anatomical basis sustaining the thalamic modulation of basal ganglia circuits has long been characterized, the functional significance of these transverse circuits still remain to be properly accommodated within the basal ganglia model, both under normal conditions as well as in situations of dopaminergic depletion. However, the recent demonstration of primary (e.g., non-dopamine related) neurodegenerative phenomena restricted to the CM-Pf in Parkinson's disease (PD) has renewed interest in the role played by the caudal intralaminar nuclei in the pathophysiology of PD. Concomitantly, evidence has become available of increased metabolic activity in the caudal intralaminar nuclei in rodent models of PD. Finally, CM-Pf neurosurgery in patients suffering from PD has produced contrasting outcomes, indicating that a consensus is still to be reached regarding the potential usefulness of targeting the caudal intralaminar nuclei to treat movement disorders of basal ganglia origin.

Chronic lymphocytic leukemia (CLL) has few known modifiable risk factors. Recently, circadian disruption has been proposed as a potential contributor to lymphoid neoplasms' etiology. Serum melatonin levels have been found to be significantly lower in CLL subjects compared with healthy controls, and also, CLL prognosis has been related to alterations in the circadian molecular signaling. We performed the first investigation of an association between night shift work and CLL in 321 incident CLL cases and 1728 population-based controls in five areas of Spain. Participants were interviewed face-to-face by trained interviewers to collect information on sociodemographic factors, familial, medical and occupational history, including work shifts and other lifestyle factors. We used logistic regression models adjusted for potential confounders to estimate odds ratios (OR) and 95% confidence intervals (CI). Seventy-nine cases (25%) and 339 controls (20%) had performed night work. Overall, working in night shifts was not associated with CLL (OR = 1.06; 95% CI = 0.78-1.45, compared with day work). However, long-term night shift (>20 years) was positively associated with CLL (OR(tertile 3 vs . day-work) = 1.77; 95% = 1.14-2.74), although no linear trend was observed (P trend = 0.18). This association was observed among those with rotating (OR(tertile 3 vs . day-work) = 2.29; 95% CI = 1.33-3.92; P trend = 0.07), but not permanent night shifts (OR(tertile 3 vs . day-work) = 1.16; 95% CI = 0.60-2.25; P trend = 0.86). The association between CLL and long-term rotating night shift warrants further investigation.

<h3>Abstract</h3> Serum antibodies IgM and IgG are elevated during COVID-19 to defend against viral attack. Atypical results such as negative and abnormally high antibody expression were frequently observed whereas the underlying molecular mechanisms are elusive. In our cohort of 144 COVID-19 patients, 3.5% were both IgM and IgG negative whereas 29.2% remained only IgM negative. The remaining patients exhibited positive IgM and IgG expression, with 9.3% of them exhibiting over 20-fold higher titers of IgM than the others at their plateau. IgG titers in all of them were significantly boosted after vaccination in the second year. To investigate the underlying molecular mechanisms, we classed the patients into four groups with diverse serological patterns and analyzed their two-year clinical indicators. Additionally, we collected 111 serum samples for TMTpro-based longitudinal proteomic profiling and characterized 1494 proteins in total. We found that the continuously negative IgM and IgG expression during COVID-19 were associated with mild inflammatory reactions and high T cell responses. Low levels of serum IgD, inferior complement 1 activation of complement cascades, and insufficient cellular immune responses might collectively lead to compensatory serological responses, causing overexpression of IgM. Serum CD163 was positively correlated with antibody titers during seroconversion. This study suggests that patients with negative serology still developed cellular immunity for viral defense, and that high titers of IgM might not be favorable to COVID-19 recovery.

Glial cells are key players in the initiation of innate immunity in neurodegeneration. Upon damage, they switch their basal activation state and acquire new functions in a context and time-dependent manner. Since modulation of neuroinflammation is becoming an interesting approach for the treatment of neurodegenerative diseases, it is crucial to understand the specific contribution of these cells to the inflammatory reaction and to select experimental models that recapitulate what occurs in the human disease. Previously, we have characterized a region-specific activation pattern of CD11b+ cells and astrocytes in the α-synuclein overexpression mouse model of Parkinson´s disease (PD). In this study we hypothesized that the time and the intensity of dopaminergic neuronal death would promote different glial activation states. Dopaminergic degeneration was induced with two administration regimens of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), subacute (sMPTP) and chronic (cMPTP). Our results show that in the sMPTP mouse model, the pro-inflammatory phenotype of striatal CD11b+ cells was counteracted by an anti-inflammatory astrocytic profile. In the midbrain the roles were inverted, CD11b+ cells exhibited an anti-inflammatory profile and astrocytes were pro-inflammatory. The overall response generated resulted in decreased CD4 T cell infiltration in both regions. Chronic MPTP exposure resulted in a mild and prolonged neuronal degeneration that generated a pro-inflammatory response and increased CD4 T cell infiltration in both regions. At the onset of the neurodegenerative process, microglia and astrocytes cooperated in the removal of dopaminergic terminals. With time, only microglia maintained the phagocytic activity. In the ventral midbrain, astrocytes were the main phagocytic mediators at early stages of degeneration while microglia were the major phagocytic cells in the chronic state. In this scenario, we questioned which activation pattern recapitulates better the features of glial activation in PD. Glial activation in the cMPTP mouse model reflects many pathways of their corresponding counterparts in the human brain with advanced PD. Altogether, our results point toward a context-dependent cooperativity of microglia/myeloid cells and astrocytes in response to neuronal damage and the relevance of selecting the right experimental models for the study of neuroinflammation.

Immunophenotypic analysis of peripheral blood leukocytes from patients with multiple sclerosis (MS) showed a profile reflecting a state of activation and differentiation of monocytes. A subset of CD16+ monocytes with high HLA-DR expression was more prominent in patients with MS than in healthy subjects. The presence of the inducible form of nitric oxide synthase (iNOS) in these differentiated and activated monocytes freshly obtained from patients with MS was demonstrated by immunocytochemistry and flow cytometry analysis with two different antibodies. Incubation of lymphomononuclear cells from healthy volunteers in the presence of an immunomodulating peptide (NVLGAPKKLNESQAV) led to stimulation and maturation of monocytes manifested by changes in phenotype and an increase in both iNOS mRNA and protein, as well as HLA-DR expression. In this case also iNOS was expressed mainly on subsets of CD16+ monocytes with high HLA-DR expression. NO produced by human monocytes seems to have a function in the upregulation of membrane HLA-DR. These results are suggestive of a role for monocytic iNOS in the autoimmune response underlying the pathogenesis of multiple sclerosis.

The adenosine A(2A)-dopamine D(2) receptor heteromer is one of the most studied receptor heteromers. It has important implications for basal ganglia function and pathology. Recent studies using Bioluminescence and Sequential Resonance Energy Transfer techniques shed light on the role of Ca(2+) in the modulation of the quaternary structure of the A(2A)-D(2) receptor heteromer, which was found to depend on the binding of calmodulin (CaM) to the carboxy-terminus of the A(2A) receptor in the A(2A)-D(2) receptor heteromer. Importantly, the changes in quaternary structure correlate with changes in function. A Ca(2+)/CaM-dependent modulation of MAPK signaling upon agonist treatment could be observed in cells expressing A(2A)-D(2) receptor heteromers. These studies provide a first example of a Ca(2+)-mediated modulation of the quaternary structure and function of a receptor heteromer.

The external segment of the globus pallidus (GPe) in humans and the equivalent structure in rodents, the globus pallidus (GP), influence signal processing in the basal ganglia under normal and pathological conditions. Parvalbumin (PV) immunoreactivity defines 2 main neuronal subpopulations in the GP/GPe: PV-immunopositive cells that project mainly to the subthalamic nucleus and the internal segment of the GP and PV-negative cells that mainly project to the striatum. We evaluated the number of neurons in the GP/GPe in animal models of Parkinson disease. In rats, dopaminergic denervation with 6-hydroxydopamine (6-OHDA) provoked a significant decrease in the number of GP neurons (12% ± 4%, p < 0.05), which specifically affected the PV subpopulation. A similar trend was observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. Markers of GABAergic activity (GAD65 and GAD67 mRNA) were not different from those of controls in 6-OHDA-lesioned rats. Taken together, these findings provide evidence for nondopaminergic neuronal cell loss in the basal ganglia of 6-OHDA-lesioned rats and suggest that a similar loss may occur in the MPTP monkey. These data suggest that in patients with Parkinson disease, the loss of GABAergic neurons projecting to the subthalamic nucleus may contribute to the hyperactivity of this nucleus despite the absence of gross alterations in GAD mRNA expression.

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are regulators of normal B-cell development and survival. We investigated their role in chronic lymphocyticleukemia (CLL) by relating serum protein levels and CLL cell mRNA expression with clinical factors and disease progression. In patients with CLL, BAFF serum levels were significantly lower than in controls (0.64 ng/mL vs. 0.77 ng/mL, p = 0.014), and APRIL serum levels were significantly higher (4.10 ng/mL vs. 1.84 ng/mL, p = 0.041). CLL cells expressed BAFF and APRIL mRNA at lower levels than normal B-cells. Low BAFF serum levels were significantly correlated with a high blood lymphocyte count and advanced clinical stage, whereas APRIL levels were correlated with CD38 expression. In a multivariate analysis, the combined analysis of BAFF and APRIL serum levels emerged as an independent predictor of disease progression.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized with highly variable clinical course. The most common chromosomal abnormalities in CLL, using conventional and molecular cytogenetics, are trisomy 12, del(13)(q14), del(11)(q22–23), del(17)(p13), and del(6)(q21). Whereas the prognostic marker such as IGHV mutational status remains stable during course of the diseases, chromosomal aberrations may be acquired over time. The aim of this study was to determine the incidence, and biological significance of clonal evolution (CE) using conventional and molecular cytogenetics and its relationship with prognostic markers such as CD38, ZAP70, and the mutational status of IGHV and NOTCH1 . One hundred and forty‐three untreated CLL patients were included in the study. The median time interval between analyses was 32 months (range 6–156 months). Forty‐seven patients (33%) had CE as evidenced by detection of new cytogenetic abnormalities during follow‐up. CE was not correlated with high expression of ZAP70, unmutated IGHV genes or NOTCH1 mutations. Multivariate analysis revealed that CE and IGHV mutation status had a significant impact on TFS. The combination of conventional and molecular cytogenetics increased the detection of CE, this phenomenon probably being a reflection of genomic instability and conferring a more aggressive clinical course. © 2013 Wiley Periodicals, Inc.

Preventable risk factors for chronic lymphocytic leukemia (CLL) remain largely unknown. The aim of this study was to evaluate the association between adherence to nutrition-based guidelines for cancer prevention and CLL, in the MCC-Spain case–control study. A total of 318 CLL cases and 1293 population-based controls were included in the present study. The World Cancer Research Fund/American Institute for Cancer Research (WCRC/AICR) score based on the 2018 recommendations for cancer prevention (on body fatness, physical activity, and diet) was constructed. We used logistic regression analysis adjusting for potential confounders. Individuals in the highest tertile of the WCRF/AICR score had an odds ratio for CLL of 1.25 (95 % CI 0.91; 1.73) compared with individuals with low adherence (p-trend = 0.172). Each point increment in the score was associated with an OR for CLL of 1.06 (95 % CI 0.91; 1.23). Analyses by severity of disease did not show significant heterogeneity of effects. Overall, our results do not support an association between the WCRF/AICR score and CLL, yet we might have been limited by statistical power and study design to detect modest associations. Further research, ideally with a prospective design, long follow-up, and including additional lymphoma subtypes, is warranted to confirm the impact of composite healthy lifestyle behaviors on lymphoma risk.

Hereditary xerocytosis (HX) is a rare haemolytic disease due to dehydrated red blood cells (RBCs). A unique feature of this syndrome is that affected members often show normal or near normal haemoglobin levels despite clinical and laboratory evidence of mild to moderate haemolysis. The diagnostic clue is the association of markedly increased RBC Na+ + K+ fluxes with low total cation (Na+ + K+) content. 11 patients of six unrelated families of Spanish origin with HX have been studied from clinical, genetical and biological points of view. In addition, we have investigated the sensitivity of RBC membrane to heat at three different incubation times (15, 30 and 60 min) and two different temperature values (46 degrees C and 49 degrees C). Under these conditions control RBCs (50 normal subjects) exhibited at 49 degrees C and 30 min a maximum of 30% fragmented RBCs. This value increased to 80% after 60 min of incubation. In contrast, patients with HX showed significantly lower percentages of fragmented RBCs at both 30 and 60 min of incubation (maximum 10% and 30%, respectively). In an attempt to determine if increased heat stability was unique to HX RBCs, several other congenital membranopathies with haemolytic anaemia were also studied. The degree of fragmentation, except in one case of HPP (which was strongly increased), did not differ from the control group. Electrophoretic studies of membrane proteins performed in RBCs of all the patients with HX did not explain any qualitative nor quantitative abnormality. In addition to its physiopathological interest, study of RBC heat stability, together with other haematological parameters (increased MCHC and decreased RBC osmotic fragility), may be useful for HX diagnosis, especially in laboratories which are not equipped to evaluate RBC membrane permeability.

The fusion protein, promyelocytic leukemia-retinoic acid receptor (PML-RAR)alpha, generated by the t(15;17) translocation has an abnormal cellular distribution with colocalization of RARalpha and PML proteins. We analyzed the immunostaining pattern of PML protein using the PG-M3 monoclonal antibody directed against the amino terminal portion of PML (retained in wild-type PML and PML-RARalpha fusion protein) in the diagnosis of acute promyelocytic leukemia (APL). In addition, we compared this test with other methods for detecting the PML-RARalpha fusion gene. A normal immunostaining pattern was observed in nonmyeloid disorders and in 78 of 111 acute myeloid leukemias (AMLs). A microgranular pattern was observed in 25 AMLs, all corresponding to APL. These results were concordant with the reverse transcriptase-polymerase chain reaction results for PML-RARalpha fusion gene. Only 1 case positive for the PML-RARalpha transcript showed a normal protein pattern by immunocytochemistry. PML immunostaining was helpful to rapidly differentiate 7 cases with borderline characteristics and to obtain the diagnosis in 2 cases with scarce material. The effectiveness and low cost of this technique support its routine use as a first-line procedure in the differential diagnosis of AML.

Glial cell line-derived neurotrophic factor (GDNF) neuroprotective effect on dopaminergic neurons has been described in vitro and in vivo, turning up as a promising drug for the treatment of Parkinson's disease. Unglycosylated bacteria-obtained GDNF has already been successfully delivered for a long period of time through an infusion pump directly to the putamen of Parkinsonian patients. Nevertheless, improved distribution and safety issues need to be solved and alternative strategies to long-term delivery seem necessary. The use of glycosylated GDNF could eliminate some safety concerns regarding the presence of antibodies against exogenous unglycosylated GDNF used for the treatment. Therefore, we have chosen a mammalian expression system as a source of glycosylated GDNF. In the present work, we describe the purification of recombinant rat GDNF from the culture media of baby hamster kidney (BHK) cells through several purification steps. Highly pure N-glycosylated recombinant GDNF has been obtained similar to the endogenous protein. Furthermore, the purified protein is biologically active when tested its ability to induce PC12 neurite outgrowth.

Dendritic cells (DC) play a critical role in the regulation of alloimmune responses and might influence the outcome of allogeneic stem cell transplantation (allo-SCT). We studied the clinical relevance of early reconstitution of DC after reduced-intensity conditioning allo-SCT (allo-RIC).This study included 79 adult patients undergoing allo-RIC from HLA-identical siblings. Peripheral blood samples were drawn from patients at 1 month (+1m) and 3 months (+3m) after the transplant. DC were identified as positive for HLA-DR and negative for CD3, CD19, CD14 and CD56. The expression of CD33, CD123 and CD16 was used to identify myeloid DC, plasmacytoid DC and CD16(+) DC subpopulations, respectively.Patients whose DC count at +1m was lower than the median had a higher probability of treatment-related mortality (TRM) (60% vs 12%; p=0.02), poorer overall survival (OS) (15% vs 45%; p=0.002) and worse event-free survival (EFS) (20% vs 38%; p=0.03). A multivariate analysis confirmed that low DC counts had a detrimental effect on OS (RR 3.2; p=0.007), relapse (RR 4.1; p=0.01), and EFS (RR 6; p=0.001). Low CD16(+) DC counts were observed to have a detrimental effect on EFS, which was due to both a higher incidence of deaths caused by infections (50% vs 0%, p=0.05) and a higher incidence of relapse (57% vs 50%; p=0.03). Indeed, the number of CD16(+) DC at +3 m was the most important prognostic factor for EFS (RR 6; p=0.001). Interpretations and Conclusions This study shows the clinical importance of DC recovery, especially of the CD16(+) DC subset, in the outcome of patients treated with allo-RIC.

Long non-coding RNAs (lncRNAs) comprise a family of non-coding transcripts that are emerging as relevant gene expression regulators of different processes, including tumour development. To determine the possible contribution of lncRNA to the pathogenesis of follicular lymphoma (FL) we performed RNA-sequencing at high depth sequencing in primary FL samples ranging from grade 1-3A to aggressive grade 3B variants using unpurified (n = 16) and purified (n = 12) tumour cell suspensions from nodal samples. FL grade 3B had a significantly higher number of differentially expressed lncRNAs (dif-lncRNAs) with potential target coding genes related to cell cycle regulation. Nine out of the 18 selected dif-lncRNAs were validated by quantitative real time polymerase chain reaction in an independent series (n = 43) of FL. RP4-694A7.2 was identified as the top deregulated lncRNA potentially involved in cell proliferation. RP4-694A7.2 silencing in the WSU-FSCCL FL cell line reduced cell proliferation due to a block in the G1/S phase. The relationship between RP4-694A7.2 and proliferation was confirmed in primary samples as its expression levels positively related to the Ki-67 proliferation index. In summary, lncRNAs are differentially expressed across the clinico-biological spectrum of FL and a subset of them, related to cell cycle, may participate in cell proliferation regulation in these tumours.

Merkel cell polyomavirus (MCPyV) has been suspected to cause chronic lymphocytic leukaemia (CLL) but previous data are inconsistent. We measured seroreactivities of nine polyomaviruses (MCPyV, BKPyV, JCPyV, LPyV, KIPyV, WUPyV, HPyV-6, HPyV-7 and TSPyV) in 359 CLL cases and 370 controls using bead-based multiplex serology technology. We additionally tested two herpesviruses (HSV-1 and CMV). Associations between disease and viral seroreactivities were assessed using logistic regression. All human viruses showed high seroprevalences (69-99%) against structural proteins in controls but significantly lower viral seroprevalences in cases (58-94%; OR range = 0.21-0.70, P value < 0.05), except for MCPyV (OR = 0.79, 95% CI = 0.54-1.16). Lower seroreactivity levels were observed among CLL subjects, with significant differences already observed at early stages of disease, unrelated to treatment status. Seroreactivities against polyomavirus related oncoproteins were almost null. Our data suggest no association for MCPyV polyomavirus with CLL development and an unlikely association for other polyomaviruses tested.

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to be highly effective in patients with chronic lymphocytic leukemia (CLL) and is approved for CLL treatment. Unfortunately, resistance and intolerance to ibrutinib has been observed in several studies, opening the door for more specific BTK inhibitors. CC-292 (spebrutinib) is a BTK inhibitor with increased specificity for BTK and less inhibition of other kinases. Our in vitro studies showed that CC-292 potently inhibited B-cell receptor signaling, activation, proliferation and chemotaxis of CLL cells. In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC-292 reduced tumor load and normalized tumor-associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC-292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC-292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.

The presence of inducible nitric oxide synthase (iNOS) in fresh monocytes from patients with Graves'disease was demonstrated for the first time. Immunophenotypic analysis showed a profile reflecting a state of activation and differentiation of monocytes. Incubation of lymphomononuclear cells from healthy volunteers in the presence of synthetic peptides with sequences related to thyroid autoantigens (TSH receptor, thyroid peroxidase, or thyroglobulin) led to a stimulation of monocytes manifested by a change in phenotype and expression of iNOS. This expression did not take place on isolated monocytes, unless products associated with Th1 activity were present in the medium. Active peptides contained a characteristic “2-6-11” motif already described [López-Moratallaet al.(1995)Biochim. Biophys. Acta1265,181–188]. These results are suggestive of a new role for autoantigens in the pathogenesis of Graves’ disease: that of inducing the expression of iNOS and activating the monocyte possibly underlying the autoimmune response.

Acrolein is a potent fixative that provides both excellent preservation of ultrastructural morphology and retention of antigenicity, thus it is frequently used for immunocytochemical detection of antigens at the electron microscopic level. However, acrolein is not commonly used for fluorescence microscopy because of concerns about possible autofluorescence and destruction of the luminosity of fluorescent dyes. Here we describe a simple protocol that allows fine visualization of two fluorescent markers in 40-mum sections from acrolein-perfused rat brain. Autofluorescence was removed by pretreatment with 1% sodium borohydride for 30 min, and subsequent incubation in a 50% ethanol solution containing 0.3% hydrogen peroxide enhanced fluorescence labeling. Thus, fluorescence labeling can be used for high-quality detection of markers in tissue perfused with acrolein. Furthermore, adjacent acrolein-fixed sections from a single experiment can be processed to produce high-quality results for electron microscopy or fluorescence labeling.

The administration of glial cell line-derived neurotrophic factor (GDNF) has emerged as a promising strategy for the treatment of several diseases of the nervous system as Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury and nerve regeneration as well as ocular diseases and drug addictions. A procedure for the purification of human recombinant glycosylated GDNF using a mammalian expression system as the source of the protein is discussed in the present paper. The neurotrophic factor was purified using cation exchange chromatography and gel filtration. A human cell line was chosen as the source of therapeutic protein, since a recombinant protein with a structure and glycosylation pattern equivalent to the native form is desirable for its prospective therapeutic utilization. The activity of the highly pure protein obtained was confirmed with a cell-based bioassay. The purified protein is suitable for its in vivo evaluation in animals and for possible subsequent clinical application.

Human glial cell line-derived neurotrophic factor (hGDNF) is a very promising protein for the treatment of Parkinson's disease and other neurodegenerative disorders. The present work describes a quick and simple method to obtain a high amount of purified hGDNF using a mammalian cell-derived system. The method is based on the high expression level provided by a Semliki Forest virus vector and its ability to induce a strong shut-off of host-cell protein synthesis in mammalian cells. As a result, hGDNF is the only protein present in the supernatant and can be efficiently purified by a single chromatographic step. Using this system it was possible to eliminate other secreted proteins from the culture medium, like insulin-like growth factor-5, which are hard to remove using other hGDNF production methods. Purified hGDNF presents a complex glycosylation pattern typical of mammalian expression systems and is biologically active. This protocol could be extended to other secreted proteins and could be easily scaled up for industrial purposes.

To the editor: We have read with interest the letter by Baliakas et al[1][1] on the impact of MYD88 mutation in IGHV mutated (M-IGHV) chronic lymphocytic leukemia (CLL) patients. They reported a frequency of MYD88 -mutated patients of 4% in a series of 558 M-IGHV CLL, and found that M-IGHV CLL

Molecular studies of α‐thalassaemias have revealed defects at different steps in the process of α‐gene expression. It is not surprising, therefore, that in some cases a single mutation or small deletion can result in a structurally abnormal haemoglobin that produces the α‐thalassaemia phenotype. In this report we describe a new unstable α‐globin variant, Hb Lleida, in a Spanish patient with α‐thalassaemia trait. The mutation was detected by single‐strand conformation polymorphism in the third exon of the α 2 ‐globin gene. Direct sequence analysis of the α‐globin gene showed a 12 bp deletion as the only defect of the α 2 ‐ and α 1 ‐globin genes. The propositus was revealed to be a heterozygous carrier, and two alleles were separated by electrophoresis. This deletion causes the loss of four aminoacid residues (from codon 113 to 116) and would be expected to produce an unstable haemoglobin, as a shorter α‐globin chain variant is created with 137 amino acids instead of 141 amino acids present in a normal α‐globin chain. However, no abnormal haemoglobin was found by either isoelectric focusing or haemoglobin electrophoresis. Since the deletion affects an aminoacid residue (114 Pro) involved in α 1 ‐β 1 ‐globin chain contacts, the interaction required for efficient Hb assembly is also compromised. The resulting unstable α‐globin chain is rapidly catabolized and unsuitable for haemoglobin tetramer formation, causing an α‐thalassaemia trait phenotype in the heterozygous patient.

Neuroanatomical tracing when considered as an isolated method produces relatively straightforward answers. Although single-, double- or even triple-tracing paradigms produce valuable data on the organization of brain circuits, the final outcome often is too simplistic since it is not possible to elucidate the activity of these circuits. In this regard, emerging technologies contribute with additional information about the status of neuronal circuits. The laser-guided capture microdissection microscope (LCM) allows the accurate dissection of small brain areas under the microscope that could be further analyzed for gene expression or proteomics. In order to elucidate the gene expression of a given circuit of interest, we have developed a combination of methods comprising (i) fluorescent non-radioactive in situ hybridization for the detection of vGLUT2 mRNA expression combined with retrograde tracing with Fluoro-Gold (FG; analysis performed under the confocal microscope) and (ii) laser-guided capture microdissection of brain areas containing neurons retrogradely labeled with FG followed by the measurement of changes in mRNA levels encoding for vGLUT2 by real-time PCR. Our goal was to detect changes in gene expression of the thalamostriatal pathway in unilaterally 6-OHDA lesioned rats. Taking advantage of this procedure, we found a three-fold increase in vGLUT2 mRNA expression within thalamic neurons projecting to the dopamine-depleted striatum when compared with the activity of the thalamic neurons innervating the control striatum.

The proportion of dopamine D(2)(High) receptors has been reported to increase after cocaine self-administration without a significant change in the total number of D(2) receptors. In the present article, the data of competition by dopamine of [(3)H]domperidone binding to striatal samples from naïve and cocaine-addicted animals (Briand et al., [2008] Eur Neuropsychopharmacol 18:551-556) are analyzed assuming that D(2) receptors are constitutive dimers. The results show another way of interpreting those previous results and lead to the finding that cocaine affects agonist affinity and may strongly affect the receptor-receptor cooperation between dimers, a result that can contribute to dopamine supersensitivity.

Evidence suggesting a relationship between neuroendocrine and immune systems is steadily growing. We demonstrate now that inducible nitric oxide synthase (iNOS) is expressed in human peripheral blood monocytes after incubation of lymphomononuclear cells in the presence of β-endorphin, a neuropeptide released by the pituitary in response to mental or physical stress or by activated lymphocytes. β-endorphin raised cAMP level in monocytes. The possible relationship between cAMP and iNOS expression on monocytes was investigated. Immunostaining for iNOS decreased, when besides β-endorphin an inhibitor of protein kinase A (H-89) was added to the medium at the beginning of the incubation. The cAMP level raised by β-endorphin was lowered by naloxone, which also reduced slightly iNOS expression. These results clearly point to the monocyte as a link between neuroendocrine and immune systems, an observation of potential relevance in our understanding of how stress and autoimmunity could be interconnected.

Dendritic cells (DC) play a key role in initiating immune reactions after allogeneic stem cell transplantation. The two main peripheral blood DC populations are myeloid (DC1) and lymphoplasmacytoid (DC2). A new subset of myeloid DC, expressing CD16, has been identified. We analyzed the number and CD86 expression of DC subsets in peripheral blood of 18 healthy donors, before and after granulocyte colony-stimulating factor (G-CSF) and in the inoculum of allogeneic peripheral blood transplants (allo-PBT; n=100) and allogeneic bone marrow transplants (allo-BMT; n=22). Granulocyte colony-stimulating factor administration increased the median number of DC1 (P=0.0007), of DC2 (P<0.0001) and of DC CD16+ (P=0.0001). Granulocyte colony-stimulating factor administration was also associated with a significant decrease of CD86 expression on DC1 (P=0.0003) and with a trend for an increase on DC CD16+ (P=0.07). Recipients of allo-PBT received similar quantities of DC1 and higher doses of DC2 and DC CD16+ than recipients of allo-BMT (P=0.5; P=0.0001; P<0.0001, respectively). Granulocyte colony-stimulating factor modifies the number of DC in peripheral blood and the expression of the costimulatory molecule CD86. This resulted in a different composition of DC2 and especially of DC CD16+ in the harvests, which might explain some of the differences observed in allogeneic reactions after allo-PBT with respect to allo-BMT.

Clinical and metabolic studies were performed in four members of a Spanish family with partial (50%) 6 phosphogluconate dehydrogenase (6PGD) deficiency. In all cases the activities of 6 phosphogluconolactone (6PGL) and glutathione reductase (GR) were normal, and the molecular characterization performed in the partially purified 6PGD from the propositus showed normal kinetic and electrophoretic patterns. Two females (the propositus and her sister) suffered from a well-compensated chronic nonspherocytic hemolytic anemia (CNSHA) and exhibited decreased RBC glutathione (GSH) stability with increased oxidative susceptibility, defined by enhanced malonyldialdehyde (MDA) generation "in vitro." The other two members of the family (the propositus's mother and brother) were clinically asymptomatic. In the propositus and her sister, RBC metabolism exhibited a markedly abnormal concentration of glycolytic intermediates, mainly characterized by striking increases in fructose 1,6 bisphosphate (50-fold), dihydroxiacetone-phosphate (20-fold) and glyceraldehyde 3-phosphate (tenfold). Although the precise mechanism of the hemolysis in the two patients is unknown, the enhanced oxidative threat observed in their RBCs may interfere in some way with the glycolytic pathway function, leading to a marked increase in certain metabolic intermediates located before the glyceraldehyde 3 phosphate dehydrogenase (GA3PD) step. Since it seems that GA3PD half-life is modulated by fluctuations of the cytosolic redox status, an "in situ" approach was simulated by using permeabilized RBCs. In these conditions, GA3PD activity was significantly lower in the propositus and her sister than in the asymptomatic members of the family and the simultaneous normal control.

Several different deletions underlie the molecular basis of α-thalassemia. The most common α-thalassemia determinant in Spain is the rightward deletion (−α3.7). To our knowledge, however, no cases of α-thalassemia due to nondeletional mutations have so far been described in this particular Mediterranean area. Here, we report the existence of nondeletional forms of α-thalassemia in ten Spanish families. The α2-globin gene was characterized in ten unrelated patients and their relatives only when the presence of deletional α-thalassemia was ruled out. The α2-globin gene analysis was performed using the polymerase chain reaction (PCR) followed by restriction enzyme analysis or by allele-specific priming. This allowed the identification of a 5-base pair (bp) deletion at the donor site of IVS I (αHphα) in 9 cases and the α2 initiation codon mutation (αNcoα) in one case. Although these α2-globin gene mutations are found in other Mediterranean areas, our results demonstrate their presence in the Spanish population and suggest that the αHphα/αα genotype is probably the most common nondeletional form of α-thalassemia in Spain. © 1996 Wiley-Liss, Inc.

Early T-cell precursor lymphoblastic leukaemia (ETP-ALL), accounting for 5–16% of T-cell acute lymphoblastic leukaemia (T-ALL) cases, has been proposed as a provisional entity in the recent World Health Organization classification and is associated with higher treatment refractoriness and worse overall survival than T-ALL (Arber et al, 2016). The diagnosis is based on immunophenotypic features (CD7+, CD8−, CD1a−, CD5−/low with myeloid/stem cell marker expression) and the gene expression pattern resembles an immature haematopoietic progenitor or leukaemic stem cell. NOTCH1 and FBXW7 mutations are less frequent than in T-ALL (Zhang et al, 2012). As the cell origin of ETP-ALL has been related to a common myeloid precursor, some studies suggest better responses with acute myeloid leukaemia (AML)-type regimens (Patrick et al, 2014; Conter et al, 2016). Furthermore, targeted therapies, such as FLT3 or JAK inhibitors need to be explored in ETP-ALL, given the high frequency of mutations activating these pathways (Neumann et al, 2012; Maude et al, 2015). Herein we report four consecutive patients with ETP-ALL diagnosed at our centre over a 10-year period, focusing on the salvage therapy received after being refractory to standard chemotherapy (Table 1). FLT3-ITD wt RUNX1 mut (c.3188_3189insTCCC p.Gln1063fs) FLT3-ITD (21 bp) and RUNX1 mut (c.155_156insTATTCACC p.Met52fs) NOTCH, FBXW7, NRAS and KRAS wt PETHEMA LAL-AR-2003 Partial response PETHEMA LAL-AR-2011 Refractory PETHEMA LAL-AR-2011 MRD (+) response PETHEMA LAL-AR-2011 Refractory 1. FLAG-IDA ×2 MRD (−) CR 1. FLAG-IDA ×1 MRD (−) CR 1. FLAG-IDA ×1 MRD (−) CR 1. FLAG IDA 21 MRD (+) CR 2. HDAC + Sorafenib MRD (−) CR UCB – 4/6 match TBI/Cy/ATG Haploidentical TBI/Fludarabine Related donor TBI/Cy Related donor TBI/Cy No relapse Dead; +3 months (Infection) No relapse Dead; +6 months (Infection) No relapse Alive (14+ months) No relapse Alive (5+ months) Patient 1, a 31-year-old man, presented with a mediastinal mass and a white blood cell (WBC) count of 14 × 109/l (86% blasts, fulfilling ETP-ALL criteria), without central nervous system (CNS) involvement. The patient was enrolled in the Programa Español de Tratamientos en Hematología (PETHEMA) high-risk adult ALL (LAL-AR) 2003 regimen (4-week induction therapy with vincristine, daunorubicin 45 mg/m2, prednisone and L-asparaginase 10 000 iu/m2). However, at day +14, bone marrow (BM) showed 95% blasts; therefore the induction regimen was changed to mitoxantrone (12 mg/m2 × 3 doses) and high-dose cytarabine (HDAC, 2 g/m2 × 2), which reduced blasts to 23% at the end of induction. Salvage therapy with FLAG-IDA (fludarabine 25 mg/m2 × 4 daily, cytarabine 2 g/m2, idarubicin 10 mg/m2 × 3 doses) was administered and the patient achieved complete response (CR), without detectable minimal residual disease (MRD) by flow cytometry. The patient received a myeloablative allogeneic haematopoietic cell transplantation (alloHCT) with unrelated cord blood, but died from a toxoplasmic encephalitis, 4 months after transplant while in MRD-negative CR. Patient 2 was a 34-year-old man who presented with pancytopenia. WBC count was 3 × 109/l (70% blasts), fulfilling ETP-ALL criteria, and the patient started PETHEMA LAL-AR-2011 induction regimen (same scheme as PETHEMA LAL-AR-2003). BM evaluation on Day 14 showed 78% blasts, leading to modification of induction therapy with FLAG-IDA, which achieved a MRD-negative CR at Day +28. A myeloablative alloHCT from a haploidentical family donor was performed but the patient died from an infectious pneumonia after 6 months while in MRD-negative CR. Patient 3, a 39-year-old man, presented with a mediastinal mass and presence of 0·6% leukaemic cells, fulfilling ETP-ALL criteria, in the BM aspirate. Mutations in NOTCH1, FBXW7, PTEN, KRAS and NRAS were excluded. The patient started protocol PETHEMA LAL-AR-2011, with a reduction of the mediastinal mass but persistence of 0·06% blasts at Day +28. Therapy was changed to FLAG-IDA, resulting in a MRD-negative CR after one course. Following a myeloablative alloHCT from a human leucocyte antigen (HLA)-identical related donor, the patient has maintained a MRD-negative CR for 15 months. Patient 4 was a 33-year-old man who presented with pancytopenia. WBC count was 3·5 × 109/l (80% blasts, fulfilling ETP-ALL criteria). Molecular tests demonstrated the presence of a FLT3 internal tandem duplication (FLT3-ITD), with a FLT3-ITD/FLT3 wild type (wt) ratio of 0·08, a RUNX1 mutation and absence of mutations in NOTCH1, FBXW7, PTEN, KRAS and NRAS. The CNS was also involved, and the patient was started on the PETHEMA LAL-AR-2011 protocol and triple intrathecal chemotherapy. BM assessment at Day +14 showed 95% blasts and FLAG-IDA was initiated. After one cycle, a 2% ETP-ALL cell population persisted in BM, together with a FLT3-ITD/FLT3wt ratio of 0·03. Subsequently, an additional course of HDAC combined with the FLT3 inhibitor sorafenib (400 mg po bid) was given from day +8. The patient achieved MRD-negative status after 30 days, and sorafenib was maintained until a myeloablative HLA-matched sibling donor alloHCT was performed. Following transplant, sorafenib was reintroduced and the patient remains in MRD-negative CR at 6 months from alloHCT. These cases confirm the high rate of refractoriness to standard therapy reported previously (Coustan-Smith et al, 2009; Bond et al, 2017; Jain et al, 2017). Nevertheless, 3 patients achieved MRD-negative status with FLAG-IDA, whereas one required additional HDAC treatment with sorafenib. Interestingly, all patients maintained MRD-negative CR after transplantation, although two died due to a non-disease related cause and follow-up in the remaining patients is short. The optimal induction regimen for ETP-ALL has not yet been addressed, but given our observation, a prospective trial exploring response to FLAG-IDA is warranted. Although considered an early T-ALL subtype, the cell of origin in ETP-ALL is debatable. Moreover, the mutational profile of ETP-ALL shows mutations recurrently observed in AML, including histone-modifying genes, haematopoietic transcription factors and RAS signalling, whereas frequent gene mutations observed in T-ALL (NOTCH1/FBXW7 and PTEN or RAS) are lacking (Zhang et al, 2012; Bond et al, 2017). In addition, the ETP-ALL gene expression signature shares features with AML subtypes and some studies have shown good responses using myeloid-like regimens, such as conventional doses of cytarabine with 6-mercaptopurine and cyclophosphamide (Patrick et al, 2014; Conter et al, 2016). This background warrants exploration of AML-type regimens in clinical practice. Mutational screening can unravel potential therapy targets, such as FLT3-ITD. The benefit of FLT3 inhibitors has been demonstrated in AML with midostaurin and sorafenib. Based on this experience, we administered sorafenib to Patient 4, with a favourable response. Given that FLT3-ITD is highly infrequent in ALL, clinical use of other tyrosine kinase inhibitors according to previously-identified activating signalling mutations deserve investigation (Neumann et al, 2013). The role of alloHCT in ETP-ALL is controversial, with some reports suggesting a comparable outcome to other T-ALL subtypes for ETP-ALL patients who are submitted to alloHCT in CR (Bond et al, 2017). Nonetheless, the role of alloHCT for this entity, as well as transplant-related variables, should be specifically addressed in a multicentre setting. In conclusion, we report a poor response to initial chemotherapy in four ETP-ALL patients, who were successfully salvaged with IDA-FLAG regimen or HDAC plus sorafenib. Confirmation of this preliminary observation as well as the role of targeted agents should be prospectively addressed in well-designed trials. AB, MG and AO-C collected the clinical data. MLG, DC, MA, MC and NV-G performed the diagnostic tests. AB, M-DB and JE wrote the manuscript. All authors reviewed the manuscript. The authors declare that there is no conflict of interest regarding the publication of this manuscript.