Delayed Disease Onset Research Articles

The Helminth Parasite Heligmosomoides polygyrus Attenuates EAE in an IL-4Rα-Dependent Manner.

Helminth parasites are effective in biasing Th2 immunity and inducing regulatory pathways that minimize excessive inflammation within their hosts, thus allowing chronic infection to occur whilst also suppressing bystander atopic or autoimmune diseases. Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory lesions within the central nervous system; there are very limited therapeutic options for the progressive forms of the disease and none are curative. Here, we used the experimental autoimmune encephalomyelitis (EAE) model to examine if the intestinal helminth Heligmosomoides polygyrus and its excretory/secretory products (HES) are able to suppress inflammatory disease. Mice infected with H. polygyrus at the time of immunization with the peptide used to induce EAE (myelin-oligodendrocyte glycoprotein, pMOG), showed a delay in the onset and peak severity of EAE disease, however, treatment with HES only showed a marginal delay in disease onset. Mice that received H. polygyrus 4 weeks prior to EAE induction were also not significantly protected. H. polygyrus secretes a known TGF-β mimic (Hp-TGM) and simultaneous H. polygyrus infection with pMOG immunization led to a significant expansion of Tregs; however, administering the recombinant Hp-TGM to EAE mice failed to replicate the EAE protection seen during infection, indicating that this may not be central to the disease protecting mechanism. Mice infected with H. polygyrus also showed a systemic Th2 biasing, and restimulating splenocytes with pMOG showed release of pMOG-specific IL-4 as well as suppression of inflammatory IL-17A. Notably, a Th2-skewed response was found only in mice infected with H. polygyrus at the time of EAE induction and not those with a chronic infection. Furthermore, H. polygyrus failed to protect against disease in IL-4Rα−/− mice. Together these results indicate that the EAE disease protective mechanism of H. polygyrus is likely to be predominantly Th2 deviation, and further highlights Th2-biasing as a future therapeutic strategy for MS.

Genetic inactivation of RIP1 kinase does not ameliorate disease in a mouse model of ALS.

RIP1 kinase is proposed to play a critical role in driving necroptosis and inflammation in neurodegenerative disorders, including Amyotrophic Lateral Sclerosis (ALS). Preclinical studies indicated that while pharmacological inhibition of RIP1 kinase can ameliorate axonal pathology and delay disease onset in the mutant SOD1 transgenic (SOD1-Tg) mice, genetic blockade of necroptosis does not provide benefit in this mouse model. To clarify the role of RIP1 kinase activity in driving pathology in SOD1-Tg mice, we crossed SOD1-Tgs to RIP1 kinase-dead knock-in mice, and measured disease progression using functional and histopathological endpoints. Genetic inactivation of the RIP1 kinase activity in the SOD1-Tgs did not benefit the declining muscle strength or nerve function, motor neuron degeneration or neuroinflammation. In addition, we did not find evidence of phosphorylated RIP1 accumulation in the spinal cords of ALS patients. On the other hand, genetic inactivation of RIP1 kinase activity ameliorated the depletion of the neurotransmitter dopamine in a toxin model of dopaminergic neurodegeneration. These findings indicate that RIP1 kinase activity is dispensable for disease pathogenesis in the SOD1-Tg mice while inhibition of kinase activity may provide benefit in acute injury models.

Prediction of the Age at Onset of Spinocerebellar Ataxia Type 3 with Machine Learning.

In polyglutamine (polyQ) disease, the investigation of the prediction of a patient's age at onset (AAO) facilitates the development of disease-modifying intervention and underpins the delay of disease onset and progression. Few polyQ disease studies have evaluated AAO predicted by machine-learning algorithms and linear regression methods. The objective of this study was to develop a machine-learning model for AAO prediction in the largest spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) population from mainland China. In this observational study, we introduced an innovative approach by systematically comparing the performance of 7 machine-learning algorithms with linear regression to explore AAO prediction in SCA3/MJD using CAG expansions of 10 polyQ-related genes, sex, and parental origin. Similar prediction performance of testing set and training set in each models were identified and few overfitting of training data was observed. Overall, the machine-learning-based XGBoost model exhibited the most favorable performance in AAO prediction over the traditional linear regression method and other 6 machine-learning algorithms for the training set and testing set. The optimal XGBoost model achieved mean absolute error, root mean square error, and median absolute error of 5.56, 7.13, 4.15 years, respectively, in testing set 1, with mean absolute error (4.78 years), root mean square error (6.31 years), and median absolute error (3.59 years) in testing set 2. Machine-learning algorithms can be used to predict AAO in patients with SCA3/MJD. The optimal XGBoost algorithm can provide a good reference for the establishment and optimization of prediction models for SCA3/MJD or other polyQ diseases. © 2020 International Parkinson and Movement Disorder Society.

Novel Selenium-based compounds with therapeutic potential for SOD1-linked amyotrophic lateral sclerosis.

BackgroundAmyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits.MethodsWe have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model.FindingWe have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds.InterpretationOur finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19).FundingProject funding was supported by the 10.13039/100000971ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the 10.13039/100009950Ministry of Education, Culture, Sports, Science and Technology (10.13039/501100001700MEXT), Japan.

Preclinical characterization of itacitinib (INCB039110), a novel selective inhibitor of JAK1, for the treatment of inflammatory diseases

Pharmacological modulation of the Janus kinase (JAK) family has achieved clinically meaningful therapeutic outcomes for the treatment of inflammatory and hematopoietic diseases. Several JAK1 selective compounds are being investigated clinically to determine their anti-inflammatory potential. We used recombinant enzymes and primary human lymphocytes to assess the JAK1 specificity of itacitinib (INCB039110) and study inhibition of signal transducers and activators of transcription (STAT) signaling. Rodent models of arthritis and inflammatory bowel disease were subsequently explored to elucidate the efficacy of orally administered itacitinib on inflammatory pathogenesis. Itacitinib is a potent and selective JAK1 inhibitor when profiled against the other JAK family members. Upon oral administration in rodents, itacitinib achieved dose-dependent pharmacokinetic exposures that highly correlated with STAT3 pharmacodynamic pathway inhibition. Itacitinib ameliorated symptoms and pathology of established experimentally-induced arthritis in a dose-dependent manner. Furthermore, itacitinib effectively delayed disease onset, reduced symptom severity, and accelerated recovery in three distinct mouse models of inflammatory bowel disease. Low dose itacitinib administered via cannula directly into the colon was highly efficacious in TNBS-induced colitis but with minimal systemic drug exposure, suggesting localized JAK1 inhibition is sufficient for disease amelioration. Itacitinib treatment in an acute graft-versus-host disease (GvHD) model rapidly reduced inflammatory markers within lymphocytes and target tissue, resulting in a marked improvement in disease symptoms. This is the first manuscript describing itacitinib as a potent and selective JAK1 inhibitor with anti-inflammatory activity across multiple preclinical disease models. These data support the scientific rationale for ongoing clinical trials studying itacitinib in select GvHD patient populations.

Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints.

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that <25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

3016 – SECRETORY MPP3 REINFORCE MYELOID DIFFERENTIATION TRAJECTORY AND AMPLIFY MYELOID CELL PRODUCTION

Hematopoietic stem cells (HSC) and their downstream lineage-biased multipotent progenitors (MPP) tailor blood production and control myelopoiesis on demand. However, we are still lacking the resolution of myeloid differentiation trajectories and cellular heterogeneity in MPP populations. Here, we show that myeloid-biased MPP3 are composed of several molecular subsets, with a distinct population of myeloid-primed cells with high endoplasmic reticulum (ER) volume and FcgR expression. Interestingly, FcgR+/ERhi MPP3 secrete high levels of IL-6 and TNFa cytokines upon inflammatory stimuli, and have faster division rates than ERlo MPP3. Moreover, condition media containing cytokines secreted by stimulated MPP3 promotes myeloid cell production from naïve HSCs, MPP3, and MPP4. These results suggest that FcgR+/ERhi MPP3 represent a transitional population toward granulocyte/macrophage progenitor (GMP) commitment, which could function as a self-reinforcing amplification compartment for myeloid differentiation in stress conditions. We next use our inducible Scl-tTA:TRE-BCR/ABL (BA) mouse model of myeloproliferative neoplasm (MPN) to investigate their role in leukemia. We find a massive expansion of FcgR+/ERhi MPP3 associated with a molecular remodeling of the MPP3 compartment and constitutive secretion of a unique set of cytokines, distinct from the ones produced in stress conditions. In particular, Galectin-3 (Gal3) is highly expressed by leukemic MPP3, and BA mice deficient for Gal3 (BA:Gal3-/-) show significantly reduced numbers of FcgR+/ERhi MPP3 and GMPs, and delayed disease onset. These results demonstrate the importance of FcgR+/ERhi MPP3 in amplifying myeloid cell production. Together, they identify a novel regulatory function for a subset of secretory MPP3 that controls myeloid differentiation through lineage-priming and cytokine production.

Absence of Subcerebral Projection Neurons Is Beneficial in a Mouse Model of Amyotrophic Lateral Sclerosis.

ObjectiveRecent studies carried out on amyotrophic lateral sclerosis patients suggest that the disease might initiate in the motor cortex and spread to its targets along the corticofugal tracts. In this study, we aimed to test the corticofugal hypothesis of amyotrophic lateral sclerosis experimentally.Methods Sod1 G86R and Fezf2 knockout mouse lines were crossed to generate a model that expresses a mutant of the murine Sod1 gene ubiquitously, a condition sufficient to induce progressive motor symptoms and premature death, but genetically lacks corticospinal neurons and other subcerebral projection neurons, one of the main populations of corticofugal neurons. Disease onset and survival were recorded, and weight and motor behavior were followed longitudinally. Hyper‐reflexia and spasticity were monitored using electromyographic recordings. Neurodegeneration and gliosis were assessed by histological techniques.ResultsAbsence of subcerebral projection neurons delayed disease onset, reduced weight loss and motor impairment, and increased survival without modifying disease duration. Absence of corticospinal neurons also limited presymptomatic hyper‐reflexia, a typical component of the upper motoneuron syndrome.InterpretationMajor corticofugal tracts are crucial to the onset and progression of amyotrophic lateral sclerosis. In the context of the disease, subcerebral projection neurons might carry detrimental signals to their downstream targets. In its entirety, this study provides the first experimental arguments in favor of the corticofugal hypothesis of amyotrophic lateral sclerosis. ANN NEUROL 2020;88:688–702

N-Acylethanolamine Acid Amidase contributes to disease progression in a mouse model of multiple sclerosis

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.

Muscle BDNF: a potential therapeutic target for Kennedy's disease.

Muscle BDNF: a potential therapeutic target for Kennedy's disease.

Common Variants Coregulate Expression of GBA and Modifier Genes to Delay Parkinson's Disease Onset.

Background GBA mutations are numerically the most significant genetic risk factor for Parkinson's disease (PD), yet these mutations have low penetrance, suggesting additional mechanisms.ObjectivesThe objective of this study was to determine if the penetrance of GBA in PD can be explained by regulatory effects on GBA and modifier genes.MethodsGenetic variants associated with the regulation of GBA were identified by screening 128 common single nucleotide polymorphisms (SNPs) in the GBA locus for spatial cis‐expression quantitative trail locus (supported by chromatin interactions).ResultsWe identified common noncoding SNPs within GBA that (1) regulate GBA expression in peripheral tissues, some of which display α‐synuclein pathology and (2) coregulate potential modifier genes in the central nervous system and/or peripheral tissues. Haplotypes based on 3 of these SNPs delay disease onset by 5 years. In addition, SNPs on 6 separate chromosomes coregulate GBA expression specifically in either the substantia nigra or cortex, and their combined effect potentially modulates motor and cognitive symptoms, respectively.ConclusionsThis work provides a new perspective on the haplotype‐specific effects of GBA and the genetic etiology of PD, expanding the role of GBA from the gene encoding the β‐glucocerebrosidase (GCase) to that of a central regulator and modifier of PD onset, with GBA expression itself subject to distant regulation. Some idiopathic patients might possess insufficient GBA‐encoded GCase activity in the substantia nigra as the result of distant regulatory variants and therefore might benefit from GBA‐targeting therapeutics. The SNPs’ regulatory impacts provide a plausible explanation for the variable phenotypes also observed in GBA‐centric Gaucher's disease and dementia with Lewy bodies. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, LLC on behalf of International Parkinson and Movement Disorder Society.

Pertussis toxin treatment promotes a B cell driven disease during TH17 EAE

Abstract Pertussis toxin (PTX) is an enigmatic co-adjuvant that is required for disease induction in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the functional role of PTX during adoptive transfer TH17-EAE. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested and cultured from MOG35–55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. Recipient mice were treated with PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX delayed disease onset but did not alter disease severity or incidence. We found that PTX reduced trafficking of myeloid cells and inflammatory T helper cells in the CNS. Importantly, PTX treatment increased the number of B cells in the brain with increased antigen presenting function, suggesting an altered disease toward a B cell-dependent pathology. When disease progression was compared between WT and μMT mice treated with PTX or PBS, there was no significant difference in TH17-EAE in the PBS treated groups. However, PTX treatment significantly delayed disease onset, severity and incidence in the μMT mice vs. WT. In conclusion, our current data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype and therefore promotes a B cell driven disease. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study B cell activity during multiple sclerosis.

NAD+ attenuates experimental autoimmune encephalomyelitis through induction of CD11b+ gr-1+ myeloid-derived suppressor cells.

Objective: To investigate the effects of nicotinamide adenine dinucleotide (NAD+) on the pathogenesis of the animal model for multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE).Methods: EAE model was induced by myelin oligodendrocyte protein (MOG 35-55). Clinical scores of EAE were measured in mice with or without NAD+ treatment. Hematoxylin and Eosin (HE) and Luxol Fast Blue (LFB) staining were performed to assess inflammation and demyelination, respectively. Expressions of target proteins were measured by Western blot. The numbers of myeloid-derived suppressor cells (MDSCs) were measured by immunofluorescent staining and flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of inflammatory cytokine in serum.Results: NAD+ treatment could decrease inflammatory cells and demyelination foci, attenuate the clinical scores of EAE and slightly delay disease onset. Western blot showed that NAD+ treatment up-regulated the expression of phosphorylated-STAT6 (p-STAT6) and SIRT1. Besides, NAD+ treatment up-regulated the expression of p-IκB and down-regulated the expression of p-NF-κB. In addition, NAD+ treatment could increase the numbers of CD11b+ gr-1+ MDSCs and the expression of Arginase-1. Moreover, NAD+ treatment up-regulated the expressions of IL-13 and down-regulated the expression of IFN-γ and IL-17.Conclusions: The present study demonstrated that NAD+ treatment may induce the CD11b+ gr-1+ MDSCs to attenuate EAE via activating the phosphorylation of STAT6 expression. Therefore, NAD+ should be considered as a potential novel therapeutic strategy for MS.

New Hope for Delaying Clinical Onset of Rheumatoid Arthritis: Early Intervention with Rituximab

Rheumatoid arthritis (RA) is a highly prevalent autoimmune disease that affects 16 million people globally. It is caused by an inflammatory autoimmune response that results in swelling of the joints and chronic pain. While we know that RA operates via the immune system, the specific mechanisms of RA pathogenesis are not fully understood, making diagnosis and treatment options limited. Rituximab, a monoclonal CD20 antibody, is a current form of RA treatment that specifically targets autoreactive B-cells to help mitigate the symptoms of RA at the clinical stage. Gerlag et al. (2019) outline a preventative window of opportunity for preclinical RA intervention with rituximab and identified two predictive biomarkers through exploratory methods. Their findings demonstrate that early administration of rituximab during preclinical RA delays disease onset and impedes its progression. This timeframe for intervention offers a promising first step for future studies investigating RA mechanisms and early treatments.

Spinal Motoneuron TMEM16F Acts at C-boutons to Modulate Motor Resistance and Contributes to ALS Pathogenesis

Neuronal Ca2+ entry elicited by electrical activity contributes to information coding via activation of K+ and Cl- channels. While Ca2+-dependent K+ channels have been extensively studied, the molecular identity and role of Ca2+-activated Cl- channels (CaCCs) remain unclear. Here, we demonstrate that TMEM16F governs a Ca2+-activated Cl- conductance in spinal motoneurons. We show that TMEM16F is expressed in synaptic clusters facing pre-synaptic cholinergic C-boutons in α-motoneurons of the spinal cord. Mice with targeted exon deletion in Tmem16f display decreased motor performance under high-demanding tasks attributable to an increase in the recruitment threshold of fast α-motoneurons. Remarkably, loss of TMEM16F function in a mouse model of amyotrophic lateral sclerosis (ALS) significantly reduces expression of an activity-dependent early stress marker and muscle denervation, delays disease onset, and preserves muscular strength only in male ALS mice. Thus, TMEM16F controls motoneuron excitability and impacts motor resistance as well as motor deterioration in ALS.

The influence of metallothionein treatment and treadmill running exercise on disease onset and survival in SOD1G93A amyotrophic lateral sclerosis mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterised by the degeneration of motor neurons innervating skeletal muscle. The mechanisms underlying neurodegeneration in ALS are not yet fully elucidated, and with current therapeutics only able to extend lifespan by a matter of months there is a clear need for novel therapies to increase lifespan and patient quality of life. Here, we evaluated whether moderate-intensity treadmill exercise and/or treatment with metallothionein-2 (MT2), a neuroprotective protein, could improve survival, behavioural or neuropathological outcomes in SOD1G93A familial ALS mice. Six-week-old female SOD1G93A mice were allocated to one of four treatment groups: MT2 injection, i.m.; moderate treadmill exercise; neither MT2 nor exercise; or both MT2 and exercise. MT2-treated mice survived around 3% longer than vehicle-treated mice, with this mild effect reaching statistical significance in Cox proportional hazards analysis once adjusted for potential confounders. Mixed model body weight trajectories over time indicated that MT2-treated mice, with or without exercise, reached maximum body weight at a later age, suggesting a delay in disease onset of around 4% compared to saline-treated mice. Exercise alone did not significantly increase survival or delay disease onset, and neither exercise nor MT2 substantially ameliorated gait abnormalities or muscle strength loss. We conclude that neither exercise nor MT2 treatment was detrimental in female SOD1G93A mice, and further study could determine whether the mild effect of peripheral MT2 administration on disease onset and survival could be improved via direct administration of MT2 to the central nervous system.

A Novel Targeted Approach to Achieve Immune System Reset: CD45-Targeted Antibody Drug Conjugates Enable Autologous HSCT and Ameliorate Disease in Preclinical Autoimmune Disease Models

Resetting the immune system through autologous hematopoietic stem cell transplant (autoHSCT) is a highly effective treatment in patients with autoimmune diseases (AID). AutoHSCT achieved long-term remission in patients with relapsed refractory and secondary progressive multiple sclerosis (Muraro 2017), superior to their previous standard of care (Burt 2019). AutoHSCT in scleroderma patients achieved superior outcomes in two randomized studies (Tyndall 2014, Sullivan 2018). These impressive results are achieved by both eradication of autoreactive immune effector cells and re-establishment of a self-tolerant immune system, i.e., immune system reset. However, only a fraction of eligible patients undergo autoHSCT, in part due to toxicity associated with current conditioning regimens that remove the disease-causing cells. To enable more patients to benefit from immune reset without debilitating chemotherapy, we generated novel anti-human CD45 ADCs that cross react with NHP and evaluated these for the ability to deplete hematopoietic cells in vitro and in vivo (Fig.1). In vitro the CD45-ADC efficiently killed human CD34+ progenitors and peripheral CD3+ T cells from both healthy donors and MS patients. In vivo in humanized NSG mice, single doses of the CD45-ADCs were well-tolerated and led to depletion of human hematopoietic cells in BM (Fig. 1A) and periphery. In NHPs, single doses of CD45-ADCs were well tolerated and depleted BM HSCs and peripheral lymphocytes (Fig.1B). CD45-ADC treatment of hNSGs with sclerodermatous xenoGVHD resulted in resolution of symptoms (Fig. 1C). To model the complete immune reset approach in mouse models of AID, we generated a tool anti-mouse CD45 ADC. A single-dose enabled full myeloablation (>99% depletion of LT-HSCs) and complete donor chimerism with congenic HSCT (>90% chimerism at 16 weeks). In an adoptive transfer model of type I diabetes, treatment with a single dose of CD45-ADC and congenic HSCT led to disease prevention. In a murine model of MS EAE, a single dose of the CD45-ADC followed by congenic HSCT enabled full donor chimerism; treatment prior to disease onset significantly delayed disease onset and reduced disease severity; treatment after disease onset also halted progression of symptoms. These data demonstrate that CD45-ADC conditioning followed by congenic HSCT is sufficient for full myeloablation and immune reset. Experiments are ongoing, and evaluation of this ADC in murine models of diabetes and arthritis will be presented. These results indicate that targeted immune depletion with a single dose of CD45-ADC may be sufficient to enable auto-HSCT and immune reset in multiple AID indications. Targeted conditioning with CD45-ADC may enable more patients to benefit from immune reset through removal of pathogenic cells and autoHSCT without the morbidity and mortality associated with current chemotherapeutic conditioning.

Protocol for the Use of the Ketogenic Diet in Preclinical and Clinical Practice.

Many age-related diseases are associated with metabolic abnormalities, and dietary interventions may have some benefit in alleviating symptoms or in delaying disease onset. Here, we review the commonly used best practices involved in applications of the ketogenic diet to facilitate its translation into clinical use. The findings reveal that better education of physicians is essential for applying the optimum diet and monitoring its effects in clinical practice. In addition, investigators should carefully consider potential confounding factors prior to commencing studies involving a ketogenic diet. Most importantly, current studies should improve their reporting on ketone levels as well as on the intake of both macro- and micronutrients. Finally, more detailed studies on the mechanism of action are necessary to help identify potential biomarkers for response prediction and monitoring, and to uncover new drug targets to aid the development of novel treatments.

Targeting T cell Metabolism to Combat Autoimmunity: Implications for the Future of Type 1 Diabetes Therapeutics

Abstract Advances in the field of immunometabolism have indicated that cellular metabolic programs control T cell differentiation and effector functions. As CD4+ T cells are significant contributors to destruction of the insulin-secreting β cell in Type 1 Diabetes (T1D), strategies to limit autoreactive T cell activation and promote durable tolerance have been explored in an effort to prevent or delay disease onset. Interestingly, substantial evidence indicates that autoreactive T cells are at a heightened activation status associated with dysregulation of metabolic pathways, with distinct nutrient preferences and needs of their own. While largely unexplored in T1D, specific metabolic pathways have been exploited to correct known defects in several autoimmune diseases including but not limited to Systemic Lupus Erythematosus (SLE), Multiple Sclerosis (MS), and Rheumatoid Arthritis (RA). These therapeutic strategies have been focused on targeting glycolysis, which is the major pathway used by activated T cells as it is required for acquisition of effector functions. Use of glycolysis inhibitors such as 2-Deoxy-d-glucose (2-DG), have shown impressive success in limiting T cell mediated damage that could prevent or reverse onset of autoimmunity. More importantly, while specificity has been a major hurdle for use of immunotherapies, regulating immunity by targeting metabolism resulted in selective targeting of autoreactive T cells while leaving global immune function intact, a phenomenon commonly referred to as “cellular selectivity based on demand”. This review will outline examples of metabolic regulation success in autoimmunity and provide evidence to support use of these inhibitors in T1D, where new therapeutic strategies are desperately needed.

Cromolyn sodium delays disease onset and is neuroprotective in the SOD1G93A Mouse Model of amyotrophic lateral sclerosis

Accumulating evidence suggests that neuroinflammatory processes are implicated in the initiation and progression of amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated an increase in microgliosis and astrogliosis in the lumbar spinal cord of SOD1G93A transgenic mice before the onset of symptoms, a neuroinflammatory response which correlated with disease progression. Importantly, early stage homeostatic microglia enhanced motor neuron survival, while pro-inflammatory microglia were toxic to motor neurons in the SOD1G93A mice. Recent studies from our group have demonstrated that cromolyn sodium, an FDA approved compound, exerts neuroprotective effects in mouse models of Alzheimer’s disease by altering microglial cell activation. Here, we tested the neuroprotective and anti-inflammatory effects of cromolyn sodium in the SOD1G93A mouse model of ALS. Our results indicate that cromolyn sodium treatment significantly delayed the onset of neurological symptoms, and improved deficits in PaGE performance in both male and female mice, however, there was only an effect on survival in female mice. Furthermore, there was a significant increase in motor neuron survival in the lumbar spinal cord as well as a significant decrease in the denervation of the neuromuscular junction of the tibialis anterior muscle in cromolyn treated transgenic SOD1G93A mice. Lastly, cromolyn treatment decreased the expression of pro-inflammatory cytokines/chemokines in the lumbar spinal cord and plasma and decreased mast cell degranulation in the tibialis anterior muscle of transgenic SOD1G93A mice. Together, these findings suggest that cromolyn sodium provides neuroprotection in the SOD1G93A mice by decreasing the inflammatory response.