Prokineticin System Is a Pharmacological Target to Counteract Pain and Its Comorbid Mood Alterations in an Osteoarthritis Murine Model

Osteoarthritis (OA) is the most prevalent joint disease associated with chronic pain. OA pain is often accompanied by mood disorders. We addressed the role of the Prokineticin (PK) system in pain and mood alterations in a mice OA model induced with monosodium iodoacetate (MIA). The effect of a PK antagonist (PC1) was compared to that of diclofenac. C57BL/6J male mice injected with MIA in the knee joint were characterized by allodynia, motor deficits, and fatigue. Twenty-eight days after MIA, in the knee joint, we measured high mRNA of PK2 and its receptor PKR1, pro-inflammatory cytokines, and MMP13. At the same time, in the sciatic nerve and spinal cord, we found increased levels of PK2, PKR1, IL-1β, and IL-6. These changes were in the presence of high GFAP and CD11b mRNA in the sciatic nerve and GFAP in the spinal cord. OA mice were also characterized by anxiety, depression, and neuroinflammation in the prefrontal cortex and hippocampus. In both stations, we found increased pro-inflammatory cytokines. In addition, PK upregulation and reactive astrogliosis in the hippocampus and microglia reactivity in the prefrontal cortex were detected. PC1 reduced joint inflammation and neuroinflammation in PNS and CNS and counteracted OA pain and emotional disturbances

468 glp compliant non clinical safety and biodistribution of a recombinant aav2 8 vector administered intravenously for treatment of mucopolysaccharidosis type vi

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disorder caused by deficiency of the enzyme arylsulfatase B (ARSB), which results in widespread accumulation and excretion of toxic glycosaminoglycans. We recently developed a successful gene therapy approach based on a single systemic administration of AAV2/8 that targets liver of MPS VI animal models. In view of a gene therapy clinical trial for MPS VI, we performed GLP-compliant non-clinical studies to assess the safety and biodistribution of AAV2/8. TBG. hARSB, a recombinant AAV2/8 vector encoding human ARSB (hARSB) under the control of the thyroxine-binding globulin promoter (TBG). We used transgenic C57/BL6-TgARSBC91S mice that overexpress an inactive hARSB C91S mutant and are thus immune tolerant to hARSB. Mice were treated with either AAV2/8.TBG. hARSB or the vehicle alone, as control. Toxicity was evaluated on day 15 (D15) and 180 (D180) after systemic injection of 2×1013 gc/kg, which is 10X the highest dose proposed for the clinical study [20males(M)+20females(F)/treatment/timepoint]. No mortality, abnormal clinical signs and alteration in body weight, body temperature and food intake were observed through the study. Similarly, no clinically relevant changes in blood chemistry and hematology were found in treated mice compared to controls. Histopathology revealed thyroid epithelial hypertrophy in AAV-treated mice. AAV2/8.TBG. hARSB biodistribution and expression was evaluated on D15 and D180 at the dose of 2×1012 gc/kg, which is 1X the highest dose proposed for the clinical study (5M+5F/treatment/timepoint). Although vector DNA was present in all organs on D15, it was sequestered mainly in liver at levels at least 3 logs higher than those found in other organs. Vector DNA declined on D180, but remained high in liver. Accordingly, hARSB was mainly expressed stably in liver, supporting TBG tissue specificity. Vector DNA was found in gonads of both sexes at 3 logs lower than in liver. A robust reduction of vector DNA was observed on D180. A supportive study conducted in male rabbits showed that vector shedding in semen was only transient, which suggests that the risk of inadvertent germline transmission of AAV2/8. TBG.hARSB is minimal at least in male animals. An in situ hybridization study is ongoing in ovaries to elucidate AAV localization. Finally, AAV DNA was only transiently present in plasma, urine and stools of mice (up to D37, D2 and D14, respectively), which minimizes the potential risk associated with transmission to third parties and/or the environment. In conclusion, these studies show a safe profile of intravenous administrations of AAV2/8. TBG.hARSB and pave the way for the phase I/II clinical trial

Concise Review: Chronic Myeloid Leukemia: Stem Cell Niche and Response to Pharmacologic Treatment

Nowadays, more than 90% of patients affected by chronic myeloid leukemia (CML) survive with a good quality of life, thanks to the clinical efficacy of tyrosine kinase inhibitors (TKIs). Nevertheless, point mutations of the ABL1 pocket occurring during treatment may reduce binding of TKIs, being responsible of about 20% of cases of resistance among CML patients. In addition, the presence of leukemic stem cells (LSCs) represents the most important event in leukemia progression related to TKI resistance. LSCs express stem cell markers, including active efflux pumps and genetic and epigenetic alterations together with deregulated cell signaling pathways involved in self-renewal, such as Wnt/β-catenin, Notch, and Hedgehog. Moreover, the interaction with the bone marrow microenvironment, also known as hematopoietic niche, may influence the phenotype of surrounding cells, which evade mechanisms controlling cell proliferation and are less sensitive or frankly resistant to TKIs. This Review focuses on the role of LSCs and stem cell niche in relation to response to pharmacological treatments. A literature search from PubMed database was performed until April 30, 2017, and it has been analyzed according to keywords such as chronic myeloid leukemia, stem cell, leukemic stem cells, hematopoietic niche, tyrosine kinase inhibitors, and drug resistance. Stem Cells Translational Medicine 2018

Unsupervised neural networks as a support tool for pathology diagnosis in MALDI-MSI experiments:A case study on thyroid biopsies

Artificial intelligence is getting a foothold in medicine for disease screening and diagnosis. While typical machine learning methods require large labeled datasets for training and validation, their application is limited in clinical fields since ground truth information can hardly be obtained on a sizeable cohort of patients. Unsupervised neural networks - such as Self-Organizing Maps (SOMs) - represent an alternative approach to identifying hidden patterns in biomedical data. Here we investigate the feasibility of SOMs for the identification of malignant and non-malignant regions in liquid biopsies of thyroid nodules, on a patient-specific basis. MALDI-ToF (Matrix Assisted Laser Desorption Ionization -Time of Flight) mass spectrometry-imaging (MSI) was used to measure the spectral profile of bioptic samples. SOMs were then applied for the analysis of MALDI-MSI data of individual patients' samples, also testing various pre-processing and agglomerative clustering methods to investigate their impact on SOMs' discrimination efficacy. The final clustering was compared against the sample's probability to be malignant, hyperplastic or related to Hashimoto thyroiditis as quantified by multinomial regression with LASSO. Our results show that SOMs are effective in separating the areas of a sample containing benign cells from those containing malignant cells. Moreover, they allow to overlap the different areas of cytological glass slides with the corresponding proteomic profile image, and inspect the specific weight of every cellular component in bioptic samples. We envision that this approach could represent an effective means to assist pathologists in diagnostic tasks, avoiding the need to manually annotate cytological images and the effort in creating labeled datasets

Hematopoietic reconstitution dynamics of mobilized- and bone marrow-derived human hematopoietic stem cells after gene therapy

Mobilized peripheral blood is increasingly used instead of bone marrow as a source of autologous hematopoietic stem/progenitor cells for ex vivo gene therapy. Here, we present an unplanned exploratory analysis evaluating the hematopoietic reconstitution kinetics, engraftment and clonality in 13 pediatric Wiskott-Aldrich syndrome patients treated with autologous lentiviral-vector transduced hematopoietic stem/progenitor cells derived from mobilized peripheral blood (n = 7), bone marrow (n = 5) or the combination of the two sources (n = 1). 8 out of 13 gene therapy patients were enrolled in an open-label, non-randomized, phase 1/2 clinical study (NCT01515462) and the remaining 5 patients were treated under expanded access programs. Although mobilized peripheral blood- and bone marrow- hematopoietic stem/progenitor cells display similar capability of being gene-corrected, maintaining the engineered grafts up to 3 years after gene therapy, mobilized peripheral blood-gene therapy group shows faster neutrophil and platelet recovery, higher number of engrafted clones and increased gene correction in the myeloid lineage which correlate with higher amount of primitive and myeloid progenitors contained in hematopoietic stem/progenitor cells derived from mobilized peripheral blood. In vitro differentiation and transplantation studies in mice confirm that primitive hematopoietic stem/progenitor cells from both sources have comparable engraftment and multilineage differentiation potential. Altogether, our analyses reveal that the differential behavior after gene therapy of hematopoietic stem/progenitor cells derived from either bone marrow or mobilized peripheral blood is mainly due to the distinct cell composition rather than functional differences of the infused cell products, providing new frames of references for clinical interpretation of hematopoietic stem/progenitor cell transplantation outcome.</p

Real-life Diagnostic and Therapeutic Approach to CLL: A New Proposal from an Expert Panel in Tuscany Region

BACKGROUND: In the last years genomic and somatic alterations have shown to play a pivotal role in the pathogenesis of chronic lymphocytic leukemia (CLL) and new prognostic factors have been identified accordingly.AIM: To describe a real-life diagnostic and therapeutic approach to CLL that takes into account the role of genomic and somatic prognostic factors in the risk stratification of developing progressive disease, and treatment decision.METHODS: This new proposal has been developed and validated by ten key opinion leaders from Tuscany Region during two Expert Meetings. The approach suggested comes from their experience in daily clinical practice and is supported by guidelines recommendations, clinical trials results, and drugs prescribing conditions in Italy.RESULTS: Beside TP53 deletion or mutated status, the Expert Panel highlighted the importance of the IGHV mutation status characterization, since the diagnosis, in order to identify patients who will have a more aggressive progression. Furthermore, just before starting treatment, to obtain useful prognostic information and indication in the selection of the therapy, they recommend cytogenetic analysis for the detection of del(11q), trisomy 12, del(13q), del(17p), conventional karyotyping of stimulated CLL cells, TP53 sequencing, and molecular genetic analysis to detect IGHV mutation status.CONCLUSIONS: The Expert Panel recognized the limitations associated with traditional staging systems in identifying patients who will have a more aggressive disease course and predicting response to treatment and suggested a real-life diagnostic and therapeutic approach to CLL to update the current patient management in light of recent advances that have improved understanding of CLL

Efficacy and safety of venous angioplasty of the extracranial veins for multiple sclerosis. Brave Dreams Study (Brain Venous Drainage Exploited Against Multiple Sclerosis): study protocol for a randomized controlled trial.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a disabling progressive course. Chronic cerebrospinal venous insufficiency (CCSVI) has recently been described as a vascular condition characterized by restricted venous outflow from the brain, mainly due to blockages of the internal jugular and azygos veins. Despite a wide variability among studies, it has been found to be associated with MS. Data from a few small case series suggest possible improvement of the clinical course and quality of life by performing percutaneous balloon angioplasty (PTA) of the stenotic veins.Study design and methodsThis is a multicenter, randomized, parallel group, blinded, sham-controlled trial to assess the efficacy and safety of PTA. Participants with relapsing remitting MS or secondary progressive MS and a sonographic diagnosis of CCSVI will be enrolled after providing their informed consent. Each participant will be centrally randomized to receive catheter venography and PTA or catheter venography and sham PTA. Two primary end points with respect to efficacy at 12 months are (1) a combined end point obtained through the integration of five functional indicators, walking, balance, manual dexterity, bladder control, and visual acuity, objectively measured by instruments; and (2) number of new brain lesions measured by T2-weighted MRI sequences. Secondary end points include annual relapse rate, change in Expanded Disability Status Scale score, proportion of patients with zero, one or two, or more than two relapses; fatigue; anxiety and depression; general cognitive state; memory/attention/calculus; impact of bladder incontinence; and adverse events. Six hundred seventy-nine patients will be recruited. The follow-up is scheduled at 12 months. Patients, treating neurologists, trained outcome assessors, and the statistician in charge of data analysis will be masked to the assigned treatment. DISCUSSION: The study will provide an answer regarding the efficacy of PTA on patients' functional disability in balance, motor, sensory, visual and bladder function, cognitive status, and emotional status, which are meaningful clinical outcomes, beyond investigating the effects on inflammation. In fact, an important part of patients' expectations, sustained and amplified by anecdotal data, has to do precisely with these functional aspects.Trial registrationClinicaltrials.gov NCT01371760