Preclinical assessment of antigen-specific chimeric antigen receptor regulatory T cells for use in solid organ transplantation.

A primary goal in transplantation medicine is the induction of a tolerogenic environment for prevention of transplant rejection without the need for long-term pharmacological immunosuppression. Generation of alloantigen-specific regulatory T cells (Tregs) by transduction with chimeric antigen receptors (CARs) is a promising strategy to achieve this goal. This publication reports the preclinical characterization of Tregs (TR101) transduced with a human leukocyte antigen (HLA)-A*02 CAR lentiviral vector (TX200) designated to induce immunosuppression of allograft-specific effector T cells in HLA-A*02-negative recipients of HLA-A*02-positive transplants. In vitro results demonstrated specificity, immunosuppressive function, and safety of TX200-TR101. In NOD scid gamma (NSG) mice, TX200-TR101 prevented graft-versus-host disease (GvHD) in a xenogeneic GvHD model and TX200-TR101 Tregs localized to human HLA-A*02-positive skin transplants in a transplant model. TX200-TR101 persisted over the entire duration of a 3-month study in humanized HLA-A*02 NSG mice and remained stable, without switching to a proinflammatory phenotype. Concomitant tacrolimus did not impair TX200-TR101 Treg survival or their ability to inhibit peripheral blood mononuclear cell (PBMC) engraftment. These data demonstrate that TX200-TR101 is specific, stable, efficacious, and safe in preclinical models, and provide the basis for a first-in-human study.

HITS-CLIP in various brain areas reveals new targets and new modalities of RNA binding by fragile X mental retardation protein.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the functional deficiency of the fragile X mental retardation protein (FMRP), an RNA-binding protein involved in translational regulation of many messenger RNAs, playing key roles in synaptic morphology and plasticity. To date, no effective treatment for FXS is available. We searched for FMRP targets by HITS-CLIP during early development of multiple mouse brain regions (hippocampus, cortex and cerebellum) at a time of brain development when FMRP is most highly expressed and synaptogenesis reaches a peak. We identified the largest dataset of mRNA targets of FMRP available in brain and we defined their cellular origin. We confirmed the G-quadruplex containing structure as an enriched motif in FMRP RNA targets. In addition to four less represented motifs, our study points out that, in the brain, CTGKA is the prominent motif bound by FMRP, which recognizes it when not engaged in Watson-Crick pairing. All of these motifs negatively modulated the expression level of a reporter protein. While the repertoire of FMRP RNA targets in cerebellum is quite divergent, the ones of cortex and hippocampus are vastly overlapping. In these two brain regions, the Phosphodiesterase 2a (Pde2a) mRNA is a prominent target of FMRP, which modulates its translation and intracellular transport. This enzyme regulates the homeostasis of cAMP and cGMP and represents a novel and attractive therapeutic target to treat FXS.

The KDEL receptor couples to Gαq/11 to activate Src kinases and regulate transport through the Golgi.

Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Gα(q/11) which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic.

Design and synthesis of a multivalent fluorescent folate-calix[4]arene conjugate: cancer cell penetration and intracellular localization.

A novel fluorescently labeled folate conjugate in which four folic acid units are covalently conjugated with a 7-nitro-benzofurazan fluorophore by means of a calix[4]arene platform was synthesized by using a Cu-catalyzed azide-alkyne cycloaddition reaction (click chemistry). The synthesized construct (FA-C4-NBD) was characterized by mass spectrometry, NMR and fluorescence spectroscopy. Confocal fluorescence microscopy experiments were carried out to evaluate the cell penetration ability of FA-C4-NBD on normal and cancer cells. The cellular uptake of FA-C4-NBD proceeds via folate receptor-mediated endocytosis. FA-C4-NBD is internalized into HeLa cancer cells which express high levels of folate receptors, whereas the uptake into fibroblast NIH3T3 cells which have very low expression levels of folate receptors is negligible. The involvement of the folate receptor was corroborated by competition tests with free folic acid. Co-localization analysis with different organelle markers indicated that FA-C4-NBD is not eliminated by recycling towards the outside of the cell, but accumulates intracellularly in the endo-lysosomal system.

Methionine sulfoxide reductase A down-regulation in human breast cancer cells results in a more aggressive phenotype.

Breast cancer is one of the most frequent of human malignancies, and it is therefore fundamental to identify the underlying molecular mechanisms leading to cancer transformation. Among other causative agents in the development of breast cancers, an important role for reactive oxygen species (ROS) has emerged. However, most studies on the role of ROS in cancer have not reached specific conclusions, and many issues remain controversial. In the present study, we show that methionine sulfoxide reductase A (MsrA), which is known to protect proteins from oxidation and which acts as a ROS scavenger, is down-regulated in a number of breast cancers. Moreover, levels of MsrA correlate with advanced tumor grade. We therefore investigated the functional role of MsrA in breast cancer cells. Our data show that reduction of MsrA levels results in increased cell proliferation and extracellular matrix degradation, and consequently in a more aggressive cellular phenotype, both in vivo and in vitro. We also show that the underlying molecular mechanisms involve increased ROS levels, resulting in reduction of phosphatase and tensin homolog deleted on chromosome ten protein (PTEN), and activation of the phosphoinositide 3-kinase pathway. In addition, MsrA down-regulation results in up-regulation of VEGF, providing additional support for tumor growth in vivo.

A Pilot Study on Early-Onset Schizophrenia Reveals the Implication of Wnt, Cadherin and Cholecystokinin Receptor Signaling in Its Pathophysiology.

Early-Onset Schizophrenia (EOS) is a very rare mental disorder that is a form of schizophrenia occurring before the age of 18. EOS is a brain disease marked by an early onset of positive and negative symptoms of psychosis that impact development and cognitive functioning. Clinical manifestations commonly include premorbid features of Autism Spectrum Disorder (ASD), attention deficits, Intellectual Disability (ID), neurodevelopmental delay, and behavioral disturbances. After the onset of psychotic symptoms, other neuropsychiatric comorbidities are also common, including obsessive-compulsive disorder, major depressive disorder, expressive and receptive language disorders, auditory processing, and executive functioning deficits. With the purpose to better gain insight into the genetic bases of this disorder, we developed a pilot project performing whole exome sequencing of nine trios affected by EOS, ASD, and mild ID. We carried out gene prioritization by combining multiple bioinformatic tools allowing us to identify the main pathways that could underpin the neurodevelopmental phenotypes of these patients. We identified the presence of variants in genes belonging to the Wnt, cadherin and cholecystokinin receptor signaling pathways.

Bilateral internal carotid artery occlusions in a pediatric patient with refractory acute myeloid leukemia.

Thromboembolism is a well-known complication of cancer including acute myeloid leukemia (AML) especially in patients with high myeloblast counts. However, spontaneous vaso-occlusion in the main arteries is very rare especially in patients with low blast counts and no pre existing vasculopathy. We report the case of a 3-year-old male with refractory AML who developed spontaneous bilateral internal carotid artery occlusion with diffuse cerebral infarcts. Strokes are rarely secondary to spontaneous carotid artery thrombosis and few cases have been reported in the literature.

Reduction of Fmr1 mRNA Levels Rescues Pathological Features in Cortical Neurons in a Model of FXTAS.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a rare disorder associated to the presence of the fragile X premutation, a 55-200 CGG repeat expansion in the 5' UTR of the FMR1 gene. Two main neurological phenotypes have been described in carriers of the CGG premutation: (1) neurodevelopmental disorders characterized by anxiety, attention deficit hyperactivity disorder (ADHD), social deficits, or autism spectrum disorder (ASD); and (2) after 50 years old, the FXTAS phenotype. This neurodegenerative disorder is characterized by ataxia and a form of parkinsonism. The molecular pathology of this disorder is characterized by the presence of elevated levels of Fragile X Mental Retardation 1 (FMR1) mRNA, presence of a repeat-associated non-AUG (RAN) translated peptide, and FMR1 mRNA-containing nuclear inclusions. Whereas in the past FXTAS was mainly considered as a late-onset disorder, some phenotypes of patients and altered learning and memory behavior of a mouse model of FXTAS suggested that this disorder involves neurodevelopment. To better understand the physiopathological role of the increased levels of Fmr1 mRNA during neuronal differentiation, we used a small interfering RNA (siRNA) approach to reduce the abundance of this mRNA in cultured cortical neurons from the FXTAS mouse model. Morphological alterations of neurons were rescued by this approach. This cellular phenotype is associated to differentially expressed proteins that we identified by mass spectrometry analysis. Interestingly, phenotype rescue is also associated to the rescue of the abundance of 29 proteins that are involved in various pathways, which represent putative targets for early therapeutic approaches.

The KDEL receptor signalling cascade targets focal adhesion kinase on focal adhesions and invadopodia.

Membrane trafficking via the Golgi-localised KDEL receptor activates signalling cascades that coordinate both trafficking and other cellular functions, including autophagy and extracellular matrix degradation. In this study, we provide evidence that membrane trafficking activates KDEL receptor and the Src family kinases at focal adhesions of HeLa cells, where this phosphorylates ADP-ribosylation factor GTPase-activating protein with SH3 domain, ankyrin repeat and PH domain (ASAP)1 and focal adhesion kinase (FAK). Previous studies have reported extracellular matrix degradation at focal adhesions. Here, matrix degradation was not seen at focal adhesions, although it occurred at invadopodia, where it was increased by KDEL receptor activation. This activation of KDEL receptor at invadopodia of A375 cells promoted recruitment and phosphorylation of FAK on tyrosines 397 and 861. From the functional standpoint, FAK overexpression inhibited steady-state and KDEL-receptor-stimulated extracellular matrix degradation, whereas overexpression of the FAK-Y397F mutant only inhibited KDEL-receptor-stimulated matrix degradation. Finally, we show that the Src and FAK activated downstream of KDEL receptor are part of parallel signalling pathways. In conclusion, membrane-traffic-generated signalling via KDEL receptor activates Src not only at the Golgi complex, but also at focal adhesions. By acting on Src and FAK, KDEL receptor increases invadopodia-mediated matrix degradation.

New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons.

Fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and a leading cause of autism, results from the loss of expression of the Fmr1 gene which encodes the RNA-binding protein Fragile X Mental Retardation Protein (FMRP). Among the thousands mRNA targets of FMRP, numerous encode regulators of ion homeostasis. It has also been described that FMRP directly interacts with Ca2+ channels modulating their activity. Collectively these findings suggest that FMRP plays critical roles in Ca2+ homeostasis during nervous system development. We carried out a functional analysis of Ca2+ regulation using a calcium imaging approach in Fmr1-KO cultured neurons and we show that these cells display impaired steady state Ca2+ concentration and an altered entry of Ca2+ after KCl-triggered depolarization. Consistent with these data, we show that the protein product of the Cacna1a gene, the pore-forming subunit of the Cav2.1 channel, is less expressed at the plasma membrane of Fmr1-KO neurons compared to wild-type (WT). Thus, our findings point out the critical role that Cav2.1 plays in the altered Ca2+ flux in Fmr1-KO neurons, impacting Ca2+ homeostasis of these cells. Remarkably, we highlight a new phenotype of cultured Fmr1-KO neurons that can be considered a novel cellular biomarker and is amenable to small molecule screening and identification of new drugs to treat FXS.

New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes.

Cytoplasmic FMRP interacting protein 1 (CYFIP1) is a candidate gene for intellectual disability (ID), autism, schizophrenia and epilepsy. It is a member of a family of proteins that is highly conserved during evolution, sharing high homology with its Drosophila homolog, dCYFIP. CYFIP1 interacts with the Fragile X mental retardation protein (FMRP, encoded by the FMR1 gene), whose absence causes Fragile X syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE regulatory complex (WRC), thus representing a link between translational regulation and the actin cytoskeleton. Here, we present data showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members, not only by post-translational mechanisms, as previously hypothesized. Moreover, we studied the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in two animal models - fly and mouse. We show that these two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, our results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction.

A Golgi-based KDELR-dependent signalling pathway controls extracellular matrix degradation.

We recently identified an endomembrane-based signalling cascade that is activated by the KDEL receptor (KDELR) on the Golgi complex. At the Golgi, the KDELR acts as a traffic sensor (presumably via binding to chaperones that leave the ER) and triggers signalling pathways that balance membrane fluxes between ER and Golgi. One such pathway relies on Gq and Src. Here, we examine if KDELR might control other cellular modules through this pathway. Given the central role of Src in extracellular matrix (ECM) degradation, we investigated the impact of the KDELR-Src pathway on the ability of cancer cells to degrade the ECM. We find that activation of the KDELR controls ECM degradation by increasing the number of the degradative structures known as invadopodia. The KDELR induces Src activation at the invadopodia and leads to phosphorylation of the Src substrates cortactin and ASAP1, which are required for basal and KDELR-stimulated ECM degradation. This study furthers our understanding of the regulatory circuitry underlying invadopodia-dependent ECM degradation, a key phase in metastases formation and invasive growth.

Clinical presentation of parvovirus B19 infection in children with aplastic crisis.

The records of 22 children with parvovirus B19-induced aplastic crisis were reviewed. The group consisted of 16 children with sickle cell hemoglobinopathies and 6 with hereditary spherocytosis. Children presented to the hospital 0.5 to 8 days (mean, 2.4 days) after the onset of symptoms. The children with sickle-cell disease presented earlier (mean, 1.4 days) than did children with hereditary spherocytosis (mean, 5 days; P = 0.02. Fever was the most common symptom, occurring in 73% of children. Rash did not occur in either group. Reticulocyte counts began to rise 1 week after onset of illness associated with a rise in parvovirus B19-specific IgG antibody. These data suggest that parvovirus B19 infection in children with sickle-cell hemoglobinopathies and heredity spherocytosis differs from infection in normal children.

Insertion of MLL sequences into chromosome band 5q31 results in an MLL-AF5Q31 fusion and is a rare but recurrent abnormality associated with infant leukemia.

MLL gene rearrangements leading to production of MLL fusion proteins are commonly detected in infant leukemia patients; the most common MLL fusion associated with infant leukemia is the MLL-AF4 fusion. A single case of chromosomal rearrangement leading to production of an MLL fusion with AF5Q31, a gene structurally similar to AF4, has been detected recently in the malignant cells of an infant leukemia patient. We have identified a second case of MLL-AF5Q31 fusion, arising from an insertion of MLL sequences into chromosome 5, also in an infant leukemia patient. Because MLL and AF5Q31 are transcribed in opposite orientations, a simple balanced chromosomal translocation cannot produce a fusion protein, and complex chromosomal rearrangements such as insertions and inversions are required to produce an MLL-AF5Q31 fusion protein. This report demonstrates that chromosomal insertion of MLL sequences is a rare but recurrent abnormality associated with infant leukemia.

Mycoplasma disease and acute chest syndrome in sickle cell disease.

BACKGROUND: Acute chest syndrome (ACS) is the leading cause of hospitalization, morbidity, and mortality in patients with sickle cell disease. Radiographic and clinical findings in ACS resemble pneumonia; however, etiologies other than infectious pathogens have been implicated, including pulmonary fat embolism (PFE) and infarction of segments of the pulmonary vasculature. The National Acute Chest Syndrome Study Group was designed to identify the etiologic agents and clinical outcomes associated with this syndrome. METHODS: Data were analyzed from the prospective study of 671 episodes of ACS in 538 patients with sickle cell anemia. ACS was defined as a new pulmonary infiltrate involving at least 1 complete segment of the lung, excluding atelectasis. In addition, the patients had to have chest pain, fever >38.5C, tachypnea, wheezing, or cough. Samples of blood and deep sputum were analyzed for evidence of bacteria, viruses, and PFE. Mycoplasma pneumoniae infection was determined by analysis of paired serologies. Detailed information on patient characteristics, presenting signs and symptoms, treatment, and clinical outcome were collected. RESULTS: Fifty-one (9%) of 598 episodes of ACS had serologic evidence of M pneumoniae infection. Twelve percent of the 112 episodes of ACS occurring in patients younger than 5 years were associated with M pneumoniae infection. At the time of diagnosis, 98% of all patients with M pneumoniae infection had fever, 78% had a cough, and 51% were tachypneic. More than 50% developed multilobar infiltrates and effusions, 82% were transfused, and 6% required assisted ventilation. The average hospital stay was 10 days. Evidence of PFE with M pneumoniae infection was seen in 5 (20%) of 25 patients with adequate deep respiratory samples for the PFE assay. M pneumoniae and Chlamydia pneumoniae was found in 16% of patients with diagnostic studies for C pneumoniae. Mycoplasma hominis was cultured in 10 (2%) of 555 episodes of ACS and occurred more frequently in older patients, but the presenting symptoms and clinical course was similar to those with M pneumoniae. CONCLUSIONS: M pneumoniae is commonly associated with the ACS in patients with sickle cell anemia and occurs in very young children. M hominis should be considered in the differential diagnosis of ACS. Aggressive treatment with broad-spectrum antibiotics, including 1 from the macrolide class, is recommended for all patients as well as bronchodilator therapy, early transfusion, and respiratory support when clinically indicated.

Regionalizacão da agricultura do Estado do Paraná, Brasil / Agriculture regionalization of Paraná State, Brazil

Este trabalho apresenta uma caracterizacão da agricultura do estado do Paraná com base em trinta e cinco variáveis extraídas do Censo Agropecuário de 1995/96 e duas variáveis extraídas do Mapa de Aptidão Agrícola das Terras. As mesmas dizem respeito ao acesso à terra, uso da terra, uso de tecnologias químicas e mecânicas, uso de capital, relacões de trabalho, terceirizacão, fertilidade natural dos solos e possibilidade de mecanizacão. A Análise Fatorial com trinta e sete variáveis permitiu descrever os sete fatores principais que explicaram 66,7 por cento da variância. A Análise de Agrupamento dos valores dos sete fatores permitiu classificar dez tipos de municípios, organizados em nove mesorregiões.

Estimativas das famílias potenciais beneficiárias de programas de assentamentos rurais no Brasil / Estimation of potential beneficiaries families of rural settlements in Brazil

Relata que há grande divergência, na literatura, sobre as famílias a serem beneficiadas por programas de política agrária no Brasil. Percebe que há uma convergência de que três grupos de famílias são prioritários: os de trabalhadores assalariados sem-terra, os de trabalhadores com acesso precário à terra, tais como os parceiros, arrendatários e posseiros, e os de proprietários-minifundistas que ocupam áreas abaixo do mínimo necessário para garantir a sobrevivência da família. Procura estimar o número potencial para cada um desses subgrupos de família, tendo o cuidado de identificar as diferenças conceituais entre as duas fontes e suas implicações. Relata que os resultados mostram que o público potencial beneficiário de ações fundiárias no Brasil varia em um intervalo entre 3,1 milhões e 6,4 milhões de famílias, o que depende da fonte e dos critérios utilizados. Observa que a maioria dessas famílias reside na região Nordeste, seguida pelas regiões Sudeste e Sul.