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Macrophage Fcgamma receptors (FcgammaRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites. We examined FcgammaR signaling and phagocytic function in bone marrow-derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn. Many FcgammaR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases 1 and 2. Significant reduction of IgG-dependent phagocytosis was not seen in hck(-)(/)-fgr(-)(/)- or lyn(-)(/)- cells, although the single mutant lyn(-)(/)- macrophages did manifest signaling defects. Thus, Src family kinases clearly have roles in two events leading to FcgammaR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization. Since FcgammaR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.

Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking. Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors.

Introduction: An understanding of the clinimetric properties of clinical assessments, including their constraints, is critical to sound clinical study and trial design. Utilizing data from Enroll-HD-a global, prospective HD observational study and clinical research platform-we examined several well-established HD clinical assessments across all stages of disease for evidence of instrument constraints, specifically floor/ceiling effects, to inform selection of appropriate instruments for use in future studies/trials and identify gaps in instrument utility over the life-course of the disease. Material and Methods: Analyzing publicly available data from 6,614 HD gene-expansion carriers (HDGECs), we grouped participants into deciles based on baseline CAP score, which ranged from 26 to 229. We used descriptive statistics to characterize data distribution for 25 outcome measures (encompassing motor, function, cognition, and psychiatric/behavioral domains) in each CAP decile. A skewness statistic threshold of ±2 was defined a priori to indicate floor/ceiling effects. Results: We found evidence of floor/ceiling effects in the early premanifest stages of disease for most motor and function assessments (e.g., TMS, TFC) and select cognitive tasks (MMSE, Trail Making tests). Other cognitive assessments, and the HADS-SIS scales, performed well ubiquitously, with no evidence of floor/ceiling effects at any disease stage. Floor/ceiling effects were evident at every disease stage for certain assessments, including PBA-s measures. Ceiling effects were apparent for DCL from onset stages onwards, as expected. Discussion: Developing instruments sensitive to subtle differences in performance at the earlier stages of the disease spectrum, particularly in motor and function domains, is warranted., Competing Interests: NG-K is member of the Enroll-HD Clinical Trials Committee, CHDI. KA received $2,000 payment for her involvement in the Enroll-HD Scientific Oversight Committee. She has also received travel funding to attending Enroll-HD SOC meetings, including those where this manuscript was planned and discussed. Through Georgetown University, KA also receives funding from CHDI as site principal investigator for the Georgetown MedStar Enroll-HD clinical site. CF-A was an employee at CHDI Management and was employed by company ClinMed LLC. BL has served on Scientific Advisory Boards for sRNAlytics, the Huntington's Disease Society of America, and the Huntington Society of Canada. JS is the chair of the Scientific Oversight Committee of Enroll-HD, for which she receives an annual stipend of $2,000 from CHDI Foundation. Through Monash University, JS also receives funding from CHDI as site principal investigator of Monash University/Calvary Health Care Bethlehem Hospital Enroll-HD clinical site. CS and JW are employed by CHDI Management, Inc. as advisors to CHDI Foundation, Inc. CHDI Foundation, Inc. is a non-profit biomedical research organization exclusively dedicated to collaboratively developing therapeutics that substantially improve the lives of individuals with Huntington's disease. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abreu, Ware, Georgiou-Karistianis, Leavitt, Fitzer-Attas, Lobo, Fernandes, Handley, Anderson, Stout and Sampaio.)

Background: Changes in phosphodiesterase 10A enzyme levels may be a suitable biomarker of disease progression in Huntington's disease., Objectives: To evaluate phosphodiesterase 10A PET imaging as a biomarker of HD progression using the radioligand, [ 18 F]MNI-659., Methods: The cross-sectional study (NCT02061722) included 45 Huntington's disease gene-expansion carriers stratified into four disease stages (early and late premanifest and Huntington's disease stages 1 and 2) and 45 age- and sex-matched healthy controls. The primary analysis compared striatal and pallidal phosphodiesterase 10A availability between Huntington's disease gene-expansion carriers and healthy controls as assessed by [ 18 F]MNI-659 binding. We assessed changes in phosphodiesterase 10A expression using several PET methodologies and compared with previously proposed measures of Huntington's disease progression (PET imaging of D 2/3 receptors and anatomical volume loss on MRI). The longitudinal follow-up study (NCT02956148) continued evaluation of phosphodiesterase 10A availability in 35 Huntington's disease gene-expansion carriers at a mean of 18 months from baseline of the cross-sectional study., Results: Primary analyses revealed that phosphodiesterase 10A availability in caudate, putamen, and globus pallidus was significantly lower in Huntington's disease gene-expansion carriers versus healthy controls across all stages. Striatal and pallidal phosphodiesterase 10A availability progressively declined in the premanifest stages and appeared to plateau between stages 1 and 2. The percentage decline of phosphodiesterase 10A availability measured cross-sectionally between Huntington's disease gene-expansion carriers and healthy controls was greater than that demonstrated by D 2/3 receptor availability or volumetric changes. Annualized rates of phosphodiesterase 10A change showed a statistically significant decline between the cross-sectional study and follow-up., Conclusions: [ 18 F]MNI-659 PET imaging is a biologically plausible biomarker of Huntington's disease progression that is more sensitive than the dopamine-receptor and volumetric methods currently used. © 2020 International Parkinson and Movement Disorder Society., (© 2020 International Parkinson and Movement Disorder Society.)

Phosphodiesterase 10A enzyme (PDE10A) is an important striatal target that has been shown to be affected in patients with neurodegenerative disorders, particularly Huntington´s disease (HD). PDE10A is expressed on striatal neurones in basal ganglia where other known molecular targets are enriched such as dopamine D 2/3 receptors (D 2/3 R). The aim of this study was to examine the availability of PDE10A enzyme in relation with age and gender and to compare those changes with those related to D 2/3 R and volumes in different regions of the basal ganglia. As a secondary objective we examined the relative distribution of D 2/3 R and PDE10A enzyme in the striatum and globus pallidus. Forty control subjects (20F/20M; age: 44±11y, age range 27-69) from an ongoing positron emission tomography (PET) study in HD gene expansion carriers were included. Subjects were examined with PET using the high-resolution research tomograph (HRRT) and with 3T magnetic resonance imaging (MRI). The PDE10A radioligand 18 F-MNI-659 and D 2/3 R radioligand 11 C-raclopride were used. The outcome measure was the binding potential (BP ND ) estimated with the two-tissue compartment model ( 18 F-MNI-659) and the simplified reference tissue model ( 11 C-raclopride) using the cerebellum as reference region. The PET data were corrected for partial volume effects. In the striatum, PDE10A availability showed a significant age-related decline that was larger compared to the age-related decline of D 2/3 R availability and to the age-related decline of volumes measured with MRI. In the globus pallidus, a less pronounced decline of PDE10A availability was observed, whereas D 2/3 R availability and volumes seemed to be rather stable with aging. The distribution of the PDE10A enzyme was different from the distribution of D 2/3 R, with higher availability in the globus pallidus. These results indicate that aging is associated with a considerable physiological reduction of the availability of PDE10A enzyme in the striatum. Moreover as result of the analysis, in the striatum for both the molecular targets, we observed a gender effect with higher BP ND the female group., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The Attenuation of Disease progression with Azilect GIven Once-daily (ADAGIO) delayed-start study demonstrated a benefit of early-start treatment with rasagiline 1 mg/day versus delayed-start treatment in PD. This follow-up study aimed to assess whether these benefits persist and the clinical progression rate during long-term naturalistic treatment., Methods: The ADAGIO Follow-Up study was initiated approximately 26 months after completion of the ADAGIO study. Patients were followed for 3 years and were treated in an open-label manner with rasagiline 1 mg/day and any other PD treatment that was deemed appropriate. Changes from follow-up baseline to study end in UPDRS scores, and the emergence of clinical milestones (including unsteady gait and/or balance impairment, falls, freezing of gait, and cognitive decline) were assessed., Results: The study enrolled 683 patients (58% of the full ADAGIO cohort and 72% of ADAGIO completers). At baseline, mean time from diagnosis was 46.9 months and UPDRS total score was 25.6 units. There were no significant differences in UPDRS total or subscale scores or time to any milestone between patients who were in the original ADAGIO early-start group versus those in the delayed-start group. At study end, patients (total cohort) had worsened by a mean ± standard deviation of 6.0 ± 11.6 UPDRS total units, 3.3 ± 8.6 UPDRS motor units and 2.0 ± 4.0 UPDRS activities of daily living (ADL) units. Overall, 43.6% of patients had onset of unsteady gait/balance impairment, 35.7% had fallen, 26.2% had freezing of gait, and 33.1% had cognitive decline., Conclusions: The ADAGIO Follow-Up study failed to demonstrate long-term benefits of early-start rasagiline treatment in the prior ADAGIO study. Clinically important milestones occurred in a substantial proportion of patients. © 2016 International Parkinson and Movement Disorder Society., (© 2016 International Parkinson and Movement Disorder Society.)

Background: The study of complex neurodegenerative diseases is moving away from hypothesis-driven biological methods toward large scale multimodal approaches, requiring standardized collaborative efforts. Enroll-HD exemplifies such an integrated clinical research platform, designed and implemented to meet the research and clinical needs of Huntington's disease (HD). The aim of this study was to describe the unique organization of Enroll-HD and report baseline data analyses of its core study., Methods: The Enroll-HD platform incorporates electronic data capture, biosampling, and a longitudinal observational study spanning four continents (ClinicalTrials.gov Identifier: NCT01574053). The primary study population includes HD gene expansion carriers (HDGECs; CAG expansion ≥36), subdivided into manifest/premanifest HD. The control population consists of genotype-negative first-degree relatives and family controls not genetically related. The study includes 10 core clinical assessments covering motor, cognitive, and behavioral domains., Results: This data set comprises 1,534 participants (HDGEC = 1,071; controls = 463). Participant retention was high; 42 participants prematurely withdrew from the study. Mean ± standard deviation SD CAG repeat size was 43.5 ± 3.5 for HDGECs and 19.8 ± 3.4 for controls. Motor and behavioral assessments identified numerical differences between controls and HDGECs (manifest > premanifest > controls). Functional and independence assessments were generally similar for the premanifest and control groups with overlap in range of scores obtained. For the majority of cognitive tests, there were large differences between participants with manifest HD and all other groups., Conclusions: These first data from the Enroll-HD clinical research platform demonstrate the maturity and potential of the platform in collecting high-quality, clinically relevant data. Future data sets will be substantially larger as the platform expands longitudinally and regionally.

Background: Multiple system atrophy is a complex neurodegenerative disorder for which no effective treatment exists. We aimed to assess the effect of rasagiline on symptoms and progression of the parkinsonian variant of multiple system atrophy., Methods: We did this randomised, double-blind, placebo-controlled trial between Dec 15, 2009, and Oct 20, 2011, at 40 academic sites specialised in the care of patients with multiple systemic atrophy across 12 countries. Eligible participants aged 30 years or older with possible or probable parkinsonian variant multiple system atrophy were randomly assigned (1:1), via computer-generated block randomisation (block size of four), to receive either rasagiline 1 mg per day or placebo. Randomisation was stratified by study centre. The investigators, study funder, and personnel involved in patient assessment, monitoring, analysis and data management were masked to group assignment. The primary endpoint was change from baseline to study end in total Unified Multiple System Atrophy Rating Scale (UMSARS) score (parts I and II). Analysis was by modified intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00977665., Findings: We randomly assigned 174 participants to the rasagiline group (n=84) or the placebo group (n=90); 21 (25%) patients in the rasagiline group and 15 (17%) in the placebo group withdrew from the study early. At week 48, patients in the rasagiline group had progressed by an adjusted mean of 7·2 (SE 1·2) total UMSARS units versus 7·8 (1·1) units in those in the placebo group. This treatment difference of -0·60 (95% CI -3·68 to 2·47; p=0·70) was not significant. 68 (81%) patients in the rasagiline group and 67 (74%) patients in the placebo group reported adverse events, and we recorded serious adverse events in 29 (35%) versus 23 (26%) patients. The most common adverse events in the rasagiline group were dizziness (n=10 [12%]), peripheral oedema (n=9 [11%]), urinary tract infections (n=9 [11%]), and orthostatic hypotension (n=8 [10%])., Interpretation: In this population of patients with the parkinsonian variant of multiple system atrophy, treatment with rasagiline 1 mg per day did not show a significant benefit as assessed by UMSARS. The study confirms the sensitivity of clinical outcomes for multiple system atrophy to detect clinically significant decline, even in individuals with early disease., Funding: Teva Pharmaceutical Industries and H Lundbeck A/S., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The ADAGIO study investigated whether rasagiline has disease-modifying effects in Parkinson's disease. Rasagiline 1 mg per day, but not 2 mg per day, was shown to be efficacious in the primary analysis. Here, we report additional secondary and post-hoc analyses of the ADAGIO study., Methods: ADAGIO was a placebo-controlled, double-blind, multicentre, delayed-start study, in which 1176 patients with untreated early Parkinson's disease were randomly assigned to receive rasagiline 1 mg or 2 mg per day for 72 weeks (early-start groups) or placebo for 36 weeks followed by rasagiline 1 mg or 2 mg per day for 36 weeks (delayed-start groups). We assessed the need for additional antiparkinsonian therapy and changes in non-motor experiences of daily living and fatigue scales (prespecified outcomes) and changes in unified Parkinson's disease rating scale (UPDRS) scores and subscores in placebo and active groups (post-hoc outcomes). The ADAGIO study is registered with ClinicalTrials.gov, number NCT00256204., Findings: The need for additional antiparkinsonian therapy was reduced with rasagiline 1 mg (25 of 288 [9%] patients) and 2 mg (26 of 293 [9%]) versus placebo (108 of 593 [18%]; odds ratio for 1 mg rasagiline vs placebo 0·41, 95% CI 0·25-0·65, p=0·0002; 2 mg rasagiline vs placebo 0·41, 0·26-0·64, p=0·0001). At week 36, both doses significantly improved UPDRS motor subscores compared with placebo (1 mg rasagiline mean difference -1·88 [SE 0·35]; 2 mg rasagiline -2·18 [0·35]; both p<0·0001) and activities of daily living subscores (ADL; 1 mg rasagiline -0·86 [0·18]; 2 mg rasagiline -0·88 [0·18]; both p<0·0001), and 1 mg rasagiline significantly improved UPDRS mentation subscore (-0·22 [0·08]; p=0·004). At week 72, the only significant difference between early-start and delayed-start groups was for ADL subscore with the 1 mg dose (-0·62 [0·29]; p=0·035). When assessed for the effect on non-motor symptoms at week 36, both doses showed benefits on the Parkinson fatigue scale versus placebo (1 mg rasagiline mean difference -0·14 [SE 0·05], p=0·0032; 2 mg rasagiline -0·19 [0·05], p<0·0001), and the 1 mg dose showed benefits on the scale for non-motor experiences of daily living compared with placebo (mean difference -0·33 [0·17]; p=0·049). The rate of progression of total UPDRS score for patients in the placebo group was 4·3 points [SE 0·3] over 36 weeks, with extrapolation to about 6 units per year. In the placebo group, patients with the lowest quartile of baseline UPDRS scores (≤14; n=160) progressed more slowly than did those with highest scores (>25·5; n=145; mean difference -3·46 [SE 0·77]; p<0·0001)., Interpretation: These findings show that rasagiline delayed the need for symptomatic antiparkinsonian drugs and emphasise the contribution of the UPDRS ADL in the response of the rasagiline 1 mg per day early-start versus delayed-start group. The rate of UPDRS deterioration was less than was anticipated from previous studies and correlated with baseline severity. Understanding of the pattern of UPDRS deterioration is essential to assess disease modification., Funding: Teva Pharmaceutical Industries and H Lundbeck A/S., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study was designed to follow the long-term efficacy, safety, and tolerability of rasagiline for Parkinson's disease (PD) with data collected from all patients who had ever taken rasagiline during the 12-month TEMPO monotherapy trial (N = 398) and subsequent open-label extension. Patients were followed for up to 6.5 years with a mean of 3.5 +/- 2.1 years. After 12 months, additional PD medications were added as required. Of patients remaining in the trial at 2 years, 46% were maintained on rasagiline monotherapy. The majority of patients received a dopamine agonist prior to levodopa as the first additional dopaminergic agent. Analysis using a Kaplan-Meier method indicated that by 5.4 years only 25% of patients progressed to Hoehn & Yahr stage III. Rasagiline was well tolerated, with 11.3% of patients (45/398) withdrawing because of an adverse event. Rasagiline therapy for PD was effective, well tolerated, and safe in this long-term trial.

The purpose of this study to compare the long-term clinical outcome of early versus delayed rasagiline treatment in early Parkinson's disease (PD). Subjects (N = 404) were randomly assigned to initial treatment with rasagiline (early-start group) or placebo for 6 months followed by rasagiline (delayed-start group) in the TEMPO study. Subjects who chose to participate in an open-label extension (N = 306) continued to receive rasagiline as well as other PD medications as needed. Average (+/-SD) duration in the study was 3.6 +/- 2.1 years; 177 subjects received rasagiline for > or =5.0 years. Over the entire 6.5-year follow-up period, the adjusted mean difference in change from baseline in total UPDRS scores was 2.5 units (SE 1.1; P = 0.021) or 16% (SE 5.7; P = 0.006) in favor of the early-start versus delayed-start rasagiline group. Although the interaction between treatment and time was significant, values for the early-start group were better than the delayed-start group across all time points. Significantly less worsening (percent change) in total UPDRS scores was observed in the early-start group at the time points 0.5, 1.5, 2.0, 3.0, 4.5, 5.0, and 5.5 years (P < 0.05). Compared to delayed start, early initiation of rasagiline provided long-term clinical benefit, even in the face of treatment with other dopaminergic agents. This might reflect enduring benefits due to neuroprotection or effects on compensatory mechanisms in early PD.

Protein tyrosine kinases (PTKs) transmit activation signals in almost every cell type, including immune effector cells. The aberrant or constitutive activation of PTKs can often cause neoplastic transformation. The use of chimeric receptors based on PTKs may enable us to elucidate the signaling pathways of normal immune cells and other cell types, and the abnormal events that can lead to malignant transformation. In this review, we focus on antigen specific chimeric PTKs in which antibody-derived scFv are joined to the Syk family of PTKs. These chimeric receptors yielded reagents that can selectively redirect immune effector cells and specifically activate them to produce cytokines or lyse their target. The advantages of using such PTK-based chimeras to redirect lymphocytes to tumor targets and their potential as an immunotherapeutic approach to malignant disease is discussed.

T cells of tumor bearers often show defective TCR-mediated signaling events and, therefore, exhibit impaired immune responses. As such, patients with heavy tumor burden are often not amenable to adoptive T cell therapy. To overcome this limitation, we have developed a chimeric receptor that joins an extracellular single chain Fv (scFv) of a specific Ab for Ag recognition to an intracellular protein tyrosine kinase (PTK) for signal propagation. Stimulation through the scFv-PTK receptor should bypass defective TCR-proximal events and directly access the T cell's effector mechanisms. In this study we describe the optimization of a scFv-PTK configuration, leading to complete T cell activation. The cytosolic PTK Syk is superior to its family member, Zap-70, for intracellular signaling. As a transmembrane (TM) domain, CD4 performs better than CD8 when plastic-immobilized Ag serves as a stimulator. However, when APC are used to trigger chimeric receptors, the need for a flexible spacer between the scFv and TM domains becomes apparent. The CD8alpha-derived hinge successfully performs this task in chimeric scFv-Syk receptors regardless of its cysteine content. A cytotoxic T cell hybridoma expressing chimeric receptor genes composed of scFv-CD8(hinge)-CD8(TM)-Syk or scFv-CD8(hinge)-CD4(TM)-Syk is efficiently stimulated to produce IL-2 upon interaction with APC and specifically lyses appropriate target cells in a non-MHC-restricted manner.

Functional PDGFalpha receptors are selectively expressed on highly lung-metastasizing clones of the 3LL Lewis lung carcinoma, but not on low-mestastatic clones. The highly metastatic clones are also growth induced in vitro by PDGF and lung conditioned medium. To investigate whether modification of PDGFalpha receptor expression or function can affect metastatic capability, we transfected cells of a low-metastatic 3LL clone with a full length PDGFalpha receptor gene and cells of a highly-metastatic clone with a truncated kinase domain PDGFalpha receptor gene. Introduction of the full length PDGFalpha receptor conferred upon low-metastatic cells the ability to grow in vitro in the presence of PDGF-AA and to colonize the lung in experimental and spontaneous metastases assays. Conversely, introduction of a truncated version of the PDGFalpha receptor into highly metastatic cells reduced their metastatic load to control levels. Accordingly, their responses to PDGF-AA, including growth stimulation and receptor autophosphorylation, were reduced. These results demonstrate that PDGFalpha receptor expression and function can control the capacity of tumor cells to generate metastases in the lung. The response of this receptor to lung-derived PDGF-like factors may define a paracrine mode of metastatic cell growth in the target organ.

The protein tyrosine kinase Syk is activated upon engagement of immune recognition receptors. We have focused on the identification of signaling elements immediately downstream to Syk in the pathway leading to T cell activation. To circumvent T cell receptor (TCR). CD3 activation of Src family kinases, we constructed a signaling molecule with an extracellular single chain Fv of an anti-TNP antibody, attached via a transmembrane region to Syk (scFv-Syk). In a murine T cell hybridoma, direct aggregation of chimeric Syk with antigen culminates in interleukin-2 production and target cell lysis. Initially, it causes an increase in the association between scFv-Syk and the cytosolic protein Cbl and subsequently promotes tyrosine phosphorylation of Cbl. Interestingly, although both Cbl and phospholipase C-gamma (PLC-gamma) are phosphorylated in this hybridoma upon TCR.CD3 cross-linking, these two events are uncoupled in scFv-Syk-transfected cells, in which we were unable to detect antigen-driven PLC-gamma phosphorylation. These results support a model in which Syk can initiate and directly activate the T cell's signaling machinery and position Cbl as a primary tyrosine kinase substrate in this pathway. Furthermore, for efficient PLC-gamma phosphorylation to occur in these cells, the combined actions of different tyrosine kinase families may be required.