Delivery of PAMAM dendrimers and dendriplexes across natural barriers (blood-brain barrier and placental barrier) in healthy pregnant mice.

Gene therapy is an important tool for treating fetal diseases that allows for the delivery and integration of therapeutic genes into the genome of cells carrying mutations. Nanomolecules, like PAMAM dendrimers, have recently come into wider use for carrying vectors as they have several advantages over viral vectors. Namely, (1) tunable size and surface chemistry, (2) uniform size, (3) the ability to target specific tissues, and (4) the ability to carry large biomolecules and drugs. Recently, we demonstrated that 4th generation (G4) PAMAM dendrimer with a cystamine core and a non-toxic surface having 90% -OH and 10% -NH2 groups (D-Cys) could cross the blood-brain barrier following injection into the bloodstream. In the current study, as a proof of concept, we delivered the dendrimers alone (D-Cys) tagged with Cy5.5 (D-Cys-cy5.5) to healthy pregnant C57BL/6J mice to determine the fate of these dendrimers in the pregnant mice as well as in the fetus. Systematic diffusion of the D-Cys-cy5.5 was evaluated on gestational day 17 (3 days after injection) using in vivo imaging. This revealed that the dendrimer was taken up into circulation and away from the injection site. Analysis of sections by fluorescence microscopy showed that D-Cys-cy5.5 was able to successfully cross the maternal blood-brain barrier. However, analysis of the fetal brains failed to detect dendrimers in the central nervous system (CNS). Instead, they appeared to be retained in the placenta. This is one of the first studies to analyze the distribution of surface-modified PAMAM dendrimer in the pregnant mouse and fetus following systemic injection.

Characteristics and outcomes associated with CD2 and CD25 expression on bone marrow mast cells in patients with systemic mastocytosis.

Not available.

Scatter estimation and correction using time-of-flight and deconvolution in x-ray medical imaging.

Objective.Time-of-flight (TOF) scatter rejection requires a total timing jitter, including the detector timing jitter and the x-ray source's pulses width, of 50 ps or less to mitigate most of the effects of scattered photons in radiography and CT imaging. However, since the total contribution of the source and detector to the timing jitter can be retrieved during an acquisition with nothing between the source and detector, it can be demonstrated that this contribution may be partially removed to improve the image quality.Approach.A scatter correction method using iterative deconvolution of the measured time point-spread function estimates the number of scattered photons detected in each pixel. To evaluate the quality of the estimation, GATE was used to simulate the radiography of a water cylinder with bone inserts, and a head and torso in a system with total timing jitters from 100 ps up to 500 ps full-width-at-half-maximum (FWHM).Main results.With a total timing jitter of 200 ps FWHM, 89% of the contrast degradation caused by scattered photons was recovered in a head and torso radiography, compared to 28% with a simple time threshold method. Corrected images using the estimation have a percent root-mean square error between 2% and 14% in both phantoms with timing jitters from 100 to 500 ps FWHM which is lower than the error achieved with scatter rejection alone at 100 ps FWHM.Significance.TOF x-ray imaging has the potential to mitigate the effects of the scattering contribution and offers an alternative to anti-scatter grids that avoids loss of primary photons. Compare to simple TOF scatter rejection using only a threshold, the deconvolution estimation approach has lower requirements on both the source and detector. These requirements are now within reach of state-of-the-art systems.

Interactions between eosinophils and IL-5Rα-positive mast cells in nonadvanced systemic mastocytosis.

BACKGROUND: Bidirectional interactions between eosinophils and mast cells (MCs) have been reported in various allergic diseases. Bone marrow (BM) eosinophilia, and to a lesser extent blood eosinophilia, is common in systemic mastocytosis (SM), but its significance remains unknown. OBJECTIVE: We described blood and BM eosinophil characteristics in SM. METHODS: A large collection of BM biopsy samples was analyzed using immunohistochemical staining and whole-slide imaging. Eosinophil and extracellular granules were detected by eosinophil peroxidase (EPX) staining and MCs by KIT staining. Complementary analyses were conducted using flow cytometry and immunofluorescence. RESULTS: Eosinophil infiltrates and large areas of eosinophil degranulation were observed within or around BM MC infiltrates in SM. EPX staining surface, highlighting intact eosinophils and eosinophil degranulation, was higher in nonadvanced SM (n = 37 BM biopsy samples) compared with both controls (n = 8, P = .0003) and advanced SM (n = 24, P = .014). In nonadvanced SM, positive correlations were observed between serum tryptase levels and percentages of eosinophil counts in BM aspirations (Spearman r coefficient r = 0.38, P = .038), eosinophils count in BM biopsy samples (r = 0.45, P = .007), EPX staining (r = 0.37, P = .035), and eosinophil degranulation (r = 0.39, P = .023). Eosinophil counts in BM biopsy samples also correlated with MC counts (r = 0.47, P = .006) and KIT staining surface (r = 0.49, P = .003). BM MCs expressed IL-5 receptor and other usual eosinophil cytokine/chemokine receptors, and blood eosinophils displayed several increased surface markers compared with controls, suggesting an activated state. CONCLUSION: Our data suggest possible cross talk between MCs and eosinophils, supporting MC tryptase release and MC activation-related symptoms. This suggests a rationale for targeting eosinophils in nonadvanced SM not fully controlled by other therapies.

Presymptomatic Targeted Circuit Manipulation for Ameliorating Huntington's Disease Pathogenesis.

Early stages of Huntington's disease (HD) before the onset of motor and cognitive symptoms are characterized by imbalanced excitatory and inhibitory output from the cortex to striatal and subcortical structures. The window before the onset of symptoms presents an opportunity to adjust the firing rate within microcircuits with the goal of restoring the impaired E/I balance, thereby preventing or slowing down disease progression. Here, we investigated the effect of presymptomatic cell-type specific manipulation of activity of pyramidal neurons and parvalbumin interneurons in the M1 motor cortex on disease progression in the R6/2 HD mouse model. Our results show that dampening excitation of Emx1 pyramidal neurons or increasing activity of parvalbumin interneurons once daily for 3 weeks during the pre-symptomatic phase alleviated HD-related motor coordination dysfunction. Cell-type-specific modulation to normalize the net output of the cortex is a potential therapeutic avenue for HD and other neurodegenerative disorders.

Unilateral Administration of Surface-Modified G1 and G4 PAMAM Dendrimers in Healthy Mice to Assess Dendrimer Migration in the Brain.

Polyamidoamine (PAMAM) dendrimers are nanoparticles that have a wide scope in the field of biomedicine. Previous evidence shows that the generation 4 (G4) dendrimers with a 100% amine surface (G4-NH2) are highly toxic to cells in vitro and in vivo due to their positively charged amine groups. To reduce the toxicity, we modified the surface of the dendrimers to have more neutral functional groups, with 10% of the surface covered with -NH2 and 90% of the surface covered with hydroxyl groups (-OH; G4-90/10). Our previous in vitro data show that these modified dendrimers are taken up by cells, neurons, and different types of stem cells in vitro and neurons and glial cells in vivo. The toxicity assay shows that these modified dendrimers are less toxic compared with G4-NH2 dendrimers. Moreover, prolonged dendrimer exposure (G1-90/10 and G4-90/10), up to 3 weeks following unilateral intrastriatal injections into the striatum of mice, showed that dendrimers have the tendency to migrate within the brain via corpus callosum at different rates depending on their size. We also found that there is a difference in migration between the G1 and G4 dendrimers based on their size differences. The G4 dendrimers migrate in the anterior and posterior directions as well as more laterally from the site of injection in the striatum compared to the G1 dendrimers. Moreover, the G4 dendrimers have unique projections from the site of injection to the cortical areas.

Using microdialysis to monitor dopaminergic support of limb-use control following mesencephalic neurosphere transplantation in a rodent model of Parkinson's Disease.

Controlled nigrostriatal dopamine release supports effective limb use during locomotion coordination that becomes compromised after this pathway deteriorates in Parkinson's Disease (PD). How dopamine release relates to active ongoing behavior control remains unknown. Restoring proper release strategy appears important to successful PD treatment with transplanted dopamine-producing stem cells. This is suggested by apparently distinct behavioral support from tonic or phasic release and corresponding requirements of requisite afferent control exhibited by intact nigrostriatal neurons. Our laboratory previously demonstrated that transplanted dopaminergic cells can elicit skilled movement recovery known to depend on phasic dopamine release. However, efforts to measure this movement-related dopamine release yielded seemingly paradoxical, incongruent results. In response, here we explored whether those previous observations derived from rapid reuptake transport into either transplanted cells or residual, lesion-surviving terminals. We confirmed this using minimal reuptake blockade during intrastriatal microdialysis. After unilateral dopamine depletion, rats received transplants and were subjected to our swimming protocol. Among dopamine-depleted and transplanted rats, treatment supported restoration of limb movement symmetry. Interestingly, subsequent reuptake-restricted microdialysis confirmed distinct swimming-induced dopamine increases clearly occurred among these lesioned/transplanted subjects. Thus, phasic firing control appears to contribute to transplant-derived recovery in Parkinsonian animals.

Efficacy and safety of mammalian target of rapamycin inhibitors in systemic mastocytosis: A nationwide French pilot study.

Systemic mastocytosis (SM) corresponds to a rare and heterogeneous spectrum of diseases characterized by the accumulation of atypical mast cells (MCs). Advanced mastocytosis (Adv-SM) is associated with poor survival; in contrast, patients with non-advanced SM (non-Adv-SM) usually have a normal life expectancy but may experience poor quality of life. Despite recent therapeutic progress including tyrosine kinase inhibitors, new treatment options are needed for refractory and/or intolerant patients with both severely symptomatic and Adv-SM. In vitro, the mTOR pathway is activated in MCs from patients bearing the KIT D816V mutation. Furthermore, rapamycin induces the apoptosis of KIT D816V MCs selectively. In this nationwide study, we report the outcomes of patients diagnosed with SM and treated with a mammalian target of rapamycin inhibitor (imTOR) within the French National Reference Center for mastocytosis (CEREMAST). All patients registered were relapsing, treatment-refractory, or ineligible for other cytoreductive therapy. Non-Adv-SM patients received imTOR as a monotherapy (rapamycin/everolimus), and Adv-SM patients received imTOR as a monotherapy or in combination with cytarabine. The objective response rate (ORR) in non-Adv-SM was 60% (partial response in 40% and major response in 20%), including reductions in skin involvement, mediator release symptoms, and serum tryptase. In the Adv-SM group, the ORR was 20% (including one major response and one partial response, both in patients with a KIT D816V mutation), which enabled a successful bridge to allogeneic stem cell transplantation in one patient. Our results suggest that imTOR treatment has potential benefits in patients with SM harboring a KIT D816V mutation.

Comprehensive Genetic Profiling Reveals Frequent Alterations of Driver Genes on the X Chromosome in Extranodal NK/T-cell Lymphoma.

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm with male dominance and a poor prognosis. A better understanding of the genetic alterations and their functional roles in ENKTCL could help improve patient stratification and treatments. In this study, we performed a comprehensive genetic analysis of 178 ENKTCL cases to delineate the landscape of mutations, copy number alterations (CNA), and structural variations, identifying 34 driver genes including six previously unappreciated ones, namely, HLA-B, HLA-C, ROBO1, CD58, POT1, and MAP2K1. Among them, CD274 (24%) was the most frequently altered, followed by TP53 (20%), CDKN2A (19%), ARID1A (15%), HLA-A (15%), BCOR (14%), and MSN (14%). Chromosome X losses were the most common arm-level CNAs in females (∼40%), and alterations of four X-linked driver genes (MSN, BCOR, DDX3X, and KDM6A) were more frequent in males and females harboring chromosome X losses. Among X-linked drivers, MSN was the most recurrently altered, and its expression was lost in approximately one-third of cases using immunohistochemical analysis. Functional studies of human cell lines showed that MSN disruption promoted cell proliferation and NF-κB activation. Moreover, MSN inactivation increased sensitivity to NF-κB inhibition in vitro and in vivo. In addition, recurrent deletions were observed at the origin of replication in the EBV genome (6%). Finally, by integrating the 34 drivers and 19 significant arm-level CNAs, nonnegative matrix factorization and consensus clustering identified two molecular groups with different genetic features and prognoses irrespective of clinical prognostic factors. Together, these findings could help improve diagnostic and therapeutic strategies in ENKTCL. Significance: Integrative genetic analyses and functional studies in extranodal NK/T-cell lymphoma identify frequent disruptions of X-linked drivers, reveal prognostic molecular subgroups, and uncover recurrent MSN alterations that confer sensitivity to NF-κB inhibition.

Allogeneic haematopoietic cell transplantation for advanced systemic mastocytosis: Best practice recommendations on behalf of the EBMT Practice Harmonisation and Guidelines Committee.

Systemic Mastocytosis (SM) is a multifaceted clinically heterogeneous disease. Advanced SM (AdvSM) comprises three entities: aggressive SM (ASM), mast cell leukaemia (MCL) and SM with an associated hematologic neoplasm (SM-AHN), the latter accounting for 60-70% of all AdvSM cases. Detection of a disease-triggering mutation in the KIT gene (esp. KIT D816V) in >90% of the patients with ASM or SM-AHN has led to a significant improvement in therapeutic options by the implementation of two KIT-targeting kinase inhibitors: midostaurin and avapritinib. Although complete remissions have been reported, neither of these targeted agents is 'curative' in all patients and the duration of responses varies. The median overall survival, depending on the WHO subtype and scoring result, is approximately 1 to 4 years. Although the European Competence Network on Mastocytosis (ECNM) and American Initiative in Mast Cell Diseases (AIM) consensus groups recommend allogeneic haematopoietic cell transplantation (allo-HCT) in drug-resistant and other high-risk patients, there is a relative lack of information to guide clinicians on which patients with AdvSM should be considered for transplant, and how KIT inhibitors may fit into the transplant algorithm, including their use pre- and post-transplant to optimise outcomes. Following the generation of an expert panel with a specialist interest in allo-HCT and mastocytosis, these best practice recommendations were generated according to the European Society for Blood and Marrow Transplantation (EBMT) Practice Harmonisation and guidelines and ECNM methodology. We aim to provide a practical, clinically relevant and up-to-date framework to guide allo-HCT in AdvsM in 2024 and beyond.

Histological characterization of liver involvement in systemic mastocytosis.

BACKGROUND AND AIMS: Systemic mastocytosis (SM) is characterized by the accumulation of atypical mast cells (MCs) in organs. Liver histology of SM has been marginally described and accurate histological classification is critical, given the consequences of aggressive SM diagnosis. We aimed to describe the histological features associated with liver SM using updated tools. METHODS: Using the database of the French Reference Centre for Mastocytosis, we retrospectively identified patients with a liver biopsy (LB) and a diagnosis of SM. All LB procedures were performed according to the local physician in charge and centrally reviewed by an expert pathologist. RESULTS: A total of 28 patients were included: 6 had indolent SM, 9 had aggressive SM, and 13 had SM with an associated hematologic neoplasm. Twenty-five (89%) patients presented hepatomegaly, and 19 (68%) had portal hypertension. The LB frequently showed slight sinusoid dilatation (82%). Fibrosis was observed in 3/6 indolent SM and in almost all advanced SM cases (21/22), but none of them showed cirrhosis. A high MC burden (>50 MCs/high-power field) was correlated with elevated blood alkaline phosphatase levels (p = .030). The presence of portal hypertension was associated with a higher mean fibrosis grade (1.6 vs. 0.8 in its absence; p = .026). In advanced SM, the presence of nodular regenerative hyperplasia (NRH) was associated with decreased overall survival (9.5 vs. 46.3 months, p = .002). CONCLUSIONS: MC infiltration induced polymorphic hepatic lesions and the degree of fibrosis is associated with portal hypertension. NRH identifies a poor prognosis subgroup of patients with advanced SM. Assessing liver histology can aid in SM prognostic evaluation.

Inducible Knockout of 14-3-3ß Attenuates Proliferation and Spheroid Formation in a Human Glioblastoma Cell Line U87MG.

Glioblastomas (GBs) are the most common and malignant brain tumors in adults. A protein encoded by the gene YWHAB, 14-3-3ß, is commonly found to be upregulated throughout the initiation and progression of GB. The 14-3-3ß has oncogenic roles in several different types of cancer cells through interactions with proteins such as Bad, FBI1, Raf-1, Cdc25b, and others. Previous RNA interference studies have shown that 14-3-3ß promotes proliferation, cell cycle progression, and migration and invasion of GB cells. However, despite the many oncogenic functions of 14-3-3ß, a CRISPR/Cas9 knockout model of 14-3-3ß has not been investigated. This study confirmed previous findings and showed that siRNA inhibition of 14-3-3ß results in reduced cellular proliferation in a human glioblastoma cell line, U87MG. We also used a YWHAB Tet-On CRISPR/Cas9 U87MG cell line that, upon doxycycline induction, leads to robust Cas9 expression and subsequent knockout of 14-3-3ß. Using this model, we show that loss of 14-3-3ß significantly reduces cellular proliferation and spheroid formation of U87MG cells.

Correction: Bowers et al. Tart Cherry Extract and Omega Fatty Acids Reduce Behavioral Deficits and Gliosis in the 5xFAD Mouse Model of Alzheimer's Disease. Brain Sci. 2021, 11, 1423.

There is an error in the title of the published paper [...].

French guidelines for the etiological workup of eosinophilia and the management of hypereosinophilic syndromes.

Eosinophilic-related clinical manifestations are protean and the underlying conditions underpinning eosinophilia are highly diverse. The etiological workup of unexplained eosinophilia/hypereosinophilia can be challenging, and can lead sometimes to extensive, inappropriate, costly and/or invasive investigations. To date, guidelines for the etiological workup and management of eosinophilia are mainly issued by hematologists, and thus mostly cover the scope of clonal hypereosinophilic syndromes (HES). Here, thanks to an extensive literature review, and thanks to the joint work of a large panel of experts involving physicians from both adult and pediatric medicine and from various subspecialties (as well as a representative of a patients' association representative), we provide recommendations for both the step-by step diagnostic workup of eosinophilia (whether unexplained or within specific contexts) as well as the management and follow-up of the full spectrum of eosinophilic disorders (including clonal, reactive, lymphocytic and idiopathic HES, as well as single-organ diseases). Didactic prescription summaries intended to facilitate the prescription of eosinophil-targeted drugs are also provided, as are practical diagnostic and therapeutic algorithms. Lastly, this set of recommendations also includes a summary intended for general practitioners, as well as an overview of the therapeutic patient education program set up by the French reference center for HES. Further updates will be mandatory as new validated information emerges.

The Effectiveness and Safety of Pharmaceutical-Grade Cannabidiol in the Treatment of Mastocytosis-Associated Pain: A Pilot Study.

Mastocytosis patients often experience a number of symptoms, including mastocytosis-associated pain that is difficult to manage due to resistance to usual antalgic treatments and/or the patient's poor tolerance. Mastocytosis patients display significantly higher levels of indoleamine-2,3-dioxygenase-1 (IDO1) activity, leading to hyperactivation of the N-methyl-D-aspartate receptor. As cannabidiol (CBD) is known to inhibit IDO1's enzymatic activity, we hypothesized that pharmaceutical-grade CBD is an effective treatment for mastocytosis-associated pain. Patients with non-advanced mastocytosis and refractory pain were eligible for inclusion in this observational pilot study. CBD was initiated at 50 mg/day and increased to a maximum of 900 mg/day. Pain was scored on a 0-to-10 numerical rating scale (NRS). A total of 44 patients were included over a 2-year period. The median dose of CBD prescribed was 300 mg/day (range: 50-900 mg/day). Elevated liver enzymes were observed in one patient. The mean ± standard deviation NRS pain score decreased significantly from 7.27 ± 1.35 before treatment to 3.78 ± 1.99 after 3 months of treatment (p < 0.0001). Fifteen patients (34%) were able to discontinue all their previous antalgic medications. CBD treatment might be a safe, effective treatment for mastocytosis-associated pain and its use requires confirmation in a randomized, controlled trial.

Efficacy of a short sandwich protocol, methotrexate, gemcitabine, L-asparaginase and dexamethasone chemotherapy combined with radiotherapy, in localised newly diagnosed NK/T-cell lymphoma: A French retrospective study.

Extranodal NK/T-cell lymphoma, nasal type is a rare and aggressive form of lymphoma, historically associated with poor prognosis. We report here the results of a retrospective multi-centre study evaluating the efficacy of MGAD (methotrexate, gemcitabine, L-asparaginase and dexamethasone) regimen (two cycles) combined with 'sandwich' radiotherapy in 35 patients with localised newly diagnosed extranodal NK/T-cell lymphoma. Thirty-two patients (91%) reached complete remission. With a long median follow-up of 59.6 months, progression-free and overall survival at 2 and 5 years were 71%, 80% and 53%, 73%, respectively. Around one third of the patients experienced relapse within a median time of 14.5 months. Side-effects were manageable with grades 3-4 cytopenias, mucositis and infection in 50%, 24% and 21% of the cases, respectively. Monitoring of asparaginase activity was performed in 13 patients and showed inactivation of the drug in seven (54%) patients. Our results indicate that a short therapy by sandwich MGAD chemoradiotherapy is a tolerable and effective treatment option in localised newly diagnosed extranodal NK/T-cell lymphoma patients.

Reduced peripheral blood dendritic cell and monocyte subsets in MDS patients with systemic inflammatory or dysimmune diseases.

BACKGROUND: Systemic inflammatory and autoimmune diseases (SIADs) occur in 10-20% of patients with myelodysplastic syndrome (MDS). Recently identified VEXAS (Vacuoles, E1 enzyme, X-linked, Autoinflammatory, Somatic) syndrome, associated with somatic mutations in UBA1 (Ubiquitin-like modifier-activating enzyme 1), encompasses a range of severe inflammatory conditions along with hematological abnormalities, including MDS. The pathophysiological mechanisms underlying the association between MDS and SIADs remain largely unknown, especially the roles of different myeloid immune cell subsets. The aim of this study was to quantitatively evaluate peripheral blood myeloid immune cells (dendritic cells (DC) and monocytes) by flow cytometry in MDS patients with associated SIAD (n = 14, most often including relapsing polychondritis or neutrophilic dermatoses) and to compare their distribution in MDS patients without SIAD (n = 23) and healthy controls (n = 7). Most MDS and MDS/SIAD patients had low-risk MDS. Eight of 14 (57%) MDS/SIAD patients carried UBA1 somatic mutations, defining VEXAS syndrome.Compared with MDS patients, most DC and monocyte subsets were significantly decreased in MDS/SIAD patients, especially in MDS patients with VEXAS syndrome. Our study provides the first overview of the peripheral blood immune myeloid cell distribution in MDS patients with associated SIADs and raises several hypotheses: possible redistribution to inflammation sites, increased apoptosis, or impaired development in the bone marrow.