Combined Regulatory T-Lymphocyte and IL-2 Treatment Is Safe, Tolerable, and Biologically Active for 1 Year in Persons With Amyotrophic Lateral Sclerosis

BACKGROUND AND OBJECTIVES: In a phase 1 amyotrophic lateral sclerosis (ALS) study, autologous infusions of expanded regulatory T-lymphocytes (Tregs) combined with subcutaneous interleukin (IL)-2 were safe and well tolerated. Treg suppressive function increased and disease progression stabilized during the study. The present study was conducted to confirm the reliability of these results. METHODS: Participants with ALS underwent leukapheresis, and their Tregs were isolated and expanded in a current Good Manufacturing Practice facility. Seven participants were randomly assigned in a 1:1 ratio to receive Treg infusions (1 × 10 RESULTS: The Treg/IL-2 treatments were safe and well tolerated, and Treg suppressive function was higher in the active group of the RCT. A meaningful evaluation of progression rates in the RCT between the placebo and active groups was not possible due to the limited number of enrolled participants aggravated by the COVID-19 pandemic. In the 24-week OLE, the Treg/IL-2 treatments were also safe and well tolerated in 8 participants who completed the escalating doses. Treg suppressive function and numbers were increased compared with baseline. Six of 8 participants changed by an average of -2.7 points per the ALS Functional Rating Scale-Revised, whereas the other 2 changed by an average of -10.5 points. Elevated levels of 2 markers of peripheral inflammation (IL-17C and IL-17F) and 2 markers of oxidative stress (oxidized low-density lipoprotein receptor 1 and oxidized LDL) were present in the 2 rapidly progressing participants but not in the slower progressing group. DISCUSSION: Treg/IL-2 treatments were safe and well tolerated in the RCT and OLE with higher Treg suppressive function. During the OLE, 6 of 8 participants showed slow to no progression. The 2 of 8 rapid progressors had elevated markers of oxidative stress and inflammation, which may help delineate responsiveness to therapy. Whether Treg/IL-2 treatments can slow disease progression requires a larger clinical study (ClinicalTrials.gov number, NCT04055623). CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that Treg infusions and IL-2 injections are safe and effective for patients with ALS

Spectral Analysis of the Primary Flight Focal Plane Arrays for the Thermal Infrared Sensor

Thermal Infrared Sensor (TIRS) is a (1) New longwave infrared (10 - 12 micron) sensor for the Landsat Data Continuity Mission, (2) 185 km ground swath; 100 meter pixel size on ground, (3) Pushbroom sensor configuration. Issue of Calibration are: (1) Single detector -- only one calibration, (2) Multiple detectors - unique calibration for each detector -- leads to pixel-to-pixel artifacts. Objectives are: (1) Predict extent of residual striping when viewing a uniform blackbody target through various atmospheres, (2) Determine how different spectral shapes affect the derived surface temperature in a realistic synthetic scene

Period and chemical evolution of SC stars

The SC and CS stars are thermal-pulsing AGB stars with C/O ratio close to unity. Within this small group, the Mira variable BH Cru recently evolved from spectral type SC (showing ZrO bands) to CS (showing weak C2). Wavelet analysis shows that the spectral evolution was accompanied by a dramatic period increase, from 420 to 540 days, indicating an expanding radius. The pulsation amplitude also increased. Old photographic plates are used to establish that the period before 1940 was around 490 days. Chemical models indicate that the spectral changes were caused by a decrease in stellar temperature, related to the increasing radius. There is no evidence for a change in C/O ratio. The evolution in BH Cru is unlikely to be related to an on-going thermal pulse. Periods of the other SC and CS stars, including nine new periods, are determined. A second SC star, LX Cyg, also shows evidence for a large increase in period, and one further star shows a period inconsistent with a previous determination. Mira periods may be intrinsically unstable for C/O ~ 1; possibly because of a feedback between the molecular opacities, pulsation amplitude, and period. LRS spectra of 6 SC stars suggest a feature at wavelength > 15 micron, which resembles one recently attributed to the iron-sulfide troilite. Chemical models predict a large abundance of FeS in SC stars, in agreement with the proposed association.Comment: 14 pages, 20 figures. MNRAS, 2004, accepted for publication. Janet Mattei, one of the authors, died on 22 March, 2004. This paper is dedicated to her memor

Fractalkine Signaling Regulates the Inflammatory Response in an α-Synuclein Model of Parkinson Disease

<div><p>Background</p><p>Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer’s disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1 (fractalkine) and its receptor (CX3CR1) have important roles in modulating neuroinflammation.</p><p>Methods</p><p>To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an <i>in vivo</i> mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and <i>in vitro</i> phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn.</p><p>Results</p><p>We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn.</p><p>Conclusion</p><p>Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD.</p></div

CX3CR1-/- primary microglia show decreased internalization of α-syn and phagocytosis <i>in vitro</i>.

<p>(A) DIC and fluorescent images from primary microglia isolated from WT and CX3CR1-/- following exposure to Nile Red fluorescent microspheres for 30 minutes. Scale = 10um. (B) Quantification of the average number of beads internalized per microglia and statistically quantified using Student’s t-test (p = 0.0019). (C) Internalization of aggregated α-SYN (anti-Syn204, red) in primary microglia (IBA1 blue) from WT and CX3CR1-/- mice. Scale = 10um. Images taken using Leica Microsystems TCS SP5 Visible-Upright Confocal microscope.</p

The effect of CX3CR1-/- on α-syn-induced TH+ neuron loss at 6 months post transduction of AAV2-SYN.

<p>(A) TH+ neuron chromogen staining (Ni-DAB) of the ipsilateral SNpc 6 months post transduction in AAV2-GFP or AAV2-SYN injected WT and CX3CR1-/- mice. (B) Unbiased stereological cell counts of TH+ neurons in the ipsilateral SNpc from WT and CX3CR1-/- mice at 6 months post transduction of AAV2-GFP or AAV2-SYN. Unbiased cell counts are reported as a percent of the contralateral side. Two-way ANOVA with Sidak’s multiple comparisons test (p<0.001) (n = 7–9 mice per group). Images captured using Nikon Eclipse E800 microscope.</p

CX3CR1-/- primary microglia show decreased antigen processing in <i>in vitro</i>.

<p>(A) Antigen processing of DQ Ovalbumin (red) is decreased in CX3CR1-/- primary microglia compared to WT primary microglia following a 30 minute treatment. Scale = 10um. (B) ImageJ quantification of corrected total cell fluorescence (CTCF) of processed DQ ovalbumin (red). Using chamber slides for the experiment, each chamber is used as an independent variable with 4–5 pictures per chamber including 5–8 microglia per picture. 4 chambers (1 slide) used per treatment group and experiment replicated 3 times. Students t-test used for statistical analysis (p = 0.0358). Images taken using Leica Microsystems TCS SP5 Visible-Upright Confocal microscope.</p

MHCII expression and IgG deposition is attenuated in CX3CR1-/- mice following α-SYN overexpression.

<p>(A) MHCII expression (red) in the ipsilateral SNpc (TH blue) following 4 week AAV2-SYN transduction (green). Scale bar = 25um. (B) Quantification of MHCII expression at 4 weeks post transduction of AAV2-GFP or AAV2-SYN in WT and CX3CR1-/- mice. Ipsilateral SNpc sections were rated by a reader blinded to the treatment group. Blinded ratings displayed as medians with ranges with a rating of 4 being highest MHCII expression. n = 5-6/group, p = 0.0146 using Kruskal-Wallis test with Dunn’s multiple comparison test. (C) IgG deposition (red) in ipsilateral SNpc (TH green) following 4 weeks AAV2-SYN transduction in WT and CX3CR1-/- mice. Scale bar = 125um. Images taken using Leica Microsystems TCS SP5 Visible-Upright Confocal microscope.</p

Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson’s disease

Abstract Background Parkinson’s disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD. Methods In vitro, we utilized microglia cultures from WT or CIITA −/− mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA −/− mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration. Results Using ICC and DQ-ovalbumin, we show that CIITA −/− microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration. Conclusion Our data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD

α-Synuclein fibrils recruit peripheral immune cells in the rat brain prior to neurodegeneration

Abstract Genetic variation in a major histocompatibility complex II (MHCII)-encoding gene (HLA-DR) increases risk for Parkinson disease (PD), and the accumulation of MHCII-expressing immune cells in the brain correlates with α-synuclein inclusions. However, the timing of MHCII-cell recruitment with respect to ongoing neurodegeneration, and the types of cells that express MHCII in the PD brain, has been difficult to understand. Recent studies show that the injection of short α-synuclein fibrils into the rat substantia nigra pars compacta (SNpc) induces progressive inclusion formation in SNpc neurons that eventually spread to spiny projection neurons in the striatum. Herein, we find that α-synuclein fibrils rapidly provoke a persistent MHCII response in the brain. In contrast, equivalent amounts of monomeric α-synuclein fail to induce MHCII or persistent microglial activation, consistent with our results in primary microglia. Flow cytometry and immunohistochemical analyses reveal that MHCII-expressing cells are composed of both resident microglia as well as cells from the periphery that include monocytes, macrophages, and lymphocytes. Over time, α-Synuclein fibril exposures in the SNpc causes both axon loss as well as monocyte recruitment in the striatum. While these monocytes in the striatum initially lack MHCII expression, α-synuclein inclusions later form in nearby spiny projection neurons and MHCII expression becomes robust. In summary, in the rat α-synuclein fibril model, peripheral immune cell recruitment occurs prior to neurodegeneration and microglia, monocytes and macrophages all contribute to MHCII expression